The effect of calcium on cardiac phosphorylase

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The effect of calcium on cardiac phosphorylase activation, contractile force and cyclic AMP in euthyroid and hyperthyroid rat hearts1 ELIZABETH J. HARTLEY'AND JOHN H. MCNEILE~ Division of Bhar~n41c.olo~y and Toxicology, Faculty of Blzarnzaceutica&Sciences, Univer,rity of B r i r i ~ IColunlbia, ~ V~znr.or~vrr, B.C., runudu V 6 T 1 W 5 Received February 19, 1976

HAWTEEY, E. J., and M c N ~ I L I ,1. , H. 1976. The effcct of calcium on cardiac phosphorylase activation, contractile force and cyclic AMP in euthyroid and hyperthyroid rat hearts. Can. J. Physiol. Pharmacol. 54, 590-595. Calcium chloride injected into isolated perfused rat hearts produced a positive inotropic effect and increased the Icvels of phosphorylase a (EC 2.4.1.1). The increase in enzyme activity lagged behind the inotropic effect. Pretreatment of animals with thyroid hormone cnhanced the ability of noradrenaline to activate phospkorylasc but did not affect the inotropic or phosphorylase activating eRect of calcium. Thyroid hormone pretreatment did enhance the chronotropic efTect of calcium. Calcium did not affect the cardiac levels of cyclic AMP. It is concluded that calcium can activate phosphorylase by a mechanism other than cyclic AMP and that the enhancement of adrcnergic arnine-induced phosphorylase activation by thyroid hormone is not a calcium mediated event.

It has now been well established that thyroid hormone pretreatment does not enhance the inotropic or chronotropic effect of the adrenergic amines (Benfey and Varma 1963; Margolius and Gaffney 1965; van der Schoot and Moran 1965). However, it is also clear that the hypertllyroid state does sensitize the heart and other tissues to the lipolytic and glycogenolytic actions of adrenergic drugs (Hornbrook et al. 1965; Hess and ShanfeId 1965; McNeill and Brody 1968; Krishna et aE. 1968; McNeiIl et al. 1969; Young and McNeill 1974). In the heart the ability of adrenergic amines to activate glycogen phosphorylase ( 1,4-a-D-glucan :orthophosphate a-glucosyltransferase (EC 2.4.1.1 ) is greatly enhanced in hyperthyroid animals. The mechanism for the enhancement has been extensively investigated but is, as yet, unknown. It is not due to blockade of amine uptake (McNejll and Brody 1968) or to a reserpinelike supersensitivity (McNeill 1969 ). Suggestions that thyroid hormone may affect the synthesis or breakdown of cyclic AMP (Hornbrook et ab. 1965) have not stood the test of 'Supported by a grant from the British Columbia Heart Foundation. 'Prcscnt address: Department of Pharmacology, Chelsea College of Science, University of London, L.ondon, England. T o whom reprint requests should be addressed.

experimental investigation ( McNcill et ak. 8969; McNejll 1969; Young and McNeill 1974). Reports that adenylate cyclase (ATB pyrophosphate-lyase (cyclizing) (EC 4.6.1.1 ) ) could be stimulated directly by thyroxine have proved tnlc for cat heart ( k v e y and Epstein, 1968) but thyroid hormones do not appear capable of directly increasing the activity sf adenylate cyclase obtained from rat heart (MeNeil1 c.t al. 1978 ) . It has been suggested (Young and McNeill 11974) that thyroid horr-none might alter the response of phosphorylase to calcium. Calcium can activate phosphorylase in the heart (Friesen et ul. 1967) and the alteration of the response to calcium thus has a potential for explaining thyroid supersensitivity. Thyroid hormone is known to affect some aspects of calcium metabcjlisnl in the heart. Report by Suko ( 4 97 1) and Nayler et al. ( 1971 ) have indicated that sarcoplasmic reticulum isolated from hyperthyroid animals accumulated and exchanged calcium more readily than did sarcoplasmic reticulum from euthyroid animals. The increased release of bound calcium by an agonist like noradrenaline could thus account for the supersensitivity noted. The present study was undertaken to investigate the effect of thyroid horinone pretreatment on cardiac phosphorylase activation by calcium. Contractile force changes and

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previously chilled in a mixture of dcshol - Dry Ice. The hearts were then stored at -80 "C until assayed. Phosphorylase was measured in the direction of glycogen synthesis as previously described by McNeilH and Hrody (1966). Since total phosphsrylase did not change all results are presentcd as percentage phosphorylase a which is: [(phosphorylase activity in the absence of AMP)/(phosphorylase activity in the presence of AMP)] x 100. The hearts were assayed for cyclic AMP rnsing a modification of the proteinbinding assay of Gilman (11970) rnsing a cyclic AMP kit (TRK, 432 Amersham!Searle, Oakville. a n t . ) . Animals were made hyperthyroid by the smbcutaneous injection of 3.3',fif-triiodo-L-thyronine (500 pg/kg) in alkaline saline administered daily for 3 days. This treatment has previously been demonstrated to make rats hyperthyroid (McNeill et a!. 1969). Statistical analysis was done by the Student t test for rnnpaired data for phosphor~~lase a and cyclic AMP and paired data for the positive inotropic effect (Lewis 1966). A probability sf