Inhibitory Effect Of Sodium Nitroprusside Research Articles

Sodium Nitroprusside Inhibits Detrusor Muscle Contraction through G-protein Mediated Inhibition of Intracellular Ca2+ Release

Background: Nitric oxide (NO) physiology is very complex in detrusor smooth muscle (DSM). While NO relaxes detrusor in some species, in contrary, it produces contraction in other. NO donor sodium nitroprusside (SNP) was found to inhibit CCh-induced contraction in goat DSM in earlier studies, but it is not known how SNP produces inhibitory action on goat DSM. Therefore, the aim of this in vitro study was to investigate the mechanism of inhibitory action of SNP pertaining to involvement of G- protein and other second messenger on goat DSM. Methods: Goat detrusor muscle strips collected from local abattoir were mounted in a thermostatically controlled (37o±0.5oC) organ bath (20 ml capacity) containing physiological solution. Following 1 hr of equilibration, carbachol (CCh) (10-5 M) induced sub-maximal contraction was elicited both in the absence and presence of SNP (10-5 M). Involvement of NO and guanylyl cyclase was assessed using nitric oxide synthase inhibitor L-NAME (10-5 M) and guanylyl cyclase inhibitor ODQ (10-5 M). Experiments were also carried out in the presence of G-protein activator aluminium fluoride (AlF) (10-5 M), protein kinase C (PKC) activator phorbol-12-myristate (PMA) (10-5 M), nifedipine, low Ca2+ PSS, theophylline and Zero Ca2+ PSS. Result: Inhibitory effect of SNP (10-5 M) on CCh-induced contraction on goat DSM was reversed by nitric oxide synthase inhibitor L-name (10-5 M) and guanylyl cyclase inhibitor ODQ (10-5 M). Prior incubation of the tissues with AlF and PMA also reversed the SNP mediated inhibition. On the other hand, nifedipine and low Ca2+ PSS; and theophylline and zero Ca2+ PSS potentiated the SNP-mediated inhibition. Thus, the present study shows that SNP elicited inhibitory effect on goat DSM is both NO-dependent and cGMP-dependent. In addition, the result shows that SNP inhibits G-protein coupled PKC leading to inhibition of both Ca2+ entry and intracellular Ca2+ release with a resultant decrease in intracellular Ca2+.

MRP4 over-expression has a role on both reducing nitric oxide-dependent antiplatelet effect and enhancing ADP induced platelet activation

The impact of inhibition of multidrug resistance protein 4 (MRP4) on nitric oxide (NO) resistance and on ADP-induced platelet aggregation is unknown. The aim of this investigation was to verify whether platelet NO resistance correlates with MRP4 expression and evaluate whether this can be reduced by in vitro MRP4 inhibition mediated by cilostazol. Moreover, we assessed if inhibition of MRP4-mediated transport reduces ADP-induced platelet reactivity. The inhibitory effect of sodium nitroprusside (SNP), a NO-donor that enhances cyclic guanosine monophosphate (cGMP) cytosolic concentration, was assessed in platelets obtained from aspirin treated patients and in a control population. The inhibitory effect of SNP was evaluated by ADP-induced aggregation in SNP-treated platelets. The impact of MRP4 on ADP-induced platelet aggregation was performed in high on aspirin residual platelet reactivity (HARPR) patients and compared to healthy volunteers (HV), and a control cohort (CTR). In aspirin-treated patients with high levels of MRP4, reduced SNP inhibition was found compared to those with low levels of MRP4. MRP4 inhibition by cilostazol significantly reduced ADP-induced platelet aggregation in HARPR population, and to a lesser extent in HV and CTR populations. In conclusion, cilostazol can mitigate the hyper-reactive platelet phenotype of HARPR patients by reducing residual ADP-induced platelet aggregation and increasing NO-dependent endothelial antiplatelet effects.

Beneficial effects of silymarin against nitric oxide-induced oxidative stress on cell characteristics of bovine oviduct epithelial cell and developmental ability of bovine IVF embryos

The antioxidative effects of silymarin were examined against nitric oxide-induced oxidative stress on cell characteristics of bovine oviduct epithelial cell (BOEC) and developmental rates of bovine in vitro fertilisation (IVF) embryos. The cell viability and morphology in BOEC which were treated with sodium nitroprusside (SNP) declined, as concentrations of SNP increase. The additions of silymarin 50, 100 and 200 µM into SNP 1000 µM increased the cell viability inhibited by SNP 1000 µM alone, but addition of silymarin 500 µM into SNP 1000 µM group did not recover the inhibitory effect of SNP 1000 µM (p<0.05). The levels of malondialdehyde (MDA), as the indicator of lipid peroxidation (LPO) in the treatments of SNPs increased with dose-dependant manners (p<0.05), whereas additions of silymarin 50, 100 and 200 µM into SNP 1000 µM significantly decreased MDA levels by 1.6, 1.4 and 1.3 folds of control (p<0.05). Antioxidant and Bcl-2 genes were expressed in control or SNP 1000 µM plus silymarin 200 µM treatment group, whereas pro-apoptotic genes (caspase-3 and Bax) were expressed only in SNP treatment groups. When bovine IVF embryos were co-cultured with BOEC supplemented with silymarin in the presence or absence of SNP, the development ability to blastocyst in 200 µM of silymarin alone was the highest among all treatments. These results suggest that silymarin has positive effects on cell characteristics such as viability, morphology and LPO of BOEC, and the increase of bovine IVF embryo development rate might be through antioxidative and anti-apoptotic actions.

Effect of nitric oxide on epithelial ion transports in noncystic fibrosis and cystic fibrosis human proximal and distal airways

The airways of patients with cystic fibrosis (CF) exhibit decreased nitric oxide (NO) concentrations, which might affect airway function. The aim of this study was to determine the effects of NO on ion transport in human airway epithelia. Primary cultures of non-CF and CF bronchial and bronchiolar epithelial cells were exposed to the NO donor sodium nitroprusside (SNP), and bioelectric variables were measured in Ussing chambers. Amiloride was added to inhibit the Na(+) channel ENaC, and forskolin and ATP were added successively to stimulate cAMP- and Ca(2+)-dependent Cl(-) secretions, respectively. The involvement of cGMP was assessed by measuring the intracellular cGMP concentration in bronchial cells exposed to SNP and the ion transports in cultures exposed to 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (ODQ), or to 8Z, a cocktail of 8-bromo-cGMP and zaprinast (phosphodiesterase 5 inhibitor). SNP decreased the baseline short-circuit current (I(sc)) and the changes in I(sc) induced by amiloride, forskolin, and ATP in non-CF bronchial and bronchiolar cultures. The mechanism of this inhibition was studied in bronchial cells. SNP increased the intracellular cGMP concentration ([cGMP](i)). The inhibitory effect of SNP was abolished by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO scavenger (PTIO) and ODQ and was partly mimicked by increasing [cGMP](i). In CF cultures, SNP did not significantly modify ion transport; in CF bronchial cells, 8Z had no effect; however, SNP increased the [cGMP](i). In conclusion, exogenous NO may reduce transepithelial Na(+) absorption and Cl(-) secretion in human non-CF airway epithelia through a cGMP-dependent pathway. In CF airways, the NO/cGMP pathway appears to exert no effect on transepithelial ion transport.

Expression of nitric oxide synthase isoforms in different stages of buffalo ( Bubalus bubalis) ovarian follicles: Effect of nitric oxide on in vitro development of preantral follicle

The present study was designed to investigate the expression of nitric oxide synthase (NOS) isoforms in buffalo ovarian preantral (PFs), antral (AFs) and ovulatory (OFs) follicles (Experiment 1); effect of NO on in vitro survival and growth of PFs (Experiment 2) and NOS activity in immature oocytes by NADPH-diaphorase test (Experiment 3). In Experiment 1, NOS isoforms (neuronal, inducible and endothelial) were localized immunohistochemically; mRNA and protein expression was analyzed by semi-quantitative RT-PCR and western blot, respectively. In Experiment 2, PFs were isolated by micro-dissection method from buffalo ovaries and cultured in 0 (control), 10 −3, 10 −5, 10 −7 and 10 −9 M sodium nitroprusside (SNP). PFs were further cultured with 10 −5 M SNP + 1.0 mM N ω-nitro-L-arginine methyl ester (L-NAME) or 1.0 μg/ml hemoglobin (Hb) to examine the reversible effect of SNP. Immunohistochemical studies demonstrated that inducible nitric oxide synthase (iNOS) immunoreactivity was predominantly localized in granulosa and theca cells whereas, neuronal (nNOS) and endothelial (eNOS) nitric oxide synthase in the theca, granulosa and cumulus cells of PFs, AFs and OFs. The amount of mRNA as well as protein of nNOS and eNOS was found similar between different stages of follicles. In contrast, higher level of iNOS mRNA was observed in OFs and protein in the AFs. Higher doses of SNP (10 −3, 10 −5, 10 −7 M) inhibited (P < 0.05) while, lower dose of SNP (10 −9 M) stimulated (P < 0.05) the survival, growth, and antrum formation of PFs. The inhibitory effects of SNP were reversed by Hb, while L-NAME was not found effective. In conclusion, expression of NOS isoforms mRNA and protein in PFs, AFs, and OFs and NOS enzyme activity in immature follicular oocytes suggest a role for NO during ovarian folliculogenesis in buffalo. NO plays a dual role on growth and survival of PFs depending on its concentration in the culture medium.

Inactivation of Membrane Surface Ecto-5′-nucleotidase by Sodium Nitroprusside in C6 Glioma Cells

Ecto-5'-nucleotidase (NT5E), a predominant enzyme that produces extracellular adenosine from AMP, plays an important role in a variety of physiological and pathophysiological processes. This study was performed to identify agents that affect NT5E activity using C6 glioma cells. When cells were incubated with sodium nitroprusside (SNP), phorbol 12-myristate 13-acetate, forskolin, lipopolysaccharide, or interferon-γ, only SNP inhibited NT5E activity in a time- and concentration-dependent manner (IC(50) = 1.2 µM). The inhibitory effect of SNP was long-lasting even after SNP washout; and its action was not mimicked by nitric oxide generating agents, 8-bromo cyclic GMP, ferricyanide, ferrocyanide, or sodium cyanide. SNP did not change NT5E mRNA level or membrane surface protein expression. Similar to SNP, Fe(2+) inhibited NT5E activity, but to a lesser extent. Although Fe(2+) is known to increase oxidative stress, Fe(2+)-mediated oxidative stress was not involved in SNP inhibition of NT5E because the inhibition of NT5E by SNP was not affected by superoxide dismutase and catalase. In contrast, addition of Zn(2+), an essential metal co-factor of NT5E activity, prevented SNP from inhibiting NT5E. These results suggest that SNP disrupts a critical Zn(2+)-dependent enzyme activity and might be useful as a pharmacological tool for inhibiting NT5E.

Interactive effect of nitric oxide and brassinosteroids on photosynthesis and the antioxidant system of Lycopersicon esculentum

An interactive effect of nitric oxide and 24-epibrassinolide (EBL) or 28-homobrassinolide (HBL) was investigated in Lycopersicon esculentum Mill. cv. K-21. Seeds prior to sowing were exposed to sodium nitroprusside (SNP) (0, 10−5 M or 1 M), a nitric oxide donor, for 8 h, and the resulting seedlings were subsequently sprayed with 10−8 M EBL or HBL at 30-day stage. The analysis of plant samples after 24 h of the EBL or HBL spray showed that the lower SNP concentration (10−5 M) favored the growth, pigment content, photosynthetic and enzymatic activities, and also improved the antioxidant system. However, 1 M SNP had inhibitory impact on most of the indices, except the antioxidant system. Brassinosteroids (EBL or HBL), on the other hand, had a stimulatory impact on the aforementioned indices. The inhibitory effect of SNP (1 M) was neutralized by the application of BRs, where EBL was more effective than HBL.

Regulation of TGF‐β1 gene transcription in human prostate cancer cells by nitric oxide

Overexpression of transforming growth factor (TGF)-beta1 is associated with advanced prostate cancer. Our previous studies showed an inverse correlation between the expressions of TGF-beta1 and inducible nitric oxide synthase (iNOS) in prostatic tumors in mice. The purpose of this study was to investigate regulation of TGF-beta1 expression in human prostate cancer cells by nitric oxide (NO). Expression of TGF-beta1 in the three well-characterized lines of human prostate cancer cells (PC-3MM2, LNCaP, and DU145) was determined by using the enzyme-linked immunoabsorbance assay (ELISA), real-time reverse-transcriptase PCR (RT-PCR), nuclear run-on, and promoter activity analyses. Expression of both TGF-beta1 protein and mRNA was inhibited in both dose- and time-dependent manners by NO donors sodium nitroprusside (SNP), S-nitroso-N-acetylpenicilamine (SNAP), S-nitrosoglutathione (GSNO), and (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide (NOR-1) and by transfection of iNOS. The inhibitory effects of SNP and iNOS on TGF-beta1 expression were reduced in cells treated with NO scavengers N-dithiocarboxysarcosine (DTCS), 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), and hemoglobin, or with the iNOS inhibitor N-methyl-arginine (NMA). SNP downregulated the in vitro transcription of TGF-beta1 mRNA, inhibited TGF-beta1 promoter activity, but had no significant effects on TGF-beta1 mRNA stability. These results show that NO downregulates TGF-beta1 expression in prostate cancer cells at transcription level by suppressing the de novo synthesis of TGF-beta1 mRNA.

Angiotensin-(1-7) Enhances Anti-Aggregatory Effects of the Nitric Oxide Donor Sodium Nitroprusside

Patients with ischemic heart disease have platelets that are resistant to the anti-aggregatory effects of nitric oxide (NO) donors. This NO resistance is associated with increased whole blood superoxide radical (O2-) content. Angiotensin II (Ang II) has been shown to augment O2- formation. Recent studies have demonstrated that angiotensin-(1-7) [Ang-(1-7)] has opposite actions to those of Ang II in the vasculature. This study compares the effects of Ang-(1-7) and Ang II on platelet aggregation and platelet responsiveness to the NO donor sodium nitroprusside (SNP). Platelet aggregation was induced by the thromboxane A2 mimetic U46619 (1-5 micromol/L), and the inhibitory effects of SNP (10 micromol/L) on the rate and extent of aggregation were quantified. Ang II did not induce aggregation, but 10-100 nmol/L Ang II potentiated U46619-induced aggregation by 21+/-6% in the absence and by 26+/-9% in the presence of SNP (P<0.01 for both), in blood samples from 8 normal subjects. By contrast, Ang-(1-7) alone did not affect platelet aggregation, but 10-100 nmol/L Ang-(1-7) potentiated the anti-aggregatory effects of SNP in blood samples from both normal subjects (n=17) and patients with acute coronary syndromes (n=17). This effect of Ang-(1-7) was bimodal, and at higher concentrations of Ang-(1-7), potentiation was abolished. The maximum incremental effects of Ang-(1-7) on inhibition of aggregation were 25+/-4% and 28+/-5%, for rate and extent of aggregation respectively (P<0.01 for both), corresponding to a 2.3-fold potentiation of the anti-aggregatory effect of SNP. Platelets from patients were resistant to the anti-aggregatory effect of SNP, but potentiation of SNP effects by Ang-(1-7) was similar for patients and normal subjects. Thus, Ang-(1-7) potentiates the anti-aggregatory effects of NO donor, and may therefore counteract platelet NO resistance that accompanies cardiovascular disease.

The inhibitory effect of sodium nitroprusside on HIF-1 activation is not dependent on nitric oxide-soluble guanylyl cyclase pathway

Adaptation to hypoxia and maintenance of O 2 homeostasis involve a wide range of responses that occur at different organizational levels in the body. One of the most important transcription factors that activate the expression of O 2-regulated genes is hypoxia-inducible factor 1 (HIF-1). Nitric oxide (NO) mediates a variety of biological effects including relaxation of blood vessels and cytotoxicity of activated macrophages. We investigated the effect of the clinically used nitrates nitroglycerin (NTG), isosorbide dinitrate (ISDN), and sodium nitroprusside (SNP) on HIF-1-mediated transcriptional responses to hypoxia. We demonstrate that among the three nitrates, only SNP inhibits HIF-1 activation in response to hypoxia. In contrast, NTG or ISDN does not affect HIF-1 activity. SNP inhibits the accumulation of HIF-1α, the regulatory subunit of HIF-1, and the transcriptional activation of HIF-1α via a mechanism that is not dependent on either NO or soluble guanylate cyclase.

Modulation of dural nociceptor mechanosensitivity by the nitric oxide-cyclic GMP signaling cascade.

The role of the nitric oxide (NO)-cGMP signaling cascade in modulation of peripheral nociception is controversial. Although behavioral studies have suggested both pro- and anti-nociceptive effects, little is known about the direct action of this signaling cascade on primary afferent nociceptive neurons that mediate these behaviors. Here, using single-unit recordings, we examined the direct effect of NO-cGMP signaling on spontaneous activity and mechanical responses of nociceptive afferents that innervate the dura mater. We found that the NO donor sodium nitroprusside (SNP), when applied topically to the neuronal receptive field, induced both sensitization and inhibition of the mechanical responses, albeit in different populations of neurons, which could be distinguished based on their baseline mechanical thresholds. SNP, however, did not change the level of spontaneous activity. Administration of the cGMP analogue 8-pCPT-cGMP mimicked only the inhibitory effect. When SNP was co-applied with either an inhibitor of guanylyl cyclase or a cGMP blocker, sensitization never occurred, and the inhibitory effect of SNP could also be blocked. Our findings suggest that NO can either increase or decrease the mechanical responsiveness of nociceptors and that its action might depend, in part, on the baseline level of neuronal excitability. Our results also implicate cGMP in mediating the inhibitory effect of NO.

Effects of SNP, ouabain, and amiloride on electrical potential profile of isolated sheep pleura.

The fluid and solute transport properties of pleural tissue were studied by using specimens of intact visceral and parietal pleura from adult sheep lungs. The samples were transferred to the laboratory in a Krebs-Ringer solution at 4 degrees C within 1 h from the death of the animal. The pleura was then mounted as a planar sheet in a Ussing-type chamber. The results that are presented in this study are the means of six different experiments. The spontaneous potential difference and the inhibitory effects of sodium nitroprusside (SNP), ouabain, and amiloride on transepithelial electrical resistance (R(TE)) were measured. The spontaneous potential difference across parietal pleura was 0.5 +/- 0.1 mV, whereas that across visceral pleura was 0.4 +/- 0.1 mV. R(TE) of both pleura was very low: 22.02 +/- 4.1 Omega. cm2 for visceral pleura and 22.02 +/- 3.5 Omega. cm2 for parietal pleura. There was an increase in the R(TE) when SNP was added to the serosal bathing solution of parietal pleura and to the serosal or mucosal bathing solution in visceral pleura. The same was observed when ouabain was added to the mucosal surface of visceral pleura and to either the mucosal or serosal surface of parietal pleura. Furthermore, there was an increase in R(TE) when amiloride was added to the serosal bathing solution of parietal pleura. Consequently, the sheep pleura appears to play a role in the fluid and solute transport between the pleural capillaries and the pleural space. There results suggest that there is a Na+ and K+ transport across both the visceral and parietal pleura.

Modulation by nitric oxide (NO) of the intensity of non-quantum mediator secretion in neuromuscular junctions in rats.

Experiments on rat diaphragm muscle showed that the nitric oxide (NO) donors sodium nitroprusside SNP) and S-nitroso-N-acetylpenicillamine (SNAP). as well as L-arginine. a substrate for NO synthesis. decreased the level of muscle fiber hyperpolarization (the H effect) after blockade of cholinoceptors on the postsynaptic membrane by d-tubocurarine in conditions of irreversible inhibition of acetylcholinesterase with armine. Conversely, disruptions to NO synthesis in muscle fibers by the NO synthase blocker NG-nitro-L-arginine methyl ester (L-NAME) led to increases in the H effect both in vitro and in vivo. Inactivated solutions of sodium nitroprusside and inactive forms of arginine and NAME (D-arginine. D-NAME) had no effect on the magnitude of the H effect, while hemoglobin, which efficiently binds NO molecules, blocked the inhibitory effects of sodium nitroprusside. SNAP, and L-arginine on the magnitude of the H effect. All these points provide evidence that NO can function as a modulator of non-quantum mediator release in the neuromuscular junctions of warm-blooded animals.

Nitric oxide inhibits Ca(2+) mobilization through cADP-ribose signaling in coronary arterial smooth muscle cells.

The present study was designed to determine whether the cADP-ribose-mediated Ca(2+) signaling is involved in the inhibitory effect of nitric oxide (NO) on intracellular Ca(2+) mobilization. With the use of fluorescent microscopic spectrometry, cADP-ribose-induced Ca(2+) release from sarcoplasmic reticulum (SR) of bovine coronary arterial smooth muscle cells (CASMCs) was determined. In the alpha-toxin-permeabilized primary cultures of CASMCs, cADP-ribose (5 microM) produced a rapid Ca(2+) release, which was completely blocked by pretreatment of cells with the cADP-ribose antagonist 8-bromo-cADP-ribose (8-Br-cADPR). In intact fura 2-loaded CASMCs, 80 mM KCl was added to depolarize the cells and increase intracellular Ca(2+) concentration ([Ca(2+)](i)). Sodium nitroprusside (SNP), an NO donor, produced a concentration-dependent inhibition of the KCl-induced increase in [Ca(2+)](i), but it had no effect on the U-46619-induced increase in [Ca(2+)](i). In the presence of 8-Br-cADPR (100 microM) and ryanodine (10 microM), the inhibitory effect of SNP was markedly attenuated. HPLC analyses showed that CASMCs expressed the ADP-ribosyl cyclase activity, and SNP (1-100 microM) significantly reduced the ADP-ribosyl cyclase activity in a concentration-dependent manner. The effect of SNP was completely blocked by addition of 10 microM oxygenated hemoglobin. We conclude that ADP-ribosyl cyclase is present in CASMCs, and NO may decrease [Ca(2+)](i) by inhibition of cADP-ribose-induced Ca(2+) mobilization.

Nitric Oxide Inhibits Inducible Nitric Oxide Synthase mRNA Expression in RAW 264.7 Macrophages

Using cultured murine RAW 264.7 macrophages, the present study investigates the influence of nitric oxide (NO) on the expression of the inducible NO synthase (iNOS) enzyme at the transcriptional level. Incubation of cells with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) led to a marked increase in iNOS mRNA levels. Inhibition of LPS/IFN-γ-induced NO synthesis with the L-arginine analogue NG-monomethyl-L-arginine (L-NMMA) was accompanied by a significant up-regulation of iNOS mRNA that was reversed in the presence of the NO donor sodium nitroprusside (SNP). Treatment of cells with SNP alone decreased LPS/IFN-γ-induced iNOS mRNA levels in a concentration-dependent manner. The inhibitory effect of SNP on iNOS mRNA expression was not prevented by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of the soluble guanylyl cyclase. In agreement with this finding, incubation of cells with the membrane-permeable cyclic GMP analogue 8-bromo cyclic GMP left LPS/IFN-γ-induced iNOS mRNA expression virtually unaltered. Together, our results demonstrate that both iNOS-derived and exogenous NO exert an inhibitory effect on the expression of iNOS by a mechanism independent of the soluble guanylyl cyclase/cyclic GMP pathway. In conclusion, NO may control the extent of iNOS mRNA expression by a negative autoregulatory feedback.

Nitric oxide induces the synthesis of vascular endothelial growth factor by rat vascular smooth muscle cells.

Vascular endothelial growth factor (VEGF) is known to induce the release of nitric oxide (NO) from endothelial cells. However, the effect of NO on VEGF synthesis is not clear. Accordingly, the effect of endogenous and exogenous NO on VEGF synthesis by rat vascular smooth muscle cells (VSMCs) was investigated. Two in vitro models were used: (1) VSMCs stimulated to produce NO by treatment with interleukin (IL)-1beta (10 ng/mL) and (2) VSMCs lipotransfected with pKecNOS plasmid, containing the endothelial constitutive NO synthase (ecNOS) cDNA. The synthesis of NO was inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME, 2 to 5 mmol/L) or diaminohydroxypyrimidine (DAHP, 2.5 to 5 mmol/L), inhibitors of NOS and GTP cyclohydrolase I, respectively. Some cells treated with L-NAME or DAHP were supplemented with L-arginine (10 mmol/L) or tetrahydrobiopterin (BH(4); 100 micromol/L), respectively. In addition, we studied the effect of sodium nitroprusside (SNP; 10 and 100 micromol/L) and chemically related compounds, potassium ferrocyanide and ferricyanide, on VEGF generation. IL-1beta induced iNOS expression and NO generation and significantly upregulated VEGF mRNA expression and protein synthesis. L-NAME and DAHP totally inhibited NO generation and decreased the IL-1beta-upregulated VEGF synthesis by 30% to 40%. Supplementation with L-arginine or BH(4) increased NO generation by L-NAME- or DAHP-treated cells, and VEGF synthesis was augmented by addition of BH(4). The cells generating NO after pKecNOS transfection released significantly higher amounts of VEGF than cells transfected with control plasmids. Inhibition of NO generation by L-NAME decreased VEGF synthesis. In contrast to the effect of endogenous NO, we observed the inhibition of VEGF synthesis in the presence of high (10 or 100 micromol/L) concentrations of SNP. This effect was mimicked by chemically related ferricyanide and ferrocyanide compounds, suggesting that the inhibitory effect of sodium nitroprusside may be mediated by an NO-independent mechanism. The results indicate that endogenous NO enhances VEGF synthesis. The positive interaction between endogenous NO and VEGF may have implications for endothelial regeneration after balloon angioplasty and for angiogenesis.

Effects of atrial natriuretic peptide and sodium nitroprusside on epidermal growth factor-stimulated wound repair in rabbit corneal epithelial cells

Purpose. Treatment of rabbit corneal epithelial cells (RCEC) with epidermal growth factor (EGF) stimulates cell proliferation and wound repair in a cell culture model system. Studies have also shown that atrial natriuretic peptide (ANP) and sodium nitroprusside (SNP), a nitric oxide-generating agent, inhibit proliferation of a variety of cell types. The aim of the present work was to examine whether ANP or SNP has any effect on EGF-stimulated proliferation of RCEC involved in wound repair. Methods. The SV-40 immortalized RCEC were cultured in 24-well plates until they became confluent. Wounds of uniform size (8 mm diameter) were created and the cells allowed to grow in the presence and absence of EGF and/or other agents. At prescribed time intervals, the cells were stained by Giemsa and the wound areas digitized and quantified by Sigma Image Scan System. The cGMP contents in RCEC, treated with or without ANP or SNP, were measured by radioimmunoassay. Results. Addition of EGF (1–100 ng/ml) to RCEC stimulated cell proliferation which significantly reduced the time required for wound closure. Addition of ANP (1 nM to 10 µM) or SNP (10 µM to 1 mM), in the presence of EGF, dose-dependently inhibited the growth factor-stimulated wound closure in RCEC. When added alone to the cells, ANP or SNP increased cGMP accumulation in a dose-dependent manner. Addition of ANP (1 µM) or SNP (1 mM) to primary corneal epithelial cells, in the presence and absence of EGF, also inhibited the wound closure with a corresponding increase in cGMP contents. Treatment of the cells with ODQ (10 nM to 10 µM), a soluble guanylate cyclase inhibitor, dose-dependently decreased the SNP-induced accumulation of cGMP, and reversed the inhibitory effect of SNP on EGF-stimulated wound closure. Addition of membrane-permeable cGMP analog, 8-bromo-cGMP, to RCEC inhibited the EGF-stimulated wound closure in a dose-dependent manner. Treatment of RCEC with mitomycin C (5 µM) exerted a marked inhibitory effect on wound closure in the presence and absence of EGF, and also abrogated the inhibitory effect of 8-bromo-cGMP on wound closure in the EGF-treated and untreated cells. Conclusions. The results demonstrate that ANP and SNP inhibit the EGF-stimulated wound repair in RCEC. The effect of these agents is mediated via activation of guanylate cyclases that generate cGMP. Cyclic GMP appears to exert its inhibitory effect at the level of cell proliferation and not cell migration. The data suggest an important role for cGMP-dependent protein kinase in proliferation of RCEC stimulated by EGF.

Role of nonselective cation channels as Ca 2+ entry pathway in endothelin-1-induced contraction and their suppression by nitric oxide

The present study was carried out to clarify the role of nonselective cation channels as a Ca 2+ entry pathway in the contraction and the increase in [Ca 2+] i induced by endothelin-1 in endothelium-denuded rat thoracic aorta rings, and their suppression by nitric oxide (NO). In Ca 2+-free medium, the endothelin-1-induced contraction was suppressed to about 20% of control values, although the increase in [Ca 2+] i became negligible. The contraction and the increase in [Ca 2+] i monitored by fura 2 fluorescence were unaffected by a blocker of L-type voltage-operated Ca 2+ channels nifedipine. A blocker of nonselective cation channels 1-[ β-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1 H-imidazole·HCl(SK&F 96365) suppressed the endothelin-1-induced contraction and increase in [Ca 2+] i to the level similar to that after removal of extracellular Ca 2+. SK&F 96365 had no further effect on the endothelin-1-induced contraction in the absence of extracellular Ca 2+. The endothelin-1-induced contraction and increase in [Ca 2+] i were abolished by a donor of NO sodium nitroprusside. The effects of another NO donor 3-morpholinosydnonimine (SIN-1) were also tested and yielded essentially similar results to those for sodium nitroprusside on the endothelin-1-induced contraction. Furthermore, the inhibitory effects of sodium nitroprusside could be blocked with a guanylate cyclase inhibitor 1 H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) at 30 μM. These findings suggest that Ca 2+ entry through nonselective cation channels but not voltage-operated Ca 2+ channels plays a critical role in the endothelin-1-induced increase in [Ca 2+] i and the resulting contraction and that inhibition by NO of the endothelin-1-induced contraction is mainly the result of blockade of Ca 2+ entry through these channels.

Cyclic GMP inhibits cytoplasmic Ca 2+ oscillation by increasing Ca 2+-ATPase activity in rat megakaryocytes

The regulatory effects of cyclic GMP on purinoceptor-operated cytoplasmic Ca 2+ oscillation of rat megakaryocytes were investigated by using whole-cell patch-clamp technique. ATP-induced oscillatory K + currents though Ca 2+-activated K + channels ( I KCas) were depressed by pretreatment with the guanylate cyclase activator, sodium nitroprusside, and a stable membrane-permeable cGMP analogue, 8-bromo-cGMP. The inhibition by sodium nitroprusside was blocked by treatment with a cyclic nucleotide-dependent protein kinase inhibitor, N-[2-(methylamino)]-5-isoquinolinesulfonamide·HCl (H-8) (10 μM), but not by a selective cAMP-dependent-protein kinase inhibitor, Rp-cAMPS (100 μM). The oscillatory I KCa directly evoked by intracellular d-myo-inositol-trisphosphate (IP 3) perfusion was also inhibited by the application of sodium nitroprusside. The inhibitory effect of sodium nitroprusside disappeared when the ATP-induced oscillatory I KCa was changed to a monophasic sustained I KCa current by inhibition of Ca 2+-ATPase. These results suggested that cGMP depressed Ca 2+ mobilization by improving Ca 2+-ATPase activity by phosphorylation.

Ouabain Produces Diverse Excitatory Effects on Afferent Baroreceptor Nerve Activity in SHR and WKY Animals

The effect of acute ouabain treatment was evaluated on afferent baroreceptor nerve activity in spontaneously hypertensive rats (SHR) compared with Wistar Kyoto rats (WKY). Under urethane anesthesia (1.2 mg/Kg) the discharge of the recurrent laryngeal nerve was utilized as index of arterial baroreceptor activity (BNA) in rats with the ipslateral vagus cut at a proximal level. The ouabain (30 μg, i.v.) treatment produced an excitatory effect on BNA without changes in basal arterial pressure in both groups studied. This effect was larger in SHR (92±10%) than WKY (37±4%, P < 0.01)The arterial pressor response to phenylephrine was similar in both SHR and WKY before (20±1 and 22±1.2 mmHg) and after (18±1.4 and 20±2 mmHg, respectively) ouabain. The BNA under phenylephrine-induced peaks of high arterial pressure was significantly higher in SHR (61±15%) than in WKY (41±5% P < 0.01) but after ouabain treatment the opposite was observed (31±5 vs. 61±4% P < 0.01). The inhibitory effects of sodium nitroprusside on arterial pressure and BNA were similar in SHR and WKY groups both before and after the ouabain treatmentThese data indicate an excitatory effect of ouabain on baroreceptor nerve activity in normotensive and markedly in hypertensive rats which could contribute to the reflex arterial pressure regulation, besides the known inotropic action on the heart