This grant application proposes to study muscarinic stimulation under several conditions. In the first specific aim the applicant will determine the mechanism for muscarinic inhibition of the heart in the presence of sympathetic stimulation. Questions to be studied will include the role of cGMP and its function to promote cGs-PDE and to activate G-kinase in muscarinic inhibition of cAMP stimulated calcium currents in ventricular myocytes. Specific inhibitors of CGs-PDE and G-kinase will be used. In the second portion of specific aim #1 the applicant will use the specific inhibitor of cGMP-production, LY83583 or pertussis toxin to determine the role of cGMP inhibition by muscarinic agonists. In the aim A3 the applicant will study the role of protein phosphatase in the inhibition of Ica and contraction in ventricular myocytes. Specifically the applicant will determine whether okadaic acid, an inhibitor of phosphatase one and 2A blocks inhibition of Ica and contractions by carbamylcholine and cGMP.
In specific aim 2 the applicant will study the mechanism for the stimulation of the heart by muscarinic agonists in the presence of sympathetic activation. Specifically the applicant will determine whether, in the presence of low doses of isoproterenol, carbamylcholine increases intracellular calcium transients and contraction in conjunction with an increase in Ica and the role of cGMP, effects of cGMP inhibitors and inhibition of cGMP production, LY83583, on the positive inotropic response. The applicant will determine whether selective blockers of cGMP-inhibited PDE, milrinone, interfere with the stimulatory effect of muscarinic agonists. If no effect of PDE blockers on the stimulatory response to muscarinic stimulation are seen, the applicant will study whether or not a pertussis toxin insensitive stimulation of IP3 production is involved in this process. In the third specific aim the applicant will study the mechanism for muscarinic stimulation of the heart in the absence of sympathetic activation. The applicant will determine what subtype of the muscarinic receptor is involved in the positive inotropic effect of muscarinic agonist and the role of sodium calcium exchange in this process.
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