No abstract

Peroxynitrite (ONOO-), which is formed from the reaction ofnitric oxide (NO) and superoxide (O2), has been suggestd to be responsible for some of the cytotoxic effects ofthese molecules. When protonated, ONOO-gives rise to hydroxyl (OH') Nitric oxide (NO) synthesized from L-arginine is known to be an important mediator in several physiological systems. However, when generated in large quantities for long periods, mainly during immunological reactions, NO is cytostatic/cytotoxic for invading microorganisms and cancer cells and might be also damaging for the tissues that generate it (1).The mechanism of NO-induced cytostasis/cytotoxicity includes direct inhibition of mitochondrial respiration, aconitase activity, and DNA synthesis (2). However, another potential route of NO toxicity is through its interaction with other molecules, mainly superoxide anion (O°). It was originally demonstrated that NO generated from vascular cells or from leukocytes interacts with O°, leading to the reduction of its vasorelaxant and platelet antiaggregatory actions (3-5).

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 < 20 nM) higher than that reported for purified sGC. Deactivation of NO-stimulated sGC activity, studied by trapping free NO with hemoglobin, took place within seconds (or less) rather than the minute time scale reported for the purified enzyme. Measurement of the rates of accumulation and degradation of cGMP were used to follow the activity of sGC over time. The peak activity, occurring within seconds of adding NO, was swiftly followed by desensitization to a steady-state level 8-fold lower. The same desensitizing profile was observed when the net sGC activity was increased or decreased or when cGMP breakdown was inhibited. Recovery from desensitization was relatively slow (half-time ‫؍‬ 1.5 min). When the cells were lysed, sGC desensitization was lost. Analysis of the transient cGMP response to NO in human platelets showed that sGC underwent a similar desensitization. The results indicate that, in its natural environment, sGC behaves much more like a neurotransmitter receptor than had been expected from previous enzymological studies, and that hitherto unknown sGC regulatory factors exist. Rapid sGC desensitization, in concert with variations in the rate of cGMP breakdown, provides a fundamental mechanism for shaping cellular cGMP responses and is likely to be important in decoding NO signals under physiological and pathophysiological conditions. N itric oxide (NO) performs numerous physiological functions, including relaxation of smooth muscle, inhibition of platelet aggregation, and neural communication in the brain (1, 2). A major receptor for NO is the enzyme, soluble guanylyl cyclase (sGC), which catalyzes the production of the effector molecule, cGMP from GTP (3, 4).Compared with neurotransmitter receptors or related adenylyl and guanylyl cyclases (5, 6), the NO receptor enzyme appears rather unremarkable. It is composed of two different subunits (␣ and ␤), but only two isoforms have been shown to exist at the protein level: the ␣1␤1 isoform, which is expressed widely, and the ␣2␤1 isoform present in human placenta (7-9). Also, sGC appears to lack the functional complexity exhibited by related enzymes or receptors. For example, there is no established mechanism for regulation of the enzyme (e.g., by phosphorylation) and, on activation by NO, purified sGC generates cGMP at a constant rate for long periods of time (10, 11). Furthermore, the two naturally occurring sGC isoforms possess very similar functional and pharmacological properties (9).How sGC responds to NO in living cells, however, has not been investigated, nor is it understood why different cells display very different patterns of NO-stimulated cGMP accumulation ranging from a transient spike-like response (12) to a more slowly developing plateau (13). Here we have analyzed the kinetics of NO...