The force and rate of the heart beat are regulated through release of acetylcholine (Ach) and norepinephrine (NE) by the autonomic nervous system, however, the molecular mechanisms of this regulation are poorly understood. While it is certain that NE acts partly through the cyclic-AMP system the mechanisms of Ach action are unknown. An understanding of these mechanisms is important for understanding the normal functioning heart and for understanding cardiac disease. For example, T.N. James and others have implicated cardiacneuropathies with certain kinds of sudden death. Ach decreases heart rate by activating a hyperpolarizing background potassium current and decreases the force of contraction by decreasing calcium entry into the cell. The slow onset (-100 msec delay) and long duration (several seconds) in response to a brief pulse of Ach suggest a multistep mechanism with, possibly, one or more enzymatic reactions. The experiments described by this research proposal will test the hypothesis that the action of Ach in the heart is dependent on coupling of the muscarinic cholinergic receptor to a specific GTP binding protein distinct from the GTP protein associated with beta-adrenergic stimulation of adenylate cyclase. The overall approach will be to inhibit the GTP protein with a toxin from Bordetella pertussis or elevate and lower the intracellular levels of GTP. The effects of these treatments on Ach activation of potassium current, inhibition of calcium current and inhibition of calcium current increased by the action of NE will be measured in single cardiac cells of the frog atrium and sinus venosus using the sensitive patch clamp technique in the whole-cell recording configuration. The results from this work will contribute to the understanding of the molecular mechanisms which regulate the heart beat and facilitate an understanding of cholinergic mechanisms elsewhere in the body.
