Effect of carnosine on the activation of human platelet soluble guanylate cyclase by sodium nitroprusside and protoporphyrin IX

Effect of carnosine on the activation of soluble guanylate cyclase by sodium nitroprusside and protoporphyrin IX was studied using human platelet 105000 g supernatants and partially purified heme-deficient guanylate cyclase preparations. In experiments with 105000 g supernatants, carnosine (1 mM) inhibited the enzyme activation by nitroprusside by about 70%. With the partially purified heme-deficient guanylate cyclase, the enzyme activation by nitroprusside was lowered by 86%, and the remaining insignificant stimulatory effect remained unchanged upon carnosine addition. The stimulatory effect of protoporphyrin IX on the partially purified heme-deficient enzyme preparation did not differ from that observed with the 105000 g supernatant; carnosine addition had no effect on activation of guanylate cyclase by protoporphyrin IX. It was concluded that the inhibitory effect of carnosine on the ability of the enzyme to be activated by nitroprusside is due to the interaction of carnosine with guanylate cyclase, and that it is heme directed.     

The role of carnosine in the function of soluble of guanylate cyclase]

The effect of carnosine on activation of human platelet soluble guanylate cyclase has been studied in 105,000 g supernatants and partially purified haem-deficient enzyme preparations. In the 105,000 g supernatant carnosine (1 mM) inhibited (by about 70%) the enzyme activation caused by sodium nitroprusside. In partially purified haem-deficient guanylate cyclase preparations the inhibition of enzyme activation by sodium nitroprusside was 86%; further addition of carnosine had no effect on the enzyme activity. The strength of the activating effect of protoporphyrin IX on partially purified haem-deficient guanylate cyclase did not differ from that for the 105,000 g supernatant; this stimulating effect did not change after carnosine addition. A conclusion is drawn that the inhibiting effect of carnosine on the ability of guanylate cyclase to be activated by sodium nitroprusside is due to the dipeptide interaction with the guanylate cyclase haem.     

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.     

Soluble guanylate cyclase in molecular mechanisms underlying the therapeutic action of drugs

The influence of ambroxol (a mucolytic agent) on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside (SNP) and Sin-1) were investigated. Ambroxol in the range of concentrations from 0.1 to 10 ??M had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the SNP-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC₅₀ values of 3.9 and 2.1 ??M, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX. The influence of artemisinin (an antimalarial agent) on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1?100 ??M) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the SNP-induced activation of human platelet guanylate cyclase with the IC₅₀ value of 5.6 ??M. Artemisinin (10 ??M) also inhibited (by 71 ± 4.0%) the activation of the enzyme by a thiol-dependent NO-donor, the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 ??M), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the signaling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin.     

Nitric oxide. Potentiation of NO-dependent activation of soluble guanylate cyclase: (Patho)physiological and pharmacotherapeutic importance

The review highlights the molecular mechanism underlying the physiological effects of nitric oxide (NO), the role of signaling system: NO-soluble guanylate cyclase-cyclic 3′,5′-guanosine monophosphate (cGMP) in the realization of NO action. This review considers data on basic chemical characteristics of guanylate cyclase, such as the subunits structure, isoforms, modern concepts of the catalytic and regulatory centers of this enzyme. Realization of physiological effects of NO by guanylate cyclase depends on its heme prostetic group. NO-dependent activation of guanylate cyclase may be synergistically increased by a new NO-independent, allosteric activator of soluble guanylate cyclase-YC-1-(benzyl indasol derivative). Special attention is paid to the data on guanylate cyclase sites responcible for binding of the enzyme with YC-1 and the possible molecular mechanism underlying the synergistic increase of NO-dependent activation of soluble guanylate cyclase by YC-1. New compounds of endogenous nature capable to potentiate and synergistically increase the activation of guanylate cyclase by NO-donors have been found and investigated. The important physiological, pharmacotherapeutical and pathophysiological significance of this new fact is discussed.     

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.     

Potentiation of YC-1 activation of soluble guanylate cyclase by NO donors and the increase of the synergistic effect of YC-1 on the NO-dependent activation of the enzyme by 1,2,3-triazolyl-1,2,5-oxadiazole derivatives

The influence of (1H-1,2,3-triazol-1-yl)-1,2,5-oxadiazole derivatives: 4-amino-3-(5-methyl-4-ethoxycarbonyl-(1H-1,2,3-triazol-1-yl)-1,2,5-oxadiazole (TF₄CH₃) and 4,4′-bis(5-methyl-4-ethoxycarbo-nyl-1H-1,2,3-triazol-1-yl)-3,3′-azo-1,2,5-oxadiazole (2TF₄CH₃) on stimulation of human platelet soluble guanylate cyclase by YC-1, NO donors (sodium nitroprusside, SNP, and spermine NONO) and on a synergistic increase of NO-dependent activation of the enzyme in the presence of YC-1 has been investigated. Both compounds increased guanylate cyclase activation by YC-1, potentiated guanylate cyclase stimulation by NO donors and increased the synergistic effect of YC-1 on the NO-dependent activation of soluble guanylate cyclase. The similarity in the properties of the examined 1,2,3-triazol-1-yl-1,2,5-oxadiazole derivatives with that of YC-1 and a possible mechanism underlying the recognized properties of compounds used are discussed.     

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.     

Electron spin resonance study of the role of NO . catalase in the activation of guanylate cyclase by NaN3 and NH2OH. Modulation of enzyme responses by heme proteins and their nitrosyl derivatives.

The role of NO . catalase in the activation of partially purified soluble guanylate cyclase of rat liver by NaN3 and NH2OH was examined by electron spin resonance (ESR) spectroscopy. Equilibration of bovine liver catalase with NO resulted in formation of a paramagnetic species exhibiting a three-line ESR spectrum similar to that of NO . catalase. This paramagnetic complex produced concentration-dependent stimulation of preparations of partially purified guanylate cyclase that were devoid of detectable endogenous heme content. The stimulation of partially purified guanylate cyclase by NO . catalase was similar to that obtained with NO . hemoglobin and with NO . cytochrome P-420 prepared by reaction of hepatic microsomes of phenobarbital-treated rats with NO. By contrast, these same enzyme preparations did not respond to NO or catalase alone. Addition of hematin or hemoglobin plus a reducing agent to purified guanylate cyclase restored enzyme responsiveness to NO and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but not to NaN3 or NH2OH. Responses to the latter agents were restored by catalase and potentiated by a H2O2-generating system. Formation of the NO . catalase complex was evident by ESR spectroscopy in test solutions containing NaN3 or nh2oh, catalase, and a glucose-glucose oxidase, H2O2-generating system. The presence of NO . catalase correlated well with the ability of test solutions to activate purified guanylate cyclase. These results provide evidence for catalase-dependent NO generation from NaN3 and NH2OH under conditions leading to guanylate cyclase activation. Preformed NO . hemoglobin or NO . cytochrome P-420 also activated heme-deficient partially purified guanylate cyclase. The ability of several preformed NO . heme protein complexes, but not NO, to stimulate heme-deficient guanylate cyclase supports the concept that formation of the paramagnetic nitrosyl . heme complex, mediated by either enzymatic or nonenzymatic reactions, is a common and essential step in the process by which NO or NO-forming compounds activate guanylate cyclase. In the absence of the NO ligand, both hemoglobin and catalase suppress the stimulatory effects of the corresponding NO . heme proteins on guanylate cyclase. Release of each heme protein from the NO . heme protein complex occurs more rapidly under aerobic compared to anaerobic conditions. However, hemoglobin is approximately 2000 times more effective as an inhibitor of NO . hemoglobin stimulation of guanylate cyclase than is catalase as an inhibitor of NO . catalase action. This finding may explain the more pronounced decline in the rate of cGMP generation in air in the presence of NO . hemoglobin compared to NO . catalase. The results imply that guanylate cyclase responses to activators that can form NO are determined by both the stimulatory activity of the endogenous heme acceptors of NO and the relative inhibitory effects of the unliganded heme proteins present.     

Activation of soluble guanylate cyclase by NO-hemoproteins involves NO-heme exchange. Comparison of heme-containing and heme-deficient enzyme forms

The mechanism of activation of soluble guanylate cyclase purified from bovine lung by high molecular weight, nitrosyl-hemoprotein complexes is reported. Heme-containing, heme-deficient, and heme-reconstituted forms of guanylate cyclase were studied. Nitric oxide (NO) and nitroso compounds activated heme-containing and heme-reconstituted enzymes (over 50-fold), with an accompanying shift in the Soret absorption peak from 431 to 398 nm, but failed to activate or alter the spectral characteristics of heme-deficient enzyme. In contrast, preformed NO-hemoprotein complexes as well as low molecular weight NO-heme activated all forms of guanylate cyclase. Heme-deficient guanylate cyclase was first reacted with excess amounts of NO-hemoglobin, NO-myoglobin, or NO-catalase and then rapidly separated from the NO-hemoprotein by column chromatography. Spectrophotometric analysis indicated that the NO-heme moiety was transferred from each of the NO-hemoproteins to heme-deficient guanylate cyclase. Approximately 1 mol of NO-heme was bound per mol of holoenzyme and the specific activity of this enzyme form was over 50-fold greater than that of unreacted, heme-deficient enzyme. NO-heme was tightly bound to guanylate cyclase as no transfer of enzyme-bound NO-heme to apohemoglobin was evident. Enzyme activated by NO-hemoproteins closely resembled, kinetically, that activated by NO or NO-heme. In contrast, reactions between heme-deficient guanylate cyclase and hemoproteins did not result in heme transfer, whereas heme alone rapidly reconstituted the enzyme. These observations indicate that soluble guanylate cyclase can be readily reconstituted with, and thereby activated by, NO-heme through an exchange reaction with NO-hemoproteins.     

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.     

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.     

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.     

Regulation of soluble guanylate cyclase activity by porphyrins and metalloporphyrins

Alterations of the chemical structure of protoporphyrin IX markedly altered the activation of soluble guanylate cyclase purified from bovine lung. Hydrophobic side chains at positions 2 and 4 and vicinal propionic acid residues at positions 6 and 7 of the porphyrin ring (protoporphyrin IX, mesoporphyrin IX) were essential for maximal enzyme activation (Ka = 7-8 nM; Vmax = 6-8 mumol of cGMP/min/mg). Substitution of hydrophobic with polar groups (hematoporphyrin IX, coproporphyrin III), or with hydrogen atoms ( deuteroporphyrin IX), and methylation of propionate residues resulted in decreased enzyme stimulation. Stimulatory porphyrins increased the Vmax and the apparent affinities of enzyme for MgGTP and uncomplexed Mg2+. An open central core in the porphyrin ring was essential for enzyme activation. The pyrrolic nitrogen adduct, N-phenylprotoporphyrin IX, was inhibitory and competitive with protoporphyrin IX (KI = 73 nM). Similarly, metalloporphyrins inhibited enzymatic activity and ferro-protoporphyrin IX (KI = 350 nM), zinc-protoporphyrin IX (KI = 50 nM) and manganese-protoporphyrin IX (KI = 9 nM) were competitive with protoporphyrin IX. Inhibitory porphyrins and metalloporphyrins also prevented enzyme activation by S-nitroso-N- acetylpenicillamine and NO. Guanylate cyclase reconstituted with such porphyrins required higher concentrations of protoporphyrin IX for further activation and were not activated by NO. Thus, porphyrins, metalloporphyrins, and NO appeared to interact at a common binding site on guanylate cyclase. This common site is likely that which normally binds heme and, therefore, NO-heme when the heme-containing enzyme is exposed to NO. Thus, NO and nitroso compounds may react with enzyme-bound heme to generate a modified porphyrin which structurally resembles protoporphyrin IX in its interaction with guanylate cyclase.     

Activation of soluble guanylate cyclase by arachidonic acid and 15-lipoxygenase products

The activity of soluble guanylate cyclase can be increased by exposure of the enzyme to arachidonic acid or to some oxidized metabolites of the fatty acid. We have tried to determine whether activation of the enzyme by arachidonate requires that the fatty acid be converted to an oxidized metabolite, either by a possible trace contaminant of a lipoxygenase or by guanylate cyclase itself, which contains a heme moiety. Soluble guanylate cyclase purified from bovine lung was activated 4-6-fold by arachidonic acid. This activation was not dependent on the presence of oxygen in the incubation medium. No detectable metabolites of arachidonic acid were formed during incubation with soluble guanylate cyclase. Addition of soybean lipoxygenase to the incubation did not increase activation by arachidonic acid. The inhibitors of lipoxygenase activity, nordihydroguaiaretic acid and eicosatetraynoic acid, had direct effects on soluble guanylate cyclase and interfered with its activation by arachidonate, whereas another lipoxygenase inhibitor, BW 755 C, did not. The data suggest that arachidonic acid increases the activity of guanylate cyclase by direct interaction with the enzyme rather than by being converted to an active metabolite.     

Inhibition of NO-dependent soluble human platelet guanylate cyclase by isatin

Isatin (indole-dione-2,3) is an endogenous indole that exhibits a wide spectrum of biological and pharmacological activities. Physiologically relevant concentrations of isatin (ranged from 1 nM to 10 μM) did not influence basal activity of soluble human platelet guanylate cyclase (sGC), but caused a bell-shaped inhibition of the NO-activated enzyme. Inhibition of the NO-dependent activation by isatin did not depend on a chemical nature of the NO donors. The inhibitory effects of ODC (a heme-dependent inhibitor of sGC) and isatin were non-additive suggesting that the inhibitory effect of isatin may involve the heme binding domain (possibly heme iron) and experiments with hemin revealed some isatin-dependent changes in its spectrum. Isatin also inhibited sGC activation by the allosteric activator YC-1. It is suggested that the bell shaped inhibition of the NO-dependent activation of sGC by isatin may be attributed to complex interaction of isatin with the heme binding domain and the allosteric YC-1-binding site of sGC.     

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.     

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.     

Nitric oxide regulation of mitochondrial oxygen consumption II: Molecular mechanism and tissue physiology

Nitric oxide (NO) is an intercellular signaling molecule; among its many and varied roles are the control of blood flow and blood pressure via activation of the heme enzyme, soluble guanylate cyclase. A growing body of evidence suggests that an additional target for NO is the mitochondrial oxygen-consuming heme/copper enzyme, cytochrome c oxidase. This review describes the molecular mechanism of this interaction and the consequences for its likely physiological role. The oxygen reactive site in cytochrome oxidase contains both heme iron (a₃) and copper (Cu_B) centers. NO inhibits cytochrome oxidase in both an oxygen-competitive (at heme a₃) and oxygen-independent (at Cu_B) manner. Before inhibition of oxygen consumption, changes can be observed in enzyme and substrate (cytochrome c) redox state. Physiological consequences can be mediated either by direct "metabolic" effects on oxygen consumption or via indirect "signaling" effects via mitochondrial redox state changes and free radical production. The detailed kinetics suggest, but do not prove, that cytochrome oxidase can be a target for NO even under circumstances when guanylate cyclase, its primary high affinity target, is not fully activated. In vivo organ and whole body measures of NO synthase inhibition suggest a possible role for NO inhibition of cytochrome oxidase. However, a detailed mapping of NO and oxygen levels, combined with direct measures of cytochrome oxidase/NO binding, in physiology is still awaited. mitochondria; cytochrome oxidase