The objective of these studies is to determine the sites and mechanisms of action of selected neuropeptides that are candidate neurotransmitters in the sympathetic control of the gastrointestinal tract and its vasculature. These peptides include the opioids enkephalin and dynorphin and the cholecystokinins. Two primary techniques will be used in abdominal sympathetic ganglia and mesenteric blood vessels of the guinea pig to investigate the role of these peptides in sympathetic function. Intracellular microelectrode recording in prevertebral sympathetic ganglia and mesenteric blood vessels in in vitro preparations which maintain natural attachments between the ganglia and the end organs will be used to characterize the responses to neuropeptides. Patch-voltage clamp recording from prevertebral ganglionic neurons in primary tissue culture will be used to determine the cellular mechanisms of action of the neuropeptides on sympathetic neurons. Neuropeptides will be administered to ganglion-blood vessel, ganglion-gut and single neuron preparations both by superfusion and by pressure ejection from micropipettes. Presynaptic efferent and afferent nerve fibers entering the abdominal prevertebral sympathetic ganglia which innervate the gastrointestinal tract will be stimulated electrically. In the ganglia, the neuropeptides will be evaluated for their effects on the excitability of ganglionic neurons and on the release of neurotransmitters from presynaptic nerve fibers. In the blood vessels, the effects on the excitability of vascular smooth muscle cells and on the amplitude of nerve-evoked potentials in vascular smooth muscle cells will be compared in arteries and veins. To determine if these neuropeptides are released from nerve fibers the responses of ganglionic neurons to application of these peptides will be compared to the response of these same neurons to electrical stimulation of the nerve fibers. With patch-voltage clamp recording we will test the hypothesis that neuropeptides decrease a potassium conductance to produce the characteristic depolarization. With whole cell voltage clamp recording the effects of neuropeptides on macroscopic inward and outward currents will be determined. With single channel recording the reversal potential, single channel conductance and the ionic specificity of the channels affected by application of neuropeptides will be determined. The use of specific receptor antagonists either commercially available or developed by other participants in the Program will enable determination of the specific receptor subtypes subserving the responses. All of the functions of the gastrointestinal tract are modulated by the sympathetic nervous system. Because peptides are so prevalent within the sympathetic nervous system innervating the gastrointestinal tract, the elucidation of the role of these peptides which have been implicated as neurotransmitters will contribute to our understanding pathology of the gastrointestinal tract, especially conditions involving malfunction of the sympathetic nervous syst .
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