This application seeks to investigate the mechanisms by which opiates and other inhibitory neurotransmitters modulate synaptic transmission. in particular it has been shown that neuropeptide Y (NPY), a widely distributed neuropeptide, can inhibit neurotransmitter release throughout the central and peripheral nervous systems. We have demonstrated that NPY is a powerful inhibitor of Ca2+ currents in a variety of neurons. In the present application we shall propose experiments designed to investigate the way in which NPY, opiates and norepinephrine modulate Ca2+ signals in rat myenteric plexus neurons in vitro. We shall investigate whether inhibition of the Ca2+ current by these neurotransmitters involves a G-protein. The specificity of receptor/G-protein/Ca2+ channel interactions will be explored by reconstituting the system in pertussis toxin treated neurons and Xenopus oocytes using purified or recombinant G-protein alpha-subunits of various types, some of which will carry specific mutations. We shall also investigate whether the same neurotransmitters also modulate K+ channels in myenteric plexus neurons and whether the specificity of G-protein mediated coupling in this case is the same or different as that for receptor/Ca2+ channel interactions. We shall also use a combined patch clamp/microfluorimetry technique to investigate the effects of inhibitory neurotransmitters on [Ca2+](i) transients in myenteric plexus neurons. We shall attempt to elucidate those processes that are important for buffering [Ca2+](i) increases elicited by physiological stimuli and to analyze how these various processes can be regulated by inhibitory neurotransmitters. These investigations should be of importance in understanding the cellular basis of opiate action in the nervous system.
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