magnesium-gtp and 3-(5--hydroxymethyl-2--furyl)-1-benzylindazole

Soluble guanylate cyclase (sGC) is highly activated by nitric oxide (NO) and 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 therefore 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 deactivation takes several hours at 20 degrees C. However, recent reports indicate that Mg-GTP, oxyhemoglobin, and reducing and oxidizing agents could deactivate 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 degrees C. Our results show that Mg-GTP, the substrate of NO-sGC, has no influence on the deactivation. Similarly, reducing agents glutathione and dithiothreitol shortened 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-carboxyphenyl)-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 other hand, YC-1, an activator of sGC, stabilized the activated enzyme, preventing NO dissociation, as was evident from the inability of oxyhemoglobin or CPTIO to deactivate NO-sGC. Calcium, which is known to inhibit NO-sGC, also inhibited the effects of oxyhemoglobin and CPTIO, slowing down the deactivation 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 together, 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 cell.

Topics: Animals; Calcium; Cattle; Cytosol; Dithiothreitol; Enzyme Activation; Enzyme Activators; Enzyme Stability; Free Radical Scavengers; Glutathione; Guanosine Triphosphate; Guanylate Cyclase; Indazoles; Nitric Oxide; Oxyhemoglobins; Reducing Agents; Solubility; Substrate Specificity