Naunyn-Schmiedeberg's Arch Pharmacol (1981) 317:13 - 18

Naunyn-Schmiedeberg's

Archivesof Pharmacology �9 Springer-Verlag 1981

In Kitten Ventricular Myocardium, the Inotropic Potency of an Agonist is Determined by Both Its Intrinsic Activity for the Adenylyl Cyclase and Its Affinity for the fl-Adrenoceptors

Alberto J. Kaumann

Klinische Physiologie, Physiologisches Institut der Universitfit, Universit~itsstrasse 1, D-4000 Dtisseldorf, Federal Republic of Germany

Summary. Three /~-adrenoceptor-related parameters were measured for 23 agonists on kitten ventricular myocardium: inotropic potencies (ECso'S) on papil lary muscles, intrinsic activities for st imulation of the adenylyl cyclase in membrane particles, and affinities for/3-adrenoceptors labelled with 3H- ( - ) -p rop rano lo l in membrane particles.

For catecholamines a dissociation between inotropic potencies and fi-adrenoceptor affinities was observed. Inotropic potencies are 1 . 3 - 1 . 8 orders of magnitude higher than affinities. This dissociation between inotropic activation and binding is similar for (+) - and ( - ) -enant iomers of catecholamines, despite the higher affinities of the latter. The dissociation is considerably smaller for dopamine and dobut- amine, which suggests a role of the /~-OH group in the phenomenon. The effect is also smaller with resorcinols than with catechols; it decreases further, or is not observed at all, when one of the ring hydroxyl groups is replaced or absent. With most agonists the degree of dissociation appears to be directly propor t ional to the degree of adenylyl cyclase stimulation. Abou t 1/4 of maximum adenylyl cyclase stimu- lation suffices to activate the inotropic processes fully. It is suggested that both the magnitude of the dissociation and the degree of adenylyl cyclase activation are indicators of the degree of change induced by agonist in the conformation of the ]3-adrenoceptor.

The product ion of marked inotropic effects by noradren- aline and adrenaline with low /%adrenoceptor occupancy and little adenylyl cyclase st imulation may permit the re- gulation of heart function. Condit ions of desensitization that reduce maximum activation of the adenylyl cyclase may not necessarily reduce maximum inotropic effects of these catecholamines.

The agonist (+)-hydroxybenzylpindolol caused positive inotropic effects only at high/~-adrenoceptor occupancy. This is consistent with the idea that sufficient conformational change occurs only near receptor saturation.

Key words: Heart spare fi-adrenoceptors - Inotropism - Ad eny ly l cyclase - Intrinsic activity - Agonists

Introduction

Recently, affinity estimates for catecholamines were made from binding studies on membrane particles of kitten myocar-

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dium and compared with estimates of inotropic potency (Kaumann 1978). The main conclusion of these studies was that catecholamines were able to elicit nearly maximum inotropic effects with only small fractional /3-adrenoceptor occupancy. This conclusion received independent support with the experiments in cat papil lary muscle of Venter (1979), who used an irreversible l igand to occlude/~-adrenoceptors. When 90 % of the receptors were occluded, isoprenaline still caused maximum inotropic effects but 50 times higher con- centrations of isoprenaline were required to match inotropic effects in the absence of receptor occlusion. The experiments of Kaumann (1978) and Venter (1979) represent an example of spare receptor (Stephenson 1956) capacity.

It was recently proposed that the degree of inot ropic spare /~-adrenoceptor capacity was propor t ional to the intrinsic activity of agonists as stimulants of heart adenylyl cyclase (Kaumann 1980). The purpose of the present paper is to evaluate this proposal and to contribute some insights into the structural requirements of the phenomenon.

Methods

Experiments were carried out at 32.5 ~ C on kitten (0.6-1.4 kg, either sex) myocardium. Endogenous noradrenaline can interfere with adenylyl cyclase stimulation by ligands (Kaumann and Birnbaumer 1974a) and may also distort direct binding assays. The myocardial noradrenaline was therefore depeleted by pretreating the kittens with 5 mg/kg s.c. reserpine 20-24 h before sacrifice. The kittens were lightly anaesthetized with chloroform and the hearts rapidly removed and washed with a solution containing (mmol/1): Na + 140, K + 5, Ca 2+ 2.25, Mg 2+ I, CI- 98.5, SO~- 1, HCO~ 29, HPO]- 1, fumarate 10, pyruvate 5, L-glutamate 5, glucose 10, ethylenediamine tetraacetic acid (EDTA) disodium salt 0.04, equilibrated with 95 % 02 and 5 % CO2.

Papillary Muscles. The tissues (width less than 0.8 mm) were quickly excised from the right ventricle and set up (usually in pairs) in an apparatus with a 50 ml organ bath (Blinks 1965) containing the above solution. The tissues were attached to strain gauge transducers and peak tensions recorded. The muscles were driven at 5 s intervals with square wave pulses of 5 ms duration and currents of just threshold intensity. The tissues were stretched to a length at which maximum peak tension developed. In order to block irreversibly extraneuronal and neuronal uptake of agonists and possible interactions with myocardial c~- adrenoceptors, the muscles were incubated 2 h with 5 pmol/1 phenoxy- benzamine (Kaumann 1970, 1972, 1977; Kaumann et al. 1978). After phenoxybenzamine was washed out, 60 min were allowed for stabili- zation before a first cumulative concentration-effect curve for an agonist was determined. Increases in peak tension were measured as effects. All effects were equilibrium effects. After a maximum effect was observed, a curve for (-)-isoprenaline was determined in the presence of the highest effective concentration of agonist. The maximum concentration of (--)-

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14

isoprenaline was 20gmol/1 with all agonists, except with (+)-hy- droxybenzylpindolol where it was 200 gmol/1. The muscles were washed to remove the agonists, then incubated 90 min with 10 nmol/1 of the fl- adrenoceptor antagonist (-)-bupranolol and a second curve for the agonist determined with (-)-bupranolol (for literature on bupranolol see Wfichter et al. 1980). From these curves the - l o g ECs0 (pD2) was determined for each muscle and the 2 + SEM of pD 2 calculated for a group of tissues. A quantitative estimate of fl-adrenoceptor blockade was calculated from the shift of the concentration-effect curve by ( - ) - bupranolol. To this end, the difference of the pD 2 without ( - ) - bupranolol and the pD2 with 10 nmol/1 (-)-bupranolol was computed from individual muscles and expressed as 2_+ SEM.

The intrinsic activity (Ari6ns 1954) for inotropic effects was esti- mated in each muscle from the ratio between the maxinmm effect of an agonist over the maximum effect of (-)-isoprenaline. The 2 + SEM of the intrinsic activity was calculated (N ~> 6).

Ventricular Membrane Particles. Cell-free membrane particles were obtained by homogenization of tissues with a Polytron as described in Kaumann and Birnbaumer (1974a). After discarding a 1,000 g pellet, the supernatant was centrifuged for 30 rain at 10,000 g; the resuspended pellet was kept frozen at 80 ~ C until use. Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard.

Binding Assay. The method of Kaumann (1978) was used. fl-Adreno- ceptors were labelled with aH-(-)-propranolol (specific activity 20.91 Ci/ mmol, New England Nuclear, Boston, MA, USA). Incubations were con- ducted for 10 min. Samples contained in a volume of 100 ~tl: 3 - 6 nmol/1 3H-(-)-propranolol; (mmol/1): 64 Tris HC1 pH 7.6, 3.2 MgSO4, 0.64 EDTA; 4 0 - 6 0 btg membrane protein and agonists, where applicable. Incubations were terminated by the addition of 1 mI ice-cold buffer followed by rapid vacuum filtration on Whatman GF/A glass fiber filters and washed four times with 3 ml ice-cold washing solution (10 mmol/1 Tris- HC1, pH 8.0, 5 retool/1 MgC12). The filters were dried, treated with 0.5 ml Protosol (NEN, Dreieich, FRG) for 30 min at 60~ then cooled on ice; 8 ml of Econofluor (NEN, Dreieich, FRG) were then added and the radioactivity measured by liquid scintillation counting (efficiency, 46 ~). Non specific binding, defined as that binding observed in the presence of 100 btmol/1 (-)-isoprenaline, was less than 26 ~ of the total binding. Nonspecific binding was not significantly different in the presence of 1 ~tmol/l (-)-propranolol.

Curves for inhibition of binding (N > 4 per agonist) were carried out by using 8 - 1 2 concentrations (2 or 3 per log unit) of agonist to compete with 3H-(-)-propranolol for ~-adrenoceptors. Equilibrium dissociation constants (KD) for agonists were calculated from inhibition curves by the equation (Cheng and Prusoff 1973):

K D = ICs0/(I+[B*]/KB,), (1)

where ICso is the concentration of ligand to remove half of specifically bound 3H-(-)-propranoloi, [B*] is the concentration of 3H-(-)- propranolol used, and KB* is the equilibrium dissociation constant for 3H-(-)-propranolol estimated from direct binding ( - l o g KB,_+SD, mol/1, = 8.4+0.1, data taken from W/ichter et al. 1980). SEM for - l o g KD were calculated from W~ichter et al. (1980):

~ ' , R5 R I Z ~ / ~ C H - - C H - - N H - - R 7

.,>< '~

R t R z R~ R 4 R s R, R v

OH OH H C OH H . NDrad renaline OH OH H C OH H C ' , Adrenaline OH OH H C OH H CH2CH a N-Ethylnoradrenaline

B . .

OH OH H C OH H CH I s o p r e n a l l n e /

CH=

CH=

OH OH H C OH H C--CH= OH H ydroxybenzy lisoprenalim P

CHa

o. o . . c o . c.3 , N o r d e f r i n e

OH OH H C OH CH,CI'~ CH I s o e t h a f i n e I CH=

OH OH H C H H H Oopamine

OH OH H C H H ~H C H , C H , - - ~ ' ~ - - O H D o b u t a m i n e

CH=

OH H OH C OH H CH v r c l p r e n a l l n e /

CHa

OH H OH C OH H c - - e l l , T e r b u t a l i n e [ CHa

OH=

CH2OH OH H C O. H C - - C ~ S a l b u t a m o l I CH3

CH= I

CH.O. OH H " OH H C--CH~ P i r b u t e r o l 1 c~

CHj I

OH H C OH H CH S o t e r e n o l CH~,qO2NH /

CH~

CHa

C H , O~N . . . . . . . ~ . . . . . ~ Zintero, CH=

CH3 / CI C[ H C OH H ~H D i c h l o r o i s o p r e n a l i n e

I CH=

, c oH , e, o-Chloroisoc~ranal ine H Ct I CHa

~ Ha

I I HP IPAP HO 0 -- CH 2 - CH -- CH~ -- NH -- CH

OH CHa

CH=

' -<5- ~ n - c .2 - ~H-- CH,-- NH-- ell--ell, OH H y d r o x y b e n z y l p i n d o l o l H ICH 3

F i g . 1. Chemical structure of agonists. For further details see Methods and text

1 2 1 1 S E M o f ( - l o g K D ) = X/N[SD( logiC,0)] + ~ I ( K B , / [ B , ] ) + ~ I 2 {[SD(-'~ [SD( IogK,,*)]} 2"

Adenylyl Cyclase Assay. Incubation conditions were similar to those of Kaumann et al. (1979). Assay tubes contained in a volume of 50 gl (mmol/1): 0.1 [c~-32p]ATP (250-650 cpm/pmol), 2 MgC12, I EGTA, 0.1 ascorbic acid, 0.01 GTP, 1 3H cyclic AMP (8,000-12,000cpm), 20 creatine phosphate, 25 Tris . HC1 pH 7.6, 0.01-1.0 agonist where appropriate; (IU/ml): 15 creatine phosphokinase, 9.8 myokinase; 4 0 - 60 ~tg membrane protein. The reaction was terminated by the addition of 100 gl of a solution containing: 10 mmol/l cyclic AMP, 40 mmol/1ATP, and 1 ~ sodium dodecyl sulfate, pH 3.5. Production of cyclic AMP was quantitated by the method of Salomon et al. (1974). Intrinsic activities of agonists were calculated using (-)-isoprenaline as reference. Materials' andDrugs. Materials for the 3H-( )-propranolol binding assay were those of Kanmann (1978). For adenylyl cyclase assays, adenosine 5-

(2)

triphosphate Tris salt (ATP), guanosine 5'-triphosphate Tris salt (GTP), creatine phosphate, myokinase (adenylate kinase), adenosine-3',5'- monophosphate Tris salt (cyclic AMP) were from Sigma Chemical Co., St. Louis, MS, USA; adenosine 5'-triphosphate ([c~-32P] ATP) and adenosine-3',5'-monophosphate ([3,8-3H]cyclic AMP) were from Amersham Buchler GmbH, Braunschweig, FRG; creatine phospho- kinase was from Calbiochem., San Diego, CA, USA. Reserpine phos- phate was from Ciba-Geigy, Basel, Switzerland; phenoxybenzamine hydrochloride from Smith, Kline and French Ltd., Philadelphia, PA, USA; (-)-bupranolol hydrochloride from Sanol (Monheim, FRG).

The structures of agonists used are depicted in Fig. 1. (-)-Iso- prenaline bitartrate, (+)-isoprenaline bitartrate, (-)-N-ethylnoradren- aline . HC1, (+)-adrenaline bitartrate monohydrate, (_+)-p-chloro-

15

Table 1. fi-Adrenoceptor agonists on kitten ventricle at 32.5~ Inotropic potencies (pD 2 = - l o g ECs0), intrinsic activities and antagonism by 10 nmol/l (-)-bupranolol. Intrinsic activities for adenylyl cyclase stimulation and equilibrium dissociation constants (pKD = --log KD) for fl- adrenoceptor-agonist complexes. Dissociation between inotropic pD2's and fi-adrenoceptor pKo's

Agonist Papillary muscles (positive inotropism) Membrane particles Inotropic p D 2 - pKo

No. Intrinsic pD 2 (mol/1) pD2-(pD 2 with Direct binding Adenylyl activity* 2_+ SEM bupranolol) cyclase ~

No a pK D (tool/l) No. b Intrinsic 2 • SEM activity ~

_+ 7 S E M 2 pD2 + SEM 2 pKD

(-)-Isoprenaline (+)-Isoprenaline (-)-Noradrenaline (+)-Noradrenaline (-)-Adrenaline ( +)-Adrenaline Dopamine D obutamine ( •

zylisoprenaline (-)-N-Ethyl-

noradrenaline Nordefrine Isoetharine ( -+)-Salbutamol ( _+)-Pirbuterol ( -+)-Soterenol ( _+)-Zinterol ( _+)-Terbutaline ( -+)-Orciprenaline (-)-Dichloro-

isoprenaline ( _+)-p-Chloro-

isoprenaline (-)-HPIPAP

(Prenalterol) (+)-HPIPAP

(H 133/21) ( _+)-Hydroxy-

benzylpindolol

12 1.00 9.00_+0.14 1.28-+0.08 10 7.22-+0.10 32 1.00 1.78+_0.17 8 1 . 0 1 6.25_+0.09 1.07-+0.05 4 4.95-+0.10 12 0.78 1.30-+0.13

10 1.00 8.00_+0.14 1.19_+0.10 8 6.56+_0.04 4 0.97 1.44_+0.15 6 0.98 6.25_+0.12 1.25_+0.05 6 4.90_+0.10 8 0.91 1.35+0.16 8 1.02 8.12_+0.13 1.31_+0.09 8 6.48_+0.05 8 1.10 1.64_+0.14 6 0.99 6.52_+0.08 0.98_+0.08 6 5.04_+0.08 8 1.05 1.48_+0.11 6 0.98 4.80-+0.07 0.90_+0.08 5 4.35_+0.09 8 0.61 0.45-+0.11 6 1.00 6.97+_0.19 1.20_+0.14 6 6.30_+0.09 8 0.42 0.67-+0.21

12 1.02 8.40_+0.12 1.17_+0.11 8 7.06+_0.08 4 1.01 1.34-+0.14

7 1.00 8.18_+0.11 1.20_+0.09 6 6.93_+0.07 8 0.99 1.25_+0.13 6 0.98 8.10_+0.09 1.19+_0.05 4 6.72+_0.14 8 0.91 1.38+_0.17 6 1.03 5.91-+0.19 1.08_+0.04 4 5.30-+0.13 4 0.80 0.61_+0.23

12 0.97 6.00-+0.18 1.31 _+0.11 7 5.64_+0.05 4 0.27 0.36_+0.19 6 0.97 5.97_+0.06 0.95_+0.10 4 5.71• 4 0.37 0.26_+0.08 7 0.96 6.35_+0.19 1.23_+0.09 6 6.35_+0.09 8 0.47 0.00-+0.21 9 0.98 6.59_+0.11 1.27_+0.12 6 6.45_+0.08 8 0.61 0.14_+0.14 9 1.03 5.79+0.10 1.28_+0.08 6 4.96_+0.04 8 0.55 0.83_+0.1i

10 1.00 6.67_+0.16 1.38_+0.11 6 5.47_+0.05 4 0.63 1.20+_0.17

8 0.72 7.69_+0.17 1.20_+0.10 4 7.50_+0.10 8 0.24 0.19_+0.20

8 0.66 7.70_+0.14 1.33• 4 7.60_+0.1l 4 0.17 0.10_+0.18

8 0.82 6.74_+0.09 0.92-+0.08 4 6.76_+0.05 8 0.16 -0.02_+0.10

6 0.52 4.72+0.09 0.95+_0.10 4 4.79_+0.04 8 0.11 -0 .07+0 . t0

12 0.69 7.70_+0.12 0.96 d 8 9.11 _+0.14 8 0.11 1.41 _+0.18

a Number of concentration-competition curves b Number of assays c Basal adenylyl cyclase activity was 2.42 _+ 0.01 pmol. mg - 1. min 1 ; activity with 0.1 mmol/l (-)-isoprenaline was 8.08 + 0.10 pmol/1 �9 mg - ~ �9 min- 1. d Because of slow stimulation kinetics only one curve was determined in each muscle; 6 muscles were without (-)-bupranolol, another 6 muscles with

(-)-bupranolol e The standard error of the intrinsic activities was always smaller than 5 %

isoprenaline �9 HCI, (-)-dichloroisoprenaline. HC1, nordefrine (cobefrin) (prepared as the hydrochloride) and isoetharine - HC1 were from Sterling- Winthrop, Rensselaer, NY, USA; (-)-adrenaline bitartrate and do- pamine. HC1 fiom Serva, Heidelberg, FRG; ( _+)-hydroxybenzylisopren- aline (prepared as the hydrochloride) from Duphar, Weesp, The Netherlands; (+_)-orciprenaline - HC1 from Boehringer, Ingelheim, FRG; (+)-terbutaline sulfate, (-)-t-(4-hydroxyphenoxy)-3-isopropylamino- propan-2-oI �9 HC1 [(-)-HPIPAP, prenalterol, H133/22] and (+)- HPIPAP �9 HC1 (H 133/21) from H/issle, M61ndal, Sweden; (+)-salbuta- mol sulfate from Allen and Hanbury, Ware, Hertfordshire, GB; (_+)-hy- droxybenzylpindolol - HC1 from Sandoz, Basel, Switzerland; (+)-pir- buterol " HC1 from Pfizer, Groton, CT, USA; (• - HC1 and (_+)-soterenot - HC1 from Mead Johnson, Evansville, IN, USA; dobuta- mine �9 HC1 from Eli Lilly, Indianapolis, IN, USA.

For experiments on intact tissues 10 or 100 mmol/1 stock solutions of agonists were made with deionized, red• water containing 0.04 mmoi/1 EDTA and adjusted to pH 4 with HC1 for catecholamines. For biochemical assays all stocks of agonists were prepared in a solution containing 0.25 mmol/1 ascorbate and 2.5 mmol/1 EGTA, pH 6.

Results Papillary Muscles. Inotropic Potencies and Intrinsic Activities of Agonists ; Antagonism by (-)-Bupranolol. The resul ts are s u m m a r i z e d in Tab le 1. O f the 23 agonis t s tested, 18 were full agonis t s [i.e. in t r ins ic act ivi ty was one wi th respect to ( - ) - i soprenal ine] . All o f the ca t echo lamines , resorc inols , (+_)- s a lbu t amo l , (+_)-pirbuterol , (_+)-soterenol a n d ( • were full agon i s t s ; ( - ) - d i c h l o r o i s o p r e n a l i n e , (_+)-p-chloro- i soprenal ine , ( • p r ena l t e ro l [(--)- H P I P A P ] a n d ( + ) - H P I P A P were s t r o n g pa r t i a l agonis ts . The m o s t p o t e n t agonis t s were the ( - ) - e n a n t i o m e r s o f the cate- cho lamines , (_+) -hydroxybenzy l i soprena l ine a n d nordef r ine . The ( + ) - e n a n t i o m e r s were 1 . 6 - 2.7 orders of m a g n i t u d e less p o t e n t t h a n the i r c o r r e s p o n d i n g ( - ) - e n a n t i o m e r s . F u r t h e r - more , ( + ) - n o r a d r e n a l i n e a n d d o b u t a m i n e were 1.4 a n d 2.2 o rders of m a g n i t u d e m o r e po ten t , respectively, t h a n d o p a m i n e .

16

2s

I.C =o

I

4 E_

~ --I.C o

- - I . 5

O

y O

�9 I I I

0.5 1.0 1.2

INTRINSIC ACTIVITY FOR

ADENYLYL CYCLASE

Fig. 2. Correlation between inotropic spare ]~-adrenoceptor capacity with intrinsic activity for adenylyl cyclase stimulation by agonists in kitten ventricle. Data from Table 1. (�9 Full inotropic agonists, (O) and (A) partial inotropic agonists, (A) (_+)-hydroxybenzylpindolol (not included for correlation). The correlation coefficient was 0.9005 (P < 0.001, N = 22)

The /Lblocker (-)-bupranolol antagonized, surmoun- tably, the effects of all agonists. Concentration-effect curves for agonists with and without 10 mmol/1 (-)-bupranolol were nearly parallel and were separated by approximately one order of magnitude.

Affinities of Agonists for Ventricular fl-Adrenoceptors. All agonists competed with 3H-(-)-propranolol for /~- adrenoceptors in a concentration-dependent manner. The equilibrium dissociation constants for agonists are presented in Table 1. Whenever tested, the (-)-enantiomers exhibited a 1.4-2.3 orders of magnitude higher affinity than the cor- responding (+)-enantiomer.

Intrinsic Activity of Agonists for VentricuIar Adenylyl Cyclase. All 23 agonists significantly stimulated the adenylyl cyclase (Table 1). In every case, the maximum effect was obtained with 0.1 mmol/1 agonist. Despite vast differences in affinity, the highest intrinsic activities were obtained with the catechol- amines, regardless of enantiomer. Those agonists with mo- dified rings displayed intermediate intrinsic activities. All compounds, which were strong partial agonists (i. e. intrinsic activity > 0.5) for inotropy, were weak partial agonists (i.e. intrinsic activity < 0.2) for the adenylyl cyclase.

Correlation Between the Intrinsic Activity for the Adenylyl Cyclase and Inotropic Spare ~-Adrenoceptors. Table 1 shows that concentrations of most agonists for half maximal inotropic effects are lower than concentrations to half saturate/~-adrenoceptors. The greatest discrepancy was ob- served with those agonists with the highest intrinsic activities for the adenylyl cyclase (Table 1). Thus, the discrepancy was greatest with catecholamines and smallest with (_+)-pirbu- terol. The full inotropic agonists (+)-zinterol and (+)-sote- renol, and the inotropic partial agonists (-)-dichloro- isoprenaline, (+)-p-chloroisoprenaline, ( - ) -HPIPAP and (+)-HPIPAP did not show significant dissociation between inotropic ECso'S and binding KD's. The inotropic partial

agonist (_+)-hydroxybenzylpindolol is unique in that it dis- played a much greater affinity than inotropic potency, yet it significantly stimulated the adenylyl cyclase.

Figure 2 illustrates the correlation between the intrinsic activity for adenylyl cyclase stimulation and the discrepancy between inotropic ECso'S and /%adrenoceptor KD's. (_+)- Hydroxybenzylpindolol was omitted for the computation of the correlation.

Discussion

Lack of Influence of c~-Adrenoceptors. The presence of ~- adrenoceptors in mammalian heart may interfere with the interpretation of the inotropic potencies for some agonists, particularly noradrenaline (Kaumann 1970), adrenaline (Wagner and Brodde 1978), dopamine (Wagner et al. 1980) and prenalterol (Aass et al. 1980). Such interference is unlikely to be of consequence for two reasons: first, the irreversible e-blocker phenoxybenzamine was used systemati- cally on all papillary muscles. Second, for each agonist, the blockade produced by (-)-bupranolol agreed quantitatively with previous affinity estimates for this antagonist (Kaumann 1977, 1978; Kaumann et al. 1980; W/ichter et al. 1980). Therefore it appears that all of the agonists used acted mainly through/?-adrenoceptors.

Dependence of Conformational ~-Adrenoceptor Change on Agonist Chemistry. For (-)-isoprenaline, half maximal in- otropic response can be obtained with a concentration of agonist that produces only 2% fractional /%adrenoceptor occupancy. This dissociation between inotropic potency and actual affinity is a classical spare receptor phenomenon (Stephenson 1956). For most catecholamines the dissociation is similar; half maximum inotropic stimulation is produced with only 2 - 5 % fractional/~-adrenoceptor occupancy; the exceptions were isoetharine, dopamine and dobutamine, which required 20%, 26% and 18 % /Ladrenoceptor occu- pancy, respectively. The resorcinol derivatives, ( _+)-orcipren- aline and (+)-terbutaline, were intermediate, requiring 6 % and 13 % /Ladrenoceptor occupancy, respectively, for half maximum inotropic stimulation. (_+)-Salbutamol and (_+)- pirbuterol required 30% and 35% //-adrenoceptor occu- pancy, respectively, for half maximum inotropic stimulation. All other agonists except (+_)-hydroxybenzylpindolol caused about 50% /~-adrenoceptor occupancy for half maximum inotropic effect, i.e. did not exhibit significant spare /?- adrenoceptor capacity.

Agonists may cause marked positive inotropic effects at low /Ladrenoceptor occupancies because they produce a pronounced change in receptor conformation. On the other hand, agonists that require relatively high fi-adrenoceptor occupancies to cause positive inotropic effects, may only be able to induce small changes in receptor conformation. With this presupposition conformational change is strongest for catecholamines that possess an asymmetric carbon - regard- less of enantiomer. Conformational change is substantially smaller for dopamine than for either (+)- or ( - ) - noradrenaline. The conformational change is also weak for isoetharine, despite the presence of a/~-hydroxyl. In this case the c~-ethyl group may produce steric hindrance. It should be noted that the amino group per se of a catecholamine is not necessary to induce conformational change, though it con- tributes greatly to the overall affinity (Kaumann et al. 1977).

17

It should be emphasized that although the preceding considerations appear valid from the presented data, further experimental conditions should be tested, especially related to affinity. It is well known that GTP lowers the affinity of catecholamines for fl-adrenoceptors in membrane particles (Maguire et al. 1976; Lefkowitz et al. 1976; Hegstrand et al. 1979). In membrane particles from ventricular cells of reserpine-pretreated kittens the affinity of (-)-isoprenaline is decreased by a factor of 2.5 by GTP (Morris et al. 1981). Because the present binding data were obtained in membrane particles without exogenous GTP, the affinities of full agonists appear somewhat overestimated (i.e. the spare receptors are underestimated). Furthermore, in intact cells affinities of fl-adrenoceptors for catecholamines may even be 100 times lower than in cell-free membrane particles (Terasaki and Brooker 1978). Binding experiments on intact cells of kitten ventricle are in progress to revaluate the affinities of agonists.

Relationship Between Conformational fl-Adrenoceptor Change and Adenylyl Cyclase Activity. There is evidence supporting the assumption that fl-adrenoceptors coupled to the adenylyl cyclase and receptors mediating inotropic effects of catechol- amines are the same (Kaumann and Birnbaumer 1974a; Kaumann et al. 1980). In the preceding section it was proposed that the degree of dissociation between high inotropic potency and low fl-adrenoceptor occupancy by an agonist is proportional to the conformational receptor change. Because agonists induce close interaction between/% adrenoceptors and adenylyl cyelase, it is conceivable that the enzyme activity mirrors the change in receptor conformation. If so, the enzyme stimulation should be larger the greater the change in/?-adrenoceptor conformation (i.e., the greater the dissociation between high inotropic potency and low receptor occupancy with an agonist). This hypothesis is supported by the correlation shown in Fig. 2. The abscissa intercept of the regression and the transition from partial inotropic agonist (filled circles) to full inotropic agonist (open circles) suggests that only about 1/4 of maximum possible stimulation of adenylyl cyclase is necessary to cause maximal possible inotropism.

Venter (1979) observed in cat heart that the maximum rate of cAMP accumulation with isoprenaline was 7.8 times slower when 90 ~o of the fl-adrenoceptors were occluded than when all receptors were available. The data of Venter that maximum inotropic effects with isoprenaline are still ob- served with reduced Vmax of cAMP accumulation is consistent with the present and previous data (Kaumann and Birnbaumer 1974a, 1976; Kaumann et al. 1978).

On the Meaning of Inotropic Spare fl-Adrenoceptors. A biochemical cascade (that includes adenylyl cyclase) may connect the signal caused by a change in fl-adrenoceptor conformation to the contractile proteins and thus generate greater contractile strength. However, this cascade is not necessarily the same for stimulation of other receptors coupled to adenylyl cyclase. Thus, prostaglandin PGE1 stimulates a ventricular adenylyl cyclase without causing positive inotropic effects (Kaumann an Birnbaumer 1974b; Hayes et al. 1980). Consequently, the fl-adrenoceptor- adenylyl cyclase complex must be arranged strategically so that an initial protein of the biochemical cascade can utilize selectively cyclic AMP only from fl-adrenoceptor activation but not from PGE1 receptor activation.

The cyclic AMP generated by an agonist-induced change in fl-adrenoceptor conformation manifests itself as a signal that is further amplified by the heart cell. This amplification must result in the saturation of some steps in the cascade beyond the adenylyl cyclase, thus producing a ceiling in inotropic effects. Since only 1/4 of maximum adenylyl cyclase activity is required for maximum inotropic effects ofcatechol- amines, the heart may still be able to contract maximally under conditions of desensitization (i.e. during or after overstimulation with catecholamines). This concept is sup- ported by the observation that chronotropic and inotropic concentration-effect curves for a catecholamine are shifted one log unit to the right by desensitization without a decrease in maximum effects but with concomitant reduction to 1/2 of maximal possible stimulation of adenylyl cyclase (Kaumann and Birnbaumer 1976).

Inotropic Stimulation at Only High fl-Adrenoceptor Occupancy. The estimate of affinity of (+_)-hydroxybenzyl- pindolol ( - l o g KD, tool/l, 9.1) for aH-(-)-propranolol- labelled /%adrenoceptors agrees with functional estimates ( - l o g KD, mol/l, 9.0) of (_+)-hydroxybenzylpindolol for/~- adrenoceptors mediating inotropic effects of catecholamines in kitten papillary muscles (Kaumann et al. 1978). Unlike all other agonists used in this report, (+_)-hydroxybenzylpin- dolol has to occupy most available fl-adrenoceptors (96 ~ - as estimated from direct binding) to cause its half maximum inotropic effect. A similar pattern was already detected from the comparison of blockade and stimulation of inotropism, chronotropism and adenylyl cyclase (Kaumann et al. 1978). Stimulation at only high fl-adrenoceptor occupancies has also been observed with pindolol and its analogs (Kaumann 1973 ; Kaumann et al. 1979; Kaumann and Blinks 1980; Bearer et al. 1980).

Acknowledgements. I thank Leslie Scott, Gill Muncke, Gudrun Gfinther, Sibille Holzmann and Sylvia Berger for technical help with portions of this work. I am grateful to Tom H. Morris for help with the adenylyl cyclase assay. The agonists and (-)-bupranolol were kindly furnished by the manufactures. This work was supported by grant SFB 30 Kardiologie D 5 of the Deutsche Forschungsgemeinschaft.

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Received October 14, 1980 / Accepted April 23, 1981