Nitric oxide gas (NO) increased guanylate cyclase [GTP pyrophosphate-yase (cyclizing), EC 4.6.1.21 activity in soluble and particulate preparations from various tissues. The effect was dose-dependent and was observed with all tissue preparations examined. The extent of activation was variable among different tissue preparations and was greatest (19-to 33-fold) with supernatant fractions of homogenates from liver, lung, tracheal smooth muscle, heart, kidney, cerebral cortex, and cerebellum. Smaller effects (5-to 14-fold) were observed with supernatant fractions from skeletal muscle, spleen, intestinal muscle, adrenal, and epididymal fat. Activation was also observed with partially purified preparations of guanylate cyclase. Activation of rat liver supernatant preparations was augmented slightly with reducing agents, decreased with some oxidizing agents, and greater in a nitrogen than in an oxygen atmosphere. After activation with NO, guanylate cyclase activity decreased with a half-life of 3-4 hr at 4°but re-exposure to NO resulted in reactivation of preparations. Sodium azide, sodium nitrite, hydroxylamine, and sodium nitroprusside also increased guanylate cyclase activity as reported previously. NO alone and in combination with these agents produced approximately the same degree of maximal activation, suggesting that all of these agents act through a similar mechanism. NO also increased the accumulation of cyclic GMP but not cyclic AMP in incubations of minces from various rat tissues. We propose that various nitro compounds and those capable-of forming NO in incubations activate guanylate cyclase through a similar but undefined mechanism. These effects may explain the high activities of guanylate cyclase in certain tissues (e.g., lung and intestinal mucosa) that are exposed to environmental nitro compounds.

The particulate enzyme responsible for the synthesis of endothelium-derived relaxing factor has been purified from cultured and native (noncultured) bovine aortic endothelial cells. Purification of the solubilized particulate enzyme preparation by affinity chromatography on adenosine 2',5'-bisphosphate coupled to Sepharose followed by Superose 6 gel filtration chromatography resulted in a single protein band after denaturing polyacrylamide gel electrophoresis that corresponded to -135 kDa. The enzyme activity in the various fractions was assayed by its stimulatory effect on soluble guanylyl cyclase of rat fetal lung fibroblasts , by the formation of L-citrulline from L-arginine, by measuring nitrite/nitrate formation, and by bioassay on endotheliumdenuded vascular strips. Endothelium-derived relaxing factor synthase was purified 3419-fold from the crude particulate fraction of cultured bovine aortic endothelial cells with a 12% recovery (RFL-6 assay). Purified endothelium-derived relaxing factor synthase required L-arginine, NADPH, Ca2 , calmodulin, and 5,6,7,8-tetrahydrobiopterin for full activity.Endothelial cells synthesize endothelium-derived relaxing factor (EDRF) from L-arginine (1, 2). The pharmacological and biochemical properties of EDRF are mimicked by nitric oxide (NO) (3) or NO-containing compounds (4). These agents activate soluble guanylyl cyclase (5, 6) thereby increasing cGMP and causing relaxation of vascular smooth muscle (7-9), inhibition of platelet aggregation (10), and other effects (9, 11). EDRF/NO synthase has been purified and characterized from brain (12-14), polymorphonuclear neutrophils (15), and endotoxin/cytokine-induced macrophages (16,17). The endothelial, brain, and neutrophil EDRF/NO synthases are constitutive enzymes whereas the macrophage activity is expressed only after induction with endotoxin and/or a cytokine. The constitutive enzyme from brain is soluble and Ca2+/calmodulin-regulated whereas the enzyme from neutrophils has been described as soluble and Ca2+-dependent, but not calmodulin-dependent. Analysis with denaturing gel electrophoresis revealed a single band corresponding to 155 kDa and 150 kDa for the brain (12) and neutrophil (15) In endothelial cells, a particulate Ca2+/calmodulin-regulated enzyme accounts for >95% of the total EDRF synthase activity (18,19). We now report the purification to homogeneity (denaturing molecular mass of 135 kDa) of this constitutive particulate EDRF synthase and characterize the enzyme as NADPH-and (6R)-5,6,7,8-tetrahydrobiopterin (BH4)-dependent. Some of these observations have been reported in abstract form (20,21).

The present study investigates the hypothesis that endothelium-dependent relaxation of vascular smooth muscle may be mediated through the formation of cGMP. Relaxation of the rat thoracic aorta to acetylcholine, histamine, and Ca++ ionophore A23187 was associated with increased levels of cGMP in a time- and concentration-dependent manner, whereas cAMP levels were unaltered. Removal of the endothelium prevented relaxation to these agents and prevented the increased levels of cGMP. Removal of the endothelium after exposure to acetylcholine only partially decreased the elevated levels of cGMP, suggesting that the changes in cGMP occurred within the smooth muscle cells. Eicosatetraynoic acid, an inhibitor of lipoxygenase and cyclooxygenase, and quinacrine, an inhibitor of phospholipase, prevented and reversed acetylcholine-induced relaxation, respectively, and inhibited acetylcholine-induced increased levels of cGMP. In contrast, sodium nitroprusside-induced relaxation and increased levels of cGMP were independent of the presence of the endothelium, exposure to eicosatetraynoic acid, and quinacrine. The present results support the hypothesis that vascular smooth muscle relaxation induced by some agents is dependent on the presence of the endothelium and is mediated through the formation of an endothelial factor that increases cGMP levels in smooth muscle.

Nitric oxide (NO), a simple free radical gas, elicits a surprisingly wide range of physiological and pathophysiological effects. NO interacts with soluble guanylate cyclase to evoke many of these effects. However, NO can also interact with molecular oxygen and superoxide radicals to produce reactive nitrogen species that can modify a number of macromolecules including proteins, lipids, and nucleic acids. NO can also interact directly with transition metals. Here, we have reviewed the non--3',5'-cyclic-guanosine-monophosphate-mediated effects of NO including modifications of proteins, lipids, and nucleic acids.

Although cyclic guanosine monophosphate (GMP)' was first described in biological samples more than two decades ago, its role in some physiological processes has only become apparent in the past few years (see references [1][2][3][4]. This relatively slow development is probably attributable to the low concentrations of the nucleotide in tissues, the complex and insensitive methods available during the early studies, and the biases many investigators had regarding its possible functions. The latter was undoubtedly influenced by the many similarities of the cyclic GMP system with that of cyclic AMP and the attention cyclic AMP has received during this period. While analogies and similarities between these two cyclic nucleotide systems do exist, the cyclic GMP system presents more complexities due to the existence of several isoenzymes responsible for its synthesis.It is known that the conversion of guanosine triphosphate (GTP) to cyclic GMP is catalyzed by at least two isoenzyme forms of guanylate cyclase. The kinetic, physicochemical, and antigenic properties of the cytosolic and membrane-associated isoenzymes are quite different (see references 2, 4). The relative abundance of the soluble and particulate enzyme is variable in different tissues and species. While intestinal mucosa and retina possess predominately the particulate isoenzyme and platelets contain the soluble isoenzyme, most tissues such as vascular smooth muscle have both isoenzymes. Furthermore, the regulation of each of these isoenzymes is quite different. The soluble enzyme appears unique in that it can be activated by reactive free radicals such as nitric oxide (5), and probably hydroxyl free radical (6) and some porphyrins (7,8). On the other hand, the particulate isoenzyme can be activated with agents such as Escherichia coli heat-stable enterotoxin (9-1 1), atriopeptins (12, 13), and hemin (14). Cations, thiols, other redox agents, and detergents also have complex effects on the activity of both isoenzymes (2).Studies with the kinetic characterization of these isoenzymes led to the present understanding of the role of cyclic GMP in smooth muscle relaxation. Azide, added to inhibit GTPase activity in crude enzyme preparations, was found to activate the enzyme (15). While some hormones, autocoids, and other agents were able to increase cyclic GMP accumulation in intact tissues, these agents had no effects on guanylate cyclase activity in broken Receivedfor publication 9 January 1986.1. Abbreviations used in this paper: EDRF, endothelial-derived relaxant factor; GMP, guanosine monophosphate; GTP, guanosine triphosphate; ST, Escherichia coli heat-stable enterotoxin. cell preparations. It was reasoned that an understanding ofazide activation of guanylate cyclase could lead to understanding the mechanisms of hormonal regulation of the enzyme and some functions of cyclic GMP. Some of these predictions have been fulfilled. The stimulatory effects of azide were dependent upon the presence of heme-containing proteins in guanylate cyclase incubations s...