Naunyn-Schmiedeberg's Arch Pharmacol (1982) 320:119 - 129 Naunyn-Schmiedeberg's

Archives of Pharmacology �9 Springer-Verlag 1982

Cultured Heart Cells as a Model for fl-Adrenoceptors in a Heart Pacemaker

Chronotropic Spare fl-Adrenoceptors and Spare Adenylyl Cyclase for (--)-Isoprenaline but not for (--)-Dichloroisoprenaline in Rat Cardiocytes

Alberto J. Kaumann with the technical assistance of Gill Muncke and Leslie Scott

Lehrstuhl fiir Klinische Physiologic, Physiologisches Institut der Universit~t, UniversitS.tsstrasse 1, D-4000 D/isseldorf, Federal Republic of Germany

Summary. Spontaneously beating rat cardiocytes were cul- tured. The beating frequency and cellular cyclic AMP was measured. Adenylyl cyclase and fl-adrenoceptors (labeled with 3H(-)-propranolol) were assayed in a cardiocyte homogenate. Work with spontaneously beating right atria of reserpine-pretreated rats and with chicken cardiocytes is also presented.

1. (-)-Isoprenaline (ISO) and (-)-dichloroisoprenaline (DCI) increased the beating frequency of rat cardiocytes with ECs o'S of 0.4 nmol/1 and 15 nmol/1, respectively; the intrinsic activity of DCI was 0.8. DCI antagonized surmountably the effects of ISO; an equilibrium dissociation constant K e of 20 nmol/1 was estimated. On rat atria the ECso of DCI was 20 nmol/1, the intrinsic activity 0.7 and the Kp 15 nmol/1. The quantitative agreement of chronotropic DCI data in car- diocytes and atria shows that cardiocytes are a convenient model for the study of fl-adrenoceptors in a heart pacemaker.

2. DCI and low concentrations (< 1 nmol/1) of ISO did only increase cellular cyclic AMP of rat cardiocytes in the presence of 1 mmol/1 3-isobutyl-l-methyl-xanthine (MIX). The accumulation of cellular cyclic AMP induced by ISO (with or without MIX) or DCI was not linear with time. The ECso'S were 62nmol/1 for ISO and 30nmol/1 for DCI; the intrinsic activity of DCI was 0.1. Stimulation of the adenylyl cyclase of a cardiocyte homogenate by ISO was linear with time; the ECs0 for ISO was 79 nmol/1.

3. The positive chronotropic effects and effects on cel- lular cyclic AMP of ISO and DCI were antagonized sur- mountably by (-)-bupranolol. Equilibrium dissociation constants K B of 0.2-0.3nmol/1 were estimated for (-)-bupranolol. These KB'S agree with KB values from myocardial tissues, suggesting that the effects of ISO and DCI on rat cardiocytes are mediated through the same /Lad- renoceptors.

4. 3H-(-)-Propranolol bound in saturable manner and stereo-selectively to rat cardiocyte fl-adrenoceptors (equilib- rium dissociation constant KD = 4 nmol/1). (--)-Bupranolol, ISO and DCI competed with 3H-(-)-propranolol for fl-ad- renoceptors with KD'S of 0.7 nmol/1, 107 nmol/1 and 26 nmol/1, respectively.

5. Concentrations of ISO that cause near maximal chro- notropic effects in rat cardiocytes are considerably below those required for a comparable degree of fl-adrenoceptor saturation (spare receptors) and adenylyl cyclase stimulation (spare cyclase). Contrarily, when DCI occupies fl-ad- renoceptors, chronotropic effects and cyclic AMP effects

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

appear closely associated. An average of 135 fl-adrenoceptor- DCI complexes are needed to cause the same chronotropic effect as one fl-adrenoceptor-ISO complex.

6. The beating frequency of chicken cardiocytes was only marginally and inconstantly increased by ISO. However, ISO increased cellular cyclic AMP in chicken cardiocytes; the effect was antagonized by 10nmol/1 (-)-bupranolol and partially surmounted by ISO. The data are consistent with the existence of fl-adrenoceptors in chicken cardiocytes. Both /?-adrenoceptors and adenylyl cyclase appear poorly coupled to ionic channels responsible for pacemaker activity in chicken cardiocytes.

Key words: fl-Adrenoceptor occupancy - Chronotropism - Adenylyl cyclase - Spare fl-adrenoceptors - fl-Adre- noceptor - Pacemaker coupling

Introduction Some myocardial effects of catecholamines are postulated to occur through a fl-adrenoceptor-mediated increase in cellular cyclic AMP. Support for this comes from the mimicry of catecholamine stimuli with intracellular application of cyclic AMP or with the extracellular administration of N6-2-O - dibutyryl-3',5'-cyclic AMP (for critical reviews of the evid- ence see Tsien 1977; Scholz 1980).

Quantitative evidence indicates that inotropic fl-ad- renoceptors are the same as adenylyl cyclase coupled fl-adre- noceptors. Thus, affinities of antagonists and partial agonists for /~-adrenoceptors mediating inotropic effects of (-)-iso- prenaline are nearly the same as their affinities for fl-ad- renoceptors causing stimulation of membrane adenylyl cyc- lase by (-)-isoprenaline (Kaumann and Birnbaumer 1974; Kaumann et al. 1978, 1980). Contrarily, concentration effect curves for inotropic effects in heart tissues do not overlap with curves for stimulation of membrane adenylyl cyclase. Inotropic curves are situated at 30- to 1000-times lower concentrations of catecholamines than curves for adenylyl cyclase stimulation (Kaumann and Birnbaumer 1974, 1976), even if cyclase activity is amplified with guanylnucleotides (Kaumann et al. 1978). The absence of a measurable rise in cyclic AMP during inotropic effects at low catecholamine concentrations raises the question about the intermediate role of cyclic AMP. It appears that inotropism is activated by catecholamines at low fl-adrenoceptor occupancy and mar- ginal increases of cyclic AMP (Kaumann 1978, 1981 ; Venter 1979).

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The overall relationship of/~-adrenoceptor occupancy to adenylyl-cyclase stimulation and chronotropic effects of cat- echolamines is not well understood. It is difficult to obtain quantitative Information on changes in cellular cyclic AMP and/%adrenoceptor occupancy solely in sinoatrial pacemaker cells. Biochemical measurements in atrial tissues or mem- branes would be spurious due to the abundance of cells without physiological pacemaker function. In order to seek information on the relation of chronotropic effects of ligands to /%adrenoceptor occupancy and cyclic AMP we have chosen the model of spontaneously beating cultured rat cardiocytes shown to possess chronotropic/~-adrenoceptors (Kaumann and Wittmann 1975; Kaumann et al. 1977). To minimize possible c~-adrenoceptor-mediated effects in cul- tured heart cells (Wallukat and Wollenberger 1979) (-)-iso- prenaline was chosen as a /~-agonist. (-)-Dichloro- isoprenaline (DCI) was used as a partial agonist. The affinity of these ligands was estimated from competition with aH-(-)- propranolol for receptor binding. Increases of cellular and membrane-bound cyclic AMP and beating rate were quan- tified as/~-adrenoceptor mediated effects of agonists.

In order to compare p-adrenoceptors mediating increases in the rate of beating of cultured cardiocytes with sinoatrial /%adrenoceptors we determined the intrinsic activity and affinity of DCI both in cardiocytes and spontaneously beating atria of adult rats. The affinity of DCI was also estimated from its antagonism to the effects of (-)-iso- prenaline.

Several authors described positive chronotropic effects of catecholamines on cultured heart cells of chicken and mam- mals (Wollenberger 1964; Boder and Johnson 1972; Goshima 1974; Kaumann and Wittmann 1975; Kaumann et al. 1977). However, workers from two laboratories could not observe any chronotropic effects of catecholamines in cul- tured chicken heart cells (Kaufmann et al. 1969; Sperelakis 1972). Kaufmann et al. (1969) attributed the lack of chrono- tropic effects of adrenaline to enzymatic digestion of the /%adrenoceptors by the trypsin used to disaggregate chicken heart cells. We have reexamined the hypothesis of /~-ad- renoceptor digestion by measuring the effects of (-)-iso- prenaline on the rate of beating and on cellular cyclic AMP of chicken cardiocytes.

Methods

Rat Cell Cultures. The method was that of Kaumann and Wittmann (1975) with some modifications. Cultures were made from 1 - 3 days old WistarII rats. The rats were sacrificed by cervical dislocation. After cleaning the thorax with 70 ~o alcohol the procedure was further carried out under aseptic conditions. The thorax was opened, heart exposed and ventricle removed. The ventricles were immersed at room temperature in a physiological solution containing penicillin 250,000IU/1, streptomycin 0.1 g/1 and phenol red 10mg/1. The composition of the solution was (mmol/1) : Na + 140, K + 5, M g 2+ l, C1- 94, SO 2- 1, HCO;- 29, HPO 2- 1, fumarate 10, pyruvate 5,1-glutamate 5, glucose 10. Water was deionized and twice distilled, no calcium was added to the solution. Ventricles of up to 30 rats were collected in above solution at room temperature and washed with three changes of solution to remove all visible blood. The solution was removed after each wash with a Pasteur pipette and discarded. The ven- tricles were then finely minced with scissors (cut into 6 - 8

pieces per ventricle) and again washed in 3 changes of solution. Heart cells were disaggregated in physiological solution containing 0.125 ~ trypsin. The minced ventricles and 20 ml of trypsin solution were placed in a bottle together with a Teflon covered magnet. The bottle was placed in an incubator on a magnetic stirrer which was set to cause gentle agitation at 37 ~ C. The trypsin solution was removed and discarded after 5 min and the minced ventricles were re- suspended in 30ml trypsin solution and returned to the incubator. Heart cells were than allowed to disaggregate for 3 0 - 60 min. The cell suspension was removed with a Pasteur pipette and placed in a centrifuge tube. 30 ml trypsin solution was added to the remaining minced ventricles which were returned for 10min to the incubator and further suspended cells removed. Trypsin was added for additional 10min periods 7 - 8 times to the residual ventricles until it w a s possible to draw up remaining tissue in a Pasteur pipette. The united cell suspensions were centrifuged at 200 x g for 10 min. The supernatant was discarded. The cell pellet was re- suspended in Waymouth 's (1959) medium (modified with glutamine 350 mg/1 without NaHCO3) (Flow Laboratories, Bonn, F R G ; Waymouth 's MB 752/1 medium) supplemented with 10 ~ horse serum and containing penicillin 250,000 IU/1, streptomycin 0.1g/l, phenol red 10mg/1 and NaHCO3 2.24 g/1. After resuspending, the cells were centrifuged at 200 • g for 10 min to remove remaining traces of trypsin. The

supernatant was discarded and the cells were resuspended in Waymouth 's medium. The cell suspension was kept at 37 ~ C in the incubator until all cells were disaggregated. The cell suspensions were pooled and filtered through a G glass filter (Schott, Mainz, FRG). The filtrate was centrifuged at 200 • g for 10 min. The supernatant was decanted and the cell pellet was resuspended in Waymouth 's medium to give a density of about 500,000 cells per IN. Cells were planted in plastic cell culture flasks with 25 cm 2 or 75 cm 2 growth area. Flasks were kept in an incubator at 37 ~ C with a mixture of air and CO2 to maintain a pH of 7.2. Lids were loosely applied to the flasks to allow the circulation of gases. Medium was changed after 24 h and thereafter every second day.

Chicken Cell Culture. Chicken eggs were incubated at 37 ~ C and 95 ~ humidity in an incubator. Cultures were made from 1 1 - 1 3 days old embryonic hearts. After taking out the embryo, heart cells were prepared as described by Kaumann and Wittmann (1975). As chicken heart cells disaggregate with greater ease than rat heart cells, only 5 successive 7 min trypsinization steps were performed.

Monitoring of the Beating Rate of Heart Cells. Contractions of rat and chicken cardiocytes were recorded on a 2 channel Watanabe recorder using a Leitz (Wetzlar, FRG) Diavert inverted microscope equipped with a dynamic cytophoto- meter (Kaufmann et al. 1969), a photo camera and television camera (Kaumann et al. 1977). Recordings were from cell clusters or incipient monolayers. During the course of an experiment all recordings were obtained from the same cell beating synchroneously within a cluster of cells.

Preparation of Cell Membranes. Beating cells were harvested at confluency (usually 5 days of culture). Membranes from cultured cells were prepared by decanting medium and rinsing the cell surface of a culture flask with ice cold 0.9 ~ NaC1 solution. The cells were then gently removed from the flask surface with a rubber-covered rod and 2 ml saline. All further

121

steps were on ice. The cells were washed to remove medium by centrifuging twice at 300 • g for 10min at 0 ~ C. After decanting the supernatant, the cell pellet was resuspended in 25 mmol/1 Tris HC1 with 5 mmol/1 ethylenediamine tetra- acetic acid disodium salt (EDTA) at pH 8 and gently homoge- nized (10 strokes) in a glass Potter homogenizer. The cell suspension was centrifuged at 5,000 • for 20min. After resuspension of the pellet in the buffer followed by a second homogenization, the suspension was filtered through jap- anese silk. The membrane filtrate was centrifuged at 5,000 • g for 30rain and after resuspension in buffer again filtered through japanese silk and rapidly frozen in liquid nitrogen. The membranes were stored at - 8 0 ~ C.

Cyclic AMP Test. Preparation of samples (modification of the method of Brown et al. 1971), injection of drugs, freezing of cultures, protein determination (Lowry et al. 1951), assay of cellular cyclic AMP (Gilman 1970) and its recovery, were as in Kaumann et al. (1977). Times of incubation of ligands are given under Results.

Adenylyl Cyclase Assay. Enzyme activity was determined at 32.5 ~ C by measuring the conversion of c~-[32p]ATP to cyclic [32p]adenosine-3',5'-monophosphate ([32p]cyclic AMP) and isolating the [32p]cyclic AMP by the method of Salomon et al. (1974). Membrane particles were incubated for 10 min in 50 gl medium containing 0.1 mmol/1 e-[32p]ATP (specific activity 130-239cpm/pmol), 2mmol/1 MgCI2, 1retool/1 EGTA, 0.1 mmol/1 ascorbic acid, 10 gmol/1 GTP, 1 retool/1 [3H]cyclic AMP (10,000 cpm), 20 mmol/1 creatine phosphate, 15 IU/ml creatine kinase, 9.8 IU/ml myokinase, 25mmol/ Tris HC1, pH 7.5, and (-)-isoprenaline as indicated under Results. The reaction was stopped by addition of 100gl of a solution containing 10 mmol/1 cyclic AMP, 40 mmol/1 ATP and 1% sodium dodecyl sulphate.

Binding Assay. The method of Kaumann (1978) was used. Incubations were made at 32.5~ C for 10min in 100gl with 3H-(-)-propranolol [specific activity 20.91 Ci/mmole, New England Nuclear, Dreieich, FRG (NEN)], 72mmol/1 Tris HC1 pH7.6, 3.6mmol/1 MgSO4, 0.72retool/1 EDTA, 26 to 194 gg membrane protein per test tube, with or without cold fl-adrenoceptor ligand. After diluting the reaction with 1 ml of cold washing solution (50mmol/1 Tris HC1 pH 8.0 and 20 mmol/1 MgCIE) the medium was filtered under vacuum through a 2.4cm GF/B Whatman filter paper placed on a precooled glass frit. The membranes stuck to the filter were washed 4 times with 2 ml icecold washing solution. Less than 9 s were required from the addition of 1 ml washing solution until finishing the last 2 ml wash. The filter papers were dried, heated for 30 min at 60 ~ C in 0.5 ml protosol (NEN), cooled, neutralized with 50~tl glacial acetic acid, soaked in 10ml econofluor (NEN) and counted for radioactivity with 49 % efficiency. Membrane protein was determined with the procedure of Lowry et al. (1951). Specifically bound 3H-(-)-propranolol was calculated from

Mol bound =

cpm plus the background cpm; recovery of membrane protein on the filter paper was 65 %. Non-specific binding was not significantly different when 0.2mmol/1 (-)-isoprenaline, 1 gmol/1 (-)-propranolol or 1 gmol/1 (-)-bupranolol were used as competing agents.

Isolated Right Atria. Tissues of reserpine-pretreated (/0 mg/kg ip, 24 h) Wistar II rats (160 - 180 g, either sex) were used at 32.5 ~ C. The rats were sacrificed by a blow on the head. The hearts were rapidly removed and washed free from blood with modified Krebs solution; the solution was de- scribed under rat cell cultures but contained also 2.25 mmol/1 CaC12 and 40 gmol/1 EDTA, and was equilibrated with 95 % 0 2 and 5 % CO2. The hearts were dissected in oxygenated solution at room temperature. Right atria were set up in pairs in a 50 ml bath and apparatus described by Blinks (1965). The atria were suspended at a resting tension just sufficient for measurable development of tension to avoid increases in spontaneous rate by stretching the sinoatrial node (Blinks 1956). The rate of atrial beating was recorded via strain gauge transducers on a 4 channel Watanabe recorder.

Chronotropic Concentration-Effect Curves. Cumulative curves for (-)-isoprenaline and (-)-dichloroisoprenaline were determined by the sequential addition of the drugs to the cell culture or isolated organ bath in amounts that increased the total concentration in steps of 0.2 to 0.5 log units. Enough time was alowed for each effect to reach equilibrium. Positive chronotropic effects of the drugs were expressed in beats/min in both cells and atria. SEM of the mean effects were calculated.

Equilibrium Dissociation Constants for fl-Adrenoceptor- Ligand Complexes. Functional Estimates. Constants for the antagonist (-)-bupranolol and the partial agonist (-)-di- chloroisoprenaline were estimated with the use of equieffec- tive concentration ratios (CR) of agonist [(-)-isoprenaline] in the presence and absence of blocking ligand. For the antag- onist (B) the constant KB was calulated from

K B = [BI/(CR - 1) (2)

after verifying - whenever possible - that the double log plot of (CR - t) against concentrations [B] of B did show a slope near unity (Arunlakshana and Schild 1959). Because K B is independent of the receptor occupancy YA by the agonist, CR of equation (2) can be taken at any equieffective level regardless of the YA associated with that effect.

For the concentration [P] of the partial agonist, the fractional receptor occupancy YP was estimated from t h e slope of the relation ofequieffective concentrations of agonist (A2) in the absence of [P] as a function of concentrations (A3) in the presence of [P] (Colquhoun 1973; Rang 1973; Kaumann and Marano 1973) yp = 1 -- slope. (3)

In some cells yp was estimated from (Stephenson 1956)

yp = 1 Az--A1 A3 '

total cpm bound - non-specific cpm bound

efficiency (cpm/dpm) �9 specific activity (Ci/mol) �9 2.22 x 1012 (dpm/Ci) �9 recovery

(4)

(1)

where non-specific cpm were the sum of the membrane bound counts not removed by 0.2 mmol/1 (-)-isoprenaline, 1 gmol/1 (-)-propranolol or 1 l~mol/1 (-)-bupranolol, plus filter bound

where A1 is the concentration of full agonist causing the same effect as [P]. Equations (3) and (4) are only valid if all used concentrations of agonist (Ai) are smaller than the equilibrium

122

constant KA (i.e. A1 < KA, Stephenson 1956). The equilib- rium constant Kp was calculated from

Kp = [e]/yp - [P]. (5)

Direct Binding Estimates. The equilibrium constant K o for fl-adrenoceptor-bound 3H-(-)-propranolol was estimated from l/slope of a Scatchard (1949) plot. The KD for a cold ligand was calculated from (Cheng and Prusoff 1973)

K D = I C s o 1 + Ks, J '

where ICs0 is the concentration of cold ligand causing half maximal removal of specifically bound 3H-(-)-propranolol. [B*] is the concentration of aH-(-)-propranolol used and Ks, is the equilibrium dissociation constant for 3H-(-)-pro- pranolol estimated from direct saturation binding with Scatchard (1949) analysis. The IC5o was estimated from concentration curves of cold ligand competing with ra- dioligand for membrane fl-adrenoceptors. To this end at least 8 concentrations of cold ligand were used per curve.

Statistics. Different equilibrium constants were compared by a group t-test. Statistical significance (P < 0.05) of difference of data from the same cell or tissue were estimated by paired t-test. The slope of equation (3) was calculated as the slope from a linear regression of A2 = intercept + slope' A3 fitted with weights ([A3] + ECs0 of A3) -4 (Marano and Kaumann 1976). The standard error of the mean (SEM) of - l o g KD(pKD) was calculated from equation (8) of W/ichter et al. (1980).

Materials. 2.5 ~ Trypsin in modified Hanks balanced salt solution (Flow Lab, 7-024); horse serum, screened for mycoplasma and bacteriophage (Flow Lab, 4-012D). Sera and trypsin solutions were stored at - 2 0 ~ C, the remaining media at 4 ~ C. Culture flasks were Falcon flasks. Materials for the cyclic AMP (cAMP) and adenylyl cyclase were those of Kaumann et al. (1977). ~-[3zP]ATP was purchased from Amersham Buchler, Braunschweig, FRG.

Drugs. (-)-Isoprenaline bitartrate dihydrate, (-)-dichlo- roisoprenaline (DCI) HC1 (Sterling Winthrop, Reffsselaer, NY, USA); (-)-bupranolol HC1 (K1255) (Sand, Monheim, FRG); (-)-propranolol HC1, (+)-propranolol HC1 (ICI); 3-isobutyl-l-methylxanthin (MIX) (Ega Chemie, Steinheim, FRG); Reserpine (Ciba-Geigy, Basel, Switzerland). Stock solutions for (-)-is oprenaline (10 - 100 mmol/1) were made up in 0.04 retool/1 EDTA and enough HC1 for a pH of 4.0; further dilutions were made in 0.04mmol/1 EDTA. The stock 0.1 tool/1 of MIX was prepared in redistilled water and its pH adjusted to 8.0 with N H 3. The administration of 0.04 mmol/1 EDTA solution or N H 3 containing solution to cells did not modify the rate of beating.

Results

Chronotropic Effects of Agonists in Rat Cardiocytes

Positive Chronotropic Effects of (--)-Isoprenaline and (-)-Di- chloroisoprenaline. Figure 1 (lower panel) shows that (-)-di- chloroisoprenaline [(--)-DCI] increased the beating rate of cardiocytes with an intrinsic activity of 0.8 with respect to the maximum effect of (-)-isoprenaline, suggesting that ( - ) -DCI is a partial agonist. The maximum effect of ( - ) -DCI was significantly smaller (P < 0.001) than the maximum effect of

�9 60 r . / e - ~ . . . . ; 1200 I (-} -Isoprenaline ~ r " / ~ - ~ f 1

I E ~J

t 'E �9 6 0 0

o ~ 201

/ (-)" Dichl~176 a, '~ ' . -~""J l~ ' I

/ * / / . . / 1 ~~ o

- 11 10 9 8 7 6 5 4 5o ~

-LOG. CONCENTRATION mol/I

t l ,0

t l .0 - -

o6g

0

Fig. 1. The effects of (-)-isoprenaline and (-)-dichloroisoprenaline on rat cardiocytes. Comparison of concentration-effect curves for chrono- tropism (e) and increases of cellular cyclic AMP (O) with curves (broken lines) for fractional fl-adrenoceptor occupancy (y). For chronotropism only one cumulative concentration-effect curve for an agonist was determined per culture (symbols represent ~ _+ SEM of n = 8 for each agonist). The basal frequency was not significantly different for the two groups. Cyclic AMP determinations were performed in cultures prein- cubated 15min with 1 mmol/1 3-isobutyl-l-methylxanthine. Each con- centration of (-)-isoprenaline and (-)-dichloroisoprenaline was in- cubated I rain and 5 min, respectively. Each cyclic AMP value represents the 2 + SEM of 4 - 6 cultures. Fractional receptor occupancies were calculated with the dissociation equilibrium constants for the agonists estimated from direct binding data of Fig. 9 and Table 1

(-)-isoprenaline. Comparison of ECsos (Table 1) shows that (-)-isoprenaline was 40 times more potent than (-)-DCI.

The onset of the chronotropic effect of ( - ) -DCI was fast. Half times of onset were 36s with 20nmol/1 and 16s with 1 gmol/1 (-)-DCI. Equilibrium effects were observed in 2.5 rain with 20 nmol/1 and in 1 rain with 1 btmol/1 (-)-DCI. The effects of ( - ) -DCI were stable during a 30 min obser- vation period.

Competitive Antagonism of the Positive Chronotropic Effects 'of (-)-D CI by (-) -Bupranolol. The fl-blocker (-)-bupranolol (1-40nmol /1) antagonized competitively the effects of ( - ) -DCI (experiments not shown). ( - ) -DCI surmounted the blockade caused by (-)-bupranolol. Concentration-effect curves of ( - ) -DCI were parallel in the presence and absence of (-)-bupranolol. The slope of a Schild-plot from 5 experiments was 0.95 (Fig. 2). An average pKB(--log tool/1 KB) of 9.50 (Table 2) was estimated for (-)-bupranolol with equation (2).

The Affinity of (-)-DCI for fl-Adrenoceptors Mediating Chronotropic Effects of (-)-Isoprenaline. As expected from a partial agonist, ( - ) -DCI antagonized the effects of (-)-iso- prenaline. Figure 3 represents an experiment from which the dissociation equilibrium constant Kp for ( - ) -DCI was esti- mated with equations (4) and (5). In 3 experiments of this kind pKp(-logKpmol/1) were 7.80, 7.63 and 7.65. In two ad- ditional experiments, pKp values of 7.91 and 7.51 for ( - ) -DCI were estimated from equations (3) and (5). An average pKp of 7.70 (Tab led was estimated for ( - ) -DCI from the 5 experiments.

Positive Chrono tropic Effects and Affinity of (--)-D CI for Rat Sinoatrial fl-Adrenoeeptors

As on cardiocytes, ( - ) -DCI was also a partial agonist on spontaneously beating right atria of adult rats. The onset of

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Table 1. Relationships between potencies (pD2 = - log mol/1 ECs0) of agonists as stimulants of adenylyl cyclase and chronotropism, and equilibrium dissociation constants (pKD = log mol/1KD; pKv = -- log tool/1 Kv) for agonist-fi-adrenoceptor complexes in rat cardiocytes

Agonist pK D pK e Chronotropic pD 2 - pK D Chronotropic pD2 Cellular Adenylyl cyclic cyclase

• SEM n[ AMP pD 2

pD2 2 • SEM n 2 + SEM n

• 2 + SEMpKD

(-)-Isoprenaline 9.43 _+ 0.21 (6)" 7.21" 7.10 b 6.96 • 0.07 (12) ~ - - 2.46 • 0.22 (-)-Dichloro- 7.82 • 0.18 (5)" 7.50 ~ - 7.59 • 0.05 (16) ~ 7.70 • 0.07 (5) a 0.23 + 0.19

isoprenaline

From the experiments of Fig. 1 b From the experiments of Fig. 8 ~ From the experiments of Fig. 9. KD values were calculated from equation (6) a Kv was calculated from equation (5), using 30 - 600 nmol/1 (-)-DCI, from experiments as in Fig. 4 (this paper) and Fig. 12 of Kaumann et al. (1978)

Table 2. Estimates of equilibrium dissociation constants ( - log mol/1) for the @)-bupranolol-fl-adrenoceptor complex in rat cardiocytes

Species Competing ligand Chronotropic effects Cellular cyclic AMP Direct binding

2 + S E M n ~ • n E • n

Rat (-)-isoprenaline 9.50 _+ 0.08" 7 9.47 r 2 -- (-)-dichloroisoprenaline 9.54 -]- 0.05 b 5 9.57 __+ 0.08 r 3 -- 3H-(--)-propranolol -- -- 9.16 • 0.11 a

From Kanmann and Wittmann (1975) b Experiments not shown ~ Data from Fig. 6 d Data from Fig. 9

3

I ._o

13E = 2 o

E 8 C

8 1

o,

0 10

~A~.~- (-) - DCI (-)-DCI cAMP onot ropism

S[.OPE = 1.11 v SLOPE - 0 95

~ s o p r e : i ne cAMP

/2,~- enaline ism # I SLOPE=0.96 I I

9 8 7 -LOG. [(-)-Bupranolol] mol/I

Fig. 2. Schild plots of (-)-bnpranolol as antagonist of the effects of (-)-DCI and (--)-isoprenaline on cellular cyclic AMP and beating rate of rat cardiocytes. Circles are data from chronotropic effects of (-)-DCI. The broken line represents a plot with chronotropic data of (-)-iso- prenaline taken from Kaumann and Wittmann (1975). The data of the effects of (-)-isoprenaline (0, II) and (-)-DCI (A, A, ~ ) on cellular cyclic AMP are from Fig. 6

the pos i t ive c h r o n o t r o p i c effect was s lower in a t r i a t h a n in cardiocytes . H a l f t imes o f onse t were 11.1 + 3.3 m i n (n = 3) for 10nmol/1, 2 . 7 + 0 . 2 m i n (n = 3) for 100nmol /1 a n d 2.0 _+0.2ra in (n = 4) for 1000nmol /1 ( - ) - D C I . F i g u r e 4 ( top

pane l ) shows t h a t the in t r ins ic ac t iv i ty for the pos i t ive c h r o n o t r o p i c effect o f ( - ) - D C I was 0.7 wi th respect to ( - ) - i sop rena l ine . As expec ted f r o m a pa r t i a l agonis t , ( - ) - D C I a n t a g o n i z e d s u r m o u n t a b l y the c h r o n o t r o p i c effects of ( - ) - i s o p r e n a l i n e (Fig. 4, b o t t o m panel) . A n average Kp of 17nmol /1 was e s t ima ted for the ( - ) - D C I - f l - a d r e n o c e p t o r

"7 .r E

>- (.9 Z UJ

ee"

U -

130-

120-

110-

100

. ~

~ o . . . . . i !

A1A2 A3 Ou~

- Log. [(-)-Isoprenaline] mol/I

Fig. 3. Antagonism by (-)-DCI of the chronotropic effect of (-)-iso- prenaline in a 5-day old culture of rat cardiocytes. Effects of (-)-iso- prenaline in the absence (�9 and presence (@) of 0.1 ~tmol/1 (-)-DCI. After administration (-)-isoprenaline, the medium was changed 4 times and @)-DCI administered 12min before challenging again the cardiocytes with (-)-isoprenaline. A1, A2 and A 3 represent (-)-isoprenaline con- centrations. The fractional fl-adrenoceptor occupancy Y~)-DCI and dissociation equilibrium constant K()_Dc~ were estimated from

A 2 - A 1 equations (4) and (5), respectively. Y(-)-DcI = 1 -

A3 Y(->DCI = 0.818 ; K(_)~DC I = 22 nmol/1

complex f r o m e q u a t i o n s (3) a n d (5) (see a lso inset a n d legend to Fig. 4). The pKp (7.81 _+ 0.11) a n d the - l o g ECsomol /1 (7.70 _+ 0.17) were n o t s igni f icant ly different . The recep to r o c c u p a n c y curve o f ( - ) - D C I , ca lcu la ted f r o m its Kp, m a t c h e d closely the expe r imen t a l po in t s for the pos i t ive c h r o n o t r o p i c effect (Fig. 4, t op panel , l ine t h r o u g h c losed circles).

124

z 350

~ 310

~ . 27G

23C

(-)-ISO & l P ~ ]1.0

10 9 8 7 6 5 -LOG. mol/I CONCENTRATION

z 34O

a0~

b

220

i ~ mo --

l ~ 2 ~ . . (, 2011 400 [(-)-ISOPRE NALINE ] (_) _DC I nlTIOl/I

-LOG. [E-)-ISOPRENALINE] mol/I

Fig. 4. Relationship between stimulation and blockade caused by (-)- DCI in right atria of reserpine-pretreated (10 mg/kg ip, 24 h) rats. Top panel: A concentration-effect curve for (-)-DCI (@) was determined up to 1 ~tmol/1; to evaluate the intrinsic activity of (-)-DCI, a concentration- effect curve for (-)-isoprenaline (O) was obtained in the presence of 1 gmol/1 (-)-DCI. Symbols represent s _+ SEM of 4 atria; (A) is the initial rate before the administration of (-)-DCI was begun, Y( )-oci represents the fractional/~-adrenoceptor occupancy calculated from equation (5), using an average K r for (-)-DCI estimated from the experiment displayed in the bottom panel. Bottom panel: Two concentration-effect curves for (-)-isoprenaline were determined. After the first curve (�9 the atria were incubated 45 min with 1 p.mol/1 (-)-DCI before the second curve (@) for (-)-isoprenaline was determined in the presence of (-)-DCI. Symbols represent 2 + SEM of 4 atria, (O) and (A) the basal beating rates before the first curve for (-)-isoprenaline and before the administration of @)-DCI. The inset shows individual plots of equieffective concentrations of (-)-isoprenaline from each of the 4 atria. Fractional/~-adrenoceptor occupancies Y(-)-oci calculated from equation (3) were: 0.974 for (A), 0.983 for (0), 0.986 for (@) and 0.992 for (zX)

A n a t tempt to study positive chronot ropic effects of ( - ) - D C I in the presence of the phosphodiesterase inhibi tor 3 - i sobuty l - l -methylcanth ine (MIX) failed. 1 mmol/1 M I X caused arrhythmia. 0.1 mmol / l M I X enhanced cellular beat- ing rate bu t ( - ) - D C I caused ar rhythmic beat ing in the presence of MIX.

Increases of Cellular Cyclic AMP by (-)-Isoprenaline and (-)-DCI in Rat Cardiocytes. The basal con ten t of cyclic A M P varied greatly between different cell batches (Table 3) ; effects of agonists on several batches were therefore expressed as percentage of basal cyclic A M P content . However, variabil i ty was small between different cultures f rom the same batch of hearts (Table 3). Wi th in the same batch, cellular cyclic A M P did no t change significantly between the 5th and the 7th day of culture, regardless of whether the cultures were exposed to med ium con ta in ing or no t con ta in ing horse serum 1/2h before assay. Low concent ra t ions (0.1 - 0.4 nmol/1) of (-)- iso- prenal ine that enhanced the beat ing rate of cardiocytes (Fig. 1) did no t modify their con ten t of cyclic A M P (Table 3). Higher concent ra t ions of ( - ) - i soprenal ine (1 - 10 nmol/1) caused t ransi tory increases in cellular cyclic A M P (Fig. 5). 1 gmol/1 ( - ) - D C I was ineffective in raising cellular cyclic A M P (Table 3), a l though it increased the rate of bea t ing of cardiocytes.

Because ( - ) - D C I and low concent ra t ions of (-)- iso- prenal ine were ineffective in increasing cellular cyclic A M P , the phosphodiesterase inhib i tor 3 - i sobuty l - l -methylxan th ine (MIX) was used. Phosphodiesterase may have hydrolyzed cyclic A M P formed with ( - ) - D C I or low ( - ) - i soprenal ine concentra t ions . 1 mmol/1 M I X increased per se cellular cyclic A M P unt i l the 5th min of incuba t ion (Table 3); bu t appeared no t to increase it further between the 5th m in and 25th min (experiments no t shown).

Table 3 shows that in the presence of 1 mmol/1 M I X both 1 gmol/1 ( - ) - D C I and 0.1 nmol/1 ( - ) - i soprenal ine caused significant increases in cellular cyclic A M P . However, even in the presence of MIX, ( - ) - i soprenal ine ( 0 . 1 - 1 0 nmol/1) only caused t ransi tory increases in cellular cyclic A M P (Fig. 5).

Table 3. The effect of (-)-DCI and (-)-isoprenaline on the cellular content of cyclic AMP in rat cardiocytes

Condition Incubation Cell cyclic AMP time (rain) pmol. mg- 1 protein

s +_ SEM (n)

Significance of difference between control and agonist-treated

A a Control b 1 6.8 + 1.0 (3) 0.1 nmol/l (-)-isoprenaline 1 7.1 + 1.5 (4)

B ControP 1 8.6 + 1.1 (4) 0.4nmol/1 (-)-isoprenaline 1 7.1 + 1.4 (4)

C lmmol/1MIX ~ 15min Control b 1 67.1 _+ 5.1 (6) 0.1 nmol/1 (--)-isoprenaline 1 99.0 + 5.5 (4)

D lmmol/1 MIX 5 min Control b 5 65.0_+ 5.3 (7) 0.1 nmol/1 (-)-isoprenaline 5 82.0 + 12.5 (4)

E Control b 5 31.6+_ 1.8 (26) 1 gmol/1 (-)-DCI 5 28.9 + 1.7 (14) lmmol/1MIX 15min Control b 5 75.0+ 8.6 (11)

1 p, mol/1 (-)-DCI 5 197.0 _+ 25.3 (7)

P < 0.005

P < 0.001

a Cultures from the same batch of hearts indicated by capital letters b 0.4gmol/1 EDTA c MIX is 3-isobutyl-l-methylxanthine

125

"o 4 0 -

'7

-6 3 0 =

< 20- .o_

o 15- -5 ---- 10- 8 .E

5-

o-

2 0 0 0 -

1500 - 5

3/~r~ ~- ~r 1~ mol/l

1o0o-

3

5 0 0 -

3 3

10-8 mol/ I ~ 10-9

0 -

15rnin PREINCUBATION with l mmol/f MIX

�9 / 10 -5 mol/I

/ 4 3

10-8 mol/I

INCUBATION TIME (min) with (-)-ISOPRENALINE

Fig. 5. Kinetics of the increase of cellular cyclic AMP caused by (-)- isoprenaline in rat cardiocytes. The influence of 3-isobutyl-1- methylxanthine (MIX) is shown on the right panel. Each symbol represents 2 + SEM of the indicated number of cultures. The basal content of cyclic AMP was measured in triplicates or quadruplicates at min 0, 1, 2, 3 and 5 and found not to change with time (experiments not shown)

Cellular accumulat ion of cyclic A M P induced by (-)-iso- prenaline or ( - ) -DCI was never linear with time, regardless of concentration (Fig. 5). For this reason it was difficult to determine concentration-effect curves for the agonists. A first approximat ion was at tempted by taking the cyclic A M P values of 1 min and 5min incubation times for (-)- isoprenaline and ( - ) -DCI , respectively, in MIX-treated cells. Concentration-effect curves of this kind are shown in Fig. 1. Under these conditions, (-)- isoprenaline (10gmol/1) ( - ) -DCI (10gmol/1) caused 23-fold and 2.6-fold increases in cellular cyclic AMP, respectively. The intrinsic activity of ( - ) -DCI with respect to (-)- isoprenaline is 0.1. The ECs0 for effects on cellular cyclic A M P of (-)- isoprenaline was 2 log units higher than the ECs0 for its chronotropic effects (Table 1, Fig. 1). Contrari ly to (-)-isoprenaline, the ECs0's for chronotropic effects and cyclic A M P effects for ( - ) -DCI were similar (Table 1, Fig. 1).

It is assumed that the effects of (-)- isoprenaline and ( - ) -DCI on cellular cyclic A M P are mediated through the same /~-adrenoceptor. The results of Fig. 6 support this assumption. The antagonist ( - ) -bupranolo l qaused sur- mountable and concentrat ion-dependent blocl~ade of the effects of (-)- isoprenaline and ( - ) -DCI . Concentrat ion-rat ios represented in the Schild plot of Fig. 2 show that:

1. the effects of (-)- isoprenal ine and ( - ) -DCI are antago- nized to similar extent by ( - ) -bupranolo l ;

2. the antagonism appears simple competitive because the slope of the Schild plot is close to one;

3. the antagonism by ( - ) -bupranolo l of positive chrono- tropic effects and cyclic A M P effects for each (-)-iso- prenaline and (--)-DCI is similar, suggesting that both classes of effect are mediated through the same @)-bupranolol /%adrenoceptor.

Because kinetics for the accumulation of cellular cyclic A M P with (-)- isoprenal ine were not linear, a cell membrane homogenate was prepared and the adenylyl cyclase activity assayed. Both basal and (-)- isoprenaline-st imulated cyclic A M P product ion were linear up to 20 min incubation periods

15(

<

"~ 10(

~o~ ~o ..<.

3 4

6 6 4

1 3 1 1 1 1 1 1 1 1 1 0 9 8 7 6 9 8 7 6 5 [(-)-ISOPRENALINE]~-)-Dichloroisoprenaline]

-LOG. mol/I Fig. 6. Surmountable antagonism by (-)-bupranolol of the increase of cellular cyclic AMP caused by (-)-isoprenaline and (-)-DCI in rat cardiocytes. Each symbol represents 2 + SEM of the indicated number of cultures. Cultures were incubated I min voith the indicated concentrations of (-)-isoprenaline. Cultures with (-)-DCI received 1 mmol/1 3-isobutyl- 1-methylxanthine 15 rain before a 5 min incubation with the indicated concentrations of @)-DCI. (-)-Bupranolol was preincubated 20 min before the administration of isoprenaline or (-)-DCI. Concentration- ratios of (-)-isoprenaline and (-)-DCI were taken at the 100 % and 115 % effect levels, respectively, and are represented in the Schild plot of Fig. 2. (-)-Bupranolol nmol/l (O) 1; (E) 20; (Z5) 2; (A) 14; (~-) 60

10 pmol/I '~ 4( ( - ) - I S O P R E ?

o a0

o.

~ 2s ~176176

1C

I I I I I 5 10 15 20 25

T I M E (min)

Fig. 7. Acumulation of cyclic AMP as a function of time in a rat cardiocyte homogenate. 360 gl of membrane suspension (final protein content 64.9 mg/l) was added to 840 gl of adenylyl cyclase medium containing no (0) or 10 pmol/l (�9 (-)-isoprenaline. Both components were preincubated at 32.5 ~ C for 5 min and incubation was continued at this temperature. 50 gl aliquots were removed at the times indicated and the reaction was terminated with 100 gl of stopping solution. Specific activity of the ~-3zp-ATP (0.1 mol/l) was 239cpm/pmol. For further details see Methods

(Fig. 7) in these membranes. Under these conditions, a concentration-effect curve for (-)- isoprenaline exhibited an EC5o of ( - log mol/1) 7.1 (Fig. 8).

Direct Binding Estimates of the Affinity of (-)-Isoprenaline, (-)-DCI and Other Ligands for Rat Cardiocyte ~-Adrenoceptors

3H-(-) -Propranolol was used to label /3-adrenoceptors. 6 nmol/1 3H-(--)-propranolol bound rapidly (q/2 = 28 _+ 2 s,

126

.'F. 25 E #- 1.0 ~=

20 o

0.5

�9

6 . 1 0 - I I I 0 u"

10 9 8 7 6 5 4 -LOG. [(-)-ISOPRENALINE] mol/I

Fig. 8. Stimulation by @)-isoprenaline of the adenylyl cyclase of a rat cardiocyte homogenate at 32.5 ~ C. Membrane suspension (final protein concentration 110 mg/l) was added to adenylyl cyclase medium in a final volume of 50 ~tl. Specific activity of the eAzP-ATP (0.t mmol/1) was 130 cpm/pmol. Each point represents a single determination. The line represents the fractional occupancy curve for (-)-isoprenaline calculated from the experiments of Fig. 9 and data of Table 1

n = 16) to membranes (experiment not shown). 70 % of the bound 3H-(- ) -propranolol was rapidly displaced by 0.1 mmol/1 (-)- isoprenal ine (hi2 = 65_+ 17s, n = 4), or 2 gmol/1 ( - ) -propranolo l (tl/2 = 84 + 17 s, n = 4). Other bind- ing characteristics of 3H-(- ) -propranolol were examined as shown in Fig. 9. The (-)- isoprenal ine-removable component of bound 3H-(- ) -propranolol depends in a saturable manner on concentration. The Scatchard plot (inset to top panel of Fig. 9) yielded an estimate for Ko of 4.2 nmol/1 ( - l o g 8 . 3 8 ) and of 228 fmol/mg protein binding sites removable with 0.1 mmol/1 (-)- isoprenaline. Figure 9 (bot tom panel) illus- trates that binding to these sites was stereoselective, as expected from fi-adrenoceptors. The stereoisomers of pro- pranolol were used to inhibit the binding of 3H-(-)- propranolol . The pKD's ( - - log tool/1KD, 2 _+ SEM) were 8.76 _+0.08 ( n = l l ) for ( - ) -propranolo l and 6.25_+0.02 (n = 4) for (+)-propranolol . This yields a stereoselectivity rat io of 324, which is similar to values obtained from previous binding (Kaumann 1978) and from functional studies (Kaumann et al. 1980). ( - ) -Bupranolo l was twice as potent as ( - ) -propranolo l for inhibit ion of 3H-(- ) -propranolol binding (Fig. 9, bot tom); the pKD was 9.16 _+ 0.11 (see also Table 2). The pKD's of ( - ) -DCI and (-)- isoprenal ine were 7.59 _+ 0.05 and 6.96 _+ 0.07, respectively (Table 1).

Increase of Cellular Cyclic AM P by (-)-Isoprenaline in Chicken Cardiocytes. Antagonism by (--)-Bupranolol

In 20 cultures of chicken cardiocytes (age 1 - 8 days) we were unable to detect consistent positive chronotropic effects of (-)-isoprenaline. Small effects of (-)- isoprenal ine appeared to occur in only 5 cultures (results not shown); however, it was not possible to detect a concentration-dependence, al though the effect was antagonized by ( - ) -bupranolol . This inconstant result left open the question of existence off i-adrenoceptors in chicken cardiocytes. We therefore investigated a possible effect of (-)- isoprenal ine through fi-adrenoceptors coupled to the adenylyl cyclase. To this end, we determined cellular cyclic A M P after 1 min incubations with (-)-isoprenaline. Cellular cyclic A M P was increased by (-)- isoprenal ine in a con- centrat ion-dependent manner (Fig. 10). 10nmol/1 ( - ) -bu-

200 F /

,5o I_ ) / ......- . . . . . . .

-6 "7 ,,f g E IO0 e - / / ~ ~ -]40 E ~'1'-

$ l i f o " 6 ' loo 200 0 ~ ~ BOUND (fmol . rag-l)

0 P" ' ' I I 0 5 10 15 20 [3H-(-)-PROPRANOLOL] nmol/I

o ~ ___o~.- = [ ~ " ~ - ' ~ , ~ "~X,~,.,-Pro,,,~

e sol- \ / 4- I (-)'Prop ran~176 ~ . ~ " ~

~ 1001 I I ~ o 10 9 8 7 6 5 4

-Log. Concentration mol/I

Fig.9. Saturation binding of 3H-(-)-propranolol (top panel) and in- hibition by ligands (bottom panel) in a homogenate of rat cardiocytes. Top panel: membrane suspension (320 mg/1) was incubated in 100 pl of incubation medium for 10min at 32.5~ with the indicated concen- trations of 3H-(-)-propranolol. Each point represents the mean + SEM of quadruplicate. Symbols without vertical bars have SEM smaller than size of symbol. (O) Specific binding; (�9 non-specific binding (obtained in the presence of 0.2mmol/1 (-)-isoprenaline). The inset shows the resultant Scatchard plot, which yields an estimate for K D of 4.2nmol/1 and for Bma x of 228fmol/mg. Bottom panel: membrane suspension (340 gg/ml) was incubated in 100 gl incubation medium for I0min at 32.5~ with 4.1-5.8nmol/1 3H-(-)-propranolol and the indicated concentrations of unlabeled ligands. Each symbol represents the mean of n determinations; the SEM was always smaller than the size of the symbol, n was t 1 for (-)-propranolol, 4 for (+)-propranolol, 4 for (-)-bu- pranolol, 16 for (-)-dichloroisoprenaline [(-)-DCI] and 12 for (-)-iso- prenaline [(-)-ISO]

pranolol (25 min preincubation) shifted the concentration- effect curve to the right (Fig. 10).

Discussion

Rat Cardiocytes as a Model for fl-Adrenoceptor-Mediated Regulation of Pacemaker Activity

Beating cultured heart cells represent an appropria te model for the study of the mode of action of fi-adrenoceptor agonists. Due to the virtual absence of intercellular barriers, diffusion paths between the medium and the biophase (Furchgot t 1955) can be neglected. Half times of onset for the chronotropic onset of ( - ) -DCI were approximately 10 times faster in cardiocytes than in beating right atr ia (this paper) or kitten left atr ia (Kaumann and Blinks 1980). The culture procedure did not modify the characteristic effects of ( - ) -DCI . Thus, the intrinsic activity, the affinity and the close association between the receptor occupancy curve and concentration-effect curve for the chronotropic effects of ( - ) -DCI were remarkably similar in cardiocytes and right atria.

Our data show that the stimulating effects of (--)-DCI on cellular beating rate and cyclic A M P were antagonized by the fi-blocker ( - ) -bupranolo l to the same extent as the effects of (-)- isoprenal ine (see also Kaumann and Wit tmann 1975),

127

200, ~ 4 r 4 64

nmol/I ~f~ I (-)-Bupranolol o 1/"~

- LOG. [(-}-IsoprenalineJ mol/I Fig. 10. Increase of cellular cyclic AMP by (-)-isoprenaline and antagonism by (-)-bupranolol in chicken cardiocytes. Cells were prein- cubated 25 min at 35 ~ C without and with (-)-bupranolol. Each concen- tration of (-)-isoprenaline was incubated for I min. Symbols represent 2 +_ SEM of determinations; number of cultures is indicated near symbol

.8 loo "6 �9 =o

Table 4. Chronotropic efficacies (eA) of (-)-isoprenaline on rat cardiocytes

A Beats/rain nmol/1 eAb

[(-)-Isoprenaline] a [(__)_DCI]a

t0 0.036 1.6 199 20 0.23 5.5 92 30 0.40 50.0 188 40 2.0 79.0 43

a Equieffective concentrations of the agonists were estimated from the average chronotropic concentration-effect curves of Fig. 1

KA [P] b eA . . . . ep; the K A value (110 nmol/1) for (-)-iso-

[A] [el + K e prenaline was taken from Table 1; the Kp value (23 nmol/l) for (-)- DCI was the average of the Kp and K D values given under Table 1 ; ep was taken as one

,suggesting that all three ligands act through the same fl-adrenoceptors. Furthermore, the affinity estimates for (-)-bupranolol were similar for rat cardiocyte fl-adrenoceptors and sinoatrial fl-adrenoeeptors of rat (Kaumann et al. 1977) and other species (Kaumann et al. 1980).

Because ( - ) -DCI is not a substrate of catechol-O-methyl transferase (COMT) but the effects of (-)-isoprenaline and ( - ) -DCI are antagonized similarly by (-)-bupranolol, COMT appears not to limit significantly the concentration of (-)-isoprenaline near cardiocyte fi-adrenoceptors. COMT does interfere with the blocking pattern of fl-blockers in other systems (for instance kitten papillary muscle, Kaumann et al. 1978).

Chronotropic Spare fl-Adrenoceptors for (-)-Isoprenaline in Rat Cardiocytes

In order to cause nearly maximum chronotropic effects, (-)-isoprenaline appears to occupy only 15~o of the fl-adrenoceptors (Fig. 1, Table1). The data are therefore consistent with the existence of a spare receptor phenomenon (Stephenson 1956) for the chronotropic effects of (-)-iso- prenaline. On the other hand, as expected from a partial agonist (Stephenson 1956), ( - )-DCI caused nearly maximum effects with high fl-adrenoceptor occupancy (Fig. 1, Table 1). The data permit a rough estimation of the chronotropic afficacy eA for (--)-isoprenaline (with the efficacy of ( - ) -DCI taken as one). The values ofeA range from 43 to 199 (Table 4), with an average of 135. Thus, on average, approximately 135 (-)-DCI-fl-adrenoeeptor complexes are required in order to cause the same positive chronotropic effect as on (-)-iso- prenaline-fl-adrenoceptor complex.

Chronotropic spare fl-adrenoceptors of rat cardiocytes resemble inotropic spare fl-adrenoceptors of kitten ventricle. Equilibrium dissociation constants K o of 60 nmol/1 (receptors labeled with 3H-(-)-propranolol, Kaumann 1981) or 80nmol/1 (receptors labeled with 3H-(-)-bupranolol in the presence of GTP (Morris et al. 1981) were estimated for the (-)-isoprenaline-fl-adrenoceptor complex of kitten ventricle. These estimates agree with the Ko of 110 nmol/1 for (-)-iso- prenaline for rat fi-adrenoceptors (Table 1). 6 -20nmol /1 (-)-isoprenaline cause nearly maximum inotropic effects in

kitten papillary muscle (Kaumann 1978). At these concen- trations (-)-isoprenaline appears to occupy only 7 - 2 0 ~o of kitten ventricular fl-adrenoceptors which agrees with the 15 ~o receptor fraction occupied by (-)-isoprenaline for nearly maximum chronotropic effects in rat cardiocytes.

GTP lowers the affinity of (-)-isoprenaline for fl-ad- renoceptors (Maguire et al. 1976; Lefkowitz et al. 1976; Hegstrand et al. 1979). Because no exogeneous GTP was added to our cardiocyte membranes during binding assays, the affinity estimate for (-)-isoprenaline may be higher in membrane particles than on intact cardiocytes presumed to contain optimal concentrations of GTP. The affinity of (-)-isoprenaline was estimated for fl-adrenoceptors with a binding assay in intact rat cardiocytes by Porzig and Reuter (personal communication). Surprisingly, however, they found a slightly higher affinity of (-)-isoprenaline for fl-adrenoceptors of intact cardiocytes (KD 32 nmol/1) than we found on cardiocyte membrane particles (K D 110nmol/l). Because of the rough agreement of affinity estimates for (-)-isoprenaline-fl-adrenoceptor complexes of intact car- diocytes and cardiocytes membranes, our estimates ofchrono- tropic spare receptors and efficacy for (-)-isoprenaline appear to be realistic.

A Spare Adenylyl Cyclase for the Chronotropic Effects of (-)-Isoprenaline in Rat Cardiocytes ?

Our data appear not to support straightforwardly the second messenger hypothesis which demands that cyclic A MP nec- essarily causes the positive chronotropic effects of catechol- amines. Low concentrations (0A-0.4nmol/1) of (-)-iso- prenaline and I pmol/1 ( - ) -DCI cause positive chronotropic effects but did not modify the cellular content of cyclic AMP. Only in the presence of the phosphodiesterase inhibitor MIX these concentrations of the drugs and also lower concen- trations of (--)-DCI caused significant increases in cellular cyclic AMP. Chronotropic and cellular cyclic AMP kinetics differed. While 1 nmol/1 (-)-isoprenaline causes a stable increase in cellular beating from the 1st to the 5thmin of administration, a significant increase of cellular cyclic AMP was only observed at the 1st min but not at the 2nd, 3rd or 5thmin (Kaumann et al. 1977; Figure5 this paper). For unknown reasons, the kinetics of cellular accumulation of

128

cyclic A M P with (-)- isoprenal ine or ( - ) -DCI are not linear, even in the presence of the phosphodiesterase inhibitor MIX. The onset of chronotropic effects of ( - ) - D C I (without MIX) is faster than the onset of non-linear accumulat ion of cellular cyclic A M P (not shown). All this evidence suggests that if cyclic A M P is involved in causing cells to beat faster with (-)- isoprenal ine or ( - ) -DCI , our method does not detect the cellular compar tment where cyclic A M P is theoretically increased to cause membrane permeabil i ty changes leading to increased beating rate.

Al though the evidence discussed does not provide a quantitative f ramework for the involvement of cyclic A M P in the chronotropic effects of ( - )- isoprenal ine or ( - ) -DCI , some other evidence permits arguments in favour of the second messenger hypothesis. The positive chronotropic effects and the cyclic A M P increase observed with both (-)- isoprenaline or ( - ) -DCI are antagonized quantitatively to similar extent by ( - ) -bupranolol , suggesting that the same class of f l-adrenoceptor mediates both effects. The affinity estimates for ( - ) -bupranolo l on cardiocytes agree with previous estimates on heart tissues (Kaumann et al. 1980; Wfichter et al. 1980) and membrane particles (Kaumann and Birnbaumer 1974; Morr is et al. 1981), strongly suggesting that the cardiocytes contain/~-adrenoceptors not different from those of heart tissues. As in tissues (Kaumann and Birnbaumer 1974, 1976), cardiocytes also exhibited a lower sensitivity to the st imulat ion of adenylyl cyclase than for chronotropic st imulation by (-)- isoprenaline. The discrepancy in sensitivity does not appear to be an artifact due to non-linear kinetics of cellular cyclic A M P accumulat ion because the same low sensitivity for ( -)- isoprenal ine was also observed on the cardiocyte adenylyl cyclase with linear kinetics of (-)- isoprenal ine stimulation.

The difference between chronotropic sensitivity and cyclase sensitivity to (-)- isoprenal ine of cardiocytes is similar to the difference between inotropie sensitivity and cyclase sensitivity in kitten ventricle (Kaumann 1981). For both systems approximately a/4 of maximum enzyme st imulation by (-)- isoprenal ine suffices to cause nearly maximum chro- notropic or inotropic effects.

F rom simple theoretical analysis Str ickland and Loeb (1981) demand an obligatory separat ion of agonist binding and biological response. They assumed that the format ion of cyclic A M P is propor t ional to the number of receptors occupied by the agonist. In their model, the degree of separat ion between low ECso (i.e. high biological potency) and high K A (i.e. low receptor binding affinity) of an agonist is directly related to the affinity of cyclic A M P for its effector (protein kinase) and to the total amount of agonist receptors. Str ickland and Loeb quote a variety of hormone receptor systems (corticotropin, lutropin, vasopressin, etc.) that me- diate biological responses at agonist concentrations below those required for a comparable degree of receptor saturat ion or adenylyl cyclase stimulation. Rat cardiocytes (chrono- tropic response - this paper) and cat papil lary muscle (inotropic response - Kaumann 1981) contain B-adrenocep- tor systems which also appear to behave according to the hypothesis of Str ickland and Loeb.

Chicken Cardiocytes Possess fi-Adrenoceptors

A positive chronotropic effect of catecholamines on chicken cardiocytes was described by Wollenberger (1964); inconstant and marginal effects were seen by us (this paper)

and no effect could be detected by Kaufmann et al. (1969) or Sperelakis (1972). The difference among laboratories may be due to differences in culture conditions. Kaufmann et al.- (1969) at t r ibuted the lack of positive chronotropic effects to tryptic digestion of fi-adrenoceptors. Al though our results tend to agree with those of Kaufmann et al. and Sperelakis, we also demonstra ted that (-)- isoprenal ine enhances cellular cyclic AMP. The effect is antagonized by the fi-blocker ( - ) -bupranolo l to an extent expected from the affinity of ( - ) -bupranolo l for mammal ian fi-adrenoceptors (Kaumann et al. 1980; W~ichter et al. 1980; Morris et al. 1981). The evidence is consistent with the existence off l-adrenoceptors in our chicken cardiocytes, but inconsistent with the suggestion that the trypsin-treatment digested the fi-adrenoceptors (Kaufmann et al. 1969). For unknown reasons, activation of both fl-adrenoceptors and adenylyl cyclase by (-)- iso- prenaline appears to be coupled poorly to the ionic channels of chicken cardiocytes.

Acknowledgements. The author is grateful to Tom Morris for help with the adenylyl cyclase assay, to Sonja Wtrner and Rainer Wittmann for help with preliminary experiments, to Sibille Holzmann for typing the manuscript, to Sylvia Berger for photography, to Dr. H. Porzig (University of Bern, Switzerland) for reading the manuscript and to the Deutsche Forschungsgemeinschaft (SFB 30, Cardiologie, D 5) for sup- porting this research.

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Received February 11, 1982/Accepted May 13, 1982