Sub-second Kinetics of the Nitric Oxide Receptor, Soluble Guanylyl Cyclase, in Intact Cerebellar Cells

Abstract

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. Nitric oxide (NO)1 performs diverse biological functions, ranging from relaxation of smooth muscle and inhibition of platelet aggregation to neural signaling in the peripheral and central nervous systems (1, 2). The principal receptor mediating the physiological actions of NO is the cGMP-synthesizing enzyme, soluble guanylyl cyclase (sGC). Exposure to NO increases the sGC catalytic activity up to several hundred-fold (3-5), and the resulting cellular accumulation of cGMP can engage a number of downstream targets, including cGMP-dependent protein kinase (6), cGMP-regulated phosphodiesterases (7), and cyclic nucleotide-gated ion channels (8) to bring about the various biological effects.Since the purification of sGC in the late 1970s, much knowledge of the structure and the mechanism of activation of the enzyme by NO has accrued (9). The enzyme is an ␣␤-heterodimer with a prosthetic heme group, the NO binding site, attached to the ␤-subunit. Somewhat unexpectedly by comparison with analogous receptors, sGC exhibits limited molecular heterogeneity; only 2 ␣-and 2 ␤-subunits have been identified, and only 2 isoforms have so far been found to exist at the protein level as follows: ␣ 1 ␤ 1 , which is widespread, and ␣ 2 ␤ 1 , which is found in human placenta (10). The two isoforms appear to have similar functional and pharmacological properties (10).As with any other signaling molecule, knowledge of the kinetic properties of the receptor is a prerequisite for understanding how the signals are decoded. When studied in tissue homogenates or in its purified form, sGC exhibits simple Michaelis-Menten-type kinetics. Consequently, in the presence of NO, excess substrate (GTP), and cofactor (Mg 2ϩ ), sGC synthesizes cGMP at a constant rate over long periods, more in ...

On the activation of soluble guanylyl cyclase by nitric oxide

“…Recent evidence has revealed that the NO receptor behaves substantially differently within intact cells from its behavior in the test tube. In particular, the rate of deactivation is Ϸ25-fold faster than even the highest estimate for purified sGC (27), and the receptor exhibits a desensitizing profile of catalytic activity (28). The mechanistic details of this discrepancy in behavior are unknown at present.…”

Kinetics of a Cellular Nitric Oxide/cGMP/Phosphodiesterase-5 Pathway

“…The receptors are ␣␤-heterodimers, of which two main isoforms, ␣ 1 ␤ 1 and ␣ 2 ␤ 1 , exist at differing levels in different tissues (5). Studies on purified receptors (6) and intact cells (7) suggest that activation of GC occurs very rapidly, within a few ms or less of adding NO, and complete deactivation in cells (upon removal of NO) requires only about 500 ms (7). Furthermore, when studied in isolation, the receptors are highly sensitive NO detectors, with only about 1 nM being needed to evoke half-maximal GC activity (8).…”

Bioanalytical profile of the l-arginine/nitric oxide pathway and its evaluation by capillary electrophoresis

“…Briefly, purified sGC in vitro has an EC 50 of 85-250 nM of NO [217,218], is fully activated by NO within a few milliseconds [219], remains constantly active upon available substrate and ligand, and deactivates in a seconds-to-minutes range after NO disappears from the reaction scene [220,221]. sGC in vivo exhibits EC 50 of 45 nM of NO and an approximately 25-fold faster deactivation [222]. Downstream cGMP signaling cascades include stimulation of G-family protein kinases, altering cGMP-gated ion channels, and modulation of cGMP-binding phosphodiesterases [206].…”