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.     

Requirement of thiols for activation of coronary arterial guanylate cyclase by glyceryl trinitrate and sodium nitrite possible involvement of S-nitrosothiols

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 nitropruside, none of the thiols tested enhanced activation by nitric oxide. S-Nitrosocysteine, which is formed when cysteine reacts with either NO₂^? 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 trinitrate 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     

Activation of purified soluble guanylate cyclase by arachidonic acid requires absence of enzyme-bound heme

The mechanism by which arachidonic acid activates soluble guanylate cyclase purified from bovine lung is partially elucidated. Unlike enzyme activation by nitric oxide (NO), which required the presence of enzyme-bound heme, enzyme activation by arachidonic acid was inhibited by heme. Human but not bovine serum albumin in the presence of NaF abolished activation of heme-containing guanylate cyclase by NO and nitroso compounds, whereas enzyme activation by arachidonic acid was markedly enhanced. Addition of heme to enzyme reaction mixtures restored enzyme activation by NO but inhibited enzyme activation by arachidonic acid. Whereas heme-containing guanylate cyclase was activated only 4- to 5-fold by arachidonic or linoleic acid, both heme-deficient and albumin-treated heme-containing enzymes were activated over 20-fold. Spectrophotometric analysis showed that human serum albumin promoted the reversible dissociation of heme from guanylate cyclase. Arachidonic acid appeared to bind to the hydrophobic heme-binding site on guanylate cyclase but the mechanism of enzyme activation was dissimilar to that for NO or protoporphyrin IX. Enzyme activation by arachidonic acid was insensitive to Methylene blue or KCN, was inhibited competitively by metalloporphyrins, and was abolished by lipoxygenase. Whereas NO and protoporphyrin IX lowered the apparent Km and Ki for MgGTP and uncomplexed Mg2+, arachidonic and linoleic acids failed to alter these kinetic parameters. Thus, human serum albumin can promote the reversible dissociation of heme from soluble guanylate cyclase and thereby abolish enzyme activation by NO but markedly enhance activation by polyunsaturated fatty acids. Arachidonic acid activates soluble guanylate cyclase by heme-independent mechanisms that are dissimilar to the mechanism of enzyme activation caused by protoporphyrin IX.     

Activation of purified guanylate cyclase by nitric oxide requires heme. Comparison of heme-deficient, heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung

Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containing forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 microM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent Km for GTP in the presence of excess Mn2+ or Mg2+ was 10 microM and 85-120 microM, respectively, for unactivated guanylate cyclase. The apparent Km for GTP in the presence of Mn2+ was not altered but the Km in the presence of Mg2+ was lowered to 58 microM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg2+ or Mn2+. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO and nitroso compounds.     

Relaxation of bovine coronary artery and activation of coronary arterial guanylate cyclase by nitric oxide, nitroprusside and a carcinogenic nitrosoamine.

The principal objective of this study was to test the hypothesis that nitroprusside relaxes vascular smooth muscle via the reactive intermediate, nitric oxide (NO), and that the biologic action of NO is associated with the activation of guanylate cyclase. Nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and NO elicit concentration-dependent relaxation of precontraced helical strips of bovine coronary artery. Nitroprusside, MNNG and NO also markedly activate soluble guanylate cyclase from bovine coronary arterial smooth muscle and, thereby, stimulate the formation of cyclic GMP. Three heme proteins, hemoglobin, methemoglobin and myoglobin, and the oxidant, methylene blue, abolish the coronary arterial relaxation elicited by NO. Similarly, these heme proteins, methylene blue and another oxidant, ferricyanide, markedly inhibit the activation of coronary arterial guanylate cyclase by NO, nitroprusside and MNNG. The following findings support the view that certain nitroso-containing compounds liberate NO in tissue:heme proteins, which cannot permeate cells, inhibit coronary arterial relaxation elicited by NO, but not by nitroprusside or MNNG; the vital stain, methylene blue, inhibits relaxation by NO, nitroprusside and MNNG; heme proteins and oxidants inhibit guanylate cyclase activation by NO, nitroprusside and MNNG in cell-free mixtures. The findings that inhibitors of NO-induced relaxation of coronary artery also inhibit coronary arterial guanylate cyclase activation suggest that cyclic GMP formation may be associated with coronary arterial smooth muscle relaxation.     

Properties of purified soluble guanylate cyclase activated by nitric oxide and sodium nitroprusside

Highly purified rat lung soluble guanylate cyclase was activated with nitric oxide or sodium nitroprusside and the degree of activation varied with incubation conditions. With Mg2+ as the action cofactor, about 2- to 8-fold activation was observed with nitric oxide or sodium nitroprusside alone. Markedly enhanced activation (20-40 fold) was observed when 1 muM hemin added to the enzyme prior to exposure to the activating agent. The activation with hemin and sodium nitroprusside was prevented in a dose-dependent manner by sodium cyanide. The level activation was also increased by the addition of 1 mM dithiothreitol, but unlike hemin which had no effect on basal enzyme activity, dithiothreitol led to a considerable increase in basal activity. Activated guanylate cyclase decayed to basal activity within one hour at 2 degrees C and the enzyme could be reactivated upon re-exposure to nitroprusside or nitric oxide. Under basal conditions, Michaelis-Menten kinetics were observed, with a Km for GTP of 140 muM with Mg2+ cofactor. Following activation with nitroprusside or nitric oxide, curvilinear Eadie-Hofstee transformations of kinetic data were observed, with Km's of 22 MuM and 100 MuM for Mg-GTP. When optimal activation (15-40 fold) was induced by the addition of hemin and nitroprusside, multiple Km's were also seen with Mg-GTP and the high affinity form was predominant (22 MuM). Similar curvilinear Eadie-Hofstee transformations were observed with Mn2+ as the cation cofactor. These data suggest that multiple GTP catalytic sites are present in activated guanylate cyclase, or alternatively, multiple populations of enzyme exist.     

Activation of purified guanylate cyclase by nitric oxide requires heme comparison of heme-deficient,heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung

Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containg forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 μM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent K_m for GTP in the presence of excess Mn²⁺ or Mg²⁺ was 10 μM and 85–120 μM, respectively, for unactivated guanylate cyclase. The apparent K_m for GTP in the presence of Mn²⁺ was not altered but the K_m in the presence of Mg²⁺ was lowered to 58 μM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg²⁺ or Mn²⁺. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO nitroso compounds.     

Reversible inactivation of guanylate cyclase by mixed disulfide formation

Highly purified preparations of guanylate cyclase from rat lung were inactivated by several disulfide compounds in a time- and dose-dependent manner. Cystamine and cystine were the most potent disulfides tested, but other compounds which contained the cysteamine moiety (NH2CH2CH2S-), including pantethine and oxidized coenzyme A, were also able to partially inactivate the enzyme. In addition to the decrease in basal activity (measured with either Mg2+-GTP or Mn2+-GTP), disulfide-inhibited enzyme was activated to a lesser extent by nitric oxide. Treatment with dithiothreitol or other reducing agents restored basal activity and increased the level of cGMP production following nitric oxide activation. Control enzyme samples exhibited a single GTP Km of 25 microM or 150 microM with Mn2+ or Mg2+, respectively. However, cystamine-treated enzyme showed these same Km values as well as an additional GTP Km of 2 to 3 microM using either metal ion as cofactor. When [35S]cystine was incubated with purified enzyme, radioactivity was incorporated into the trichloroacetic acid-precipitable protein, and the counts were released following dithiothreitol treatment. In addition, [35S]cystine-labeled enzyme co-migrated with native guanylate cyclase on nondenaturing polyacrylamide gels. These data indicate that mixed disulfides can be formed between guanylate cyclase and certain naturally occurring compounds, and that disulfide formation leads to a reversible loss of enzyme activity.     

Ascorbate activates soluble guanylate cyclase via H2O2-metabolism by catalase

Conditions necessary for the activation by ascorbic acid of soluble guanylate cyclase purified from bovine lung have been examined. Ascorbic acid (0.1-10 mM) did not directly activate the enzyme, nonetheless, pronounced activation by ascorbate (3-10 mM) was observed in incubation mixtures containing 1 microM bovine liver catalase. Superoxide dismutase (SOD) and mannitol did not affect the catalase-dependent activation of guanylate cyclase elicited by ascorbate, suggesting that superoxide anion and hydroxyl radical were not mediating the activation of the enzyme. However, SOD enhanced the relatively low level activation of the enzyme elicited by catalase in the absence of added ascorbate. Pronounced inhibition (both with and without added ascorbate) was observed of catalase-dependent activation of guanylate cyclase by either ethanol (100 mM) or a fungal catalase preparation. Neither ethanol nor fungal catalase inhibited activation of guanylate cyclase by S-nitrosyl-N-acetyl-penicillamine (SNAP), a source of the nitric oxide free radical. These observations indicate that autoxidation of ascorbic acid or thiols present with the guanylate cyclase preparation leads to generation of H2O2, and its metabolism by bovine liver catalase mediates the concomitant activation of guanylate cyclase. The mechanism of activation appears to be associated with the presence of Compound I of catalase and to be inhibited by superoxide anion.     

ANF stimulation of detergent-dispersed particulate guanylate cyclase from bovine adrenal cortex

Particulate guanylate cyclase from bovine adrenal cortex can be stimulated by ANF. A 2-fold stimulation of the enzyme was obtained with 100 nM ANF and a half-maximal stimulation, with a 5 nM dose. The stimulation by ANF persisted for at least 30 min. Various detergents, such as Triton X-100, Lubrol PX, cholate, CHAPS, digitonin and zwittergent, stimulated several-fold the activity of particulate guanylate cyclase. However, only Triton X-100 dispersed particulate guanylate cyclase without affecting its response to ANF. The dose-response curve of ANF stimulation of the particulate and the Triton X-100 dispersed enzyme was similar. The dispersion of a fully responsive guanylate cyclase to ANF will help us to uncover the type of interactions between guanylate cyclase and ANF. It will also be used as a first step for the purification of an ANF-sensitive particulate guanylate cyclase.     

Restoration of the responsiveness of purified guanylate cyclase to nitrosoguanidine, nitric oxide, and related activators by heme and hemeproteins. Evidence for involvement of the paramagnetic nitrosyl-heme complex in enzyme activation.

Purification of soluble guanylate cyclase activity from rat liver resulted in loss of enzyme responsiveness to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), nitroprusside, nitrite, and NO. Responses were restored by addition of heat-treated hepatic supernatant fraction, implying a requirement for heat-stable soluble factor(s) in the optimal expression of the actions of the activators. Addition of free hematin, hemoglobin, methemoglobin, active or heat-inactivated catalase partially restores responsiveness of purified guanylate cyclase to MNNG, NO, nitrite, and nitroprusside. These responses were markedly potentiated by the presence of an appropriate concentration of reducing agent (dithiothreitol, ascorbate, cysteine, or glutathione), which maintains heme iron in the ferro form and favors formation of paramagnetic nitrosyl . heme complexes from the activators. High concentrations of heme or reducing agents were inhibitory, and heme was not required for the expression of the stimulatory effects of Mn2+ or Mg2+ on purified guanylate cyclase. Preformed nitrosyl hemoglobin (10 micron) increased activity of the purified enzyme 10- to 20-fold over basal with Mn2+ as the metal cofactor and 90- to 100-fold with Mg2+. Purified guanylate cyclase was more sensitive to preformed NO-hemoglobin (minimally effective concentration, 0.1 micron) than to MNNG (1 micron), nitroprusside (50 micron), or nitrite (1 mM). A reducing agent was not required for optimal stimulation of guanylate cyclase by NO-hemoglobin. Maximal NO-hemoglobin-responsive guanylate cyclase was not further increased by subsequent addition of NO, MNNG, nitrite, or nitroprusside. Activation by each agent resulted in analogous alterations in the Mn2+ and Mg2+ requirements of enzyme activity, and responses were inhibited by the thiol-blocking agents N-ethylmaleimide, arsenite, or iodoacetamide. The results suggest that NO-hemoglobin, MNNG, NO, nitrite, and nitroprusside activate guanylate cyclase through similar mechanisms. The stimulatory effects of preformed NO-hemoglobin combined with the clear requirements for heme plus a reducing agent in the optimal expression of the actions of MNNG, NO, and related agents are consistent with a role for the paramagnetic nitrosyl . heme complex in the activation of guanylate cyclase.     

Purification and properties of heme-deficient hepatic soluble guanylate cyclase: effects of heme and other factors on enzyme activation by NO, NO-heme, and protoporphyrin IX

Purified hepatic soluble guanylate cyclase (EC 4.6.1.2) had maximal specific activities in the unactivated state of 0.4 and 1 μmol cyclic GMP min^?1 mg protein^?1, when MgGTP and MnGTP, respectively, were used as substrates. The apparent K_m for GTP was 85 or 10 μm in the presence of excess Mg²⁺ or Mn²⁺, respectively. Guanylate cyclase purified as described was deficient in heme but could be readily reconstituted with heme by reacting enzyme with hematin and excess dithiothreitol at 4 °C and pH 7.8. Unpurified guanylate cyclase was activated 20- to 84-fold by NO, nitroso compounds, NO-heme, and protoporphyrin IX. The purified enzyme was only slightly (2- to 3-fold) activated by NO and nitroso compounds but was markedly (50-fold) activated by NO-heme and protoporphyrin IX, achieving maximal specific activities of 10 μmol cyclic GMP min^?1 mg protein^?1. Enzyme activation by NO and nitroso compounds was restored by addition of hematin or by reconstitution of guanylate cyclase with heme. Excess hematin, however, inhibited enzyme activity. A partially purified heat-stable factor (activation-enhancing factor) was found to enhance (2- to 35-fold) enzyme activation without directly stimulating guanylate cyclase. In the presence of optimal concentrations of hematin, enzyme activation was still increased (2-fold) by the activation-enhancing factor but not by bovine serum albumin. Guanylate cyclase was markedly inhibited by SH reactive agents such as cystine, o-iodosobenzoic acid, periodate, and 5,5′-dithiobis (2-nitrobenzoic acid). In addition, CN^? and FMN inhibited enzyme activation by NO-heme, but not by protoporphyrin IX, and did not affect basal enzymatic activity. Hepatic soluble guanylate cyclase appears to possess SH groups required for catalysis and to require heme and/or other unknown factors for the full expression of enzyme activation by NO and nitroso compounds.     

Guanylate cyclase from bovine lung. Evidence that enzyme activation by phenylhydrazine is mediated by iron-phenyl hemoprotein complexes

The mechanism of activation of soluble guanylate cyclase purified from bovine lung by phenylhydrazine is reported. Heme-deficient and heme-containing forms of guanylate cyclase were studied. Heme-deficient enzyme was activated 10-fold by NO but was not activated by phenylhydrazine. Catalase or methemoglobin enabled phenylhydrazine to activate guanylate cyclase 10-fold and enhanced activation by NO to over 100-fold. Heme-containing enzyme was activated only 3-fold by phenylhydrazine but over 100-fold by NO. Added hemoproteins enhanced enzyme activation by phenylhydrazine to 12-fold without enhancing activation by NO. Reducing or anaerobic conditions inhibited, whereas oxidants enhanced enzyme activation by phenylhydrazine plus catalase, and KCN had no effect. In contrast, enzyme activation by NO and NaN3 was inhibited by oxidants or KCN. NaN3 required native catalase, whereas phenylhydrazine also utilized heat-denatured catalase for enzyme activation. Thus, the mechanism of guanylate cyclase activation by phenylhydrazine differed from that by NO or NaN3. Guanylate cyclase activation by phenylhydrazine resulted from an O2-dependent reaction between phenylhydrazine and hemoproteins to generate stable iron-phenyl hemoprotein complexes. These complexes activated guanylate cyclase in the absence of O2, but lost activity after acidification, basification, or heating. Gel filtration of prereacted mixtures of [U-14C]phenylhydrazine plus hemoproteins resulted in co-chromatography of radioactivity, protein, and guanylate cyclase stimulating activity, and yielded a phenyl-hemoprotein binding stoichiometry of four under specified conditions (one phenyl/heme). [14C]Phenyl bound to heme-containing but not heme-deficient guanylate cyclase and binding correlated with enzyme activation. Moreover, reactions between enzyme and iron-[14C] phenyl hemoprotein complexes resulted in the exchange or transfer of iron-phenyl heme to guanylate cyclase and this correlated with enzyme activation.     

Conditions of activation of guanylate cyclase from human platelets

Preincubation (50 min, 0 degree C) with nitroprusside increases 12-fold the activity of human platelet guanylate cyclase. The stimulating effect of nitroprusside is enhanced two-fold by dithiothreitol (2 mM) and by 60% by hemoglobin (20 micrograms/ml). Storage of guanylate cyclase preparations (105000 g supernatant) for 2-3 days at 4 degrees C causes a progressive increase of the enzyme activity and diminishes the stimulating effect of nitroprusside. After storage of guanylate cyclase preparations for 3 days, hemoglobin (20 micrograms/ml) augments the stimulating effect of nitroprusside by 130%. It is concluded that the degree of activation of guanylate cyclase by nitroprusside reflects the functional state of the enzyme.     

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.     

YC-1-like potentiation of nitric oxide-dependent activation of soluble guanylate cyclase by adrenochrom

The influence of adrenochrome and YC-1 activation of human platelet soluble guanylate cyclase was investigated. Adrenochrome (0.1–10.0 μM) had no effect on the basal activity, but it potentiated in a concentration- dependent manner the spermine NONO-induced activation of this enzyme. Adrenochrome also sensitized guanylate towards nitric oxide (NO) and produced the leftward shift of the spermine NONO concentration response curve. Addition of adrenochrome decreased the YC-1-induced leftward shift of the spermine NONO concentration response curve. Adrenochrome also inhibited enzyme activation byYC-1. Thus, synergistic activation of NO-stimulated guanylate cyclase activity by adrenochrome represents a new biochemical effect of this compound and indicates that adrenochrome may act as an endogenous regulator of the NO-dependent stimulation of soluble guanylate cyclase. This new property of adrenochrome, similar to YC-1 but more effective, should be taken into consideration especially under conditions of adrenochrome overproduction in the body.     

Activation of soluble guanylate cyclase from rat lung by incubation or by hydrogen peroxide.

A 37,000 X g supernatant fraction prepared from fat lung homogenate demonstrated a 2- to 3-fold increase in guanylate cyclase activity after incubation at 30 degrees for 30 min (preincubation). Treatment of the supernatant fraction with Triton X-100 increased activity to approximately the same extent as preincubation, but would not increase the activity after preincubation. By chromatography on Sepharose 2B, before and after preincubation, it was demonstrated that the increase in activity was only associated with the soluble guanylate cyclase, and not the particulate enzyme. Activation by preincubation required O2. It was completely inhibited by thiols such as 2-mercaptoethanol, and by bovine serum albumin, KCN, and sodium diethyldithiocarbamate. These inhibitors suggested a copper requirement for activation, and this was confirmed by demonstrating that 20 to 60 muM CuCl2 could relieve the inhibition by 0.1 mM sodium diethyldithiocarbamate. 2-Mercaptoethanol inhibition could also be reversed by removal of the thiol on a Sephadex G-25 column, however, this treatment partially activated the enzyme. Addition of 2-mercaptoethanol to a preincubated preparation would not reverse the activation. H2O2 was found to activate guanylate cyclase, either by its generation in the lung supernatant with glucose oxidase and glucose, or by its addition to a preparation in which the catalase was inhibited with KCN. KCN or bovine serum albumin was able to partially inhibit activation by glucose oxidase plus glucose, however, larger amounts of glucose oxidase could overcome that inhibition, indicating a catalytic role for Cu2+ at low H2O2 concentrations. No direct evidence for H2O2 formation during preincubation could be found, however, indirect evidence was obtained by the spectrophotometric detection of choleglobin formation from hemoglobin present in the lung supernatant fluid. The H2O2 is believed to result from the reaction of oxyhemoglobin with ascorbate.     

Fatty acid activation of guanylate cyclase from fibroblasts and liver.

Sodium arachidonate and sodium oleate increased particulate guanylate cyclase activity from homogenates of Balb 3T3 cells or rat liver. The fatty acids were about equipotent and were maximally effective at about 100 μm concentrations. Higher concentrations were less effective or inhibitory. Activation was similar in an air or nitrogen atmosphere and was unaltered by KCN, aspirin, or indomethacin. The dose-response curve was shifted to the right when arachidonate was preincubated prior to its addition to guanylate cyclase assays. Agents that facilitate fatty acid oxidation and the formation of malonyldialdehyde during preincubation such as glutathione, hemoglobin, Mn²⁺, Fe³⁺, or lipoxygenase shifted the dose-response curve further to the right. In contrast, agents that decreased or prevented arachidonate oxidation and malonyldialdehyde formation during preincubation such as butylated hydroxyanisole, propyl gallate, hydroquinone, and diphenylfuran prevented the shift in the dose-response curve or in some instances shifted the dose-response curve to the left. Activation of guanylate cyclase by arachidonate was reversed by the addition of lipoxygenase to incubations. These studies indicate that unsaturated fatty acids and not their oxidation products activate particulate enzyme from Balb 3T3 cells. The mechanism of fatty acid activation appears to be different from activation by nitro compounds. Fatty acids but not nitro compounds activated fibroblast preparations, and the effect of fatty acids in contrast to the activation by nitroprusside in liver preparations was not prevented with Lubrol PX.     

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.     

Synthesis of nitric oxide in the bovine retina.

In the absence of light, high concentrations of cGMP open ion channels in the plasma membranes of rod outer segments. The source of stimulation of retinal guanylate cyclase is not known. Nitric oxide is a potent stimulator of guanylate cyclase in other cell systems. The present data demonstrate that nitric oxide synthase, an enzyme responsible for the production of nitric oxide, is present in retina, and specifically in the rod outer segments. This enzyme uses L-arginine as a substrate and is NADPH- and calcium- dependent. L-arginine-derived nitric oxide may be a source of activation of guanylate cyclase in the retina.