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     

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.     

Cytochrome P-450 mediated biotransformation of organic nitrates

The vascular biotransformation of organic nitrates appears to be a prerequisite for their action as vasodilators. In the current study, we assessed the involvement of cytochrome P-450 in the denitration of glyceryl trinitrate and the enantiomers of isoidide dinitrate. Denitration of organic nitrates by the microsomal fraction of rat liver was NADPH dependent and followed apparent first-order kinetics. Under aerobic conditions, the t1/2 of D-isoidide dinitrate was significantly shorter than that of L-isoidide dinitrate (11.9 vs. 14.1 min, p less than or equal to 0.05), which is consistent with the greater potency of the D-enantiomer for vasodilation. Under anaerobic conditions, the denitration of glyceryl trinitrate was very rapid (t1/2 approximately 30 s). Organic nitrate biotransformation was inhibited by carbon monoxide, SKF 525A, and dioxygen. This suggests that the biotransformation of organic nitrates can occur through the direct interaction with the heme moiety of cytochrome P-450. The biotransformation of glyceryl trinitrate was catalyzed preferentially by those isoenzymes induced by phenobarbital. The biotransformation of glyceryl trinitrate was regioselective for 1,3-glyceryl dinitrate formation except in phenobarbital-induced microsomes under aerobic conditions, in which preferential formation of 1,2-glyceryl dinitrate occurred. These data suggest that cytochrome P-450 is involved in the biotransformation of organic nitrates and raises the possibility that vascular cytochrome P-450 may play a role in the mechanism-based biotransformation of organic nitrates, the result of which is vascular smooth muscle relaxation.     

Endothelium-derived relaxing factor (nitric oxide) has protective actions in the stomach

The role that nitric oxide, an endothelium-derived relaxing factor, may play in the regulation of gastric mucosal defence was investigated by assessing the potential protective actions of this factor against the damage caused by ethanol in an ex vivo chamber preparation of the rat stomach. Topical application of glyceryl trinitrate and sodium nitroprusside, which have been shown to release nitric oxide, markedly reduced the area of 70% ethanol-induced hemorrhagic damage. Topical application of a 0.01% solution of authentic nitric oxide also significantly reduced the severity of mucosal damage. Pretreatment with indomethacin precluded the involvement of endogenous prostaglandins in the protective effects of these agents. The protective effects of NO were transient, since a delay of 5 minutes between NO administration and ethanol administration resulted in a complete loss of the protective activity. The protection against ethanol afforded by 10 micrograms/ml nitroprusside could be completely reversed by intravenous infusion of either 1% methylene blue or 1 mM hemoglobin, both of which inhibit vasodilation induced by nitric oxide. Intravenous infusion of 1% methylene blue significantly increased the susceptibility of the mucosa to damage induced by topical 20% ethanol. These results indicate that ethanol-induced gastric damage can be significantly reduced by nitric oxide. The mechanisms underlying the protective actions of nitric oxide are unclear, but may be related to its vasodilator or anti-aggregatory properties.     

Diaphorase can metabolize some vasorelaxants to NO and eliminate NO scavenging effect of 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)

Diaphorase was studied as a possible oxidoreductase participating in NO production from some vasorelaxants. In the presence of NADH or NADPH, diaphorase can convert selected NO donors, glycerol trinitrate (GTN) and formaldoxime (FAL) to nitrites and nitrates with NO as an intermediate. This activity of diaphorase was inhibited by diphenyleneiodonium (DPI) (inhibitor of some NADPH-dependent flavoprotein oxidoreductases), while it remained uninhibited by NG-nitro-L-arginine methyl ester (inhibitor of NO synthase) 7-Ethoxyresorufin (inhibitor of cytochrome P-450 1A1 and cytochrome P-450 NADPH-dependent reductase) inhibited the conversion of GTN only. Existence of NO as an intermediate of the reaction was supported by results of electron paramagnetic resonance spectroscopy. In addition to its ability to affect the above mentioned NO donors, diaphorase was able to reduce 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) and thus to eliminate its NO scavenging effect. This activity of diaphorase could also be inhibited by DPI. The reaction of diaphorase with GTN and PTIO was not affected by superoxide dismutase (SOD) or catalase. Reaction of FAL with diaphorase was lowered with SOD by 38 % indicating the partial participation of superoxide anion probably generated by the reaction of diaphorase with NADH or NADPH. Catalase had no effect. Diaphorase could apparently be one of the enzymes participating in the metabolism of studied NO donors to NO. The easy reduction and consequent elimination of PTIO by diaphorase could affect its use as an NO scavenger in biological tissues.     

Influence of dinitroglycerol and nitroethyleneglycol lipid derivatives on spectral parameters of human hemoglobin

The interaction of organic nitrates (nitroethyleneglycol, dinitroglycerol, and their esters with arachidonic acid) with oxyhemoglobin and methemoglobin has been studied. Addition of nitroethyleneglycol and dinitroglycerol to oxyhemoglobin is accompanied by a modest but significant increase in oxidation rate of the heme protein to the high-spin ferri-form--methemoglobin. Arachidonoylglycerol dinitrate exerts a similar but more pronounced effect on hemoglobin: a molar excess of this dinitrate induces the transformation of a significant portion of oxyhemoglobin to methemoglobin, whereas arachidonoylnitroethyleneglycol is inactive. Arachidonoylglycerol dinitrate also induces changes in the spectral characteristics of methemoglobin; this may be due to disintegration of the methemoglobin with the loss of heme. The data demonstrate that some organic nitrates can interact with hemoglobin; this should be taken into account when using the oxyhemoglobin technique for measuring nitric oxide generation from these compounds.     

Stoichiometry of the reaction of oxyhemoglobin with nitrite.

During the reaction of oxyhemoglobin (HbO2) with nitrite, the concentration of residual nitrite, nitrate, oxygen, and methemoglobin (Hb+) was determined successively. The results obtained at various pH values indicate the following stoichiometry for the overall reaction: 4HbO2 + 4NO2- 4H+ leads to 4Hb+ + 4NO3- + O2 + 2H2 O (Hb denotes hemoglobin monomer). NO2- binds with methemoglobin noncooperatively with a binding constant of 340 M-1 at pH 7.4 and 25 degrees C. Thus, the major part of Hb+ produced is aquomethemoglobin, not methemoglobin nitrite, when less than 2 equivalents of nitrite is used for the oxidation.     

Increased permeability of the blood-brain barrier following administration of glyceryl trinitrate in common carp (Cyprinus carpio L.)

Nitric oxide (NO) has been implicated in the pathogenesis of migraine and treatment with its exogenous donor glyceryl trinitrate (GTN) represents widely accepted experimental "migraine model". In this study, glyceryl trinitrate was administered intraperitoneally to carps, serum nitrite and nitrate levels were determined, permeability of blood-brain barrier was investigated, and histological changes of brain tissue were analyzed. Serum nitrite and nitrate levels displayed characteristic biphasic pattern with moderate initial increase and maximal terminal increase, suggesting the GTN-induced endogenous NO synthesis. Increased permeability of the blood-brain barrier in GTN-treated animals was determined based on Evans blue capillary leakage into the brain tissue. Histological analysis revealed changes consistent with vasodilatation and oedema. Our study strongly supports the importance of the NO role in the pathogenesis of migraine attacks and increase in blood-brain barrier permeability during the attack. The study has also provided evidence that this mechanism of action is conserved to the lower vertebrate.     

Comparison of neuronal and endothelial isoforms of nitric oxide synthase in stably transfected HEK 293 cells

The neuronal and endothelial isoforms of nitric oxide (NO) synthase (nNOS and eNOS, respectively) both catalyze the production of NO but are regulated differently. Stably transfected HEK 293 cell lines containing nNOS, eNOS, and a soluble mutant of eNOS were therefore established to compare their activity in a common cellular environment. NOS activity was determined by measuring L-[3H]citrulline production in homogenates and intact cells, the conversion of oxyhemoglobin to methemoglobin, and the production of cGMP. The results indicate that nNOS is more active than eNOS, both in unstimulated as well as calcium-stimulated cells. Under basal conditions, the soluble mutant of eNOS appeared to be slightly more active than wild-type eNOS in terms of NO and cGMP formation, suggesting that membrane association may be crucial for inhibition of basal NO release but is not required for stimulation by Ca2+-mobilizing agents. The maximal activity of soluble guanylate cyclase was significantly reduced by transfection with wild-type eNOS due to downregulation of mRNA expression. These results demonstrate that nNOS and eNOS behave differently even in an identical cellular environment.     

The interaction between nitrogen oxides and hemoglobin and endothelium-derived relaxing factor

Among nitrogen oxides, NO and NO₂ are free radicals and show a variety of biological effects. NO₂ is a strongly oxidizing toxicant, although NO, not oxidizing as NO₂, is toxic in that it interacts with hemoglobin to form nitrosyl-and methemoglobin. Nitrosylhemoglobin shows a characteristic electron spin resonance (ESR) signal due to an odd electron localized on the nitrogen atom of NO and reacts with oxygen to yield nitrate and methemoglobin, which is rapidly reduced by methemoglobin reductase in red cells. NO was found to inhibit the reductase activity. Part of NO inhaled in the body is oxidized by oxygen to NO₂, which easily dissolves in water and converts to nitrite. The nitrite oxyhemoglobin autocatalytically after a lag. The mechanism of the oxidation, particularly the involvement of superoxide, was controversial. The stoichiometry of the reaction has now been established using nitrate ion electrode and a methemoglobin free radical was detected by ESR during the oxidation. Complete inhibition of the autocatalysis by aniline or aminopyrine suggests that the radical catalyzes conversion of nitrite to NO₂, which oxidizes oxyhemoglobin. Recently NO was shown to be one of endothelium- derived relaxing factors and the relaxation induced by the factor was inhibited by hemoglobin and potentiated by superoxide dismutase.     

Role of NADPH and the NADPH-dependent methemoglobin reductase in the hydroxylase activity of human erythrocytes

Human erythrocytes were shown previously to catalyze the oxyhemoglobin-requiring hydroxylation of aniline, and the reaction was stimulated apparently preferentially by NADPH in the presence of methylene blue (K. S. Blisard and J. J. Mieyal,J. Biol. Chem.254, 5104, 1979). The current study provides a further characterization of the involvement of the NADPH-dependent electron transport system in this reaction. In accordance with the role of NADPH, the hydroxylase activity of erythrocytes or hemolysates from individuals with glucose-6-phosphate dehydrogenase deficiency (i.e., with diminished capacity to form NADPH) displayed decreased responses to glucose or glucose 6-phosphate, respectively, in the presence of methylene blue in comparison to samples from normal adults; maximal activity could be restored by direct addition of NADPH to the deficient hemolysates. Kinetic studies of the methylene blue-stimulated aniline hydroxylase activity of normal hemolysates revealed a biphasic dependence on NADPH concentrations: a plateau was observed at relatively low concentrations (K_m^NADPH ~ 20 μm), whereas saturation was not achieved at the higher concentrations of NADPH. The latter low efficiency phase (i.e., at the higher concentrations of NADPH) could be ascribed to a direct transfer of electrons from NADPH to methylene blue to hemoglobin. The high efficiency phase suggested involvement of the NADPH-dependent methemoglobin reductase; accordingly 2′-AMP, an analog of NADP⁺, effectively inhibited this reaction, but the pattern was noncompetitive. This behavior is suggestive of a mechanism by which both NADPH and methylene blue are substrates for the reductase and interact with it in a sequential fashion. The kinetic patterns observed for variation in NADPH concentration at several fixed concentrations of methylene blue, and vice versa, are consistent with this interpretation.     

The kinetic differences between sodium nitrite, amyl nitrite and nitroglycerin oxidation of hemoglobin

The effect of sodium nitrite, amyl nitrite and nitroglycerin (glyceryl trinitrate) on the hemoglobin of adult erythrocytes was examined in vitro. Both amyl nitrite and nitroglycerin reacted immediately with oxyhemoglobin to effect oxidation into methemoglobin while sodium nitrite required an inductionary period (lag phase) prior to the reaction. Kinetic studies of the biomolecular rate law for each of the preceding reaction's reactionary periods (log phases) allowed rate constant calculations to be made. The values are 1.14 x 10(4) M-1 min-1, 7.45 x 10(4) M-1 min-1, and 3.50 x 10(1) M-1 min-1 for sodium nitrite, amyl nitrite and nitroglycerin, respectively. A comparison of the amyl nitrite and nitroglycerin rate constants reveals that amyl nitrite is approximately 2000-fold more toxic to oxyhemoglobin than nitroglycerin. These oxidant's effect on in vitro hemoglobin solutions are comparable since both reactions approximate to rectangular hyperbolae. Sodium nitrite reacts about 300-fold faster with oxyhemoglobin than does nitroglycerin. However, the sodium nitrite reaction proceeds in a sigmoidal fashion which makes a strict comparison between these compounds relative toxicities less clear cut.     

Paramagnetic centers formed in mouse blood gamma-irradiated at 77K

The method of low-temperature ESR-spectroscopy was used to study the primary products of radiation injury of mouse blood. It was shown that when blood samples were exposed to gamma-radiation at 77 K radicals of water, lipids and proteins and also oxyhemoglobin adducts (Fe3+--O2(2-)) were formed. It was shown that oxyhemoglobin electron adducts were protonated during stepwise annealing to form complexes (Fe3+--HO2-). High spin methemoglobin is probably the final product of transformations of the oxyhemoglobin complexes.     

A common enzyme may be responsible for the conversion of organic nitrates to nitric oxide in vascular microsomes.

We compared the nitric oxide (NO)-generating behavior of nitroglycerin (NTG), pentaerythritol trinitrate (PEtriN) and isosorbide dinitrate (ISDN), in the microsomal preparation of bovine coronary artery smooth muscle cells. The comparative NO generating activities among these nitrates were consistent with their relative reported vasodilating activities. Consistent with our previous observations with NTG, 400 microM bromosulfophthalein did not affect NO generation from PEtriN and ISDN in vascular microsomes while 400 microM 1-chloro-2,4-dinitrobenzene completely inhibited NO generation from these nitrates. Gel filtration chromatography with solubilized microsomes of bovine aortic smooth muscle cells showed the primary activity of NO generation from all three nitrates to be eluted at about 200 kD, consistent with that found with solubilized microsomes from the bovine coronary artery microsomes. These results suggest that organic nitrates may be converted to NO by one common enzyme in vascular microsomes.     

Possible contribution of oxyhemoglobin to the iron-induced hemolysis simultaneous effect of iron and hemoglobin on lipid peroxidation

The mechanism of iron toxicity in iron overloaded patients is not well established. A hypothesis was put forward that free radical processes are involved. Our earlier study indicates that iron-induced hemolysis is preceded by peroxidation of the membrane lipids. In the present work the simultaneous effect of iron and hemoglobin on lipid peroxidation was studied. It was found that in hemoglobin-containing liposome suspensions Fe2+ in concentrations above 10(-5) M inhibits the peroxidation, while Fe3+ drastically potentiates it, with concomitant transformation of oxyhemoglobin to methemoglobin. The experiments with scavengers of activated oxygen indicate superoxide anion radical (O-.2), hydroxyl radical (OH.) and singlet oxygen (1O2) participation. The possible mechanism of the phenomenon is discussed. A conclusion is drawn that the toxic effect of Fe3+ may be associated not only with iron--membrane interaction, but also with increased methemoglobin formation and O-.2 release.     

Nitric oxide formation during microsomal hepatic denitration of glyceryl trinitrate: involvement of cytochrome P-450

Glyceryl trinitrate was denitrated by rat liver microsomes in the presence of NADPH with formation of a mixture of glyceryl dinitrates and glyceryl mononitrates. The highest activity was obtained under anaerobic conditions and the reaction was inhibited by O2 indicating that it is a reductive denitration. It was also inhibited by CO, metyrapone and miconazole showing that it was catalyzed by cytochrome P-450. Finally the formation of the cytochrome P-450-Fe(II)-NO complex during this reaction was shown by visible spectroscopy. These data demonstrate that microsomal reductive denitration of glyceryl trinitrate is catalyzed by cytochrome P-450 and can be involved in the formation of the endothelium-derived relaxing factor (EDRF = nitric oxide).     

Conversion of oxyhemoglobin to methemoglobin by organic and inorganic reductants

Human oxyhemoglobin is converted to methemoglobin by a wide array of organic and inorganic reductants. Depending upon the concentration and nature of the reductant, varying amounts of deoxyhemoglobin are produced. The general overall sequence is: FeO2 leads to (1) FeIII leads to (2) FeII. The intermediacy of methemoglobin can be demonstrated by direct spectral observation and by cyanide trapping. For organic reductants, the second-order rate constants for (1) vary from greater than 300 (phenylhydroxylamine) to 1.4 X 10(-4) M-1 s-1 (malononitrile). Generally the rates parallel the ease of hydrogen abstraction by iron-bound oxygen from the substrate, and simply hydrocarbons are reactive. Rates for these processes have been ascertained with recrystallized protein, lysed cells, and intact human erythrocytes. At room temperature oxyhemoglobin quantitatively converts benzaldehyde to benzoic acid and hydroquinone to benzoquinone. Rates for inorganic species (process 1) range from greater than 7 X 10(3) (chromous ion) to 0.015 M-1 s-1 (ferrocyanide). Ferrous ion rapidly deoxygenates oxyhemoglobin by direct attack on the oxy complex but methemoglobin is not an intermediate with this reagent. Taken together the results support the theoretical prediction that reductants should oxidize oxyhemoglobin, and they demonstrate at least some degree of radical character to the oxy complex.     

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.     

NO donors. Part 18: Bioactive metabolites of GTN and PETN—Synthesis and vasorelaxant properties

The vasodilators glyceryl trinitrate (GTN) and pentaerythrityl tetranitrate (PETN) are supposed to be degraded in vivo to the lower nitrates PETriN, PEDN, PEMN, 1,2-GDN, 1,3-GDN, 1-GMN, and 2-GMN. We synthesized these bioactive metabolites as reference compounds for pharmacokinetic studies. The use of HPLC-methods for monitoring the stepwise reduction of PETN to lower nitrates and the syntheses of the glyceryl dinitrates proved advantageous. Furthermore, we measured the vasorelaxant properties of all metabolites by performing organ bath experiments with porcine pulmonary arteries. In general, the vasodilator potency increases with the number of nitrate moieties in the compound.     

The special behavior of equine erythrocytes connected with the methemoglobin regulation

Erythrocytes from thoroughbred horses were submitted to total (80-90%) and partial (25-40%) oxidation of hemoglobin by sodium nitrite. The ability of these cells to reduce methemoglobin to hemoglobin in the presence of either glucose, glucose plus methylene blue or lactate was investigated. The results were compared with those ones obtained for human erythrocytes. Under total oxidation: the horse erythrocytes need longer incubation time with glucose or glucose plus methylene blue than human erythrocytes for reducing the methemoglobin; methylene blue did not enhance methemoglobin reduction in the equine erythrocytes, as occurred in human erythrocytes; for horses, lactate was a more efficient substrate in promoting methemoglobin reduction. The reduction of methemoglobin by equine erythrocytes under partial oxidation was very quick in any of the incubation media. The results can explain the incongruity between the previously reported inability of equine erythrocytes to reduce methemoglobin and the lack of methemoglobinemias in equine veterinary practice.