In vitro cytotoxicity of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine, towards cells from human oral tissue.

The cytotoxicity of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP), towards cultured human cells from oral tissue was evaluated. The toxicity of SNAP to Smulow-Glickman gingival epithelial cells was correlated with the liberation of nitric oxide, as N-acetyl-D,L-penicillamine, the SNAP metabolites, N-acetyl-D,L-penicillamine disulfide and nitrite, and preincubated (denitrosylated) SNAP did not affect viability. Comparing equimolar concentrations of various nitric oxide donors, cytotoxicity appeared to be inversely related to the relative stability (i.e., half-life) of the test compound; the sequence of cytotoxicity for a 4 hr exposure was S-nitrosoglutathione>spermine NONOate> SNAP>DPTA NONOate>DETA NONOate. Intracellular reduced glutathione (GSH) was lowered in S-G cells exposed to SNAP. Pretreatment of the cells with the GSH depleter, 1,3-bis-(chloroethyl)-1-nitrosourea (BCNU), enhanced the toxicity of SNAP Similar findings of enhanced sensitivity to SNAP were noted with gingival fibroblasts and periodontal ligament cells pretreated with BCNU. The toxicity of SNAP towards the gingival epithelial cells was decreased by cotreatment with the antioxidants, N-acetyl-L-cysteine, L-ascorbic acid, and (+)-catechin. Cells exposed to SNAP exhibited nuclear aberrations, including multilobed nuclei and multinucleation. SNAP-induced cell death was apparently by apoptosis, as noted by fluorescence microscopy and DNA agarose gel electrophoresis.     

In vitro Cytotoxicity of the Nitric Oxide Donor,S-Nitroso-N-acetyl-peraciUamine,towards Cells from Human Oral Tissue

Abstract: The cytotoxicity of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP), towards cultured human cells from oral tissue was evaluated. The toxicity of SNAP to Smulow-Glickman gingival epithelial cells was correlated with the liberation of nitric oxide, as N-acetyl-D, L-penicilIamine, the SNAP metabolites, N-acetyl-D, L-penicillamine disulfide and nitrite, and preincubated (denilrosylated) SNAP did not affect viability. Comparing equimolar concentrations of various nitric oxide donors, cytotoxicity appeared to be inversely related to the relative stability (i.e., half-life) of the test compound; the sequence of cytotoxicity for a 4 hr exposure was S-nitrosoglutathione>>spermine NONOate> SNAP>DPTA NONOate>>DETA NONOate. Intracellular reduced glutathione (GSH) was lowered in S-G cells exposed to SNAP. Pretreatment of the cells with the GSH depleter, 1, 3-bis-(chloroethyl)-1-nitrosourea (BCNU), enhanced the toxicity of SNAP. Similar findings of enhanced sensitivity to SNAP were noted with gingival fibroblasts and periodontal ligament cells pretreated with BCNU. The toxicity of SNAP towards the gingival epithelial cells was decreased by cotreatment with the antioxidants, N-acetyl-L-cysteine, L-ascorbic acid, and (+)-catechin. Cells exposed to SNAP exhibited nuclear aberrations, including multilobed nuclei and multinucleation. SNAP-induced cell death was apparently by apoptosis, as noted by fluorescence microscopy and DNA agarose gel electrophoresis.     

Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-dependent toxicity

Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 and in HepG2 E47 cells which express CYP2E1. Nitric oxide (NO) participates in the regulation of various cell activities as well as in cytotoxic events. NO may act as a protectant against cytotoxic stress or may enhance cytotoxicity when produced at elevated concentrations. The goal of the current study was to evaluate the effect of endogenously or exogenously produced NO on AA toxicity in liver cells with high expression of CYP2E1 and assess possible mechanisms for its actions. Pyrazole-induced rat hepatocytes or HepG2 cells expressing CYP2E1 were treated with AA in the presence or absence of an inhibitor of nitric oxide synthase L-N(G)-Nitroarginine Methylester (L-NAME) or the NO donors S-nitroso-N-acetylpenicillamine (SNAP), and (Z)-1-[-(2-aminoethyl)-N-(2-aminoethyl)]diazen-1-ium-1,2-diolate (DETA-NONO). AA decreased cell viability from 100% to 48+/-6% after treatment for 48 h. In the presence of L-NAME, viability was further lowered to 23+/-5%, while, SNAP or DETA-NONO increased viability to 66+/-8 or 71+/-6%. The L-NAME potentiated toxicity was primarily necrotic in nature. L-NAME did not affect CYP2E1 activity or CYP2E1 content. SNAP significantly lowered CYP2E1 activity but not protein. AA treatment increased lipid peroxidation and lowered GSH levels. L-NAME potentiated while SNAP prevented these changes. Thus, L-NAME increased, while NO donors decreased AA-induced oxidative stress. Antioxidants prevented the L-NAME potentiation of AA toxicity. Damage to mitochondria by AA was shown by a decline in the mitochondrial membrane potential (MMP). L-NAME potentiated this decline in MMP in association with its increase in AA-induced oxidative stress and toxicity. NO donors decreased this decline in MMP in association with their decrease in AA-induced oxidative stress and toxicity. These results indicate that NO can be hepatoprotective against CYP2E1-dependent toxicity, preventing AA-induced oxidative stress.     

Role of cyclic GMP on inhibition by nitric oxide donors of human eosinophil chemotaxis in vitro

1. This study was designed to investigate the effects of the nitric oxide (NO) donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) on N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP, 1 x 10(-7) M)-induced human eosinophil chemotaxis, cyclic guanosine-3',5'-monophosphate (cGMP) levels, protein nitration and cytotoxicity. 2. Human eosinophils were exposed to SNP, SIN-1 and SNAP (0.001-1.0 mM) for either short (10 min) or prolonged (90 min) time periods. Exposition of eosinophils with these NO donors significantly inhibited the eosinophil chemotaxis irrespective of whether cells were exposed to these agents for 10 or 90 min. No marked differences were detected among them regarding the profile of chemotaxis inhibition. 3. Exposition of eosinophils to SNP, SIN-1 and SNAP (0.001-1.0 mM) markedly elevated the cGMP levels above basal levels, but the 90-min exposition resulted in significantly higher levels compared with the 10-min protocols (5.3+/-0.6 and 2.6+/-0.2 nM 1.5 x 10(6) cells(-1), respectively). The cGMP levels achieved with SNAP were greater than SNP and SIN-1. 4. The NO donors did not induce cell toxicity in any experimental condition used. Additionally, eosinophils exposed to SNP, SIN-1 and SNAP (1.0 mM each) either for 10 or 90 min did not show any tyrosine nitration in conditions where a strong nitration of bovine serum albumin was observed. 5. Our findings show that inhibitory effects of fMLP-induced human eosinophil chemotaxis by NO donors at short or prolonged exposition time were accompanied by significant elevations of cGMP levels. However, additional elevations of cGMP levels do not change the functional profile (chemotaxis inhibition) of stimulated eosinophils.     

Nitric oxide up-regulates aldose reductase expression in rat vascular smooth muscle cells: a potential role for aldose reductase in vascular remodeling

Acceleration of the polyol pathway under hyperglycemia is among the mechanisms implicated in the pathogenesis of diabetic complications. Although aldose reductase (AR), the rate-limiting enzyme in this pathway, is a target for pharmacological intervention of diabetic complications, the clinical efficacy of AR inhibitors has not been consistently proved. Because nitric oxide (NO) plays important roles in vascular hemodynamics and inflammatory responses that are affected under diabetic conditions, the interaction of NO with AR was investigated with rat aortic smooth muscle cells. Spontaneous NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamin e, elicited a dose-dependent increase in AR mRNA to a maximum of 7-fold in 12 h. The activity of AR was elevated after 10 h of SNAP treatment. These effects of NO donors were suppressed by the addition of 2-(trimethylammoniophenyl)-4,4,5, 5-tetramethylimidazoline-1-oxy 3-oxide, a scavenger of NO. Induction of AR mRNA by SNAP was completely abolished by actinomycin D or cycloheximide, but unaffected by guanylate cyclase inhibitors or genistein, a tyrosine kinase inhibitor. Pretreatment of the cells with N-acetyl-L-cysteine significantly suppressed the SNAP-induced up-regulation of AR mRNA. Under normal glucose conditions, inclusion of the AR inhibitor ponalrestat augmented the cytotoxic effect of SNAP on the cells. The level of AR mRNA also was elevated in a murine macrophage cell line RAW 264.7 stimulated with lipopolysaccharide and interferon-gamma. Inhibition of NO synthesis completely abolished the increase in AR mRNA in the stimulated cells. The up-regulation of AR by NO in the vascular lesions may modulate NO-induced cell death and the ensuing vascular remodeling during inflammatory responses.     

Nitric Oxide Donors Enhance Rectal Absorption of Macromolecules in Rabbits

Purpose. The objective of this investigation is to evaluate the potential of nitric oxide (NO) donors as a new class of absorption enhancers which may act on intestinal epithelial cells through epithelial actions of the chemical mediator, NO. Methods. Suppositories containing NO donors and insulin were administered into the rabbit rectum. After administration of the suppository, blood samples were collected from the auricular vein. The plasma insulin and glucose concentrations were determined. Results. The NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP, 4 mg) induced a significant increase in the rate of insulin absorption from the rectum. Administration of a suppository containing SNAP without insulin affected neither the plasma insulin nor the plasma glucose concentration. Other NO donors, NOR1 and NOR4, also induced increases in the insulin absorption. The absorption enhancement effect of SNAP was inhibited by coadministration of the NO scavenger carboxy-PTIO. SNAP also enhanced FITC-dextran (MW 4,000) absorption. Little cytotoxicity of SNAP (3.0 mg/ml) as assessed in terms of the rate of lactate dehydrogenase (LDH) release from Caco-2 cells was detected for 2 h of incubation. Conclusions. These findings suggest that NO enhanced macromolecular absorption from the rectum without mucosal cell damage, and that NO donors can act as potent absorption enhancers.     

Comparative effects of several nitric oxide donors on intracellular cyclic GMP levels in bovine chromaffin cells: correlation with nitric oxide production.

1. Sodium nitroprusside, S-nitroso-N-acetyl-D,L-penicillamine, Spermine NONOate and DEA NONOate raised cyclic GMP levels in bovine chromaffin cells in a time and concentration dependent manner with different potencies, the most potent being DEA/NO with an EC50 value of 0.38 +/- 0.02 microM. 2. Measurements of NO released from these donors revealed that DEA/NO decomposed with a half-life (t1/2) of 3.9 +/- 0.2 min. The t1/2 for SPER/NO was 37 +/- 3 min. SNAP decomposed more slowly (t1/2 = 37 +/- 4 h) and after 60 min the amount of NO produced corresponded to less than 2% of the total SNAP present. The rate of NO production from SNAP was increased by the presence of glutathione. 3. For DEA/NO and SPER/NO there was a clear correlation between nitric oxide production and cyclic GMP increases. Their threshold concentrations were 0.05 microM and maximal effective concentration between 2.5 and 5 microM. 4. For SNAP, threshold activation was seen at 1 microM, whereas full activation required a higher concentration (500-750 microM). The dose-response for SNAP increases in cyclic GMP was shifted nearly two orders of magnitude lower in the presence of glutathione. At higher concentrations an inhibition of cyclic GMP accumulation was found. This effect was not observed with either the nitric oxide-deficient SNAP analogue or other NO donors. 5. Although NO-donors are likely to be valuable for studying NO functions, their effective concentrations and the amount of NO released by them are very different and should be assessed in each system to ensure that physiological concentrations of NO are used.     

In vitro response of human gingival epithelioid S-G cells to minocycline.

Minocycline, a broad-spectrum antibiotic used in the treatment of acne and periodontal disease and to control inflammatory diseases such as rheumatoid arthritis, has recently been shown to induce a spectrum of adverse health effects. In the light of these contradictory data, this research was directed to provide basic information on the toxicology of minocycline, using in vitro cell culture models, and to evaluate its efficacy in periodontal therapies, particularly for wound healing. The human gingival epithelioid S-G cell line was used as the bioindicator. The greater toxicity of minocycline over doxycycline and tetracycline, related antimicrobial agents, probably correlated with its higher lipophilicity. The cytotoxicity of minocycline was unaffected by an S9 hepatic microsomal fraction, indicating that it is a direct-acting, rather than a metabolism-mediated, cytotoxicant. In comparative toxicity studies, much variation in the degree of sensitivity to minocycline was noted for different cell types. No correlation in the extent of sensitivity to minocycline and the physiologic state of the bioindicator cell (normal, transformed or malignant) was noted. The toxicity of minocycline to the S-G cells was dependent on its concentration and length of exposure. For a continuous 3-day exposure of the S-G cells to minocycline, the midpoint cytotoxicity (or, NR(50)) value, as quantified in the neutral red (NR) assay, was 204 microg/ml on day 1, 84 microg/ml on day 2, and 59 microg/ml on day 3. For a 1-h exposure of the S-G cells in phosphate buffered saline (PBS), the NR(50) value was 780 microg/ml minocycline. Although a 1-h exposure in PBS to 200 microg/ml minocycline exerted some toxicity, the S-G cells recovered on exposure to growth medium; irreversible, progressive damage occurred at 400 microg/ml minocycline and greater. Minocycline, at 50 microg/ml, enhanced attachment of the S-G cells to a gelatin-coated surface and cell migration towards an immobilized fibronectin gradient, both biologic parameters important in periodontal wound healing. Minocycline generally had little or no effect on production of the pro-inflammatory cytokines, interleukin-6 (IL-6) and interleukin-8 (IL-8), by non-activated S-G cells, the exception being stimulation of IL-6 at 48 h. IL-1beta, however, greatly stimulated IL-6 and IL-8 production, which was further increased by concurrent exposure to minocycline. This suggested that minocycline may enhance the ability of gingival epithelial cells to participate in the early, inflammatory phase of periodontal wound healing. The limitation of minocycline efficacy to a rather narrow window of concentration, centering about 50 microg/ml, and primarily for short-term exposures may possibly explain, in part, the contradictory clinical data on the health effects of this drug.     

Opposite effects of nitric oxide donors on DNA single strand breakage and cytotoxicity caused by tert-butylhydroperoxide

1. The effects of three different NO donors on tert-butylhydroperoxide (tB-OOH)-induced DNA cleavage and toxicity were investigated in U937 cells.
2. Treatment with S-nitroso-N-acetyl-penicillamine (SNAP, 1–30 μM), while not in itself DNA-damaging, potentiated the DNA strand scission induced by 200 μM tB-OOH in a concentration-dependent fashion. The enhancing effects of SNAP were observed with two different techniques for the assessment of DNA damage. Decomposed SNAP was inactive. S-nitrosoglutathione (GSNO, 300 μM) and (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NO, 1 mM) also increased DNA cleavage generated by tB-OOH and these responses, as well as that mediated by SNAP, were prevented by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide (PTIO).
3. SNAP neither inhibited catalase activity nor increased the formation of DNA lesions in cells exposed to H₂O₂. Furthermore, SNAP did not affect the rate of rejoining of the DNA single strand breaks generated by tB-OOH.
4. Under the conditions utilized in the DNA damage experiments, treatment with tB-OOH alone or associated with SNAP did not cause cell death. However, SNAP as well as GSNO markedly reduced the lethal response promoted by millimolar concentrations of tB-OOH and these effects were abolished by PTIO. Decomposed SNAP was inactive.
5. It is concluded that low levels of NO donors, which probably release physiological concentrations of NO, enhance the accumulation of DNA single strand breaks in U937 cells exposed to tB-OOH. This NO-mediated effect appears to (a) not depend on inhibition of either DNA repair (which would increase the net accumulation of DNA lesions by preventing DNA single strand break removal) or catalase activity (which would also enhance the net accumulation of DNA lesions since H₂O₂ is one of the species mediating the tB-OOH-induced DNA cleavage) and (b) be caused by enforced formation of tB-OOH-derived DNA-damaging species. In contrast to these results, similar concentrations of NO prevented cell death caused by millimolar concentrations of tB-OOH. Hence, DNA single strand breakage generated by tB-OOH in the absence or presence of NO does not represent a lethal event.

     

Differential effects of nitric oxide donors on basal and electrically evoked release of acetylcholine from guinea-pig myenteric neurones.

1. The effects of the nitric oxide (NO) donors, 3-morpholino-sydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside on basal and electrically evoked release of [3H]-acetylcholine were studied in myenteric plexus longitudinal muscle preparations of the guinea-pig small intestine preincubated with [3H]-choline. 2. The NO donors concentration-dependently increased basal release of [3H]-acetylcholine. The increase in release was calcium-dependent and was prevented in the presence of tetrodotoxin. Superoxide dismutase (150 u ml-1) potentiated the effect of SIN-1. The selective inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 0.01-1 microM), antagonized the facilitatory effect of SNAP. 8-Bromo cyclic GMP and the cyclic GMP-specific phosphodiesterase inhibitor, zaprinast (both 0.1-1 mM), also enhanced basal [3H]-acetylcholine release. The effect of 10 microM SNAP was significantly enhanced in the presence of zaprinast. 3. The NO donors concentration-dependently inhibited the electrically evoked release of [3H]-acetylcholine, whereas 8-bromo cyclic GMP and zaprinast enhanced the evoked release. The inhibition of acetylcholine release by SNAP was not affected by ODQ (0.01-1 microM). 4. It is concluded that NO stimulates basal acetylcholine release from myenteric neurones through activation of guanylyl cyclase. In addition, NO inhibits the depolarization evoked release of acetylcholine by a presynaptic mechanism unrelated to cyclic GMP. The data imply that NO is not only an inhibitory transmitter to intestinal smooth muscles but also a modulator of cholinergic neurotransmission in the myenteric plexus.     

The effect of nitric oxide donors on haemodynamics and blood flow distribution in the porcine carotid circulation.

1. The role of nitric (NO) in the regulation of capillary and arteriovenous anastomotic blood flow was evaluated in the carotid circulation of the pig. For this purpose, the effect of intracarotid (i.c.) infusions of saline and two NO donors, nitroprusside sodium (NPR) and S-nitroso-N-acetylpenicillamine (SNAP) in concentrations of 3-100 micrograms min-1 was studied on systemic haemodynamics and carotid blood flow and its distribution in anaesthetized pigs with low arteriovenous anastomotic blood flow, by use of the radioactive microsphere method. 2. Apart from heart rate, which increased after both NPR and SNAP, no major changes in systemic haemodynamic variables were observed. In contrast to saline, both NPR and SNAP increased common carotid blood flow, vascular conductance and vascular pulsations dose-dependently. 3. The distribution of the carotid artery blood flow over capillary and arteriovenous anastomotic fraction remained stable after saline infusions. Both NPR and SNAP enhanced total capillary blood flow and conductance. In contrast to NPR, arteriovenous anastomotic blood flow and conductance were increased by SNAP. 4. At the tissue level, capillary blood flow increases following NPR or SNAP were reflected by an increase in both extracerebral and dural blood flow without changes in total brain blood flow. 5. These results indicate that both NO donors cause arteriolar dilatation together with enhanced vascular pulsations in the carotid circulation of the pig. Probably by way of a 'steal' phenomenon, this pronounced arteriolar dilatation limits the effect of NO donors on arteriovenous anastomoses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide- and nitric oxide donors-induced relaxation and its modulation by oxidative stress in piglet pulmonary arteries.

Inhaled nitric oxide (iNO) is widely used in the treatment of pulmonary hypertension while inhaled NO donors have been suggested as an alternative therapy. The differential susceptibility to inactivation by oxidative stress and oxyhaemoglobin of NO and two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) were analysed in isolated endothelium-denuded pulmonary arteries from 2-week-old piglets stimulated with U46619. NO, SNAP and SNP relaxed the arteries (pIC(30)=7.73+/-0.12, 7.26+/-0.17 and 6.43+/-0.13, respectively) but NO was not detected electrochemically in the bath after the addition of SNP and only at concentrations at which SNAP produced more than 50% relaxation. The sGC inhibitor ODQ (10(-6) M) or the sarcoplasmic Ca(2+)-ATPase thapsigargin (2x10(-6) M) markedly inhibited the relaxation induced by NO, SNAP and SNP. Addition of oxyhaemoglobin (3x10(-7) M) or diethyldithiocarbamate (1 mM) markedly inhibited NO- (pIC(30)=6.88+/-0.07 and 6.92+/-0.18, respectively), weakly inhibited SNAP- and had no effect on SNP-induced relaxation. Xanthine oxidase (5 mu ml(-1)) plus hypoxanthine (10(-4) M) markedly inhibited NO- (pIC(30)=6.96+/-0.12) but not SNAP- or SNP-induced relaxation. Superoxide dismutase (SOD), MnCl(2), diphenileneiodonium and exposing the luminal surface of the rings outwards (inversion) potentiated the relaxant responses of NO (pIC(30)=8.52+/-0.16, 8.23+/-0.11, 8.01+/-0.11 and 8.20+/-0.10, respectively). However, SOD did not modify the NO detected by the electrode and had no effect on SNAP- or SNP-induced relaxation. Therefore, the kinetics and local distribution of NO release of NO donors influence the susceptibility to the scavenging effects of oxyhaemoglobin and superoxide.     

In vitro response of human gingival epithelial S-G cells to resveratrol

WST-1 (mitochondrial dehydrogenase activities). Arrest of cell growth, due to inhibition of DNA synthesis, may explain the leveling of toxicity between day 2 and 3 for a 3-day continuous exposure to resveratrol. Irreversible damage to cell proliferation was noted in S-G cells exposed to 75-150 microM resveratrol for 2 days and then subsequently maintained for another 3 days in resveratrol-free medium. The cytotoxicity of resveratrol was neither potentiated nor ameliorated in the presence of an hepatic S9 microsomal fraction. The cytotoxicity of hydrogen peroxide to S-G cells was lessened by N-acetyl-L-cysteine and quercetin, but not by resveratrol. For nitric oxide, only N-acetyl-L-cysteine reduced toxicity. The ability of resveratrol to function as an antioxidant was, therefore, not noted under these test conditions.     

Hyperoxia attenuates the inhibitory effect of nitric oxide donors on HIF prolyl-4-hydroxylase-2: Implication on discriminative effect of nitric oxide on HIF prolyl-4-hydroxylase-2 and collagen prolyl-4-hydroxylase

Prolyl 4-hydroxylases (P4Hs), such as collagen prolyl-4-hydroxylases (CPHs) and hypoxia inducible factor prolyl-4-hydroxylases (HPHs), have recently been recognized as promising drug targets for the treatment of fibrotic and ischemic diseases. CPHs and HPHs catalyze identical metabolic reactions, yet lead to quite different physiological consequences, collagen synthesis and the regulation of oxygen homeostasis. Selective modulation of the two enzymes should provide a therapeutic benefit upon pharmacotherapy. In an in vitro VHL capture assay, hydroxylation of the 19mer HIF peptide (corresponding to HIF-1α residues 556–574) by HPH-2 was effectively prevented by nitric oxide (NO) donors, (±)-S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione. The NO donors also caused inhibition of HPHs and accumulation of nonhydroxylated HIF-1α protein in A549 human lung adenocarcinoma cells. Hyperoxia (100% O₂) attenuated both NO donor-induced accumulation of HIF-1α and inhibition of HPH-mediated hydroxylation. In the presence of a proteasome inhibitor, MG132, the hyperoxia-mediated degradation of HIF-1α was deterred and hydroxylated HIF-1α was detected. SNAP, while being an effective inhibitor of proline 4-hydroxylation of HIF-1α by HPH-2, did not diminish proline hydroxylation of collagen by CPHs. Our data suggest that NO inhibits HPH-2 via competing with dioxygen and that the discriminative effect of NO on CPHs and HPH-2 is attributable to the difference in the affinity of the two enzymes toward dioxygen.     

The liver-selective nitric oxide donor O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) protects HepG2 cells against cytochrome P450 2E1-dependent toxicity

HepG2 cells expressing CYP2E1 (E47 cells) are more susceptible to toxicity by arachidonic acid (AA) or after glutathione depletion with an inhibitor of glutamate-cysteine ligase, l-buthionine-(S,R)-sulfoximine (BSO), compared with control HepG2 cells (C34 cells). The ability of nitric oxide (NO) to protect against CYP2E1-dependent toxicity has not been evaluated. We therefore studied the ability of O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), a liver-selective NO donor, to protect against CYP2E1-dependent toxicity and compared this with protection by chemical NO donors. E47 cells incubated with V-PYRRO/NO produced NO, whereas C34 cells did not. Incubation of E47 cells with 50 microM AA or 100 microM BSO for 2 days resulted in a 50% loss of cell viability. VPYRRO/NO (1 mM) blocked this toxicity of AA and BSO by a mechanism involving NO release via CYP2E1 metabolism of VPYRRO/NO. NO scavengers hemoglobin and 2-(4-carboxophenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide blocked the protective effects of V-PYRRO/NO. V-PYRRO/NO inhibited CYP2E1 activity and production of reactive oxygen species, whereas hemoglobin prevented these events. AA and BSO induced lipid peroxidation and decreased mitochondrial membrane potential; both of these effects were blocked by V-PYRRO/NO. Unlike V-PYRRO/NO, the chemical donors spermine/NO and (S)-nitroso-N-acetylpenicillamine release NO directly when added to the medium; however, they could partially protect against the CYP2E1-dependent toxicity. These results suggest that VPYRRO/NO protects HepG2 cells against CYP2E1-dependent toxicity through inhibition of CYP2E1-derived reactive oxygen species production and lipid peroxidation by the generated NO and that this compound may be valuable in protecting against CYP2E1-dependent toxicity via liver P450-specific generation of NO.     

Feedback inhibition of nitric oxide synthase activity by nitric oxide.

1. A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-gamma, 10 u ml-1) plus lipopolysaccharide (LPS, 10 ng ml-1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2. The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3. The decline in the NO synthase activity was significantly slowed down in cells cultured in a low L-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4. The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5. SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6. The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.     

Enhanced expression of haem oxygenase-1 by nitric oxide and antiinflammatory drugs in NIH 3T3 fibroblasts

1. Haem oxygenase-1 (HO-1) can exert protective effects against oxidative stress and inflammation. Fibroblasts participate in inflammatory responses where they produce high levels of prostaglandins (PGs) and nitric oxide (NO). However, little is known of the presence of HO-1 in these cells and the possible interactions among these pathways. Incubation of cells with NO donors, spermine nonoate (SPNO) and S-nitroso-N-acetylpenicillamine (SNAP), induced a dose- and time-dependent expression of HO-1 protein. 2. NO donors increased basal PGE(2) release although they reduced PGE(2) accumulated in the medium and cyclo-oxygenase (COX) activity when cells were stimulated with lipopolysaccharide (LPS). COX-2 protein was weakly induced by SPNO in basal conditions and in the presence of LPS a synergy for HO-1 and COX-2 protein expression was observed. 3. Our results indicate that reactive oxygen species participate in the inductive effect of NO donors or LPS on HO-1 expression, whereas endogenous NO production may play a role in the mechanism of the synergy exhibited by SPNO and LPS on HO-1 and COX-2 expression. In this system, zinc protoporphyrin IX did not affect nitrite levels but reduced COX activity. 4. The selective COX-2 inhibitors SC58125 and NS398 as well as the non-selective COX inhibitor, indomethacin, strongly reduced PGE(2) synthesis and showed a synergy with NO donors in HO-1 and COX-2 induction. Addition of PGE(2) had no effect, suggesting a mechanism independent of PGs formation. 5. In inflammatory conditions a number of factors could cooperate to induce HO-1 and COX-2, with a positive regulation by COX inhibitors.     

SNAP and SIN-1 increase brain production of kynurenic acid

The influence of nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN-1), on the central production of an endogenous glutamate receptor antagonist, kynurenic acid, was evaluated in vitro. In cortical slices, SNAP and SIN-1 potently increased the extracellular concentration of kynurenic acid. A free radical scavenger, l-ascorbate reversed this effect. Neither SNAP nor SIN-1 altered the activity of kynurenic acid biosynthetic enzymes, kynurenine aminotransferases (KAT I and II). These data reveal a novel aspect of the brain response to studied herein NO donors and suggest that in the milieu containing NO-related free radicals the formation of kynurenic acid is enhanced.     

Differential effects of intrathecally and intracerebroventricularly administered nitric oxide donors on noxious mechanical and thermal stimulation.

Involvement of nitric oxide (NO) in nociceptive transmission is well documented. However, there is controversy concerning the exact role of NO in mediation of nociception at different levels of the nervous system. Most studies agree that NO promotes hyperalgesia at the level of the spinal cord. Conversely, at supraspinal sites exogenously applied NO has been found to be both pro- and antinociceptive. In light of this discrepancy, the aim of the present study was to compare the effects of NO donors on nociceptive transmission at spinal and supraspinal sites of the central nervous system using mechanical (paw pressure; PP) and thermal (tail-flick; TF) noxious stimulation. Four NO donors which release NO through different mechanisms were used: S-nitrosoglutathione (SNOG; 3-600 nmol), S-nitroso-N-acetylpenicillamine (SNAP; 0.18-4.5 nmol), hydroxylamine (HYD; 60-1200 nmol) and 3-morpholino-sydnonimine (SIN-1; 490-970 nmol). They were injected intrathecally (i.t.) or intracerebroventricularly (i.c.v.) to male Wistar rats and nociceptive thresholds were evaluated in TF and PP tests. It was found that NO donors administered i.t. or i.c.v. produced a dose-dependent hyperalgesia in the PP test. The hyperalgesia induced by mechanical stimuli was stronger after i.t. than after i.c.v. administration of NO donors. The SIN-1-induced hyperalgesia, as evaluated by teh PP test, was reversed by i.t. pretreatment with haemoglobin (1.5-4 nmol) a NO scavenger, and methylene blue (267-1070 nmol) a guanylate cyclase and NO synthase inhibitor, suggesting that NO exerts its action by facilitating cyclic guanosine 3',5'-monophosphate (GMP) formation. Unlike in the PP test, SNAP and SNOG had no effect on the nociceptive threshold in the TF test, and only SIN-1 administered i.t. produced a weak hyperalgesia in that test, while HYD caused a mild but significant prolongation of the TF reflex. The above data show that NO produces hyperalgesia principally in response to noxious mechanical stimuli. This effect seems to be predominantly mediated in the spinal cord, however, it occurs at both levels of the central nervous system.     

Irsogladine maleate potentiates the effects of nitric oxide on activation of cAMP signalling pathways and suppression of mesangial cell mitogenesis

BACKGROUND AND PURPOSE: Deficiency in nitric oxide (NO) is a major factor leading to deterioration and progression of certain glomerular diseases. Agents enhancing NO availability and potentiality are renoprotective. Irsogladine maleate (IM), an anti-ulcer drug, is reported to improve gastric blood flow via NO-dependent mechanisms. We, therefore, asked whether and how IM interacted with NO on glomerular mesangial cells. EXPERIMENTAL APPROACH: Mesangial cells were exposed to IM and NO donors. Activation of cAMP signalling pathways was assessed by intracellular cAMP, phosphorylation of VASP, activation of the cAMP response element (CRE) and expression of CRE-regulated proteins. KEY RESULTS: IM alone did not affect cell proliferation. However, it greatly enhanced the growth-inhibitory effect of NO donor S-nitroso-N-acetylpenicillamine (SNAP). IM acted synergistically with NO on suppression of mitogen-activated protein kinase activation, induction of gap junction protein connexin43, increase of intracellular cAMP, and phosphorylation of VASP. With the use of the CRE-SEAP-based reporting system, IM and SNAP cooperatively activated cAMP response elements (CRE). A similar activation of cAMP was induced by IM with two different NO donors, the sGC activator Bay 41-2272 and the cGMP analogue 8-bromo-cGMP. The effects of SNAP and IM on cAMP activation were mimicked by phosphodiesterase 3 (PDE3) and PDE4 inhibitors. In addition, IM markedly augmented cytokine-induced expression of iNOS, production of NO and activation of CRE. CONCLUSION AND IMPLICATIONS: The effects of NO were greatly potentiated by IM through synergistic activation of cAMP pathway. Combined therapy with IM and NO may be developed for certain renal diseases.