Central and ganglionic nicotinic acetylcholine receptors (nAChR) may play a role in the etiology of some important acute and chronic manifestations of alcohol abuse. In a number of species, including humans, nAChR-mediated epinephrine release from the adrenal medulla is enhanced by ethanol, an effect which may lead to cardiac hypertrophy in chronic consumers. Elsewhere in the sympathetic nervous system ethanol inhibits nAChR-mediated norepinephrine release. Such paradoxical actions are also observed in the CNS, where chronic ethanol treatment leads to increases in nAChR number in some brain regions, but decreases in others. Our hypothesis is that ethanol's effects in vivo are dependent on the rate and duration of ACh stimulation. It augments responses to low frequency stimuli, such as that maintaining sympathetic tone, but under continuous stimulation it enhances desensitization or down-regulation. Neuronal nAChR's are poorly characterized, but supporting evidence comes from comparing ethanol's actions to those of longer alcohols (and anesthetics) at closely related nAChR's of muscle endplates and electric organs of fish. Unlike other anesthetics, ethanol enhances the initial channel opening response to acetylcholine (ACh), but like other anesthetics, it also accelerates the desensitization of nAChR during prolonged exposure to ACh.
Out first aim i s to characterize in detail ethanol's effects on all the established conformational transitions (ACh binding, channel opening and closing, fast and slow desensitization, and ACh self- inhibition) of the nAChR, using nAChR-rich vesicles from Torpedo electroplaque. Will this allow one to describe the phenomena outlined above? Our second aim is to determine the underlying mechanisms of ethanol's actions at nAChR. Kinetic and pharmacological criteria (including pressure reversal) will be used to distinguish non-specific from specific actions. Spin-labeled lipids will probe the state of nAChR's membrane environment in ethanol's presence. The high concentration and purity of nAChR in Torpedo vesicles makes them ideal for these studies. However, to relate our findings more directly to mammalian physiology, our third aim is to test effects observed in Torpedo vesicles in a nAChR-containing mammalian neuronal cell line, PC12.
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