Stimulation of m1 muscarinic receptors is associated with a second messenger cascade that previous workers have proposed proceeds as follows: Stimulated receptors coupled via G proteins activate phospholipase C stimulating the production of IP3 which liberates Ca+2 from cellular stores which stimulates NOS to hydrolyze L-arginine and form NO and L-citrulline. NO stimulates the enzyme guanylate cyclase to form cyclic GMP. The applicant presents hypotheses and data to support the hypotheses that this scheme may not be totally correct, at least in a causal sense. In particular, the accumulation of L-citrulline occurs over many minutes while the levels of NO and cyclic GMP rise and fall transiently. The applicant plans to use a newly developed microelectrode to accurately and sensitively measure NO and to correlate the time courses and dose-response curves for these three parameters in the hopes of more fully understanding the interrelationships. Additionally, the applicant proposes that the stimulation of NOS has an early and a late phase with the former being mediated by IP3-dependent and the latter being mediated by IP3-independent pathways. Preliminary data presented supports this hypothesis and a series of experiments to further test it are proposed. The applicant also proposes to study the phenomenon of agonist-induced desensitization of NOS. This desensitization appears to occur rapidly (minutes) and is manifested by a blunted cyclic GMP response. On the other hand, in this same rapid time frame, receptor stimulation of L-citrulline accumulation is not blunted. Experiments will be conducted in which NO is directly measured and this parameter compared to the others with the goal to test the hypothesis that agonist-induced desensitization of NO generation is due to a decrease in the coupling of the receptor to one or more of the steps in the signalling pathway involved in the elevation of Ca+2. Finally, a phenomenon is described in which cells incubated for 48 hours with agonist have an increased response to rechallenge by agonist. Experiments to examine the mechanism of this anomalous result are proposed. It is hypothesized, and the preliminary data support, that an increase in NOS protein and perhaps an increase in the sensitivity of the enzyme to Ca+2 are involved.
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