Inhibition of nitric oxide-activated guanylyl cyclase by calmodulin antagonists.

BACKGROUND AND PURPOSE: Nitric oxide (NO) controls numerous physiological processes by activation of its receptor, guanylyl cyclase (sGC), leading to the accumulation of 3'-5' cyclic guanosine monophosphate (cGMP). Ca(2+)-calmodulin (CaM) regulates both NO synthesis by NO synthase and cGMP hydrolysis by phosphodiesterase-1. We report that, unexpectedly, the CaM antagonists, calmidazolium, phenoxybenzamine and trifluoperazine, also inhibited cGMP accumulation in cerebellar cells evoked by an exogenous NO donor, with IC(50) values of 11, 80 and 180 microM respectively. Here we sought to elucidate the underlying mechanism(s). EXPERIMENTAL APPROACH: We used cerebellar cell suspensions to determine the influence of CaM antagonists on all steps of the NO-cGMP pathway. Homogenized tissue and purified enzyme were used to test effects of calmidazolium on sGC activity. KEY RESULTS: Inhibition of cGMP accumulation in the cells did not depend on changes in intracellular Ca(2+) concentration. Degradation of cGMP and inactivation of NO were both inhibited by the CaM antagonists, ruling out increased loss of cGMP or NO as explanations. Instead, calmidazolium directly inhibited purified sGC (IC(50)= 10 microM). The inhibition was not in competition with NO, nor did it arise from displacement of the haem moiety from sGC. Calmidazolium decreased enzyme V(max) and K(m), indicating that it acts in an uncompetitive manner. CONCLUSIONS AND IMPLICATIONS: The disruption of every stage of NO signal transduction by common CaM antagonists, unrelated to CaM antagonism, cautions against their utility as pharmacological tools. More positively, the compounds exemplify a novel class of sGC inhibitors that, with improved selectivity, may be therapeutically valuable.

Sub-second kinetics of the nitric oxide receptor, soluble guanylyl cyclase, in intact cerebellar cells.

Soluble guanylyl cyclase (sGC) catalyzes cGMP synthesis and serves as a physiological receptor for nitric oxide (NO). Recent evidence indicates that key properties of sGC within cells differ from those of purified sGC. We have devised a technique for resolving NO-stimulated sGC activity in cells on a sub-second time scale, enabling the first quantitative description of the kinetics of the enzyme within its natural environment. Upon release of NO from a caged derivative, sGC became activated without any lag observable at a 20-ms sampling time. Deactivation of sGC on removal of NO occurred with a rate constant of 3.7 s(-)(1), which is 25-fold faster than the fastest estimate for purified sGC. Desensitization of sGC occurred with a time constant of 6.9 s at an estimated 70 nm NO and became faster at a higher concentration, indicating that NO accelerates desensitization. The concentration-response curve for NO consequently became increasingly bell-shaped with time, a phenomenon that causes the apparent potency of NO to increase with time. The results indicate that sGC within cells behaves in a highly dynamic fashion, allowing the NO-cGMP pathway to operate within a kinetic framework more resembling that of neurotransmission than the properties of purified sGC suggest.

"cAMP-specific" phosphodiesterase contributes to cGMP degradation in cerebellar cells exposed to nitric oxide.

Nitric oxide (NO) functions as a diffusible messenger in the central nervous system and elsewhere, exerting many of it physiological effects by activating soluble guanylyl cyclase, so increasing cellular cGMP levels. Hydrolysis of cyclic nucleotides is achieved by phosphodiesterases (PDEs) but the enzyme isoforms responsible for degrading cGMP in most cells have not been identified. We have devised a method for quantitatively monitoring the rate of breakdown of cGMP within intact cells and have applied it to rat cerebellar cell suspensions previously stimulated with NO. In contrast to previous findings in cultured cerebellar cells, there was no evidence from the use of selective inhibitors that PDE 1 participated importantly in cGMP hydrolysis. Moreover, procedures expected to increase PDE 1 activity by raising cytosolic Ca2+ concentrations (neurotransmitter agonists, Ca2+ ionophore) failed to influence cGMP breakdown. Instead, through the use of inhibitors selective for different PDE families, two isoforms were implicated: a "cGMP-specific" PDE (PDE 5), inhibited by sildenafil and zaprinast, and a "cAMP-specific" PDE (PDE 4), inhibited by low concentrations of rolipram and Ro-20-1724 and by milrinone. An explanation is offered for a participation of PDE 4 based on the high estimated intracellular cGMP concentration (approximately 800 microM) and the low affinity of the enzyme for cGMP. In accordance with predictions, recombinant PDE 4 was shown to hydrolyze high cGMP concentrations in a rolipram-sensitive manner. The widespread use of rolipram to test for a specific involvement of cAMP in cellular phenomena must therefore be questioned.

Rapid desensitization of the nitric oxide receptor, soluble guanylyl cyclase, underlies diversity of cellular cGMP responses.

A major receptor for nitric oxide (NO) is the cGMP-synthesizing enzyme, soluble guanylyl cyclase (sGC), but it is not known how this enzyme behaves in cells. In cerebellar cells, NO (from diethylamine NONOate) increased astrocytic cGMP with a potency (EC(50)