Presence Of S-nitroso-N-acetylpenicillamine Research Articles

Nitrosylation of tissue transglutaminase enhances fibroblast migration and regulates MMP activation

In wound healing, the TG2 enzyme plays a dual functional role. TG2 has been shown to regulate extracellular matrix (ECM) stabilization by its transamidase activity while increasing cell migration by acting as a cell adhesion molecule. In this process, nitric oxide (NO) plays a particularly important role by nitrosylation of free cysteine ​​residues on TG2, leading to the irreversible inactivation of the catalytic activity.In this study, transfected fibroblasts expressing TG2 under the control of the tetracycline-off promoter were treated with NO donor S-nitroso-N-acetyl penicillamine (SNAP) to analyze the interplay between NO and TG2 in the regulation of cell migration/invasion as well as TGF-β1-dependent MMP activation. Our results demonstrated that inhibition of TG2 cross-linking activity by SNAP promoted the migration and invasion capacity of fibroblasts by hindering TG2-mediated TGF-β1 activation. While the inhibition of TG2 activity by NO downregulated the biosynthesis and activity of MMP-2 and MMP-9, that of MMP-1a and MMP-13 was shown to be upregulated in a TGF-β1-dependent manner under the same conditions. In the presence of SNAP, interaction of TG2 with its cell surface binding partners Integrin-β1 and Syndecan-4 was reduced, which was paralleled by an increase in TG2 and PDGF association. These findings suggests that migratory phenotype of fibroblasts can be regulated by the interplay between nitric oxide and TG2 activity.

Nitric oxide stimulates a PKC-Src-Akt signaling axis which increases human immunodeficiency virus type 1 replication in human T lymphocytes

Human immunodeficiency virus (HIV) infections are typically accompanied by high levels of secreted inflammatory cytokines and generation of high levels of reactive oxygen species (ROS). To elucidate how HIV-1 alters the cellular redox environment during viral replication, we used human HIV-1 infected CD4+T lymphocytes and uninfected cells as controls. ROS and nitric oxide (NO) generation, antioxidant enzyme activity, protein phosphorylation, and viral and proviral loads were measured at different times (2–36 h post-infection) in the presence and absence of the NO donor S-nitroso-N-acetylpenicillamine (SNAP). HIV-1 infection increased ROS generation and decreased intracellular NO content. Upon infection, we observed increases in copper/zinc superoxide dismutase (SOD1) and glutathione peroxidase (GPx) activities, and a marked decrease in glutathione (GSH) concentration. Exposure of HIV-1 infected CD4+T lymphocytes to SNAP resulted in an increasingly oxidizing intracellular environment, associated with tyrosine nitration and SOD1 inhibition. In addition, SNAP treatment promoted phosphorylation and activation of the host's signaling proteins, PKC, Src kinase and Akt. Inhibition of PKC leads to inhibition of Src kinase strongly suggesting that PKC is the upstream element in this signaling cascade. Changes in the intracellular redox environment after SNAP treatment had an effect on HIV-1 replication as reflected by increases in proviral and viral loads. In the absence or presence of SNAP, we observed a decrease in viral load in infected CD4+T lymphocytes pre-incubated with the PKC inhibitor GF109203X. In conclusion, oxidative/nitrosative stress conditions derived from exposure of HIV-1-infected CD4+T lymphocytes to an exogenous NO source trigger a signaling cascade involving PKC, Src kinase and Akt. Activation of this signaling cascade appears to be critical to the establishment of HIV-1 infection.

Phosphodiesterase Type 5 Is Not Upregulated by Tadalafil in Cultures of Human Penile Cells

Tadalafil, a long-acting phosphodiesterase type 5 (PDE5) inhibitor, improves the erectile response by inhibiting cyclic guanosine monophosphate (cGMP) breakdown. Sustained higher levels of cGMP may hypothetically upregulate PDE5 expression and/or activity and lead to tachyphylaxis. We have investigated whether PDE5 upregulation occurs in vitro in cultures of human penile cells subjected to long-term incubation with increasing concentrations of tadalafil in the presence of a nitric oxide (NO) donor. Human corpora cavernosa smooth muscle cells (CSMC) and tunica albuginea fibroblasts (TAF) primary cultures were characterized by immunocytochemistry and Western blot, and incubated with graded concentrations of tadalafil for 14 days, adding S-nitroso-N-acetyl penicillamine (SNAP) as an NO donor for the last 24 hours or at time zero, and cGMP levels were measured. Incubations were repeated for 7, 10, and 14 days, in the presence of SNAP, and PDE5 was estimated by Western blot, and at 14 days, by immunocytochemistry combined with quantitative image analysis, and by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Constructs of the human PDE5A promoter expressing luciferase were cloned and transfected into CSMC, and promoter activation by 8-deoxybromo-cGMP (8-Br-cGMP) was measured by luminometry. Incubations of CSMC with SNAP and tadalafil up to 14 days did not upregulate PDE5 mRNA or protein levels. With TAF, PDE5 protein was also not upregulated despite a slight increase in mRNA levels. PDE5 enzyme activity was unaffected by tadalafil in either CSMC or TAF. No upregulation of the PDE5 promoter was observed with up to 2 mM 8-Br-cGMP. Long-term incubation of human penile cells with tadalafil at concentrations above the in vitro IC50, and around the in vivo Cmax utilized in the clinical setting, did not upregulate PDE5A expression nor decrease cGMP levels. These data suggest that PDE5 upregulation is unlikely to occur in vivo on long-term tadalafil treatment.

Regulation of lens aldose reductase activity by nitric oxide

To examine the regulation of aldose reductase (AR) activity by nitric oxide (NO) in human lens epithelial cells (HLEC), cultured rat lens, and normal and diabetic rat lens, we have incubated HLEC or cultured rat lenses with 1 m m of the NO donors S-nitroso- N-acetylpenicillamine (SNAP) or S-nitrosoglutathione (GSNO), and the AR activity and sorbitol content were measured. Non-diabetic and diabetic (treated with streptozotocin 65 mg kg −1 body wt, i.p.) rats were injected with the nitric oxide synthase (NOS) inhibitor, l-NAME (50 mg kg −1 body wt day −1, ×10 days i.p.) or NOS substrate, l-arginine (200 mg kg −1 body wt day −1, ×10 days i.p.). In a separate group of rats, a nitroglycerin (NG)-patch that releases 200 ng min −1 NO was applied to the dorsal neck region. After 10 days of treatment, the lenses were removed and their AR activity and sorbitol content were measured. Incubation of HLEC with SNAP or GSNO reduced AR activity. A similar reduction in AR activity and sorbitol accumulation was observed when diabetic and non-diabetic rat lenses were cultured in the presence of SNAP and GSNO. Total protein-SSG in diabetic lens was lower compared to normal lens. Treatment of diabetic and non-diabetic rats with l-NAME enhanced AR activity and sorbitol accumulation, whereas NG patch and l-arginine significantly decreased AR activity and sorbitol accumulation in diabetic lenses compared to non-diabetic. Increased S-glutathiolation of AR was observed in the presence of SNAP. These results suggest that decreased glutathiolation of cellular proteins in diabetic rat lens compared to non-diabetic lens is related to decreased NO availability in diabetic rats which would decrease GSNO. Restoring the NO levels in diabetic animals increases glutathiolation of cellular proteins, inhibits AR activity and prevents sorbitol accumulation. Exogenous delivery of NO may represent a potentially useful strategy for preventing or delaying diabetic cataractogenesis and the development of other diabetic complications.

The role of overproduction of nitric oxide in apoptosis of BALB/c mice macrophages infected with leishmania major in vitro.

Nitric oxide (NO) derived from activated macrophages has been shown to be crucial for the host's leishmanicidal activities. Excess NO, however, can induce apoptosis in some cell types, including macrophages. In the present investigation, we studied the role of NO in inducing apoptosis of BALB/c mice macrophages infected with Leishmania major in vitro. The peritoneal macrophages were harvested and cultured with or without L.major in the presence of a donating reagent (s-Nitroso-N-Acetylpenicillamine (SNAP)) or an inhibitor of NO synthase (NG -Methyl-L-Arginine (NMMA)). The concentration of NO in culture supernatants was measured after 18 hours incubation. Simultaneously, macrophages undergoing apoptosis were identified by fluorescence and electron microscopy. The results showed an increase in apoptosis rate in parallel to nitrite production in macrophages cultured in the presence of SNAP. Although macrophages infected with L.major had no significant increase in NO production, they showed a significant increase in apoptosis rate. Besides, macrophages cultured with NMMA, had a decreased NO production but the apoptosis rate increased. Therefore, mechanisms involved in apoptosis induction in the last two groups may be different from NO overproduction.

Preincubation with atrial natriuretic peptide protects NG108-15 cells against the toxic/proapoptotic effects of the nitric oxide donor S-nitroso- N-acetylpenicillamine.

The TUNEL method is used to quantify the proapoptotic effects of an NO donor, S-nitroso- N-acetylpenicillamine (SNAP), in NG108-15 cells. Unlike sodium nitroprusside used in previous studies, SNAP does not release cyanide along with NO, thus NO toxicity was determined without concurrent cyanide toxicity. The present study also determined if pretreatment with ANP could protect against NO-induced apoptosis in NG108-15 cells. Cell death at 24 h following SNAP treatment was associated with apoptotic DNA fragmentation. SNAP at 0.5, 0.75, 1.0, and 2.0 mM caused significant (P<0.05) increases in the percentage of TUNEL-labeled cells from a control of 0.90% to 6.19%, 6.36%, 7.25%, and 15.1%, respectively. Thus, SNAP caused concentration-dependent induction of apoptosis in NG108-15 cells. SNAP-induced apoptosis was confirmed by morphological changes and increased levels of polynucleosome-sized fragments of DNA assessed by capillary electrophoresis. Preincubation for 24 h with ANP at 0.01, 0.1, and 1.0 microM, before the SNAP, significantly (P<0.05) decreased the percentage of labeled cells from 7.25% to 5.10%, 4.36%, and 3.24% in the presence of SNAP (1 mM) and from 15.1% to 7.91%, 6.64%, and 5.60% in the presence of SNAP (2 mM), respectively, representing protection of 24.0%, 34.0%, and 57.0% against SNAP (1 mM) and 26.0%, 37.0%, and 50.9% against SNAP (2 mM). Thus, prior activation of a cGMP-mediated neuroprotective mechanism induced by ANP appears to counterbalance, at least partially, the proapoptotic effects of excess NO. This neuroprotective mechanism involving cGMP may be especially important in protecting against the development of neurodegenerative diseases in which excess NO is thought to contribute to neuronal apoptosis.

The cardioprotective and mitochondrial depolarising properties of exogenous nitric oxide in mouse heart.

Nitric oxide (NO) is reported to be both protective and detrimental in models of myocardial ischaemia/reperfusion injury, which may be concentration dependent. Our objective was to characterise this dichotomy using the nitric oxide donor, S-nitroso N-acetyl penicillamine (SNAP) in isolated perfused mouse heart and isolated mouse cardiac mitochondria. To determine the effect of nitric oxide concentration on myocardial viability, isolated mouse hearts were subjected to 35 min global ischaemia and 30 min reperfusion in the presence of SNAP (0.02-20 microM). To determine whether NO mediated protection was via opening of the putative mitochondrial K(ATP) channel and/or free radical synthesis, SNAP perfused hearts were also treated with the mitochondrial K(ATP) channel blocker, 5-hydroxy decanoate (5-HD) and the free-radical scavenger, N-(2-mercaptopropionyl)-glycine (MPG). This data was correlated with mitochondrial membrane potential (Delta Psi(m)), measured with the potentiometric dye, tetra-methyl rhodium methyl ester (TMRM), in isolated mitochondria,by flow cytometry. SNAP dose-dependently attenuated infarct size, with maximal protection observed at 2 microM (17+/-4% versus controls 32+/-3%, P<0.01). At greater concentrations however, protection was lost with infarct sizes tending towards control at 20 microM (29+/-3%). These results were paralleled by changes in Delta Psi(m) in the isolated mitochondria: Delta Psi(m) depolarisation peaking with 1 microM SNAP (26+/-4% shift in TMRM fluorescence, P<0.01); at greater concentrations, this relationship was lost. The mitochondrial K(ATP) channel blocker, 5-HD, resulted in both abrogation of SNAP infarct size reduction and concomitant loss of Delta Psi(m) depolarisation in the mitochondria. MPG however did not influence the cardioprotective properties of SNAP. We demonstrate that nitric oxide can mediate cardioprotection in a dose-dependent fashion by an effect that may be related to Delta Psi(m). Both cardioprotection and Delta Psi(m) changes are sensitive to 5-HD and the cardioprotection appears independent of free-radical synthesis.

Enhancement of Fibroblast Collagen Synthesis by Nitric Oxide

Fibroblasts can be stimulated by cytokines to synthesize nitric oxide (NO, nitrogen monoxide), while wound-derived fibroblasts synthesize NO spontaneously. Since wound fibroblasts are phenotypically characterized by greater collagen synthesis when compared to fibroblasts derived from noninjured tissue, we hypothesized that there may be a correlation between wound-induced NO synthesis and enhanced collagen production. To study the role of NO on collagen metabolism, normal dermal fibroblasts were cultured in the presence or absence of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) and their collagen metabolism was studied on the transcriptional as well as translational level. Fibroblast collagen synthesis was enhanced by 74.3 ± 18.2 and 87.5 ± 28.2% in the presence of 100 and 400 μM SNAP, respectively. This effect was not due to increased collagen type I or type III gene transcription. Cellular proliferation measured by thymidine incorporation was significantly decreased in the presence of SNAP, indicating that the increased collagen production was due to a net increase of collagen synthesis by the cells. Investigation of the collagen breakdown pathway showed that neither collagenase gene expression nor collagenase protein expression was affected by SNAP. The results of this study demonstrate for the first time that NO enhances collagen synthesis, most likely at a posttranslational level.

Nitric Oxide Inhibits Neonatal Hepatocyte Oxidative Metabolism

Liver function is frequently impaired in neonates with sepsis. Nitric oxide (NO) is thought to be a mediator of organ dysfunction and liver oxidative metabolism during sepsis. The authors developed an in vitro model to investigate the effect of NO and the combined effect of NO plus H2O2 on neonatal hepatocyte oxidative metabolism. Hepatocytes were isolated from neonatal rats. Oxygen consumption was measured polarographically. In Study A, cells were exposed to S-Nitroso-N-acetylpenicillamine (SNAP), an NO donor, at various concentrations. In study B, myxothiazol and oligomycin, inhibitors of mitochondrial respiration, were added to investigate the site of action of NO. In study C, hepatocytes were incubated in the presence of both SNAP (300 micromol/L) and H2O2 (1.5 mmol/L). In study D, morphological alterations induced by NO and NO plus H2O2 were investigated by hepatocyte electron microscopy. In study A, SNAP caused a dose-dependent decrease in oxygen consumption. A significant inhibition was reached at 300 micromol/L SNAP. In study B, the lack of further inhibition when SNAP was given together with myxothiazol indicates that NO acts intramitochondrially. Similarly, no further inhibition occurred when the NO donor was given together with oligomycin, suggesting that the effect of NO is mainly at the level of ATP synthase. In study C, concomitant addition of 300 micromol/L SNAP and 1.5 mmol/L H2O2 to hepatocytes caused further inhibition of oxygen consumption compared with either SNAP or H2O2 alone. In study D, mild alterations in hepatocyte morphology were noted in the presence of SNAP or SNAP plus H2O2. In neonatal hepatocytes, NO significantly inhibits mitochondrial oxygen consumption, possibly at the level of ATP synthase. The effect of NO is additive to that of H2O2. Morphological findings were consistent with these biochemical effects and suggest that NO and H2O2 are important mediators of liver damage during sepsis.

Tumor suppressor p53 but not cGMP mediates NO-induced expression of p21(Waf1/Cip1/Sdi1) in vascular smooth muscle cells.

Cyclin-dependent kinase inhibitor p21(Waf1/Cip1/Sdi1) has been suggested to be involved in the antiproliferative effect of nitric oxide (NO) in vascular smooth muscle cells (VSMCs). To elucidate the mechanism underlying NO-induced p21 expression, we investigated the roles of tumor suppressor p53 and the guanylate cyclase-cGMP pathway. The induction of p21 by the NO donor S-nitroso-N-acetylpenicillamine (SNAP) seemed to be due to transactivation because SNAP elevated the activity of p21 promoter but did not stabilize p21 mRNA and protein. Because SNAP did not stimulate the deletion mutant of p21 promoter that lacked p53 binding sites, we tested the involvement of p53. The expression level of p53 was down-regulated after mitogenic stimulation, whereas it was sustained in the presence of SNAP. SNAP markedly stimulated DNA binding activity of p53. Furthermore, SNAP failed to induce p21 in VSMCs obtained from p53-knock out mice and in A431 cells that contained mutated p53. The antiproliferative effect of SNAP also was attenuated in these cells. NO stimulates guanylate cyclase and its product cGMP has been shown to inhibit VSMC proliferation. However, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, did not prevent SNAP-induced p21 expression. 8-Bromo-cGMP, 3-isobutyl-1-methylxanthine, and their combination did not induce p21. Although 8-bromo-cGMP had a small antiproliferative effect, the elevation of cGMP concentration induced by SNAP was little throughout the G(1) phase. The antiproliferative effect of SNAP was not attenuated by Rp-8-bromoguanosine-3',5'-monophosphorothioate, an inhibitor of cGMP-dependent protein kinase. These results suggested that NO induces p21 through a p53-dependent but cGMP-independent pathway.

Positive and negative inotropic effects of NO donors in atrial and ventricular fibres of the frog heart.

1. The cardiac effects of the NO donors sodium nitroprusside (SNP), S-nitroso-N-acetyl-penicillamine (SNAP) and 3-morpholino-sydnonimine (SIN-1) were studied in frog fibres to evaluate the contribution of cyclic GMP-dependent mechanisms. 2. SNP and SNAP (0.1-100 microM) reduced the force of contraction in a concentration-dependent manner in atrial and ventricular fibres. This effect was associated with a reduction in the time to peak (TTP) and the time for half-relaxation of contraction (T). 3. SIN-1 (100 microM) also reduced the force of contraction in two-thirds of the atrial fibres. However, it exerted a positive inotropic effect in the remaining atrial fibres, as well as in most ventricular fibres. 4. The guanylyl cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 microM) antagonized the negative inotropic effects of SIN-1 (50 microM) and SNAP (25 microM) but had no effect on the positive inotropic response to SIN-1 (100 microM). 5. In the presence of SIN-1, superoxide dismutase (SOD, 50-200 U ml-1) either potentiated the negative inotropic effect or turned the positive inotropic effect of the drug into a negative effect. SOD had no effects when applied alone or in the presence of SNAP. 6. 6-Anilino-5,8-quinolinedione (LY 83583, 3-30 microM), a superoxide anion generator also known as a cyclic GMP-lowering agent, exerted a positive inotropic effect, which was antagonized by SOD (200-370 U ml-1) but not by ODQ (10 microM). 7. We conclude that SNP, SNAP and SIN-1 exert cyclic GMP-dependent negative inotropic effects, which are attributed to the generation of NO. In addition, SIN-1 and LY 83583 exert cyclic GMP-independent positive inotropic effects, which require the generation of superoxide anion.

Growth inhibition and radiosensitization of cultured glioma cells by nitric oxide generating agents.

The authors examined the effect of nitric oxide (NO) generating agents on the growth and radiosensitivity of cultured glioma cells. Three glioma, rat C6, and human T98G and U87 cell lines were treated with the NO generating agents, S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP). These agents released NO in the cell culture media and inhibited the growth of the glioma cells. Growth-inhibition was attenuated by hemoglobin, a known inhibitor of NO, suggesting it is mediated by NO. When C6 and T98G cells were irradiated in the presence of SNAP or SNP at 100 microM, radiosensitization was observed. SNAP at 100 microM exhibited a sensitizer enhancement ratio (SER) of 1.4 for C6 cells and 1.8 for T98G cells. SNP at 100 microM only radiosensitized T98G cells with a SER of 1.9. The effect of SNP on radiosensitization of C6 cells was unclear. We conclude that NO generating agents are potential growth inhibitors and radiosensitizers for malignant glioma cells. NO mediated radiosensitization of glioma cells by NO generating agents may offer a new therapeutic approach for malignant glioma.

Exogenous nitric oxide inhibits mesangial cell adhesion to extracellular matrix components

Interactions of mesangial cells (MCs) with components of the extracellular matrix (ECM) profoundly influence the MC phenotype, such as attachment, contraction, migration, survival and proliferation. Here, we investigated the effects of exogenous nitric oxide (NO) on the process of MC adhesion to ECM molecules. Incubation of rat MCs with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) dose- and time-dependently inhibited MC adhesion and spreading on various ECM substrata, being more pronounced on collagen type I than on collagen type IV, laminin or fibronectin. In contrast, SNAP did not inhibit MC adhesion to L-polylysine-coated plates. The inhibitory effects of SNAP were reduced by hemoglobin and enhanced by superoxide dismutase. The anti-adhesive action of SNAP was mimicked not only by other NO donors but also by 8-bromo-cGMP, and significantly reversed by the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ). Moreover, SNAP and 8-bromo-cGMP decreased the adhesion-induced phosphorylation of focal adhesion kinase (pp125FAK). In the presence of SNAP or 8-bromo-cGMP, adherent MCs exhibited disturbed organization of alpha-actin filaments and reduced numbers of focal adhesions, as shown by immunocytochemistry. In additional experiments with adherent MCs, it was found that exposure to SNAP or 8-bromo-cGMP for 12 and 24 hours induced detachment of MCs. The results indicate that exogenous NO interferes with the establishment and maintenance of MC adhesion to ECM components. This inhibitory NO effect is mediated predominantly by cGMP-signaling. Disturbance of MC attachment to ECM molecules could represent an important mechanism by which NO affects MC behavior in vitro and in vivo.

Induction of the Cyclin-dependent Kinase Inhibitor p21Sdi1/Cip1/Waf1 by Nitric Oxide-generating Vasodilator in Vascular Smooth Muscle Cells

Nitric oxide-generating vasodilators inhibit vascular smooth muscle cell proliferation. To elucidate the mechanism underlying this process, we investigated the effect of S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide-releasing agent, on the smooth muscle cell cycle. When G0 cells were stimulated with fetal bovine serum and basic fibroblast growth factor, DNA synthesis assessed by [3H]thymidine incorporation started about 15 h later. SNAP dose-dependently inhibited this incorporation, and this effect was maximal at 100 microM. This inhibition was attenuated when SNAP was added after 9-12 h. SNAP inhibited the activity of cyclin-dependent kinase 2 (Cdk2) and phosphorylation of the retinoblastoma protein, both of which usually increased from about 9 h, whereas it did not inhibit the activities of cyclin D-associated kinase(s), Cdk4, and Cdk6, which normally increased from 0-3 h. Although SNAP reduced the mRNA levels of cyclins E and A, it neither reduced their protein levels nor impaired their association with Cdk2. SNAP did not reduce the mRNA levels of cyclins G, C, and D1, Cdk2, Cdk4, and Cdk5, which were normally elevated from 0-3 h. The mRNA and protein levels of the Cdk inhibitor p21 were high in the early G1 phase, peaking at 3 h and then rapidly decreasing after 6 h. In the presence of SNAP, however, p21 expression was enhanced, and moreover, the later decrease disappeared. SNAP also increased the amount of Cdk2-associated p21. These results suggested that nitric oxide inhibits the G1/S transition by inhibiting Cdk2-mediated phosphorylation of the retinoblastoma protein and that p21 induction is involved in the Cdk2 inhibition.

Nitric oxide suppresses increases in mesangial cell protein kinase C, transforming growth factor beta, and fibronectin synthesis induced by thromboxane.

Thromboxane (TX) stimulation of fibronectin (FN) synthesis in mesangial cells (MC) is dependent on protein kinase C (PKC)-mediated increases in transforming growth factor beta (TGF beta), and is suppressed by increases in cellular cGMP. The current studies evaluate the role of cGMP-dependent and -independent actions of nitric oxide (NO) in modulating the responses of MC to the TX analogue U46619. TX-stimulated increases in PKC activity, TGF beta, and FN synthesis in MC were suppressed by either 8-Br-PET-cGMP or the NO donor S-nitroso-N-acetylpenicillamine (SNAP). By contrast, NO, but not cGMP, inhibited basal PKC activity, TGF beta bioactivity and FN synthesis. The cGMP-dependent protein kinase 1-alpha inhibitor 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphorothioate (Rp) restored the PKC, TGF beta, and the FN synthetic responses to TX when added to MC before exposure of the cells to either cGMP or SNAP. However, neither Rp nor the guanylate cyclase inhibitor Ly83583 significantly altered SNAP inhibition of basal PKC. In addition, Rp failed to alter the decreases in basal TGF beta bioactivity and FN synthesis seen in the presence of SNAP. In contrast to the FN response to U46619, cGMP and SNAP did not affect the stimulation of FN synthesis by exogenous TGF beta. The later findings are consistent with inhibitory actions of NO and cGMP at, or proximal to, U46619-induced increases in TGF beta in the suppression of TX-signaled increases in FN synthesis. Thus, NO depresses basal PKC and TGF beta bioactivity in MC by mechanisms that are largely independent of cGMP, whereas NO inhibition of these MC responses to TX is mediated primarily by increases in cGMP and activation of protein kinase 1-alpha.

MODULATION OF INTRACELLULAR Ca2+ TRANSIENTS BY NITRIC OXIDE IN AIRWAY SMOOTH MUSCLE CELLS. ▴ 2308

Nitric oxide (NO) causes tracheal smooth muscle (TSM) relaxation by inhibiting agonist-stimulated elevation of intracellular Ca2+([Ca2+]i). via cGMP-dependent mechanisms. In this study, we examined the effect of s-nitroso-n-acetylpenicillamine (SNAP), a NO donor, on the Ca2+ response to acetylcholine (ACh). Single, freshly isolated porcine TSM cells were loaded with fura-2/AM and [Ca2+]i was measured as the ratio of fura-2 emissions when excited at 340 and 380 nm, using a video fluorescence imaging system. SNAP (0.1-1.0 μM) inhibited the normal [Ca2+]i response to ACh (0.1-1.0 μM) at an extracellular Ca2+ ([Ca2+]o) of either 0 or 2.5 mM. After SNAP inhibition of [Ca2+]i response to ACh in 0 mM Ca2+, a large Ca2+ response was seen when [Ca2+]o was readjusted to 2.5 mM, indicating that Ca2+ influx was not affected by NO. After depleting the sarcoplasmic reticulum (SR) Ca2+ pool with ACh and re-exposing to 2.5 mM Ca2+ in the presence of SNAP, the subsequent ACh response in the absence of SNAP was unaffected, indicating that NO does not affect reloading of the SR. These results suggest that NO selectively affects SR Ca2+ release, but does not affect Ca2+ influx or SR reloading. Inhibition of SR Ca2+ release can lead to relaxation of TSM. Supported by NIH grants HL51736 and HL07111, Mayo Foundation, and Univ. of Minnesota. MSK was a recipient of an Abbott Senior Fellowship.

Nitric oxide raises cytosolic concentrations of Ca 2+ in cultured nodose ganglion neurons from rabbits

The effects of donors of nitric oxide (NO), namely, S-nitroso- N-acetylpenicillamine (SNAP) and E-methyl-2- E-hydroxyimino-5-nitro-6-methoxy-3-hexeneamide (NOR1), on cytosolic concentrations of Ca 2+ ([Ca 2+] i) were studied in cultured nodose ganglion neurons from newborn and young rabbits by loading with fura-2AM and microfluorometry. Application of SNAP (5 μM-1 mM) increased [Ca 2+] i in 46% of neurons tested. The threshold dose of the response was 10 μM and the response increased in a dose-dependent fashion. The increase in [Ca 2+] i at 50 μM averaged 74 ± 8% above the control value. [Ca 2+] i rose immediately after injection of SNAP and the plateau level was maintained in the presence of SNAP. NOR1, another donor of NO, increased [Ca 2+] i with an average increase of 82 ± 7% at 50 μM. Quantitation of NO gas in the solution of NOR1 by a redox chemiluminescence method revealed the constant release of 0.06 ppm NO from 5 ml of a 20 μM solution of NOR1. These data suggest that NO released from donors of NO induced an increase in [Ca 2+] i in nodose ganglion neurons and, therefore, that NO might play a role as a transmitter or a modulator in autonomic primary sensory systems.

Inhibition of low-density lipoprotein oxidation by nitric oxide Potential role in atherogenesis

The effects of nitric oxide (•NO) and nitrovasodilators on the oxidation of low-density lipoprotein (LDL) have been studied. S-Nitroso- N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) inhibited Cu 2+-and 2,2′-azobis-2-amidinopropane hydrochloride-dependent oxidation of LDL as monitored by oxygen consumption and the formation of thiobarbituric acid-reactive substances, conjugated dienes, and lipid hydroperoxides. In the case of SNP, inhibition of LDL oxidation occurred only when the incubation mixture was irradiated with visible light. SNAP, however, exerted a dose-dependent inhibition of Cu 2+-catalyzed oxidation of LDL even in the dark. Addition of •NO dissolved in deoxygenated buffer also inhibited the progression of LDL oxidation. Mouse peritoneal macrophages were less able to degrade LDL that had been oxidized in the presence of SNAP. Using an •NO electrode, it was estimated that a continuous production of •NO (⩽ 760 nM/min) could retard the progression of LDL oxidation. We propose that •NO can inhibit LDL oxidation by acting as a chain-breaking antioxidant that is capable of scavenging carbon-centered and peroxyl radicals. Biological implications of this novel •NO antioxidant property are discussed in relation to atherogenesis and contrasted to the prooxidant property of •NO when generated in the presence of superoxide.