Peroxynitrite but not nitric oxide donors destroys epinephrine: HPLC measurement and rat aorta contractility

Nitric oxide (NO) and peroxynitrite (ONOO) have been reported to destroy catecholamines. We compared the ability of NO donors and peroxynitrite to decompose epinephrine in both chemical and pharmacological experiments. Epinephrine (1 microM) was incubated with NO donors (SNAP and MAHMA NONOate) and ONOO at a concentration of 0.1 mM in phosphate buffer (pH 7.4; 0.1 M) or Krebs solution for 10 minutes at 37 degrees C. HPLC revealed that the concentration of epinephrine in the presence of NO donors was unaltered. In contrast, peroxynitrite decreased epinephrine concentration more than 20 fold. Similar relationships were obtained in the study of rat thoracic aorta ring contraction. The contractile activity (EC50) of epinephrine in control solutions and after incubation of NE with NO donors did not change. EC50 was measured at 8-10 nM in control solutions and after preincubation with NO donors. However when epinephrine was preincubated with peroxynitrite, no contractile effect was evoked. Therefore, under these experimental conditions peroxynitrite, but not NO donors, was capable of destroying epinephrine.     

Cyclic AMP and cyclic GMP independent stimulation of ventricular calcium current by peroxynitrite donors in guinea pig myocytes

We investigated the potential involvement of peroxynitrite (ONOO(-)) in the modulation of calcium current (I(Ca)) in guinea pig ventricular myocytes with the whole-cell patch clamp technique and with cyclic AMP (cAMP) measurements. Because of the short half-life of ONOO(-) at physiological pH, we induced an increase in its intracellular levels by using donors of the precursors, nitric oxide (NO) and superoxide anion (O(2) (-)). High concentrations of NO donors, SpermineNONOate (sp/NO, 300 microM) or SNAP (300 microM) increased basal I(Ca) (50.3 +/- 4.6%, n = 7 and 46.2 +/- 5.0%, n = 13). The superoxide anion donor Pyrogallol (100 microM) also stimulated basal I(Ca) (44.6 +/- 2.8%, n = 11). At lower concentration sp/NO (10 nM) and Pyrogallol (1 microM), although separately ineffective on I(Ca), enhanced the current if applied together (33.5 +/- 0.7%, n = 7). The simultaneous donor of O(2) (-) and NO, SIN-1 (500 microM), also stimulated basal I(Ca) (22.8 +/- 2.1%, n = 13). In the presence of saturating cyclic GMP (cGMP, 50 microM) in the patch pipette or of extracellular dibutyryl cGMP (dbcGMP, 100 microM), I(Ca) was still increased by SIN-1 (32.0 +/- 6.1%, n = 4 and 30.0 +/- 5.4%, n = 8). Both Manganese(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP, 100 microM) a ONOO(-) scavenger, and superoxide dismutase (SOD) (150 U/ml) reversed the stimulatory effect of SIN-1 on I(Ca) (respectively -0.6 +/- 4.1%, n = 4 and 3.6 +/- 4.3%, n = 4). Intracellular cAMP level was unaltered by SIN-1, while it was enhanced by blocking the NO-cGMP pathway with the NO synthase inhibitor L-NMMA. These results suggest that peroxynitrite donors increase cardiac calcium current without the involvement of cAMP and cGMP.     

Multiple potassium channels mediate nitric oxide-induced inhibition of rat vascular smooth muscle cell proliferation.

Several nitric oxide (NO) effects in the cardiovascular system are mediated by soluble guanylate cyclase (sGC) activation but potassium channels (KC) are also emerging as important effectors of NO actions. We investigated the relationship among vascular smooth muscle cell proliferation, NO, cyclic GMP, and KC using the A7r5 smooth muscle cell line derived from rat aorta. NO donors (two nitrosothiols, S-nitroso-acetyl-d,l-penicillamine, SNAP, and S-nitroso-glutathione, GSNO, and an organic nitrate, glyceryl trinitrate, GTN; 1-1000 microM) dose-dependently inhibited cell proliferation. ODQ (a selective inhibitor of sGC; 0.1 and 1 microM) and KT5823 (a selective inhibitor of cGMP-dependent protein kinase, 1 microM) prevented NO effects, confirming that sGC is a key target. In this report, we show that tetraethylammonium (TEA, a non-selective blocker of KC, 300 microM), and 4-aminopyridine (a selective blocker of voltage-dependent KC, 100 microM) prevented SNAP inhibitory effects on cell proliferation, whereas glibenclamide (a selective blocker of ATP-dependent KC, 1 microM) was ineffective. Iberiotoxin (a selective blocker of high conductance calcium-activated KC, 100 nM), as well charybdotoxin (a blocker of high and intermediate conductance calcium-activated KC, 100 nM) and apamine (a selective blocker of small conductance calcium-activated KC, 100 nM), blocked the antiproliferative effect induced by SNAP. NS1619 (an opener of high conductance calcium-activated KC, 1-100 microM), inhibited cell proliferation. In addition, sub-effective concentrations of ODQ (100 nM) and TEA (10 microM) synergized in blocking SNAP antiproliferative effects. Thus, voltage-dependent and calcium-activated but not ATP-dependent KC appear to have a prominent role, besides sGC activation, in NO-induced inhibition of vascular smooth muscle cell proliferation.     

Nitric oxide can differentially modulate striatal neurotransmitter concentrations via soluble guanylate cyclase and peroxynitrite formation

In vivo microdialysis was used to investigate whether nitric oxide (NO) modulates striatal neurotransmitter release in the rat through inducing cyclic GMP formation via soluble guanylate cyclase or formation of peroxynitrite (ONOO(-)). When NO donors, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 1 mM) or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (NOC-18; 1 mM), were retrodialysed for 15 min, acetylcholine (ACh), serotonin (5-HT), glutamate (Glu), gamma-aminobutyric acid (GABA), and taurine levels were significantly increased, whereas those of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) were decreased. Only effects on ACh, 5-HT, and GABA showed calcium dependency. Inhibition of soluble guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ; 100 and 200 microM) dose-dependently reduced NO donor-evoked increases in ACh, 5-HT, Glu, and GABA levels. Coperfusion of SNAP or NOC-18 with an ONOO(-) scavenger, L-cysteine (10 mM) resulted in enhanced concentrations of Glu and GABA. On the other hand, DA concentrations increased rather than decreased, and no reductions in DOPAC and 5-HIAA occurred. This increase in DA and the potentiation of Glu and GABA were calcium-dependent and prevented by ODQ. Similar to NO, infusions of ONOO(-) (10 or 100 microM) decreased DA, DOPAC, and 5-HIAA. Overall, these results demonstrate that NO increases ACh, 5-HT, Glu, and GABA levels primarily through a cyclic GMP-dependent mechanism. For DA, DOPAC, and 5-HIAA, effects are determined by levels of ONOO(-) stimulated by NO donors. When these are high, they effectively reduce extracellular concentrations through oxidation. When they are low, DA concentrations are increased in a cyclic GMP-dependent manner and may act to facilitate Glu and GABA release further. Thus, changes in brain levels of antioxidants, and the altered ability of NO to stimulate cyclic GMP formation during ageing, or neurodegenerative pathologies, may particularly impact on the functional consequences of NO on striatal dopaminergic and glutamatergic function.     

Hypoxia enhances a cGMP-independent nitric oxide relaxing mechanism in pulmonary arteries

Nitric oxide (NO) donors generally relax vascular preparations through cGMP-mediated mechanisms. Relaxation of endothelium-denuded bovine pulmonary arteries (BPA) and coronary arteries to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) is almost eliminated by inhibition of soluble guanylate cyclase activation with 10 microM 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), whereas only a modest inhibition of relaxation is observed under hypoxia (PO2 = 8-10 Torr). This effect of hypoxia is independent of the contractile agent used and is also observed with NO gas. ODQ eliminated SNAP-induced increases in cGMP under hypoxia in BPA. cGMP-independent relaxation of BPA to SNAP was not attenuated by inhibition of K+ channels (10 mM tetraethylammonium), myosin light chain phosphatase (0.5 microM microcystin-LR), or adenylate cyclase (4 microM 2',5'-dideoxyadenosine). SNAP relaxed BPA contracted with serotonin under Ca2+-free conditions in the presence of hypoxia and ODQ, and contraction to Ca2+ readdition was also attenuated. The sarcoplasmic reticulum Ca2+-reuptake inhibitor cyclopiazonic acid (0.2 mM) attenuated SNAP-mediated relaxation of BPA in the presence of ODQ. Thus hypoxic conditions appear to promote a cGMP-independent relaxation of BPA to NO by enhancing sarcoplasmic reticulum Ca2+ reuptake.     

Nitric oxide mediates protein kinase C isoform translocation in rat heart during postischemic reperfusion

It is controversial whether nitric oxide (NO) is protective or deleterious against ischemia-reperfusion injury. We examined the effect of NO on PKC isoform translocation and protection against ischemia-reperfusion injury in perfused heart. An NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester, 3.0 microM), administered only during reperfusion but not during ischemia, inhibited the translocation of PKC-alpha, -delta and -epsilon isoforms to the nucleus-myofibril fraction and the translocation of PKC-alpha to the membrane fraction after ischemia (20 min) and reperfusion (10 min) in the perfused rat heart. NO donors, 3-morpholinosydnonimine (SIN-1) or S-nitroso-N-acetylpenicillamine (SNAP) activated purified PKC in vitro. SIN-1 also induced PKC isoform translocation in perfused heart. On the other hand, PKC selective inhibitor, calphostin C (0.2 microM) or chelerythrine (1.0 microM), aggravated the contractile dysfunction of ischemic heart during reperfusion, when they were perfused during reperfusion. These data suggest that NO generated during reperfusion following ischemia activates PKC isoforms and may protect the heart against contractile dysfunction in the perfused rat heart.     

Species dependent relaxation of intrapulmonary arteries (IPA) of rabbits, dogs and humans by prostacyclin

Helically cut strips of successive IPA segments of rabbits, dogs and human patients were set up for isometric recording in vitro. High tone was produced by norepinephrine (NE, 3 microM). This tone was markedly reduced by prostacyclin (PGI2) in the secondary, tertiary and quaternary branches of human and canine pulmonary trunk. The IC50 values for PGI2 ranged from 22 to 503 nM, the human vessels being more sensitive to prostacyclin than canine IPA. Under these conditions, the primary and secondary branches of the rabbit pulmonary trunk were not relaxed by PGI2. The contractile potency of NE was determined in each pulmonary vessel studied. The secondary segments of rabbit IPA were about ten times as sensitive to NE (EC50 for NE: 38 +/- 7 nM) as compared to the secondary IPA from dogs and humans (EC50 values: 370 +/- 84 and 440 +/- 50, respectively). When high tone was induced by equieffective contractile concentrations of NE (3 microM for canine and human IPA and 0.3 microM for rabbit vessels), PGI2 was still less effective (P less than 0.01) in relaxing secondary IPA of rabbits (IC25: 220 +/- 55) than the corresponding segments of dogs and humans (IC25: 51 +/- 12 and 17 +/- 4, respectively). The difference between canine and human vessels was also significant (P less than 0.02). These results indicate that there is an interspecies difference in the sensitivity of IPA to NE and PGI2.     

Dissociation of cGMP accumulation and relaxation in myometrial smooth muscle: effects of S-nitroso-N-acetylpenicillamine and 3-morpholinosyndonimine

In guinea pig, primate and man, nitric oxide (NO)-induced regulation of myometrial smooth muscle contraction is distinct from other smooth muscles because cyclic guanosine 3',5'-cyclic monophosphate (cGMP) accumulation is neither necessary nor sufficient to relax the tissue. To further our understanding of the mechanism of action of NO in myometrium, we employed the NO donors, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosyndonimine (SIN-1) proposed to relax airway smooth muscle by disparate mechanisms involving elevation in intracellular calcium ([Ca(2+)](i)) or cGMP accumulation, respectively. Treatment of guinea pig myometrial smooth muscle with either NO donor at concentrations thought to produce maximal relaxation of smooth muscles resulted in significant elevations in cGMP that were accompanied by phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein (VASP), shown here for the first time to be present and phosphorylated in myometrium. Stimulation of myometrial strips with oxytocin (OT, 1 microM) produced an immediate increase in contractile force that persisted in the continued presence of the agonist. Addition of SNAP (100 microM) in the presence of OT relaxed the tissue completely as might be expected of an NO donor. SIN-1 failed to relax the myometrium at any concentration tested up to 300 microM. In Fura-2 loaded myometrial cells prepared from guinea pig, addition of SNAP (100 microM) in the absence of other agonists caused a significant, reproducible elevation of intracellular calcium while SIN-1 employed under the same conditions did not. Our data further support the notion that NO action in myometrium is distinct from that in other smooth muscles and underscores the possibility that discrete regional changes in [Ca(2+)](i), rather than cGMP, signal NO-induced relaxation of the muscle.     

Peroxynitrite participates in mechanisms involved in capacitation of cryopreserved cattle

The effect of peroxynitrite (ONOO(-)) on the capacitation rates of cryopreserved bull spermatozoa and the participation of protein kinases in the capacitation process were evaluated. A pool of spermatozoa from five bulls was incubated in Tyrode's albumin lactate pyruvate (TALP) medium in the presence of heparin (10 IU/ml), sodium nitroprusside (SNP, 50 nM), a nitric oxide donor or 3-morpholinosydnonimine (SIN-1, 1-20 microM), a ONOO(-) donor. The participation of ONOO(-) was evaluated at 15, 30 and 45 min and confirmed by using a specific scavenger, uric acid (2-20 mM). Spermatozoa capacitated with SIN-1 were incubated with ovarian follicular fluid of cattle to evaluate their ability to undergo acrosome reaction. The role of ONOO(-) during capacitation induced by heparin or nitric oxide was evaluated by the addition of uric acid. The participation of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK) in capacitation induced by ONOO(-) was evaluated by incubation with specific inhibitors (50 microM H-89, 0.1 microM bisindolylmaleimide I, and 3 microM genistein, respectively). Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC) and true acrosome reaction was determined by trypan blue and Differential-Interferential Contrast (DIC). SIN-1 concentrations employed had no effect on progressive motility or sperm viability. Capacitation values of 10 microM SIN-1 treatment (23+/-2%) were significantly greater with respect to the control (4.6+/-1.62%). At 15 min of incubation the greatest capacitation was observed (P<0.05), reaching a plateau between 15 and 45 min. Follicular fluid induced acrosome reaction in spermatozoa previously capacitated with 10 microM SIN-1 (P<0.05). Uric acid prevented SIN-1-induced capacitation and significantly diminished capacitation induced by heparin or SNP. The addition of PKA and PKC inhibitors failed to modify the capacitation induced by SIN-1 (27.4+/-3.85 and 24.8+/-4.75, respectively). Genistein, a PTK inhibitor, produced a significant capacitation decrease (8.6+/-5.5%). These results indicate that endogenous ONOO(-) may be generated during heparin- or SNP-induced capacitation. Exogenous ONOO(-) acts as a capacitation inducer and involves the participation of PTK, as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa.     

The survival of skeletal muscle myoblasts in vitro is sensitive to a donor of nitric oxide and superoxide, SIN-1, but not to nitric oxide or peroxynitrite alone.

The survival of skeletal muscle myoblasts in culture after exposure either to a donor of NO, sodium nitroprusside (SNP), or ethanamine, 2,2'-(hydroxynitrosohydrazono)bis-(DETA NONOate), or to a donor of both NO and O(-)(2), 3-morpholinosydnonimine hydrochloride (SIN-1), was investigated. SIN-1 reduced clonogenic survival markedly but donors of NO alone did not. The injurious effect of SIN-1 was prevented by oxyhemoglobin or by uric acid but not by superoxide dismutase. The exposure of myoblasts to authentic peroxynitrite (ONOO(-)) or to DETA NONOate in the presence of an O(-)(2)-generating system did not reduce their survival. The results show that NO or ONOO(-) alone is not detrimental to myoblast survival and suggest that SIN-1 toxicity is, at least in part, mediated by H(2)O(2) in this myoblast culture system.     

Nitric oxide and platelet energy metabolism

This study was undertaken to determine whether nitric oxide (NO) can affect platelet responses through the inhibition of energy production. It was found that NO donors: S-nitroso-N-acetylpenicyllamine, SNAP, (5-50 microM) and sodium nitroprusside, SNP, (5-100 microM) inhibited collagen- and ADP-induced aggregation of porcine platelets. The corresponding IC50 values for SNAP and SNP varied from 5 to 30 microM and from 9 to 75 microM, respectively. Collagen- and thrombin-induced platelet secretion was inhibited by SNAP (IC50 = 50 microM) and by SNP (IC50 = 100 microM). SNAP (20-100 microM), SNP (10-200 microM) and collagen (20 microg/ml) stimulated glycolysis in intact platelets. The degree of glycolysis stimulation exerted by NO donors was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or uncouplers (2,4-dinitrophenol). Neither the NO donors nor the respiratory chain blockers affected glycolysis in platelet homogenate. SNAP (20-100 microM) and SNP (50-200 microM) inhibited oxygen consumption by platelets. The effect of SNP and SNAP on glycolysis and respiration was not reduced by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-stimulated guanylate cyclase. SNAP (5-100 microM) and SNP (10-300 microM) inhibited the activity of platelet cytochrome oxidase and had no effect on NADH:ubiquinone oxidoreductase and succinate dehydrogenase. Blocking of the mitochondrial energy production by antimycin A slightly affected collagen-evoked aggregation and strongly inhibited platelet secretion. The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.     

Loss of oxidized and chlorinated bases in DNA treated with reactive oxygen species: implications for assessment of oxidative damage in vivo

Oxidative damage to DNA has been reported to occur in a wide variety of disease states. The most widely used "marker" for oxidative DNA damage is 8-hydroxyguanine. However, the use of only one marker has limitations. Exposure of calf thymus DNA to an .OH-generating system (CuCl(2), ascorbate, H(2)O(2)) or to hypochlorous acid (HOCl), led to the extensive production of multiple oxidized or chlorinated DNA base products, as measured by gas chromatography-mass spectrometry. The addition of peroxynitrite (ONOO(-)) (<200 microM) or SIN-1 (1mM) to oxidized DNA led to the extensive loss of 8-hydroxyguanine, 5-hydroxycytosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2-hydroxyadenine, 8-hydroxyadenine, and 4,6-diamino-5-formamidopyrimidine were lost at higher ONOO(-) concentrations (>200 microM). Exposure of DNA to HOCl led to the generation of 5-Cl uracil and 8-Cl adenine and addition of ONOO(-) (<200 microM) or SIN-1 (1mM) led to an extensive loss of 8-Cl adenine and a small loss of 5-Cl uracil at higher concentrations (>500 microM). An .OH-generating system (CuCl(2)/ascorbate/H(2)O(2)) could also destroy these chlorinated species. Treatment of oxidized or chlorinated DNA with acidified nitrite (NO(2)(-), pH 3) led to substantial loss of various base lesions, in particular 8-OH guanine, 5-OH cytosine, thymine glycol, and 8-Cl adenine. Our data indicate the possibility that when ONOO(-), nitrite in regions of low pH or .OH are produced at sites of inflammation, levels of certain damaged DNA bases could represent an underestimate of ongoing DNA damage. This study emphasizes the need to examine more than one modified DNA base when assessing the role of reactive species in human disease.     

Comparison of toxic effects of nitric oxide and peroxynitrite on Uronema marinum (Ciliata: Scuticociliatida)

To discover the effects of nitric oxide (NO) and peroxynitrite on Uronema marinum (a ciliate responsible for systemic scuticociliatosis in cultured olive flounder Paralichthys olivaceus), the dose-dependent inhibitory effect of NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) on the proliferation and survival of U. marinum was investigated. The inhibitory effects of exogenous superoxide dismutase (SOD) and catalase on the toxicity of SIN-1 were also investigated. After 24 h of incubation in the presence of 0.2 mM SNAP, the number of ciliates was not statistically different from that of the controls, whereas incubation in the presence of 0.5 mM SNAP reduced the number of parasites significantly to 59.1% of controls. Concentrations of SNAP higher than 0.5 mM resulted in greater reductions in the number of ciliates, but levels of generated NO far exceeded physiological ranges. The number of viable ciliates incubated for 24 h with 0.2 mM SIN-1 was reduced significantly to 25.0%, and all ciliates were killed by incubation in concentrations above 0.5 mM SIN-1. Although SOD decreased the toxic effect of SIN-1 on U. marinum, protection was not complete and did not improve after increasing the SOD concentration from 50 to 400 U ml(-1). Addition of catalase ranging from 500 to 10000 U ml(-1) completely protected U. marinum from SIN-1 toxicity. Ciliates exposed to catalase alone or catalase plus SIN-1 showed significantly higher and dose-dependent proliferation rates compared to controls. Addition of haemoglobin, ranging from 0.5 to 2.0 mg ml(-1), also protected U. marinum from SIN-1 toxicity, and increased the proliferation rate dose-dependently. In conclusion, resistance of U. marinum to oxidative and nitrative stress may allow this pathogen to withstand the NO- and oxygen-radical-dependent killing mechanisms of phagocytic cells.     

Specificity of antioxidant enzyme inhibition in skeletal muscle to reactive nitrogen species donors

Nitric oxide (*NO) and its by-products modulate many physiological functions of skeletal muscle including blood flow, metabolism, glucose uptake, and contractile function. However, growing evidence suggests that an overproduction of nitric oxide contributes to muscle wasting in a number of pathologies including chronic heart failure, sepsis, COPD, muscular dystrophy, and extreme disuse. Limited data point to the potential of inhibition various enzymes by reactive nitrogen species (RNS), including (.)NO and its downstream products such as peroxynitrite, primarily in purified systems. We hypothesized that exposure of skeletal muscle to RNS donors would reduce or downregulate activities of the crucial antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). Diaphragm muscle fiber bundles were extracted from 4-month-old Fischer-344 rats and, in a series of experiments, exposed to either (a) 0 (control), 1, or 5 mM diethylamine NONOate (DEANO: *NO donor); (b) 0, 100, 500 microM, or 1 mM sodium nitroprusside (SNP: *NO donor); (c) 0 or 2 mM S-nitroso-acetylpenicillamine (SNAP: *NO donor); or (d) 0 or 500 microM SIN-1 (peroxynitrite donor) for 60 min. DEANO resulted in a 50% reduction in CAT, GPX, and a dose-dependent inhibition of Cu, Zn-SOD. SNP resulted in significantly lower activities for total SOD, Mn-SOD isoform, Cu, Zn-SOD isoform, CAT, and GPX in a dose-dependent fashion. Two millimolar SNAP and 500 microM SIN-1 also resulted in a large and significant inhibition of total SOD and CAT. These data indicate that reactive nitrogen species impair antioxidant enzyme function in an RNS donor-specific and dose-dependent manner and are consistent with the hypothesis that excess RNS production contributes to skeletal muscle oxidative stress and muscle dysfunction.     

Nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, stimulate myoblast proliferation in vitro

Summary Nitric oxide (NO) is an inter- and intracellular messenger involved in a variety of physiologic and pathophysiologic conditions. The effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) and their effect on myoblast proliferation was examined. Both donors stimulated an increase in myoblast cell number over a range (1–10 μM) of donor concentrations. However, 50 μM SNAP inhibited myoblast proliferation. Cell numbers from cultures treated with degraded 10 μM SNAP were equivalent to the control. Therefore, it appears NO can stimulate as well as inhibit myoblast proliferation.     

Effects of endothelin-1 and nitric oxide on proliferation of cultured guinea pig bronchial smooth muscle cells

The proliferative effects of endothelin-1 (ET-1), both alone and in combination with epidermal growth factor (EGF), and the effect of nitric oxide (NO) on the cell proliferation were investigated in cultured guinea pig bronchial smooth muscle cells. ET-1 (10-100 nM) alone augmented cell proliferation, and was additive to the effect of EGF (0.48 nM) in a concentration-dependent manner. An ET(A) antagonist, BQ-123 (10 microM), reduced the cell-proliferative effect of ET-1, whereas an ET(B) antagonist, BQ-788 (10 microM), did not influence the effect. A NO donor, SIN-1 (10 nM-1 microM), reduced the cell-proliferative effect of ET-1 in a concentration-dependent manner. The effect of SIN-1 (1 microM) was partly, but significantly, reversed by a soluble guanylyl cyclase inhibitor, ODQ (1 microM). These results suggest that ET-1 acts not only as a co-mitogen with EGF but also as a mitogen alone, and that its action is mediated through activation of ET(A) receptors. Therefore, ET-1 may contribute to airway remodeling, a pathophysiological hallmark of asthma. In addition, NO, which is produced mainly in the airway epithelium and is partly mediated through cGMP-dependent pathway, may reduce the phenomenon.     

Protein thiols and glutathione influence the nitric oxide-dependent regulation of the red blood cell metabolism.

Nitric oxide (NO) can modulate red blood cell (RBC) glycolysis by translocation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPD) (EC 1.2.1.12) from the cytosolic domain of the membrane protein band 3 (cdb3) in the cytosol. In this study we have investigated which NO-reactive thiols might be influencing GAPD translocation and the specific role of glutathione. Two highly reactive Cys residues were identified by transnitrosylation with nitrosoglutathione (GSNO) of cdb3 and GAPD (K(2) = 73.7 and 101.5 M(-1) s(-1), respectively). The Cys 149 located in the catalytic site of GAPD is exclusively involved in the GSNO-induced nitrosylation. Reassociation experiments carried out at equilibrium with preparations of RBC membranes and GAPD revealed that different NO donors may form -SNO on, and decrease the affinity between, GAPD and cdb3. In intact RBC, the NO donors 3-morpholinosydnonimine (SIN-1) and peroxynitrite (ONOO(-)) significantly increased GAPD activity in the cytosol, glycolysis measured as lactate production, and energy charge levels. Our data suggest that ONOO(-) is the main NO derivative able to cross the RBC membrane, leading to GAPD translocation and -SNO formation. In cell-free experiments and intact RBC, diamide (a thiol oxidant able to inhibit GAPD activity) was observed to reverse the effect of SIN-1 on GAPD translocation. The results demonstrate that cdb3 and GAPD contain reactive thiols that can be transnitrosylated mainly by means of GSNO; these can ultimately influence GAPD translocation/activity and the glycolytic flux.     

Actions of NO donors and endogenous nitrergic transmitter on the longitudinal muscle of rat ileum in vitro: mechanisms involved

The aim of this work has been to characterize and to compare the responses of the rat ileal longitudinal muscle to the nitric oxide (NO) donors, sodium nitroprusside (SNP) and morpholinosydnonimine hydrochloride (SIN-1). SNP (10(-5)-10(-3) M) caused a contraction followed by a relaxation, both components being concentration-dependent. In contrast, SIN-1 (10(-5)-10(-4) M) caused a relaxation followed by a contraction. Neither the neural blocker tetrodotoxin (TTX) nor atropine were able to change the response to SNP, whereas nifedipine abolished its contractile component. In contrast, TTX and nifedipine diminished both the relaxation and the contraction in response to SIN-1, whereas atropine decreased only the contractile component. The specific guanylate cyclase inhibitor oxadiazolo-quinoxalin-1-one (ODQ) decreased the relaxation induced by SNP but did not modify that caused by SIN-1. The K+ channel blockers charybdotoxin, apamin and tetraethylamonium were unable to modify the response to SNP. In contrast, both TEA and apamin significantly decreased the relaxation induced by SIN- 1. The relaxation resulting from electrical field stimulation (EFS) of enteric nerves in non-adrenergic non-cholinergic conditions is mainly but not exclusively nitrergic, as incubation with the NO synthase inhibitor L-NNA markedly decreases such relaxation. EFS-induced relaxation is also sensitive to ODQ. We conclude that SNP acts mainly on smooth muscle cells activating L-type Ca2+ channels, which result in contraction, and activates the soluble guanylate cyclase, which results in relaxation. In contrast SIN-1 has mixed--neuronal and muscular--effects, the contraction being caused both by acetylcholine release from neurons and by direct activation of L-type Ca2+ channels on smooth muscle cells. SIN-1-induced relaxation is cGMP-independent and it is likely to occur as a consequence of both, neuronal release of inhibitory transmitter(s) and by activation of apamin sensitive K+ channels. The effect of the nitrergic transmitter released from enteric nerves is different from those caused by SIN-1 but shows similarities with those caused by SNP.     

Nitric oxide attenuates matrix metalloproteinase-9 production by endothelial cells independent of cGMP- or NFκB-mediated mechanisms

Cardiovascular diseases involve critical mechanisms including impaired nitric oxide (NO) levels and abnormal matrix metalloproteinase (MMP) activity. While NO downregulates MMP expression in some cell types, no previous study has examined whether NO downregulates MMP levels in endothelial cells. We hypothesized that NO donors could attenuate MMP-9 production by human umbilical vein endothelial cells (HUVECs) as a result of less NFκB activation or cyclic GMP (cGMP)-mediated mechanisms. We studied the effects of DetaNONOate (10–400 μM) or SNAP (50–400 μM) on phorbol 12-myristate 13-acetate (PMA; 10 nM)-induced increases in MMP-9 activity (by gel zymography) or concentrations (by ELISA) as well as on a tissue inhibitor of MMPs’ (TIMP)-1 concentrations (by ELISA) in the conditioned medium of HUVECs incubated for 24 h with these drugs. We also examined whether the irreversible inhibitor of soluble guanylyl cyclase ODQ modified the effects of SNAP or whether 8-bromo-cGMP (a cell-permeable analog of cGMP) influenced PMA-induced effects on MMP-9 expression. Total and phospho-NFκB p65 concentrations were measured in HUVEC lysates to assess NFκB activation. Both NO donors attenuated PMA-induced increases in MMP-9 activity and concentrations without significantly affecting TIMP-1 concentrations. This effect was not modified by ODQ, and 8-bromo-cGMP did not affect MMP-9 concentrations. While PMA increased phospho-NFκB p65 concentrations, SNAP had no influence on this effect. In conclusion, this study shows that NO donors may attenuate imbalanced MMP expression and activity in endothelial cells independent of cGMP- or NFκB-mediated mechanisms. Our results may offer an important pharmacological strategy to approach cardiovascular diseases.