REGULATION OF PHOSPHOLAMBAN AND TROPONIN-I PHOSPHORYLATION IN THE INTACT RAT CARDIOMYOCYTES BY ADRENERGIC AND CHOLINERGIC ROLES OF CYCLIC-NUCLEOTIDES, CALCIUM, PROTEIN-KINASES AND PHOSPHATASES AND DEPOLARIZATION

Protein phosphorylation was investigated in [P-32]-labeled cardiomyocy tes isolated from adult rat heart ventricles. The beta-adrenergic stim ulation (by isoproterenol, ISO) increased the phosphorylation of inhib itory subunit of troponin (TN-I), C-protein and phospholamban (PLN). S uch stimulation was largely mediated by increased adenylyl cyclase (AC ) activity, increased myoplasmic cyclic AMP and increased cyclic AMP d ependent protein kinase (A-kinase)-catalyzed phosphorylation of these proteins in view of the following observations: (a) dibutyryl-and brom o-derivatives of cyclic AMP mimicked the stimulatory effect of ISO on protein phosphorylation while (b) Rp-cyclic AMP was found to attenuate ISO-dependent stimulation. Unexpectedly, 8-bromo cyclic GMP was found to markedly increase TN-I and PLN phosphorylation. Both beta(1)- and beta(2)-adrenoceptors were present and ISO binding to either receptor was found to stimulate myocyte AC. However, the stimulation of the bet a(2)-AR only marginally increased while the stimulation of beta(1)-AR markedly increased PLN phosphorylation. Other stimuli that increase ti ssue cyclic AMP levels also increased PLN and TN-I phosphorylation and these included isobutylmethylxanthine (non-specific phosphodiesterase inhibitor), milrinone (inhibits cardiotonic inhibitable phosphodieste rase, sometimes called type III or IV) and forskolin (which directly s timulates adenylyl cyclase). Cholinergic agonists acting on cardiomyoc yte M(2)-muscarinic receptors that are coupled to AC via pertussis tox in(PT)-sensitive G proteins inhibited AC and attenuated ISO-dependent increases in PLN and TN-I phosphorylation. The in vivo PT treatment, w hich ADP-ribosylated G(i)-like protein(s) in the myocytes, markedly at tenuated muscarinic inhibitory effect on PLN and TN-I phosphorylation on one hand and, increased the beta-adrenergic stimulation, on the oth er. Controlled exposure of isolated myocytes to N-ethyl maleimide, als o led to the findings similar to those seen following the PT treatment . Exposure of myocytes to phorbol,12-myristate, 13-acetate (PMA) incre ased the protein phosphorylation, augmenting the stimulation by ISO, a nd such augmentation was antagonized by propranolol suggesting modulat ion of the beta-adrenoceptor coupledAC pathway by PMA. Okadaic acid (O A) exposure of myocytes also increased protein phosphorylation with th e results supporting the roles for type 1 and 2A protein phosphatases in the dephosphorylation of PLN and TN-I. Interestingly OA treatment a ttenuated the muscarinic inhibitory effect which was restored by subse quent brief exposure of myocytes to PMA. While the stimulation of alph a adrenoceptors exerted little effect on the phosphorylation of PLN an d TN-I, inactivation of alpha adrenoceptors by chloroethylclonidine (C EC), augmented beta-adrenergically stimulated phosphorylation. KCl-dep endent depolarization of myocytes was observed to potentiate ISO-depen dent increase in phosphorylation (incubation period 15 sec to 1 min) a s well as to accelerate the time-dependent decline in this phosphoryla tion seen upon longer incubation. Verapamil decreased ISO-stimulated p rotein phosphorylation in the depolarized myocytes. Depolarization was found to have little effect on the muscarinic inhibitory action on ph osphorylation. Prior treatment of myocytes with PMA, was found to augm ent ISO-stimulated protein phosphorylation in the depolarized myocytes . Such augmented increases were completely blocked by propranolol. For skolin also stimulated PLN and TN-I phosphorylation. Prior exposure of myocytes to forskolin followed by incubation in the depolarized and p olarized media showed that PLN was dephosphorylated more rapidly in th e depolarized myocytes. The results support the view that both cyclic AMP and calcium signals cooperatively increase the rates of phosphoryl ation of TN-I and PLN in the depolarized cardiomyocytes during beta-ad renergic stimulation. The results raise the additional possibility tha t the calcium signal may regulate the dephosphorylation of PLN in the depolarized cell. While muscarinic attenuation of beta-adrenergic acti on on protein phosphorylation was mediated, in part, by decreased AC a ctivity, and muscarinic inhibition of AC and protein phosphorylation w as not detectably influenced by the depolarization, the evidence was s een that muscarinic stimulation of dephosphorylation mechanisms are in timately involved. The postulate that the simultaneous stimulation of alpha(1)-adrenoceptors inhibits beta-adrenergic stimulation PLN and TN -I phosphorylation is supported.