Nitric oxide (NO) is thought to relax smooth muscle cells (SMC) by stimulation of guanylate cyclase, accumulation of its product cGMP and cGMP-dependent modification of several intracellular processes via cGMP-dependent protein kinase, including activation of Ca2+-dependent K+ channels (K+ Ca) and inhibition of L-type Ca2+ channels. Recently, we showed a novel pathway for NO-induced cGMP-independent relaxation of normal rabbit aorta which is mediated by the direct effect of NO on K+ Ca. Freshly dispersed SMC from rabbit aorta also have delayed rectifier (K+dr) channels and it is not clear, if K+ Ca channel is the only target for the direct effect of NO. My preliminary data shows that NO inhibits agonist-induced dihydropyridine-insensitive Ca2+ influx into SMC in a cGMP-independent manner. I found small (3 pS) calcium-conducting nonselective cation channels (and corresponding whole-cell currents) which can be activated by agonist and are inhibited by NO and nickel, but not nifedipine. These channels could mediate agonist-induced Ca2+ influx and effect of NO on it in normal SMC. The main hypothesis of this proposal is that K+ Ca, K+ dr, L-type Ca2+ and nonselective cation channels mediate the cGMP-independent NO-induced decrease in intracellular calcium (and relaxation) of SMC and play a central role in these processes. The overall goal is to characterize the two novel channel-mediated pathways for cGMP-independent NO-induced SMC relaxation. One of these pathways starts with direct cGMP-independent activation of K+ channels and via membrane hyperpolarization can inhibit Ca2+ influx through L-type Ca2+ channels. Another pathway can be mediated by inhibition by NO of non-selective cation channels which will suppress Ca2+ influx into SMC and decrease intracellular Ca2+. I propose to determine the distinct mechanisms underlying these direct pathways, their functional role and relative importance, comparing them with the indirect cGMP-mediated effects of NO.
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