The overall objective of the proposed research is to further our knowledge of the importance of adenosine derivatives at vertebrate synapses. In the preceding grant period, the applicants found that endogenous adenosine, apparently derived from neurally-released ATP, may be the cause of neuromuscular depression in the frog. The applicants also found that ATP, via an action on receptors that incorporate ion channels, is a fast excitatory neurotransmitter between mammalian neurons in culture. In the ensuing period of support, the applicants will use electrophysiological methods towards the following three specific aims: 1) to study further the possibility that endogenous adenosine derived from ATP is the mediator of neuromuscular depression in the frog by determining if quantal ATP release can be detected in conjunction with quantal acetylcholine (ACh) release. The applicants will use outside-out patches from acutely dissociated neurons in celiac ganglion to perform these experiments. These patches contain both ATP and ACh receptors. Their results from this past period of support suggest that adenosine inhibits ACh release in frog by reducing the apparent affinity of Ca2+ for a strategic component of the secretory apparatus. Is this effect mediated by an inhibition of phosphoinositide turnover, as has been suggested by others? 2) The applicants will examine the mechanism by which adenosine derivatives inhibit ACh release from mammalian motor nerve endings and its physiological importance. Is ATP the mediator of neuromuscular depression at mouse motor nerve endings and if so, what is the mechanism? Are Ca2+ currents reduced and/or is the ability of intracellular Ca2+ to promote ACh release impaired? Is a decrease in cyclic AMP concentration the mediator of inhibition as suggested previously? 3) Specific Aim 3 will study further the action of ATP as a fast excitatory transmitter. The proposed experiments on neuromuscular junctions could result in almost immediate clinical relevance. Specifically, if endogenous adenosine is the mediator of neuromuscular depression in mammalian species, then it is possible that adenosine receptor antagonists can be used to prevent the debilitating neuromuscular depression that occurs in patients with myasthenia gravis. The results with ATP will also provide valuable additional knowledge on the basic physiological properties of purinergic synapses and their relationship to nicotinic synapses in mammals.
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