Louis J. Ignarro

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 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.

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.

Possible involvement of S‐nitrosothiols in the activation of guanylate cyclase by nitroso compounds

The inhibitory effects of methemoglobin on activation of coronary arterial soluble guanylate cyclase by nitric oxide (NO), nitroprusside and N-methylN’nitro-N-nitrosoguanidine (MNNG) were described in [ 11. Also dithiothreitol (DTT) enhanced activation of coronary arterial and hepatic soluble guanylate cyclase by nitroprusside and MNNG but not by NO, and reversed the methemoglobin blockade of activation by nitroprusside and MNNG but not by NO. Indeed, DTT was required for hepatic guanylate cyclase activation by nitroprusside. These observations suggested that DTT may react directly with the nitroso compounds to promote release of NO, which may then overcome the inhibition by methemoglobin [ 11. An alternative explanation for the effects of DTT on guanylate cyclase activation was a direct nonenzymatic transfer of the NO-moiety from the nitroso compound to guanylate cyclase (or a ferroheme intermediate) in the presence of thioB. Each of the above hypotheses was tested and the data here illustrate that: (1) Thiols promote release of NO gas from MNNG, but not from nitroprusside, in aqueous neutral buffer. (2) Inhibition of guanylate cyclase activation by NO varies directly with the concentration of methemoglobin and indirectly with the amount of NO. (3) Thiols react with NO to form stable S-nitrosothiols which are potent activators 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.

Abstract 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 μ m in the presence of excess Mg 2+ or Mn 2+ , 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.

Regulation of lysosomal enzyme secretion: Role in inflammation

The purpose of this review is first to discuss the lysosomal concept as it applies to the mediation of tissue injury, and second to provide new evidence for the regulation of lysosomal enzyme secretion from human neutrophils by cyclic nucleotides, autonomic neurohormones, prostaglandins, glucocorticosteroids and calcium. Lysosomal enzymes gain access to the extracellular environment by the selective secretion of lysosome granule contents from neutrophils during cell contact with various immune reactants. Discharge of lysosomal contents results in the provocation of acute inflammation and connective tissue degradation. The immunologic secretion of lysosomal enzymes from human neutrophils requires the presence of extracellular calcium and can be modulated by several different classes of hormones, drugs and other agents. Agents that enhance lysosomal enzyme secretion also produce a concomitant accumulation of cyclic GMP (guanosine 3′, 5′-monophosphate) in neutrophils. Such substances include immune reactants, acetylcholine, prostaglandin F2α and calcium ionophores. Cyclic GMP itself also stimulates lysosomal enzyme secretion. On the other hand, inhibition of enzyme release is frequently accompanied by the accumulation of intracellular cyclic AMP (adenosine 3′, 5′-monophosphate). These effects are produced by epinephrine (catecholamines) and several prostaglandins. Cyclic AMP also inhibits lysosomal enzyme secretion. Glucocorticosteroids also inhibit enzyme secretion but this effect is accompanied by an inhibition of cyclic AMP accumulation rather than by the stimulation of cyclic AMP accumulation. The presence of cyclic GMP, cyclic AMP and the cellular processes for accumulating either nucleotide provide the human neutrophil with the means by which to modulate the secretion of lysosomal contents in response of the cells to various endogenous substances and drugs.

Activation of coronary arterial guanylate cyclase by nitric oxide, nitroprusside, and nitrosoguanidine—Inhibition by calcium, lanthanum, and other cations, enhancement by thiols

Abstract Although reports that certain vasodilate s activate soluble guanylate cyclase, especially in the presence of thiols, and elevate cyclic GMP levels in smooth muscle suggest that cyclic GMP is involved in vascular smooth muscle relaxation, earlier reports that Ca 2+ activates guanylate cyclase and that Ca 2+ -dependent contractile agents elevate cyclic GMP levels are seemingly at odds with this hypothesis. The objective of this study was to examine the effects of Ca 2+ related cations, and thiols on bovine coronary arterial soluble guanylate cyclase. Guanylate cyclase activity was detected in the presence of Mg 2+ or Mn 2+ but not of other cations. Basal activity was greater in the presence of Mn 2+ than of Mg 2+ . Activity of guanylate cyclase stimulated by nitroprusside, nitric oxide, or nitrosoguanidine, however, was greater with Mg 2+ , although the requirement of activated enzyme for Mn 2+ was reduced about 10-fold. Ca 2+ markedly inhibited guanylate cyclase activation in the presence of Mg 2+ but not of Mn 2+ . La 2+ inhibited enzyme activation in the presence of Mg 2+ or Mn 2+ . Neither Ca 2+ nor La 3+ altered basal enzymatic activity. Results that were qualitatively similar to those indicated above were observed with partially purified, heme-free, coronary arterial soluble guanylate cyclase. Nitric oxide and nitroso compounds activated partially purified enzyme, and thiols enhanced enzyme activation by nitroprusside and nitrosoguanidine without appreciably altering basal activity. Irreversible sulfhydryl binding agents such as ethacrynic acid and gold inhibited both basal and activated guanylate cyclase. These results suggest that changes in intracellular concentrations of free Ca 2+ and sulfhydryl groups could influence the rate of formation of cyclic GMP by vasodilators and that this, in turn, could alter smooth muscle tone.

Evidence that regulation of hepatic guanylate cyclase activity involves interactions between catalytic site -SH groups and both substrate and activator.

Abstract Partially purified hepatic soluble guanylate cyclase (EC 4.6.1.2) was rapidly inactivated by molecular oxygen and by low concentrations of SH oxidants at pH 7.4, a process which was prevented and reversed by dithiothreitol. Cystamine and 5,5′-dithiobis(2-nitrobenzoic acid) inhibited enzymatic activity in a time- and temperature-dependent manner. Various disulfides, SH oxidants, and thiol alkylating agents inhibited both basal guanylate cyclase activity and activity stimulated by nitric oxide, S -nitrosocysteine, and nitrosylhemoglobin. These observations indicate that guanylate cyclase possesses one or more SH groups at its catalytic site. Preincubation of guanylate cyclase with excess MgGTP or with enzyme activators markedly protected the enzyme against inhibition by various thiol reactive agents. These findings suggest that the SH groups at the catalytic site interact also with enzyme activators. 2,3-Dimercaprol, which possesses vicinal dithiols, but not dithiothreitol markedly inhibited guanylate cyclase activity at concentrations equal to or exceeding those of MgGTP. Thus, two closely juxtaposed SH groups may be located at the catalytic site. The presence of cysteine or hematin in preincubates of guanylate cyclase, to which nitric oxide was also added, was mandatory in order to enable the activated form of the enzyme to be recovered by gel filtration. Guanylate cyclase activated by S -nitrosocysteine or nitrosyl-hemoglobin was recovered in the maximally activated state, and this was prevented by preincubation of enzyme with SH oxidants or excess MgGTP. These data imply that cysteine, hematin, and their nitrosyl derivatives bind to SH groups at the catalytic site of guanylate cyclase.

Bidirectional regulation of lysosomal enzyme secretion and phagocytosis in human neutrophils by guanosine 3',5'-monophosphate and adenosine 3',5'-monophosphate.

Summary The biologic roles of guanosine 3′,5′-monophosphate (cyclic GMP) and adenosine 3′,5′-monophosphate (cyclic AMP) in the secretion of lysosomal enzymes from, and in phagocytosis by, human neu-trophils were studied. Contact between neutrophils and particulate immunologic reactants results in both phagocytosis of the particles and secretion of lysosomal enzymes. These cellular events are accompanied by the accumulation of cyclic GMP and require the presence of extracellular calcium. Acetylcholine, pilocarpine, and cyclic GMP enhance, whereas epinephrine, cyclic AMP, and/or dibutyryl cyclic AMP inhibit, both phagocytosis and lysosomal enzyme secretion. The stimulatory action of cholinergic agents and the inhibitory action of epinephrine are accompanied by the accumulation of cyclic GMP and cyclic AMP, respectively, in human neutrophils. The data suggest that cyclic GMP mediates whereas cyclic AMP inhibits the major functions of human neutrophils. Moreover, by virtue of their effects on cyclic nucleotide accumulation, autonomic neurohormones are capable of modulating human neutrophil function.

Cytidine 3′,5′-monophosphate (cyclic CMP) formation by homogenates of mouse liver☆

Cyclic CMP3 has been identified as a product of the reaction between mouse liver homogenate, CTP and Mn2+ at neutral pH and 37°. This reaction appears to be enzymatic in character in that product formation is pH-, temperature-, time-, and substrate-dependent, and is inhibited by boiling the homogenate. Cyclic CMP formation is enhanced with 0.3 mM Mn2+ or Fe2+ and inhibited with 3 mM Mn2+ or detergents. Cyclic CMP was identified as one of the reaction products by comparison with authentic compound in several systems including: chromatography on neutral alumina columns, Dowex 1-formate columns, polyethyleneimine cellulose columns and thin layer plates; crystallization to constant specific activity; radioimmunoassay.

Selective alterations in responsiveness of guanylate cyclase to activation by nitroso compounds during enzyme purification.

Partially purified, heme-free, hepatic soluble guanylate cyclase was activated by NO, S-nitrosocysteine and NO-heme complexes to the extent of 19–34-fold in the presence of 3 mM Mg2+, but only up to 2-fold in the presence of 3mM Mn2+, when the GTP concentration was 1 mM. Even with Mg2+, however, nitroprusside and nitrosoguanidine failed to activate guanylate cyclase. Dithiothreitol or cysteine and, to a much lesser extent, hematin or hemoglobin restored the capacity of nitroprusside and nitrosoguanidine to activate guanylate cyclase, and enhanced enzyme activation by NO. Restoration or enhancement of enzyme activation with thiols was unrelated to their reducing potential because nonthiol reductants such as dithionite and ascorbate failed to influence guanylate cyclase activation. Instead, thiols likely reacted with the nitroso compounds to form S-nitrosothiols, which were potent activators of guanylate cyclase. Similarly, heme-containing substances reacted with nitroso compounds to form NO-heme complexes, which are known to activate guanylate cyclase. Enzyme activation by nitroprusside and nitrosoguanidine was restored, and that by NO and NO-hemoglobin was enhanced, by addition of heated hepatic soluble fraction (devoid of guanylate cyclase activity). The heated soluble fraction appears to contain a partially heat-stable, thiol-containing component(s) of molecular weight greater than 5000, which may be in part responsible for the observed effects on enzyme activation. These data suggest that partial enzyme purification results in the removal of thiol components that are required for the full expression of guanylate cyclase activation.