Naunyn-Schmiedeberg's Arch Pharmacol (1982) 319:216- 221 Naunyn-Schmiedeberg's

Archives of Pharmacology �9 Springer-Verlag 1982

An Initial Characterization of Human Heart/~-Adrenoceptors and Their Mediation of the Positive Inotropic Effects of Catecholamines

Alberto J. Kaumann, Horst Lemoine, Tom H. Morris, and Ulrich Schwederski

Lehrstuhl ffir Klinische Physiologic, Physiologisches Institut der Universitfit und Chirurgische Klinik B, Universit~itsstrasse 1, D-4000 Dfisseldorf, Federal Republic of Germany

Summary. The positive inotropic effects of catecholamines were studied on samples of ventricular myocardium taken from patients undergoing open heart surgery. The adenylyl cyclase and binding of 3H-(-)-bupranolol were examined in membrane particles prepared from similarly obtained samples.

The equilibrium dissociation constant (Ke) for (-)-bu- pranolol was estimated in 4ways: blockade of the positive inotropic effects of catecholamines, blockade of the stimu- lation of the adenylyl cyclase by catecholamines, saturation binding of 3H-(-)-bupranolol, inhibition of the binding of 3H-(--)-bupranolol by its unlabeled stereoisomers. The es- timates of KD fall in the range 0.5-1.4nmol/1. The stereo- selectivity ratio (KD (+)-isomer/KD (--)-isomer) is 73. Both values for bupranolol are very similar in cat and man.

The inotropic potency of (-)-noradrenaline is nearly 2 orders of magnitude higher in cat heart tissues than in tissues from human hearts. The difference in inotropic potencies between species is only partially accounted for by the five-fold lower potency of (-)-noradrenaline for the human heart adenylyl cyclase as compared to the cat enzyme.

Key words: fi-Adrenoceptors of human left ventricle - Positive inotropic effects of catecholamines - Adenylyl cyclase stimulation - Blocking potency of (-)-bupranolol - direct binding of 3H-(-)-bupranolol

Introduction

Although a vast literature exists concerning the adrenocep- tors of numerous animal species, relatively little is known about those of human myocardium. Most experiments to date have been performed on the right atrial appendage (Harms 1976; Schfimann et al. 1978; Wagner et al. 1980), a tissue which is routinely excised during open heart surgery. In contrast, human ventricular myocardium is only rarely available. Despite this limitation we have undertaken a study of human ventricular myocardium because in our hands this tissue is better suited to experimental manipulation. As a tissue it is more stable and more reproducible than the right atrial appendage, and it is less prone to conduction blocks. Membrane particles prepared from human ventricular myocardium are cleaner, containing less fat and connective tissue, than those derived from human atria, and thus are better suited for biochemical studies.

Send offprint requests to A. J. Kaumann at the above address

Our goals in this study are to compare 1) fl-adrenoceptors mediating positive inotropic effects with those mediating adenylyl cyclase stimulation, 2)human myocardial fl-ad- renoceptors with those of cat. The antagonist (-)-bnpranolol was employed in the current work because it is pure an- tagonist with a large therapeutic margin (i.e. the ratio of blocking potency to cardiodepressant potency is > 5000, Kaumann et al. 1980; Kaumann and Blinks 1980), and because it has recently become available as a radioIigand (Kaumann et al. 1981a; Morris et al. 1981). (-)-Isoprenaline and the physiological catecholamines (-)-adrenaline and (-)-noradrenaline were used as agonists.

Methods

Cardiac tissues were excised from patients undergoing open heart surgery. Fentanyl was used for the induction of anaesthesia, ethrane, as anaesthetic gas and pancuronium, as muscle relaxant. Left ventricular papillary muscles and trabeculae came from patients with mitral valve lesions. Septal strips of patients with hypertrophic obstructive cardio- myopathy (HOCM) were also used. Although many patients were treated with a variety of drugs (diuretics, digitalis, etc.), none were receiving a/]-blocker at the time of surgery. The tissues were transported to the laboratory in a sealed vial with oxygenated physiological solution containing (mmol/l): 120Na +, 5K +, 2.25Ca +2, 1Mg +2, 98.5C1-, ISO2 2, 29 HCO;-, 1 HPO2 2, 0.04 EDTA, equilibrated with 95 % O2 and 5 % COz. The water was deionized and double distilled in glass.

I so la ted Tissues. Preparation of the tissues was begun within 5 rain after surgical removal. The tissues were dissected at room temperature to yield papillary muscles and trabeculae (width < 1.2ram)or septal strips (cut approximately 10 x 4 mm and < I mm thick, without causing visible damage to

the endocardial surface) which were set up at 37~ in an apparatus with a 50 ml organ bath (Blinks 1965) containing the solution described above supplemented with (mmol/1): 20Na +, 10 fumarate, 5 pyruvate, 5 L-glutamate, and 10 glucose. The muscles were attached to strain-gauge trans- ducers and driven at 5 s intervals with square-wave pulses of 5 ms duration and just-threshold voltage. The tissues were stretched to the length at which the maximum force of contraction was obtained and then recorded on 4-channel Watanabe polygraphs. In order to irreversibly block both tissue uptake of catecholamines and c~-adrenoceptors (Kaumann 1970, 1972; Wagner et al. 1980) the tissues were incubated for 2 h with 5 gmol/1 phenoxybenzamine. Further

0028-1298/82/0319/0216/$01.20

experimental procedures were begun 1 h after the phenoxy- benzamine was washed out.

In order to study the antagonism of the effects of catecholamines by a /Lblocker, the method of successive concentration-effect curves was used. Specimens prepared as described above were reasonably stable for 8 h. Despite a time-dependent decrease in both resting and peak tensions, concentration-effect curves were quite reproducible. In con- trol experiments (not shown) a small decrease in sensitivity of 0.12 _+ 0.03 and 0.29 + 0.07 log-units was noted between the first and second and between the first and third curves, respectively. Where possible, several tissues from the same ventricle were employed with one serving as the control.

Membrane Particles. Tissues were transported and dissected on ice, and membrane particles were prepared as described (Kaumann and Birnbaumer 1974) and stored at -80~ Protein content was determined by the method of Lowry et aL (1951) using bovine serum albumin as standard.

Adenylyl Cyclase Assay. Test tubes contained in the volume indicated (mmol/1): 0.1 [e-32p]-ATP (100- 700 cpm/pmol), 2 MgC12, 1 EGTA, 0.1 ascorbic acid, 0.01 GTP, i [3H]-cyclic AMP (200 cpm/nmol), 20 creatine phosphate, 25 Tris-HCI (pH 7.6), agonist and antagonist, where appropriate; and the enzymes (IU/ml) : 15 creatine phosphokinase, 9.8 myokinase. The reaction was begun by the addition of the membrane suspension and continued at 37 ~ C for the time indicated. The reaction was terminated by the addition of 100 rtl of a solution containing 10 retool/1 cyclic AMP, 40 mmol/1 ATP, and 1 sodium dodecyl sulfate, pH 3.5. The cyclic AMP formed was quantitated by double chromatography as described by Salomon et al. (1974).

Binding Experiments. The procedure was that of Morris et al. (1981). The membrane suspension was incubated at 37~ in a 200 gl volume with the indicated concentrations of [3H]-(-)- bupranolol in an incubation medium containing (mmol/1) : 25 Tris- HC1 (pH 7.6), 2 MgCI2, 1 EGTA, 0.1 ascorbic acid, 0.01 phentolamine, and where indicated, (-)-bupranolol or (+)- bupranolol. Binding observed in the presence of 100 gmol/1 (-)-isoprenaline was considered to be nonspecific. The re- action was stopped by dilution with 1 ml ice-cold incubation medium. The membrane particles were collected by vacuum on Whatman GF/A glass-fiber filters and washed 4 times with 3 ml ice-cold washing solution (10 mmol/1 Tris- HC1 (pH 8.0) and 5 retool/1 MgC12). The filters were dried, then treated with 0.5 ml Protosol (NEN) at room temperature overnight. Eight ml of Econofluor (NEN), acidified with 6.5 ml/1 glacial acetic acid, were then added and the radioactivity was measured by liquid scintillation counting. Binding is expressed in fmol/mg protein after correction for counting efficiency (46 ~) and recovery (65 ~).

Quantitative Analysis. Equilibrium cumulative concentra- tion-effect curves were determined on ventricular tissues by the sequential addition of a catecholamine to the bath in amounts that increased the total concentration in steps of 1/2 log unit. The effects of catecholamines were ex- pressed as percentages of the maximum effect obtained in the same curve. Log EClo , EC3o, ECso, EC70 and EGg0 were determined from the concentration-effect curve of each individual experiment by linear interpolation and the s +_ SEM of the log values was calculated for each effect level.

217

Absolute response in successive curves were compared by expressing all developed tensions (basal and maximum) as percentages of the maximum tension obtained in the frist curve.

The antagonism by (-)-bupranolol of the inotropic effects and adenylyl cyclase stimulating effects of catecholamines were analyzed by the equation of Arunlakshana and Schild (1959), which relates in a double-log form the degree of blockade to the concentration of blocker:

log ( C R - 1 ) = log ( [Bl) - log Ke (1)

where [B] is the concentration of blocker; KB, the blocking constant, which for a simple competitive blocker should equal the equilibrium dissociation constant; CR, the ratio of concentrations of agonist which produce equal effects in the absence and presence of blocker. The slope of a plot according to equation (1) should be one for simple competitive in- hibition. For inotropic experiments K~ was calculated from the equation:

KB = [(-)-bupranolol]

exp i o { log (CR)(_)_bu p . . . . 1ol-- log (CR)spont . . . . . . . }-- 1 (2)

To estimate KD, and Bma x data from saturation binding experiments were fitted to the equation (Morris et al. 1981):

F([3H-BU]) =Bmax {[3H-BU]/(KD, q- [3H-BU]} (3)

where KD, is the equilibrium dissociation constant, B~,a x is maximum binding, and 3H-BU is 3H-(--)-bupranolol.

The data from the binding inhibition experiments were fitted to the equation (Morris et al. 1981):

( I lt F([LI) = Bs 1 ICs0 + [L], (4)

where [L] is the concentration of inhibiting ligand; ICs0, the concentration of L that inhibits 50 ~ of the specifically bound 3H-(-)-bupranolol; Bs, the specific binding without L.

The equilibrium dissociation constant (KD) for the un- labeled ligand was calculated from the equation (Cheng and Prusoff 1973):

[3H_BUI) KD = ICs0/ 1 + ~ . ) (5)

where KD, is the equilibrium dissociation constant for 3H- BU; ICs0 , the value obtained from equation (4).

Binding data were fitted, without transformation, by a computer program (Biomedical Biostatistics Programm, 3 R, UCLA) which performed a non-linear least square analysis (for further details see Morris et al. 1981).

Materials and Drugs. Adenosine 5'-triphosphate. Tris salt (ATP); creatine phosphate; myokinase; adenosine 3'-5'- monophosphoric acid (cyclic AMP); guanosine 5'-triphos- phate, Tris salt (GTP); were from Sigma, St. Louis, MO, USA. [c~-32p]-ATP and [3,8-aH]-cyclic AMP were from Radiochemical Center, Amersham, GB; creatine phospho- kinase from Calbiochem, La Jolla, CA, USA; phentolamine (regitine mesylate) from Ciba-Geigy, Basel, Switzerland; (-)- bupranolol - HC1, (+)-bupranolol �9 HC1 (optical purity greater than 99.999~o - W~ichter et al. 1980), 3H-(-)-bu- pranolol-HC1 (specific activity 17.5Ci/mmol), from Sanol (Monheim, FRG); (-)-isoprenaline bitartrate and (-)-nor- adrenaline bitartrate monohydrate, from Sterling-Winthrop,

I O CONTROL 1 �9 2 nmol[I (-)-Bupranolol �9 40

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218

t t t t t t 0.06 0.2 0.6 2 6 20

[ ( - . ) -ADRENALINE] p rnol/ I

Fig. 1. Comparison between cumulative concentration-effect curves for (-)-adrenaline on left ventricular myoeardium of two different patients. A cuneiform portion of septum partially covered by endocardium (width 0.8mm, length 8mm) was removed from a 48 year-old male with hypertrophic obstructive cardiomyopathy (HOCM). A papillary muscle (width 0.9 ram, length 6 mm) was obtained from a 54 year-old female with a mitral lesion. Both preparations were set up in the same organ bath at 37~ and stimulated 12/rain with 1,2V (HOCM) and 0.6V (mitral lesion)

Rensaelaer, NY, USA; phenoxybenzamine. HC1, from Smith, Kline and French Labs., Philadelphia, PA, USA; (-)-adrenaline bitartrate, from Serva, Heidelberg, F R G ; ethylenediamine tetraacetic acid, disodium salt (EDTA) and ethylene bis(oxyethylenenitrilo)tetraacetic acid (EGTA), from Merck, Darmstadt, FRG.

For experiments on intact tissues 10 or 100 mmol/1 stock solutions of catecholamines were made with deionized, redistilled water containing 0.04 mmol/1 EDTA and adjusted to p H 4 with HC1. For biochemical assays all stocks of unlabeled ligands were prepared in a solution containing 2.5 mmol/1 EGTA and 0.25 mmol/1 ascorbic acid, pH 6.

R e s u l t s

Inotropic Effects of Catecholamines. The method of cumu- lative concentration-effect curves yielded satisfactory results with the three catecholamines tested: (-)-noradrenaline, (-)- adrenaline and (-)-isoprenaline (Figs. 1 and 2). Although most ventricular preparations were from patients with a mitral lesion, successful concentration-effect curves were also obtained on ventricular septal tissue from three patients with hypertrophic obstructive cardiomyopathy (HOCM). Figure 1 illustrates the effect of (-)-adrenaline on two preparations: one from a patient with HOCM and a second from a patient with a mitral lesion. The sensitivity to (-)-adrenaline was very similar; the mean ECs0 ( - log mol/1, 2 + SEM, n = number of tissues) was 6.05 ___ 0.12, n = 3 and 6.06 + 0.06, n = 10 for preparations from patients with HOCM or a mitral lesion, respectively. Mean ECs0 values for @)-isoprenaline and (-)- noradrenaline were 7.22 +_ 0.12, n = 5 and 5.92 +_ 0.15, n = 10, respectively, for preparations from patients with a mitral lesion.

Antagonism by (-)-Bupranolol. We next examined the ability of ( - ) -bupranolol to antagonize the effects of catecholamines. When sufficient tissue from a single ventricle was available we investigated simultaneously the antagonism of the effects of all three agouists. Figure 2 shows a composite of four such experiments. With each agonist ( - ) -bupranolol appeared to be a simple competitive blocker as evidenced by the sur-

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F i g . 2. A n t a g o n i s m by ( - ) - b u p r a n o l o l os the effects o f ( - ) -no radrena l ine , (-)-adrenaline and (-)-isoprenaline at 37 ~ C. Papillary muscles, trabec- ulae and ventricular strips were removed from the hearts of four patients with lesions of the mitral valve. Three tissues from each patient were set up in separate organ baths. Only one catecholamine was given to each tissue. Three successive concentration-effect curves were determined on each tissue. After determining a first curve (O) the catecholamine was washed out, the tissues were incubated for 90 min with 2 nmol/l (-)-bupranolol and a second curve (0) for catecholamine was de- termined. The drugs were washed out once more, the tissues were incubated 60 min with 40 mnol/1 (-)-bupranolol and a third curve (I) for the catecholamine was determined. Histograms represent the basal strength of contraction (black portion) and maximum effect of catechol- amine (open portion) corresponding to the first, second and third curve.The lowest panel represents a composite of Schild-plots for (-)- bupranolol as a function of the different agonists [(�9 (-)-noradren- aline; (Zx) (-)-adrenaline; ([3) (-)-isoprenaline] used in the upper panels. Concentration ratios, measured at the ECs0 levels, were un- corrected for desensitization. Bars through symbols and histograms represent the SEM

219

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0 I I I I I 9 8 7 6 9 8 7 6 5 4 3 0 5 10 15 20 25 (-)-BU (-I-ISOPRENALINE

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Fig. 3. Accumulation of cyclic AMP as a function of time. Membranes were derived from left ventricular myocardium of patients with mitral lesion. 360 gl of membrane suspension (final protein content 1148mg/1) was added to 8401~1 of adenylyl cyclase medium containing no (O) or 50 gmol/1 (O) (-)-isoprenaline. Both components were preincubated at 37~ for 5 min and incubation was continued at this temperature. 50 gl aliquots were removed at the times indicated and added to 100 gI of stopping solution. Specific activity of the ATP was 302 cpm/pmol. The lines through the symbols were fitted by eye. For further details see the Methods

Fig. 4. Antagonism by (-)-bupranolol of the stimulation of the adenylyl cyclase by (-)-isoprenaline. Estimate of the equilibrium blocking constant for (-)-bupranolol. Membranes were derived from left ventricular myocardium of patients with a mitral lesion. Membrane suspension (940 mg/1) was incubated for 10min at 37~ in 50gl of adenylyl cyclase assay medium containing the indicated concentrations of (-)-isoprenaline and (-)-bnpranolol, and 0.1 mmol/1 c~A2p-ATP (158 cpm/pmol). Each point represents a single determination. Concentration-effect curves were with- out (O) and with 2 (O), 20 (A), and 200 (el nmol/1 (-)-bupranolol. The inset shows the resultant Schild-plot. The least square fit to a straight line according to equation (1) yielded a slope of 0.91. The average equilibrium dissociation blocking constant is (- log tool/l) 9.36 + 0.06

mountable nature of its blockade and by the parallel shifts it produced in the concentration-effect curves. The resultant Schild-plots exhibit slopes near one (Fig. 2, bottom), even after correction of the data for desensitization (not shown). the equilibrium blocking constant (Ks) for (-)-bupranolol was estimated to be 1 nmol/1, the blocker being equally effective against each agonist. Individual blocking constants ( - l o g mol/1, 2 + S E M ) were 8.96+0.04, 8.97_+0.11, and 8.92 -+ 0.08 against (-)-noradrenaline, (-)-adrenaline, and (-)-isoprenaline, respectively.

Incubation Conditions for the Adenylyl Cyclase Assay. Figure 3 demonstrates that both basal and (-)-isoprenaline-stimulated adenylyl cyclase activities of human ventricular membrane particles were linear at 37~ for at least 10 min. Because of this apparent stability, all adenylyl cyclase assays were carried out for 10rain.

Potency of Catecholamines as Stimulants of the Adenylyl Cyclase. Saturating concentrations of catecholamines caused approximately a twofold increase in basal adenylyl cyclase activity (Figs. 3 and 4). The ECs0's for adenylyl cyclase stimulation were 0.2 pmol/1 for (-)-isoprenaline and 5 gmol/1 for (-)-noradrenaline. Basal adenylyl cyclase activity was only marginally decreased by (-)-bupranolol (Fig. 4). (-)-Bu- pranolol inhibited in a simple competitive manner the stimu- lation of the adenylyl cyclase produced by (-)-isoprenaline (Fig. 4) as shown by the surmountability of the blockade and by the Schild-plot, which is both linear and of unit slopel Equilibrium blocking constants for (-)-bupranolol were

estimated to be 0.5nmol/1 against (-)-isoprenaline (Fig. 4) and 0.9 nmol/t against (-)-noradrenaline (not shown).

Saturation Binding of 3 H-(-)-Bupranolol. A saturation bind- ing experiment is depicted in Fig. 5. Although nonspecific binding is high (Fig. 5A), the specific binding clearly shows a small, saturable component (Fig. 5A and B). The specific binding conforms satisfactorily to equation (3), suggesting that (-)-bupranolol interacts with a single class of fi-ad- renoceptors. The best fit to the data yields an equilibrium dissociation constant of 0.9 nmol/1 and 38 fmol of specific binding sites per mg membrane protein.

Stereoselective Binding of 3H-(-)-Bupranolol. The stereo- isomers of bupranolol were used to inhibit the binding of 3H-(-)-bupranolol (Fig. 6). The equilibrium dissociation con- stants so derived [see equations (4) and (5)] are 1.4 nmol/l and 102nmol/1 for the (-)-isomer and (+)-isomer, respectively. The affinity estimate obtained here for (-)-bupranolol agrees well with that calculated from saturation binding (cf. Figs. 5 and 6). The stereoselectivity ratio (K, (+)-isomer/KD (--)-iso- mer) is 73.

Discussion

Characterization of Human Myocardial fl-Admnoceptors. The affinity of (-)-bupranolol for human heart fi-adrenoceptors was estimated by four independent methods. (-)-Bupranolol was used to block the positive inotropic effects and the stimulation of the adenylyl cyclase produced by catechol-

220

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Fig.SA and B. Saturation binding of 3H-(-)-bupranolol. Membrane particles were derived from left ventricular myocardium of patients with mitral lesion, Membrane suspension (360 rag/l) was incubated in 200 ~tl of incubation medium for 30 rain at 37 ~ C with the indicated concentrations of 3H-(--)-bupranolol plus 0.01 retool/1 phentolamine. Each point repre- sents the mean -+ SEM of sextuplicate determinations. (A) Total binding (�9 non-specific binding (obtained in the presence of 0.1retool/1 (-)-isoprenaline) (~); specific binding (e). (B) Specific binding on an amplified scale. Bars through symbols are SEM. Symbols without bars have a SEM smaller than the symbol. The best fit of the specific binding to equation (3) yielded: - log KD = 9.03 -+0.12 (KD = 0.93nmol/1) and Bma ~ = 37.9 • 3.3 fmol/mg

o Z

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r-+ 50

:~ ( - ) - B u p r a T 03 z o ~-~

= I I I I �9 . ' ~ . , , 100 I I oj i 11 10 9 8 7 6 5

- L O G . m o l / I

Fig. 6. Inhibition of binding of ~H-(-)-bupranolol by its enantiomers. Membrane particles were derived from left ventricular myocardium of patients with mitral lesion. Membrane suspension (623 rag/l) was in- cubated in 200 gl of incubation medium for 30 rain at 37~ with 1.038 + 0.002nmol/1 ~H-(-)-bupranolol, 0.01 mmol/1 phentolamine and the indicated concentrations of unlabeled ligands. Each point represents the mean +_ SEM of quadruplicate determinations. Symbols without bars have a SEM smaller than the symbol. The data were fitted according to equation (4). The estimated ICs0's were ( - log mol/1, ff + SEM) 8.62 +0.07 and 6.75_+0.06 for (-)-bupranolol and (+)-bupranolol, re-

' spectively. Initial binding was 40.3 _+ 0.6 fmol/mg of which 20.6 • 0.3 fmol/mg (51%) was nonspecific

amines. Tri t iated @)-bupranolol was used for saturat ion binding and binding inhibit ion experiments. These methods yielded values for the equil ibrium dissociation constant in the range 0.5 - 1.4 nmol/1. The close agreement between methods serves to substantiate each individual method; in part icular it indicates that the small specific binding component seen with 3H-(- ) -bupranolol is p robably the /%adrenoceptor. In sup- por t of this assessment is the finding that the binding of tr i t iated ( - ) -bupranolo l is stereoselective (Fig. 6). The enantiomeric ratio is 73, in good agreement with previous estimates (Kaumann and Birnbaumer 1974; Kaumann 1977; Wgchter et al. 1980; Morris et al. 1981).

Since the affinities of antagonists for/%adrenoceptors are unaffected by changes in temperature (for review see Weiland et al. 1979), it appears valid to compare the affinity values o f ( - ) -bupranolo l in cat ventricle at 2 4 - 3 2 . 5 ~ with those obtained here in human ventricle at 37~ The affinity of ( - ) -bupranolo l is essentially the same for myocardial f l-adrenoceptors of man (this paper), cat and other species (see K a uma nn et al. 1979, 1980; Morris et al. 1981). This suggests that for a relatively non-selective antagonist the structures which determine affinity for the fi-adrenoceptor are similar in these species.

The blocking potency of ( - ) -bupranolo l does not vary with the choice of agonist (Fig. 2). It would appear then that either the f l-adrenoceptor popula t ion mediat ing the inotropic effects of catecholamines is homogeneous or that ( - ) -bu- pranolol does not distinguish among the hypothetical f l-adrenoceptor subtypes. Fur ther work with subtype-specific fi-blockers is in progress to test this point.

The Low Inotropic Potency of (-)-Noradrenaline on Human Ventricular Myocardium. In human heart the inotropic potency of ( - ) -noradrenal ine is 1 l~mol/1, which is 60 times lower than in kitten heart tissue (this paper, Kaumann et al. 1981b). It is unlikely that the difference is due to tissue degradation, since the tissues were prompt ly removed from the operat ing theater and set up in a nearby room. Furthermore, it is not likely that the tow potency is the result of par t ia l desensitization. Al though our labora tory animals routinely receive reserpine to deplete the endogenous stores of catecholamines, this procedure does not potentiate the effect of (-)- isoprenaline in the presence of phenoxybenzamine in cat heart (Kaumann 1970).

In membrane preparat ions the adenylyl cyclase of human ventricle is about five-fold less sensitive than the enzyme of kitten ventricle (this paper, Kaumann et al. 1981 b). It appears therefore that the bulk of the difference in tissue sensitivities is the product of steps after the adenylyl cyclase. Human heart tissues are known to use cyclic A M P to mediate the positive inotropic effects of catecholamines (Meinertz et al. 1974), but perhaps less efficiently than cat heart tissues.

Cat heart displays a large reserve capacity (Kaumann et al. 1978; Kaumann 1981) such that the maximum inotropic effects are at tained with a catecholamine at a receptor occupancy of only a few per cent (Kaumann 1978) and is still attainable, albeit at higher concentrations of agonist, even when the adenylyl cyclase is markedly desensitized (Kaumann and Birnbaumer 1976; Kaumann et al. 1978). Human heart on the other hand appears to have only a small reserve capacity (this paper and Kaumann et al. 1981b), since the inotropic potencies of catecholamines are only five-fold greater than their potencies as stimulants of the adenylyl cyclase. Thus in man, desensitization is likely to reduce the maximum inotropic response to agonists with little decrease in sensitivity. This hypothesis is currently under investiga- tion in our laboratory.

Acknowledgements. We are indebted to Prof. W. Bircks and his staff for providing the surgical biopsies, for lending us their laboratory facilities and for their constant encouragement of this work. We wish to acknowledge the technicai assistance provided by Gill Muncke, Gudrun Gfinther and Christine Jfingst, and also the help provided by Sibille Holzmann and Sylvia Berger in the preparation of the manuscript and figures. This work was supported by grant SFB 30 Kardiologie D 5 and J 3 of the Deutsche Forschungsgemeinschaft.

221

References

Arunlakshana O, Schild HO (1959) Some quantitative uses of drug antagonism Br J Pharmacol 14: 48 - 58

Blinks JR (1965) Convenient apparatus for recording contractions of isolated muscle. Appl Physiol 20:755-757

Cheng YC, Prusoff WH (1973) Relationship between the inhibition constant (K~) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22: 3099- 3108

Harms HH (1976) Isoproterenol antagonism of cardioselective beta adrenergic receptor blocking agents: A comparative study of human and guinea-pig cardiac and bronchial beta adrenergic receptors. J Pharmaeol Exp Ther 199: 329- 335

Kaumann AJ (1970) Adrenergic receptors in heart muscle: Relations among factors influencing the sensitivity of the cat papillary muscle to catecholamines. J Pharmacol Exp Ther 173: 383-398

Kaumann AJ (1972) Potentiation of the effect of isoprenaline and noradrenaline by hydrocortisone in cat heart muscle. Naunyn- Schmiedeberg's Arch Pharmacol 273: 134-153

Kaumann AJ (1977) Relaxation of heart muscle by catecholamines and by dibutyryl cyclic adenosine 3',5'-monophosphate. Similarity of /3-adrenoceptors mediating contractile and relaxant effects of cate- cholamines in kitten papillary muscle. Naunyn-Schmiedeberg's Arch Pharmacol 296: 205 - 215

Kaumann AJ (1978) On spare/~-adrenoceptors for inotropic effects of catecholamines in kitten ventricle. Naunyn-Schmiedeberg's Arch Pharmacol 305 : 97 -102

Kaumann AJ (1981) In kitten veutricular myocardium, the inotropic potency of an agonist is determined by both its intrinsic activity for the adenylyl cyclase and its affinity for the ~-adrenoceptors. Naunyn-Schmiedeberg's Arch Pharmacol 317:13 - 18

Kaumann AJ, Birnbaumer L (1974) Characteristics of the adrenergic receptor coupled to myocardial adenylyl cyclase. Stereospecifity for ligands and determination of apparent affinity constants for /~-blockers. J Biol Chem 249: 7874- 7885

Kaumann AJ, Birnbaumer L (1976) Desensitization of kitten atria to chronotropic, inotropic and adenylyl cyclase stimulating effects of (-)-isoprenaline. Naunyn-Schmiedeberg's Arch Pharmacol 311:237-248

Kaumann AJ, Blinks JR (1980) Stimulant and depressant effects of fl-adrenoceptor blocking agents on isolated heart muscle: A non- adrenornimetic positive inotropic effect. Naunyn-Schmiedeberg's Arch Pharmacol 311:205-218

Kaumann AJ, Birnbaumer L, Wittmann R (1978) Heart /3-ad- renoceptors. In: O'Malley BW, Birnbaumer L (eds) Hormone receptors, vol 3. Academic Press, New York, pp 133-177

Kaumann AJ, Morris TH, Birnbaumer L (1979) A comparison of the influence of N-isopropyl and N-tert butyl substituents on the affinity

of ligands for sinoatrial /~-adrenoceptors in rat atria and /Lad- renoceptors coupled to the adenylyl cyclase in kitten ventricle. Naunyn-Schmiedeberg's Arch Pharmacol 307 :1 - 8

Kaumann A J, McInery TK, Gilmour DP, Blinks JR (1980) Comparative assessment of/?-adrenoceptor blocking agents as simple competitive antagonists in isolated heart muscle: similarity of inotropic and chronotropic blocking potencies against isoproterenol. Naunyn- Schmiedeberg's Arch Pharmacol 31l : 219 - 236

Kaumann AJ, Morris TH, Sandrock K (1981a) ~H-(-)-bupranolol (3H-BU), a new/?-adrenoceptor radioligand: binding to cat heart ]~-adrenoceptors. N aunyn-Schmiedeberg's Arch Pharmacol 315:R59

Kaumann AJ, Lemoine H, Morris TH (1981b) Species-dependent relationship between stimulation of heart contractile strength and adenylyl cyclase activity by noradrenaline and adrenaline. J Physiol 315:18-19P

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with the Folin phenol reagent. J Biol Chem 193 : 265 - 275

Meinertz T, Nawrath H, Scholz H, Winter K (1974) Response of isolated human ventricular myocardium to cyclic AMP and its dibutyryl derivative. Klin Wochenschr 52: 701 - 703

Morris TH, Sandrock K, Kaumann AJ (1981) ~H(-)-Bupranolol, a new fl-adrenoceptor radioligand: Characterization of its binding to kitten heart /Ladrenoceptors. Naunyn-Schmiedeberg's Arch Pharmacol 317 : 19 - 25

Salomon Y, Londos C, Rodbell M (1974) A highly sensitive adenylate cyclase assay. Anal Biochem 58:541- 548

Sch/imann HJ, Wagner J, Knorr A, Reidemeister JChr, Sadony V, Schramm G (1978) Demonstration in human atrial preparations of c~-adrenoceptors mediating positive inotropic effects. Naunyn- Schmiedeberg's Arch Pharmacol 302:333 - 336

W~ichter W, M/inch U, Lemoine H, Kaumann AJ (1980) Evidence that (+)-bupranolol interacts directly with myocardial/3-adrenoceptors, Control of optical purity with differential thermal analysis. Naunyn- Schmiedeberg's Arch Pharmacol 313 : 1 - 8

Wagner J, Sch/imann HJ, Knorr A, Rohm N, Reidemeister JChr (1980) Stimulation by adrenaline and dopamine but not by noradrenaline of myocardial e-adrenoceptors mediating positive inotropie effect in human atrial preparations. Naunyn-Schmiedeberg's Arch Pharmacol 312: 99 - 102

Wetland GA, Minneman KP, Molinoff PB (1979) Fundamental differ- ence between the molecular interactions of agonists and antagonists with the/~-adrenergic receptor. Nature 281:114-117

Received July 14, 1981/Accepted February 15, 1982