The goals of the current project are to study the mechanisms which regulate the activity and levels of guanine nucleotide regulatory proteins (G proteins) in embryonic chick heart and to determine the role of the regulation of G proteins in modulating the responsiveness of the heart to muscarinic stimulation. Specifically, we will test the hypothesis that treatment with phorbol esters, prolonged exposure to muscarinic agonist results in phosphorylation or other modification of G proteins which render them incapable of coupling the muscarinic receptor to a physiologic response. We have demonstrated that under conditions where levels of the 39 Kda(alpha 39) and 41 Kda(alpha) pertussis toxin substrate were limiting, in both atrial cells pretreated with pertusssis toxin and cultures of hears 31/2days in ovo. muscarinic stimulation resulted in a positive chronotropic response. Hence we will test the hypothesis that the levels of G proteins are important in controllin the extent of coupling of muscarinic receptors to muscarinic functions which are insensitive to pertussis toxin and which include the positive chronotropic response to muscarinic stimulation such as production of diacylglycerol, activation of protein kinase C, release of arachidonic acid, and activation of Na+/H antiporter. Furthermore, we will test the hypothesis that those stimuli which increse the responsiveness of the heart to muscarinic stimulaiton such as growth of heart cells in lipoproteindepleted serum, and coculture of embryonic chick hearts 31/2 days in ovo with ciliary ganglia, do so by stimulating the appearance of increased levels of low affinity muscarinic receptors followed by the synthesis of increased levels of G proteins and the subsequent interconversion of low affinity receptors to a haigh affinity form and that the time course of the increase in new receptors and G proteins correlates with the appearance of increasedmuscarinic inhibition of adenylate cyclase, increased stimulation of K efflux, and increased sensitivity of the inotropic and chronotropic response of the heart to muscarinic stimulation. We will further test the hypothersis that the appearance of the increased levels of G protein represents new protein synthesis as demonstrated by the time course of appearance of increased levels of messenger RNAs coding for alpha39 and/or alpha41. These studies will help us understand the mechanism of regulation of muscarinic responsiveness and the role of G proteins in setting the chromotropic and inotropic state of the heart.
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