The overall significance of our proposed studies is to further the understanding of the physiological relevance of peptide action in neurotransmitter function in general and on adrenomedullary secretion in particular. Pilot studies have shown that low concentrations (1 X 10-8 M) of neuropeptide Y (NPY) inhibit nicotine stimulated adrenomedullary secretion. Our hypothesis is that NPY exerts its effect on adrenomedullary catecholamine secretion by modulating nicotinic receptor stimulation.
The specific aims are to : 1) delineate the effects and structural requirements of NPY on nicotinic receptor stimulation, 2) determine whether NPY in chromaffin cells influences secretion, 3) describe NPY binding and its effect on agonist binding to the nicotinic receptor, and 4) identify and characterize the role of G-binding proteins and phosphoinositide turnover in the modulatory action of NPY. The modulatory effect of NPY will be examined by measuring the nicotine receptor-induced secretion of epinephrine, norepinephrine and the enkephalin-like peptides (ME-IRM) using cultured bovine chromaffin cells. Catecholamine and ME-IRM secretion will be measured by high performance liquid chromatography with electrochemical detection, and with radioimmunochemical techniques using a specific antibody to (Met5)-enkephalin, respectively. NPY influence on secretion will be determined by studying it's action in the presence of various nicotinic agonists, it's action on agents stimulating secretion through other than nicotinic receptors and the effect of truncated forms of NPY and peptides possessing sequence homologies with NPY. The role of endogenous NPY will be examined through the use of antibodies and antisense RNA to NPY. The mechanism of the modulatory effect of NPY on secretion will be examined by studying the effect of NPY on nicotinic receptor binding and on changes produced in the second messengers (G-binding proteins, phosphoinositide turnover) that may be involved in the transduction of the message received during nicotinic receptor stimulation. We will employ the techniques of cell culture, radioimmunoassay, high performance liquid chromatography, antisense oligonucleotide probes, radioreceptor binding, ion-exchange chromatography and thin layer chromatography. The health related consequences of the proposed studies are far-reaching due to the varied roles suggested for neuropeptides in physiology and pathology, e.g., defective neuronal regulation leading to hypertension or mental disorders.
