Life Sciences Vol . 19, pp . 1597-1602, 1976 . Printed in the U .S .A .

Pergamon Press

INTERCONVERSION OF MYOCARDIAL ADRENOCEPTORS : ITS RELATIONSHIP TO ADENYLATE CYCLASE ACTIVATION George Kunos, Lucia Mucci and Valerie Jaeger Department of Pharmacology and Therapeutics, McGill University Montreal, Quebec, Canada (Received in final form October 4, 1976) Summary Hypothyroidism in rats induces a shift from g toward a in the properties of myocardial adrenoceptors mediating the rise in cyclic AMP (c-AMP) . The change occurs in contracting but not in quiescent myocardial preparations, it can be reversed by treating hypothyroid rats with thyroxin, and it is similar to changes in the properties of adrenoceptors mediating inotropic responses in the same preparations . The results indicate that adrenoceptors mediating the rise in c-AMP and contractility are similarly interconvertible and suggest that the a-adrenoceptor mediated rise in c-AMP is a consequence of events leading to an inotropic response rather than their cause . Considerable evidence indicates that activation of myocardial adenylate cyclase and the inotropic response to catecholamines are closely related (1), although dissociation of the two events under a number of conditions makes their cause and effect relationship unlikely (2) . Both effects can be mediated by ßadrenoceptors . Inotropic responses also have an a-adrenoceptor component (3,4) and observations of a change in the balance of a- and g-adrenoceptors mediating inotropic and chronotropic reactions at low temperatures (5,6,7,8,9,10,11) or in hypothyroidism (12,13,14) have been interpreted to indicate that a- and ßadrenoceptors are two forms of a common unit whose configuration is under metabolic control (5,11,13,15) . The present study was undertaken to determine if adrenoceptors mediating the rise in c-AMP levels are similarly interconvertible . Previous studies on am hibian and mammalian ventricular slices (16) or cardiac membrane fragments (17~ at different temperatures failed to demonstrate a change in the properties of these receptors, but the preparations were not contracting . The present results confirm the absence of an a-adrenoceptor mediated rise in c-AMP levels in a non-beating preparation, but show that adrenoceptors mediating the rise in c-AMP levels and increased contractility can be similarly modified by altered thyroid states, when c-AMP is measured in the same, beating preparations . An abstract of these results has been published (18) . Methods To facilitate comparison with earlier results on inotropic responses, treatments were the same as in our previous study (13) and c-AMP was measured in preparations identical to those used to measure contractility . Male SpragueDawley rats (180-220 g) were thyroidectomized 4 to 6 weeks before the experiments . Growth of these animals was retarded and serum thyroxin levels, measured by the method of Murphy and Pattee (19) were significantly lower (18 t 8 ng/ml) than those in weight matched controls (60 ± 4 ng/ml) or in thyroidectomized animals treated daily with 1 mg/kg thyroxin intraperitoneally, for 8 days before the experiment (91 ± 8 ng/ml) . Left atria were removed, weighed and suspended 1597

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in Krebs-Henseleit solution at 31°C, bubbled with 5 % C0 2 in 0 2 . The atria were connected to a force-displacement transducer and polygraph and resting tension was set to 0 .5 g . Preparations were either left quiescent or were stimulated through small platinum electrodes (0 .3-0 .8 V, 3 msec, 1 Hz) . Drugs were added to the bath and concentrations were expressed as the final concentration in the medium . Preliminary experiments showed that peak c-AMP levels occurred 14 sec after the administration of isoproterenol and 20 sec after phenylephrine and these times were selected for freezing of the tissue . Preparations were frozen by a Wollenberger clamp at acetone-dry ice temperature and were immediately homogenized in ice-cold 5 % trichloroacetic acid . c-AMP concentrations were determined by a competitive protein-binding assay (20) and were expressed as pmole per mg wet tissue . Recovery of unlabelled c-AMP added to the homogenate was 8595 % . In a few experiments, purification of tissue extracts on a Biorad AG 1-X8 formate column yielded c-AMP levels not significantly different from those obtained in unpúrified extracts . The experimental protocol was similar in experiments, where cyclic GMP (c-GMP) levels were determined in spontaneously beating pairs of atria . Peak c-GMP levels occurred 14 sec after isoproterenol or noradrenaline and 20 sec af ter phenylephrine . The atria were homogenized in ice-cold 5 % trichloroacetic acid and c-GMP was separated on a Biorad AG -X8 formate column (21) and determined by radioimmunoassay (22) . Recovery of 3 H-c-GMP added to the homogenate was 55-65 % . Several concentrations of the agonists were tested and the data in Table 3 represent responses to maximally effective concentrations . Differences between mean cyclic nucleotide concentrations obtained in the presence of a drug and in its absence were analyzed by the unpaired t test, and differences with a P va~.ué of less than 0 .05 were considered significant . Results Isoproterenol and phenylephrine produced concentration-dependent increases in c-AMP levels in contracting atria from both control and hypothyroid animals . Fig . 1 shows that the maximal effect of isoproterenol in both groups was an in crease in c-AMP levels of approximately 3-fold, but the threshold concentration that produced a rise was 10 times lower in control atria than in those from hypothyroid rats : Phenylephrine only increased c-AMP levels by 70 to 100 %and, in contrast to isoproterenol, produced a significant increase in c-AMP levels in a 5 times higher concentration in control than in hypothyroid preparations (see also Fig . 2) . Similar reciprocal changes in the concentrations of the two drugs producing half-maximal responses (~ig . 1, vertical lines) 2 resulted in a decrease of their potency ratio from 2 X 10 in controls to 6 X 10 in hypothyroids, and this decrease was similar to the decrease in the ratio of the inotropic ~otencies of the two agoni~ts, calculated from earlier results (13) (2 .2 X 10 in controls and 1 .1 X 10 in hypothyroids) .In both groups, however, the potency of isoproterenol was approximately 10-fold lower and the potency of phenylephrine 50 to 80-fold lower for increasing c-AMP levels than for increasing force . i

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Similar reciprocal changes were observed with the antagonists phento~amine and propranolol (Table 1), although in equimolar concentrations (10 - M) propranolol was more effectlVe than metoprolol and its inhibition of responses to phenylephrine remained significant in hypothyroid preparations . Quiescent control preparations responded to phenylephrine in the same way as contracting atria (Table 2) : the rise in c-AMP was inhibited by propranolol but not by POB . However, phenylephrine did not significantly alter c-AMP levels in quiescent atria from hypothyroid rats, either in the presence or in the absence of POB . Maximal increases in c-AMP in response to isoproterenol were the same in these as in the contracting preparations . Since it has been recently proposed that a-adrenoceptor responses of the myocardium are mediated through c-GMP and that the observed changes in the effects of catecholamines on c-GMP with temperature may explain temperature-in duced changes in inotropic and chronotropic responses (10), we measured the effects of adrenergic drugs on c-GMP levels in spontaneously beating pairs of atria from rats in different thyroid states . The results shown in Table 3 are in agreement with those reported by others (10) . The rise in c-GMP in control atria in response to agonists suggested the involvement of a-adrenoceptors : phenylephrine produced a greater increase than did noradrenaline and isoproterenol was ineffective . The increase in c-GMP was completely inhibited, however, by low concentrations of either a- or ß-adrenoceptor blocking drugs . In hypothyroid preparations phenylephrine lost its effectiveness and isoproterenol remained ineffective, leaving noradrenaline the only agonist that produced a rise in c-GMP . This effect was again inhibited by both POB and propranolol . Effective inhibition of the c-GMP response by both a- and ß-adrener is blocking drugs in different htyroid states or at different temperatures (10~ does not allow this response to be characterized in terms of specific receptors . The rise in c-GMP in response to adrenergic a onists may be related to some event, possibly the mobilization of calcium (24~, that can follow activation of either a- (25) or ß-adrenoceptors (26) . Discussion Earlier observations have shown that hypothyroidism in rats can shift the balance of myocardial adrenoceptors mediating inotropic responses from ß toward a (12,13) . The present data indicate that the receptors involved with adenylate cyclase are similarly affected by altered thyroid state and both results are compatible with the concept that a- and B-adrenoceptors represent interconvertible forms of a metabolically controlled single unit (5,11,13) . Our observation that phenylephrine can increase c-AMP levels in contracting myocardial preparations through activation of ß-adrenoceptors confirms similar fìndings reported by others (27,28), and correlates with the predominantly 9-adrenergic inotropic effect of phenylephrine in atrial preparations of euthyroid animals (29,30) . The observation that phenylephrine did not increase c-AMP concentrations in non-beating preparations from thyroidectomized rats also agrees with a similar absence of response reported for non-contracting cardiac preparations tested at low temperatures (16,17) and suggests that in the non-contracting myocardium a-adrenoceptor stimulation is not associated with an increase in c-AMP production . On the other hand, an a-receptor mediated rise in c-AMP could be detected in contracting atria from hypothyroid rats, whereas the effectiveness of g-adrenergic drugs was reduced, which appears to reflect an interconversion of a- and g-adrenoceptors, similar to that indicated by results with inotropic responses (13) . There are, however, quantitative differences between the two responses . In euthyroid animals POB and phentolamine moderately inhibited the effect of phenylephrine on contractility (13,14), but not on CAMP levels . On the other hand, propranolol was reported to inhibit the effect of phenylephrine on c-AMP but not on contractility in perfused hearts of hypothyroid rats (28) . These observations and the greater potency ratio of isoproterenol and phenylephrine calculated from c-AMP measurements than from inotropic

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responses may indicate a greater dominance of E3-adrenoceptors in the former response, both in control and in hypothyroid pre arations . Although the present and previous data (13~ indicate that the receptors involved with adenylate cyclase and those associated with the contractile process are similarly affected by hormonal factors, they do not prove or disprove the relationship of enzyme activation and inotropism . However, the finding that the a-adrenoceptor mediated rise in c-AMP required contractility may suggest that this effect is a consequence of events leading to an inotropic response rather than their cause . Acknowledgement This work was supported by a grant from the Medical Research Council . References 1 . 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 .

E .W . SUTHERLAND, G .A . ROBISON and R .W . BUTCHER, Circulation 37 279-306(1968) . B .E . SOBEL and S .E . MAYER, Circulation Res . 32 4 ~3j. D .G . WENZEL and J .L . SU, Arch . íßt. Pharmacodn . Ther . _160 379-389 (1966) . W.C . GOVIER, Life Sci . 6 ~-1365 (1 67 . G . KUNOS and M . SZENTIVÂNYI, Nature (Lond .) 217 1077-1078 (1968) . G .A . BUCKLEY and C .C . JORDAN, Bra J.~harmacol . 38 394-398 (1970) . G . KUNOS, M .S . YONG and M . NICKERSON, Nature new Biól . _241 119-120 (1973) . M .N .E . NARRI, Acta Pharmacol . Tox . 33 2~~9 -(T9T3 R . TIRRI, M .N .E . HARRÌ an~L . LÂITINÉN, Acta ~Ph~siol . Scand . _90 260-266(1974) . S .M . AMER and J .E . BYRNE, Nature (Lond .) 256 421-424 (1975 . G. KUNOS and M . NICKERSON, J . Ph suol . ( Lond .) 256 23-40 (1976) . H. NAKASHIMA, K. MAEDA, A . SEK Y and Y . HAGINO,Japan . J . Pharmacol . 21 819-825 (1971) . G. KUNOS, I . VERMES-KUNOS and M . NICKERSON, Nature (Lond .) _250 779-781(1974) . G. KUNOS, Brit . J . Pharmacol . In press . J.L . MATHENY andR .P . LQUIST, Arch . int .Pharmacodyn . Ther . 25 8 4-10 (1975) . B.G . BENFEY, G . KUNOS and M . NICKERSON,Brit . J . Pharmacy- 51253-257 (1974) . M .G . CARON and R .J . LEFKOWITZ, Nature (Lond .) X49 5~ 8-260 (1~4) . G. KUNOS, Proc . 6th Int . Congr . Pharmacy-Helsinki, 1975, p . 62 . B.E .P . MURP-I~and C.J.PATTEE, J. CTin.endocr . Metab . 24 187-196 (1964) . 03 5-31(1970) . A.G . GILMAN, Proc . Natl . Acad . Sci . USA, 6 G. SCHULTZ, J~ -HARDMAN, K . SCHl1LTZ,C.E.BAIRD and E .W . SUTHERLAND, Proc . Natl . Acad . Sci . USA, 70 1721-1725 (1973) . A.L . STEINER~.SPAGLIARI, . L .R . CHASE and D.M . KIPNIS, _J . Bio1 . Chem . _247 1114-1120 (1972) . B. JOHANSSON, Eur . J . Pharmacol . 24 194-204 (1973) . G. SCHULTZ, J .G .HARDMAN, K . SCHULTZ, C .E . BAIRD and E .W . SUTHERLAND, Proc . Natl . Acad . Sci . USA, 70 3889-3893 (1973) . Z. SEL~NGER,S . BARI,S . EIMERL and M. SCHRAMM, J. Biol . Chem . 248 369-372 (1973) . E . CARMELIET and J . VEREECKE, Pflue er's Arch . 313 300-315 (1969) . J . McNEILL and S .C . VERMA, _J . P armacol . ~ex . Thér . 187 296-299 (1973) . J-B . OSNES and I . l~1YE, _Adv . _in Cyc is Nuc1 . Res . _5 41433 Raven Press, New York (1974) . R.F . FURCHGOTT, Fed. Proc . 29 1352-1361 (1970) . J. WAGNER and D.REINHARDT,~rch . Pharmacol . 282 295-306 (1974) .

Interconversion of myocardial adrenoceptors: its relationship to adenylate cyclase activation.

Life Sciences Vol . 19, pp . 1597-1602, 1976 . Printed in the U .S .A . Pergamon Press INTERCONVERSION OF MYOCARDIAL ADRENOCEPTORS : ITS RELATIONSHI...
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