The long range objectives of these studies is to determine the biochemical and molecular mechanisms by which neuronal stimuli, ion channels and autonomic receptors interact to modulate cardiac function and cardiac excitability. The goals of these studies are to study the interaction of muscarinic agonist with the receptor and the guanine nucleotide regulatory protein in mediating a physiologic response and to determine 1) the role of high and low affinity muscarinic receptors, 2) the role of guanine nucleotide regulatory proteins in coupling the receptor to a physiologic response, 3) the role of parasympathetic innervation of the heart in inducing the development of cardiac responsiveness to muscarinic stimulation. Comparison of the muscarinic response in cultures of embryonic chick atrium and ventricle will allow us to study two muscarinic effects in separate tissues and to test the hypothesis: 1) that binding of muscarinic agonsit to low affinity sites in the ventricle is coupled to a decrease in c-AMP levels, a decrease in Ca+2 permeability as measured by 45Ca+ uptake, a shortening of the action potential duration and a negative inotropic response 2) that binding of muscarinic agonist to high affinity sites in atrium is coupled to an increase in K+ permeability as measured by 42K+ efflux a hyperpolarization of the resting membrane potential and a negative chronotropic response and a shortening of the duration of the action potential, a secondary decrease in 45Ca+ uptake and a negative inotropic effect. 3) That the inhibitory guanine nucleotide regulatory protein (Ni) can exist in several states characterized by differences in hormone specific GTPase activity, affinity for GTP as measured by 8-azido-GTP binding and differences in amino acid sequence or post-translational modification and that only a specific state of Ni is capable of coupling the muscarinic receptor to inhibition of adenylate cyclass activity. These studies should increase our understanding of the interaction of ion channels, autonomic receptors and the factors which couple the receptor to a physiologic response in determining the chronotropic and inotropic state of the heart and in the genesis of arrhythmias.
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