Cyclic GMP-dependent Mechanism Research Articles

Nitrosothiols in the Immune System: Signaling and Protection

In the immune system, nitric oxide (NO) has been mainly associated with antibacterial defenses exerted through oxidative, nitrosative, and nitrative stress and signal transduction through cyclic GMP-dependent mechanisms. However, S-nitrosylation is emerging as a post-translational modification (PTM) involved in NO-mediated cell signaling. Precise roles for S-nitrosylation in signaling pathways have been described both for innate and adaptive immunity. Denitrosylation may protect macrophages from their own S-nitrosylation, while maintaining nitrosative stress compartmentalized in the phagosomes. Nitrosothiols have also been shown to be beneficial in experimental models of autoimmune diseases, mainly through their role in modulating T-cell differentiation and function. Relationship between S-nitrosylation, other thiol redox PTMs, and other NO-signaling pathways has not been always taken into account, particularly in the context of immune responses. Methods for assaying S-nitrosylation in individual proteins and proteomic approaches to study the S-nitrosoproteome are constantly being improved, which helps to move this field forward. Integrated studies of signaling pathways in the immune system should consider whether S-nitrosylation/denitrosylation processes are among the PTMs influencing the activity of key signaling and adaptor proteins. Studies in pathophysiological scenarios will also be of interest to put these mechanisms into broader contexts. Interventions modulating nitrosothiol levels in autoimmune disease could be investigated with a view to developing new therapies.

Relative importance of calcium-activated potassium channels in nipradilol-induced aortic relaxation in rats.

Nipradilol (CAS 81486-22-8), a vasodilatory beta-blocker, has been shown to dilate smaller vessels than nitroglycerin does, and the vasodilative effects of nipradilol have been reported to be less mediated by cyclic GMP (guanosine monophosphate) than those of nitroglycerin. To test the hypothesis that cyclic GMP-independent potassium channels have a larger role in nipradilol-induced aortic relaxation than cyclic GMP-dependent mechanisms, the effects of a potassium channel blocker, tetraethylammonium (TEA, CAS 56-34-8), and of a guanylate cyclase inhibitor, methylene blue (MB, CAS 61-73-4), on nipradilol-induced aortic relaxation were investigated and compared with those on nitroglycerin-induced aortic relaxation in isolated rat aortic rings. Relaxation response was expressed as percent relaxation, which is a percentage of the tension developed by 10(-7) mol/l norepinephrine. Nitroglycerin and nipradilol similarly relaxed the aortic ring in a concentration-dependent manner (10(-9)-10(-4) mol/l). In contrast, desnitronipradilol, a nipradilol analogue which has no nitroxy group, induced almost no aortic relaxation. TEA at 10(-3) mol/l, which is selective for calcium-activated potassium channels, inhibited the aortic relaxation induced by nipradilol (10(-5) mol/l) to a significantly greater extent than that induced by nitroglycerin (10(-5) mol/l) (% relaxation: 30.0 +/- 6.8 vs. 51.1 +/- 6.1%, p < 0.05). MB (10(-5) mol/l) suppressed the relaxation by nitroglycerin slightly but not significantly more than that by nipradilol. (% relaxation: 54.7 +/- 9.9 vs. 64.6 +/- 5.7%). The combination of TEA and MB almost completely eliminated the relaxation induced by nipradilol as well as by nitroglycerin. Thus, cyclic GMP-independent calcium activated potassium channels may be more involved in the aortic relaxation by nipradilol than that by nitroglycerin in rats.

Effect of exogenous nitric oxide on murine immune response induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide

Elevated nitric oxide (NO) has been associated with destructive periodontal disease. The aim of the present study was to test the hypothesis that exogenous NO may inhibit a protective immune response to Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) in a murine model. Mice of the BALB/c strain were sham immunized, immunized with A. actinomycetemcomitans LPS, treated with S-nitroso-N-acetyl penicillamine (SNAP; a NO donor) and immunized with A. actinomycetemcomitans LPS or treated with SNAP plus 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) and immunized with A. actinomycetemcomitans LPS. All animals were then challenged subcutaneously with viable A. actinomycetemcomitans. The serum-specific immunoglobulin G (IgG) subclasses and both interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) as well as splenic inducible nitric oxide synthase (iNOS) activity before and after bacterial challenge were assessed. The diameter of skin lesions was determined. Groups of mice were treated with l-N(6)-(1-iminoethyl)-lysine (l-NIL), an iNOS inhibitor, or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor, prior to injections with SNAP and/or A. actinomycetemcomitans LPS, and the skin lesions were assessed. Treatment with SNAP increased the iNOS activity, suppressed both serum-specific IgG2a and IFN-gamma levels, and delayed the healing of the lesions. These SNAP-induced immune alterations were restored by treatment with carboxy-PTIO. Pretreatment with l-NIL resulted in partial healing, whereas pretreatment with ODQ induced a delayed healing of the lesions. The present study suggests that exogenous NO may suppress a protective T helper 1-like murine immune response to A. actinomycetemcomitans LPS by an endogenous NO-independent but a cyclic GMP-dependent mechanism.

Inhibition of pacemaker currents by nitric oxide via activation of ATP-sensitive K+ channels in cultured interstitial cells of Cajal from the mouse small intestine

We investigated the role of nitric oxide (NO) in pacemaker activity and signal mechanisms in cultured interstitial cells of Cajal (ICC) of the mouse small intestine using whole cell patch-clamp techniques at 30 degrees C. ICC generated pacemaker potential in the current clamp mode and pacemaker currents at a holding potential of -70 mV. (+/-)-S-nitroso-N-acetylpenicillamine (SNAP; a NO donor) produced membrane hyperpolarization and inhibited the amplitude and frequency of the pacemaker currents, and increased resting currents in the outward direction. These effects were blocked by the use of glibenclamide (an ATP-sensitive K+ channel blocker), but not by the use of 5-hydroxydecanoic acid (a mitochondrial ATP-sensitive K+ channel blocker). Pretreatment with ODQ (a guanylate cyclase inhibitor) almost blocked the NO-induced effects. The use of cell-permeable 8-bromo-cyclic GMP also mimicked the action of SNAP. However, the use of KT-5823 (a protein kinase G inhibitor) did not block the NO-induced effects. Spontaneous [Ca2+]i oscillations in ICC were inhibited by the treatment of SNAP, as seen in recordings of intracellular Ca2+ ([Ca2+]i). These results suggest that NO inhibits pacemaker activity by the activation of ATP-sensitive K+ channels via a cyclic GMP dependent mechanism in ICC, and the activation of ATP-sensitive K+ channels mediates the inhibition of spontaneous [Ca2+]i oscillations.

Increased Apoptosis in First Trimester Extravillous Trophoblasts from Pregnancies at Higher Risk of Developing Preeclampsia

Preeclampsia complicates 5 to 10% of pregnancies and is a leading cause of maternal and fetal mortality and morbidity. Although the cause is unknown, inadequate invasion and remodeling of maternal uterine arteries by extravillous trophoblasts (EVTs) in the first trimester is a common feature. Uterine spiral artery resistance as detected by Doppler ultrasound is commonly used in the second trimester to identify pregnancies destined to develop preeclampsia. Correlation between high uterine resistance and the failure of trophoblast invasion has been reported as early as 12 weeks. However, the reason for this failure has not been established. Understanding the processes involved would significantly improve our diagnostic potential. In this study, we correlated increased first trimester uterine artery resistance with a biological abnormality in trophoblast function. EVTs derived from high-resistance pregnancies were more sensitive to apoptotic stimuli than those from normal-resistance pregnancies. Survival of EVTs from high-resistance pregnancies could be increased by nitric oxide, whereas inhibition of nitric oxide in cells from normal-resistance pregnancies increased apoptotic sensitivity. This predates the onset of symptoms by several weeks and provides evidence for a mechanism responsible for the incomplete uterine vessel remodeling and the differences in artery resistance between preeclamptic and normal pregnancies.

Nitric oxide blocks hKv1.5 channels by S-nitrosylation and by a cyclic GMP-dependent mechanism

This study was undertaken to analyze whether nitric oxide (NO) modulates the human potassium channel hKv1.5, which generates the ultrarapid delayed rectifier current (IKur) that determines the height and duration of atrial action potentials. Currents were recorded using the whole-cell patch-clamp in Ltk- cells expressing hKv1.5 channels. The NO donors (+/-)-S-Nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside, a NO solution, and l-Arginine inhibited hKv1.5 currents in a concentration-dependent manner. The NO concentration in the cell chamber was monitored using a sensor, and the IC50 for NO-induced hKv1.5 block was 340+/-70 nM. SNAP also inhibited the IKur recorded in mouse ventricular myocytes. The NO effects were partially mediated by the activation of the soluble guanylate cyclase (sGC)/cGMP/cGMP-dependent protein kinase (PKG) pathway. The biotin-switch assay demonstrated the presence of S-nitrosylated cysteines (Cys) on the hKv1.5 protein in SNAP-treated cells. Molecular modeling of hKv1.5 channel structure suggests that S-nitrosylation of Cys331 (segment 2, S2) and Cys346 (S2) would be stabilized by hydrogen bridge bonds with Ile262 (S1) and Arg342 (S2), respectively. NO inhibits the hKv1.5 current by a cGMP-dependent mechanism and by the S-nitrosylation of the hKv1.5 protein, an effect that contributes to shaping the human atrial action potentials.

Nitric oxide inhibits neutrophil migration by a mechanism dependent on ICAM-1: Role of soluble guanylate cyclase

In the present study, we addressed the role of intercellular adhesion molecule type 1 (ICAM-1/CD54) in neutrophil migration to inflammatory site and whether the inhibitory effect of nitric oxide (NO) upon the neutrophil rolling, adhesion and migration involves down-modulation of ICAM-1 expression through a cyclic GMP (cGMP) dependent mechanism. It was observed that neutrophil migration induced by intraperitoneal administration of endotoxin (LPS), carrageenan (Cg) or N-formyl peptide (fMLP) in ICAM-1 deficient (ICAM-1-/-) is similar to that observed in wild type (WT) mice. The treatment of mice with NO synthase (NOS) inhibitors, NG-nitro-l-arginine, aminoguanidine or with a soluble guanylate cyclase (sGC) inhibitor, ODQ enhanced LPS- or Cg-induced neutrophil migration, rolling and adhesion on venular endothelium. These parameters induced by LPS were also enhanced by 1400 W, a specific iNOS inhibitor, treatment. On the other hand, the treatment of the mice with S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, reduced these parameters induced by LPS or Cg by a mechanism sensitive to ODQ pretreatment. The NOS inhibitors did not enhance LPS-, Cg- or fMLP-induced migration and adhesion in ICAM-1-/- mice. Moreover, genetic (iNOS-/- mice) or pharmacological inhibition of NOS or of sGC enhanced LPS-induced ICAM-1 expression on mesenteric microcirculation vessels of WT mice. By contrast, SNAP reduced the ICAM-1 expression by a mechanism dependent on cGMP. In conclusion, the results suggest that although during inflammation, ICAM-1 does not contribute to neutrophil migration, it is necessary for the down-modulatory effect of inflammation-released NO on the adhesion and transmigration of neutrophils. Moreover, these NO effects are mediated via cGMP.

Megakaryocyte apoptosis: sorting out the signals

British Journal of Pharmacology (2005) 145, 271–273. doi:10.1038/sj.bjp.0706202

Urocortin-induced relaxation in the human internal mammary artery

Urocortin, a potent vasodilator, plays physiological or pathophysiological roles in the cardiovascular system. However, little is known about its action in human vascular tissues. The present study was designed to investigate the vascular effect of urocortin on human internal mammary artery (IMA) in vitro and the possible underlying mechanisms. Human IMA was obtained from patients undergoing coronary artery bypass grafting. The isolated IMA rings were mounted in organ baths and changes in isometric tension were measured by using Grass force-displacement transducer. Corticortropin-releasing factor-receptors (CRF-R) were also analyzed in the IMA by using RT-PCR analysis. In 9,11-dideoxy-11alpha,9alpha-epoxy-methanoprostaglandin F(2alpha) (U46619)-precontracted endothelium-intact rings, urocortin induced concentration-dependent relaxations with pD(2) of 8.69+/-0.11 and this effect was markedly reduced in endothelium-denuded rings. Relaxations to urocortin in endothelium-intact rings were attenuated to the same extent after treatment with N(G)-nitro-l-arginine (l-NNA) and 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one (ODQ). Urocortin-induced relaxations were also inhibited by treatment with putative K(+) channel blockers, such as tetraethylammonium (TEA(+)), charybdotoxin (CTX), and iberiotoxin (IBX). In endothelium-denuded rings, treatment with TEA(+), CTX, or IBX attenuated relaxation to urocortin as well as sodium nitroprusside (SNP). The bands for CRF-R1, CRF-R2alpha, and CRF-R2beta mRNAs were observed in both endothelium-intact and endothelium-denuded human IMA. Urocortin produced both endothelium-dependent and -independent relaxation in human IMA rings. The endothelium-dependent component primarily involves the release of endothelial nitric oxide (NO) that in turn stimulates Ca(2+)-activated K(+) channels in vascular smooth muscle via cyclic GMP-dependent mechanisms. CRF-R1, CRF-R2alpha, and CRF-R2beta mRNAs are present in the human IMA.

Nitric oxide protects human extravillous trophoblast cells from apoptosis by a cyclic GMP-dependent mechanism and independently of caspase 3 nitrosylation

Apoptosis is thought to play an important regulatory role in placental development and inappropriate trophoblast apoptosis has been implicated in complications of pregnancy such as pre-eclampsia. Here we show that apoptosis of a human extravillous trophoblast-derived cell line (SGHPL-4) can be regulated by nitric oxide (NO). Nitric oxide produced exogenously by the addition of NO donors was able to delay or inhibit apoptosis induced by a combination of tumour necrosis factor α and actinomycin D and to suppress the activity of caspase 3. Treatment with hepatocyte growth factor (HGF) stimulated expression of the inducible isoform of NO synthase and was also able to protect SGHPL-4 cells from caspase 3 activation and apoptosis. The inhibition of basal NO production with NO synthase inhibitors was shown to sensitise cells to apoptotic stimuli and to reduce the level of endogenous caspase 3 nitrosylation. The anti-apoptotic effects of NO in these extravillous trophoblast cells appear to be mediated through the production of cyclic GMP as inhibitors of soluble guanylate cyclase inhibited the protective effect of both HGF and NO donors.

Contribution of bradykinin and nitric oxide to AT2 receptor-mediated differentiation in PC12 W cells.

We investigated the effect of angiotensin II on intracellular cyclic GMP content and neurite outgrowth as an indicator of cell differentiation in PC12 W cells. Neurite outgrowth was examined by phase-contrast microscopy. Outgrown neurites were classified as small, medium and large, and were expressed as neurites per 100 cells. Angiotensin II (10-7 m) increased the outgrowth of medium and large neurites by mean +/- SEM 20.2 +/- 2.3 and 6.6 +/- 1.4 compared with 1.66 +/- 0.5 and 0.1 +/- 0.06 neurites per 100 cells in control. Cellular cyclic GMP content increased by 50-250% with angiotensin II at concentrations of 10-6-10-4 m. Both blockade of AT2 receptors and of nitric oxide synthase markedly reduced angiotensin II-induced neurite outgrowth and cyclic GMP production. In contrast, B2 receptor blockade had no effect or even increased these angiotensin II effects. Sodium nitroprusside and 8-bromo-cyclic GMP both mimicked the effects of angiotensin II on cell differentiation. The protein kinase G inhibitor KT-5823 inhibited the neurite outgrowth induced by both angiotensin II and 8-bromo-cyclic GMP. Our results demonstrate that angiotensin II can stimulate cell differentiation in PC12 W cells by nitric oxide-related and cyclic GMP-dependent mechanisms. The effects of angiotensin II on cell differentiation and cyclic GMP production were mediated via the AT2 receptor and further enhanced by bradykinin B2 receptor blockade.

Nebivolol: a selective β 1-adrenergic receptor antagonist that relaxes vascular smooth muscle by nitric oxide- and cyclic GMP-dependent mechanisms

Nebivolol is a highly selective β 1-adrenergic receptor antagonist that also possesses vasodilator properties that are attributed largely to nitric oxide (NO). The objective of the present study was to elucidate in more detail the mechanisms by which nebivolol relaxes vascular smooth muscle. In the canine species, nebivolol caused relaxation of isolated precontracted rings of coronary artery and pulmonary artery largely by endothelium-dependent, NO-dependent, and cyclic GMP-dependent mechanisms. Vasorelaxation was inhibited by N G-methylarginine, and this inhibition was reversed by addition of excess l-arginine. Moreover, the vasorelaxant responses to nebivolol were markedly inhibited by oxyhemoglobin, methylene blue, and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), whereas vasorelaxation was enhanced by zaprinast. Rat aortic ring preparations, however, relaxed in response to nebivolol by both endothelium-dependent and endothelium-independent mechanisms, both involving NO, and cyclic GMP. Endothelium-dependent and endothelium-independent vasorelaxation were inhibited by oxyhemoglobin, methylene blue, and ODQ. However, only endothelium-dependent vasorelaxation in response to nebivolol was inhibited by N G-methylarginine. Additional experiments ruled out other endothelium-independent vasorelaxant mechanisms. In conclusion, the vasodilator responses to nebivolol involve NO and cyclic GMP in both vascular endothelial and smooth muscle cells.

Effect of Nitric Oxide on Arachidonic Acid Release from Human Amnion-like WISH Cells

In order to clarify the possible interactions between nitric oxide (NO) and arachidonic acid (AA) pathways, human amnion-like WISH cells were perifused to measure the effects of the following substances on [3H]arachidonic acid release: (1) sodium nitroprusside (SNP), a nitric oxide donor; (2) 1,1,1-trifluoromethyl-6,9,12,15-heicosatetraen-2-one, a cytosolic phospholipase A2 (cPLA2) inhibitor; (3)l -arginine, the substrate of nitric oxide synthase (NOS); (4) 3-(5′-Hydroxymethyl-2′-furyl)-1-benzylindazole and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one, activator and inhibitor of soluble guanylyl cyclase, respectively; (5) a membrane-permeable non-hydrolyzable analogue of guanosine-3′,5′-cyclic monophosphate (cGMP). Furthermore, the effect of SNP on prostaglandin E2 (PGE2) release was tested. Exogenous and endogenous NO, as well as the guanylyl cyclase activator and cGMP analogue, significantly increased [3H]arachidonic acid release. Both soluble guanylyl cyclase and PLA2 inhibitors counteracted SNP response. Exogenous NO increased PGE2 release, although to a much lesser degree compared with arachidonic acid release.Our results indicate that NO stimulates AA release in WISH cells by activating PLA2 through a cyclic GMP-dependent mechanism.

Differential role of endothelium in hawthorn fruit extract-induced relaxation of rat cerebral, coronary, carotid, and aorta

The present study was aimed to examine the role of endothelium in the relaxant effect of hawthorn fruit extract of Crataegus pinnatifida in four different types of rat arteries, posterior cerebral communicating artery, right descending coronary artery, common carotid artery, and aorta. In <TEX>$9,11-dideoxy-11{\alpha}$</TEX>, <TEX>$9{\alpha}-epoxy-methanoprostaglandin$</TEX> <TEX>$F_{2{\alpha}}$</TEX> (U46619)-preconstricted arterial rings except for aorta, the extract produced endothelium-independent relaxations with similar potency. This relaxation was unaffected by pretreatment with <TEX>$100\;{\mu}M\;N^G-nitro-L-arginine$</TEX> methylester (L-NAME, the nitric oxide synthase inhibitor), <TEX>$3\;{\mu}M$</TEX> 1H-[l,2,4]oxadiazolo<TEX>$[4,2-{\alpha}]$</TEX>quinoxalin-1-one (ODQ, the guanylate cyclase inhibitor), or <TEX>$10\;{\mu}M$</TEX> indomethacin (the cyclooxygenase inhibitor). Putative <TEX>$K^+$</TEX> channel blockers (charybdotoxin plus apamin or glibenclamide) did not affect the extract-induced relaxation in cerebral or coronary artery rings. In contrast, in rat aortic rings the extract produced significantly smaller relaxant response in endothelium-denuded rings than that in endothelium-intact rings. Pretreatment with L-NAME or ODQ abolished the extractinduced endothelium-dependent aortic relaxation, whilst indomethacin <TEX>$(3\;{\mu}M)$</TEX> had no effect. The present results indicate that hawthorn fruit extract possesses a vasorelaxing effect in cerebral, coronary and carotid arteries and this effect is independent of the presence of a functional endothelium. However, the extract-induced endothelium-dependent relaxation in rat aorta was mediated through endothelial nitric oxide and cyclic GMP-dependent mechanisms, suggesting that active components in the extract may act on endothelium to stimulate release of nitric oxide in large conduit arteries of the rats.

Urocortin-induced endothelium-dependent relaxation of rat coronary artery: role of nitric oxide and K+ channels.

1. The mechanisms underlying the vasodilator response to urocortin are incompletely understood. The present study was designed to examine the role of endothelial nitric oxide and Ba(2+)-sensitive K(+) channels in the endothelium-dependent component of urocortin-induced relaxation in the rat left anterior descending coronary artery. 2. Urocortin induced both endothelium-dependent and -independent relaxation with respective pD(2) of 8.64+/-0.03 and 7.90+/-0.10. Removal of endothelium reduced the relaxing potency of urocortin. In rings pretreated with 10(-4) M N(G)-nitro-L-arginine methyl ester, 10(-5) M methylene blue or 10(-5) M ODQ, the urocortin-induced relaxation was similar to that observed in endothelium-denuded rings. L-Arginine (5x10(-4) M) antagonized the effect of N(G)-nitro-L-arginine methyl ester. 3. The relaxant response to urocortin was reduced in endothelium-intact rings preconstricted by 3.5x10(-2) M K(+) and abolished when extracellular K(+) was raised to 5x10(-2) M. Pretreatment with 10(-4) M BaCl(2) significantly inhibited urocortin-induced relaxation. Combined treatment with 10(-4) M BaCl(2) plus 10(-4) M N(G)-nitro-L-arginine methyl ester did not cause further inhibition. In urocortin (10(-8) M)-relaxed rings, BaCl(2) induced concentration-dependent reversal in vessel tone. Tertiapin-Q (10(-6) M) also attenuated urocortin-induced relaxation. In contrast, BaCl(2) did not alter urocortin-induced relaxation in endothelium-denuded rings. 4. In endothelium-denuded rings, hydroxylamine- and nitroprusside-induced relaxation was inhibited by 10(-4) M BaCl(2), but not by 10(-6) M tertiapin-Q. 5. The endothelium of the coronary artery was moderately stained with the antiserum against urocortin. 6. Taken together, the present results indicate that the urocortin-induced endothelium-dependent relaxation of rat coronary arteries is likely attributable to endothelial nitric oxide and subsequent activation of Ba(2+)- or tertiapin-Q-sensitive K(+) channels. The urocortin-induced endothelium-dependent relaxation appears to be mediated by cyclic GMP-dependent mechanisms.

Pharmacology and Physiology of Perivascular Nerves Regulating Vascular Function: Neurogenic Cerebral Vasodilation Mediated by Nitric Oxide

In cerebral arteries isolated from most of mammals, nerve stimulation produces relaxations in contrast to contractions in peripheral arteries. The relaxant mechanism is found to be non-adrenergic and non-cholinergic, but the neurotransmitter is not clarified until recently. Based on several functional and histological studies with isolated cerebral arteries, nitric oxide (NO) is now considered to be a neurotransmitter of the vasodilator nerve and the nerve has been called a nitroxidergic (nitrergic) nerve. Upon neural excitation, calcium influxed through N-type Ca2+ channels activates neuronal NO synthase, and then NO is produced by the enzyme from L-arginine. The released NO activates soluble guanylate cyclase in smooth muscle cells, resulting in relaxation with a cyclic GMP-dependent mechanism. The functional role and neuronal pathway have also been investigated in anesthetized dogs and Japanese monkeys. The nitroxidergic (nitrergic) nerves innervating the circulus arteriosus, including the anterior and middle cerebral and posterior communicating arteries, are found to be postganglionic nerves originated from the ipsilateral pterygopalatine ganglion and tonically dilate cerebral arteries in the resting condition. Our findings suggest that the nitroxidergic (nitrergic) nerve plays a physiologically important role to maintain a steady blood supply to the brain.

Nitric oxide regulates human eosinophil adhesion mechanisms in vitro by changing integrin expression and activity on the eosinophil cell surface.

1. The nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), inhibits both rat and human eosinophil chemotaxis in vitro. Here, the role of nitric oxide (NO) in human eosinophil cell surface integrin expression and function was investigated. 2. Human peripheral blood eosinophils were treated with L-NAME (0.01 - 1.0 mM) and their adhesion to human fibronectin and serum observed. Adhesion of cells to fibronectin and serum increased by 24.0+/-4.6 and 43.8+/-4.7%, respectively, when eosinophils were treated with 1.0 mM L-NAME. Increased adhesion by L-NAME could be abolished when cells were co-incubated with VLA-4- and Mac-1-specific monoclonal antibodies (mAbs). 3. The NO donor, sodium nitroprusside (2.5 mM), significantly inhibited eosinophil adhesion to fibronectin and serum by 34.3+/-4.5 and 45.2+/-5.6%, respectively. This inhibition was accompanied by a 4 fold increase in the levels of intracellular cyclic GMP. 4. Flow cytometrical analysis demonstrated that L-NAME induced an increased expression of CD11b (Mac-1) on the eosinophil cell surface of 36.3+/-7.4%. L-NAME had no effect upon CD49d (VLA-4) expression. 5. Treatment of human eosinophils, in vitro, with H-[1,2,4] oxadiazolo quinoxalin-1-one (ODQ) (0.1 mM), an inhibitor of soluble guanylate cyclase, also significantly increased eosinophil adhesion to fibronectin and serum by 73.5+/-17.9 and 91.7+/-12.9%, respectively. This increase in adhesion could also be inhibited by co-incubation with the Mac-1 and VLA-4-specific mAbs. 6. In conclusion, results indicate that NO, via a cyclic GMP-dependent mechanism, inhibits the adhesion of human eosinophils to the extracellular matrix (ECM). This inhibition is accompanied by a decrease in the expression and function of the eosinophil's adhesion molecules, in particular, the expression of the Mac-1 integrin and the function of the VLA-4 integrin.

Nitric Oxide Activates the Sarcolemmal KATPChannel in Normoxic and Chronically Hypoxic Hearts by a Cyclic GMP-dependent Mechanism

Chronic myocardial hypoxia results in elevated nitric oxide (NO) production and increased current through the sarcolemmal KATPchannel. We hypothesized these two processes are related and determined whether NO alters the electrophysiology of Purkinje fibers obtained from rabbits (n=12/group) raised in a normoxic (FIO2=0.21) or hypoxic (FIO2=0.12) environment from birth to 9 days of age. Action potential duration (APD)90was shorter (112±3 ms v 126±3 ms) and maximum diastolic potential (MDP) was more negative (−84±2 mV v−80±1 mV) in hypoxic hearts compared with normoxic controls. In normoxic hearts the NO donors, S-nitrosoglutathione (GSNO) 50 μM and spermine NONOate (50μM ) shortened APD90and increased MDP to levels present in chronically hypoxic hearts. This effect was completely abolished by the KATPchannel blocker glibenclamide (3 μM) and by a nitric oxide trap, Carboxy-PTIO (100 μM). The NO carrier glutathione (50 μM) and decomposed spermine NONOate had no effect on APD90or MDP. GSNO had no effect in hypoxic hearts; however, when GSNO was combined with glibenclamide APD90increased, and MDP decreased to normoxic values. 8-Bromo cGMP (100 μM) shortened APD90and increased MDP to levels present in chronically hypoxic hearts. This effect was abolished by glibenclamide. A soluble guanylyl cyclase inhibitor, ODQ (10 μM), had no effect on action potentials in normoxic hearts but in hypoxic hearts resulted in an increase in APD90to levels present in normoxic hearts and a decrease in MDP. The effect of ODQ could not be reversed by GSNO. We conclude that NO activates the sarcolemmal KATPchannel in normoxic and chronically hypoxic hearts by a cyclic GMP-dependent mechanism.

Nitric oxide attenuates angiotensin II-induced activation of p70 S6 kinase and protein synthesis in rat cardiomyocytes via cyclic GMP-dependent mechanisms Masafumi Kitakaze, Hiroshi Asanuma, Shoji Sanada, Masanori Asakura, Hisakazu Ogita, Yulin Liao, Koichi Node, Seiji Takashima, Tsunehiko Kuzuya, Masatsugu Hori. Osaka University, Osaka, Japan

Nitric oxide attenuates angiotensin II-induced activation of p70 S6 kinase and protein synthesis in rat cardiomyocytes via cyclic GMP-dependent mechanisms Masafumi Kitakaze, Hiroshi Asanuma, Shoji Sanada, Masanori Asakura, Hisakazu Ogita, Yulin Liao, Koichi Node, Seiji Takashima, Tsunehiko Kuzuya, Masatsugu Hori. Osaka University, Osaka, Japan

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

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