Differential nature of cross-talk among three G-coupled receptors regulating adenylyl cyclase in rat cardiomyocytes chronically exposed to receptor agonists.

Chronic exposure of cells to cognate agonists has been established to cause homologous desensitization of G protein-coupled receptors. In this work, we show that exposure of adult rat cardiomyocytes to isoproterenol (ISO) for 24 h led to the desensitization of beta-adrenoceptor (beta-AR) coupled adenylyl cyclase (AC) activity, which was associated with an increased inhibition of AC by M2-muscarinic receptor (MR) agonist, carbachol (Cch), and a decreased inhibition of AC by A1-adenosine receptor (AdR) agonist, N6-phenylisopropyladenosine (R-PIA). Chronic exposure of cells to Cch caused the desensitization of M2-MR-coupled AC, decreased the inhibitory action of R-PIA on AC and increased ISO-stimulated AC, while chronic exposure to R-PIA caused the desensitization of A1-AdR-coupled AC and modestly increased ISO-stimulated AC without any significant effect on Cch inhibition of the enzyme. Thus, chronic exposure of cardiomyocytes revealed for the first time a more complex and differential nature of cross-talk among the three major G-coupled receptors in modulating AC.

Phosphorylation of inhibitory subunit of troponin and phospholamban in rat cardiomyocytes: modulation by exposure of cardiomyocytes to hydroxyl radicals and sulfhydryl group reagents.

Myocytes were isolated from rat heart ventricles and then incubated with [32P]-sodium phosphate to label intracellular ATP stores. Incubations of the [32P]-labelled cardiomyocytes with a beta-adrenoceptor agonist isoproterenol (10 microM) and with a plant diterpene forskolin (100 microM) which directly stimulates adenylyl cyclase increased the phosphorylation of an inhibitory subunit of troponin (TN-I) and phospholamban (PLN). Brief exposure (1 min) of labelled myocytes to the hydroxyl radical generating system (H2O2 plus FeCl2) decreased markedly the stimulatory action of isoproterenol and forskolin on TN-I and PLN phosphorylation. Similar exposure of myocytes to 5-5'-dithiobis-nitrobenzoic acid (DTNB) a sulfhydryl oxidizing reagent exerted little inhibitory effect on the isoproterenol or forskolin stimulated TN-I and PLN phosphorylation. In contrast exposure of myocytes to low concentrations (< 50 microM) of N-ethylmaleimide (NEM) a sulfhydryl alkylating reagent augmented the stimulatory effect of isoproterenol on TN-I and PLN phosphorylation. The results further showed that brief treatment of myocytes to H2O2 plus FeCl2 markedly decreased isoproterenol-, but not forskolin-, stimulated cyclic AMP accumulation in the myocytes. The stimulatory action of NEM on the isoproterenol-stimulated TN-I and PLN phosphorylation appeared related to greater increase in the isoproterenol-stimulated cyclic AMP accumulation in the NEM-treated cardiomyocytes. The results are consistent with the postulate that hydroxyl radical exposure of cardiomyocytes blunts the beta-adrenoceptor-mediated stimulation of adenylyl cyclase leading to decreased phosphorylation of TN-I and PLN and imply that such alterations account in part the reported depressed rate of relaxation of the myocardium exposed to oxygen free radicals.

Protein phosphorylation in rat cardiac microsomes: effects of inhibitors of protein kinase A and of phosphatases.

The phosphorylation of rat cardiac microsomal proteins was investigated with special attention to the effects of okadaic acid (an inhibitor of protein phosphatases), inhibitor 2 of protein phosphatase 1 and inhibitor of cyclic AMP-dependent protein kinase (protein kinase A). The results showed that okadaic acid (5 microM) modestly but reproducibly augmented the protein kinase A-catalyzed phospholamban (PLN) phosphorylation, although exerted little effect on the calcium/calmodulin kinase-catalyzed PLN phosphorylation. Microsomes contained three other substrates (M(r) 23, 19 and 17 kDa) that were phosphorylated by protein kinase A but not by calcium/calmodulin kinase. The protein kinase A-catalyzed phosphorylation of these three substrates was markedly (2-3 fold) increased by 5 microM okadaic acid. Calmodulin was found to antagonize the action of okadaic acid on such phosphorylation. Protein kinase A inhibitor was found to decrease the protein kinase A-catalyzed phosphorylation of microsomal polypeptides. Unexpectedly, inhibitor 2 was also found to markedly decrease protein kinase A-catalyzed phosphorylation of phospholamban as well these other microsomal substrates. These results are consistent with the views that protein phosphatase 1 is capable of dephosphorylating membrane-associated phospholamban when it is phosphorylated by protein kinase A, but not by calcium/calmodulin kinase, and that under certain conditions, calcium/calmodulin-stimulated protein phosphatase (protein phosphatase 2B) is also able to dephosphorylate PLN phosphorylated by protein kinase A. Additionally, the observations show that protein phosphatase 1 is extremely active against the three protein kinase A substrates (M(r) 23, 19 and 17 kDa) that were present in the isolated microsomes and whose state of phosphorylation was particularly affected in the presence of dimethylsulfoxide. Protein phosphatase 2B is also capable of dephosphorylating these three substrates.

Altered responsiveness of guanylyl cyclase to nitric oxide following treatment of cardiomyocytes with S-nitroso-D,L-acetylpenicillamine and sodium nitroprusside.

The stimulation of cardiomyocyte guanylyl cyclase by nitric oxide (NO)-donor drugs was examined before and after exposure of these cells to the NO-donor drugs: S-nitroso-d,l-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP). Short- (2-hr) and long-term (24-hr) exposure attenuated the maximal stimulation of GC by either SNAP or SNP by up to 80% ("desensitization"). However this "desensitization" of the myocardial GC was atypical in nature in that the reduction in maximal NO-stimulated GC activity was associated with an increase in the affinity of the GC towards either NO-donor, a finding not as yet reported. There was also evidence of "cross-desensitization" of GC (e.g., SNAP exposure decreasing the stimulatory effect of SNP). Further, this is the first time that SNAP-induced desensitization of GC has been observed.

Alterations in inotropy, nitric oxide and cyclic GMP synthesis, protein phosphorylation and ADP-ribosylation in the endotoxin-treated rat myocardium and cardiomyocytes.

To evaluate the effects of the in vivo endotoxin treatment of the rat on (1) the contractile responses in the subsequently isolated papillary muscle to adrenergic and cholinergic agonists and (2) the biochemical parameters (cyclic GMP, nitric oxide synthesis, protein phosphorylation and ADP-ribosyslation) in the subsequently isolated cardiomyocytes. Following the in vivo endotoxin treatment (4 mg/kg i.p., 18 h), contractile responses to increasing amounts of isoprenaline or to increasing amounts of oxotremorine in the presence of a fixed amount of isoprenaline were determined in isolated papillary strips. Activities of nitric oxide synthase, guanylyl cyclase, as well as phosphorylation of phospholamban and troponin-inhibitory subunit, and pertussis toxin-catalyzed and endogenous ADP-ribosylations were determined in the intact cardiomyocytes and subcellular fractions. The increase in the force of contraction by isoprenaline was reduced, while its inhibition by oxotremorine was greater in the endotoxin-treated papillary strips. The activities of both nitric oxide synthase, primarily of the inducible form of the enzyme, and cytosolic guanylyl cyclase were higher while the phosphorylations of both phospholamban and troponin-inhibitory subunit were of lesser magnitude in the cardiomyocytes following the in vivo endotoxin treatment. Pertussis toxin-catalyzed ADP-ribosylation of the 41 kDa polypeptide, which is the alpha subunit of Gi, was also decreased. The results of the present study support the postulate that alterations in both the cyclic AMP and cyclic GMP signalling cascade contribute to the myocardial dysfunction caused by endotoxin and cytokines.

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.

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)