Adenylyl Cyclase, a Coincidence Detector for Nitric Oxide

Abstract

Nitric oxide (NO) donors inhibit hormone-and forskolin-stimulated adenylyl cyclase activity in purified plasma membrane preparations from N18TG2 neuroblastoma cells. Northern blot analyses indicate that the predominant isoform of adenylyl cyclase in N18TG2 cells is the type VI. Our experiments eliminate all the known regulatory proteins for this isoform as possible targets of NO. NO decreases the V max of the enzyme without altering the K m for ATP. Occupancy of the substrate-binding site protects the enzyme from the inhibitory effects of NO, suggesting that the conformation of the enzyme determines its sensitivity. The inhibition is reversed by reducing agents, implicating a Cys residue(s) as the target for nitric oxide and an S-nitrosylation as the underlying modification. These findings implicate NO as a novel cellular regulator of the type VI isoform of adenylyl cyclase. Nitric oxide (NO)1 has been attributed roles in a variety of cellular activities throughout the body. For example, NO has been implicated as a regulator of vasodilation, synaptic plasticity, and immune defense. In each case, however, NO has potentially deleterious effects should its production be disturbed, as occurs in excitotoxicity and ischemia (reviewed in Refs. 1-3). To understand the means by which NO achieves its paradoxical effects, and eventually to control its actions, attempts have focused on characterizing the enzymes responsible for NO production (NOS), identifying target molecules that are altered by NO, and specifying the underlying mechanism(s) by which NO alters those targets.One target of NO is the soluble guanylyl cyclase. It is the subsequent increase in cGMP levels that is believed to mediate NO-induced vasodilation. Given that an activation of soluble guanylyl cyclase occurs almost universally in response to NO, changes in cGMP have received primary consideration as the mechanism by which NO acts. However, the multiplicity of NO actions are unlikely to be explained by a common mechanism and a number of laboratories have suggested alternative targets of NO. Generally, cellular components have been implicated as targets of NO based largely on the ability of NO or NO-releasing compounds to alter their activities in vitro. Thus, whether they are altered in intact cells in response to NO, or what the possible physiological consequences may be, remain speculative. Our approach to discerning how NO functions has been to examine its effects on intact cells. We previously demonstrated that NO inhibits the production of cAMP pulses in Dictyostelium discoideum and does so independently of any changes in guanylyl cyclase activity (4, 5). The sum of the data indicated that NO specifically alters either a regulatory domain of the adenylyl cyclase itself or a distinct regulatory moiety. We have also observed that the addition of NO gas or NO donor compounds to cultures of N18TG2 neuroblastoma cells inhibits G s -coupled and forskolin-stimulated cAMP accumulation (6). NO-mediated inhibition of forskolin-stimulated cAMP production is ...

Inducible nitric oxide synthase modulates lipolysis in adipocytes

“…We observed that inhibition of adenylyl cyclase activity and PKA completely blocked the lipolytic effect of iNOS inhibition, implying that NO suppresses lipolysis by reducing cAMP and PKA activation. Interestingly, previous reports have shown that NO inhibits adenylyl cyclase activity (29,30). Thus, the NO donor sodium nitroprusside was found to decrease forskolin-induced activation of type VI adenylyl cyclase.…”

The cholinergic influence on the mesenteric ganglion affects the liberation of ovarian steroids and nitric oxide in oestrus day rats: characterization of an ex vivo system

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Zinc Inhibition of cAMP Signaling

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