Nitric oxide decreases cytosolic free calcium in Balb/c 3T3 fibroblasts by a cyclic GMP-independent mechanism.

The purpose of this study was to investigate the effects of NO on cytosolic calcium levels in Balb/c 3T3 fibroblasts that were previously shown to lack soluble guanylate cyclase activity. Authentic NO as well as two NO-generating vasodilators, S-nitroso-N-acetyl-penicillamine and isosorbide dinitrate, decreased cytosolic calcium in these fibroblasts. The effect of NO and S-nitroso-N-acetylpenicillamine was concentration-dependent and, for the most part, reversible. Since S-nitroso-N-acetylpenicillamine did not increase either cGMP or cAMP, NO did not increase cGMP, and 8-bromo-cGMP did not alter cytosolic free calcium, we conclude that NO decreases cytosolic free calcium by a cyclic nucleotide-independent mechanism in Balb/c 3T3 fibroblasts.     

Clearance receptor-binding atrial natriuretic peptides inhibit mitogenesis and proliferation of rat aortic smooth muscle cells.

The current studies were designed to explore the effects of C-receptor-binding atrial natriuretic peptide analogues on serum-induced mitogenesis in cultured rat aortic smooth muscle cells. To this end, rANF99-126 and a series of truncated (rANF103-126, rANF103-125), ring-deleted (des[Gln116, Ser117, Gly118, Leu119, Gly120]rANF102-121-NH2 (c-ANF) and linear des(Cys105, Cys121)rANF104-126 peptide analogues were used. The latter two peptides have been reported to be selective for the ANF-C receptor. In cells subcultured between passage 3 to 19, rANF99-126, rANF103-126, and rANF103-125 concentration-dependently (0.1-1000 nM) inhibited serum-induced (3H) thymidine incorporation with maximal inhibition observed at 1 microM for each peptide (approximately 40, 31 and 56%) respectively. Furthermore, des[Cys105, Cys121]rANF104-126 inhibited serum-induced (3H)thymidine incorporation concentration-dependently without altering basal or elevated cellular cAMP or cGMP levels. Moreover, the reduction in thymidine incorporation was associated with inhibition of serum-induced clonal cell proliferation. In contrast, c-ANF failed to inhibit serum-induced mitogenesis, yet at a concentration of 100 nM it antagonized the antimitogenic effects of des[Cys105, Cys121]rANF104-126 or rANF99-126 without having any effect on basal or elevated cellular cyclic nucleotide levels. We conclude that the antimitogenic effect of atrial peptides is mediated through interaction with the ANF-C receptor and may be independent of changes in cellular cyclic nucleotide levels.     

Regulation of NHE3 by nitric oxide in Caco-2 cells

The effect of nitric oxide (NO) on Na+/H+ exchange (NHE) activity was investigated utilizing Caco-2 cells as an experimental model. Incubation of Caco-2 cells with 10(-3) M S-nitroso-N-acetylpenicillamine (SNAP), a conventional donor of NO, for 20 min resulted in a approximately 45% dose-dependent decrease in NHE activity, as determined by assay of ethylisopropylamiloride-sensitive 22Na uptake. A similar decrease in NHE activity was observed utilizing another NO-specific donor, sodium nitroprusside. SNAP-mediated inhibition of NHE activity was not secondary to a loss of cell viability. NHE3 activity was significantly reduced by SNAP (P < 0.05), whereas NHE2 activity was essentially unaltered. The effects of SNAP were mediated by the cGMP-dependent signal transduction pathway as follows: 1) LY-83583 and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), specific inhibitors of soluble guanylate cyclase, blocked the inhibitory effect of SNAP on NHE; 2) 8-bromo-cGMP mimicked the effects of SNAP on NHE activity; 3) the SNAP-induced decrease in NHE activity was counteracted by a specific protein kinase G inhibitor, KT-5823 (1 microM); 4) chelerythrine chloride (2 microM) or calphostin C (200 nM), specific protein kinase C inhibitors, did not affect inhibition of NHE activity by SNAP; 5) there was no cross activation by the protein kinase A-dependent pathway, as the inhibitory effects of SNAP were not blocked by Rp-cAMPS (25 microM), a specific protein kinase A inhibitor. These data provide novel evidence that NO inhibits NHE3 activity via activation of soluble guanylate cyclase, resulting in an increase in intracellular cGMP levels and activation of protein kinase G.     

Lack of association of epidermal growth factor-, insulin-, and serum-induced mitogenesis with stimulation of phosphoinositide degradation in BALB/c 3T3 fibroblasts

The hypothesis that inositol phospholipid degradation is a step in the mechanism by which epidermal growth factor (EGF) stimulates mitogenesis in confluent monolayers of quiescent BALB/c 3T3 fibroblasts was tested. The maximum mitogenic response (a nearly 30-fold increase in incorporation of [3H]thymidine) occurred at 1 ng/ml EGF (0.16 nM). This degree of stimulation corresponded to 60% of that elicited by 10% serum. To determine whether EGF stimulated formation of inositol phosphates via degradation of polyphosphoinositides, the intracellular levels of [3H] inositol phosphates and [3H]phosphoinositides were determined after EGF addition to BALB/c 3T3 fibroblasts prelabeled with [3H]inositol. These experiments were performed under conditions designed to mimic exactly those conditions used to study mitogenesis. The results demonstrated that 10% serum or 10 ng/ml of platelet-derived growth factor, but not as much as 50 ng/ml EGF or 10 micrograms/ml insulin, increased the levels of inositol phosphates via degradation of phosphoinositides in the presence of 10 mM Li+. The serum-induced effects occurred in 30 s, the earliest time investigated. Phorbol dibutyrate (100 nM), alone or in conjunction with EGF (10 ng/ml), failed to stimulate inositol phospholipid degradation. However, phorbol dibutyrate inhibited the serum-induced stimulation. Finally, fetal bovine serum dialyzed so as to retain peptide mitogens lost almost 70% of the capacity to stimulate degradation of inositol phospholipids while remaining as mitogenic as the control serum. Thus, stimulation of inositol phospholipid degradation is an unlikely component in the mechanism by which EGF and probably insulin and serum stimulate mitogenesis in BALB/c 3T3 fibroblasts.     

In vivo exposure to carbon monoxide causes delayed impairment of activation of soluble guanylate cyclase by nitric oxide in rat brain cortex and cerebellum

Carbon monoxide induces delayed neurological and neuropathological alterations, including memory loss and cognitive impairment. The bases for the delay remain unknown. Activation of soluble guanylate cyclase by nitric oxide modulates some forms of learning and memory. Carbon monoxide binds to soluble guanylate cyclase, activating it but interfering with its activation by nitric oxide. The aim of this work was to assess whether exposure of rats to carbon monoxide alters the activity of soluble guanylate cyclase or its modulation by nitric oxide in cerebellum or cerebral cortex. Rats exposed chronically or acutely to carbon monoxide were killed 24 h or 7 days later. Acute carbon monoxide exposure decreased cyclic guanosine monophosphate (cGMP) content and reduced activation of soluble guanylate cyclase by nitric oxide. Cortex was more sensitive than cerebellum to chronic exposure, which reduced activation of soluble guanylate cyclase by nitric oxide in cortex. In cerebellum, chronic exposure induced delayed impairment of soluble guanylate cyclase activation by nitric oxide. Acute exposure effects were also stronger at 7 days than at 24 h after exposure. This delayed impaired modulation of soluble guanylate cyclase by nitric oxide may contribute to delayed memory loss and cognitive impairment in humans exposed to carbon monoxide.     

Differential antimitogenic effectiveness of atrial natriuretic peptides in primary versus subcultured rat aortic smooth muscle cells: Relationship to expression of ANF-C receptors

Previous studies have shown that atrial natriuretic peptides inhibit mitogenesis in subcultured aortic smooth muscle cells by a mechanism that appears to be mediated via the C-type or “clearance” receptor. In the current study, we have compared the antimitogenic effect of these peptides in serum-stimulated primary aortic smooth muscle cell cultures and in subcultured cells. A series of atrial peptides, including rANF_99–126, rANF_103–126, and rANF_103–125, were only poorly antimitogenic in serum-stimulated primary cultures, whereas des[Cys¹⁰⁵, Cys¹²¹] rANF_104–126 which binds selectively to the ANF-C receptors had no antimitogenic activity. In contrast, in subcultured cells (between subcultures 3 and 25), rANF_99–126, rANF_103–126, rANF_103–126, Cys¹¹⁶rANF_102–116, and des[Cys¹⁰⁵, Cys¹²¹]rANF_104–126 inhibited serum-induced [³H]thymidine incorporation (IC₅₀ in the range of 10–50 nM), with maximal inhibition of 40–70%. The lack of antimitogenic activity in primary cultures did not appear to be related to the lack of cGMP elevation elicited by atrial peptides or to an inherent insensitivity to the action of antimitogens, because primary cultures were responsive to the cGMP-elevating effect of atrial peptides and the cells were more rather than less sensitive to the antimitogenic effect of the nitric-oxide-vasodilator, SNAP, as compared to subcultured cells. Analysis of the affinity and binding capacity of freshly isolated aortic membranes, and primary or secondary cultures for [¹²⁵I]rANF_99–126, revealed that the number of ANF receptors increased by tenfold, following subculture. Moreover, subcultured cells contained receptors with increased binding affinity for peptide analogues selective for the ANF-C-type type receptor. Covalent cross-linking studies with (¹²⁵I)rANF_99–126 confirmed that membranes prepared from fresh aortae predominantly expressed the ANF-A/guanylate cyclase receptor, whereas in subcultured cells the predominantly cross-linked protein was the ANF-C-type receptor, with receptors in primary cultures occupying an intermediate position. These results suggest that the binding and antimitogenic activity of atrial peptides in aortic smooth muscle cells depends on the phenotypic state of these cells. Moreover, the increased antimitogenic potency of atrial peptides in secondary cultures may reflect increased expression of the ANF-C-type receptors. © 1993 Wiley-Liss, Inc.     

Constitutive and permissive roles of nitric oxide activity in embryonic ciliary cells

Embryos of Helisoma trivolvis exhibit cilia-driven rotation within the egg capsule during development. In this study we examined whether nitric oxide (NO) is a physiological regulator of ciliary beating in cultured ciliary cells. The NO donor S-nitroso-N-acetylpenicillamine (SNAP; 1-1,000 microM) produced a dose-dependent increase in ciliary beat frequency (CBF). In contrast, the nitric oxide synthase (NOS) inhibitor 7-nitroindazole (10 and 100 microM) inhibited the basal CBF and blocked the stimulatory effects of serotonin (100 microM). NO production in response to serotonin was investigated with 4,5-diaminofluorescein diacetate imaging. Although SNAP (100 microM) produced a rise in NO levels in all cells, only 22% of cells responded to serotonin with a moderate increase. The cGMP analog 8-bromo-cGMP (8-Br-cGMP; 0.2 and 2 mM) increased CBF, and the soluble guanylate cyclase inhibitor LY-83583 (10 microM) blocked the cilioexcitatory effects of SNAP and serotonin. These data suggest that NO has a constitutive cilioexcitatory effect in Helisoma embryos and that the stimulatory effects of serotonin and NO work through a cGMP pathway. It appears that in Helisoma cilia, NO activity is necessary, but not sufficient, to fully mediate the cilioexcitatory action of serotonin.     

Nitric oxide and nitric oxide-generating compounds inhibit hepatocyte protein synthesis.

Hepatocytes are stimulated to produce nitric oxide (NO.) from L-arginine in response to conditioned Kupffer cell medium or a combination of cytokines. Associated with the production of NO.in hepatocytes, there is a profound decrease in total protein synthesis ([3H]leucine incorporation). This report demonstrates that authentic NO.and the NO.-generating compound S-nitroso-N-acetylpenicillamine inhibit hepatocyte total protein synthesis in a reversible and concentration-dependent fashion. In parallel with the suppression of hepatocyte total protein synthesis, authentic NO.inhibits the production of two specific hepatocyte proteins, albumin and fibrinogen, without influencing the quantity of albumin mRNA. Although authentic NO.induces a rapid increase in cGMP levels in hepatocytes, the addition of the cGMP analog 8-bromoguanosine 3':5' cyclic monophosphate to unstimulated HC cultures does not reproduce the inhibition of total protein synthesis. These data show that NO.is the hepatocyte L-arginine metabolite that inhibits protein synthesis. Furthermore, these findings indicate that NO.does not inhibit hepatocyte protein synthesis solely through the activation of soluble guanylate cyclase but appears to affect a translational or posttranslational process.     

Antiproliferative effect of nitric oxide on rat glomerular mesangial cells via inhibition of mitogen-activated protein kinase.

The effect of nitric oxide (NO) donors and lipopolysaccharide (LPS) on the proliferation of rat glomerular mesangial cells was characterized. Exogenous application of a NO donor inhibited serum-induced proliferation in a time- and dose-dependent manner. S-Nitrosoglutathione (GSNO) also increased cGMP generation and arachidonic acid release, but it did not cause any measurable increase in the cytosolic Ca2+ concentration. Chelation of cytosolic Ca2+ or inhibition of mitogen-activated protein kinase (MAPK) kinase had an inhibitory effect on proliferation, but neither enhanced the antiproliferative effect of GSNO. In contrast, inhibition of guanylate cyclase or phospholipase A2 had no effect on proliferation, but partially reversed GSNO-induced antiproliferation by approximately 98 and 65%, respectively. GSNO did not cause cell death. Incubation of cells with LPS induced endogenous NO generation and had an antiproliferative effect. LPS-induced antiproliferation was reversed completely by inhibition of nitric oxide synthase and partially by inhibition of guanylate cyclase or phospholipase A2. GSNO or LPS inhibited serum-induced MAPK activation, and both effects were partially reversed by inhibition of guanylate cyclase or phospholipase A2. Inclusion of 8-bromo-cGMP or arachidonic acid in the growth medium resulted in a similar antiproliferative effect. In conclusion, in rat glomerular mesangial cells, MAPK inhibition and an antiproliferative effect could be induced by either an increase in the cellular concentration of NO or exposure of the cells to LPS. Part of the effect of NO was attributable to the increased cellular cGMP generation and arachidonic acid release.     

A novel biological effect of atrial natriuretic hormone: inhibition of mesangial cell mitogenesis

We have investigated the effect of atrial natriuretic hormone on serum-induced mitogenesis in cultured rat mesangial cells. Synthetic peptides, atriopeptin 28 and atriopeptin 24, dose-dependently decreased thymidine incorporation, with a half-maximal effect at approximately 1 nM and a maximal inhibition of approximately 60%. Moreover, atriopeptin 28 significantly decreased the clonal proliferation of mesangial cells. Atriopeptin 28 also decreased resting cytosolic Ca but had no effect on the increase induced by serum, relative to the lower baseline established by atriopeptin 28. Nevertheless, the overall effect of atriopeptin 28 on Ca was to attenuate the serum-induced increase, relative to the original resting level. These results therefore provide evidence for a novel biological effect of atrial natriuretic hormone and suggest that the antimitogenic effect may be mediated by atriopeptin-induced alterations of intracellular Ca dynamics. We speculate that atrial natriuretic hormone may be a modulator of mesangial cell mitogenesis in vivo.     

Amyloid-β inhibits No-cGMP signaling in a CD36- and CD47-dependent manner

Amyloid-β interacts with two cell surface receptors, CD36 and CD47, through which the matricellular protein thrombospondin-1 inhibits soluble guanylate cyclase activation. Here we examine whether amyloid-β shares this inhibitory activity. Amyloid-β inhibited both drug and nitric oxide-mediated activation of soluble guanylate cyclase in several cell types. Known cGMP-dependent functional responses to nitric oxide in platelets and vascular smooth muscle cells were correspondingly inhibited by amyloid-β. Functional interaction of amyloid-β with the scavenger receptor CD36 was indicated by inhibition of free fatty acid uptake via this receptor. Both soluble oligomer and fibrillar forms of amyloid-β were active. In contrast, amyloid-β did not compete with the known ligand SIRPα for binding to CD47. However, both receptors were necessary for amyloid-β to inhibit cGMP accumulation. These data suggest that amyloid-β interaction with CD36 induces a CD47-dependent signal that inhibits soluble guanylate cyclase activation. Combined with the pleiotropic effects of inhibiting free fatty acid transport via CD36, these data provides a molecular mechanism through which amyloid-β can contribute to the nitric oxide signaling deficiencies associated with Alzheimer's disease.     

Activation of soluble guanylate cyclase by nitrovasodilators is inhibited by oxidized low-density lipoprotein

In the presence of oxidized low-density lipoprotein the stimulatory effects of nitric oxide, sodium nitroprusside and S-nitrosoglutathione on soluble guanylate cyclase partially purified from bovine platelets were diminished in a concentration-dependent manner with IC50 values around 100 micrograms total cholesterol/ml. This inhibitory effect was potentiated about 10-fold when the enzyme was pre-incubated with the lipoprotein for 10 minutes at 37 degrees C which indicates a direct interaction of the lipoprotein with the guanylate cyclase. As oxidized low-density lipoprotein is present in the wall of atherosclerotic arteries, we suggest that the impaired response of atherosclerotic blood vessels to vasodilators may be due to a diminished activation of smooth muscle guanylate cyclase.     

Nitric oxide decreases lung liquid production via guanosine 3',5'-cyclic monophosphate

We studied the role of cGMP in nitric oxide (NO)-induced changes in lung liquid production (J(v)) in chronically instrumented fetal sheep. Forty-five studies were done in which J(v) was measured by a tracer dilution technique. Left pulmonary arterial flow (Q(lpa)) was measured by a Doppler flow probe. There were two series of experiments. In the first, we gave 8-bromo-cGMP, a cGMP analog, by either the pulmonary vascular or intraluminal route; in the second, we used agents to inhibit or enhance endogenous cGMP activity. When infused directly into the pulmonary circulation, 8-bromo-cGMP significantly increased Q(lpa) but had no effect on J(v). Conversely, when instilled into the lung liquid, 8-bromo-cGMP had no effect on Q(lpa) but significantly reduced J(v). Inhibition of guanylate cyclase activity with methylene blue totally blocked, whereas phosphodiesterase inhibition with Zaprinast significantly enhanced, the effect of instilled NO on J(v). Thus the reduction in lung liquid caused by NO appears to be mediated by cGMP, perhaps through a direct effect on the pulmonary epithelium.     

Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine.

Sodium nitroprusside, nitroglycerin, sodium azide and hydroxylamine increased guanylate cyclase activity in particulate and/or soluble preparations from various tissues. While sodium nitroprusside increased guanylate cyclase activity in most of the preparations examined, the effects of sodium azide, hydroxylamine and nitroglycerin were tissue specific. Nitroglycerin and hydroxylamine were also less potent. Neither the protein activator factor nor catalase which is required for sodium azide effects altered the stimulatory effect of sodium nitroprusside. In the presence of sodium azide, sodium nitroprusside or hydroxylamine, magnesium ion was as effective as manganese ion as a sole cation cofactor for guanylate cyclase. With soluble guanylate cyclase from rat liver and bovine tracheal smooth muscle the concentrations of sodium nitroprusside that gave half-maximal stimulation with Mn2+ were 0.1 mM and 0.01 mM, respectively. Effective concentrations were slightly less with Mg2+ as a sole cation cofactor. The ability of these agents to increase cyclic GMP levels in intact tissues is probably due to their effects on guanylate cyclase activity. While the precise mechanism of guanylate cyclase activation by these agents is not known, activation may be due to the formation of nitric oxide or another reactive material since nitric oxide also increased guanylate cyclase activity.     

Chronic exposure to aluminium impairs the glutamate-nitric oxide-cyclic GMP pathway in the rat in vivo.

Aluminium is neurotoxic and is considered a possible etiologic factor in Alzheimer's disease, dialysis syndrome and other neurological disorders. The molecular mechanism of aluminium-induced impairment of neurological functions remains unclear. We showed that aluminium impairs the glutamate-nitric oxide-cGMP pathway in cultured neurons. The aim of this work was to assess by in vivo brain microdialysis whether chronic administration of aluminium in the drinking water (2.5% aluminium sulfate) also impairs the glutamate-nitric oxide-cGMP pathway in the cerebellum of rats in vivo. Chronic exposure to aluminium reduced NMDA-induced increase of extracellular cGMP by ca 50%. The increase in extracellular cGMP induced by the nitric oxide generating agent S-nitroso-N-acetylpenicillamine was higher (240%) in rats treated with aluminium than in controls. Immunoblotting experiments showed that aluminium reduced the cerebellar content of calmodulin and nitric oxide synthase by 34 and 15%, respectively. Basal activity of soluble guanylate cyclase was decreased by 66% in aluminium-treated rats, while the activity after stimulation with S-nitroso-N-acetylpenicillamine was similar to controls. Basal cGMP in the cerebellar extracellular space was decreased by 50% in aluminium-treated rats. These results indicate that chronic exposure to aluminium reduces the basal activity of guanylate cyclase and impairs the glutamate-nitric oxide-cGMP pathway in the animal in vivo.     

Nitric oxide decreases renal medullary Na+, K+-ATPase activity through cyclic GMP-protein kinase G dependent mechanism.

The aim of this study was to investigate the effect of nitric oxide on renal Na+,K(+)-ATPase and ouabain-sensitive H+,K(+)-ATPase activities. The study was performed in male Wistar rats. The investigated substances were infused under general anaesthesia into abdominal aorta proximally to the renal arteries. The activity of ATPases was assayed in isolated microsomal fraction. NO donor, S-nitroso-N-acetylpenicillamine (SNAP), infused at doses of 10(-7) and 10(-6)mol/kg/min decreased medullary Na+,K(+)-ATPase activity by 29.4% and 45.2%, respectively. Another NO donor, spermine NONOate, administered at the same doses reduced Na+,K(+)-ATPase activity in the renal medulla by 31.7% and 46.5%, respectively. Neither of NO releasers had any effect on Na+,K(+)-ATPase in the renal cortex and on either cortical or medullary ouabain-sensitive H+,K(+)-ATPase. Infusion of NO precursor, L-arginine (100 micromol/kg/min), decreased medullary Na+,K(+)-ATPase activity by 32.2%, whereas inhibitor of nitric oxide synthase, L-NAME (10 nmol/kg/min), increased this activity by 20.7%. The effect of synthetic NO donors was mimicked by 8-bromo-cGMP and blocked by inhibitors of soluble guanylate cyclase, ODQ or methylene blue, as well as by specific inhibitor of protein kinase G, KT5823. In addition, inhibitory effect of either SNAP or 8-bromo-cGMP on medullary Na+,K(+)-ATPase was abolished by 17-octadecynoic acid (17-ODYA), which inhibits cytochrome P450-dependent metabolism of arachidonic acid. These data suggest that NO decreases Na+,K(+)-ATPase activity in the renal medulla through the mechanism involving cGMP, protein kinase G, and cytochrome P450-dependent arachidonate metabolites. In contrast, NO has no effect on Na+,K(+)-ATPase in the renal cortex and on either cortical or medullary ouabain-sensitive H+,K(+)-ATPase.     

Maxi-circles and mini-circles in kinetoplast DNA from trypanosoma cruzi

Glyceryl trinitrate specifically required cysteine, whereas NaNO2 at concentrations less than 10 mM required one of several thiols or ascorbate, to activate soluble guanylate cyclase from bovine coronary artery. However, guanylate cyclase activation by nitroprusside or nitric oxide did not require the addition of thiols or ascorbate. Whereas various thiols enhanced activation by nitroprusside, none of the thiols tested enhanced activation by nitric oxide. S-Nitrosocysteine, which is formed when cysteine reacts with either NO-2 or nitric oxide, was a potent activator of guanylate cyclase. Similarly, micromolar concentrations of the S-nitroso derivatives of penicillamine, GSH and dithiothreitol, prepared by reacting the thiol with nitric oxide, activated guanylate cyclase. Guanylate cyclase activation by S-nitrosothiols resembled that by nitric oxide and nitroprusside in that activation was inhibited by methemoglobin, ferricyanide and methylene blue. Similarly, guanylate cyclase activation by glyceryl trinitrae plus cysteine, and by NaNO2 plus either a thiol or ascorbate, was inhibited by methemoglobin, ferricyanide and methylene blue. These data suggest that the activation of guanylate cyclase by each of the compounds tested may occur through a common mechanism, perhaps involving nitric oxide. Moreover, these findings suggest that S-nitrosothiols could act as intermediates in the activation of guanylate cyclase by glyceryl trinitrate, NaNO2 and possibly nitroprusside.     

Cyclic GMP metabolism in relation to the regulation of cell growth in BALB/c3T3 cells.

BALB/c3T3 cell homogenates have guanylate cyclase in both 105000 g paniculate and soluble fraction. The activity in particulate fraction was much higher than that in the soluble fraction. Both enzyme activities were 2- to 3-fold greater in the resting state (G0 phase) than in the logarithmically growing state. In addition, cGMP-phosphodiesterase activity was 2-fold greater in resting than in growing cells. When G0-arrested cells entered into the G1 phase by serum addition, cGMP levels rapidly increased, whereas guanylate cyclase activities did not change within 30 min after serum addition. Four hours after serum addition, these activities had, however, decreased to one third and remained at that low level throughout the G1 phase. The relationship between cell growth and guanylate cyclase activity is discussed.     

Effects of thiols, sugars, and proteins on nitric oxide activation of guanylate cyclase.

Purification of soluble guanylate cyclase from rat liver resulted in an apparent loss of enzyme activation by nitric oxide that could be restored by dithiothreitol. methemoglobin, bovine serum albumin, or sucrose. Although hemoglobin also permitted some activation with nitric oxide, the effect of other agents to restore enzyme activation was prevented with hemoglobin. As a result of enzyme purification, there is an alteration of the dose-response relationship for nitric oxide activation. After partial enzyme purification, relatively high concentrations of nitric oxide that were stimulatory in crude enzyme preparations had no effect on enzyme activity. However, partially purified or homogeneous enzyme was activated by lower concentrations of nitric oxide. The bell-shaped dose-response curve for nitric oxide was shifted to the left with guanylate cyclase purification. The addition of dithiothreitol, methemoglobin, bovine serum albumin, or sucrose to enzyme markedly broadens the dose-response curve for nitric oxide. Thus, the apparent loss of responsiveness to nitric oxide with purification is a function of increased sensitivity of guanylate cyclase to nitric oxide. Increased sensitivity to nitric oxide with enzyme purification probably results from the removal of heme, proteins, and small molecules that can serve as scavengers or sinks for nitric oxide and prevent excessive oxidation of the enzyme.     

Potentiation of NO-dependent activation of soluble guanylate cyclase by 5-nitroisatin and the antiviral agent arbidol

Isatin (indole-dione-2,3) is an endogenous indole that exhibits a wide spectrum of biological and pharmacological activities. The effect of isatin derivatives, 5-nitroisatin and arbidol (an antiviral agent) on spermine NONO-induced activation of human platelet soluble guanylate cyclase has been investigated. 5-Nitroisatin and arbidol had no effect on basal activity, but synergistically increased in a concentration-dependent manner the spermine NONO-induced activation of this enzyme. 5-Nitroisatin and arbidol, like YC-1, sensitized guanylate cyclase towards nitric oxide (NO) and produced a leftward shift of the spermine NONO concentration response curve. However, both compounds did not influence the activation of guanylate cyclase by YC-1 and did not change the synergistic increase of spermine NONO-induced activation of soluble guanylate cyclase in the presence of YC-1. This suggests that 5-nitroisanin and arbidol did not compete with YC-1. Addition of isatin did not change the synergistic increase in the spermine NONO-induced guanylate cyclase activation by 5-nitroisatin and arbidol and did not influence a leftward shift of the spermine NONO concentration response curve produced by these compounds. These data suggest lack of competitive interaction between isatin and both its derivatives used.