title: Regulation of phospholamban and troponin-I phosphorylation in the intact rat cardiomyocytes by adrenergic and cholinergic stimuli: roles of cyclic nucleotides, calcium, protein kinases and phosphatases and depolarization.
abstract: Protein phosphorylation was investigated in [32P]-labeled cardiomyocytes isolated from adult rat heart ventricles. The beta-adrenergic stimulation (by isoproterenol, ISO) increased the phosphorylation of inhibitory subunit of troponin (TN-I), C-protein and phospholamban (PLN). Such stimulation was largely mediated by increased adenylyl cyclase (AC) activity, increased myoplasmic cyclic AMP and increased cyclic AMP dependent protein kinase (A-kinase)-catalyzed phosphorylation of these proteins in view of the following observations: (a) dibutyryl-and bromo-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 beta 2-AR only marginally increased while the stimulation of beta 1-AR markedly increased PLN phosphorylation. Other stimuli that increase tissue cyclic AMP levels also increased PLN and TN-I phosphorylation and these included isobutylmethylxanthine (non-specific phosphodiesterase inhibitor), milrinone (inhibits cardiotonic inhibitable phosphodiesterase, sometimes called type III or IV) and forskolin (which directly stimulates adenylyl cyclase). Cholinergic agonists acting on cardiomyocyte M2-muscarinic receptors that are coupled to AC via pertussis toxin(PT)-sensitive G proteins inhibited AC and attenuated ISO-dependent increases in PLN and TN-I phosphorylation. The in vivo PT treatment, which ADP-ribosylated Gi-like protein(s) in the myocytes, markedly attenuated muscarinic inhibitory effect on PLN and TN-I phosphorylation on one hand and, increased the beta-adrenergic stimulation, on the other. Controlled exposure of isolated myocytes to N-ethyl maleimide, also led to the findings similar to those seen following the PT treatment. Exposure of myocytes to phorbol, 12-myristate, 13-acetate (PMA) increased the protein phosphorylation, augmenting the stimulation by ISO, and such augmentation was antagonized by propranolol suggesting modulation of the beta-adrenoceptor coupled AC pathway by PMA. Okadaic acid (OA) exposure of myocytes also increased protein phosphorylation with the results supporting the roles for type 1 and 2A protein phosphatases in the dephosphorylation of PLN and TN-I. Interestingly OA treatment attenuated the muscarinic inhibitory effect which was restored by subsequent brief exposure of myocytes to PMA. While the stimulation of alpha adrenoceptors exerted little effect on the phosphorylation of PLN and TN-I, inactivation of alpha adrenoceptors by chloroethylclonidine (CEC), augmented beta-adrenergically stimulated phosphorylation. KCl-dependent depolarization of myocytes was observed to potentiate ISO-dependent increase in phosphorylation (incubation period 15 sec to 1 min) as well as to accelerate the time-dependent decline in this phosphorylation seen upon longer incubation. Verapamil decreased ISO-stimulated protein phosphorylation in the depolarized myocytes. Depolarization was found to have little effect on the muscarinic inhibitory action on phosphorylation. Prior treatment of myocytes with PMA, was found to augment ISO-stimulated protein phosphorylation in the depolarized myocytes. Such augmented increases were completely blocked by propranolol. Forskolin also stimulated PLN and TN-I phosphorylation. Prior exposure of myocytes to forskolin followed by incubation in the depolarized and polarized media showed that PLN was dephosphorylated more rapidly in the depolarized myocytes. The results support the view that both cyclic AMP and calcium signals cooperatively increase the rates of phosphorylation of TN-I and PLN in the depolarized cardiomyocytes during beta-adrenergic stimulation. (ABSTRACT TRUNCATED)
authors.affiliation: Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Canada.
authors.forename: P V
authors.initials: PV
authors.lastname: Sulakhe
authors.forename: X T
authors.initials: XT
authors.lastname: Vo
mesh_terms: D000262:Adenylyl Cyclases / Q000502:physiology; D000322:Adrenergic Agonists / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D008297:Male; D008564:Membrane Potentials; D009206:Myocardium / Q000378:metabolism*; D009712:Nucleotides, Cyclic / Q000502:physiology*; D010703:Phorbol Esters / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D011494:Protein Kinases / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D011942:Receptors, Adrenergic, alpha / Q000502:physiology*; D011943:Receptors, Adrenergic, beta / Q000502:physiology*; D011976:Receptors, Muscarinic / Q000502:physiology*; D015398:Signal Transduction; D014336:Troponin / Q000378:metabolism*; D019210:Troponin I; D014700:Verapamil / Q000494:pharmacology; D000099211:Phospholamban
chemical_list: D000262:Adenylyl Cyclases; D000322:Adrenergic Agonists; D002118:Calcium; D002121:Calcium Channel Blockers; D002135:Calcium-Binding Proteins; D018679:Cholinergic Agonists; D017868:Cyclic AMP-Dependent Protein Kinases; D009712:Nucleotides, Cyclic; D010703:Phorbol Esters; D010726:Phosphodiesterase Inhibitors; D010749:Phosphoprotein Phosphatases; D011493:Protein Kinase C; D011494:Protein Kinases; D011942:Receptors, Adrenergic, alpha; D011943:Receptors, Adrenergic, beta; D011976:Receptors, Muscarinic; D014336:Troponin; D019210:Troponin I; D014700:Verapamil; D000099211:Phospholamban