The low-affinity site of the β1-adrenoceptor and its relevance to cardiovascular pharmacology

Pharmacology & Therapeutics 118 (2008) 303–336

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Pharmacology & Therapeutics

j ourna l homepage: www.e lsev ie r.com/ locate /pharmthera

The low-affinitysiteof theβ1-adrenoceptorand its relevance tocardiovascularpharmacology

Alberto J. Kaumann a,⁎, Peter Molenaar b,c,⁎a Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Physiology Building, Cambridge, Cambridge CB2 3EG, UKb School of Life Sciences, Queensland University of Technology, Brisbane 4001, Australiac Department of Medicine, The Prince Charles Hospital, University of Queensland, Chermside 4032, Australia

⁎ Corresponding authors.E-mail addresses: [email protected] (A.J. Kau

β1AR, β1-adrenoceptor, β2AR, β2-adrenoceptorβ1LAR, low-affinity β1-adrenoceptor binding siteβ1HAR, high-affinity β1-adrenoceptor binding sitecAMP, cyclic AMPCGP12177, 4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-oneCHO, Chinese Hamster OvaryCRE, cAMP response elementCYP, cyanopindololERK, extracellular signal-regulated kinaseIBMX, 3-isobutyl-1-methylxanthineL-NAME,NG-nitro-L-arginine methyl esterNCPA, non-conventional partial agonistL-NMA,NG-methyl-L-arginineL-NMMA,NG-monomethyl-L-arginineMAPK, mitogen-activated protein kinasePDE, phosphodiesterasePKA, cAMP-dependent protein kinasepKD, −log molar equilibrium dissociationconstant for radioligand

0163-7258/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.pharmthera.2008.03.009

A B S T R A C T
A R T I C L E I N F O
Keywords:
Affinity states
β-Adrenoceptor blocking agof catecholamines, but at h

Arrhythmiasβ-BlockersBucindololBupranololCa2+ currentsCa2+ transientsCardiovascularCGP12177[3H]-(−)-CGP12177HeartHuman heartHuman heart failureHeart rateMutagenesisNon-conventional partial agonistsPindololPhosphodiesterasesPolymorphismCardiac and recombinantβ1- and β2-adrenoceptors

ents (β-blockers) that at low concentrations antagonize cardiostimulant effectsigh concentrations also cause cardiostimulation, have been appearing since the

late 1960s. These cardiostimulant β-blockers, coined non-conventional partial agonists, antagonize theeffects of catecholamines through a high-affinity site (β1HAR), but cause cardiostimulation mainly through alow-affinity site (β1LAR) of the myocardial β1-adrenoceptor. The experimental non-conventional partialagonist (−)-CGP12177 increases cardiac L-type Ca2+ current density and Ca2+ transients, shortens actionpotential duration but augments action potential plateau, increases heart rate and force, as well as causesarrhythmic Ca2+ transients and arrhythmic cardiocyte contractions. Other β-blockers, which do not causecardiostimulation, consistently have lower affinity for β1LAR than β1HAR. These sites were verified and thecardiac pharmacology of non-conventional partial agonists confirmed on recombinant β1-adrenoceptors andon β1-adrenoceptors overexpressed into the heart. A targeted mutation of Asp138 to Glu138 virtuallyabolished the pharmacology of β1HAR but left intact the pharmacology of β1LAR. Non-conventional partialagonists may be beneficial for the treatment of peripheral autonomic neuropathy but probably due to theirarrhythmic propensities, may be harmful for the treatment of chronic heart failure.

© 2008 Elsevier Inc. All rights reserved.

Abbreviations:

mann), [email protected] (P. Molenaar).

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http://dx.doi.org/10.1016/j.pharmthera.2008.03.009

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pKi, −log molar equilibrium dissociationconstant for a non-radioactive liganddetermined by competitionwith a radioligandpKB, −log molar equilibrium dissociation constantfor an antagonist estimated from competitiveblockade of the effects of an agonistpKP, −log molar equilibrium dissociation constantfor a partial agonist estimated from competitiveblockade of the effects of a full agonistputative β4AR, putative β4-adrenoceptorPTX, pertussis toxinpEC50, −log molar concentration causing halfmaximal effectsSPAP, secreted placental alkaline phosphatase

304 A.J. Kaumann, P. Molenaar / Pharmacology & Therapeutics 118 (2008) 303–336

TMD, transmembrane domain

Contents

1. Historical survey. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3041.1. 1966–1983 Identification of non-conventional partial agonists . . . . . . . . . . . . . . . . . . . . . . 3041.2. 1984 Initial mechanistic clues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3051.3. 1989–1996 Putative β4AR — a working hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3051.4. 1996 to present . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310

2. β1AR high (H) and low (L) affinity binding sites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3162.1. Evidence from cardiac β1AR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3162.2. Evidence from recombinant β1AR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3182.3. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318

3. Cardiovascular pharmacology of non-conventional partial agonists . . . . . . . . . . . . . . . . . . . . . . . 3183.1. Coupling to the cAMP pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3183.2. Sinoatrial tachycardia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3183.3. Atrial contractility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3203.4. Ventricular contractility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3203.5. Ventricular Ca2+ currents, Ca2+ transients and action potentials.

Are (−)-CGP12177-evoked arrhythmias mediated through β1LAR? . . . . . . . . . . . . . . . . . . . 3213.6. Effects on human β1AR overexpressed into rat heart . . . . . . . . . . . . . . . . . . . . . . . . . . 3213.7. Effects of antagonists: a lead to cardiac β1LAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3223.8. Vascular pharmacology of non-conventional partial agonists . . . . . . . . . . . . . . . . . . . . . . 3233.9. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

4. Pharmacology of non-conventional partial agonists at recombinant β1AR and plausible cardiac relevance . . . 3244.1. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

5. Structural considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3265.1. H and L binding sites or conformational states? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3275.2. Mutation of the Asp138-β1AR to Glu138-β1AR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3285.3. Mutation of the β1AR to β1(β2TMD V)AR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3295.4. The Arg389Gly polymorphism differently affects the function of recombinant β1HAR and β1LAR . . . . 3295.5. Possible insights from the 5-HT1A receptor for β-hydroxyl

and ether oxygen binding of phenoxypropanolamines to β1AR . . . . . . . . . . . . . . . . . . . . . 3305.6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

6. Clinical relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3306.1. Beneficial effects of non-conventional partial agonists . . . . . . . . . . . . . . . . . . . . . . . . . . 3306.2. Harmful effects of non-conventional partial agonists . . . . . . . . . . . . . . . . . . . . . . . . . . 3316.3. Plausible beneficial effects of β-blockers through β1LAR . . . . . . . . . . . . . . . . . . . . . . . . . 3326.4. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

7. Conclusions and outlook. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

1. Historical survey

1.1. 1966–1983 Identification of non-conventional partial agonists

The clinically successful introduction of the β-blocker propranolol bySir James Black at Imperial Chemistry Industries (ICI) in the 60s (Blacket al., 1965) prompted considerable “me too” efforts by other drugcompanies. Some of the β-blockers such as the indoleamine derivativepindolol of the Sandoz Company turned out to also possess small ormoderate agonist properties. While working as a postdoctoral fellowbetween 1966 and 1968 in the laboratory of John Blinks at Harvard, one ofus (AJK) aimed at comparing experimentally the therapeutic ratio and

intrinsic agonist activities of a series ofβ-blockers on isolated tissues fromfeline and guinea pig hearts. To compare the therapeutic ratio, the“affinity” of the blocker forβ-adrenoceptors, i.e. the blocker concentrationwhich inprinciple occupied half of the receptor population or equilibriumdissociation constant KB, was estimated from the antagonism of the car-diostimulant effects of (−)-isoprenaline and compared with the thresholdβ-blocker concentration that caused a reduction in contractile force(cardiodepression). During these studies it was found that several β-blockers produced cardiostimulant effects that were smaller than theeffects ofmaximally effective concentrations of (−)-isoprenaline, andweretherefore considered to be partial agonists. Among the partial agonistswere the experimental compounds DCI (dichloroisoprenaline) and INPEA

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(1-(4-nitrophenyl)-1-hydroxy-2-isopropylaminoethane) as well as pro-nethalol, practolol and pindolol. Unlike full agonists, which can producemaximum effects at very low receptor occupancy (spare receptors,Stephenson, 1956), partial agonists were considered at that time tosaturate a receptor population at the concentration that caused theirmaximum effects. It was therefore expected from the prevailing receptortheory that the KP of a partial agonist should match the concentrationproducing half maximum effects (EC50). This was indeed found to be thecase for DCI, INPEA and practolol, which were considered classical orconventional partial agonists. However, pindolol was unusual because itdid not behave as a classical partial agonist.

Pindolol concentrations causing cardiostimulation and half maximalagonist effectswere considerablygreater (at least one order ofmagnitude)than the KB determined against (−)-isoprenaline. Later alprenolol andoxprenolol were also found to possess non-classical behaviour, similar topindolol, prompting the classification into two forms of β-blockers withstimulant intrinsic activity, namely classical and non-conventional partialagonists. A non-conventional partial agonist causes receptor activationonly at high receptor occupancies (Kaumann,1973). Although this earlyconcept has later been refinedwith thediscoveryof highand lowaffinitystates of the β1-adrenoceptor (β1AR) by Pak and Fishman (1996), theconcept and term of non-conventional partial agonist has beenincreasingly used in the literature since 1973.

Perhaps not surprisingly, other indoleamines or compounds closelyrelated chemically to pindolol, were subsequently also found to be non-conventional partial agonists. These compounds included tert-butylpin-dolol, hydroxybenzylpindolol, iodo-hydroxybenzylpindolol (Fig. 1), car-azolol and tert-butylcarazolol (Kaumann et al., 1979; Bearer et al., 1980)(Table 1). Interestingly, hydroxybenzylpindolol and p-cloroisoprenalinewere shown to stimulate the adenylyl cyclase as a non-conventionalpartial agonist and classical partial agonist respectively (Fig. 2), in feline

Fig. 1. Non-conventional partial agonist activity of iodo-hydroxybenzylpindolol (I-HYP)in 3 regions of feline heart. Concentration–effect curves of (−)-isoprenaline (ISO), I-HYPand ISO in the presence of I-HYP 0.6 μM on spontaneously beating right atrium(sinoatrial pacemaker), as well as electrically paced left atrium and right ventricularpapillary muscle. (−)-ISO is a full agonist at β1HAR and β2AR in feline heart. Occupancyof receptors (y, broken lines) was calculated with KP values (○), estimated from thesurmountable antagonism by I-HYP of the effects of ISO (Bearer et al., 1980). Note thatreceptor occupancy curves, normalised to maximum I-HYP effects are situated at lowerI-HYP concentrations than the I-HYP curves for cardiostimulation.

cardiac membranes in test tube experiments (Kaumann et al., 1978). Thisbiochemical system was also activated by catecholamines through β-adrenoceptorswhichwere blocked byβ-blockerswith virtually the sameaffinity observed in intact feline cardiac tissues (Kaumann & Birnbaumer,1973,1974; Kaumann et al.,1980). Somenon-indoleaminergicβ-blockers,such as alprenolol, oxprenolol and pronethalol also were non-conven-tional partial agonists (Kaumann & Blinks, 1980b).

In 1983 Staehelin introduced a highly hydrophilic benzimidazolonecompound, CGP12177, as a β-blocker and radioligand (Staehelin et al.,1983). (±)-CGP12177 also possessed marked partial agonist activity onfeline myocardium (Kaumann, 1983). Interestingly, (±)-CGP12177 hadan agonist potency that was 2 orders of magnitude lower than itsblocking potency in three cardiac regions of feline heart, as seenpreviously with other non-conventional partial agonists (Table 1). Since1983 CGP12177 has been a valuable tool towards the understanding ofthe interaction between non-conventional partial agonists and βAR.

1.2. 1984 Initial mechanistic clues

Quantitative analysis of pindolol-evoked cardiostimulation revealedbiphasic concentration–effect curves that extended over 4 log concentra-tion units (Kaumann & Blinks, 1980a), suggesting complex mechanisms.The first question was whether the pindolol-evoked cardiostimulanteffects were mediated through βAR. Propranolol (1 μM, the maximumpossible non-depressant concentration) antagonized the low-sensitivitycomponent of cardiostimulation less than the relatively high-sensitivitycomponent (Kaumann&Blinks,1980a). Interpretationof these resultswasdifficult and inconclusive because the above studies had been carried outwith racemicpindolol andnoplausiblemechanismwas envisioned for thedissociation between blockade and stimulation with non-conventionalpartial agonists (Kaumann & Blinks, 1980b). This situation became clearerwith the use of the individual enantiomers, (−)-pindolol and (+)-pindololon guinea pig atrium (Walter et al., 1984). Both enantiomers hadcardiostimulant effects. It was found that (−)-pindolol elicited cardiosti-mulationwith a biphasic concentration–effect curve that extended over 4log units. The high-sensitivity (H) component but not the low-sensitivity(L) component was antagonized by the β1AR-selective antagonist (−)-bisoprolol. The L-component was, however, antagonized with moderatepotency by the β-blocker (−)-bupranolol, shown to have high affinity forboth cardiac β1AR and β2AR (Morris et al., 1981; Lemoine et al., 1985).Neither component was affected by the β2AR-selective antagonistICI118,551 (Walter et al., 1984). In contrast, the concentration–effectcurve for (+)-pindolol was monophasic and its cardiostimulant effectswere prevented by the β2AR-selective antagonist ICI118,551, i.e. mediatedthrough β2-adrenoceptors. Walter et al. (1984) concluded that thecardiostimulation caused by low concentrations of (−)-pindolol ismediated through a high-affinity (H) β1AR population which mediatesantagonism of the effects of (−)-isoprenaline, but the cardiostimulationcaused by high concentrations of (−)-pindolol is mediated through a low-affinity (L) receptor. (Fig. 3). However, it was not clear at that time forwhich βAR subtype (−)-pindolol had low affinity.

1.3. 1989–1996 Putative β4AR — a working hypothesis

The evidence accumulated with non-conventional partial agonistsled to the suggestion that the heart could express a third β-adrenoceptor,in addition to co-existingβ1ARandβ2AR (Kaumann,1989). The proposalsummarised the properties of this hypothetical receptor as activated bythe agonists (−)-pindolol and related indoleamines, aswell as CGP12177,and other β-blockers such as oxprenolol and (−)-alprenolol. The effectsof these non-conventional partial agonistswere resistant to blockade bypropranolol and bisoprolol but blocked by moderately low (1 μM)concentrations of (−)-bupranolol (Walter et al., 1984; Kaumann, 1989).

Shortly after the proposal of a third cardiac β-adrenoceptor (Kaumann,1989) the cloning of the β3AR was reported (Emorine et al., 1989). Thiscreated a great deal of confusion at that time because several non-

Table 1Agonist potencies (pEC50) and affinity estimates (pKB, pKP) of non-conventional partial agonists (NCPA) for β1HAR and β1LAR

NCPA Species System/tissue Binding radioligand Stimulation Intrinsic activitya pEC50 pKβ1H pKβ1L pKβ1H-pEC50d or pKβ1H-pKβ1L

e References

[3H]-(−)-CGP12177 Human Recombinant β1AR [3H]-(−)-CGP12177 9.3b 6.6b 2.7e Joseph et al., 2004a

(−)-CGP12177 cAMP 0.13 7.6 9.3b 1.7e

cAMP 0.13 7.6 9.9c 2.3d

Arg389β1AR [125I](−)-CYPK cAMP 0.06 7.6 9.4 1.8e Joseph et al., 2004bGly389β1AR cAMP 1.1 8.2 9.2 1.0e

Right atrium Inotropic 0.11 7.3 Kaumann, 1996Inotropic 0.15h 7.3h

Right atrium [3H]-(−)-CGP12177 9.4f 7.1b,f 2.3e Sarsero et al., 20038.9g 7.2b,g 1.7e

Inotropic 0.13f 7.2f 9.4f 2.2d

Inotropic 0.23f,h 7.3f,h 8.9g 1.6d

Lusitropic 0.35f,h 7.4f,h

Inotropic 0.68f,h,i 7.7f,h,i 9.4f 1.7d

Inotropic 0.08g,h 7.0g,h 8.9g 1.9d

Inotropic 0.59g,h,i 7.4g,h,i 8.9g 1.5d

Left atrium Inotropic 0.40g,h,i 7.4g,h,i

Right ventricular Inotropic 0.34g,h,i 7.4g,h,i

trabeculae Lusitropic 0.70g,h,i 7.5g,h,i

Left ventricle Inotropic 0.21g,h,i 7.5g.h.i

trabeculae Lusitropic 0.72g,h,i 7.4g,h,i

(±)-CGP12177 Recombinant β1AR Adenylyl cyclase 0.45 7.8 Konkar et al., 2000a0.43 7.9 Konkar et al., 2000b

[3H]-(−)-CGP12177 9.8 2.1d Baker et al., 2003cAMP 0.63 7.7CRE luciferase 0.38 7.4 Baker, J.G., 2005

(±)-CGP12177 Cat Right atrium Chronotropic 0.80 7.9 10.1c 2.2 Kaumann, 1983Left atrium Inotropic 0.75 7.8 9.6c 1.8d

Right ventricularpapillary muscle Inotropic 0.64 7.2 9.2c 2.0d

(−)-CGP12177 Rat Right atrium Chronotropic 0.60 7.3 Kaumann and Molenaar, 1996Left atrium Inotropic 0.60 7.6

[3H]-(−)-CGP12177 Left atrium [3H]-(−)-CGP12177 9.4b 7.5b 1.9e Sarsero et al., 1998Left ventricle 9.3b 7.2b 2.1e Sarsero et al., 1999

(−)-CGP12177 Left ventricularpapillary muscle Inotropic 7.6 1.7d

(±)-CGP12177 Right atrium Chronotropic 0.65 7.0 Santos and Spadari-Bratfisch, 2001[3H]-(−)-CGP12177 Guinea pig ventricle [3H]-(−)-CGP12177 9.9b 7.5b 2.4e Floreani et al., 2005(±)-Carteolol Ventricle [3H]-(−)-CGP12177 9.4b 7.1b 2.3e

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Ventricle Adenylylcyclase

0.25 6.2

Right atrium Chronotropic 9.0c

0.30 6.1 2.9Left atrium Inotropic 9.2c

0.14 5.9 3.3(−)-Pindolol Human Ventricle [3H]-(−)-Bupranolol 9.4b Kaumann and Lobnig, 1986

Right ventricular 9.0c

trabeculaeRight atrium Inotropic 9.1c

Right atrium Inotropic 0.33 6.5 6.5c 2.6d Joseph et al., 2003Recombinant β1AR cAMP 0.02 6.5

Guinea pig Right atrium Chronotropic 0.2 7.0 9.6c 2.6d Walter et al., 1984(±)-Pindolol Cat Right atrium Chronotropic 0.5 7.8 9.2c 1.4d Kaumann and Blinks, 1980a, 1980b

Rat Right atrium Chronotropic 0.25 8.0 8.8c 0.8d Kaumann et al., 1979(±)-Hydroxy Cat Right atrium Chronotropic 0.8 8.1 9.0c 0.9d Kaumann et al., 1978Benzylpindolol Left atrium Inotropic 0.7 7.6 9.1c 1.5d

Right ventricular Inotropic 0.5 7.4 9.0c 1.6d

papillary muscleVentricle Adenylyl

cyclase0.1 7.6 9.0c 1.2d

Ventricle Adenylylcyclase

9.3c Bearer et al., 1980

(±)-Iodo-hydroxybenzylpindolol

Right atrium Chronotropic 0.7 7.3 9.2c 1.9d Bearer et al., 1980 and Fig. 1Left atrium Inotropic 0.5 7.3 9.5c 2.1d


papillary muscleVentricle Adenylyl

cyclase9.3c

(continued on next page)(continued on next page)

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Table 1 (continued)

NCPA Species System/tissue Binding radioligand Stimulation Intrinsic activitya pEC50 pKβ1H pKβ1L pKβ1H-pEC50d or pKβ1H-pKβ1Le References

(±)-Iodocyanopindolol Left atrium Inotropic 0.7 7.4 10.8c 3.4d Kaumann, 1983


papillary muscle(±)-Alprenolol Right atrium Chronotropic 0.2 6.8 8.6c 1.8d Kaumann and Blinks, 1980b

(−)-Alprenolol Right atrium Chronotropic 0.2 7.4 8.9c 1.5d Kaumann and Blinks, 1980b(±)-Tert-butylpindolol Rat Right atrium Chronotropic 0.2 7.9 9.3c 2.4d Kaumann et al., 1979

(±)-Carazolol Rat Right atrium Chronotropic 0.1 7.0 9.9c 2.9d Kaumann et al., 1979

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(±)-Tert-butylcarazolol Rat Right atrium Chronotropic 0.1 7.4 9.8c 2.4d Kaumann et al., 1979

(±)-Bucindolol Human Ventricle [125I]-(−)-CYP 8.4b Hershberger et al., 1990Ventriculartrabeculae

Inotropic ~7.4j ~1.0 Maack et al., 2003

Atrialtrabeculae

Inotropic ~0.1 ~6.3 ~9.0c ~2.7 Kaumann and Molenaar, unpublished

aIntrinsic activity with respect to (−)-isoprenaline or maximum activity of (−)-isoprenaline=1.0.bpKβ1H and/or pKβ1L estimated from binding assays.cAffinity estimate (pKB or pKp) estimated from antagonism.pKβ1H estimated from binding assays — dpEC50 from functional assays or epKβ1L from binding assays.fNon-failing heart.gHeart failure.h(−)-propranolol (200 nM) present.iIBMX (60 μM) present.jAfter desensitization to (−)-isoprenaline followed by resensitization by metoprolol 30 μM.kCYP = cyanopindolol.Chemical structures re-drawn from Molenaar et al. (1997a, 1997b) and Mannhold (2005), ⁎enantiomeric carbon.

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Fig. 2. GMP-P(NH)P (10 μM)-evoked amplification of marginal stimulation of adenylylcyclase by partial agonists (top panel) and relation between adenylyl cyclase stimulationand fractionalβARoccupancy (bottompanels) in feline ventricularmembranes. Significantstimulation of the adenylyl cyclase was observed with DCI ((−)-dichloroisoprenaline), p-Cl((±)-p-chloroisoprenaline) and HYP ((±)-hydroxybenzylpindolol), but not with OXP ((−)-oxprenolol), H87/07 ((−)-p-2-methoxyethoxyphenoxy-3-isopropylamino-2-propanol)and PIND ((−)-pindolol) (top panel). Concentration–effect curves (solid lines) of (±)-p-chloroisoprenaline and (±)-hydroxybenzylpindolol for adenylyl cyclase stimulation in thepresence of GMP-P(NH)P, expressed as % of stimulation of 100 μM (−)-isoprenaline (ISO)(bottom panels; numbers at curve midpoint are pEC50). Broken lines represent receptoroccupancy curves (y), calculatedwith the corresponding pKP values (cross with horizontalerrors), estimated from the competitive antagonismof the effects of (−)-isoprenaline by thepartial agonists. Please notice that the concentration–effect curves for the partial agonists,shown in the bottom panel, were obtained between the baseline, greatly amplified byGMP-P(NH)P, and the maximum effects of the partial agonists in the presence of GMP-P(NH)P shown in the top panel. This method revealed two distinct patterns of action ofpartial agonists. For further details see Kaumann et al. (1978). Fig. 3. Comparison of concentration–effect curves (solid lines) for sinoatrial tachycardia

(shown as increases in beating rate, as a fraction of (−)-isoprenaline-evoked effects) andreceptor occupancy (yP) curves (broken lines) for (−)-pindolol. yP curves were calculatedfrom the antagonism of the effects of (−)-noradrenaline (pKB and pKP values) on right atriaand inhibition of [3H]-(−)-bupranolol binding by (−)-pindolol (pKD values) to ventricularβ1-adrenoceptors of guinea pig. Please note the existence of a high-potency componentand low-potency component of the positive chronotropic effects of (−)-pindolol, as well ashigh- (pKD) and low- (pKDx) affinity binding sites. Data of Walter et al. (1984).


conventional partial agonists also caused agonist effects mediated throughβ3AR, resistant to blockade by propranolol. After the Kaumann (1989)paper, laboratories started to use bupranolol as an antagonist andbupranolol also blocked agonist effects mediated through β3AR (asreviewed by Arch & Kaumann, 1993), which further enhanced confusion.

Therefore many authors wrongly assumed that the third cardiac β-adrenoceptor was identical to the cloned β3-adrenoceptor. Furthermore,other blockers, CGP20712Awhichwas showntohavehighaffinity forβ1AR(Kaumann, 1986; Buxton et al., 1987; Molenaar & Summers, 1987) andICI118,551whichhadhighaffinity forβ2AR(Buxtonetal.,1987;Kaumann&Lemoine, 1987; Molenaar & Summers, 1987) had considerably loweraffinities at both β3AR (Emorine et al.,1989; Baker, J. K., 2005) and the thirdcardiac β-adrenoceptor (Kaumann & Molenaar, 1996). It has emerged thatthere is broad, but not absolute agreement between affinities ofantagonists at the cloned β3AR (Hoffmann et al., 2004, Baker, J. K.,2005) and the third cardiac β-adrenoceptor (Table 2). This mightsuggest a similarity of critical structural features that allow binding ofantagonists to the third cardiac β-adrenoceptor (β1LAR) and β3AR,however this needs to be verified with mutagenesis studies.

One argument against the identity of the two receptors as ‘β3AR’ wasthat agonists selective forβ3AR failed toexert agonist andantagonist effectsin cardiac preparations or did so viaβ1ARorβ2AR (Arch &Kaumann,1993;Kaumann &Molenaar, 1996; Kaumann et al., 1997; Molenaar et al., 1997a;Malinowska & Schlicker, 1996, 1997; Sennitt et al., 1998). Criteria forclassification of β3AR were proposed by Arch and Kaumann (1993): 1.stimulation by β3AR selective agonists, 2. simulation by non-conventionalpartial agonists, 3. resistance to blockade by antagonists that possess onlyhigh affinity for β1AR and β2AR. In the heart criterion 2. and 3. but not 1.were met. A fourth criterion for β3AR mediated effects was added later, 4.blockadebyβ3AR selective antagonists (Molenaar&Kaumann,1997). Fig. 4illustrates that β3AR agonists are devoid of stimulant or depressant effectsand fail to antagonize the agonist effects of (−)-CGP12177 on humanventricle in thepresenceof IBMX.Experiments in theabsenceof IBMXhavealso demonstrated a lack of stimulant and depressant effects of β3ARagonists onhumanventricle (Molenaaret al.,1997a). Theseexperimentsdonot confirm the ventricular cardiodepressant effects described for β3ARagonists by Gauthier et al. (1996) on human ventricle.

To differentiate the third cardiac β-adrenoceptor from β3AR, theterm putative β4-adrenoceptor was used for the former receptor(Kaumann, 1997; Kaumann & Molenaar, 1997; Molenaar et al., 1997a).Conclusive experiments, carried out in β3AR knockout mice, demon-strated that the cardiostimulant effects of (−)-CGP12177 persisted,ruling out mediation through β3AR (Kaumann et al., 1998).

1.4. 1996 to present

The work of Pak and Fishman (1996) initiated the present prevailingconcept that the putative β4-adrenoceptor is a low-affinity site of theβ1AR. Using recombinant human and ratβ1AR, transfected into hamsterovary cells (CHO), they provided the following data: 1. (±)-CGP12177 atlowconcentrations antagonized the (−)-isoprenaline-evoked increase incellular cAMP but concentrations approximately 2 log units highercaused concentration-dependent increases in cAMP (Fig. 5). The agonistactivity of CGP12177 was proportional to β1AR density. 2. The agonist

Table 2Comparison of affinity estimates of clinically used β-blockers and CGP20712A for β1HAR and β1LAR of heart and recombinant β1AR at physiological densities

β-Blocker Species System/tissue Stimulation Bindingradioligand

NCPAa Catecholamine pKβ1H pKβ1L pKβ1H-pKβ1L

Reference

(−)-Bupranolol Human Ventricle [3H]-(−)-Bupranolol

9.0 Kaumann et al., 1982

Human Ventricle Adenylyl cyclase (−)-Isoprenaline 9.4Human Left ventricle Inotropic (−)-

Noradrenaline9.0

Human Right atrium Inotropic (−)-CGP12177 7.3 1.7 Kaumann, 1996Recombinant β1AR [3H]-(−)-CGP12177 9.4 Joseph et al., 2004a

cAMP (−)-Isoprenaline 9.4(−)-CGP12177 7.6 1.8

Recombinant β1AR CREd luciferase (−)-Noradrenaline

8.7 Baker, J. G., 2005

Ferret Left ventricle [125I]-(−)-CYPb 8.5 Lowe et al., 2002Right ventricular Inotropic (−)-Isoprenaline 9.1papillary muscle (−)-CGP12177 7.5 1.6

(±)-CGP12177 7.3 1.4Guinea pig Right atrium Chronotropic (−)-

Noradrenaline8.9 Lemoine and Kaumann, 1982

Guinea pig Right atrium Chronotropic (−)-Pindololc ~7.0 1.9 Walter et al., 1984Rat Left atrium [3H]-(−)-CGP12177 8.2 6.6 1.6 Sarsero et al., 1998

Right atrium Chronotropic (−)-CGP12177 6.4 Kaumann and Molenaar, 1996Left atrium Inotropic (−)-CGP12177 6.8Ventricle [3H]-(−)-CGP12177 9.5 5.8 3.7 Sarsero et al., 1999Left ventricular Inotropic (−)-CGP12177 7.0papillary muscle (±)-

Cyanopindolol6.7

(−)-Propranolol Human Right atrium Inotropic (−)-Noradrenaline

8.5 Gille et al., 1985

Adenylyl cyclase (−)-Noradrenaline

8.8

Recombinant β1AR Adenylyl cyclase (−)-Isoprenaline 8.2 Konkar et al., 2000a(±)-CGP12177 6.7 1.5

Recombinant β1AR (−)-Isoprenaline 9.1 Joseph et al., 2004a(−)-CGP12177 7.0 2.1 Joseph et al., 2004a

Propranolol Recombinant β1AR CRE luciferase (−)-Noradrenaline

8.3 Baker, J. G., 2005

(±)-CGP12177 6.4 1.9(−)-Propranolol Ferret Ventricle [125I]-(−)-CYP 8.3 Lowe et al., 2002

Inotropic (−)-Isoprenaline 9.0(−)-CGP12177 6.2 2.8

Rat Right atrium Chronotropic (−)-Noradrenaline

8.3 Kaumann and Molenaar, 1996

(−)-CGP12177 6.1 2.2Rat Recombinant β1AR Adenylyl cyclase (−)-Isoprenaline 8.6 Konkar et al., 2000a

(±)-CGP12177 7.0 1.6


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Table 2 (continued)



Reference

(−)-Pindolol Human Right atrium Inotropic (−)-Noradrenaline

9.1 Kaumann and Lobnig, 1986

(−)-CGP12177 6.5 2.6 Joseph et al., 2003

Recombinant β1AR cAMP (−)-Isoprenaline 8.6 Joseph et al., 2003(−)-CGP12177 6.3 2.3

[3H]-(−)-CGP12177 9.7 Joseph et al., 2004a(−)-Isoprenaline 9.3

(−)-CGP12177 6.5 2.8(−)-Atenolol Human Recombinant β1AR [3H]-(−)-CGP12177 7.6 Joseph et al., 2004a

cAMP (−)-Isoprenaline 7.4cAMP (−)-CGP12177 4.4 3.0

Atenolol Recombinant β1AR CRE luciferase (−)-Noradrenaline

6.8 Baker, J. G., 2005

(±)-CGP12177 3.8 3.0(−)-Atenolol Ferret Ventricle [125I]-(−)-CYP 5.9 Lowe et al., 2002


(±)-Metoprolol Human Recombinant β1AR [3H]-(−)-CGP12177 7.9 Joseph et al., 2004acAMP (−)-Isoprenaline 8.2

(−)-CGP12177 5.4 2.8

CRE luciferase (−)-Noradrenaline

7.4 Baker, J. G., 2005

(±)-CGP12177 5.3 2.1Ferret Ventricle [125I]-(−)-CYP 6.6 Lowe et al., 2002


(−)-Timolol Human Recombinant β1AR 9.0 Joseph et al., 2004acAMP (−)-Isoprenaline 9.2

(−)-CGP12177 6.4 2.8

Table 2 (continued)312

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Ferret Ventricle [125I]-(−)-CYP 8.5 Lowe et al., 2002Inotropic (−)-Isoprenaline 9.1

(−)-CGP12177 6.3 2.8(±)-Carvedilol Human Recombinant β1AR [3H]-(−)-CGP12177 9.4 Joseph et al., 2004a

cAMP (−)-Isoprenaline 9.9(−)-CGP12177 7.6 2.3


9.5 Baker, J. G., 2005

(±)-CGP12177 7.3 2.2Ferret Ventricle [125I]-(−)-CYP 8.9 Lowe et al., 2002

Inotropic (−)-Isoprenaline 8.1 Lowe et al., 1999(−)-CGP12177 6.8 1.3

(±)-Oxprenolol Human Recombinant β1AR [3H]-(−)-CGP12177 8.1 Joseph et al., 2004acAMP (−)-Isoprenaline 8.2

(−)-CGP12177 6.1 2.1

(±)-Sotalol Human Recombinant β1AR [3H]-(−)-CGP12177 6.3 Joseph et al., 2004acAMP (−)-Isoprenaline 6.0

(−)-CGP12177 3.9 2.1


5.8 Baker, J. G., 2005

(±)-CGP12177 3.7 2.1

Ferret Ventricle [125I]-(−)-CYP 5.3 Lowe et al., 2002


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Table 2 (continued)



Reference


(±)-Bisoprolol Human Recombinant β1AR 8.3 Joseph et al., 2004acAMP (−)-Isoprenaline 7.7

(−)-CGP12177 5.7 2.0


(−)-CGP12177 5.1 3.0(±)-Betaxolol CRE luciferase (−)-Noradrenaline 8.3 Baker, J. G., 2005

(±)-CGP12177 5.7 2.6

(±)-Nadolol Human Recombinant β1AR [3H]-(−)-CGP12177 8.0 Joseph et al., 2004acAMP (−)-Isoprenaline 8.1

(−)-CGP12177 6.2 1.9

CRE luciferase (−)-Noradrenaline 7.4 Baker, J. G., 2005(±)-CGP12177 5.9 1.5


(−)-CGP12177 5.7 1.5

Table 2 (continued)

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(±)-CGP20712A Human Recombinant β1AR Adenylyl cyclase (−)-Isoprenaline 8.1 Konkar et al., 2000a(±)-CGP12177 6.9 1.2

(−)-Isoprenaline 8.0 Konkar et al., 2000b(±)-CGP12177 7.4 0.6

CRE luciferase (−)-Noradrenaline 9.2 Baker, J. G., 2005(±)-CGP12177 7.1 2.1

Rat Right atrium Chronotropism (−)-Noradrenaline 9.4 Kaumann, 1986(−)-CGP12177 6.4 3.0 Kaumann and Molenaar, 1996

Ventricle Inotropism (−)-CGP12177 6.6 Sarsero et al., 1999Inotropism (±)-Cyanopindolol 6.3

Recombinant β1AR Adenylyl cyclase (−)-Isoprenaline 8.1 Konkar et al., 2000a(±)-CGP12177 7.3 0.8

Recombinant β1AR Adenylyl cyclase (−)-Isoprenaline 8.3 Konkar et al., 2000b(±)-CGP12177 7.4 0.9

(±)-Bucindolol Human Right atrium Inotropic (−)-Isoprenaline ~9.0(−)-CGP12177 ~6.0 ~3.0 Kaumann and Molenaar,

unpublished

Catecholamines

Noradrenaline Isoprenaline

Chemical structures re-drawn from Dooley et al. (1986) and Mannhold (2005), ⁎enantiomeric carbon.a Non-conventional partial agonist.b Cyanopindolol.c Biphasic concentration–effect curve for (−)-pindolol; (−)-bupranolol antagonism of the effects of the low potency component, resistant to antagonism by (−)-bisoprolol.d CRE = cAMP response element.

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Fig. 4. Lack of effect of β3AR agonists on contractile force and on the effects of (−)-CGP12177 in human right ventricular trabeculae from heart failure patients undergoingtransplantation. Electrically stimulated right ventricular trabeculaewere incubatedwith 10 μMnadolol for 45min, followed by 0.1 μM β3AR agonist, BRL 37344, CL 316243, SR 58611Aor ZD 2079 for 20min. No effect on contractile forcewas observed and the concentration of β3-adrenoceptor agonist raised to a final concentration of 1 μM. After 20min no significanteffect on contractile force was observed. IBMX (60 μM)was then added to the tissue bath and after equilibration concentration–effect curves were established to (−)-CGP12177. Therewas no significant difference in pEC50 values for the positive inotropic effects of (−)-CGP12177 in the absence (closed symbols) or presence (open symbols) of 1 μM β3AR agonists. Dataare from n trabeculae indicated in the figure, from 5 heart failure patients.


effects of (±)-CGP12177 were antagonized by the β1AR-selectiveCGP20712A (with an estimated pKB ~7.7) andwith 80-fold lower potencyby β2AR-selective ICI118,551. 3. [3H]-(−)-CGP12177 labelled a high-affinitysite with pKD=9.7. (±)-CGP12177 competed with [125I]-(−)-cyanopindololwith high affinity (pKi=9.5) for 90% of the binding sites and with a 2 logunit lower affinity (pKi=7.7) for the remaining 10% of the binding sites. 4.As expected fromtheβ1AR, racemicCGP12177wasa100-foldmorepotentagonist than R-(+)-CGP12177. Pak and Fishman (1996) concluded thatCGP12177 caused blockade of agonist effects through the high-affinitystate (βlHAR) and agonist effects through the low-affinity state (βlLAR)coupled to Gs protein. These properties essentially mimicked thedissociation between stimulation and blockade found previously withCGP12177 on isolated cardiac tissues (Kaumann, 1983, 1989; Arch &Kaumann,1993). Thework of Pak and Fishman (1996)was confirmed andexpandedwith recombinant β1AR by Konkar et al. (2000a,b), Joseph et al.(2003, 2004a,b), Baker et al. (2003), Baker J. G. (2005) and Baker J. K.(2005). TheworkofPakandFishman (1996) and its confirmation, togetherwith work on myocardium from β1AR knockout mice, promptedrecognition that non-conventional partial agonists caused cardiostimula-tion through βlLAR and blockade of the effects of catecholamines throughβlHAR (Kaumann, 2000; Granneman, 2001; Kaumann et al., 2001).

2. β1AR high (H) and low (L) affinity binding sites

Throughout this reviewweuse the followingnomenclature for the−logmolar equilibrium dissociation constant between β1AR and a ligand: pKD,for a radioligand; pKi, for a non-radioactive ligand that competes with aradioligand for receptors; pKB, for an antagonist, estimated fromcompetitive blockade of the effects of an agonist; pKP, for a partial agonist,estimated from competitive blockade of the effects of a full agonist.

2.1. Evidence from cardiac β1AR

Thefirst evidence for two distinct binding sites for a non-conventionalpartial agonist was published by Walter et al. (1984), demonstrating that

(−)-pindolol competed with [3H]-(−)-bupranolol binding with high(pKi=9.1) and very low affinity (pKi=4.9) in membranes of guinea pigventricle. Theaffinities of (−)-bupranolol and (−)-pindolol for thevery low-affinity site are too low to account for the β1LAR pharmacology, but couldbe relevant to the vascular pharmacology of non-conventional partialagonists (see Section 3.8). The high-affinity populationwas interpreted asbeingβ1ARbut thenatureof thevery low-affinity sitewasnotunderstood.

Recognition of the seminal contribution of Pak and Fishman(1996), demonstrating dissociation between stimulation and blockadewith CGP12177, took time because it had escaped our and other'sattention. In addition, Pak and Fishman (1996) were unaware of thedissociation between stimulation and blockade reported for CGP12177on cardiac tissues (Kaumann, 1983, 1989; Arch & Kaumann, 1993) thatwas in line with their data with recombinant β1AR.

Before the work of Pak and Fishman (1996) came to our attention, westudied the binding of [3H]-(−)-CGP12177 on rat left atrial membraneparticles.We noticed that (−)-propranolol (500 nM), a concentration 200-fold greater than its affinity for human β1AR (pKB=8.5, atrial receptors,Gille et al., 1985; pKi=8.5, recombinant receptors, Hoffmann et al., 2004),removed significantly less bound radioligand from the membranes thannon-radioactive (−)-CGP12177. Since the cardiostimulant effects of (−)-CGP12177 are resistant to blockade by 200 nM (−)-propranolol (Kaumann,1996), we rationalised that the (−)-propranolol-resistant and (−)-CGP12177-sensitive binding component could represent the receptorpopulation that mediated the cardiostimulant effects of (−)-CGP12177.[3H]-(−)-CGP12177 binding to rat atrial membranes was biphasic with ahigh-affinity site (pKD=9.4, 35% of total specific sites) to (−)-propranolol-inhibitable receptors and a low-affinity site (pKD=7.6, 65% of total specificsites) to (−)-propranolol-resistant receptors (Fig. 6). The high-affinity siteand low-affinity site were interpreted as corresponding to β1/β2AR andputative β4AR respectively (Sarsero et al., 1998). The atrial binding datawere confirmed and similar biphasic bindingwas observedwith [3H]-(−)-CGP12177 on ventricular membranes with pKD=9.0 for β1/β2AR andpKD=7.3 for putative β4AR (Sarsero et al., 1999). In contrast to the workwith human recombinant β1AR of Pak and Fishman (1996) reporting that

Fig. 5. Similarities of the pattern of depressant and stimulant effects of CGP12177 on humanrecombinant β1AR (top panel) and human atrium (bottom panel). The top panel showsinhibition by low (±)-CGP12177 concentrations of the cAMP signal caused by 2 nM (−)-isoprenaline followed by increases of cAMP produced by high (±)-CGP12177 concentrations(Fig. 1 of Pak & Fishman 1996, with permission of the authors). The bottom panel illustratescardiodepression at low (−)-CGP12177 concentrations and cardiostimulation at high (−)-CGP12177 concentrations in human atrial trabeculae (n=7 trabeculae from 6 patients,adapted from Sarsero et al., 2003with kind permission of Springer Science+Businessmedia).The depressant effects of CGP12177 are due to blockade of β1HAR, activated by (−)-isoprenaline at recombinant β1AR or endogenous noradrenaline released from atrialtrabecular nerves which could be prevented by pre-incubation of trabeculae with (−)-propranolol 200 nM (n=108 trabeculae from 86 patients). Note (−)-propranolol does notblockbut actuallyenhances the stimulanteffects of (−)-CGP12177. The stimulanteffects of (−)-CGP12177 occur throughβ1LAR. ‘Fade’ of the tissue response to (−)-CGP12177 (N600 nM) canbe prevented by pre-incubationwith the PDE3 inhibitor cilostamide (Kaumann et al., 2007).

Fig. 6. Individual [3H]-(−)-CGP12177 saturation binding experiments to rat atrial membraneparticles carriedoutunder threedifferentexperimental conditions to revealβ1HAR+β2ARandβ1LAR.A) Biphasic specific [3H]-(−)-CGP12177 binding at lowconcentrations toβ1HAR+β2ARsandhigher concentrations toβ1LARswith non-specific binding (NSB) determinedwith 20 μM(−)-CGP12177, B) [3H]-(−)-CGP12177 binding to β1HAR+β2ARs with (NSB) determined with500 nM (−)-propranolol and C) [3H]-(−)-CGP12177 binding carried out in the presence of500 nM (−)-propranolol to β1LAR with NSB determined with 20 μM (−)-CGP12177. Adaptedfrom Sarsero et al. (1998).


only 10% of the binding sites labelled by 30 pM [125I]-(−)-cyanopindololhad lowaffinity, rat atrial low-affinity binding siteswere 65% of total [3H]-(−)-CGP12177 binding (Sarsero et al., 1998, 1999). The difference inrelative densities is probably due to the relatively low affinity of[125I]-(−)-cyanopindolol for the β1LAR, compared to β1HAR. Althoughthe affinity of [125I]-(−)-cyanopindolol for β1LAR has not been directlydetermined, it is likely to be considerably lower than at β1HAR based oncomparison of affinities of a large number of other compounds atβ1HARand β1LAR including its de-iodonated derivative (±)-cyanopindolol(Tables 1 and 2). The affinity of (±)-cyanopindolol is 1000-fold lower atβ1LAR (pKi=7.4 rat ventricle and pKi=7.5 rat atrium, estimated frominhibition of [3H]-(−)-CGP12177 binding, Sarsero et al., 1999) than theaffinity of [125I]-(−)-cyanopindolol for β1HAR. Another difference wasthat the low-affinity component appeared to be sensitive to guaninenucleotides with recombinant β1AR (Pak & Fishman,1996) but not withrat atrial β1AR (Sarsero et al., 1999). It was therefore not unambiguouslyclear whether the putative β4AR binding sites of Sarsero et al. (1998,1999) corresponded to the low-affinity site of recombinant β1AR.

Inhibition of [3H]-(−)-CGP12177 binding by (−)-propranolol and (−)-bupranolol revealed two binding sites eachwith high-affinity pKi values of8.6 and 8.2 and low-affinity pKi values of 5.3 and 6.6 respectively on ratatrial membranes (Sarsero et al., 1998, 1999). The pattern of inhibition of[3H]-(−)-CGP12177 binding with 3 non-conventional partial agonists wasmore complex, revealing 3 binding sites of high affinity (H), lowaffinity (L)

and very low affinity (vL) (the latter two estimated in the presence of (−)-propranolol (500 nM) respectively: (±)-cyanopindolol (pKH=10.8, pKL=7.5,pKvL=5.0), (±)-carazolol (pKH=10.0, pKL=7.7, pKvL=4.0) and (−)-pindolol(pKH=8.7, pKL=7.1, pKvL=4.1, Sarsero et al., 1999). Three bindingpopulations were also detected on rat ventricle for (±)-cyanopindololwith pKH=10.0, pKL=7.4 and pKvL=5.1). The pKH valueswere interpretedas corresponding toβ1/β2AR and the pKL values to putativeβ4AR but themeaning of pKvL values was unknown (Sarsero et al., 1999).

A high- and low-affinity population for [3H]-(−)-CGP12177 wasdetected in murine ventricle with pKH=9.2 and pKL=7.0 (in thepresence of 500 nM (−)-propranolol) (Kaumann et al., 1998, 2001). Thehigh-affinity binding site, but not the low-affinity binding site,disappeared in β1/β2AR double knockout mice, casting doubt aboutthe significance of the low-affinity binding sites (Kaumann et al.,2001). In contrast, both binding sites persisted in β2AR knockout micewith pKH=9.3 and pKL=6.7 (Kaumann et al., 2001). High- and low-affinity binding sites for [3H]-(−)-CGP12177 were verified in humanatrium with pKH=9.4 and pKL=7.1 (Sarsero et al., 2003; Table 1). Asexpected from down-regulation of β1AR in heart failure (Bristow et al.,1982, 1986), the density of both high- and low-affinity binding sites


was reduced to approximately half in atria from failing hearts (Sarseroet al., 2003).

The retention of the low-affinity population for [3H]-(−)-CGP12177binding in theventricle fromβ1/β2ARdouble knockoutmice (Kaumannetal., 2001) is puzzling. Although the apparent affinity-estimate (pKD ~7.0)was expected for β1LAR, the finding rules out β1LAR. The low-affinityestimate was biased because the high non-specific ventricular binding of[3H]-(−)-CGP12177 in the β1/β2AR double knockout mice prevented thedetermination of complete saturation assays. The low-affinity siteappeared to have an 11–22-fold greater capacity than the capacity ofthe high-affinity site (pKD=9.4) corresponding to β1HAR (Kaumann et al.,2001). In contrast, the low-affinity sites, attributed to β1LAR andestimated in rat and humanmyocardium, had a 2–5-fold greater bindingcapacity than the β1HAR sites (Sarsero et al., 1998, 1999, 2003).

Treatment with hydrocortisone (initial dose of 100 mg kg−1 scfollowed by once daily 50 mg kg−1 sc) increased the density of bothβ1AR and β2AR, labelled with [3H]-(−)-CGP12177, in rat atria up to the6th day of treatment (Myslivecek et al., 2003). These authors alsomeasured the influence of hydrocortisone treatment on the (−)-propranolol-resistant binding of [3H]-(−)-CGP12177, as an estimate ofβ1LAR, with the method of Sarsero et al. (1998). The β1LAR densitytended to increase at the third day of hydrocortisone treatment, butsignificantly decreased below control levels by the sixth day ofhydrocortisone treatment (Myslivecek et al., 2003).

Two saturable binding sites for [3H]-(−)-CGP12177 were reportedon guinea pig ventricle (Floreani et al., 2005). The estimated affinitieswith pKi values of 9.9 and 7.5 are consistent with labelling of β1HARand β1LAR respectively. The non-conventional partial agonist carteolol(Chiba, 1979; Takayanagi et al., 1989) competed with [3H]-(−)-CGP12177 for β1HAR and β1LAR with pKi values of 9.4 and 7.1respectively. These data are similar to binding characteristics of [3H]-(−)-CGP12177 in rat myocardium (Sarsero et al., 1998, 1999), humanatrium (Sarsero et al., 2003) and human recombinant β1AR (Joseph etal., 2004a), illustrating similarities between the 3 species.

2.2. Evidence from recombinant β1AR

Direct evidence for a low-affinity binding site for [3H]-(−)-CGP12177 was obtained from recombinant β1AR, expressed atphysiological density (101 fmol mg−1) in CHO cells (Joseph et al.,2004a). [3H]-(−)-CGP12177 caused biphasic saturation binding withpKD=9.3 and pKD=6.6, corresponding to β1HAR and β1LAR respec-tively. The density of β1LAR low-affinity sites was 5-fold greater thanthat of β1HAR. As expected from its low affinity, [3H]-(−)-CGP12177dissociated 26 times faster from the β1LAR site than β1HAR site.

2.3. Summary

The non-conventional partial agonist [3H]-(−)-CGP12177 binds totwo saturable populations in mammalian heart. The properties ofthese binding sites are consistent with β1HAR and β1LAR in the rat,guinea pig and human myocardium. Binding of [3H]-(−)-CGP12177 toβ1HAR and β1LAR was confirmed with human recombinant β1AR.

3. Cardiovascular pharmacologyof non-conventional partial agonists

The dissociation between low agonist potency and high β1ARblocking potency of non-conventional partial agonists (Table 1), hasbeen observed in murine, rat, guinea pig, feline and human cardiactissues (reviewed previously (Kaumann,1989,1997, 2000; Kaumann &Molenaar 1997; Molenaar et al., 1997a; Arch & Kaumann 1993)) aswell as ferret heart (Lowe et al., 1999, 2002). Here we review theexperimental biochemical, physiological and pharmacological evi-dence of the effects of non-conventional partial agonists on differentcardiac regions and blood vessels.

3.1. Coupling to the cAMP pathway

Since it was shown that hydroxybenzylpindolol stimulated theadenylyl cyclase of feline ventricularmembranes at high concentrationswhile antagonizing the effects of (−)-isoprenaline at low concentrations(Fig. 2; Kaumann et al., 1978), it became likely that non-conventionalpartial agonists enhance cardiac contractility through a cAMP-depen-dent pathway. As expected, (−)-CGP12177 increased cAMP levels(Kaumann et al., 1997) and stimulated cAMP-dependent protein kinase(PKA) (Kaumann & Lynham, 1997) in rat left atrium. Consistent with aninvolvement of cAMP, the non-selective inhibitor of phophodiesterases(PDE), 3-isobutyl-1-methylxanthine (IBMX), potentiated the positiveinotropic and chronotropic effects of (−)-CGP12177 (Kaumann &Lynham,1997) in rat atria. To unravel the PDE isoenzymewhich limitedthe effects of (−)-CGP12177, Vargas et al. (2006) found that bothcilostamide-inhibitable PDE3 and rolipram-inhibitable PDE4, acting inconcert, prevented the positive inotropic effects and cAMP-enhancingeffects of CGP12177 in rat right ventricle. We had previously observedthat (−)-CGP12177did not enhance the contractilityof rat left ventricularpapillary muscles (Kaumann & Molenaar, 1996) unless PDEs wereinhibited with IBMX (Sarsero et al., 1999).

The positive inotropic effects of (−)-CGP12177 on human atrialtrabeculae (Kaumann, 1996; Kaumann & Molenaar, 1997; Sarseroet al., 2003) occur without a detectable increase in cAMP levels or PKAstimulation (Sarsero et al., 2003). In the presence of IBMX, however,(−)-CGP12177 increased both cAMP and PKA activity, although only byapproximately 10% and 25% respectively, of the maximum effect of(−)-isoprenaline. These findings are consistent with: i. the relativelysmall intrinsic activity of (−)-CGP12177 as an inotropic partial agonist(Kaumann, 1996; Sarsero et al., 2003) and ii. a protective role of PDEsagainst cardiostimulation by non-conventional partial agonists.

Positive inotropic responses to (−)-CGP12177onhumanatriumtend tofade (Kaumann, 1996; Sarsero et al., 2003), possibly due to avid cAMPhydrolysis. To investigate the role of the twomost important cardiac PDEisoenzymes PDE3 and PDE4, the PDE3-selective inhibitor cilostamide andPDE4-specific inhibitor rolipramwereused. Cilostamide, butnot rolipram,increased (−)-CGP12177 responses andprevented fade, consistent withan important role of PDE3 but not of PDE4 (Kaumann et al., 2007).The human atrial responses to (−)-noradrenaline, mediated throughβ1AR, were also potentiated (Christ et al., 2006) and fade prevented bycilostamide but not by rolipram (Kaumann et al., 2007). Thus, itappears that in human atrium PDE3 but not PDE4 limits the positiveinotropic effects of both (−)-noradrenaline through (β1HAR) and (−)-CGP12177 (through β1LAR). This is in marked contrast to rat rightventricle, in which only PDE4 reduces the effects of (−)-noradrenalinethrough β1HAR but both, PDE3 and PDE4, acting in concert, prevent theeffects of CGP12177 through β1LAR (Vargas et al., 2006).

(±)-Bucindolol, a β-blocker that causes tachycardia at relativelyhigh concentrations (Deitchman et al., 1980), caused a small increaseof cAMP in human ventricular preparations at 100 nM (Andreka et al.,2002), a concentration approximately 100-fold greater than its Ki

(Hershberger et al., 1990; Maack et al., 2000) or KB (Table 1, Kaumann& Molenaar unpublished) for β1HAR. (±)-Bucindolol (10 μM) alsocaused a small increase of cAMP (14% of the increase caused by (−)-isoprenaline) in rat ventricular cardiomyocytes (Willette et al., 1999).

The non-conventional partial agonist carteolol (Chiba, 1979; Takaya-nagi et al., 1989; Floreani et al., 2004, 2005) stimulated ventricularadenylyl cyclase of guinea pig ventricle with intrinsic activity 0.2 withrespect to (−)-isoprenaline and pEC50=6.2 (Floreani et al., 2005, Table 1).

3.2. Sinoatrial tachycardia

Tachycardia induced by β-blockers with agonist properties wasreported in reserpine-pretreated rats, listed here in decreasing orderof intrinsic activity: DCI, pindolol (LB46), practolol, INPEA, oxprenolol,pronethalol and alprenolol (Barrett & Carter, 1970). The dissociation


between low stimulation potency and high βAR blocking potency wasfirst discovered on spontaneously beating feline atria (Kaumann,1973) and verified in all species investigated so far.

The non-conventional partial agonists (±)-pindolol, (±)-alprenololand (±)-oxprenolol cause sinoatrial tachycardia with intrinsic activities0.5, 0.2 and 0.4 respectively compared to (−)-isoprenaline, on felineisolated right atria (Kaumann, 1973; Kaumann & Blinks, 1980a, 1980b).The concentration–effect curves for (±)-pindolol are biphasic and onlythe high-sensitive component was antagonized by (±)-propranolol(Kaumann & Blinks, 1980a). (±)-Pindolol, (±)-oxprenolol (Kaumann &Blinks, 1980b) and (−)-pindolol (Walter et al., 1984) also cause sinoatrialtachycardia in guineapig right atria butwith lower intrinsic activity thanin feline atria. (±)-Pindolol, (±)-oxprenolol, (±)-pronethalol, (±)-alpre-nolol and (−)-alprenolol exhibited dissociation between low-potencystimulation and high-potency blockade. The dissociation (pKB–pEC50)between stimulation (pEC50) and blockade of βAR (estimated from theantagonism of the effects of (−)-isoprenaline) was 0.6 log units for both(−)-oxprenolol and (+)-oxprenolol (Kaumann & Blinks, 1980b). Thestereoselectivity of the low-potency stimulant effects of oxprenolol isconsistent with mediation through a βAR mechanism.

(±)-Pindolol and its tert-butyl analogue, (±)-tert-butylpindolol causedsinoatrial tachycardia on rat right atria at slightly greater concentrationsthan expected from their affinity, estimated from the antagonism of (−)-isoprenaline-evoked tachycardia. (±)-Carazolol and (±)-tert-butylcarazo-lol caused tachycardia at considerably greater concentrations (3 logunits) than their affinity for β1AR (pKB values), estimated from theantagonism of (−)-isoprenaline-evoked sinoatrial tachycardia andventricular adenylyl cyclase stimulation (Kaumann et al., 1979). (±)-Cyanopindolol elicited sinoatrial tachycardia in rat right atria with alower intrinsic activity than (−)-CGP12177 (Kaumann & Molenaar, 1996;Sarsero et al., 1999). (±)-Cyanopindolol was therefore used to antagonizethe positive chronotropic effects of (−)-CGP12177. The pEC50=7.3–7.7 of(±)-cyanopindolol matched the pKP=7.4. Therefore, the estimatedreceptor occupancy curve, calculated with the pKP, closely fitted theconcentration–effect curve of (±)-cyanopindolol (Sarsero et al., 1999).

(−)-CGP12177 caused (−)-propranolol-resistant sinoatrial tachycardiain rat atria (Kaumann & Molenaar 1996) as confirmed by Cohen et al.(1999), Kompa and Summers (1999), Santos and Spadari-Bratfisch (2001)andSantos et al. (2003, 2005)with racemicCGP12177. Santos et al. (2005)compared the responses to catecholamines and CGP12177 in rat rightatrium from rats which had previously undergone surgical sinoatrialdenervation. Sinoaortic denervation disrupts baroreceptor-mediatedcontrol of heart rate and blood pressure and induces chronotropicsubsensitivity to βAR agonists (Zanesco et al., 1997) as also found forCGP12177 (Santos et al., 2005). They reported the puzzling finding thatsinoaortic denervation-induced subsensitivity to (−)-isoprenaline and(−)-noradrenaline persisted for 1 week but sensitivity to CGP12177recovered by 48 h. Furthermore, they showed that the β2AR-selectiveantagonist ICI118,551 (5–50 nM) or pertussis toxin (PTX)-treatment (toinactivate Gi coupling of β2AR) enhanced the reduced responses toCGP12177 but (−)-propranolol (200 nM) caused antagonism with highpotency, 48 h after sinoaortic denervation. ICI118,551 (50 nM), (−)-propranolol (200 nM) or PTX-treatment (see also Kompa & Summers1999) did not affect sinoatrial tachycardia of CGP12177 in normal rats.

Santos et al. (2005) suggested that under their conditions of 48 hpost sinoaortic denervation, CGP12177 also activates β2AR and thatthese receptors reduced the response through β1LAR via activation ofGi. However, (−)-propranolol (200 nM) also blocks β2AR and shouldhave caused recovery of sensitivity as Santos et al. (2005) observedwith ICI118,551. Instead 200 nM (−)-propranolol antagonized theeffects of CGP12177 in atria from rats with sinoaortic denervationwhich is inconsistent with the interpretation of Santos et al. (2005).Also the low potency of CGP12177 (pEC50=6.33–6.95) is not consistentwith a β2AR mechanism since CGP12177 can cause increases in cAMPat low concentrations (Pak & Fishman, 1996; Baker et al., 2002;Molenaar et al., 2007). Therefore, the intriguing results reported by

Santos et al. (2005) must await independent verification beforeinterpretations are ventured.

In a rat model of stress-induced changes of sinoatrial function,repeated footshock stress, Bassani and De Moraes (1988) demon-strated an increase of the chronotropic potency of both (−)-isoprenalineand (−)-adrenaline through β2AR but unchanged chronotropic potencyof (−)-noradrenaline through β1AR in isolated right atria. Santos andSpadari-Bratfisch (2001) investigated the positive chronotropic effects of(±)-CGP12177 using the stressmodel of Bassani andDeMoraes (1988) andfound that the positive chronotropic potency was unaltered compared tonon-stressed rats, consistent with unmodified potency of (−)-noradrena-line reported by Bassani and De Moraes (1988). Thus, repeated footshockstress does not appear to change the function of both sinoatrial β1HAR,activated by (−)-noradrenaline, and β1LAR, activated by (−)-CGP12177.

CGP12177-evoked sinoatrial tachycardia was particularly pro-nounced in murine, feline and ferret right atria. (−)-CGP12177 had anintrinsic activity of 0.6 compared to (−)-isoprenaline on murine atria(Kaumann et al., 1998). (±)-CGP12177 caused sinoatrial tachycardiawith intrinsic activity 0.8 in feline right atria (Kaumann, 1983;Kaumann, 2000; Table 1). The CGP12177-evoked tachycardia occurredat concentrations considerably higher (~2 log units) than its affinity forβ1AR activated by catecholamines. The in vitro evidence of dissociationbetween stimulation and blockade in feline and rat sinoatrial pace-maker, suggests but does not prove that sinoatrial tachycardia by non-conventional partial agonists presumably occurs through the β1LAR.(−)-CGP12177 elicited (−)-propranolol-resistant sinoatrial tachycardiaon ferret right atriumwith pEC50=7.5–7.9 and intrinsic activity 0.8–1.0compared to (−)-isoprenaline (Lowe et al., 1999, 2002).

The dissociation between low-potency stimulation (tachycardia)and high-potency β1AR blockadewith (±)-pindolol and (±)-oxprenololbut not with DCI and practolol (for in vitro data see above) wasconfirmed in catecholamine-depleted rats (Bilski et al., 1979). Thesinoatrial tachycardia by (−)-CGP1277 and (±)-cyanopindolol,observed in vitro (see above), was confirmed in vivo in the pithedrat (Malinowska & Schlicker, 1996, 1997).

In the perfused rabbit heart (±)-CGP12177 (500 nM) increased sino-atrial rate by 67% of the increase caused by (−)-isoprenaline (54 nM) butthe effects of β-blockers on the response of (±)-CGP12177 were notinvestigated (Younget al., 2002). Both (−)-isoprenalineand (±)-CGP12177also increase the Mg2+-efflux from perfused rabbit hearts (Young et al.,2002), but the relevance to the agonist-evoked tachycardia is not clear.

Deitchman et al. (1980) were one of the earliest groups (albeit un-knowingly) to provide an in vivo demonstration that a β-blocker((±)-bucindolol) is a non-conventional partial agonist. In anaesthethiseddogs bucindolol (3 mg kg−1) elicited significant tachycardia, resistant toantagonismbypropranolol. On theotherhand, bucindolol at 0.3mgkg−1

only produced marginal tachycardia but caused a 2 log unit shift of thechronotropic dose–response curve for (−)-isoprenaline, illustrating thedissociation between stimulation and blockade. 3 mg kg−1 bucindololalso caused a small decrease in blood pressure which may haveproduced reflex tachycardia via sympathetic nerve release of noradrena-line. However this was not the case since the tachycardia persisted incatecholamine-depleted dogs due to pre-treatment with reserpine.Furthermore, Deitchman et al. (1980) also reported that bucindololelicited a propranolol-resistant tachycardia in normotensive consciousdogs. In addition, Deitchman et al. (1980) showed that both bucindololand pindolol caused concentration-dependent tachycardia in catecho-lamine-depleted, reserpine-pretreated anaesthetised rats, but propra-nolol antagonized surmountably these effects. Bucindolol alsoincreased heart rate in renal-hypertensive dogs (Deitchman et al.,1980) as observed also before with (±)-pindolol (Himori et al.,1977). The pindolol-evoked tachycardia in renal-hypertensive dogswas not affected by propranolol (Himori et al., 1977).

Bucindolol evoked tachycardia in reserpine-pretreated rats (Mar-wood & Stokes, 1986) and pithed rats (Willette et al., 1999). Willetteet al. (1999) reported that the bucindolol-evoked tachycardia was


attenuated by carvedilol, propranolol and bisoprolol. Bucindolol in-creased murine sinoatrial heart rate. Tachycardia was more pro-nounced with intrinsic activity 0.4 compared to (−)-isoprenaline, intransgenic mice overexpressing β1AR. The bucindolol-evoked tachy-cardia was blocked by (−)-propranolol (Maack et al., 2003), makinginvolvement of β1LAR questionable. However, the high concentrationof (−)-propranolol (1 μM) used probably also blocked β1LAR (Kaumann& Molenaar, 1996).

(±)-Pindolol elicits tachycardia inman, particularlywhen the activityof the sympathetic nerve system is low. Man In't Veld and Schalekamp(1981), Man In't Veld et al. (1982), Clark et al. (1982), and Benditt et al.(1999) found that (±)-pindolol caused sinoatrial tachycardia in patientswith peripheral autonomic neuropathy and undetectable plasmanoradrenaline (Clark et al., 1982). During diurnal high sympatheticactivity pindolol can cause bradycardia through antagonism of theheart-rate enhancing effects of endogenous noradrenaline and adrena-line at β1AR and β2AR. Sympathetic activity fades nocturnally therebyfacilitating the manifestation of (±)-pindolol-evoked increases in heartrate (Fitscha et al., 1982; Kantelip et al., 1984; Channer et al., 1994).

Early work from Chiba (1979) reported evidence, obtained from ablood-perfused canine atrial preparation, that carteolol antagonizedthe positive chronotropic effects of noradrenaline at low concentra-tions but caused cardiostimulation at high carteolol concentrations.Takayanagi et al. (1989) confirmed in anaesthetised rats that carteololcaused sinoatrial tachycardia with intrinsic activity 0.88 with respectto (−)-isoprenaline and a dissociation between blockade and stimula-tion of approximately 3 log units reported by Chiba (1979). Furtherevidence that carteolol was a non-conventional chronotropic partialagonistwasprovidedbyFloreani et al. (2004) for rat sinoatrial node (pEC50~5.2) and Floreani et al. (2005) for guinea pig sinoatrial node (pEC50=6.1)(Table 1). As expected from mediation through the β1LAR, the positivechronotropic effects to carteolol in rat and guinea pig atria were resistantto blockade by 100 nM (−)-propranolol, a concentration that antagonizedthe effects of (−)-isoprenaline (Floreani et al., 2004, 2005) through β1HAR.

Carteolol increased heart rate in healthy volunteers and the effectwas not prevented, but actually enhanced by the β1AR-selectiveblocker bisoprolol (Bruck et al., 2004). Based on the sensitization ofβ2AR-mediated inotropic responses, discovered in human atrialtrabeculae to occur in patients chronically treated with β1AR-selectiveblockers (Hall et al., 1990), Bruck et al. (2004) attributed the carteolol-evoked tachycardia to be mediated through β2AR, presumablysensitized by bisoprolol but did not rule out mediation through β1LAR.

3.3. Atrial contractility

The positive inotropic effects of non-conventional partial agonistsshow variability between species. The variabilitymay reflect differencesin the physiological coupling of β1LAR, including the cAMP cascade andphosphodiesterases. For example, while inotropically relevant cAMPformed afterβ1LAR activation appears to be hydrolysed by PDE3 and notPDE4 in human atrium (Kaumann et al., 2007), it is hydrolysed by bothPDE3 and PDE4 in rat right ventricle (Vargas et al., 2006).

(±)-CGP12177 is a non-conventional partial agonist with intrinsicactivity 0.75 compared to (−)-isoprenaline in feline left atria (Kaumann,1983, 2000). The inotropic potency (pEC50=7.8) was 1.8 log units lowerthan the affinity, estimated from antagonism of the effects of (−)-isoprenaline (pKP=9.6) for atrial β-adrenoceptors (Table 1, Kaumann,1983, 2000). (−)-CGP12177 has (−)-propranolol (200 nM)-resistant agonistactivity on rat left atria with intrinsic activity 0.6 compared to (−)-isoprenaline (Kaumann&Molenaar,1996; Sarseroet al.,1999). Thepositiveinotropic effects and PKA signals of (−)-CGP12177 are potentiated by thenon-selective PDE inhibitor IBMXon rat atria (Kaumann & Lynham,1997).

In the human right atrium, (−)-CGP12177 decreases contractility atlow concentrations and increases contractility at high concentrations(Fig. 5; Kaumann, 1996; Sarsero et al., 2003). The cardiodepressanteffect of low (−)-CGP12177 concentrations is due to blockade of the

β1HAR-mediated effects of endogenous noradrenaline, released witheach pacing stimulus. Blockade of the β1HAR with (−)-propranololprevents the cardiodepressant effect and exaggerates the cardiosti-mulant effects of (−)-CGP12177 (Kaumann, 1996; Sarsero et al., 2003;Fig. 5). (−)-CGP12177 increases contractile force in the presence of(−)-propranolol (200 nM) with pEC50=7.3 and intrinsic activity 0.15–0.23compared to (−)-isoprenaline and causes hastening of relaxation(Kaumann, 1996; Sarsero et al., 2003; Table 1). The intrinsic activity of(−)-CGP12177 for positive inotropic effects was increased to 0.68 by PDEinhibitionwith IBMX (Table 1) and under these conditions it was accom-panied by small increases of cAMP levels and PKA activity (Sarsero et al.,2003). (−)-CGP12177 (60 nM) antagonized the positive inotropic effects of(−)-noradrenaline through β1HAR with pKB 9.7 (Joseph et al., 2003).

In ferret left atrium, (−)-CGP12177 increased contractility withpEC50=7.2–7.4 and an intrinsic activity of 0.4–0.5 (compared to (−)-iso-prenaline) which was resistant to blockade by (−)-propranolol (200 nM)(Lowe et al., 1999, 2002).

In feline left atrium (±)-iodo-hydroxybenzylpindolol increasedcontractility with pEC50=7.3 and intrinsic activity of 0.5 compared to(−)-isoprenaline, but antagonized the effects of (−)-isoprenalinewith apKP=9.5 (Fig. 1, Bearer et al., 1980). (−)-Pindolol produced a biphasicconcentration–effect curve on feline left atriumwith intrinsic activity0.15 compared to (−)-isoprenaline (Kaumann & Lobnig, 1986).

(−)-Pindolol does not enhance human atrial contractility unlessphosphodiesterases are inhibited, but potently antagonizes the effectsof (−)-noradrenaline with pKB=9.1 (Kaumann & Lobnig, 1986). In thepresence of the non-selective PDE inhibitor IBMX (20 μM), (−)-pindolol decreases contractility at low concentrations and increasescontractility at high concentrations (Joseph et al., 2003). The cardio-depressant effect of low (−)-pindolol concentrations is due to blockadeof the β1HAR-mediated effects of endogenous noradrenaline, releasedwith each pacing stimulus. Blockade of the β1HAR with (−)-propra-nolol prevents the cardiodepressant effect but exaggerates the cardio-stimulant effects of (−)-pindolol (Joseph et al., 2003). (−)-Pindolol is apartial agonist with intrinsic activity 0.5 compared to (−)-CGP12177 inthe presence of both IBMX and (−)-propranolol (Fig. 9). Under theseconditions, (−)-pindolol also hastens atrial relaxation, as expectedfrom an involvement of cAMP (Joseph et al., 2003).

Bucindolol (10 μM) increased contractile force of right atrialtrabeculae from patients without heart failure undergoing coronarybypass surgery, in the presence of a low concentration of forskolin(0.3 μM), propranolol (1 μM) and L-NMA (100 μM) with intrinsicactivity ~0.5 compared to (−)-CGP12177 (Bundkirchen et al., 2002).The effect of bucindolol was prevented by bupranolol (10 μM)(Bundkirchen et al., 2002). However, in the absence of forskolinbucindolol does not increase contractile force of human atrialtrabeculae and concentrations greater than 1 μM of (±)-bucindololdecrease force in a concentration-dependent manner (Molenaar &Kaumann, unpublished experiments). Bucindolol caused smallincreases in contractile force with intrinsic activity ~0.1 and ~0.2with respect to (−)-isoprenaline and (−)-CGP12177 respectively in thepresence of (−)-propranolol (200 nM) and IBMX (60 μM) (Table 1).Bucindolol antagonized the effects of (−)-isoprenaline (in the absenceof (−)-propranolol) and (−)-CGP12177 (in the presence of 200 nM(−)-propranolol) with pKB ~9.0 and pKB ~6.0 respectively (Table 2).

Carteolol increased guinea pig atrial contractility with a 2000-foldlower potency than its ability to antagonize the effects of (−)-isopre-naline (Floreani et al., 2005; Table 1). The positive inotropic effects ofcarteolol were resistant to blockade by 100 nM (±)-propranolol, con-sistent with mediation through β1LAR (Floreani et al., 2005).

3.4. Ventricular contractility

The positive inotropic effects of non-conventional partial agonistsare usually, but not always (the converse occurs in ferret), lesspronounced in ventricular trabeculae than in atrial trabeculae of rat,


cat andman. This trend could be due to lower β1AR densities and greaterphosphodiesterase activities in ventricle than atrium. For example in therat, (−)-CGP12177 and (±)-cyanopindolol on left ventricular myocardium(Kaumann & Molenaar, 1996) and (±)-CGP12177 on right ventricularpreparations (Vargas et al., 2006) failed to enhance contractility. However,in the presence of the phosphodiesterase inhibitor, IBMX, CGP12177 and(±)-cyanopindolol increased rat ventricular contractility (Sarsero et al.,1999). PDE3 inhibition with cilostamide and PDE4 inhibition withrolipram, administered separately or in combination, uncovered positiveinotropic effects in rat right ventricular myocardium (Vargas et al., 2006).Similarly, on human ventricle (−)-CGP12177 only produces marginal in-creases in contractility but in the presence of IBMX it has considerablymore pronounced effects (Table 1, Sarsero et al., 2003). The non-con-ventional partial agonists (±)-alprenolol, (±)-oxprenolol and (±)-pindolol,which exert atrial and sinoatrial cardiostimulation, do not produce po-sitive inotropic effects in feline right ventricular papillary muscles(Kaumann et al., 1980), but whether phosphodiesterase inhibition canuncover cardiostimulation has not been investigated.

(±)-Iodo-hydroxybenzylpindolol increased contractility in felineright ventricular papillary muscle with intrinsic activity 0.4 comparedto (−)-isoprenaline and pEC50=7.1, and antagonized the effects of(−)-isoprenaline with a pKP=8.7 (Fig. 1, Table 1). (±)-CGP12177increased feline ventricular contractility with intrinsic activity 0.6compared to (−)-isoprenaline and pEC50=7.2, and antagonized theeffects of (−)-isoprenaline with a pKP=9.2 (Kaumann, 1983, 2000).(−)-CGP12177 increased contractile force of ferret right and leftventricular papillary muscles with intrinsic activities of 0.7–0.8 and0.4 and pEC50=7.8 and 7.6 respectively (Lowe et al., 1999, 2002).

In the presence of (−)-propranolol (200 nM), (−)-CGP12177 causeda small increase in contractile force of human right ventriculartrabeculae that was greatly enhanced by IBMX, consistent with aprotective role of phosphodiesterases against β1LAR stimulation(Sarsero et al., 2003). As expected from a cAMP-dependent pathwayand in the presence of both (−)-propranolol and IBMX, (−)-CGP12177hastened ventricular relaxation by shortening both the time to peakcontraction and time to reach 50% relaxation with pEC50=7.4 (Sarseroet al., 2003, Table 1). In line with the in vitro findings of Sarsero et al.(1999, 2003), Zakrzeska et al. (2005) confirmed in vivo in pithedvagotomized rats that both (±)-CGP12177 and (±)-cyanopindololcaused positive inotropic and lusitropic effects through measure-ments of maximum rates of intraventricular pressure rise and decline.

Bundkirchen et al. (2002) claimed but did not demonstrate that, asfor human atrial myocardium, bucindolol (10 μM) also increasedcontractile force of left ventricular trabeculae from failing humanhearts, in the presence of a low concentration of forskolin (0.3 μM),propranolol (1 μM) and L-NMA (NG-methyl-L-arginine,100 μM). Othersreported bucindolol to produce variable effects on human leftventricular trabeculae, obtained from failing hearts, causing eithercardiodepression or cardiostimulation (Maack et al., 2000). Morerecently Maack et al. (2003) found that bucindolol did not modifyventricular contractility, arguing that this was due to β1AR desensi-tization caused by advanced heart failure. To resensitize β1AR, Maacket al. (2003) incubated the tissues for 90 min with 30 μM metoprolol,which caused marked depression of contractility. Following washout,bucindolol (1 μM) increased contractility by 43% (Maack et al., 2003).

Carteolol increased ventricular contractility and systolic pressure inhealthy volunteers (Bruck et al., 2004). The increase in systolic pressurewas not antagonized, but actually was increased by bisoprolol. Thecardiostimulant effects of carteolol were attributed to mediationthrough cardiacβ2AR, butmediation throughβ1LAR cannot be ruled out.

3.5. Ventricular Ca2+ currents, Ca2+ transients and action potentials.Are (−)-CGP12177-evoked arrhythmias mediated through β1LAR?

The effects of (−)-CGP12177 on L-type Ca2+ current, ICaL, are complex(Freestone et al., 1999). (−)-CGP12177 alone only caused inconsistent

increases in ICaL density in murine ventricular cardiomyocytes. Afterinactivation of Gi protein by incubation of cardiomyocytes with pertussistoxin, (−)-CGP12177 (100 nM) produced a small increase of ICaL (11%). Inthe presence of (−)-propranolol, (−)-CGP12177 actually reduced ICaL,plausibly due to inverse agonism, as also observed with the non-conventional partial agonist pindolol on guinea pig ventricular cardio-cytes (Mewes et al., 1993). In the presence of both (−)-propranolol(200 nM) and IBMX (6 μM), (−)-CGP12177 (100 nM) increased ICaL withintrinsic activity 0.2 compared to (−)-isoprenaline. Unexpectedly, lowconcentrations of (−)-CGP12177 (1–100 nM) increased Ca2+-transientswith intrinsic activity 0.3 compared to (−)-isoprenaline. (−)-CGP12177hastened the decline of Ca2+ transients in a concentration-dependentmanner (1–100 nM), expected from PKA-catalysed phosphorylation ofphospholamban and ensuing enhanced Ca2+ pumping back into thesarcoplasmic reticular stores by theCa2+-dependentATPase. The increasesin Ca2+ transients were prevented by (−)-bupranolol (1 μM) but not by(−)-propranolol (200 nM). Lownanomolar concentrations of (−)-CGP12177also elicited arrhythmic Ca2+ transients, consistent with early and late af-tertransients, probably due to early and late afterdepolarisations. (−)-Iso-prenaline elicited similar arrhythmic transients, but surprisingly at 40-foldhigher concentrations (pEC50=7.8) than (−)-CGP12177 (pEC50=9.4).

The findings of Freestone (1999) on murine cardiocytes are puzzlingbecause the extremely potent (nanomolar) effects of (−)-CGP12177 onCa2+ transients are not completely consistent with mediation throughthe β1LAR. Furthermore, the arrhythmic Ca2+ transients, evoked by(−)-CGP12177, are 40-fold more potent than those elicited by (−)-iso-prenaline presumably mediated through the β1HAR. This paradoxicalfinding needs clarification. An involvement of β1HAR and β2AR can beexcluded because the (−)-CGP12177-evoked arrhythmic Ca2+ transientswere resistant to blockade by (−)-propranolol (200 nM). An involvementof β1AR is likely because (−)-bupranolol prevented (−)-CGP12177-induced arrhythmias. The paradox is not due to an artefact inherent incardiocyte preparation because it is consistent with pronounced actionpotential changes observed in ferret ventricle (Lowe et al., 1998).(−)-Noradrenaline and (−)-adrenaline, acting through β1AR and β2AR, andalso (−)-CGP12177 in the presence of (−)-propranolol, caused abbreviationof action potential duration, but prolongation of the action potentialplateau (Fig. 7). The action potential shortening and plateau prolongationare consistent respectively, with activation of potassium channels andincrease in ICaL. Remarkably, Lowe et al. (1998) found that (−)-CGP12177was more potent than (−)-noradrenaline, acting through ferret β1HAR, ateliciting action potential shortening and plateau prolongation consistentwith the (−)-CGP12177-evoked arrhythmic Ca2+ transients in murine car-diocytes (Freestone et al., 1999). We speculate that these unusual findingsare eitherdue to a special conformational changeof theβ1LAR site oroccurthrough induction of another distinct (−)-propranolol-resistant state.

(−)-CGP12177wasnotonlymorepotent than (−)-isoprenaline, butwasalso as efficient as (−)-isoprenaline in eliciting arrhythmic Ca2+ transients,despite causingonly less than20%of themaximumincrease in ICaL densityand 30% of the maximum increase in Ca2+ transient caused by (−)-iso-prenaline (Freestone et al., 1999). To account for this second paradox, itwas assumed that Ca2+-induced Ca2+ release was more efficient throughthe β1AR site and/or conformation activated by (−)-CGP12177 than theβ1HAR activated by (−)-isoprenaline (Freestone et al., 1999).

3.6. Effects on human β1AR overexpressed into rat heart

Lewis et al. (2004) confirmed with (±)-CGP12177 on human β1AR(overexpressed into rat heart) several of the pharmacologic propertiesof (−)-CGP12177, established from work with human, ferret, rat andmurine ventricular and atrial myocardium (Kaumann, 1996; Kaumann& Lynham,1997; Freestone et al., 1999; Lowe et al., 2002; Sarsero et al.,1999, 2003; Kaumann et al., 2001). (±)-CGP12177 increased contrac-tility of rat ventricular myocytes with intrinsic activity 0.3 comparedto (−)-isoprenaline; the effects were antagonized by bupranolol,accompanied by an increase of cAMP and potentiated by IBMX.

Fig. 8. Comparison of pKB values, obtained from the antagonism of the effects of(−)-isoprenaline,withpKBvaluesobtained fromtheantagonismof theeffects of (−)-CGP12177,both as a function of pKi values obtained from inhibition of radioligand binding by clinicallyused β-blockers on ferret heart and human recombinant β1AR. The pKi values matched pKBvalues vs (−)-isoprenaline, consistent with mediation through β1HAR. In contrast, the pKBvalues vs (−)-CGP12177 were consistently lower than the pKi values, consistent withmediation through β1LAR. Data taken and plotted from Lowe et al. (2002) and Joseph et al.(2004a).

Fig. 7. Comparison of the effects of (−)-CGP12177 ((−)-CGP) in the presence of (−)-propranolol(200nM), (−)-noradrenaline ((−)-NA) in thepresenceof ICI118,551 (50nM)and (−)-adrenaline((−)-Ad) in the presence of CGP20712A (300 nM), bold lines on A) monophasic actionpotentials and B) left ventricular developed pressure, mediated through β1LAR, β1HARand β2AR respectively in the perfused ferret heart. The efficacy rank order is (−)-NAN

(−)-CGPN (−)-Ad for action potential shorting but (−)-AdN (−)-CGPN (−)-NA for actionpotential plateau prolongation. All 3 agonists increased ventricular pressure and hastenedrelaxation. The rank order for hastening of relaxation was (−)-NAN (−)-CGP≌(−)-Ad. Ctrl =control; ISO = (−)-isoprenaline 100 µM. (For other details see Lowe et al., 1998).


Consistent with a cAMP pathway, (±)-CGP12177 hastened cardiocyterelaxation and this was partially prevented by the PKA antagonist Rp-cAMP. As expected, these effects were found to bemore pronounced incardiocytes with 18-fold (relative to endogenous population of βAR)overexpressed human β1AR; (±)-CGP12177 and (−)-isoprenaline were6-fold and 12-fold more potent agonists and the maximum effect of(±)-CGP12177 was increased. Interestingly, propranolol (1 μM) slightlyantagonized the effects of (±)-CGP12177 in cardiocytes only expres-sing rat β1AR but not in cardiocytes overexpessing human β1AR.(±)-CGP12177 elicited arrhythmic contractions in rat ventricular cardio-myocytes overexpressing human β1AR. The arrhythmic thresholdconcentration of (±)-CGP12177 (1.2 μM at uninfected rat β1AR), butnot of (−)-isoprenaline, was reduced 3-fold in rat atrial myocytes thatoverexpressed the human β1AR (0.4 μM) (Lewis et al., 2004).

3.7. Effects of antagonists: a lead to cardiac β1LAR

Early observations indicated that some β-blockers, such as(−)-bisoprolol (Walter et al., 1984) and propranolol (reviewed by

Kaumann, 1989 and Kaumann & Molenaar, 1997) failed to antagonizeeffects of non-conventional partial agonists at concentrations thatantagonized the cardiostimulant effects of catecholamines. As men-tioned above, the first β-blocker that was recognized to antagonize thecardiostimulant effects of a non-conventional partial agonist was(−)-bupranolol (Walter et al., 1984), followed later by CGP20712A (Pak &Fishman, 1996; Kaumann & Molenaar, 1996; Malinowska & Schlicker,1996). The blocking potencies of (−)-propranolol and CGP20712A are,however, considerably lower (usually approximately 2 log units) thanthe corresponding affinities for β1HAR, estimated both from antagonismof the effects of catecholamines andbindingassays (Table 2). Subsequentevidence from cardiac β1AR (ferret) and recent work from recombinantβ1AR revealed that all clinically used β-blockers investigated so far,consistently exhibit lower affinity for β1LAR than for β1HAR (Fig. 8,Table 2).

The cardiostimulant effects of non-conventional partial agonistsare facilitated by the presence of a β-blocker devoid of cardiostimulanteffects. (−)-Propranolol at 200 nM, a concentration causing 98.5%β1HAR occupancy (pKB=8.5, Gille et al., 1985; pKi =8.5, Hoffmann et al.,2004) but less than 10% β1LAR occupancy (pKiL=5.7 rat ventricle,pKiL=5.3 rat atrium, Sarsero et al., 1999), prevented the cardiode-pressant effects of low (−)-CGP12177 concentrations but increased thecardiostimulant effects of high (−)-CGP12177 concentrations onhuman atrium (Kaumann, 1996; Sarsero et al., 2003; Fig. 5). It isdifficult to completely suppress the release of endogenous noradrena-line in electrically paced cardiac tissues, especially in human atrialtrabeculae, because relatively high currents have to be used to


uniformly activate all muscle fibers. The endogenously releasednoradrenaline enhances contractile force through β1HAR in pacedatrial trabeculae. The cardiodepressant effect of (−)-CGP12177 isexplained by blockade of the atrial β1HAR, activated by endogenousnoradrenaline. When β1HAR are blocked by (−)-propranolol and theeffect of endogenous noradrenaline prevented, the effects oncontractile force of (−)-CGP12177 are entirely cardiostimulant andincreased, compared to the absence of (−)-propranolol (Kaumann,1996; Sarsero et al., 2003; Fig. 5). Similar results were obtainedBundkirchen et al. (2002) on both human atrial and ventriculartrabeculae, on which bucindolol decreased contractile force in theabsence of (−)-propranolol (1 μM) but increased force in its presence.

Another factor that may reduce or prevent cardiostimulant effectsof non-conventional partial agonists is β1AR desensitization, assuggested by Maack et al. (2003). These authors failed to findcardiostimulant effects of bucindolol in ventricular trabeculae ob-tained from failing human hearts, pretreated 90 min with (−)-iso-prenaline (1 μM) followed by washout, to further desensitize βAR. Incontrast, a 90 min pre-treatment with a very high and markedlycardiodepressant concentration (30 μM) of the slightly β1AR-selective β-blocker (±)-metoprolol, followed by washout, causedbucindolol to increase contractile force. Maack et al. (2003) arguedthat the effect of metoprolol was due to resensitization of desen-sitized β1AR.

A strong argument for the hypothesis that cardiostimulant effectsof non-conventional partial agonists are mediated through β1LARcomes not only from the agreement of cardiostimulant potencies withbinding affinity estimates but also from the agreement with affinityestimates obtained from drug antagonism.

The cardiostimulant potencies of (±)-cyanopindolol and (−)-CGP12177are similar in rat heart (Kaumann & Molenaar, 1996; Malinowska &Schlicker, 1996; Sarsero et al.,1999). The inotropic potency of (±)-cyano-pindolol on rat left atrium (pEC50=7.5, Kaumann & Molenaar, 1996)matched the affinity of (±)-cyanopindolol for the left atrial β1-adre-noceptor binding sites (pKi=7.5) from the inhibition of [3H]-(−)-CGP12177binding by (±)-cyanopindolol, while its pKi for β1HAR and β2AR was 10.8(Sarsero et al., 1999). Furthermore, binding pKi values of 7.5 and 10.0 for(±)-cyanopindolol were estimated for ventricular β1LAR and β1HARlabelled by [3H]-(−)-CGP12177 of rat ventricle (Sarsero et al., 1999). ThepKβ1L was similar to the pEC50=7.0 of (±)-cyanopindolol, estimated fromrat left ventricular papillary muscle (Sarsero et al., 1999).

(±)-Cyanopindolol is a partial agonistwith respect to (−)-CGP12177 inrat cardiac tissues (Sarsero et al., 1999). Concentration–effect curves for(±)-cyanopindolol on rat sinoatrial pacemaker, left atrium and leftventricular papillary muscles were fitted satisfactorily with thecorresponding pKP values (pKP values of 7.3, 7.4 and 7.0 respectively),estimated from the antagonism of the cardiostimulant effects of(−)-CGP12177 by (±)-cyanopindolol (Sarsero et al., 1999). Moreover,(−)-pindolol is also a partial agonist with respect to (−)-CGP12177 in thepresence of IBMX (20 μM) and (−)-propranolol (200 nM) in humanatrium (Joseph et al., 2003). (−)-Pindolol antagonized the effects of(−)-CGP12177 with a pKP=6.4, not different from the pEC50=6.5 as anon-conventional partial agonist. The concentration–effect curve for(−)-pindolol was therefore satisfactorily fitted by use of its pKP value(Fig. 9, Joseph et al., 2003). (−)-Pindolol was also a partial agonist withrespect to (−)-CGP12177 on ferret right ventricular papillary muscle andsurmountably antagonized the effects of (−)-CGP12177 with pKP=7.6similar to the pEC50=7.4 for (−)-pindolol (Lowe et al., 2002). Theagreement of the cardiostimulant potencies of (−)-pindolol and (±)-cyanopindolol with their corresponding affinity estimates (pKP values),obtained from antagonism of the effects of (−)-CGP12177, demonstratesthat these non-conventional partial agonists compete with (−)-CGP12177 for themyocardialβ1LAR site to cause cardiostimulant effects.

Another approach illustrates systematically lower affinities of β-blockers as antagonists of the cardiostimulant effects of (−)-CGP12177than as antagonists of the effects of catecholamines, which supports

competition for β1LAR and β1HAR respectively. Lowe et al. (1999)compared themode of antagonism by carvedilol (10 nM–10 μM) of theeffects of (−)-isoprenaline and (±)-CGP12177 in ferret right and leftventricular papillary muscles as well as left atrium and sinoatrialnode. Carvedilol antagonized both effects through simple competitiveinteraction (Schild-plot slope one) and pKB=8.1–8.4 vs (−)-isoprena-line and pKB=6.8–6.9 vs (±)-CGP12177. Using large antagonistconcentration ranges, Lowe et al. (2002) systematically comparedthe affinities of another 10 clinically used β-blockers as antagonists ofthe cardiostimulant effects of (−)-CGP12177 and (−)-isoprenaline onferret right ventricular papillary muscle. They also compared theblocking potency against (−)-isoprenaline, i.e. pKB values, with pKi

values, estimated from inhibition of [125I]-(−)-cyanopindolol bindingto ferret ventricle. pKB values vs (−)-isoprenaline and binding pKi

values tended to agree (Fig. 8), consistent with mediation throughβ1HAR. In contrast, pKB values vs (−)-CGP12177 were systematicallylower than both pKB values vs (−)-isoprenaline and pKi values vs [125I]-(−)-cyanopindolol (Fig. 8, Table 2), suggesting competition between(−)-CGP12177 and the β-blockers for β1LAR.

In addition to investigations of the ability of clinically used β-blockersto antagonize the cardiostimulant agonist effects of (−)-CGP12177, theβ1AR-selective experimental compound CGP20712A also antagonizesconsiderably less (~3 log units) the agonist effects of CGP12177 than ofcatecholamines (Table 2, Kaumann & Molenaar, 1996; Malinowska &Schlicker, 1996, 1997).

3.8. Vascular pharmacology of non-conventional partial agonists

Following the demonstration that (±)-cyanopindolol is a non-conventional partial agonist through the (−)-bupranolol-sensitivelow-affinity site of the β1AR on rat cardiac tissues (Kaumann &Molenaar,1996;Malinowska & Schlicker,1996), (±)-cyanopindolol wasalso used as a tool, in addition to (±)-CGP12177, on vascular tissues.(±)-Cyanopindolol (pEC50=5.4–5.6) and (±)-CGP12177 (pEC50=4.2–4.4) relaxed rings of rat aorta, precontracted by noradrenaline(Brawley et al., 2000a) or phenylephrine (Mallem et al., 2004, 2005).Brawley et al. (2000b) reported that the NO-synthase inhibitorL-NAME reduced the relaxant potencies of both (±)-cyanopindolol and(±)-CGP12177, suggesting that the non-conventional partial agonistscaused relaxation in part through atypical β1AR located on both smoothmuscle and endothelium. However,Mallemet al. (2004) failed tofindaneffect of the NO-synthase inhibitor L-NMMA and of endothelialdenudation on the relaxant potencies of both cyanopindolol and(±)-CGP12177. As expected from the low-affinity site of the β1AR, the(±)-CGP12177-evoked relaxation was resistant to blockade by (±)-propra-nolol (300 nM) (Brawley et al., 2000a) and (±)-nadolol (3 μM) (Mallemet al., 2004). The relaxant effects of (±)-CGP12177 were antagonizedby high concentrations of both bupranolol (10–100 μM) andCGP20712A (1–10 μM) (Brawley et al., 2000a).

Relaxant effects of (±)-cyanopindolol and (±)-CGP12177, resistant topropranolol (300 nM) but sensitive to blockade by bupranolol (10 μM)or (±)-CGP20712 (10 μM),were found on rat isolatedmesenteric artery,precontracted with either phenylephrine or serotonin (Kozlowskaet al., 2003). Endothelium removal reduced the relaxant potencies ofboth cyanopindolol and (±)-CGP12177 (Kozlowska et al., 2003).Mesenteric resistance vessels of the rat, precontracted with pheny-lephrine, also relaxed with high (±)-CGP12177 concentrations(pEC50=5.7) in a propranolol (100 nM)-resistant manner (Brioneset al., 2005).

Very high concentrations of (±)-cyanopindolol (pEC25=4.3) relaxedisolated human pulmonary arteries, precontracted with serotonin, inthe presence of (±)-propranolol (300 nM) and this low potency wasfurther decreased by 10 μM of either (±)-bupranolol or (±)-CGP20712(Kozlowska et al., 2006).

Carteolol relaxed phenylephrine-precontracted rat femoralarteries with a pEC50 ~5; the effects were partially antagonized by


10 μM (±)-propranolol (Floreani et al., 2004). Carteolol also relaxedendothelium-denuded rat tail arteries with pEC50 ~4; the effects werepartially antagonized by (±)-timolol 10 μM (Floreani et al., 2004).

The relaxant effects of both (±)-cyanopindolol and/or (±)-CGP12177 have been attributed to β1LAR (Mallem et al., 2004, 2005)or through atypical βAR (Brawley et al., 2000a, 2000b; Kozlowskaet al., 2003, 2006). The resistance to blockade by propranolol isconsistent but does not prove an involvement of β1LAR. Most otherevidence appears to be inconsistent. Inconsistencies on vasculartissues include: 1. Low relaxant potencies of (±)-cyanopindolol and(±)-CGP12177. 2. Lack of competition between cyanopindolol and(±)-CGP12177 for a common receptor site. 3. Low blocking potencies ofbupranolol and CGP20712.

1. As found on other species (Kaumann, 1989) including man(Kaumann, 1996), CGP12177 also causes cardiostimulation atnanomolar concentrations on rat cardiac tissues (Kaumann &Molenaar, 1996; Malinowska & Schlicker, 1996, 1997; Sarsero et al.,1999) but vascular relaxation at micromolar concentrations(Brawley et al., 2000a 2000b; Koslowska et al., 2003; Mallem etal., 2004, 2005; Briones et al., 2005). The vascular relaxant potencyof (±)-cyanopindolol (Brawley et al., 2000a,b; Kozlowska et al.,2003; Mallem et al., 2004, 2005) is also consistently lower than itscardiostimulant potency (Kaumann & Molenaar, 1996; Malinowska& Schlicker, 1996; Sarsero et al., 1999). The low vascular relaxantpotencies of (±)-CGP12177 and (±)-cyanopindolol, compared tocardiostimulation, could be due to the need of precontraction withphenylephrine or noradrenaline through α1-adrenoceptors and5-hydroxytryptamine (5-HT) possibly through 5-HT2A receptors.These contractile agonists enhance smooth muscle cell calcium sothat greater relaxant stimuli, presumably via cAMP-dependentpathways, are needed to reduce the enhanced cell calcium andensure relaxation than in the absence of contractile agonists.Therefore the concentrations of catecholamines and non-conven-tional partial agonists needed to produce relaxation are higher thanfor cardiac tissues.

2. (±)-Cyanopindolol (1 μM) did not affect the relaxing potency of(±)-CGP12177 in the rat aorta (Brawleyet al., 2000a), inconsistentwithan interactionwith a commonβAR site. In contrast, (±)-cyanopindolol(1 μM) antagonized the effects of (−)-CGP12177 in the rat left ven-tricular papillary muscle, left atrium and sinoatrial node with pKP

values of 7.0, 7.3 and 7.4 respectively, consistent with pKi=7.5 valuesfor rat ventricle and left atrium at β1AR (Sarsero et al., 1999).

3. The blocking potencies of (±)-bupranolol against (±)-CGP12177 or(±)-cyanopindolol were one order of magnitude lower on the rataorta (pKB=5.0–5.7, Brawley et al., 2000a) and mesenteric artery(pKB=5.4, Kozlowska et al., 2003) than on the rat heart (pKB=6.4–7.0 (−)-bupranolol Kaumann &Molenaar, 1996; Sarsero et al., 1999).

The quantitative discrepancies detailed in points 2 and 3 stronglydisagree with the assumption that vascular relaxation is mediatedthrough the low-affinity site (β1LAR) as described in heart. Thus, theclaim that relaxation of rat aorta is mediated through the low-affinitysite (β1LAR) of cardiac β1AR (Mallem et al., 2004, 2005) is notsupported by quantitative considerations and the nature of thevascular atypical βAR remains elusive. The very low vascular relaxantpotency of (±)-cyanopindolol could conceivably be mediated througha very low-affinity site similar to that observed in rat ventricle(pKi =5.0 for (±)-cyanopindolol and distinct from the (β1LAR) withpKi =7.4, Sarsero et al., 1999). However, this speculation is inconsistentwith the lack of competition between (±)-CGP12177 and (±)-cya-nopindolol for the relaxation of rat aorta (Brawley et al., 2000a).Another speculation is that the low blocking potency of bupranolol inrat aorta and mesenteric artery is due to an interaction with a verylow-affinity binding site detected for [3H]-(−)-bupranolol in guineapig ventricle (pKD=4.8, Walter et al., 1984). It would imply that [3H]-(−)-bupranolol, used byWalter et al. (1984) in guinea pig ventricle and

[3H]-(−)-CGP12177, used by Sarsero et al. (1999) in rat ventricle,labelled vascular binding sites. It is unknownwhether these very low-affinity sites belong to β1AR, β2AR, or β3AR or even are β-adrenoceptors at all.

One important complicating factor for vascular relaxation causedby (±)-cyanopindolol and (±)-CGP12177 is that both cause relaxationof phenylephrine-precontracted rat and human arteries throughblockade of α1-adrenoceptors (Brahmadevara et al., 2003, 2004;Kozlowska et al., 2005). Brahmadevara et al. (2004) have demon-strated that (−)-CGP12177 was a competitive antagonist (pKB=5.3) ofthe contractile effects of phenylephrine on rat aorta and that (−)-CGP12177 and (±)-cyanopindolol competed for binding of [3H]-prazosin for rat cerebral α1-adrenoceptors with pKi values of 6.3and 5.5 respectively. Furthermore, Brahmadevara et al. (2004)reported that (±)-propranolol bound to α-adrenoceptors with apKi =5.8, consistent with a pA2=5.2 vs the α1-adrenoceptor agonistmethoxamine in rabbit aortic strip (Gulati et al., 1969). (±)-Cyano-pindolol antagonizes 5-HT-evoked contractions (Kozlowska et al.,2005), presumably through blockade of 5-HT2A receptors (pKi =4.5,Hoyer, 1989) in the human pulmonary artery, thereby causingrelaxation. Therefore, physiological antagonism should probably beavoided altogether in vascular research with non-conventional partialagonists. Alternatively, blood vessel tone could be increased throughtitration with KCl in the presence of an α1-adrenoceptor blocker (toavoid interaction with α1-adrenoceptors of noradrenaline, releasedfrom adventitial nerves by KCl-evoked depolarisation), therebycausing partial depolarisation and moderately increasing vasculartone in a stable fashion as observed with rings of human extracranialarteries relaxed by calcitonin gene related peptidesα-CGRP andβ-CGRP(Verheggen et al., 2002, 2005).

3.9. Summary

The activated cardiac β1LAR couples to the Gs protein→adenylylcyclase→cAMP→PKA→phosphoprotein→Ca2+cycling→inotropism→lusitropism pathway. Cyclic AMP generated through β1LAR is hydro-lysed by both PDE3 and PDE4 in rat ventricle but only by PDE3 inhuman atrium. PDE3 protects the human heart against overstimula-tion through both β1HAR and β1LAR. Consistent with the cAMPpathway, non-conventional partial agonists produce sinoatrial tachy-cardia, increase atrial and ventricular force and hasten relaxationusually through β1LAR in a variety of species including man. Theβ1LAR-mediated cardiostimulant effects are relatively resistant toblockade by propranolol and other clinically used β-blockers. Non-conventional partial agonists can produce experimental and clinicalarrhythmias, presumably through Ca2+-induced early and late after-depolarisations. Although there is some evidence for atypical β-adre-noceptors activated by non-conventional partial agonists in bloodvessels, these receptors appear unrelated to the cardiac and recom-binant β1LAR.

At present, there is no evidence for previously proposed orendorsed interpretations that non-conventional partial agonistsrelax blood vessels through β1LAR (Kozlowska et al., 2003; Arch,2004; Mallem et al., 2004; Baker, J. G., 2005) or through anotherdistinct state of an atypical βAR (Molenaar, 2003).

4. Pharmacology of non-conventional partialagonists at recombinant β1AR and plausible cardiac relevance

Following the agonist effects demonstrated for (±)-CGP12177 onheart (Kaumann,1983), this β-blocker also showed agonist effects on fatcells. (±)-CGP12177 activates thermogenesis through adenylyl cyclasestimulation in brown fat (Mohell & Dicker, 1989). Konkar et al. (2000a)observed biphasic concentration–effect curves for (±)-CGP12177 foradenylyl cyclase stimulation in brown fat membrane particles. Withthe help of β1AR and β3AR knockout mice and recombinant β1AR


Konkar et al. (2000a) demonstrated that CGP12177-evoked stimu-lation of adipocyte adenylyl cyclase occurs through the βlLAR of Pakand Fishman (1996) at low concentrations but through β3AR athigh concentrations. Using recombinant β1AR of rat and human atphysiological densities, Konkar et al. (2000a) also confirmed thework of Pak and Fishman (1996) by showing that CGP20712Aantagonized the effects of CGP12177-evoked stimulation of adeny-lyl cyclase with a pKB ~6.9 on human β1LAR and a pKB ~7.3 on ratβ1LAR. Konkar et al. (2000a) reported that CGP20712A antagonized6-fold and 18-fold more the effects of (−)-isoprenaline than (±)-CGP12177 respectively on rat and human β1HAR. They also reportedthat (−)-propranolol was a 40-fold and 26-fold more potent antagonistof the effects of (−)-isoprenaline than of (±)-CGP12177 on humanβ1AR.

Konkar et al. (2000b) further confirmed the work of Pak andFishman (1996) by showing that the intrinsic activity of (±)-CGP12177with respect to (−)-isoprenaline was a function of rat β1AR density,being a non-conventional partial agonist with intrinsic activity=0.4 at40 fmolmg−1 receptor density but nearly a full agonist at 3.6 pmolmg−1

density. In addition to (±)-CGP12177 Konkar et al. (2000b)) found onboth recombinant rat and human β1AR that another 2 aryloxypropa-nolamines, SB251023 and LY3662884, also activated adenylyl cyclaseboth with intrinsic activity 0.3 at rat β1LAR, and intrinsic activity 0.3and 0.4 respectively at human β1LAR. Consistent with mediationthrough the β1LAR, the effects of (±)-CGP12177 (pEC50=7.9), SB251023(pEC50=4.9) and LY3662884 (pEC50=8.5)were antagonizedwith loweraffinity by (−)-propranolol (pKB ~7.0) and CGP20712A (pKB ~7.4–7.0)than the effects of several catecholamines (pKB ~8.5–8.1), mediatedthrough β1HAR. Of note, SB251023 failed to antagonize the effects of(−)-isoprenaline.

The findingswith aryloxypropanolamines led Konkar et al. (2000b)and Granneman (2001) to suggest that this class of compounds hadlower affinity for β1LAR than for β1HAR. However, as reported byLowe et al. (2002) from ferret ventricle, the ethanolamine β-blocker(±)-sotalol also antagonized the positive inotropic effects of (−)-CGP12177with lower potency than antagonizing the effects of (−)-isoprenaline(Fig. 8A; Table 2). The finding of Lowe et al. (2002) was confirmed onrecombinant human β1AR, expressed at physiological density by Josephet al. (2004a)) who reported that (±)-sotalol antagonized the agonisteffects of (−)-isoprenaline and (−)-CGP12177 with pKB=6.0 at β1HAR andpKB=3.9 at β1LAR (Table 2). Therefore, in addition to aryloxypropanola-mines, the ethanolamines can also recognize the β1LAR (Lowe et al., 2002;Joseph et al., 2004a).

As reported by Lowe et al. (2002) for the positive inotropic effectsof (−)-isoprenaline and (−)-CGP12177 on ferret ventricle (Fig. 8),Joseph et al. (2004a) also found on recombinant human β1AR that theblocking potency of 12 clinically used β-blockers was systematicallylower against the cAMP signals produced by (−)-CGP12177 than(−)-isoprenaline (Fig. 8), consistent with competition for β1HAR andβ1LAR respectively. The affinity difference between the 2 sites rangedfrom 56-fold ((−)-bupranolol) to 1000-fold ((−)-atenolol) (Table 2).

Baker et al. (2003) investigated the effects β-blockers on humanrecombinant β1AR-mediated gene transcription through β1HAR andβ1LAR sites. Comparing antagonism of increases in cAMP responseelement (CRE) by (−)-isoprenaline and (±)-CGP12177 at human β1ARtransfected at physiological density (79 fmol mg−1), Baker et al. (2003)found that atenolol, propranolol and carvedilol were 2.6, 1.4 and 1.9log units more potent respectively as antagonists of the effects of(−)-isoprenaline than (±)-CGP12177. These results are in line withprevious results of Lowe et al. (1999, 2002) showing that atenolol,propranolol and carvedilol antagonized 2.8, 2.8 and 1.3 log units morethe ventricular positive inotropic effects of (−)-isoprenaline than of(−)-CGP12177 respectively, consistent with an interaction at β1HAR andβ1LAR. Also in linewith thework of Lowe et al. (1999, 2002) and Baker etal. (2003), Joseph et al. (2004a) found that (−)-atenolol, (−)-propranololand (±)-carvedilol antagonized 3.0, 2.1 and 2.3 log units more the cAMP

signals generated by (−)-isoprenaline than by (−)-CGP12177 at humanβ1AR transfected at the physiological density of 101 fmol mg−1.

Baker et al. (2003) found that CGP20712Awas a 2.4 log units morepotent antagonist of the effects of (−)-isoprenaline than (±)-CGP12177,somewhat out of line with a 1.3–1.5 log unit selectivity of CGP20712Afor human recombinant β1HAR reported by Konkar et al. (2000a,2000b) but more in line with a 3.1 log unit difference for rat sinoatrialβ1HAR (Kaumann, 1986; Kaumann & Molenaar, 1996; Table 2).However, and for unknown reasons, the large selectivity ofCGP20712A for rat cardiac β1HAR was not observed by Konkar et al.(2000a, 2000b) at rat recombinant β1AR, on which CGP20712A wasonly 0.8–1.6 log units selective for β1HAR compared to β1LAR.

In CHO cells expressing a high β1AR density (~1 pmol mg−1) andtransfected with the reporter gene secreted placental alkalinephosphatase (SPAP), under transcriptional control of a six-CREpromoter (McDonnell et al., 1998), Baker et al. (2003) reported that(−)-isoprenaline and (±)-CGP12177 increased SPAP secretion as fullagonists. Atenolol inhibited the SPAP secretion caused by (−)-iso-prenaline and (±)-CGP12177 with pKB=6.9 and 5.2, consistent withinteraction through β1HAR and β1LAR, respectively. Interestingly,Baker et al. (2003) found that acebutolol, carvedilol and labetalolincreased SPAP production, each with intrinsic activity ~0.4 comparedto (−)-isoprenaline. Propranolol caused small increases in SPAP(intrinsic activity=0.13). Furthermore, pindolol and alprenolol pro-duced biphasic concentration–effect curves of SPAP formation withintrinsic activities of ~0.7 and ~0.5 respectively. The β-blockersmetoprolol, CGP20712A, ICI118,551, atenolol and sotalol failed to affectgene transcription. CGP20712A antagonized less the effects ofcarvedilol (pKB=8.3) than the effects of acebutolol (pKB=9.9) andlabetalol (pKB=9.1), consistent with the interaction of carvedilol withβ1LAR, in addition to β1HAR, previously reported by Lowe et al. (1999,2002) on ferret heart. CGP20712A (100 nM) only antagonized thehigh-potency component but not the low-potency component of theeffect of (−)-pindolol and alprenolol (Baker et al., 2003). In their CHO-β1AR-SPAP cells Baker et al. (2003) found that acebutolol, labetalol,carvedilol and propranolol caused small increases in cAMP levels,confirming earlier findings of Lattion et al. (1999) with (−)-propranolol(10−6 M) and carvedilol (10−5 M) on recombinant β1AR expressed inHEK-293 cells at 2.5–3 pmol mg−1.

Although both the SPAP signal and cAMP signal by (−)-propranololreported by Baker et al. (2003) are quite small, theymay be related to asmall positive inotropic effect of slow onset, observed with (±)-pro-pranolol by Blinks (1967) on feline left atrium, and it is plausible thatthe 3 effects of (−)-propranolol are mediated through β1LAR. Althoughthe SPAP and cAMP signals caused by carvedilol are somewhat greaterthan those of (−)-propranolol, there is no evidence for acutecardiostimulant effects of carvedilol on isolated myocardium, asobserved with propranolol (Blinks, 1967). On ferret isolated myocar-dium, the cardiac system that best amplifies non-conventional partialagonist effects through β1LAR (Lowe et al., 1998, 2002), carvedilol upto 10 μMwas devoid of cardiostimulant effects (Lowe et al., 1999). Therelevance to cardiac function of the SPAP and cAMP signals, evoked bycarvedilol at non-physiological high β1AR density, is unknown at thetime of this review.

The biphasic concentration–effect curve of pindolol reported withSPAP production on recombinant β1AR was also observed with cAMPby Baker et al. (2003), resembling similar biphasic concentration–effect curves found for (−)-pindolol on guinea pig sinoatrial node(Walter et al., 1984) and racemic pindolol on feline sinoatrial node(Kaumann & Blinks 1980a) and anaesthetised rats treated withreserpine (Barrett & Carter, 1970). Only the high-potency componentof the effect of (−)-pindolol was antagonized by (−)-bisoprolol (Walteret al., 1984) or (±)-propranolol (Kaumann & Blinks, 1980a). However,the low-potency component of the effects of (−)-pindolol wasantagonized by (−)-bupranolol (1 μM), as expected from an interactionwith β1LAR (Walter et al., 1984, see also Fig. 3). Both (−)-pindolol and

Fig. 9. Positive inotropic effects of (−)-pindolol andantagonismof theeffects of (−)-CGP12177by (−)-pindolol (6 µM) in the presence of (−)-propranolol (200 nM) and IBMX (20 μM) onhuman atrial trabeculae. The equilibrium dissociation constant KP was calculated and thecalculated β1LAR occupancy curve (broken line), normalised to themaximumeffect of (−)-pindolol, well fitted the data of the concentration–effect curve of (−)-pindolol. Adaptedfrom Joseph et al. (2003) with kind permission of Springer Science+Business media.


(−)-alprenolol also increased sinoatrial rate and right ventricular forceof ferret myocardium, probably through β1LAR (Lowe et al., 2002). Itwould be interesting to know whether the low-affinity component ofthe pindolol-evoked SPAP production and cAMP reported by Baker(2003) is antagonized by (−)-bupranolol and CGP20712A with apotency expected from their corresponding affinities for β1LAR.

Human atrial myocardium is less sensitive to effects mediatedthrough βAR than feline and ferret myocardium, and (−)-pindolol failsto increase force of atrial trabeculae (Kaumann & Lobnig, 1986).Inhibition of phosphodiesterases with IBMX, however, uncovers(−)-propranolol-resistant increases of contractile force by (−)-pindololin human atrial trabeculae (Joseph et al., 2003). The cardiostimulantpotencies of (−)-pindolol and (−)-CGP12177, in the presence of IBMX,on human atrium (pEC50=6.5 and 7.6 respectively) matched thecAMP-enhancing potencies (pEC50=6.5 and 7.7) at recombinantβ1AR, transfected at physiological density (101 fmol mg−1) and theeffects were resistant to (−)-propranolol (200 nM). Furthermore,(−)-pindolol was a partial agonist with respect to (−)-CGP12177 inhuman atrium (Fig. 9). As expected from competition for a common site,(−)-pindolol surmountablyantagonized the effects of (−)-CGP12177withpKP=6.4. The agreement of stimulant potencies of (−)-pindolol onhuman atrium and recombinant receptors with the blocking potencyagainst (−)-CGP12177 demonstrates competition and interaction withβ1LAR (Joseph et al., 2003). In contrast, the potency of (−)-pindolol as anantagonist of both catecholamine-evoked cardiostimulation throughhuman atrial β1AR (pKB=9.1, Kaumann & Lobnig, 1986) and catechola-mine-evoked cAMP signal through recombinant β1HAR (pKB=8.6) wassimilarly high, as expected from β1HAR (Joseph et al., 2003). However,Joseph et al. (2003) did not find evidence on human atrium andrecombinant β1AR for the (−)-bisoprolol-sensitive component of(−)-pindolol observed on guinea pig sinoatrial node (Walter et al.,1984), as well as CGP20712A-sensitive component of pindololobserved by Baker et al. (2003) at pmol mg−1 recombinant β1ARdensity. The biphasic chronotropic concentration–effect curves ofpindolol, observed in the rat (Barrett & Carter, 1970), guinea pig(Walter et al., 1984) and feline sinoatrial node (Kaumann & Blinks,1980a) are probably the result of amplification of signals throughβ1HAR and β1LAR, unperturbed by phosphodiesterases. Consistentwith this interpretation is a recent finding on murine sinoatrialnode, that the phosphodiesterase inhibitors cilostamide (PDE3-selective) and rolipram (PDE4-specific) do not modify the chron-otropic potency of (−)-noradrenaline through β1HAR, in contrast toeffects through atrial and ventricular β1HAR, controlled by PDE4(Galindo-Tovar & Kaumann, 2008).

Joseph et al. (2004a) compared the antagonism of 12 clinically usedβ-blockers as antagonists of the cAMP-enhancing effects of (−)-isoprenaline through β1HAR and (−)-CGP12177 through β1LAR onrecombinant β1AR transfected at physiological density (101 fmol mg−1).On this system Joseph et al. (2004a) found that [3H]-(−)-CGP12177bound to two saturable sites with pKD=9.3 (β1HAR) and 6.6 (β1LAR).

They then measured the affinity of the β-blockers as inhibitors of 3–5 nM (−)-[3H]-CGP12177 binding, a concentration that occupies 87 to91% of β1HAR but only up to 2% of β1LAR. As expected, the blockingpotencies of the β-blockers correlated closely with their affinities forβ1HAR (Fig. 8). In contrast, the affinity estimates obtained from theantagonism of the (−)-CGP12177-evoked cAMP signals, mediatedthrough β1LAR, were 1.75 to 3 log units lower than the bindingaffinities at β1HAR (Table 2, Fig. 8). Thus not only (−)-propranolol and(−)-bupranolol had differential affinity for the two β1AR sites,another 10 clinically used β-blockers also did.

In linewith thework of Lowe et al. (2002) and Joseph et al. (2004a),Baker J. G. (2005) subsequently reported similar affinity differences forβ-blockers at the two sites, measuring CRE luciferase production andcAMP signals produced by (−)-isoprenaline, as well as (±)-CGP12177on recombinant β1AR at physiological density (Table 2). In addition,Baker J. G. (2005) found that CGP20712A and 10 clinically used β-blockers systematically antagonized the effects of cimaterol (anagonist with skeletal muscle growth-promoting properties, Kim etal., 1992) with slightly higher affinity (0.24–0.57 log units) than theeffects of (−)-isoprenaline and (−)-noradrenaline. Baker J. G. (2005)suggested that the catecholamines may activate both binding sites,while cimaterol and CGP12177 purely activate β1HAR and β1LARrespectively. An alternative interpretation of such findings could alsobe considered. Conceivably, cimaterol uses an additional bindingpartner or different binding partners to the binding partners ofcatecholamine binding to the β1HAR, thereby causing a conformationthat slightly increases the affinity of β-blockers. In the overexpressedrecombinant β1AR-SPAP system (~1 pmol mg−1) Baker J. G. (2005) alsoreported, that in addition to cimaterol, the effects of the non-physiological agonists, xamoterol, terbutaline, formoterol, clenbuterol,salbutamol tulobuterol and fenoterol were antagonized slightly more(0.25–0.67 log units) by CGP20712A than the effects of (−)-isoprenaline.

Lowe et al. (1999) reported for ferret isolated myocardium thatcarvedilol antagonized the cardiostimulant effects of (−)-CGP12177(pKB=6.8–6.9) with lower potency than the effect of (−)-isoprenaline(pKB=8.1–8.4). Interestingly, the ~2.3 log unit lower affinity of carvedilolfor human β1LAR, compared to human β1HAR, reported by both Josephet al. (2004a)) and Baker J. G. (2005), is in line with the 1.3 log unit loweraffinity for ferret cardiacβ1LAR.Affinityestimates fromtheantagonismbya β-blocker of (−)-CGP12177-evoked cardiostimulation in human atriumis currently only available for (−)-pindolol (Joseph et al., 2003). A moregeneral comparison of the cardiac relevance of the affinity of clinicallyused β-blockers for human recombinant β1LAR was therefore mademostly with the data of Lowe et al. (2002) obtained from the antagonismby 11 clinically used β-blockers of the positive inotropic effects on ferretventricle (Fig. 8). Despite the species difference the affinity estimatesobtained at human recombinant β1LAR agree remarkably well with theaffinity estimates for ferret ventricular β1LAR. These results, takentogether with the finding that the affinity of (−)-pindolol is identical forhuman recombinant and atrial β1LAR (Joseph et al., 2003), are consistentwith the cardiac relevance of research with recombinant β1LAR.

4.1. Summary

Recombinant β1AR mimic the pharmacology of non-conventionalpartial agonists unravelled through both cardiac β1HAR and β1LAR. Atboth cardiac and recombinant β1AR, clinically used β-blockers antag-onize the effects of catecholamines through β1HAR with 1.5–3 log unithigher potency than the agonist effects of non-conventional partialagonists through β1LAR.

5. Structural considerations

Structural features of the β1AR that are critical for catecholaminebinding (Sugimoto et al., 2002) have been deduced from pioneeringwork with the β2AR (Strader et al., 1987, 1988, 1989a, 1989c; Wieland


et al., 1996; reviewed by Strader et al., 1994). Critical anchoring pointsfor amine, hydroxyl and aromatic ring structures of catecholamineshave been determined in mutagenesis studies of the β2AR andidentification of homologous amino acids of the β1-adrenoceptor bysequence alignment (Sugimoto et al., 2002, Fig. 10).

The protonated amine group of catecholamines forms an ionicbond with the carboxylate group of Asp113 β2AR in TMD III (Straderet al., 1988). The amine group of the phenoxypropanolaminecompounds, propranolol, [125I]-cyanopindolol, [3H]-alprenolol and[3H]-(−)-CGP12177 is also anchored with Asp113 β2AR (Strader et al.,1987, 1988). These phenoxypropanolamine compounds all interactwith both β1HAR and β1LAR (Tables 1 and 2). The amine group of theβ1AR selective catecholamine (−)-RO363 (Molenaar et al., 1997b) wasassumed to interact with Asp138-β1AR which corresponds withAsp113 of the β2AR (Sugimoto et al., 2002). Previously, we probablycame to a wrong conclusion that (−)-RO363 caused positive inotropiceffects in human right atrium through activation of β1LAR (Molenaaret al., 1997b) since a low-affinity component of (−)-RO363 in thepresence of CGP20712A could alternatively be interpreted to be due toan interaction with β2AR. The function of Asp113 is essential for β2ARbut the function of the β1AR homologue Asp138 is only essential forthe β1HAR but not for the β1LAR, as reviewed in Section 5.2.

TMD V of the β1AR has three serine amino acids, Ser228, Ser229and Ser232. The catechol hydroxyl groups of (−)-RO363 were assumedto bind to Ser229 and Ser232 (Sugimoto et al., 2002). The β2ARhomologues are Ser203, Ser204 and Ser207. For catecholaminebinding at β2AR, hydrogen bonds occur between the meta-hydroxyland Ser203 (Sato et al., 1999) and Ser204 (Strader et al., 1989c) andbetween the para-hydroxyl and Ser207 (Strader et al., 1989c) in TMDV. Mutation of each of the Ser groups to Ala caused a reduction inaffinity and potency of isoprenaline for adenylyl cyclase or cyclic AMPresponses (Strader et al., 1989c; Sato et al., 1999). The benzimidazo-lone and indole groups of (−)-CGP12177 and (−)-pindolol are devoid ofhydroxyl groups corresponding to catechol hydroxyl groups.

The β-hydroxyl group of the chiral carbon of catecholaminesnoradrenaline, adrenaline and isoprenaline (Table 2) forms a hydro-gen bond with Asn293 β2AR of TMD VI (Wieland et al., 1996). Thecatecholamine β-hydroxy-Asn293 hydrogen bond contributes to thedifferent affinities of the stereo-isomers of the catecholamines(Wieland et al., 1996). The β1AR homologue, Asn344 would also be

Fig. 10. Two dimensional representation of the human β1AR indicating amino acid sites of magonist activity. Asp138 is considerably more critical for the function of agonists at β1HAR comβ1HAR and β1LAR pharmacology. In comparison to Arg389 recombinant β1ARs, agonist activiappears to contribute to the formation of β1LAR since (−)-CGP12177 is a classical partial ago

anticipated to interact with the β-hydroxyl group of a phenylethano-lamine, such as noradrenaline, but not a phenoxypropranolamine(Sugimoto et al., 2002).

The aromatic group of catecholamines is predicted to be stabilizedwithvanderWaals forces byPhe341-β1AR (Sugimoto et al., 2002) on thebasis ofmutagenesis studieswith the Phe290-β2AR (Strader et al.,1994).

There are two frequently occurring polymorphic locations of thehuman β1AR, at amino acid 49 in the extracellular amino terminus andat 389 in the intracellular carboxy tail (Small et al., 2003). Thesignificance of Arg389Gly polymorphisms at recombinant β1H andβ1LARs is reviewed in Section 5.4.

5.1. H and L binding sites or conformational states?

The concept that a non-conventional partial agonist acted througha high-affinity β1AR as an antagonist and through a low-affinityreceptor as an agonist was first suggested for (−)-pindolol (Fig. 3,Walter et al., 1984). (±)-CGP12177 competed with high affinity for 90%of binding sites, and with low affinity for 10% of binding sites labelledwith [125I]-(−)-cyanopindolol in membranes from human recombi-nant β1AR, leading Pak and Fishman (1996) to suggest the existence oftwo affinity states. Since the paper of Pak and Fishman (1996) the termhigh- and low-affinity states has been endorsed (Kompa & Summers,1999; Konkar et al., 2000a, 2000b; Lowe et al., 2002; Baker et al., 2003;Baker, J. G., 2005). The concept of two affinity states implies tworeceptor conformations, the high-affinity state associated withblockade of the effects of catecholamines and the low-affinity stateassociated with agonist effects of non-conventional partial agonists.However, saturation and kinetic binding analysis revealed two distinctbinding sites for [3H]-(−)-CGP12177, β1HAR and β1LAR, both in heart(Sarsero et al., 1998, 1999, 2003) and recombinant β1AR (Joseph et al.,2004a). Clearly, if a ligand induces two distinct conformationalchanges it must be associated with different binding partners withinthe receptor protein. Since the agonist potency of (−)-CGP12177 issimilar to the binding affinity of [3H]-(−)-CGP12177 estimated forβ1LAR in cardiac and recombinant β1AR, it suggests that the postulatedconformational change occurs through this low-affinity site. Incontrast, catecholamines cause a conformational change of the β1ARthat is impeded by β-blockers and non-conventional partial agonists,including CGP12177, acting through the high-affinity site, β1HAR.

utagenic studies carried out to date to elucidate critical regions for β1LAR binding andpared to β1LAR. A natural polymorphism exists at the 389 positionwhich differentiatesty at Gly389 receptors is reduced considerably more through β1HAR than β1LAR. TMD Vnist at the mutant receptor β1(β2 TMD V)AR.

Fig. 11. Binding of [3H]-(−)-CGP12177 to the β1HAR at Asp138-β1AR (A, pKD=9.4) and toβ1LAR at Glu138-β1AR (C inset, pKD=7.6). (−)-Isoprenaline inhibited binding of [3H]-(−)-CGP12177 to Asp138-β1AR through β1HAR with pKi =6.2 (B) but enhanced [3H]-(−)-CGP12177 (58 nM) binding at Glu138-β1AR (D). T, S and NS stand for total, specific andnon-specific binding respectively. For further details see Joseph et al. (2004c).


Does elimination of one important binding partner of one siteleave the characteristics of the other site unaffected? Does theconformational change of one of the two sites modify the bindingaffinity of a non-conventional partial agonist to the other site? Beforeaddressing these questions in Sections 5.2 and 5.3, an alternativemechanism should be mentioned.

Different agonists could trigger distinct biochemical and biophysicaleffector pathways through the same receptor (Vauquelin & Van Liefde,2005) throughmechanisms not completely accountable by the functionof two distinct sites at the receptor protein. At very highly expressedrecombinant human β1AR (7–8 pmol mg−1), Galandrin and Bouvier(2006) compared the ability of several β-blockers to enhance cAMP andextracellular signal-regulated kinase (ERK) activity. As found by others(Lattion et al., 1999; Andreka et al., 2002; Baker et al., 2003), theyobserved that bucindolol and carvedilol enhanced cAMP levels; incontrast, propranolol decreased cAMP levels. However, all 3 β-blockersactivatedmitogen-activated protein kinase (MAPK) up to approximately1/4 of theeffectof (−)-isoprenaline. Interestingly the agonist potencies ofcarvedilol (cAMP pEC50=6.6, MAPK pEC50=7.5) were similar to theaffinity of carvedilol for recombinant β1LAR (pKB=7.5, Joseph et al.,2004a; pKB=7.3 Baker, J. G., 2005), consistent with activation of botheffectors through this site. The agonist potencies of propranolol (cAMPpEC50=7.5, MAPK pEC50=7.5) were slightly higher than the correspond-ing affinity estimates for β1LAR (pKB=7.0 Joseph et al., 2004a; pKB=6.4Baker, J. G., 2005). Galandrin and Bouvier (2006) proposed signallingefficacy of a receptor system needs to include specific effectors.

The results of Galandrin and Bouvier (2006) with carvedilolsuggest that perhaps the β1LAR can adopt two distinct conformationsone for the cAMP signal, the other for the MAPK signal. The resultswith propranolol of Galandrin and Bouvier (2006) are particularlystriking because propranolol was an inverse agonist for the adenylylcyclase/cAMP system but a partial agonist for the MAPK system.Although the potencies at the two systems are identical, these resultsmust imply that different β1AR conformations, not necessarily atβ1LAR, are stabilized by propranolol to mediate the measured signal atthe two effectors. The possibility that more than one class ofconformational change occurs with activation of either β1HAR orβ1LAR or even another unknown β1AR site, cannot be excluded.

5.2. Mutation of the Asp138-β1AR to Glu138-β1AR

Carrying out pioneering mutations of the recombinant β2AR,Strader et al. (1987, 1988) postulated that the protonated amino groupof both agonists and antagonists reacts directly and ionically with thecarboxylate side chain of aspartate 113 in the third transmembranedomain of the β2AR (Strader et al., 1987, 1989a). Mutation of Asp113 toGlu113 and Asn113 reduced the catecholamine potency by 3 and 4orders of magnitude respectively (Strader et al., 1988). Asn113-β2ARdid not bind to [125I]-cyanopindolol, [3H]-(−)-CGP12177 or [3H]-dihydroalprenolol (Strader et al., 1987) and the blocking potency ofpropranolol was reduced by 4 orders of magnitude with this mutationcompared to Asp113-β2AR (Strader et al., 1989a).

The β1AR homologue of β2AR's Asp113 is Asp138. If the agonisteffects of a non-conventional partial agonist are purely mediatedthrough the low-affinity binding site of the β1AR, mutation of Asp138(Fig. 10) should disrupt catecholamine-evoked effects but preserve theeffects of the non-conventional partial agonist. This was nearly thecase with the mutation of Asp138 to Glu138 of the human β1AR(Figs. 11 and 12, Joseph et al., 2004c). The high-affinity binding of [3H]-(−)-CGP12177 (pKD=9.4) at Asp138-β1AR was abolished at Glu138-β1AR (Fig. 11) and the potency of (−)-isoprenaline to enhance cAMPwas reduced 500,000-fold (Fig.12). (−)-Bupranolol (1 μM) antagonizedthe effects of (−)-isoprenaline with a pKB=9.9 at Asp138-β1AR butfailed to cause blockade at Glu138-β1AR (Fig. 12). (−)-Isoprenalineremoved bound [3H]-(−)-CGP12177 from Asp138-β1AR but not fromGlu138-β1AR (Fig. 11). A high [3H]-(−)-CGP12177 concentration

(58 nM), similar to its affinity for β1LAR (pKD=6.7, Joseph et al.,2004a), still bound to Glu138-β1AR, presumably to β1LAR (Fig. 11).These results are similar to the marked reduction of catecholaminepotency and affinity found on the equivalent mutants of Asp113-β2AR(Strader et al., 1987, 1989a). In contrast, the potency to enhance cAMPof (−)-CGP12177 was only decreased 5-fold at Glu138-β1AR(pEC50=6.7) compared to Asp138-β1AR. Furthermore, (−)-bupranololantagonized the effects CGP12177with similar affinity at Glu138-β1AR(pKB=7.1) and Asp138-β1AR (pKB=7.5) (Fig. 12). These results wouldsuggest an independent activation of β1HAR by catecholamines andβ1LAR by (−)-CGP12177 without apparent conformational cross-talkbetween the two sites. The abolishment of both high-affinity [3H]-(−)-CGP12177 binding and (−)-bupranolol-evoked antagonism of theeffects of catecholamines at Glu138-β1AR confirms the essentialimportance of Asp138 for the effects of β-blockers, including thenon-conventional partial agonist (−)-CGP12177, at β1HAR as pre-viously shown with Asp113 for the β2AR (Strader et al., 1987, 1989a).Taken together, these results with Glu138-β1AR indicate that Asp138is only an obligatory binding partner for the interactions of (−)-iso-prenaline, (−)-bupranolol and (−)-CGP12177 at β1HAR but not atβ1LAR. (−)-Isoprenaline appears to bind only with very low affinity(~1 mM) to rat atrial β1LAR (Sarsero et al., 1998), of unknownsignificance. However, (−)-isoprenaline could still modify allosteri-cally the function of the β1LAR.

Similar mutagenic studies carried out on 5HT1A receptors, theendogenous agonist 5-HTandβ-blockers includingpindolol, propranololand alprenolol provide further insights into possible β1AR mechanisms.In the studyof Ho et al. (1992), the affinity and agonist effects of 5-HTandpindololwere determined in COS-7 cells expressing thewild-type 5HT1Areceptor or cells with separate mutations of Asp82 to Asn82, Asp116 toAsn116or Ser198 toAla198. The affinity of pindololwas conserved acrossthese mutations, however the affinity of 5-HT was reduced ~200-fold(Ho et al., 1992). In functional studies in which GTPase activity wasassessed, higher concentrations of 5-HT were required in cells withmutant receptors compared towild-type. The human β1AR homologuesare Asp104, Asp138 and Ser228. The pattern of the interaction of 5-HTand pindolol with Asp116/Asn116 5-HT1A receptors (Ho et al., 1992)shows striking similarity to that described for (−)-isoprenaline and(−)-CGP12177 on Asp138/Glu138-β1AR (Joseph et al., 2004c).

Fig.12. CyclicAMP-enhancingeffects of (−)-isoprenaline and (−)-CGP12177atAsp138-β1AR(A,C) and Glu138-β1AR (B,D). (−)-Bupranolol antagonized the effects of (−)-isoprenalinethrough β1HAR (pKB=9.9) at Asp138-β1AR (A) but not Glu138-β1AR (B). In contrast, (−)-bupranolol antagonized the effects of (−)-CGP12177 at both Asp138-β1AR (pKB=7.4) andGlu138-β1AR (pKB=6.7) through β1LAR. For further details see Joseph et al. (2004c).


Joseph et al. (2004c) made one unexpected finding with theGlu138-β1AR mutation. (−)-Isoprenaline not only failed to removebound [3H]-(−)-CGP12177 but actually enhanced radioligand binding,albeit at high concentrations (Fig. 11). Interestingly, these high(−)-isoprenaline concentrations, 30–300 μM, are the same high concen-trations that manage to increase cAMP in a concentration-dependentmanner at Glu138-β1AR (compare Figs. 11 and 12). To account for theresidual agonist effects of (−)-isoprenaline at Glu138-β1AR, it isplausible that (−)-isoprenaline could still be binding with markedlyreduced affinity to the same binding pocket without the ionic reactionbetween the protonated nitrogen and the missing Asp138. Thisplausibility is supported by the observation that the nitrogen-freecatechol-analogue, 3′,4′-dihydroxy-α-methylpropiophenone (U-0521),still increases the rate of beating of singlemyocytes obtained from new-born rats as well as of sinoatrial nodal rate of adult rats which isprevented by 2–20 nM (−)-bupranolol (Kaumann et al., 1977). Due tothe high blocking potency of (−)-bupranolol, the effects of U-0521 areprobably mediated through β1HAR. However, a very high concentrationof U-0521, 3.3 mM, did not antagonize the (−)-isoprenaline-evokedstimulation of feline membrane adenylyl cyclase, demonstrating anundetectably low affinity for the wild-type β1AR. Thus, nitrogen-free U-0521 at wild-type β1AR and (−)-isoprenaline at Glu138-β1AR could stillreact with other groups of the same binding pocket, i.e. through its twocatechol hydroxyl groups with Ser 229 (homologue to Ser204 of theβ2AR) and 232 (homologue of Ser207 of the β2AR) of TMD V, as well asthrough van derWaals forces between the aromatic group and Phe 341(homologue of Phe 290 of the β2AR) of TMD VI (Sugimoto et al., 2002;see Strader et al., 1989b for β2AR). These binding sites may be sufficientfor the conformational change of β1HAR site at the wild-type β1AR,induced by U-0521, and with (−)-isoprenaline at Glu138-β1AR. Theconformational change produced by (−)-isoprenaline, through theGlu138-β1AR, may in turn have changed the conformation of theβ1LAR site, thereby enhancing the binding affinity of [3H]-(−)-CGP12177for β1LAR. This would imply that activation of the binding of (−)-isoprenaline to the ‘orthosteric’ mutated site of the Glu138-β1AR couldhave enhanced the affinity of [3H]-(−)-CGP12177 for the β1LAR throughan allosteric mechanism. It remains an open questionwhether allostericligands, interacting with the non-mutated β1HAR site, could modify thebinding of non-conventional partial agonists to the β1LAR site.

5.3. Mutation of the β1AR to β1(β2TMD V)AR

A distinguishing feature of the β1LAR agonists, (−)-CGP12177 and(−)-pindolol is the presence of the benzimidazolone and indole groupsrespectively. These chemical moieties are likely to contribute to β1LARagonist activity or at least participate in the unique pharmacology thatdifferentiates their effects at β1AR and β2AR. The nitrogens of thebenzimidazolone and indole groups could bind in the vicinity of TMDVserine groups. However, homology between β1AR and β2AR withrespect to TMDV serine groups suggests that they by themselves do notdetermine the different pharmacology of these compounds at β1AR andβ2AR. The ability of (−)-CGP12177 to accumulate cAMP in stable CHO celllineswith either the humanβ1AR,β2ARor amutantβ1AR inwhich TMDV was substituted by the β2AR (β1(β2TMD V)AR) was determined(Fig. 13). Since the β2AR does not form a low-affinity state analogous toβ1LAR (Pak & Fishman,1996; Baker et al., 2002) substitution of the β1ARTMDV by theβ2AR TMDVmight limit the ability of β1AR to form β1LAR.Indeed this was the case. The potency of (−)-CGP12177 to increase cAMPwas higher at β2AR (pEC50=9.2) and β1-(β2TMDV)AR (pEC50=8.9) thanβ1AR (pEC50=7.9), while pEC50 values for (−)-isoprenaline were notdifferent (Molenaar et al., 2007). (−)-CGP12177 is a non-conventionalpartial agonist at β1AR and causes activation of β1LAR, but is a classicalhigh-affinity partial agonist at recombinant human β2AR (Pak &Fishman, 1996; Baker et al., 2002) and human β2AR overexpressed inmurine heart (Heubach et al., 2003). The potency of (−)-CGP12177 at themutant β1(β2TMD V)AR was similar to β2AR but higher than β1ARindicating that TMD V of the β1AR is necessary for the formation of thelow-affinity state of β1AR possibly through an interaction with thebenzimidazolone group of (−)-CGP12177, however this needs to beverified. The role of specific amino acids, alone and in combination, arecurrently being investigated.

5.4. The Arg389Gly polymorphism differentlyaffects the function of recombinant β1HAR and β1LAR

Humanβ1ARpresent thecommonArg389Glypolymorphism(Fig.10)(Mason et al.,1999;Molenaar et al., 2002; Sarsero et al., 2003).Maximumadenylyl cyclase stimulation by (−)-isoprenaline is reduced to 1/3 at theGly389 variant compared to the Arg389 variant (Mason et al., 1999).Residue 389 is in the Gs protein-coupling domain of the β1AR. Gly389appears to disrupt the predictedα-helix in this region, thereby reducingcoupling to Gs protein (Small et al., 2003). Using (−)-isoprenaline and[35S]-GTP-γ-S binding Mason et al. (1999) reported reduced coupling ofGly389 receptors compared to Arg389 receptors. Joseph et al. (2004b)investigated whether the Arg389Gly polymorphism influenced theresponses to catecholamines, mediated through β1HAR, and the non-conventional agonist (−)-CGP12177,mediated throughβ1LAR, differently.Using recombinant β1AR transfected into CHO cells at physiologicaldensities (94–101 fmolmg−1) they found that themaximumcAMP signalat Gly389 receptors caused by (−)-isoprenaline was only 3% of that ofArg389 receptors. In contrast, the maximum (−)-CGP12177 signal atGly389 receptors was still 54% of that of Arg389 receptors. Thus, from anon-conventional partial agonist with low intrinsic activity (0.06) atArg389 receptors, at Gly389 receptors (−)-CGP12177 became a full agonist(intrinsic activity 1.1), compared to (−)-isoprenaline. Interestingly, thepotencyof (−)-isoprenaline and (−)-CGP12177as enhancers of cAMP levelswas increased by 2.0 and 0.6 log units at Gly389 receptors compared toArg389 receptors but the corresponding blocking potencies of (−)-propranolol against the effects of (−)-isoprenaline (pKB=8.7–8.9) and(−)-CGP12177 (pKB=6.2–6.4) were virtually unaffected by the Arg389Glypolymorphism. These results led Joseph et al. (2004b)) to conclude thatthe β1HAR and β1LAR coupled differently to Gs protein. The relativeindifference of recombinant β1LAR to the Arg389Gly polymorphism,compared to recombinant β1HAR, is reflected in human atrium in whichthe inotropic potency and intrinsic activity of (−)-CGP12177 appears to beindependent of the Arg389Gly-β1AR polymorphism (Sarsero et al., 2003).

Fig. 13. β1(β2TMD V)AR chimera prepared by substitution of 5 heterologous amino acids of TMD V β2AR (Gln197, Ile205, Val213, Val216, Ser220) into β1AR (Arg222, Val230, Cys238,Ala241, Leu245). The potency of (−)-CGP12177 for cAMP responses was higher for β2AR (pEC50=9.2), β1(β2TMDV)AR (pEC50=8.9), than β1AR (pEC50=7.9), indicating a role for TMDVin the mediation of agonist effects through β1LAR (Molenaar et al., 2007).


5.5. Possible insights from the 5-HT1A receptor for β-hydroxyland ether oxygen binding of phenoxypropanolamines to β1AR

The β-hydroxyl group of phenylethylamine catecholamines ispredicted to bind to Asn344-β1AR (Wieland et al., 1996; Sugimotoet al., 2002) however the ether oxygen instead of the β-hydroxylgroup of the phenoxypropanolamine catecholamine (−)-RO363 mayinteract with Asn344-β1AR (Sugimoto et al., 2002). The bindingpartners of the β-hydroxyl group and ether oxygen of (−)-CGP12177and (−)-pindolol at β1AR remain to be determined. The phenoxypro-panolamine compounds pindolol, propranolol and alprenolol alsobind to the 5-HT1A receptor (Guan et al., 1992). Since the 5-HT1Areceptor shares common structural domains with β2AR (Guan et al.,1992; Ho et al., 1992) it might be possible to gain further insight intothe binding mechanisms of the phenoxypropanolamine β-hydroxyland ether oxygen groups.

The affinity of the antagonists pindolol, propranolol and alprenololfor 5-HT1A receptors was reduced by 40–150-fold following mutationof the Asn385-5-HT1A receptor to Val385 in TMD VII (Guan et al.,1992). Furthermore, while [125I]-cyanopindolol had high affinity(2 nM) for the Asn385-5-HT1A it did not bind to the mutant. Hydrogenbonds are predicted to form between the ether oxygen of pindolol andN-H group of Asn385-5-HT1A and the β-OH of pindolol and CfO groupof Asn (Guan et al., 1992; López-Rodríguez et al., 2001). In contrast, theaffinities of drugs from other chemical classes, including theendogenous agonist 5-HT were hardly affected. These observationsalso reveal that binding sites for agonists and antagonists may bedifferent (Strader et al., 1987; Ho et al., 1992; Guan et al., 1992). Anantagonist can bind to the receptor in any non-productive conforma-tion (Ho et al., 1992). The discovery of separate binding sites for 5-HTand phenoxypropanolamine βAR antagonists at 5-HT1A receptors mayalso be relevant to pindolol, pindolol derivatives and CGP12177 atβ1AR since Asn385-5-HT1A is conserved at the human β1AR (Asn360-β1AR, Guan et al., 1992). Indeed it is possible that these compoundshave different binding partners to the catecholamines at β1AR andbind differently to β1HAR and β1LAR.

5.6. Summary

Initial clues are emerging that point to structural differencesbetween β1HAR and β1LAR sites. Mutation of the Asp138-β1AR to aGlu138-β1AR markedly reduced catecholamine potency (~106-fold)and eliminated β-blocker affinity through β1HAR but nearly preservedboth potency of a non-conventional partial agonist and affinity of a β-blocker at β1LAR. Very high catecholamine concentrations actuallyenhanced binding of the non-conventional partial agonist to the β1LARsite of the Glu138-β1AR, suggesting allosteric interactions betweenβ1HAR and β1LAR sites.

Since β2AR appear not to form a low-affinity state and transmem-brane domain V (TMDV) plays an important role in structure–activity,a β1AR containing the TMD V region of the β2AR was constructed. Anon-conventional partial agonist exhibited similarly higher potency atthe resultant β1(β2TMD V)AR mutant and wild-type β2AR comparedto the β1LAR, indicating an obligatory role of β1AR(TMD V) for theformation of β1LAR.

The Arg389Gly β1AR polymorphism affects catecholamineresponses through β1HAR but hardly the responses of non-conven-tional partial agonists through β1LAR, suggesting subtle differences inthe coupling to effectors of the 2 sites.

6. Clinical relevance

6.1. Beneficial effects of non-conventional partial agonists

In patients with a failure of the autonomic nerve system, catecho-lamine concentrations are decreased. The cardiostimulant effects ofpindolol, presumably mediated through β1LAR, have been found to bebeneficial for the treatment of some patients with orthostatic hypoten-sion (Frewinet al.,1980;Man In'tVeld&Schalekamp,1981,Man In't Veldet al., 1982) and neurocardiogenic syncope (Iskos et al., 1998; Bendittet al., 1999). Although promising, these studies (Frewin et al., 1980 (2patients); Man In't Veld & Schalekamp, 1981 (3 patients); Man In't Veldet al., 1982 (4 patients); Iskos et al., 1998 (31 patients)) have involved


small numbers of patients and therefore a critical evaluationof theuse ofpindolol in these conditions awaits the completion of larger, placebocontrolled randomized studies. Postural hypotension also occurs inautonomic failure associated with multiple system atrophy (MSA, ShyDrager Syndrome), a progressive degeneration of the sympathetic nervesystem, associated with increased tachycardia to (−)-isoprenaline andenhanced density of lymphocyte β2AR (Bannister et al., 1981). Davieset al. (1981) found that pindolol (5 mg thrice daily) failed to improvepostural hypotension in 5 MSA patients and appears to have causedheart failure in two of these patients. Clearly pindolol cannot only causebeneficial effects but also harmful effects as reviewed in thenext section.

6.2. Harmful effects of non-conventional partial agonists

β1HAR-mediated inotropic responses are reduced in clinicalheart failure (Bristow et al., 1982, 1986) as found also for the effectsof (−)-CGP12177 in right atria obtained from failing hearts (Sarseroet al., 2003) and in a rat model of heart failure (Kompa & Summers,1999). Heart failure reduced the intrinsic activity of (−)-CGP12177compared to (−)-isoprenaline or Ca2+, both in the absence orpresence of IBMXbutwithout affecting the inotropic potency, comparedto human right atria from non-failing hearts (Sarsero et al., 2003). As aprotective mechanism against cardiac overstimulation and exaggeratedoxygen consumption in heart failure, the observed reduction of βAR-mediated cardiostimulation in heart failure is in part due to a reductionβ1HAR density (Bristow et al., 1982, 1986) and a similar reduction ofβ1LAR density would also be expected and was found to occur (Sarseroet al., 2003). There is now consensus that the use of cardiostimulantsacting through β1AR, including non-conventional partial agonists, is notbeneficial and may actually be harmful for the prolonged treatment ofchronic heart failure. Unlike β-blockers without inotropic intrinsicactivity, the non-conventional partial agonists, bucindolol and pindolol,were found to be unsuitable for the treatment of heart failure(Cruickshank, 2007).

(−)-CGP12177 enhanced the L-type Ca2+ current (Freestone et al.,1999), markedly shortens the duration but prolongs the plateau of theventricular action potential (Lowe et al., 1998) and produces botharrhythmic Ca2+ transients (Freestone et al., 1999) and contractions(Lewis et al., 2004). Taken together, this evidence makes it likely that anon-conventional partial agonist may generate cardiac arrhythmias,mediated at least in part through β1LAR. Although the clinically usednon-conventional partial agonists bucindolol and pindolol have lowerintrinsic activities than (−)-CGP12177, they cause cardiostimulation, atleast in part, through β1LAR which may potentially be associated witharrhythmias.

Unlike metoprolol (MERIT-HF,1999), bisoprolol (CIBIS-II, 1999) andcarvedilol (Packer et al., 1996, 2001), that do reduce mortality in heartfailure, bucindolol does not (BEST, 2001). Sympathetic neuronalactivity is increased in congestive heart failure (CHF), as demonstratedwith enhanced plasma noradrenaline levels (Cohn et al., 1984).Bristow et al. (2004), analysing results from a subset of patients(18%) of the BEST trial, noticed a marked increase of plasmanoradrenaline in placebo-treated CHF patients while in bucindolol-treated patients there was a marked decrease in plasma noradrenalineby the 3rd month of treatment. Interestingly, the 18% of thebucindolol-treated patients with a marked noradrenaline declinewere at a 1.7-fold increased risk of subsequent mortality. On the otherhand, bucindolol improved the outcome of other CHF patients whichwere less dependent on sympathetic activity for haemodynamiccompensation (Bristow, 2003; Bristow et al., 2004). However,bucindolol also appears to improve survival of CHF patients carryingthe Arg389β1AR and having high basal noradrenaline levels but not inCHF patients carrying the Gly389β1AR compared to placebo (Liggettet al., 2006).

The decreased plasma noradrenaline level found in the subset of18% of the bucindolol-treated patients with a 1.7-fold increased

mortality risk (Bristow et al., 2004) could be due to enhancedclearance or to decreased noradrenaline release. It is plausible that thepatients in which bucindolol markedly reduced noradrenaline plasmalevels, it occurs through a decrease of noradrenaline release fromnerve endings through blockade of β2AR, because the affinity ofbucindolol is similar for human β1HAR and β2AR (Hershberger et al.,1990; Maack et al., 2000). Although a marked reduction in plasmanoradrenaline occurred in the 18% subset of CHF patients treated withbucindolol (Bristow et al., 2004), it still remains to be determinedwhether blockade of cardiac pre-junctional facilitory β2AR contrib-uted to this effect because experimentally bucindolol caused anincrease in [3H]-noradrenaline release in electrical field stimulatedcanine saphenous vein strips, but nevertheless blocked isoprenaline-evoked increases in [3H]-noradrenaline release (Rimele et al., 1984).

Taken together the above discussion makes interpretation of theeffects of bucindolol in CHF difficult. On balance bucindolol does notimprove survival of CHF patients (BEST, 2001; Anderson et al., 2003;Bristow et al., 2004), despite the reported increased survival of CHFpatients carrying the Arg389β1AR (Liggett et al., 2006). Althoughbucindolol increased the combined endpoint of death or hospitaliza-tion in CHF patients with class IV heart failure (Anderson et al., 2003),the mechanism of death is not known. Based on the cardiostimulationreported for bucindolol on isolated humanmyocardium (Bundkirchenet al., 2002; Maack et al., 2003; Tables 1 and 2) we find it conceivablethat bucindolol could produce cardiac arrhythmias via β1LAR, as(−)-CGP12177 does. We suggest that when the plasma noradrena-line is low and β1HAR are blocked, bucindolol could activate β1LARthereby causing cardiostimulation associated with increased oxy-gen consumption and ischaemia leading to arrhythmias in CHFpatients. Bucindolol concentrations (0.01–1 μM) which have beenshown to increase contractility of ventricular trabeculae in vitro(Maack et al., 2003) have been measured in plasma of patientstreated with 50–200 mg bucindolol for hypertension (Websteret al., 1985) and are therefore consistent with our suggestion.However, no clinical evidence has been reported for the occurrenceof partial agonist activity of bucindolol on heart rate and con-tractility in heart failure patients (BEST, 2001; Gilbert et al., 1990;Bristow et al., 1994). Nevertheless, it would be interesting to knowwhether bucindilol could cause nocturnal tachycardia in thepatients with very low noradrenaline levels (BEST, Bristow et al.,2004).

Pindolol causes cardiostimulation, such as increases in heart rateand force, thereby increasing oxygen consumption which could bedetrimental in patients with CHF and it could lead to arrhythmias. Halfmaximal (−)-pindolol-evoked cardiostimulation in human atrialtrabeculae (Joseph et al., 2003), mediated through β1LAR (Josephet al., 2003) occurs at 300 nM. Pindolol, administrated at 15 mg thricedaily, causes stable plasma elevations of unmodified pindolol of~150 nM in healthy volunteers (Schwarz, 1982), consistent withsignificant increases in contractile force of human atrial trabeculae(Fig. 9), mediated through β1LAR (Joseph et al., 2003). Furthermore, aplasma concentration of approximately 300 nM pindolol, equivalentto half maximal activation of both human atrial β1LAR andrecombinant β1LAR (Joseph et al., 2003), increases heart rate by11 beats min−1 but reduces exercise-induced tachycardia by approxi-mately 30 beats min−1 in healthy volunteers (Aellig, 1982). Since300 nM pindolol causes in vitro cardiostimulation through β1LAR, thisreceptor site may well mediate the unwanted cardiac side effects.

Pindolol has also been reported to elicit premature ventriculartachycardia in a patient with CHF (Binkley et al., 1986a, 1986b).Furthermore, in 12 patients with ischaemic heart disease withventricular tachycardia, pindolol caused an increase in ventricularectopic activity in 7 patients and precipitated CHF in 3 patients (Podrid& Lown, 1982). The cardiostimulation by pindolol becomes evident atrest, particularly at night when sympathetic neuronal activity ismarkedly reduced. It has also been suggested that the pindolol-evoked


cardiostimulation in patients who develop angina at rest and very lowexercise level may be undesirable (Kostis et al., 1984). At nightsympathetic nerve activity is quiescent and parasympathetic activity ishigh (Van de Borne et al., 1994; Vanoli et al., 1995) thereby facilitatingmanifestation of the cardiostimulant effects of a β-blocker, unopposedby blockade of activity of noradrenaline released from the sympatheticneuroneswhich during the day could have caused bradycardia, despitethe intrinsic activity. Pindolol may be particularly contraindicated inpatients with nocturnal angina because pindolol-evoked increases inheart rate are highest at night (Fitscha et al., 1982; Kantelip et al., 1984;Channer et al.,1994). Taken together, the clinical observations illustratethe pro-arrhythmic effects and potentially adverse effects of pindololin a clinical setting of coronary artery disease.

The quantitative agreement of the pindolol plasma concentrations,causing clinical cardiostimulation and potentially arrhythmias, withthe concentrations activating in vitro human atrial β1LAR andrecombinant β1LAR, points to mediation through this β1AR site.However, two additional mehanisms could also contribute to theeffects of pindolol in man. First, interaction with β1HAR, as observedwith (−)-pindolol-evoked sinoatrial tachycardia in feline (Kaumann &Blinks, 1980b) and guinea pig (Walter et al., 1984) and confirmed withrecombinant β1AR by Baker et al. (2003). However, (−)-pindolol is onlyan agonist through β1LAR but not β1HAR in human atrium (Fig. 9,Joseph et al., 2003). Second, since racemic pindolol is used in the clinic,(+)-pindolol conceivably could elicit sinoatrial tachycardia throughβ2AR, as observed in the guinea pig (Walter et al., 1984).

6.3. Plausible beneficial effects of β-blockers through β1LAR

Some clinically used β-blockers may have sufficiently high affinityfor the β1LAR so that this receptor site can be activated by clinicallyoccurring plasma concentrations. Such is the case for carvedilol, whichhas a pKB=7.56 vs (−)-CGP12177 at recombinant β1AR expressed atphysiological density (Joseph et al., 2004a). The clinically measuredplasma concentration of unbound carvedilol of 16 ng ml−1 reported inhealthy volunteers (Kindermann et al., 2004) would therefore occupyapproximately 36% of the β1LAR sites. But how would this β1LARoccupancy potentially produce beneficial effects? The COMET trial(Poole-Wilson et al., 2003) suggests that carvedilol extends survival ofheart failure patients compared to metoprolol. Based on cAMP andSPAP partial agonist effects of carvedilol but lack of agonist effects ofmetoprolol reported by Baker et al. (2003), Perez and Karnik (2005)stated that ‘a novel agonist conformation induced by carvedilol atβ1LAR, while the classical β1HAR site is desensitized, may suggest amechanism for why it is a better therapeutic for heart failure’. There is,however, a caveat against this suggestion. The cAMP and SPAP partialagonist effects of carvedilol were obtained from recombinant β1ARexpressed at the non-physiology density of ~1 pmol mg−1 protein(Baker et al., 2003). The ferret is the species whose cardiac tissues bestamplify acute signals through cardiac β1LAR (Lowe et al., 1998, 1999,2002). Although early work of Lowe et al. (1999) with ferretmyocardium showed that carvedilol antagonized the cardiostimulanteffects of (−)-isoprenaline 20 times more than those of (−)-CGP12177(both with Schild-plot of slope one), no agonist effects of carvedilolwere detected on right and left ventricular myocardium, left atriumand sinoatrial node. Furthermore, no agonist effects were observed onhuman atrial trabeculae with up to 100 nM carvedilol (Molenaar et al.,2006), a concentration that would produce 78% occupancy of β1LAR. Itcannot be discarded, however, that the chronic treatment withcarvedilol could influence the outcome of heart failure through yetunknown gene expressions. On the other hand, carvedilol's persistentblockade of and selectivity for human cardiac β2AR, compared to β1AR(Molenaar et al., 2006), could conceivably prevent adrenaline-evokedβ2AR-mediated arrhythmias (Kaumann & Sanders, 1993), therebycontributing to a better survival of heart failure patients compared toβ1AR-selective metoprolol.

6.4. Summary

Possibly acting through β1LAR, treatmentwith the non-conventionalpartial agonist pindolol is beneficial in some patients with orthostatichypotension and neurocardiogenic syncope. However, unlike someother β-blockers used successfully for the treatment of chronic heartfailure, non-conventional partial agonists are contraindicated in thiscondition, in part due to their pro-arrhythmic potential.

7. Conclusions and outlook

Abundant evidence, accumulated during 40 years, has culminatedwith the concept that a low-affinity site of the β1-adrenoceptor(β1LAR) mediates the acute cardiostimulant effects of some clinicallyrelevant β-blockers while a high-affinity site (β1HAR) mediatesblockade of the effects of catecholamines. Cardiostimulant β-blockerswith these characteristics are known as non-conventional partialagonists. As occurs with the β1HAR site, activation of the β1LAR alsoenhances cardiomyocyte cAMP levels, involved not only in cardiosti-mulation but also in leading to biochemical events that can producecardiac arrhythmias. The cardiostimulant effects of both catechola-mines and non-conventional partial agonists are greatly blunted byphosphodiesterase3 (PDE3) in human myocardium, thereby exertingcardioprotection. Unlike non-cardiostimulant β-blockers, non-con-ventional partial agonists do not prolong survival of patients withchronic heart failure. Non-cardiostimulant β-blockers antagonize thecardiostimulant effects of non-conventional partial agonists throughβ1LAR but with lower affinity than for β1HAR. Some non-conventionalpartial agonists, e.g. carvedilol, can promote cAMP-dependent genetranscription at recombinant overexpressed β1AR, presumablythrough the β1LAR site, but it is unknown whether this contributesto the beneficial effects of carvedilol in heart failure. (−)-Pindolol canelicit cardiostimulant effects through both β1HAR and β1LAR.

An equivalent pharmacology to the cardiac pharmacology of non-conventional partial agonists has been foundwith recombinantβ1AR.Asfirst demonstrated with the β2AR, the amino group of an agonist orantagonist is alsoanchored throughan ionic interaction to theβ1ARwiththe carboxylate side chain of an aspartate in the third transmembranehelix. Mutation of Asp138 to Glu138 in the β1AR virtually abolishes theβ1HAR pharmacology but nearly leaves the β1LAR pharmacology intact.Therefore, distinct binding partners must exist for ligands at the twoβ1AR sites, to be unravelled with future mutation studies. The fifthtransmembrane domain of theβ2AR (β2TMDV) is involved in the effectsof agonists but neither a low-affinity site nor non-conventional partialagonists are known for this receptor. Replacementofβ1TMDVbyβ2TMDVat theβ1AR, converts anon-conventional partial agonist into a classicalpartial agonist by enhancing its affinity for the resultant β1(β2TMD V)-AR mutant, indicating a functional participation of TMD V in β1LARpharmacology. There are also differences in the ability of β1LAR andβ1HAR to couple revealed by the Arg389Gly polymorphism. Adenylylcyclase responses mediated through β1LAR were reduced less thanβ1HAR at Gly389 compared to Arg389β1AR. These and ongoing andfuture β1ARmutagenesis studies, as well as techniques andmodels thatassess conformational changes, will deepen our understanding of thepharmacology of non-conventional partial agonists.

Acknowledgments

AJK thanks the British Heart Foundation and PM thanks The PrinceCharles Hospital Foundation and the National Health and MedicalResearch Council of Australia for support.

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The low-affinity site of the β1-adrenoceptor and its relevance to cardiovascular pharmacology

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