Is there a third heart β-adrenoceptor?

316 TiPS - August 1989 [Vol. 101

Involvement of adenosine in supraspinal actions of morphine

morphine which we feel is extremely relevant to any discus- sion of functional relationships and interactions between these compounds in the CNS.

enosine and morphine T. W. STONE, B. 8. FREDHOLM*

AND J. W. PHILLIS+

Sawynok et al.’ have recently summarized their elegant work indicating that the release of adenosine from a population of capsaicin-sensitive primary affer- ent terminals may mediate the antinociceptive effects of morphine in the dorsal horn of spinal cord. They mention in passing behavioural work that indicates the importance of a similar process at supraspinal levels. However, both neurochemical work in vitro and in vivo and electrophysiological studies in vivo performed ten years ago strongly pointed to the involvement of adenosine in morphine’s effects in brain.

this effect was prevented by the application of theophylline at iontophoretic currents that also antagonized responses to adenosine but not GABA’*‘.

The implication of this work - that morphine acted by releasing adenosine - was slpported by the demonstration that morphine, at low micromolar concentrations, enhanced the release of adenosine from brain slices induced by several depolarizing agents45 or hypoxia6, or from the surface of the cerebral cortex in anaesthetized rats following systemic adminis- tration of the opiate7. All these studies were in turn triggered by the even earlier observation by Ho et al.* that the antinociceptive effect of morphine could be antagonized by xanthines.

Department of Phnrmacofogy, University of Glasgow, Glasgow GZ2 SQQ, UK, ‘Depart- ment of Pharmncoiogy, Karolinska Insfitute, Box 60 400, S-104 01, Stockholm, Sweden, and ‘Department of Physiology, Wayne State Universify, School of Medicine, 540 E Canfield, Detroit, Ml 48201, USA.

The electrophysiological studies were carried out using ionto- phonetic applications of morphine to cells in the striatum of anaesthetized rats. Morphine suppressed the spontaneous firing of a high proportion of neurones tested and

There is thus significant evidence for involvement of adenosine in supraspinal tctions of

References 1 Sawynok, J., Sweeney, M. I. and White,

T. D. I19891 Trends Pharmacol. Sci. 10. 186-18s ’

2 Stone, T. W. and Perkins, M. N. (1979) Nature 281, 227-228

3 Perkins, M. N. and Stone, T. W. (1980) Br. 1. Pharmacol. 69,131-138

4 Stone, T. W. (1981) Br. 1. Pharmacol. 74, 171-176

5 Fredholm, 8.8. and Vemet, L. (1978) Acta Physiol. Stand. 104,502-504

6 Fredholm, 8.8. et al. (1987) in Topics and Perspectives in Adenosine Research (Gerlach, E. and Becker, B. F., eds), PP. 509-520, Springer

7 Phi&s, J. W., Jiang, 2. G., Chelack, 8. J. and Wu, P. H. (1980) Eur. I. Phammcol. 65, 97-100

8 Ho, 1. K., Loh, H. H. and Way, E. L. (1973) I. Pharmncol. Exp. Ther. 185,336-346

a third heart f3-adrenoceptor? Albert0 J. Kaumann

A series of partial agonists with high affinity for myocardial PI- and p2- adrenoceptors cause stimulant effects in heart that are resistant to blockade of I%- and $z-adrenoceptors. The concentrations of partial agonist that cause stimulant effects greatly exceed those that cause blockade. Albert0 Kaumann suggests that such non-conventional partial agonists, often analogues of pin{olol, may act through a third heart p-adrenoceptor, which resembles the &-adrenoceptor of white adipocytes and smooth muscle of airways and ileum.

Noradrenaline and adrenaline can increase mammalian heart rate and

rate is enhanced by noradren-

force through both PI- and g2- aline through P1-adrenoceptors and by adrenaline throu h both

adrenoceptors. Sinoatrial beating 54 PI- and &-adrenoceptors’- . Atria1 force is increased through both PI-

A. I. Kaumonn L Associate Director of Pharmacoloe at SK b F, Welwyn Al.6 9AR. UK, and Affiliated Lecturer at the CIinical J’h~a~~l~gy Unit, Addeenbrookes Hospital, Cambridge CB2 ZQQ, UK.

and fi2-adrenoceptors4,5; selective fi2-responsiveness is paradoxically enhanced in atria obtained from patients chronically treated with selective PI-adrenoceptor antagon-

ists6s7. Noradrenaline and adrenaline augment contractile force of human ventricle through PI- and P2-adrenoceptors5*s. The involvement of P2-adrenoceptors varies, depending on heart region: it is most important in sinoatrial node, less so in atria1 myocardium and even less so in ventricle2-5,s. On the other hand, the role of PI-adrenoceptors is similar in all three heart regions2-5,*. Some characteristics of myocardial PI- and &-adrenoceptors are summarized in Table I.

The other chronotropic and inotropic potencies of noradrenaline, adrenaline and other catecholamines are considerably greater than their corresponding affinities for heart PI- and P2-adrenoceptors owing to marked biochemical amplification in some species2=s’3. This spare receptor14 capacity is, however, small or non-existent in human ventricle15*16.

Independent evidence for the coexistence of heart PI- and p2- adrenoceptors has been provided from radio&and binding3,sJ7-20 but the relative density of P-subtypes does not always correlate with their corresponding function.

TiPS -August 1989 IVol. 1OJ

TABLE I. Ligands for heart B_adrenoceptor subtypes

317

Regional role

Agonists

Br ‘I& I& SA node - atrium -ventricle SA node > atrium > ventricle SA node > atrium > ventricle

(-)-catecholamines (noradrenaline) (-)-denopaminea.b

(-)-catecholamines (adrenaline) procaterol (SA node)’

(-)-catecholamines?

Partial agonists (f)-xamoteroP*d (-)-practoloP

Antagonists (f)-COP-20712A‘“~”

SA node, sinoatrial node

procaterol’,i (+)-pindololk (f)-salbutamol (SA node)‘”

ICI-1 16551 “.O

(+&$l$md related indoleaminesP.k

(-)-alprenolol

(-)-bupranolol (t#

“Lemoine, H. et al. (1969) Naunyn-Schmiedeberg’s Arch. PharmacoI. 339,113-l 29; b Bing, Ft. J. et a/. (1964) Current Ther. Res. 36, 1127- 1144:‘Lemoine. H. eta/. (1969) 3. Cardiovasc. Pharmacol. 13,105-117; “Nuttall, A. and Snow, H. M. (1962) Br. ,/. Pharmacol. 77.361-366; OKaumann, A. J. and Marano, M. (1962) Naunyn-Schmiedeberg’s Arch. Pharrnacol. 316, 192-201; ‘Kaumann, A. J. (1966) P:a~myn- Schmiedeberg’e Arch. Pharmacol. 332, 4OWO9; gKaumann, A. J. and Lemoine, H. (1967) Naunyn-Schrniedebqk Arch. Pharmacol. 335, 403411: h Dooley, 0. J. et al. (1966) Eur. J. Pharrnacol. 130.137-139; ’ Kaumann, A. J. et a/. (1963) J. Recept. Res. 3.61-70; 1 Yabuuchi, Y. (1977) Bf. J. Pharmaco!. 61,513-521; ‘Walter, M. et al. (1964) Naunyn-Schmiedebegk Arch. Phannacol. 327,159-l 75; I Kaumann, A. J. et al. (1976) in Hormone Recepfors (Vol. 3) (Bimbaumer, L. and O’Malley, B. W.. eds), pp. 133-177, Academic Press; m Hall, J. A. et al. Circ. Res. (in press); ” Bilski, A. el al. (1963) J. Cardiovasc. Phannacol. 55.430-437; o Lemoine, H. et a/. (1965) Naunyn-Schmiedebergk Arch. Pharmacol. 331,4&51; P Kaumann, A. J. (1963) Z. Kardiol. 72.63-62.

Conventional partial agonists and PI- and &adrenoceptors

There are no ;fare receptors for a partial agonist . When a partial agonist has produced a maximal effect, which is smaller than the maximal effect of a full agonist, virtually all receptors are occu- pied14. As a reasonable approxi- mation one would expect the equilibrium dissociation constant K,, i.e. the concentration at which 50% of the receptors are occupied,

to mat& the concentration of the partial agonist causing half maxi- mum stimulant effects (i.e. EC&. This pattern, expected for conventional partial agonists, has been observed on a variety of heart tissues from different mammals regardless of the degree of selec- tivity of the ligands for PI- or fi2- adrenoceptors (Table II; Fig. 1). The corresponding K,s and E&s of 13 partial agonists differed by less than 0.5 log unit and clustered

symmetrically around a line of identity within an affinity range of 6 log units.

Independent evidence for the interaction of partial agonists with heart PI-adrenoceptors [xamoterol, KL-105, (-)-practolol, (-)-dichloroisoprenaline] or &- adrenocep!ors [salbutamol, procaterol, (+)-pindolol] has been provided by blocking the stimulant effects with selective PI- or &- adrenoceptor antagonists, by the

TABLE ll. Agreement between blocking and stimulant potency for conventional partial agonists

Partial agonist Receptor Species Tissue Stlmulatlon lntrlnric pK, pDz pK,,qD, Ref. activity

(-)-Dichloroieoprenaline rat cardiocyte chronotropic 0.6 7.7 7.6 -0.1 a rat cardiocyte adenylyl cyclase 0.1 7.6 7.5 a rat right atrium chronotropic 7.6 7.7 8::

(+)-Dichloroisoprenaline guinea-pig right atrium chronotropic ::3’ 0.4 b:c (-)-Praclolol cat right atrium chronotropic g 3:: -0.1 d (-)-Xamoterol cat right atrium chronotropic ::: 7:a -0.4 e

cat left atrium inotropic 7.6 :: -0.5 e cat papillary musde inotropic ::: 717 -0.2 e cat ventricle adenylyl cyclase 0.1 3:; 7.1 -0.4 e

Dopamine man ventricle inotropic :::

3.4 z:: 3.6

0.1 man ventricle adenylyl cyclase 0.2 1

(f)-H-67/67 cat right atrium chronotropic 7.3 7.0 0.3 b,c

BI $2 guinea-pig right atrium chronotropic :? 6.9 7.0 -0.1 b,c (f)-KL-105 Bi cat right atrium chronotropic 0:5 6.9 0.3

Bl cat left atrium inotropic 0.4 X:: 9.1 0 : Bl cat papillary muscle inotropic 9.1 6.9 0.2

Bl guinea-pig right atrium chronotropic ::; 9.2 9.1 0.1 : (-)-Oxprenolol f&$2? cat right atrium chronotropic 6.6 6.7 C

(+)-Oxprenolol right atrium chmnctropic ::t 7.2 7.1 ::: c (+)-pChloroisoprenaline right atrium chronotropic

::; 6.7

::; 6.5 -x:: h

(+)-INPEA right atrium chronotropic i,c Procaterol 82 cat papillary muscle inotropic

::: 6.2 7.7 0.5 j,k

(+)-Pindolol 82 guinea-pig right atrium chronotropic 7.2 7.0 0.2 I (+)-Salbutamol 82 guinea-pig right atrium chronotropic 0.7 5.6 6.2 -0.4 m

Ligands correspond to open symbols of Fig. 1. PK., -log K.(M), estimated as described in Refs d, h and n; pDa -log ECSo (M). ‘Kaumann, A. J. (1962) Naunyn-Schrniedebe@‘s Arch. Phannaco/. 320, 119-129, . bKaumann. A. J. and Blinks, J. Ft. (1960) Naunyn- Schmiedeberg’s Arch. PharmacoL 311, 20!&216; CKaumann, A. J. and Blinks, J. R. (1960) Naunyn-Schmiedeberg’s Arch. Phannacol. 311. 237-246; dKaumann, A. J. and Marano, M. (1962) Naunyn-Schrniedeberg’s Arch. Pharn?aco/. 316, 192-201; l Lemoine, H. et al. (1989) J. Cardiovasc. Phannacol. 13,105-l 17; f Kaumann. A. J. et al. (1969) Naunyn-Schmiedebeg’s Arch. PharmacoL 339.99-l 12;.“Lemoine. H. and Kaumann. A. J. (1962) Afaunyn-Schmiedeberg's Arch, Pharmaool. 320,130-144; “Marano. M. and Kaumann. A. J. (1976) J. PharmacoL Exp. Ther. 196.516-525; I Kaumann, A. J. et al. (1960) Naunyn-Schmiedebergk Arch. Pharmacof. 311,219-236; 1 Kaumann. A. J. et al. (1963) J. Recept. Res. 3.61-70; kKaumann. A. J. (1963) 2. Kardio/. 72, 63-62: ‘Walter, M. eta/. (1964) Naunyfl-SchmM?be@~ Arch. Phannacol. 327,159-175; mKaumann, A. J. eta/. (1976) in Hormone Receptors (Vol. 3) (Birnbaumer, L. and D’Malley, B. W.. eds). pp. 133-177. Academic Press: “Lemoine, H. and Kaumann, A. J. (1963) Naunyn-Schmiedeberg’s Amh. Phannacof. 322,ll l-120

31s TiPS - August 1989 [Vol. 101

ceptors. It has therefore bee;. Aag- gested that (-)-pindolol causes stimulant effects through atria1 receptors distinct from p,- and fiz- adrenoceptorsz4.

Several partial agonists, derived chemically from pindolol, cause stimulant effects on isolated heart tissues at concentrations up to 1000 times greater than those required to cause blockade of P-adrenoceptors (Fig. 1; Table III), suggest- ing interaction with receptors distinct from PI- and P2-adrenoceptors. For example, the affinity estimates for iodocyanopindolol are in the picomolar range for PI- and pz-adrenoceptors2s-27 but the E&S are 50-80 nM for its stimulant effects on isolated heart muscle.

use of the p,-adrenoceptor-selective agonist (-)-noradrenaline and by nonlinear analysis. The stimulant effects of xamoterol and (-)- denopamine (Table I) are specifi- cally mediated through &-adrenoceptors despite having only a modestly greater affinity for PI- than for f12-adrenoceptors. The latter do not mediate stimulant chronotropic and inotropic effects when occupied by xamoterol or denopamine, despite evidence that denopamine can stimulate adenylyl cyclase through bz- adrenoceptors (Ref. a in Table I).

Non-conventional partial agonists and atypical receptors

Evidence has been accumuiating over the last 15 years that some partial agonists, designated non- conventional, cause stimulant effects only at concentrations greatly in excess of those that cause blockade of myocardial effects of catecho1amines2,2123. The dissociation between blockade and stimulation was first observed with racemic pindo1012’ and was

later established to be due solely to its (-)-enantiomer. However, it is evident that there is no dlssoci- ation between blockade and stimulation for (+)-pindolol which acts through sinoatrial @2-adreno- ceptors24. The concentration-effect curves for the stimulant effects of (-)-pindolol on atria are biphasic with EC5,, values around In_w and 100 nM. The high-sensitivity component is mediated through both

&- and fi2-adrenoceptors because it is blocked by propranolol, by low (nM) (-)-bupranolol concentrations and by antagonists selective for &- and &-adreqoceptors. The low-sensitivity component of (-)-pindolol is not blocked by propranolol, is resistant to blockade by selective &- and pz-adrenoceptor antagonists but is blocked by high concentrations (PM) of the high affinity (nM) g-adrenoceptor antagonist ( -)-bupranolo124. These results refute the possibility that the low-sensitivity component is due to the less potent enantiomer in a racemate and to a differential involvement of &- and &-adreno-

6

8

11 10 9 8 7 6 5 4 3

fig. 1. Relationship between stimulant potencies (PO,) and affinities (pKJ of partial agonkts on isolated heati preparations. Open symbols: conventional partial agonists acting through&-adrenoceptors (0). &-adranoceptt;s (A) orpresumably tith (0); for *taits see Tab/e ft. Closed symbols: non-convent&w/p/ agonists, act&g at /east In part through atypicat i3-adrenoceptors; pindolol and related indoleamines (O), CGP- 72177 (A), alPrenolol (RI); for hrrther details see Table III.

Involvement of 5-HT receptors 5-HT causes feline sinoatrial

tachycardia that is antagonized by methysergide2-‘. Because pindolol, its analogues and 5-HT are indoleamines, they might interact with a common receptor; there is, however, as yet no evidence for this. Methysergide does not block the stimulant effects of (-)-pindolol in feline atria’l. The stimulant effects of (-)-pindolol and 5-HT are additive and neither (-)-bupranolol (1 PM) nor (-)- pindolol block the positive chronotropic effects of 5-HT in guinea-pig atria, ruling out interaction with a common receptor in this speciesz4.

Is there a heart fls-adrenoceptor? Evidence described above

demonstrates that non-conventional partial agonists cause part of their stimulant effects through heart receptors that are neither PI- nor Pz-adrenoceptors. Circum- stantial evidence suggests that the receptor for non-conventional partial agonists also exists in other tissues.

e (-)-Pindolol also acts as a non- conventional partial agonist in guinea-pig trachea by causing relaxant effects that are in part resistant to antagonism by silec- tive PI- and flz-adrenoceptor antagonists but are antagonized by micromolar concentrations of (-)-bupranolol. l The atypical lipolytic p-adrenoceptor in rat, which is activated selectively by novel agonists3’e3* has been designated a fi3-adreno- ceptors3, and resembles the heart receptor for non-conventional

TiPS -August 1989 [Vol. lo] 319

TABLE III. High blocking potency and low stimulant potency for non-conventional partial agonists

Partial agonist Species Tissue Stimulation Intrinsic pK, activity

PDZ pK,,-pDz Ref.

(-)-Pindolol

(&)-Pindolol

(f)-tert-butylpindolol (~)_Carazolol (+)-teti-butylcarazolol (t)-Hydroxybenzylpindolol

(+)-lodohydroxybenzylpindolol

(+)_lodocyanopindolol*

(+)-CGP-12177

(+)-Alprenolol (-)-Alprenolol

guinea-pig right atrium cat right atrium cat leit atrium cat right atrium rat rfaht atrium rat rat

right atrium right atrium

rat cat cat cat cat cat cat cat cat cat cat cat cat cat cat

right atrium right atrium left atrium papillary muscle ventricle right atrium left atrium papillary muscle left atrium papillary muscle right atrium left atrium papillary muscle right atrium right atrium

chronotropic chronotropic inotropic chronotropic chronotropic chronotropic chronotropic chronotropic chronotropic inotropic inotrooic adenylyl cyclase chronotropic inotropic inotropic inotropic inotropic chronotropic inotropic inotropic chronotropic chronotropic

0.2

z 0:5 0.2 0.2 0.1 0.1 0.8 0.7 0.5 0.1 0.7 0.5 0.4 0.7 0.6 0.6 0.7 0.6 0.2 0.2

9.5 9.5 9.2 9.2 6.6 9.3 9.9 9.6 9.0 9.1 9.0 8.6 9.2 9.4 8.7 10.6 10.4 10.1

>Z 8:6 8.9

7.0 6.5 ;:: 6.9 2.3 7.8 8.0 ;:“8 7.9 7.0 ::; 7.4 2.4 8.1 0.9 7.6 1.5 7.4 1.6 7.6 1.2 7.3 1.9 7.3 2.1 7.1 1.6 7.4 3.4 7.4 3.0 7.9 2.2 7.8 1.8

2.0 1.8 1.5

: b

: d d d e.f e.f e.f

f; f.g f.g g

:

: C

C

Ligands correspond to closed symbols of Fig. 1. *Non-equilibrium values for pKa (3 h incubation). Affinity estimates for iodocyanopindolol are even higher with greater than 3 h incubation periods. PK., -log K. (M). estimated as described in Refs h. i and j; pDp. -log EC, (M). a Walter, M. ef al. (1984) Naunyn-Schrniedeberg’s Arch. Pbarrnacol. 327,159-l 75: ’ Kaumann, A. J. and Lobnig. B. M. (1988) Br. J. Pharmacol. 89,207-218; sKaumann, A. J. and Blinks, J. Ft. (1980) Naunyn-Schmiedeberg’s Arch. fharmacol. 311,237-248; dKaumann, A. J. eta;. (1979) Naunyn-Schrniedeberg’s Arch. Pbarrnacol. 307, 1-8;eKaumann, A. J. eta/. (1978) in Hormone Receptors (Vol. 3) (Bimbaumer, L. and O’fvlalley, B. W., eds), pp. 133-177, Academic Press;‘Bearer, C. F. eta/. (1980) Mol. Pharrnacol. 17,328-338;sKaumann, A. J. (1983)Z. Kardiol. 72,63- 82; ’ Kaumann, A. J. and Marano, M. (1982) Naunyn-Scbmiedeberg’s Arch. PharmacoI. 318,192-201;‘Lemoine, H. and Kaumann, A. J. (1983) Naunyn-Schmiedebergk Arch. Phannacol. 322,ll l-120;fMarano, M. and Kaumann, A. J. (1976) J. fharmacol. Exp. Ther. 198.518-525.

partial agonists. Propranolol is 100 times less potent as a blocker of fat cell fi-adrenoceptors than as a blocker of fil-adrenoceptors31*32. The pindolol analogues cyanopindolol, hydroxybenzylpindolol and iodocyanopindolol act as partial agonists by causing lypolysis in rat fat cells with potencies (pDZ values around 7)2s similar to the stimulant potenc’es of the latter two compounds in heart tissues (Table III). l Longitudinal muscle of guinea- pig ileum relaxes with (-)- iaoprenaline and other agonists through atypical fSadrenoceptors that are resistant to blockade by propranololM but antagonized by relatively high concentrations of (+)-alprenolol (pK, 6.5)3s, (-)- dihydroalprenolol (p&C, 6.2)36 and (+)-cyanopindolol (p& 7.6)36. The low blocking potencies of these ligands resemble the low stimulant potencies of alprenolol and (-t)- iodocyanopindolol on heart (Table III).

cl 0 0

The existence of a third heart p- adrenoceptor is so far mainly sup- ported by the anomalous pattern of non-conventional partial agonists. These cause blockade of @I- and pz-

adrenoceptors at low concentrations and stimulant effects at relatively high concentrations. Their stimulant effects are fairly resistant to concentrations of @- adrenoceptor antagonists that saturate fil- and f12-adrenoceptors. This resistance would seem to be inconsistent with the idea that the receptor for non-conventional partial agonists is a p-adrenoceptor. However, this receptor appears to be coupled to a G, protein because hydroxybenzylpindolol can stimulate the adenylyl cyclase in feline ventricle (Table III; Ref. 2); pl- and fi2- adrenoceptors are also coupled to

t$;;:@l~~s;;~;~~~~~

tural similarity of non-conventional partial agonists to catecholamines, is consistent with the heart receptor for non-conventional partial agonists being a p- adrenoceptor subtype. Are there selective agonists and an endog- enous agonist [a (-)-catechol- amine?] for the third heart p- adrenoceptor? The answer to these questions and the search for selective and high affinity antagonists are future research goals.

Racemic pindolol can cause sinoatrial tachycardia in humans39. This may be due to the interaction

of (f )-pindolol with pz-adrenoceptors, or the interaction of (-)- pindolol with fix- and p2-adrenoceptors. Alternatively, it may be due to the interaction of (-)-pindolol with a third @-adrenoceptor. (-)-Pindolol is a partially selective Pa-adrenoceptor antagonist on human atria but, unlike its effect on feline atria, it does not cause positive inotropic effects in humans”. This finding does not necessarily rule out the existence of a third sinoatrial p-adrenoceptor in humans which could still mediate tachycardia caused by either (-)-pindolol or a pindolol metabolite. The metabolite could also activate and desensitize fi2- adrenoceptors, because these have been found to be downregulated in atria from pindolol-treated patient@).

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3 Kaumann, A. J. and Lemoine, H. (1985) Naunyn-Schmiedeberg’s Arch. Pharm~col. 31.2739

4 Gille, E., Lemoine, H., Ehle. 8. and Kaumann, A. J. (1985) Nounyn-Schmiede- berg’s Arch. Pharmacol. 331,60-70

320

5 Lemoine. H., Schonell. H. and Kaumann, A. ). (1988) Br. /. PhannncoL 95, 55-66

6 Hall. J. A., Kaumann, A. J., Wells, F. C. and Brown, M. J. (1988) Br. ]. Pharmocol. 93,116P

TiPS - August 1989 [Vol. 101

Sennitt,M. and Arch, J. R. S. (1984) Eur. 1. PharmacoL 100,309-319

33 Tan, S. and Curtis-Prior, P. 8. (1983) Int. /. Obesity 7, 409-414

34 Bond, R. A. and Clarke, D. E. (1988) Br. \. Pharmacot. 95,723-734

35 Blue, D. R., Bond, R. A. and Clarke, D. E. (1988) Br. 1. Pharmacol. 95, 541P

36 Blue, D. R. et e?, (19&S?> Br. I. ?rrarmacoZ. 96,246P

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39 Mann In’t Veld, A. J. and Schalekamp, M. A. D. H. (1981) Br. Med. 1. 282, 929-931

7 Kaumann, A. J., Hall, J. A., Murray, K. J., Wells, F. C. and Brown, M. J, (1989) Eur. Heart 1. 10 (Suppl. B), 29-37

8 Kaumann, A. J. and Lemoine, H. (1987) Naunyn-Schmiedeberg's Arch. PhormocoL 335.403411

9 Kaumann, A. J. (1978) Nounyn-Schmiede- berg’s Arch. PhnrmacoZ. 305,97-102

10 Venter, J. C. (1979) Mol. Phomracol. 16, 429-410

11 Kaumann, A. J. (1981) Nuvyn-Schmiede- berg’s Arch. Pharmacol. 317, 13-18

12 Kaumann, A. J. and Bimbaumer, L. (1974) /. Biol. C/rem. 249,7874-7885

13 Kaumann, A. J. and Bimbaumer, L. (1976) Naloryvll-ScRnliedeberg’s Arch. Pharmncof. 293, 199-202

14 Stephenson, R. P. (1956) Br. 1. PlmmacoL 14.379-393

15 Kaumann, A. J., Lemoine. H.. Morris, T. H. and Schwedenki, U. (1982) Nae~lytl-Sc~~~~~ic~cbc~S Arch. Pharnracol. 319.216221

16 Bristow. M. R. et al. (1982) N. Engf. 1. Med. 307.205-211

17 Hedberg, A., Minneman, K. P. and Molinoff, P. B. (1980) /. Pknrmacol. Exp. I-Ircr. 213, 503-508

18 Bmdde. 0. E., Karad, K., Zerkowski,

H. R., Rohm, N. and Reidemeister, J. C. (1983) Circ. Res. 53, 752-758

19 Stiles, G. L., Taylor, S. and Lefkowitz, R. J. (1983) Life Sci. 33, 467-473

20 Buxton, B. 8. F., Jones, C. R., Molenaar, P. and Summers, R. J. (1987) Br. J. Phamracol. 92, 299-310

21 Kaumann, A. J. (1973) Acta Physiol. Lot. Am. 23,235-236

22 Bilski, A., Robertsen, H. H. and Wale, J. L. (1976) Clin. Exp. Pharmcrol. Physiol. 6, l-9

23 Kaumann, A. J. and Lobnig, B. M. (1986) Br. ]. Pharntacol. 89, 207-218

24 Walter, M., Lemoine, H. and Kaumann, A. J. (1984) Naunyn-Schmiedeberg’s Arch. Phammol. 327, 159-175

25 Engel, G., Hoyer, D., Berthold, R. and Wagner, H. (1981) Naenyn-Schmiede- berg’s Arch. Pharmacol. 317. 277-285

26 Ferry, D. and Kaumann, A. J. (1987) Br. 1. Pharmacol. 90, 447-457

27 Neve, K. A., McGonigle, P. and Molinoff, P. B. (1986) J. Pharmacol. Exp. Tiler. 238, 46-53

28 Kaumann, A. J. (1983) Nnunyn-Schmiede- berg s Arch. Pharmacol. 322, R42

29 Kaumann, A. J. (1985) J. Curdiovasc. Pharmacol. 7 (Suppl. 7). S76-‘378

30 Saxena, P. R., Mylecharane, E. J. and Heiligers, J. (1985) Naunyn-Schmiede- berg’s Arch. Pharmacol. 330, 121-129

31 Arch, J. R. S. et al. (1984) Nature 309, 163-165

32 Wilson, C.. Wilson, S., Piercy, V.,

40 Michel, M. C. et al. (1988) Br. J. Pharma- col. 94,6BS-692

CGP-12177: (&)-4-(3-f-butylamino-2- hydroxypropoxy)-benzimidazol-2-one CGP-20712A: (?)-l-[2-(3-carbamoyl-4- hydroxyphenoxy)-ethylamino]-3-[4-(1- methyl-4-trifluormethyl-2-imidazolyl)- phenoxyl-2-propanol ICI-118551: erythro-(+)-1-(7-methyhndan- 4-yloxy)-3-isopmpylaminobutan-2-01 KL-105: (f)-2-(3-t-butylamino-2- hydroxypropoxy)-chlorobenzene H-87/67: (+)-4-(3-isopropylamino-2- hydroxypropoxy) (Zmethoxyethoxy) benzene INPEA: (+)-l-(4-nizophenyl)-2-isopropylamino-ethan-l-01

*+ localization and sensitivitv in vascul

J

r smooth muscle Hideaki Karaki

An increase in cytosoiic Ca ” level (fCazc]i) is a prerequisite for smooth muscle contraction. Simultaneous measurements of iCa2+li and muscle tension give direct information on the Ca2+ regulation of smooth muscle. The photoprotein aequorin and the fluorescent CaZ+ indicator fura- are widely used for this purpose. Although there are some inconsistencies between the results obtained with these two indicators, comparison between [Ca’+]i and muscle tension in vascularsmooth muscle indicates that stimulation of cw-adrenoce whereas stimulation of fi-adrenoceptors decreases, both the Ca p tars increases,

+ sensztzvzty of contractile elements and [Ca’+]i. Thus, as Hideaki Karaki explains, contrac- tility of vascularsmooth muscle may be regulated not only by lCa’+li but also by the Ca2+ sensitivity of the contractile elements.

The pivotal role of Ca2+ in the regulation of skeletal muscle con-

cytosolic Ca2+ levels ( [Ca2+] j) e.len in smooth muscle. The relation-

tractility was established in the 1960s by Ebashi (see Ref. 1). Since

ship between [Ca2+li and muscle

then, contraction has been used as tension has been investigated by,

a useful and reliable indicator of for example, radioactive 45Ca2+ flux experiments. In general, there is a good correlation between the

H. ffiraki is Professor and Chairman of the rate of 45Ca2+ influx and smooth

Department of Veterina y Pharmacology; muscle contraction. However, it is FacltIty of Agriculture, The University of Tokyo. Bmrkyo-ku. Tokyo 113, lapan.

difficult to distinguish free Ca2+ and bound Ca2+ by this method,

@ 1989. Ekvier Science Publishers Ltd. ILK) 0165 - f,,47,89/103.50

and resolution is not fast enough to follow the relatively rapid muscle contraction.

Development of the technique of injecting the photoprotein, aequorin, into cardiac cells2 achieved the goal of simultaneously measuring [Ca2’]i and muscle tension. However, microinjection cannot be applied to smooth muscle because of its smaller cell size, so a method was developed3 in which smooth muscle cell mem- branes are reversibly permeabil- ized with EGTA and the cells are loaded with aequorin. Using this method, it was found that depolar- ization with high K+ induces sustained increase in both muscle tension and [Ca2+]i (Refs 4 and 5). Unexpectedly, agonists such as phenylep,hrine induce a large tran- sient increase followed by a small sustained increase in [Ca2’li dur- ing sustained contraction*5 (Fig. 1). These results threw doubt on the simple correlation between [Ca2+]i and muscle tension in vascular smooth muscle6n7.

In the 198Os, Tsien and his col- leagues synthesized a new intra-

cellular Ca2+ indicator, quin2, which was followed by the im- proved indicators, fura- and indo-l (see Box). These indicators are widely used to examine the

Is there a third heart β-adrenoceptor?

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