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

Abstract

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...

Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments

“…Hepes is a very commonly used biological buffer and the finding that NO was consumed by this buffer in a SOD-sensitive manner implies a continuous formation of O # d − that interacts with NO. Moreover, the effect remained marked even at the NO concentrations relevant to sGC activation, which lie in the low nanomolar range [18,19]. Consequently, Hepes buffer could introduce artifacts associated with O # d − formation over a wide range of biologically relevant NO concentrations.…”

“…To determine whether Hepesdependent NO consumption occurred at NO concentrations relevant to physiological signalling, the activity of the NO receptor enzyme, sGC, was measured. Activation of sGC by NO catalyses the production of cGMP from GTP, and occurs at low nanomolar NO concentrations [18,19]. In Tris-buffered reaction mix, DETA\NO (3 µM) stimulated cGMP accumulation from sGC at a rate of 10.1p1.6 µmol\min per mg of protein and there was no significant change in the rate in the presence of SOD ( Figure 2D).…”

A potential role for extracellular nitric oxide generation in cGMP‐independent inhibition of human platelet aggregation: biochemical and pharmacological considerations

“…In all cases, DEA/NO was incubated in the platelets for 30 s prior to the addition of 300 ml of 10% trichloroacetic acid to lyse platelets and precipitate the proteins. The 30 s time point was used so that cGMP measurement occurred shortly after peak cGMP synthesis (Bellamy et al, 2000 …”

The Regulation and Physiological Roles of the Guanylyl Cyclase Receptors.

“…Guanylyl Cyclase Rapid desensitization of sGC is seen in rat cerebellar cells and human platelets [122,135]. The desensitization occurs with a time constant of 6.9 sec at estimated 70 nM NO and becomes faster and greater as NO concentration increases [135].…”

“…Cyclase In cultured rat vascular SMCs, NO reportedly decreases sGC a1 isoform expression, which may be mediated by a cGMP-inhibited cAMP-PDE (PDE3) and cAMP-dependent protein kinase (PKA) [121,122]. NO and nerve growth factor have been reported to decrease sGC al and P1 isoform mRNA levels in cultured rat pulmonary artery SMCs and rat pheochromocytoma PC 12 cells, respectively [123,124].…”