The proposed research has as its major long-term goal the understanding of the molecular basis of neurotransmission at muscarinic acetylcholine, histamine H1 and bradykinin receptors, the agonists for which are putative neurotransmitter in the CNS. In addition, a recent discovery in the applicant's laboratory has led to the inclusion of thrombin, the central enzyme in hemostasis, and its receptor in these studies. All these receptors when activated by their respective ligands stimulate cyclic GMP synthesis by a widely-studied murine neuroblastoma clone (N1E-115). The link between receptor activation and the production of cyclic GMP is being sought in these studies since our recent data show that this link is not Ca++ as is generally thought. Based in part on the results with thrombin, we plan to test the following hypothesis with clone N1E-115 cells. Receptors that mediate cyclic GMP synthesis do so by ultimately stimulating phospholipase A2 activity, liberating arachidonic acid (AA) which is then metabolized into a product that stimulates quanylate cyclase. Additionally, we hypothesize that another AA metabolite is later synthesized which inhibits the cyclase, thereby turning off the receptor-mediated response (desensitization). Studies are also proposed to characterize further the interactions of thrombin and bradykinin with their receptors on these cells. Methods to be used include cell culture; precursor labeling for measuring cyclic GMP synthesis by intact cells; chromatographic techniques including thin-layer, highperformance liquid and gas chromatography to measure changes in the membrane phospholipid profile and AA metabolism of intact cells exposed to various agonists; gas chromatography/mass spectrometry to identify putative metabolites of AA which may affect the activity of guanylate cyclase; and radioligand binding assays to characterize the interactions of thrombin and bradykinin with their receptors. In all these studies, various psychotherapeutic drugs will be tested for their effects on events that occur beyond the receptor site. These studies will increase our understanding of certain receptors that are affected by many antidepressants and neuroleptics (muscarinic and histamine H1) and one (muscarinic) that seems to be involved with memory and learning. In addition, results from this work may lead to our understanding of the basis of short-term changes in the sensitivity of certain neurotransmitter receptors.
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