Nitric oxide extends the oocyte temporal window for optimal fertilization

Deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age. In this study, we explore the role of nitric oxide (NO) on the sustenance of oocyte quality postovulation. Sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (ICSI) with cauda-epididymal spermatozoa following exposure to either the NO donor, S-nitroso-N-acetylpenicillamine (SNAP, 0.23 microM/min), an NO synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 1 mM), or an inhibitor of soluble guanylyl cyclase (sGC), 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, 100 microM); while their sibling oocytes were subjected to ICSI either before (young) or after culture for the corresponding period of time (old). Outcomes of normal fertilization, cleavage, and development to the morula and blastocyst stages were compared. Embryos from each subgroup were also subjected to TUNEL assay for apoptosis. A significant deterioration in the ability of the oocytes to undergo normal fertilization and development to morula and blastocyst stages occurred among oocytes aged in culture medium compared to their sibling cohorts subjected to ICSI immediately after ovulation (P<0.05). This deterioration was prevented in oocytes exposed to SNAP. In contrast, exposure to L-NAME or ODQ resulted in a significant compromise in fertilization and development to the morula and blastocyst stages (P<0.05). Finally, apoptosis was noted in embryos derived from aged oocytes and those exposed to L-NAME or ODQ, but not in embryos derived from young oocytes or oocytes exposed to SNAP. Thus, NO is essential for sustenance of oocyte quality postovulation.     

Nitric oxide inhibits spleen cell proliferative response after burn injury by inducing cytostasis,apoptosis, and necrosis of activated T lymphocytes: role of the guanylate cyclase

We previously showed that an overproduction of nitric oxide (NO) by macrophages was responsible for the collapse of lymphoproliferative responses after burn injury in rats. First, we demonstrate here that 10 days post-burn, the inhibition of splenocyte response to concanavalin-A results from cytostatic, apoptotic, and necrotic effects of NO on activated T cells. This was evidenced by various criteria at the levels of DNA, mitochondria, and plasma membrane. Inhibition of NO synthase by S-methylisothiourea (10 microM) normalized all the parameters. Second, we show that two soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ, restored the proliferative response in a concentration-dependent manner. LY83583 (0.5 microM) rescued T cells from apoptosis. Similar results were obtained with KT5823 (5 microM) a specific inhibitor of protein kinase G (PKG). In contrast, neither LY83583 nor KT5823 inhibited NO-induced necrosis. These results suggest that NO blocked T cells in the G1 phase and induced apoptosis through a sGC-PKG-dependent pathway and necrosis through an independent one.     

Nitric oxide regulates AKT phosphorylation and nuclear translocation in cultured retinal cells

Previous studies have shown that nitric oxide (NO) inhibits apoptosis of retinal neurons in culture through the canonical cyclic GMP/protein kinase G (PKG)-dependent pathway, but also involving multiple kinase pathways, such as phosphatidylinositol 3′ kinase (PI3k) and AKT. NO and AKT exhibit survival-promoting properties and display important roles in both CNS development and plasticity. The purpose of this study was to evaluate the effects of exogenous NO, derived from the NO donor S-nitroso-N-acetylpenicillamin (SNAP), or endogenous NO, produced from l-arginine, on AKT phosphorylation in cultured chick retinal neurons. Our results demonstrate that SNAP or l-arginine enhances AKT phosphorylation on both serine-473 and threonine-308 residues in a concentration and time-dependent manner. This effect was mediated by the activation of soluble guanylyl cyclase and PKG, since it was blocked by the respective enzyme inhibitors ODQ or LY83583 and KT5823, as well as by transduction with shRNA lentiviruses coding PKGII shRNA, and mimicked by the respective enzyme activators YC-1 and 8-Bromo cyclic GMP, and also by the cyclic GMP phosphodiesterase inhibitor zaprinast. In addition, LY294002 or wortmannin suppressed the SNAP effect, indicating the involvement of phosphoinositide 3′ kinase. Moreover, the mTOR inhibitor KU0063794 blocked SNAP-induced AKT phosphorylation at both residues, suggesting the participation of the mTORC2 complex in the process. Glutamate and NMDA also promoted AKT phosphorylation and a nitric oxide synthase inhibitor abrogated these effects, revealing a mechanism involving the activation of NMDA receptors and NO production. We have also found that SNAP and l-arginine induced AKT translocation into the nucleus of retinal neurons as well as other neuronal cell lines. SNAP also protects retinal cells from death induced by hydrogen peroxide and this effect was blocked by the phosphoinositide 3′ kinase inhibitor LY294002. We therefore conclude that NO produced from endogenous or exogenous sources promotes AKT activation and its shuttling to the nucleus, probably participating in neuronal survival pathways important during CNS development.     

Regulation of NHE3 by nitric oxide in Caco-2 cells

The effect of nitric oxide (NO) on Na+/H+ exchange (NHE) activity was investigated utilizing Caco-2 cells as an experimental model. Incubation of Caco-2 cells with 10(-3) M S-nitroso-N-acetylpenicillamine (SNAP), a conventional donor of NO, for 20 min resulted in a approximately 45% dose-dependent decrease in NHE activity, as determined by assay of ethylisopropylamiloride-sensitive 22Na uptake. A similar decrease in NHE activity was observed utilizing another NO-specific donor, sodium nitroprusside. SNAP-mediated inhibition of NHE activity was not secondary to a loss of cell viability. NHE3 activity was significantly reduced by SNAP (P < 0.05), whereas NHE2 activity was essentially unaltered. The effects of SNAP were mediated by the cGMP-dependent signal transduction pathway as follows: 1) LY-83583 and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), specific inhibitors of soluble guanylate cyclase, blocked the inhibitory effect of SNAP on NHE; 2) 8-bromo-cGMP mimicked the effects of SNAP on NHE activity; 3) the SNAP-induced decrease in NHE activity was counteracted by a specific protein kinase G inhibitor, KT-5823 (1 microM); 4) chelerythrine chloride (2 microM) or calphostin C (200 nM), specific protein kinase C inhibitors, did not affect inhibition of NHE activity by SNAP; 5) there was no cross activation by the protein kinase A-dependent pathway, as the inhibitory effects of SNAP were not blocked by Rp-cAMPS (25 microM), a specific protein kinase A inhibitor. These data provide novel evidence that NO inhibits NHE3 activity via activation of soluble guanylate cyclase, resulting in an increase in intracellular cGMP levels and activation of protein kinase G.     

Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates nitric oxide-induced endothelial cell migration and angiogenesis

To test the hypothesis that the phosphatidylinositol 3-kinase (PI3 kinase)/protein kinase Akt signaling pathway is involved in nitric oxide (NO)-induced endothelial cell migration and angiogenesis, we treated human and bovine endothelial cells with NO donors, S-nitroso-L-glutathione (GSNO) and S-nitroso-N-penicillamine (SNAP). Both GSNO and SNAP increased Akt phosphorylation and activity, which were blocked by cotreatment with the PI3 kinase inhibitor wortmannin. The mechanism was due to the activation of soluble guanylyl cyclase because 8-bromo-cyclic GMP activated PI3 kinase and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) blocked NO-induced PI3 kinase activity. Indeed, transfection with adenovirus containing endothelial cell NO synthase (eNOS) or protein kinase G (PKG) increased endothelial cell migration, which was inhibited by cotransfection with a dominant-negative mutant of PI3 kinase (dnPI3 kinase). In a rat model of hind limb ischemia, adenovirus-mediated delivery of human eNOS cDNA in adductor muscles resulted in time-dependent expression of recombinant eNOS, which was accompanied by significant increases in regional blood perfusion and capillary density. Coinjection of adenovirus carrying dnPI3 kinase abolished neovascularization in ischemic hind limb induced by eNOS gene transfer. These findings indicate that NO promotes endothelial cell migration and neovascularization via cGMP-dependent activation of PI3 kinase and suggest that this pathway is important in mediating NO-induced angiogenesis.     

Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15

Intracellular production of nitric oxide (NO) is thought to mediate the pancreatic B-cell-directed cytotoxicity of cytokines in insulin-dependent diabetes mellitus, and recent evidence has indicated that this may involve induction of apoptosis. A primary effect of NO is to activate soluble guanylyl cyclase leading to increased cGMP levels and this effect has been demonstrated in pancreatic B-cells, although no intracellular function has been defined for islet cGMP. Here we demonstrate that the NO donor, GSNO, induces apoptosis in the pancreatic B-cell line HIT-T15 in a dose- and time-dependent manner. This response was significantly attenuated by micromolar concentrations of a specific inhibitor of soluble guanylyl cyclase, ODQ, and both 8-bromo cGMP (100 μM) and dibutyryl cGMP (300 μM) were able to fully relieve this inhibition. In addition, incubation of HIT-T15 cells with each cGMP analogue directly promoted cell death in the absence of ODQ. KT5823, a potent and highly selective inhibitor of cGMP-dependent protein kinase (PKG), abolished the induction of cell death in HIT cells in response to either GSNO or cGMP analogues. This effect was dose-dependent over the concentration range of 10–250 nM. Overall, these data provide evidence that the activation of apoptosis in HIT-T15 cells by NO donors is secondary to a rise in cGMP and suggest that the pathway controlling cell death involves activation of PKG.     

Permissive contributions of NO and prostacyclin in CO-induced cerebrovascular dilation in piglets

Endogenously produced CO is an important dilator in newborn cerebrovascular circulation. CO dilates cerebral arterioles by activating Ca2+-activated K+ channels, but modulatory actions of other effectors and second messenger inputs are unclear. Specifically, the mechanisms behind the obligatory permissive roles of prostacyclin and NO are uncertain. Therefore, the present study was performed using acutely implanted, closed cranial windows in newborn pigs to address the hypothesis that the permissive roles of NO and prostacyclin in cerebrovascular dilation in response to CO involve a common mechanism. The NO donor sodium nitroprusside restored dilation in response to CO after inhibition of that dilation with the prostaglandin cyclooxygenase inhibitor indomethacin. The stable prostacyclin analog iloprost restored CO-induced dilation blocked by the NO synthase inhibitor Nomega-nitro-L-arginine. Restoration of dilation in response to CO by the cGMP-dependent phosphodiesterase inhibitor zaprinast and blockade of CO dilation by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one (ODQ) suggests involvement of the cGMP/PKG pathway. Iloprost or the cAMP-dependent dilator isoproterenol restored dilation in response to CO after ODQ administration. However, CO-induced dilation blocked by the cGMP-dependent PKG inhibitor Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine could not be reversed by administration of sodium nitroprusside, iloprost, or isoproterenol. Conversely, PKA inhibition did not block dilation in response to CO. Overall, data indicate that activation of PKG is the predominant mechanism of the permissive actions of NO and prostacyclin for CO-induced pial arteriolar dilation.     

Functional effects of C-type natriuretic peptide and nitric oxide are attenuated in hypertrophic myocytes from pressure-overloaded mouse hearts

Increases in the myocardial level of cGMP usually exert negative inotropic effects in the mammalian hearts. We tested the hypothesis that the negative functional effects caused by nitric oxide (NO) or C-type natriuretic peptide (CNP) through cGMP would be blunted in hypertrophied cardiac myocytes. Contractile function, guanylyl cyclase activity, cGMP-dependent protein phosphorylation, and calcium transients were assessed in ventricular myocytes from aortic stenosis-induced hypertrophic and age-matched control mice. Basal percentage shortening was similar in control and hypertrophic myocytes. S-nitroso-N-acetyl-penicillamine (SNAP, an NO donor, 10(-6) and 10(-5) M) or CNP (10(-8) and 10(-7) M) reduced percentage shortening in both groups, but their effects were blunted in hypertrophic myocytes. Maximal rates of shortening and relaxation were depressed at the basal level, and both reagents had attenuated effects in hypertrophy. Similar results were also found after treatment with guanylin and carbon monoxide, other stimulators of particulate, and soluble guanylyl cyclase, respectively. Guanylyl cyclase activity was not significantly changed in hypertrophy. Addition of Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (an inhibitor of cGMP-dependent protein kinase, 5 x 10(-6) M) blocked SNAP or the effect of CNP in control mice but not in hypertrophy, indicating the cGMP-dependent kinase (PKG) may not mediate the actions of cGMP induced by NO or CNP in the hypertrophic state. Calcium transients after SNAP or CNP were not significantly changed in hypertrophy. These results suggest that in hypertrophied mice, diminished effects of NO or CNP on ventricular myocyte contraction are not due to changes in guanylyl cyclase activity. The data also indicated that PKG-mediated pathways were diminished in hypertrophied myocardium, contributing to blunted effects.     

Effects of manipulating the nitric oxide/cyclic GMP pathway on bovine oocyte meiotic resumption in vitro

The objective of this study was to examine the effects of manipulating the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway on bovine oocyte nuclear maturation in vitro. Cumulus-enclosed oocytes (CEO) were recovered from abattoir-derived ovaries and cultured in M199+FCS for 7 or 21h in the presence of various molecules affecting the NO/cGMP pathway, and then fixed and stained for evaluation of the stage of nuclear maturation. Cyclic GMP levels were also measured in cumulus-oocyte complexes after 3 and 6 h of culture. The iNOS inhibitor, aminoguanidine (AG, 10 and 50 mM) and the NO donor sodium nitroprusside (SNP, 100 and 500 microM) significantly inhibited GVBD after 7h of culture. However, a lower concentration of SNP (0.01 microM) stimulated GVBD. The inhibitory effects of AG and SNP were reversible, indicating that they were not toxic effects. Although SNP (500 microM) increased cGMP levels in cumulus-oocyte complexes after 3 h of culture, the inhibitor of soluble guanylate cyclase ODQ and the protein kinase G (PKG) inhibitor KT5823 did not reverse the inhibitory effect of SNP on meiosis, suggesting that SNP does not inhibit meiosis through the cGMP/PKG pathway. Similarly, an analogue of cGMP (8-Bromo-cGMP 0.5, 1, 3, and 6 mM), as well as activation of guanylate cyclase with Protoporphyrin IX or atrial natriuretic peptide, or inhibition of the enzyme with ODQ, did not have any significant effect on GVBD after 7 h of culture, supporting the idea that the effects of AG and SNP were not due to altered cGMP levels. Atrial natriuretic peptide, Protoporphyrin IX and SNP 500 microM increased cGMP levels after 3 h but not 6 h of culture. In conclusion, soluble and particulate guanylate cyclases could be activated in bovine cumulus-oocyte complexes, but accumulation of cGMP was probably not responsible for the effects of NO on meiosis.     

Signaling pathway of nitric oxide-induced acrosome reaction in human spermatozoa

Nitric oxide (NO) has been recently shown to modulate in vitro motility, viability, the acrosome reaction (AR), and metabolism of spermatozoa in various mammalian species, but the mechanism or mechanisms through which it influences sperm functions has not been clarified. In human capacitated spermatozoa, both the intracellular cGMP level and the percentage of AR-positive cells were significantly increased after 4 h of incubation with the NO donor, sodium nitroprusside (SNP). SNP-induced AR was significantly reduced in the presence of the soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ; this block was bypassed by adding 8-bromo-cGMP, a cell-permeating cGMP analogue, to the incubation medium. Finally, Rp-8-Br-cGMPS and Rp-8-pCPT-cGMPS, two inhibitors of the cGMP-dependent protein kinases (PKGs), inhibited the SNP-induced AR. Furthermore, SNP-induced AR did not occur in Ca2+ -free medium or in the presence of the protein kinase C (PKC) inhibitor, calphostin C. This study suggests that the AR-inducing effect of exogenous NO on capacitated human spermatozoa is accomplished via stimulation of an NO-sensitive sGC, cGMP synthesis, and PKG activation. In this effect the activation of PKC is also involved, and the presence of extracellular Ca2+ is required.     

Modulation of Cl-/OH- exchange activity in Caco-2 cells by nitric oxide

The present studies were undertaken to determine the direct effects of nitric oxide (NO) released from an exogenous donor, S-nitroso-N-acetyl pencillamine (SNAP) on Cl-/OH- exchange activity in human Caco-2 cells. Our results demonstrate that NO inhibits Cl-/OH- exchange activity in Caco-2 cells via cGMP-dependent protein kinases G (PKG) and C (PKC) signal-transduction pathways. Our data in support of this conclusion can be outlined as follows: 1) incubation of Caco-2 cells with SNAP (500 microM) for 30 min resulted in approximately 50% inhibition of DIDS-sensitive 36Cl uptake; 2) soluble guanylate cyclase inhibitors Ly-83583 and (1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one significantly blocked the inhibition of Cl-/OH- exchange activity by SNAP; 3) addition of 8-bromo-cGMP (8-BrcGMP) mimicked the effects of SNAP; 4) specific PKG inhibitor KT-5823 significantly inhibited the decrease in Cl-/OH- exchange activity in response to either SNAP or 8-BrcGMP; 5) Cl-/OH-exchange activity in Caco-2 cells in response to SNAP was not altered in the presence of protein kinase A (PKA) inhibitor (Rp-cAMPS), demonstrating that the PKA pathway was not involved; 6) the effect of NO on Cl-/OH- exchange activity was mediated by PKC, because each of the two PKC inhibitors chelerythrine chloride and calphostin C blocked the SNAP-mediated inhibition of Cl-/OH- exchange activity; 7) SO/OH- exchange in Caco-2 cells was unaffected by SNAP. Our results suggest that NO-induced inhibition of Cl-/OH- exchange may play an important role in the pathophysiology of diarrhea associated with inflammatory bowel diseases.     

eNOS, nNOS, cGMP and protein kinase G mediate the inhibitory effect of pancreastatin, a chromogranin A-derived peptide, on growth and proliferation of hepatoma cells

Pancreastatin (PST), a chromogranin A-derived peptide, has an anti-insulin metabolic effect and inhibits growth and proliferation by producing nitric oxide (NO) in HTC rat hepatoma cells. When NO production is blocked, a proliferative effect prevails due to the activation a Galphaq/11-phospholipase C-beta (PLC-beta) pathway, which leads to an increase in [Ca2+]i, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. The aim of the present study was to investigate the NO synthase (NOS) isoform that mediates these effects of PST on HTC hepatoma cells and the possible roles of cyclic GMP (cGMP) and cGMP-dependent protein kinase. DNA and protein synthesis in response to PST were measured as [3H]-thymidine and [3H]-leucine incorporation in the presence of various pharmacological inhibitors: N-monomethyl-L-arginine (NMLA, nonspecific NOS inhibitor), L-NIO (endothelial nitric oxide synthase (eNOS) inhibitor), espermidine (neuronal nitric oxide synthase (nNOS) inhibitor), LY83583 (guanylyl cyclase inhibitor), and KT5823 (protein kinase G inhibitor, (PKG)). L-NIO, similarly to NMLA, reverted the inhibitory effect of PST on hepatoma cell into a stimulatory effect on growth and proliferation. Nevertheless, espermidine also prevented the inhibitory effect of PST, but there was no stimulation of growth and proliferation. When guanylyl cyclase activity was blocked, there was again a reversion of the inhibitory effect into a stimulatory action, suggesting that the effect of NO was mediated by the production of cGMP. PKG inhibition prevented the inhibitory effect of PST, but there was no stimulatory effect. Therefore, the inhibitory effect of PST on growth and proliferation of hepatoma cells may be mainly mediated by eNOS activation. In turn, the effect of NO may be mediated by cGMP, whereas other pathways in addition to PKG activation seem to mediate the inhibition of DNA and protein synthesis by PST in HTC hepatoma cells.     

Defects in cGMP-PKG pathway contribute to impaired NO-dependent responses in hepatic stellate cells upon activation

NO antagonizes hepatic stellate cell (HSC) contraction, although activated HSC in cirrhosis demonstrate impaired responses to NO. Decreased NO responses in activated HSC and mechanisms by which NO affects activated HSC remain incompletely understood. In normal rat HSC, the NO donor diethylamine NONOate (DEAN) significantly increased cGMP production and reduced serum-induced contraction by 25%. The guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) abolished 50% of DEAN effects, whereas the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) reiterated half the observed DEAN response, suggesting both cGMP-dependent protein kinase G (PKG)-dependent and -independent mechanisms of NO-mediated antagonism of normal HSC contraction. However, NO donors did not increase cGMP production from in vivo activated HSC from bile duct-ligated rats and showed alterations in intracellular Ca(2+) accumulation suggesting defective cGMP-dependent effector pathways. The LX-2 cell line also demonstrated lack of cGMP generation in response to NO and a lack of effect of ODQ and 8-BrcGMP in modulating the NO response. However, cGMP-independent effects in response to NO were maintained in LX-2 and were associated with S-nitrosylation of proteins, an effect reiterated in primary HSC. Adenovirus-based overexpression of PKG significantly attenuated contraction of LX-2 by 25% in response to 8-BrcGMP. In summary, these studies demonstrate that NO affects HSC through cGMP-dependent and -independent pathways. The HSC activation process is associated with maintenance of cGMP-independent actions of NO but defects in cGMP-PKG-dependent NO signaling that are improved by PKG gene delivery in LX-2 cells. Activating targets downstream from NO-cGMP in activated HSC may represent a novel therapeutic target for portal hypertension.     

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

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

Cyclic GMP-dependent and -independent regulation of MAP kinases by sodium nitroprusside in isolated cardiomyocytes

Sodium nitroprusside (SNP) elicits various physiological effects, in part through generation of the membrane permeable mediator nitric oxide (NO). In the heart, besides its role in regulating contractility, NO is involved in both protection from and induction of cellular damage. The present study investigated the role of SNP in the regulation of the mitogen-activated protein kinases (MAPKs) in isolated adult rat cardiomyocytes. SNP maximally activated Erk1, Erk2, p38 MAPK and MAPKAPK2 in 5-10 min. The activation of MAPKAPK2 by SNP was blocked by the soluble guanylyl cyclase inhibitor, 1H-[1, 2,4]oxadiazolol[4,3-a]quinoxalin-1-one (ODQ) and the p38 MAPK inhibitor, SB203580. The activation of Erk1 was insensitive to ODQ but completely blocked by the Mek1 inhibitor PD98059. The membrane-permeable homologue of cGMP, 8-Br-cGMP, also activated p38 MAPK (A(0.5) approximately 50 microM) but not Erk1 and Erk2. These results indicate that p38 MAPK and MAPKAPK2 are activated by SNP in cGMP-dependent pathways, while the Erk1 activation by SNP is independent of cGMP levels.     

NO-induced modulation of calcium-oscillations in pulmonary vascular smooth muscle

The effect of the nitric oxide (NO) donor sodium nitroprusside (SNP) on both [Ca(2+)](i)and mechanical activity was studied in the rat isolated pulmonary artery (RPA). In freshly isolated myocytes loaded with 1 microM indo-lacetoxymethyl ester for 30 min, short (40-60 s) application of ATP (100 microM) or ET-1 (0.1 microM) induced 3-6 cyclic rises in [Ca(2+)](i)(Ca-oscillations) of decreasing amplitude. Preincubation of cells with SNP (10-250 microM) for 10 min had no effect on the resting [Ca(2+)](i)value, but progressively abolished the oscillations. A similar effect was obtained with 8-bromo-cGMP (100-500 microM). SNP (0.001-100 microM) concentration-dependently relaxed ATP (10 mM, n = 4) and ET-1 (0.1 microM, n = 4)-precontracted RPA. 1H-[1,2,4]oxadiazolol [4,3,-a]quinoxalin-1-one (ODQ, 10 microM), a potent inhibitor of the cytosolic guanylyl cyclase, fully reversed the effect of SNP on ATP-induced [Ca(2+)](i)oscillations as well as on ATP-precontracted RPA. In contrast, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8, 10 microM), a potent inhibitor of cGMP-dependent protein kinase (PKG), did not alter the effect of SNP. Caffeine (5 mM) induced only one transient [Ca(2+)](i)-increase (n = 24), the amplitude of which was altered neither by SNP nor by 8-bromo-cGMP. Our results show that the relaxing effect of NO in RPA is related, at least in part, to its action on the Ca-signalling pathway. NO interacts with inositol trisphosphate pathway without interacting with the ryanodine-sensitive receptor. Finally, the effect of NO involves an increase in cGMP but appears independent of activation of PKG.     

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

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

Effect of nitric oxide on hyperpolarization-activated current in substantia gelatinosa neurons of rats

Central sensitization is the hyperexcitability of spinal processing after peripheral nerve injury or inflammation. This phenomenon may be associated with nitric oxide (NO) signal pathway in synapse. Here, we have investigated the effect of NO on hyperpolarization-activated inward current (I(h)) in substantia gelatinosa (SG) neurons, using the whole-cell patch clamp technique. I(h) was increased by the application of sodium nitro prusside (SNP, a NO donor) or 8Br-cGMP. The stimulatory effects of NO were abolished by guanylyl cyclase inhibitor, ODQ, suggesting that the effect of NO was mediated by cGMP. However, this effect of NO was not prevented by the pretreatment with KT5823, PKG inhibitor. Taken together, the activation of I(h) in SG neurons could be mediated by NO-cGMP dependent pathway. These results reveal an involvement of NO in excitability of SG neuron via the activation of I(h) may be associated with central sensitization.     

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

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