Rate of deactivation of nitric oxide-stimulated soluble guanylate cyclase:Influence of nitric oxide scavengers and calcium

Soluble guanylate cyclase (sGC) is highly activated by nitric oxide (NO) an d is the known mediator of the effects of NO on a variety of physiological processes. The rates at which sGC is activated and deactivated are therefor e of wide interest since they determine the duration of a tissue's response to NO. The effect of NO on smooth muscle dissipates in 1-2 min, suggesting that both activation and deactivation are fast. In vitro measurements show that the activation of sGC occurs in less than a second, while the deactiv ation takes several hours at 20 degrees C. However, recent reports indicate that Mg-GTP, oxyhemoglobin, and reducing and oxidizing agents could deacti vate the cyclase in several seconds to minutes, though the effectiveness of each of these agents is in dispute, We investigated the lifetime of NO-sGC in the cytosol of retina by monitoring its enzymatic activity at 20 OC. Ou r results show that ME-GTP, the substrate of NO-sGC, has no influence on th e deactivation. Similarly, reducing agents glutathione and dithiothreitol s hortened the half-life of NO-sGC only by about 30%. The greatest effect on the deactivation was caused by scavengers of NO: oxyhemoglobin reduced the half-life of NO-sGC from 106 min to 18 s; another NO scavenger, 2-(4-carbox yphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), reduced it to 42 s (20 degrees C). Similarly rapid deactivation was observed with the enzyme from bovine lung, immunoprecipitated enzyme from bovine retina, and heme-deficient enzyme from bovine retina reconstituted with heme. On the ot her hand, YC-1, an activator of sGC, stabilized the activated enzyme, preve nting NO dissociation, as was evident from the inability of oxyhemoglobin o r CPTIO to deactivate NO-sGC. Calcium, which is known to inhibit NO-sGC, al so inhibited the effects of oxyhemoglobin and CPTIO, slowing down the deact ivation of the enzyme. Lithium, which is also known to inhibit NO-sGC, had no effect on the deactivation rate of the enzyme. These results, taken toge ther, suggest that two factors with major impact on the lifetime of NO-sGC are the proximity to NO scavengers and the calcium concentration in the cel l.