Naunyn-Schmiedeberg's Naunyn-Schmiedeberg's Arch. Pharmacol. 302, 2 3 9 - 254 (1978)
Archivesof
Pharmacology 9 by Springer-Verlag 1978
Repdnted Issue without Advertisements
~
On the Presence and Distribution of -Adrenoceptors in the Heart of Various Mammalian Species* J. Wagner and O.-E. Brodde Pharmakologisches Institut der Universit~it Essen, Hufelandstrasse 55, D-4300 Essen 1, Federal Republic of Germany
Summary. The presence and distribution of myocardial
e-adrenoceptors in different parts of the heart of various mammalian species was investigated. For this reason experiments were performed in isolated cardiac preparations of rats, guinea pigs and cats. In order to obtain more information about the nature of the cardiac e-adrenoceptors additional experiments were undertaken at different temperatures. These studies were aimed to show whether or not a conversion of/3- to e-adrenoceptors or vice versa takes place. Moreover, we analyzed the influence of hypothyroidism on the sensitivity of e- and/3-adrenoceptors of preparations from rats fed with propylthiouracil. Finally, we tried to find out whether stimulation of these e-adrenoceptors leads to the formation of the cyclic nucleotides cAMP and cGMP. The following results were obtained: 1. In right ventricular strips of rats, guinea pigs and cats phenylephrine stimulated e-adrenoceptors. After blockade of/3-adrenoceptors the respective pDz-values of phenylephrine were 5.32, 5.99 and 5.33. The doseresponse curves obtained in the presence of eadrenolytic drugs were shifted to the right without depression of the maximum. In the rat, in addition, the pA2-values for the e-adrenolytic drugs yohimbine (5.79) and phentolamine (7.85) were determined. They were at least 0.5 log units higher than those found in the rat for other e-adrenoceptors (Van Rossum, 1965) thus supporting the view that the population of cardiac eadrenoceptors is different from that of other organs. In left ventricular strips of guinea pigs the pD2-value for the c~-mimetic effect of phenylephrine was of the same order of magnitude as that obtained in the right ventricle.
* This work was supported by the Deutsche Forschungsgemeinschaft Send offprint requests to J. Wagner at the above address
2. In the papillary muscle of the right ventricle of guinea pigs and cats phenylephrine stimulated ~adrenoceptors. 3. In the left atrium of the rat, phenylephrine stimulated myocardial e-adrenoceptors (pD2: 5.58). Also in this preparation the pA2-values for yohimbine (6.36) and phentolamine (8.21) were different from those found for other e-adrenoceptors in the rat (Van Rossum, 1965). Likewise in strips from the left atrium of the cat myocardial e-adrenoceptors are present. 4. In spontaneously beating right atria of the rat a clear-cut positive chronotropic effect mediated by stimulation of e-adrenoceptors could not be demonstrated although in 6 out of 15 preparations a small positive chronotropic effect became evident. No positive chronotropic effect at all was obtained by stimulation of c~-adrenoceptors in the cat right atrium. 5. In ventricular strips as well as in left atria from hypothyroid rats the pDz-value for the e-effect of phenylephrine was increased, in the atrium more than in the ventricle, while the pAz-values for yohimbine and phentolamine were not significantly different from the controls. Under these conditions a distinct positive chronotropic effect mediated by stimulation of a-receptors was found in the spontaneously beating rat atrium. 6. After stimulation of c~-adrenoceptors in atria and ventricular strips of normal and propylthiouraciltreated rats, as well as in strips of guinea-pig ventricles or cat atria no elevation of cAMP and c G M P was observed. 7. The concept of a single adrenoceptor convertible from e- to fl-type or vice versa was not supported by our experiments. These were carried out at different temperatures on left atria and on ventricular strips of rat hearts using the irreversible e-adrenoceptor blocking agent phenoxybenzaminel 8. The present experiments provide evidence for the existence of e-adrenoceptors in the myocardium of
0028-1298/78/0302/0239/$ 3.20
240 various mammalian species. Their stimulation produces positive inotropic effects without increases in heart rate, Key words: Myocardial c~-adrenoceptors - Rat Guinea pig - Cat - Hypothyroidism - Single adrenoceptor type.
Introduction
The first account on the existence of ~-adrenoceptors in rat myocardial strips was given by Wen:el and Su in 1966. Using the e-adrenolytic drug phentolamine, the authors showed that the positive inotropic effect of 10 -5 g/ml phenylephrine was completely abolished. Although these experiments favoured the assumption that at least the ventricle of the rat is endowed with eadrenoceptors, no exact dose-response relationship was established in the presence of fl-blocking agents. Next Govier (1967, 1968) found that a-adrenoceptors are present in the guinea-pig atrium driven at 1 Hz. Benfey and Varma (1967) confirmed for the rabbit left atrium (l Hz) the existence of c~-adrenoceptors mediating a positive inotropic effect of phenylephrine as well as that of small doses of adrenaline. However, the results concerning the atria of guinea pigs and rabbits could not be confirmed by our laboratory. In experiments on the guinea-pig left atrium driven at 1 or 2 Hz we could only demonstrate/3adrenoceptors as mediators of the positive inotropic effect of phenylephrine and not c~-adrenoceptors (Wagner and Reinhardt, 1974). Moreover, our results revealed that the positive ehronotropie effect of phenylephrine on the spontaneously beating right atria of guinea pigs and rabbits was caused by stimulation of/3adrenoceptors. On the other hand, there is no doubt as to the existence of e-adrenoceptors in the ventricle. Investigations from our laboratory on the papillary muscle of the right ventricle of rabbits (Schfimann et al., 1974) as well as in the isolated perfused rabbit heart (Wagner et al., 1974) or the cat ventricle in situ (Rodrigues-Pereira and Wagner, 1975; Wagner et al., 1975) clearly showed the presence of myocardial eadrenoceptors which are responsible at least in part for the positive inotropic effect of phenylephrine as well as for that of the endogenous amine adrenaline (Schfimann et al., 1974; Wagner et al., 1975). Furthermore, it was shown that only the positive inotropic effect of phenylephrine mediated by /3adrenoceptors was potentiated after inhibition of phosphodiesterase with theophylline or papaverine
Naunyn-Schmiedeberg'sArch. Pharmacol. 302 (1978) (Schiimann et al., 1974). The determination of Y5'cAMP clearly showed that only the stimulation of fladrenoceptors was accompanied by an increase in the level of cAMP (McNeill and Verma, 1973) whereas that of ~-adrenoceptors was not (Schfimann et al., 1975; Endoh et al., 1976a). Furthermore, the c~-sympathomimetic effect on the rabbit papillary muscle showed a clear-cut dependence on the frequency of stimulation as was demonstrated by means of methoxamine and naphazoline: the positive inotropic effect was most evident at a low rate of stimulation, i.e. at 0.5 Hz (Endoh and Schfimann, 1975). According to Nakashima et al. (1973) the sensitivity of c~-adrenoceptors is increased in atria from hypothyroid rats so that also a positive chronotropic effect elicited by stimulation of ~-adrenoceptors was visible. Moreover, in the frog heart there is strong indication for the existence of only one type of adrenoceptors which has the properties of an e-adrenoceptor at low temperature and those of a fi-adrenoceptor at higher temperatures (Kunos et al., 1973; Kunos and Nickerson, 1976). In the frog heart, the ~- and fi-agonist adrenaline is the sympathetic transmitter (Holt: et al., 1951 ; Grobecker and Holt:, 1966). The conversion of a single adrenoceptor from the fl- to the c~-type should produce a positive inotropic effect in the hibernating frog. Also for the rat atrium the possibility of the interconversion of a single adrenoceptor is discussed (Kunos, 1977). Furthermore, in left atria of hypothyroid rats the elevation of cAMP induced by phenylephrine was blocked by phenoxybenzamine thus supporting the interconversion of a single receptor (Kunos et al., 1976). The aim of the present experiments was to demonstrate the presence of myocardial a-adrenoceptors in different species of mammals, and to determine their distribution in different parts of the heart and the relationship of their stimulation to the level of cAMP and cGMP. Further series of experiments were conducted to obtain more information on the nature of the cardiac ~-adrenoceptor. By means of the irreversible eadrenolytic drug phenoxybenzamine we tried to find out if there is any reason to assume a single type of adrenoceptor in the rat heart which might alter its properties.
Materials and Methods A. Preparation of Cardiac Tissue and Experimental Procedure 1. ElectricallyDrivenStrips of the Ventricle.a) MaleSprague-Dawley rats weighing 200--240g were sacrificed by cervical dislocation. After opening of the thorax, the hearts were quickly removedand transferred into Krebs-Henseleit solution of room temperature.
J. Wagner and O.-E. Brodde: Cardiac c~-Adrenoceptors in Mammalian Species
241
Table 1. Basal tension, maximal development of tension in response to phenylephrine and - l o g ECs0-values of phenylephrine after repeated determination of the dose-response curve (DRC) on isolated electrica!ly driven (I Hz) rat atria at 30~ - log ECso
First D R C Second DRC Third DRC
6.33 • 0.18 6.06 • 0.14 "~ 5.68 • 0.09*
Developed tension (mg) control
maximal
374.5 • 41.7 275.8 _+ 32.6 ns 154.8 • 23.9*
540.3 • 50.2 565.7 • 62.0 n~ 506.7 • 50.5 ns
Given are means • S.E.M. of 6 preparations * P < 0.005 compared with the corresponding value of the first DRC
From the right ventricular wall two strips.were prepared and fixed on needle-shaped platinum electrodes, The preparations were suspended in Krebs-Henseleit solution of the following composition (raM): NaCI 119; CaC1 z 2.5; KC1 4.8; MgSO 4 1.2; KH~PO 4 1.2; NaHCO 3 24.9; glucose 10.0; ascorbic acid 0.054 bubbled with 95 ~ O 2 and 5 CO z at 30~ unless stated otherwise. The preparations were stimulated electrically by rectangular pulses of 1 Hz, 3 ms and 20 ~o above threshold voltage (1.9 • using a stimulator II HSE. The amplitude of contraction for all types of preparations was recorded with strain gauges on a Hetlige recorder. b) From the right ventricles of male guinea pigs (350-550g) strips were prepared as described above for rats. The strip preparations were driven at I Hz, 3ms and 20~~ above threshold (2.2 • 0.25 V). The preparations were stimulated by means of needleshaped platinum electrodes. c) From left ventricles o f guinea pigs strips were cut of a diameter of about 1 mm and 4.0 mm in length. These preparations were driven at 1 Hz, 3ms and 20 ~o above threshold voltage (6.8 • 0.2V) by field stimulation. d) Cats of either sex weighing from 1 . 5 - 1 . 8 kg were anaesthetized with diethyl ether. The animals were bled by cutting the carotid arteries. After opening the thorax the hearts were quickly removed, dissected and transferred into Krebs-Henseleit solution bubbled with 95 ~ O2 and 5 ~ CO2. From the wall of the right ventricle 4 strips were cut and fixed on needle-shaped platinum electrodes for stimulation by rectangular pulses of 1 Hz as described above (1.2 • 0.1 V). After the experiment the strip preparations of all species were dried to constant weights. The developed force of contraction of all preparations was calculated as g/g dry weight.
2. Electrically Driven Papillary Muscle. From right ventricles of guinea pigs as well as of cats papillary muscles were isolated and suspended in Krebs-Henseleit solution at 30~ The preparations were driven by field stimulation at 1 Hz (guinea pig: 6.6 _+ 0.3 V; cat: 5.9 • 0.04V) as described above.
3. Left Atrial Preparations. Left atria were prepared from the hearts of male Sprague-Dawley rats in Krebs-Henseleit solution at room temperature. The preparations were fixed at needle-shaped platinum electrodes for stimulation with t Hz (0.65 • 0.06 V). Strips from left atria of the kitten heart were prepared so that one atrium yielded 4 strips. The parameters of stimulation were identical with those given for rat atria. The bath temperature was kept at 30-C.
4. Spontaneously Beating Right Atrial Preparations. From rats and cats also right atria were isolated and suspended in Krebs-Henseleit solution at 30~C and bubbled with oxygen as described above. The preparations were attached to a plexiglas holder by means of a thread fixed on the vena cava inferior. The rate was derived from the mechanogram recorded by means of strain gauges on a Hellige recorder at a paper speed of 5 mm/s.
B. Treatment o f Rats with Propylthiouracil Male Sprague-Dawley rats were made hypothyroid by feeding 6propyl-2-thiouracil (PTU) for at least 6 weeks as described by Nakashima et al. (1973). The food (Altromin '~ pellets) contained 0.15~. Four rats were kept in one cage each and received 100g Altromin per day.
C. Determination o f c A M P and c G M P At a certain time after the administration of the drugs used the preparations were removed from the organ bath, blotted with filter paper and frozen immediately in liquid nitrogen. The time from the removal to the freezing of the preparation was 5 - 8 s. When this time was exceeded, tissues were discarded. The preparations were weighed and homogenized in 10 vol of 5 ~ trichloroacetic acid, at least 0.6 ml, by use of a microdismembrator (Braun, Melsungen) for 30 s. After centrifugation at 2000 g x 2 min, 100 gI of the supernatant was taken for the determination of cAMP and 400 lal for that of cGMP. The details of the procedure for extraction and determination of cAMP have been described previously (Sch/imann et al., 1975). For the determination of cGMP the s u p e r n a t a n t - after addition of 40 pl 1 N H C I - was extracted three times with ether, heated at 80 ~C for 4 rain to evaporate the residual ether and further purified by chromatography on a 0.4 x 5.0 cm column (Dowex 50 WX 8 1 0 0 200 mesh, H +-form-Serva, Heidelberg) as described by Dinnendahl (1974). The content of cAMP was determined by the protein binding method of Gilman (1970) and that of cGMP by the protein binding method of Dinnendahl (1974). Details of the procedure have been described recently (Brodde et al., in press). The levels of cAMP and cGMP were expressed as pmoles/mg wet weight. The recovery of a known amount of unlabelled cAMP (400pmoles) and cGMP (400 pmoles), respectively, added to 1 ml of 5 ~ trichloroacetic acid before the homogenization of the preparations pretreated with the given drugs as well as untreated control preparations amounted to 97.8 • 8.6 ~ (N = 11) for cyclic A M P and 76.9 • 9.6 ~ (N = 11) for cyclic GMP. Given are the uncorrected values.
Statistical Methods Since in all preparations used in this study the - log ECs0-values of the second and third dose-response curve differed from that of the first one, only the first dose- response curve for each preparation was used for statistical evaluation with the exception of the left atrium of the rat. In this preparation the - log ECs0 values as welt as the basal and maximal developed tension did not differ significantly between the first and second dose-response curve (Table 1). Therefore in the atrium of the rat two consecutive dose-response curves could be determined.
242
Naunyn-Schmiedeberg's Arch. Pharmacol. 302 (1978)
Table 2. Electrically driven (1 Hz) ventricular strip and papillary muscle of the cat: pDz-values for the positive inotropic effect of phenylephrine (PE) in the presence of e- and/or/3-adrenolytic drugs at 30~ Adrenolytic drug
Ventricular strip
Papillary muscle
(M) pD 2-value
basal tension
pD 2-value
(g/g d.w.)
maximal increase in tension (A) 2.3 • 0.2 (7)
ns
3.0+0.5 ns (6)
Pindolol, 3 x 10-8
5.33 • 0.09 (7)
3.7 • 0.4 (7)
Pindolol, 3 x 10 -8 Phentolamine, 10 .6
4.71• (5)
5.3 • (5)
Yohimbine, 10 5
5.53• (6)
ns
(g/g d.w.)
maximal increase in tension (A)
5.82 • 0.21 (6)
108.4 • 11.1 (6)
41.9 • (6)
2.5 • 0.4 "s (5)
4.79• (5)
157.6 • 16.1" (5)
76.2 • 15.8 nS (5)
7.9+_1.1"* (6)
5.57• (8)
234.0• (8)
118.0_+28.1' (8)
n~
basal tension
7.7
Given are the means + S.E.M.; number of experiments in parentheses * P < 0.05, ** P < 0.005 compared with PE in the presence of pindolol (dry weight = d.w.)
After an equilibration time of 60 rain submaximally effective doses of phenylephrine were administered. When the responses remained constant, cumulative dose-response curves were determined by adding 0.1 ml of phenylephrine solutions thus increasing the final concentration in steps of 0.5 log units. When a steady level of developed tension was reached the next higher concentration was applied. The effect of phenylephrine was considered to be maximal when an at least 10-fold increase in concentration failed to produce a further increase of developed tension. The blocking drugs were incubated for 1 h each. By plotting the dose-response curves of single experiments on semi-logarithmic paper the concentrations ranging from the E C10 to the EClo 0 were estimated. Means for these concentrations were calculated (Ari~ns, 1964). The affinity of the agonists was given as the pD2-value, that of the antagonists as the pA2-value. Means _+ S.E.M. for the pD2- and the pA2-values, were determined as described by Van Rossum (1963). For determination of the pDz-values for the a-mimetic effect of sympathomimetic drugs the /3-adrenoceptors were blocked throughout the experiments by pindolol. For determination of the /?-mimetic effects the cr adrenoceptors were blocked by either phentolamine or yohimbine. The concentrations used are given in the corresponding tables. Significant differences were calculated by means of Student's ttest.
Experiments with the Irreversible c~-Adreno.lytic Drug Phenoxybenzamine According to Furchgott (1954) the cardiac tissues were incubated at a concentration of 10 .6 M phenoxybenzamine for a period of 20 rain each, thereafter the unbound drug was washed away during half an hour. After equilibration of 1 h the dose-response curve for phenylephrine was determined either at constant or at a changed temperature. Following the period of washout for 30 min the bath temperature was lowered. After an equilibration time for 60 min at the low temperature the dose-response curve of phenylephrine was determined. Drugs Used. (-)-Phenylephrine hydrochloride, yohimbine hydrochloride (Boehringer, Ingelheiml); (+)-pindolol base (Sandoz, 1 We thank the pharmaceutical firms for generous gift of the drugs
Nfirnbergl); phentolamine hydrochloride (Ciba-Geigy, Basel1); phenoxybenzamine hydrochloride (Smith, Kline and French, Philadelphia1); 6-propyl-2-thiouracil (Kali-Chemie, Hannoverl); For assay: 3H-cAMP (specific activity 38.4 Ci/mmol, Radiochemical Centre Amersham); 3H-cGMP (specific activity 21 Ci/mmol, New England Nuclear, Dreieichenhain).
Results
A. Experiments on Isolated Cardiac Preparations 1. Electrically Driven Strips of Ventricle. In isolated strips from the right ventricle of the cat tlae pD2-value of phenylephrine for the positive inotropic effect induced by stimulation of ~- or/3-adrenoceptor was comparable while the maximal developed tension mediated by the .stimulation of c~-adrenoceptors amounted to only one third of that evoked by/3-adrenoceptors (Table 2). For the determination of the c~-effect of phenylephrine the /3-adrenoceptors were blocked by pindolol, 3 x 10- 8 M, whereas for the estimation of the /3-effect the c~-adrenoceptors mediated responses were eliminated by yohimbine, 10 .5 M. The comparison of the dose-response curve for the positive inotropic effect of phenylephrine in the absence and presence of phentolamine, indicated a competitive antagonism. The resulting pD2-value was significantly (P < 0.005) diminished in comparison to the corresponding control (Table 2). In preparations from the right ventricle of the guinea pig the pDz-value for the positive inotropic effect of phenylephrine mediated by c~-adrenoceptors amounted to 5.99 and was significantly higher than that for the/3sympathomimetic effect (Table 3). The maximal tension exerted by stimulation of either adrenoceptor did not differ significantly from each other. The pDz-value for the c~-sympathomimetic effect of phenylephrine obtained from strips of the left ventricle
J. Wagner and O.-E. Brodde: Cardiac c~-Adrenoceptors in Mammalian Species
243
Table 3. Electrically driven (1 Hz) right and left ventricular strips as well as papillary muscles of guinea pigs : pD2-values for the positive inotropic effect of phenylephrine (PE) in the presence of c~- and/or/~-adrenolytic drugs at 30 C
Right ventricular strip Adrenolytic drug (M)
pDz-value
Basal tension (g/g d.w.)
Maximal increase in tension (A)
Pindolol, 3 x 10 -7
5.99 _+0.09(5)
9.5 _+ 1.5(5)
7.2_+0.9(5)
Phentolamine, 3 x 10 6
4.67 _+0.09* (6)
9.9 _+ 1.5n~(6)
8.0 _+0.8ns(6)
6.06 _+0.19(4)
91.0 _+ 12.3(4)
19.5 _+3.4(4)
Left ventricular strip Pindolol, 3 x 10 -7
Papillary muscle Pindolol, 3 x 10 -7
6.59 + 0.17(8)
Yohimbine, 10 5
5.34 _+0.12" (6)
59.5 _+ 13.1 (8) 106.8 _+ 14.0" (6)
41.4 +_ 5.4(8) 73.2 _+ 19.2 "S(6)
Given are means • S.E.M.; number of experiments in parentheses * P < 0.005 compared with PE in the presence of pindolol (dry weight = d.w.)
Table4. Electrically driven ventricular strips of the rat (1 Hz): Influence of c~- and /Ladrenolytic drugs on the positive inotropic effect of phenylephrine in preparations of untreated rats (A) or in those'pretreated with propylthiouracil (B) at 30'C Adrenolytic drug (M)
pD2-value
Basal tension (g/g d.w.)
Maximal developed tension (A)
A. None Pindolol, 3 x 10 ~ Phentolamine, 3 • 10 -6
5.37 +_0.1" (7) 5.32 + 0.09 (7) 4.76 • 0.06* (5)
5,3.8 _+6.0 (7) 56.9 4 4.9 (7) 44.4 _+3.3 (5)
7.59 _+2.05 (7) 6.72 _+0.8 (7) 16.9 _+2.4* (5)
B. Pindolol, 3 x 10- 7 Yohimbine, 10 5
5.68 + 0.09** (11) 4.46 -+ 0.04** (4)
57.7 _+8.2 "~(1 I) 56.3 _+7.4 "s (4)
14.7 + 2.3*** (11) 8.5 _+3.1"** (4)
Given are means _+ S.E.M.; number of experiments in parentheses * P < 0.005 compared with the values in the presence of pindolol ** P
Archivesof
Pharmacology 9 by Springer-Verlag 1978
Repdnted Issue without Advertisements
~
On the Presence and Distribution of -Adrenoceptors in the Heart of Various Mammalian Species* J. Wagner and O.-E. Brodde Pharmakologisches Institut der Universit~it Essen, Hufelandstrasse 55, D-4300 Essen 1, Federal Republic of Germany
Summary. The presence and distribution of myocardial
e-adrenoceptors in different parts of the heart of various mammalian species was investigated. For this reason experiments were performed in isolated cardiac preparations of rats, guinea pigs and cats. In order to obtain more information about the nature of the cardiac e-adrenoceptors additional experiments were undertaken at different temperatures. These studies were aimed to show whether or not a conversion of/3- to e-adrenoceptors or vice versa takes place. Moreover, we analyzed the influence of hypothyroidism on the sensitivity of e- and/3-adrenoceptors of preparations from rats fed with propylthiouracil. Finally, we tried to find out whether stimulation of these e-adrenoceptors leads to the formation of the cyclic nucleotides cAMP and cGMP. The following results were obtained: 1. In right ventricular strips of rats, guinea pigs and cats phenylephrine stimulated e-adrenoceptors. After blockade of/3-adrenoceptors the respective pDz-values of phenylephrine were 5.32, 5.99 and 5.33. The doseresponse curves obtained in the presence of eadrenolytic drugs were shifted to the right without depression of the maximum. In the rat, in addition, the pA2-values for the e-adrenolytic drugs yohimbine (5.79) and phentolamine (7.85) were determined. They were at least 0.5 log units higher than those found in the rat for other e-adrenoceptors (Van Rossum, 1965) thus supporting the view that the population of cardiac eadrenoceptors is different from that of other organs. In left ventricular strips of guinea pigs the pD2-value for the c~-mimetic effect of phenylephrine was of the same order of magnitude as that obtained in the right ventricle.
* This work was supported by the Deutsche Forschungsgemeinschaft Send offprint requests to J. Wagner at the above address
2. In the papillary muscle of the right ventricle of guinea pigs and cats phenylephrine stimulated ~adrenoceptors. 3. In the left atrium of the rat, phenylephrine stimulated myocardial e-adrenoceptors (pD2: 5.58). Also in this preparation the pA2-values for yohimbine (6.36) and phentolamine (8.21) were different from those found for other e-adrenoceptors in the rat (Van Rossum, 1965). Likewise in strips from the left atrium of the cat myocardial e-adrenoceptors are present. 4. In spontaneously beating right atria of the rat a clear-cut positive chronotropic effect mediated by stimulation of e-adrenoceptors could not be demonstrated although in 6 out of 15 preparations a small positive chronotropic effect became evident. No positive chronotropic effect at all was obtained by stimulation of c~-adrenoceptors in the cat right atrium. 5. In ventricular strips as well as in left atria from hypothyroid rats the pDz-value for the e-effect of phenylephrine was increased, in the atrium more than in the ventricle, while the pAz-values for yohimbine and phentolamine were not significantly different from the controls. Under these conditions a distinct positive chronotropic effect mediated by stimulation of a-receptors was found in the spontaneously beating rat atrium. 6. After stimulation of c~-adrenoceptors in atria and ventricular strips of normal and propylthiouraciltreated rats, as well as in strips of guinea-pig ventricles or cat atria no elevation of cAMP and c G M P was observed. 7. The concept of a single adrenoceptor convertible from e- to fl-type or vice versa was not supported by our experiments. These were carried out at different temperatures on left atria and on ventricular strips of rat hearts using the irreversible e-adrenoceptor blocking agent phenoxybenzaminel 8. The present experiments provide evidence for the existence of e-adrenoceptors in the myocardium of
0028-1298/78/0302/0239/$ 3.20
240 various mammalian species. Their stimulation produces positive inotropic effects without increases in heart rate, Key words: Myocardial c~-adrenoceptors - Rat Guinea pig - Cat - Hypothyroidism - Single adrenoceptor type.
Introduction
The first account on the existence of ~-adrenoceptors in rat myocardial strips was given by Wen:el and Su in 1966. Using the e-adrenolytic drug phentolamine, the authors showed that the positive inotropic effect of 10 -5 g/ml phenylephrine was completely abolished. Although these experiments favoured the assumption that at least the ventricle of the rat is endowed with eadrenoceptors, no exact dose-response relationship was established in the presence of fl-blocking agents. Next Govier (1967, 1968) found that a-adrenoceptors are present in the guinea-pig atrium driven at 1 Hz. Benfey and Varma (1967) confirmed for the rabbit left atrium (l Hz) the existence of c~-adrenoceptors mediating a positive inotropic effect of phenylephrine as well as that of small doses of adrenaline. However, the results concerning the atria of guinea pigs and rabbits could not be confirmed by our laboratory. In experiments on the guinea-pig left atrium driven at 1 or 2 Hz we could only demonstrate/3adrenoceptors as mediators of the positive inotropic effect of phenylephrine and not c~-adrenoceptors (Wagner and Reinhardt, 1974). Moreover, our results revealed that the positive ehronotropie effect of phenylephrine on the spontaneously beating right atria of guinea pigs and rabbits was caused by stimulation of/3adrenoceptors. On the other hand, there is no doubt as to the existence of e-adrenoceptors in the ventricle. Investigations from our laboratory on the papillary muscle of the right ventricle of rabbits (Schfimann et al., 1974) as well as in the isolated perfused rabbit heart (Wagner et al., 1974) or the cat ventricle in situ (Rodrigues-Pereira and Wagner, 1975; Wagner et al., 1975) clearly showed the presence of myocardial eadrenoceptors which are responsible at least in part for the positive inotropic effect of phenylephrine as well as for that of the endogenous amine adrenaline (Schfimann et al., 1974; Wagner et al., 1975). Furthermore, it was shown that only the positive inotropic effect of phenylephrine mediated by /3adrenoceptors was potentiated after inhibition of phosphodiesterase with theophylline or papaverine
Naunyn-Schmiedeberg'sArch. Pharmacol. 302 (1978) (Schiimann et al., 1974). The determination of Y5'cAMP clearly showed that only the stimulation of fladrenoceptors was accompanied by an increase in the level of cAMP (McNeill and Verma, 1973) whereas that of ~-adrenoceptors was not (Schfimann et al., 1975; Endoh et al., 1976a). Furthermore, the c~-sympathomimetic effect on the rabbit papillary muscle showed a clear-cut dependence on the frequency of stimulation as was demonstrated by means of methoxamine and naphazoline: the positive inotropic effect was most evident at a low rate of stimulation, i.e. at 0.5 Hz (Endoh and Schfimann, 1975). According to Nakashima et al. (1973) the sensitivity of c~-adrenoceptors is increased in atria from hypothyroid rats so that also a positive chronotropic effect elicited by stimulation of ~-adrenoceptors was visible. Moreover, in the frog heart there is strong indication for the existence of only one type of adrenoceptors which has the properties of an e-adrenoceptor at low temperature and those of a fi-adrenoceptor at higher temperatures (Kunos et al., 1973; Kunos and Nickerson, 1976). In the frog heart, the ~- and fi-agonist adrenaline is the sympathetic transmitter (Holt: et al., 1951 ; Grobecker and Holt:, 1966). The conversion of a single adrenoceptor from the fl- to the c~-type should produce a positive inotropic effect in the hibernating frog. Also for the rat atrium the possibility of the interconversion of a single adrenoceptor is discussed (Kunos, 1977). Furthermore, in left atria of hypothyroid rats the elevation of cAMP induced by phenylephrine was blocked by phenoxybenzamine thus supporting the interconversion of a single receptor (Kunos et al., 1976). The aim of the present experiments was to demonstrate the presence of myocardial a-adrenoceptors in different species of mammals, and to determine their distribution in different parts of the heart and the relationship of their stimulation to the level of cAMP and cGMP. Further series of experiments were conducted to obtain more information on the nature of the cardiac ~-adrenoceptor. By means of the irreversible eadrenolytic drug phenoxybenzamine we tried to find out if there is any reason to assume a single type of adrenoceptor in the rat heart which might alter its properties.
Materials and Methods A. Preparation of Cardiac Tissue and Experimental Procedure 1. ElectricallyDrivenStrips of the Ventricle.a) MaleSprague-Dawley rats weighing 200--240g were sacrificed by cervical dislocation. After opening of the thorax, the hearts were quickly removedand transferred into Krebs-Henseleit solution of room temperature.
J. Wagner and O.-E. Brodde: Cardiac c~-Adrenoceptors in Mammalian Species
241
Table 1. Basal tension, maximal development of tension in response to phenylephrine and - l o g ECs0-values of phenylephrine after repeated determination of the dose-response curve (DRC) on isolated electrica!ly driven (I Hz) rat atria at 30~ - log ECso
First D R C Second DRC Third DRC
6.33 • 0.18 6.06 • 0.14 "~ 5.68 • 0.09*
Developed tension (mg) control
maximal
374.5 • 41.7 275.8 _+ 32.6 ns 154.8 • 23.9*
540.3 • 50.2 565.7 • 62.0 n~ 506.7 • 50.5 ns
Given are means • S.E.M. of 6 preparations * P < 0.005 compared with the corresponding value of the first DRC
From the right ventricular wall two strips.were prepared and fixed on needle-shaped platinum electrodes, The preparations were suspended in Krebs-Henseleit solution of the following composition (raM): NaCI 119; CaC1 z 2.5; KC1 4.8; MgSO 4 1.2; KH~PO 4 1.2; NaHCO 3 24.9; glucose 10.0; ascorbic acid 0.054 bubbled with 95 ~ O 2 and 5 CO z at 30~ unless stated otherwise. The preparations were stimulated electrically by rectangular pulses of 1 Hz, 3 ms and 20 ~o above threshold voltage (1.9 • using a stimulator II HSE. The amplitude of contraction for all types of preparations was recorded with strain gauges on a Hetlige recorder. b) From the right ventricles of male guinea pigs (350-550g) strips were prepared as described above for rats. The strip preparations were driven at I Hz, 3ms and 20~~ above threshold (2.2 • 0.25 V). The preparations were stimulated by means of needleshaped platinum electrodes. c) From left ventricles o f guinea pigs strips were cut of a diameter of about 1 mm and 4.0 mm in length. These preparations were driven at 1 Hz, 3ms and 20 ~o above threshold voltage (6.8 • 0.2V) by field stimulation. d) Cats of either sex weighing from 1 . 5 - 1 . 8 kg were anaesthetized with diethyl ether. The animals were bled by cutting the carotid arteries. After opening the thorax the hearts were quickly removed, dissected and transferred into Krebs-Henseleit solution bubbled with 95 ~ O2 and 5 ~ CO2. From the wall of the right ventricle 4 strips were cut and fixed on needle-shaped platinum electrodes for stimulation by rectangular pulses of 1 Hz as described above (1.2 • 0.1 V). After the experiment the strip preparations of all species were dried to constant weights. The developed force of contraction of all preparations was calculated as g/g dry weight.
2. Electrically Driven Papillary Muscle. From right ventricles of guinea pigs as well as of cats papillary muscles were isolated and suspended in Krebs-Henseleit solution at 30~ The preparations were driven by field stimulation at 1 Hz (guinea pig: 6.6 _+ 0.3 V; cat: 5.9 • 0.04V) as described above.
3. Left Atrial Preparations. Left atria were prepared from the hearts of male Sprague-Dawley rats in Krebs-Henseleit solution at room temperature. The preparations were fixed at needle-shaped platinum electrodes for stimulation with t Hz (0.65 • 0.06 V). Strips from left atria of the kitten heart were prepared so that one atrium yielded 4 strips. The parameters of stimulation were identical with those given for rat atria. The bath temperature was kept at 30-C.
4. Spontaneously Beating Right Atrial Preparations. From rats and cats also right atria were isolated and suspended in Krebs-Henseleit solution at 30~C and bubbled with oxygen as described above. The preparations were attached to a plexiglas holder by means of a thread fixed on the vena cava inferior. The rate was derived from the mechanogram recorded by means of strain gauges on a Hellige recorder at a paper speed of 5 mm/s.
B. Treatment o f Rats with Propylthiouracil Male Sprague-Dawley rats were made hypothyroid by feeding 6propyl-2-thiouracil (PTU) for at least 6 weeks as described by Nakashima et al. (1973). The food (Altromin '~ pellets) contained 0.15~. Four rats were kept in one cage each and received 100g Altromin per day.
C. Determination o f c A M P and c G M P At a certain time after the administration of the drugs used the preparations were removed from the organ bath, blotted with filter paper and frozen immediately in liquid nitrogen. The time from the removal to the freezing of the preparation was 5 - 8 s. When this time was exceeded, tissues were discarded. The preparations were weighed and homogenized in 10 vol of 5 ~ trichloroacetic acid, at least 0.6 ml, by use of a microdismembrator (Braun, Melsungen) for 30 s. After centrifugation at 2000 g x 2 min, 100 gI of the supernatant was taken for the determination of cAMP and 400 lal for that of cGMP. The details of the procedure for extraction and determination of cAMP have been described previously (Sch/imann et al., 1975). For the determination of cGMP the s u p e r n a t a n t - after addition of 40 pl 1 N H C I - was extracted three times with ether, heated at 80 ~C for 4 rain to evaporate the residual ether and further purified by chromatography on a 0.4 x 5.0 cm column (Dowex 50 WX 8 1 0 0 200 mesh, H +-form-Serva, Heidelberg) as described by Dinnendahl (1974). The content of cAMP was determined by the protein binding method of Gilman (1970) and that of cGMP by the protein binding method of Dinnendahl (1974). Details of the procedure have been described recently (Brodde et al., in press). The levels of cAMP and cGMP were expressed as pmoles/mg wet weight. The recovery of a known amount of unlabelled cAMP (400pmoles) and cGMP (400 pmoles), respectively, added to 1 ml of 5 ~ trichloroacetic acid before the homogenization of the preparations pretreated with the given drugs as well as untreated control preparations amounted to 97.8 • 8.6 ~ (N = 11) for cyclic A M P and 76.9 • 9.6 ~ (N = 11) for cyclic GMP. Given are the uncorrected values.
Statistical Methods Since in all preparations used in this study the - log ECs0-values of the second and third dose-response curve differed from that of the first one, only the first dose- response curve for each preparation was used for statistical evaluation with the exception of the left atrium of the rat. In this preparation the - log ECs0 values as welt as the basal and maximal developed tension did not differ significantly between the first and second dose-response curve (Table 1). Therefore in the atrium of the rat two consecutive dose-response curves could be determined.
242
Naunyn-Schmiedeberg's Arch. Pharmacol. 302 (1978)
Table 2. Electrically driven (1 Hz) ventricular strip and papillary muscle of the cat: pDz-values for the positive inotropic effect of phenylephrine (PE) in the presence of e- and/or/3-adrenolytic drugs at 30~ Adrenolytic drug
Ventricular strip
Papillary muscle
(M) pD 2-value
basal tension
pD 2-value
(g/g d.w.)
maximal increase in tension (A) 2.3 • 0.2 (7)
ns
3.0+0.5 ns (6)
Pindolol, 3 x 10-8
5.33 • 0.09 (7)
3.7 • 0.4 (7)
Pindolol, 3 x 10 -8 Phentolamine, 10 .6
4.71• (5)
5.3 • (5)
Yohimbine, 10 5
5.53• (6)
ns
(g/g d.w.)
maximal increase in tension (A)
5.82 • 0.21 (6)
108.4 • 11.1 (6)
41.9 • (6)
2.5 • 0.4 "s (5)
4.79• (5)
157.6 • 16.1" (5)
76.2 • 15.8 nS (5)
7.9+_1.1"* (6)
5.57• (8)
234.0• (8)
118.0_+28.1' (8)
n~
basal tension
7.7
Given are the means + S.E.M.; number of experiments in parentheses * P < 0.05, ** P < 0.005 compared with PE in the presence of pindolol (dry weight = d.w.)
After an equilibration time of 60 rain submaximally effective doses of phenylephrine were administered. When the responses remained constant, cumulative dose-response curves were determined by adding 0.1 ml of phenylephrine solutions thus increasing the final concentration in steps of 0.5 log units. When a steady level of developed tension was reached the next higher concentration was applied. The effect of phenylephrine was considered to be maximal when an at least 10-fold increase in concentration failed to produce a further increase of developed tension. The blocking drugs were incubated for 1 h each. By plotting the dose-response curves of single experiments on semi-logarithmic paper the concentrations ranging from the E C10 to the EClo 0 were estimated. Means for these concentrations were calculated (Ari~ns, 1964). The affinity of the agonists was given as the pD2-value, that of the antagonists as the pA2-value. Means _+ S.E.M. for the pD2- and the pA2-values, were determined as described by Van Rossum (1963). For determination of the pDz-values for the a-mimetic effect of sympathomimetic drugs the /3-adrenoceptors were blocked throughout the experiments by pindolol. For determination of the /?-mimetic effects the cr adrenoceptors were blocked by either phentolamine or yohimbine. The concentrations used are given in the corresponding tables. Significant differences were calculated by means of Student's ttest.
Experiments with the Irreversible c~-Adreno.lytic Drug Phenoxybenzamine According to Furchgott (1954) the cardiac tissues were incubated at a concentration of 10 .6 M phenoxybenzamine for a period of 20 rain each, thereafter the unbound drug was washed away during half an hour. After equilibration of 1 h the dose-response curve for phenylephrine was determined either at constant or at a changed temperature. Following the period of washout for 30 min the bath temperature was lowered. After an equilibration time for 60 min at the low temperature the dose-response curve of phenylephrine was determined. Drugs Used. (-)-Phenylephrine hydrochloride, yohimbine hydrochloride (Boehringer, Ingelheiml); (+)-pindolol base (Sandoz, 1 We thank the pharmaceutical firms for generous gift of the drugs
Nfirnbergl); phentolamine hydrochloride (Ciba-Geigy, Basel1); phenoxybenzamine hydrochloride (Smith, Kline and French, Philadelphia1); 6-propyl-2-thiouracil (Kali-Chemie, Hannoverl); For assay: 3H-cAMP (specific activity 38.4 Ci/mmol, Radiochemical Centre Amersham); 3H-cGMP (specific activity 21 Ci/mmol, New England Nuclear, Dreieichenhain).
Results
A. Experiments on Isolated Cardiac Preparations 1. Electrically Driven Strips of Ventricle. In isolated strips from the right ventricle of the cat tlae pD2-value of phenylephrine for the positive inotropic effect induced by stimulation of ~- or/3-adrenoceptor was comparable while the maximal developed tension mediated by the .stimulation of c~-adrenoceptors amounted to only one third of that evoked by/3-adrenoceptors (Table 2). For the determination of the c~-effect of phenylephrine the /3-adrenoceptors were blocked by pindolol, 3 x 10- 8 M, whereas for the estimation of the /3-effect the c~-adrenoceptors mediated responses were eliminated by yohimbine, 10 .5 M. The comparison of the dose-response curve for the positive inotropic effect of phenylephrine in the absence and presence of phentolamine, indicated a competitive antagonism. The resulting pD2-value was significantly (P < 0.005) diminished in comparison to the corresponding control (Table 2). In preparations from the right ventricle of the guinea pig the pDz-value for the positive inotropic effect of phenylephrine mediated by c~-adrenoceptors amounted to 5.99 and was significantly higher than that for the/3sympathomimetic effect (Table 3). The maximal tension exerted by stimulation of either adrenoceptor did not differ significantly from each other. The pDz-value for the c~-sympathomimetic effect of phenylephrine obtained from strips of the left ventricle
J. Wagner and O.-E. Brodde: Cardiac c~-Adrenoceptors in Mammalian Species
243
Table 3. Electrically driven (1 Hz) right and left ventricular strips as well as papillary muscles of guinea pigs : pD2-values for the positive inotropic effect of phenylephrine (PE) in the presence of c~- and/or/~-adrenolytic drugs at 30 C
Right ventricular strip Adrenolytic drug (M)
pDz-value
Basal tension (g/g d.w.)
Maximal increase in tension (A)
Pindolol, 3 x 10 -7
5.99 _+0.09(5)
9.5 _+ 1.5(5)
7.2_+0.9(5)
Phentolamine, 3 x 10 6
4.67 _+0.09* (6)
9.9 _+ 1.5n~(6)
8.0 _+0.8ns(6)
6.06 _+0.19(4)
91.0 _+ 12.3(4)
19.5 _+3.4(4)
Left ventricular strip Pindolol, 3 x 10 -7
Papillary muscle Pindolol, 3 x 10 -7
6.59 + 0.17(8)
Yohimbine, 10 5
5.34 _+0.12" (6)
59.5 _+ 13.1 (8) 106.8 _+ 14.0" (6)
41.4 +_ 5.4(8) 73.2 _+ 19.2 "S(6)
Given are means • S.E.M.; number of experiments in parentheses * P < 0.005 compared with PE in the presence of pindolol (dry weight = d.w.)
Table4. Electrically driven ventricular strips of the rat (1 Hz): Influence of c~- and /Ladrenolytic drugs on the positive inotropic effect of phenylephrine in preparations of untreated rats (A) or in those'pretreated with propylthiouracil (B) at 30'C Adrenolytic drug (M)
pD2-value
Basal tension (g/g d.w.)
Maximal developed tension (A)
A. None Pindolol, 3 x 10 ~ Phentolamine, 3 • 10 -6
5.37 +_0.1" (7) 5.32 + 0.09 (7) 4.76 • 0.06* (5)
5,3.8 _+6.0 (7) 56.9 4 4.9 (7) 44.4 _+3.3 (5)
7.59 _+2.05 (7) 6.72 _+0.8 (7) 16.9 _+2.4* (5)
B. Pindolol, 3 x 10- 7 Yohimbine, 10 5
5.68 + 0.09** (11) 4.46 -+ 0.04** (4)
57.7 _+8.2 "~(1 I) 56.3 _+7.4 "s (4)
14.7 + 2.3*** (11) 8.5 _+3.1"** (4)
Given are means _+ S.E.M.; number of experiments in parentheses * P < 0.005 compared with the values in the presence of pindolol ** P
