Nitric Oxide Donor Nitroprusside Research Articles

Phosphodiesterase-5 inhibitors tadalafil and sildenafil potentiate nitrergic-nerve mediated relaxations in the bladder vasculature

Recent studies suggest that lower urinary tract dysfunction may arise due to changes in local perfusion. Phosphodiesterase-5 inhibitors can improve urinary bladder blood flow, although the local mechanisms have not been fully elucidated. The aim was to pharmacologically characterise the vascular supply to the bladder and determine the mechanisms underlying the effects of the phosphodiesterase-5 inhibitors tadalafil and sildenafil. Responses of isolated rings of porcine superior vesical arteries to electrical field stimulation (EFS) were measured in the absence and presence of inhibitors of key neurotransmitter systems. Vasodilation responses to nitric oxide (NO) donors were also recorded, and the effects of phosphodiesterase-5 inhibitors on all responses determined. EFS caused biphasic responses with an initial vasoconstriction and a slower developing vasodilation. Vasoconstriction was mediated by ATP (55%) and noradrenaline (45%) release, whilst vasodilation was reduced by L-NNA (100 μM) (80%) and propranolol (1 μM) (20%). The nitrergic component was inhibited (81%) by L-NPA, a selective inhibitor of neuronal nitric oxide synthase (nNOS). Endothelial removal did not affect vasodilation. Tadalafil and sildenafil depressed noradrenaline-evoked vasoconstriction (by 26.8% and 35.5% respectively, P < 0.01), enhanced vasodilation to EFS (by 27.8% and 51.8% respectively, p < 0.01) and enhanced responses to NO donors nitroprusside, SIN-1, and SNAP, increasing pIC50 values (P < 0.01), without affecting maximal responses. In conclusion, neuronal NOS has a predominant role in regulating vascular tone of the porcine superior vesical artery and potentiation of nNO-mediated vasodilation is the primary mechanism underlying effects of phosphodiesterase-5 inhibitors in the bladder vasculature.

Lipopolysaccharide-induced inflammation increases nitric oxide production in taste buds

Inducible nitric oxide synthase (iNOS) is expressed when cells are induced or stimulated by proinflammatory cytokines and/or bacterial lipopolysaccharide (LPS). iNOS is a downstream gene of the NF-κB pathway. Our previous studies demonstrated that five Nfkb genes are expressed in mouse taste epithelium and taste organoids. However, it is unclear whether activation of the NF-κB pathway could induce iNOS gene expression and increase nitric oxide (NO) production in taste buds. In this study, we investigated the expression of iNOS mRNA and protein after LPS stimulation. Our results showed that a subset of taste bud cells and taste neurons express iNOS proteins after LPS stimulation. In addition, isolated mouse taste epithelium can release NO after exposure to LPS ex vivo. In taste behavioral tests, the NO donor nitroprusside enhanced mouse aversive responses to salty, bitter, and sour taste compounds. The enhanced aversive responses were especially strong for salty taste. In conclusion, our results suggest that iNOS and NO may play a role in the inflammation-associated taste disturbances.

Differential effects of long-term slow-pressor and subpressor angiotensin II on contractile and relaxant reactivity of resistance versus conductance arteries.

Vascular reactivity was evaluated in three separate arteries isolated from rats after angiotensin II (Ang II) was infused chronically in two separate experiments, one using a 14‐day high, slow‐pressor dose known to produce hypertension and the other using a 7‐day low, subpressor but hypertensive‐sensitizing dose. There were three new findings. First, there was no evidence of altered vascular reactivity in resistance arteries that might otherwise explain the hypertension due to the high Ang II or the hypertensive‐sensitizing effect of the low Ang II dose. Second, the high Ang II dose exerted a novel differential effect on arterial contractile responsiveness to the sympathetic neurotransmitter, norepinephrine, depending on the level of sympathetic innervation. It clearly enhanced that responsiveness in the sparsely innervated aorta but not in small mesenteric resistance arteries or the proximal (conductance) portion of the caudal artery, both of which are densely innervated. This suggests that the increased expression of alpha adrenergic receptors after long‐term exposure to Ang II as previously reported for aortic smooth muscle, is prevented in densely innervated arteries, likely due to long‐term Ang II‐mediated increase in sympathetic neural traffic to those vessels. Third, the same high dose of Ang II impaired aortic relaxation in response to the nitric oxide (NO) donor nitroprusside without impairing aortic endothelium‐dependent relaxation. NO is the main relaxing substance released by aortic endothelium. Accordingly, it is possible that this dose of Ang II is also associated with enhanced release of and/or enhanced smooth muscle responsiveness to other endothelial relaxing substances in a compensatory capacity.

Calcium and Calmodulin Are Involved in Nitric Oxide-Induced Adventitious Rooting of Cucumber under Simulated Osmotic Stress.

Osmotic stress is a major form of abiotic stress that adversely affects growth and development of plants and subsequently reduces yield and quality of crops. In this study, the effect of nitric oxide (NO) and calcium (Ca2+) on the process of adventitious rooting in cucumber (Cucumis sativus L.) under simulated osmotic stress was investigated. The results revealed that the effect of exogenous NO and Ca2+ in promoting the development of adventitious roots in cucumber seedlings under simulated osmotic stress was dose-dependent, with a maximal biological response at 10 μM NO donor nitroprusside (SNP) or 200 μM Ca2+. The application of Ca2+ chelators or channel inhibitors and calmodulin (CaM) antagonists significantly reversed NO-induced adventitious rooting, implying that endogenous Ca2+/CaM might be involved in NO-induced adventitious rooting under osmotic stress. Moreover, intracellular Ca amount was also increased by NO in cucumber hypocotyls during the development of adventitious roots under osmotic stress. This increase of endogenous Ca2+ was inhibited by NO specific scavenger 2-(4-carboxyphenyl) -4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), nitrate reductase inhibitors tungstate (Na2WO4) and sodium azide (NaN3). This gives an indication that Ca2+ might be a downstream signaling molecule in the adventitious root development by NO under osmotic condition. The results also show that NO or Ca2+ play a positive role in improving plant water status and photosynthetic system by increasing chlorophyll content and photochemical activity in leaves. Furthermore, NO and Ca2+ treatment might alleviate the negative effects of osmotic stress by decreasing membrane damage and reactive oxygen species (ROS) production by enhancing the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). Therefore, Ca2+/CaM may act as a downstream signaling molecule in NO-induced development of adventitious root under simulated osmotic stress through improving the photosynthetic performance of leaves and activating antioxidative system in plants.

Associations of the alternative pathway and classical pathway of complement activation with incident metabolic syndrome: The CODAM study

Inducible nitric oxide synthase (iNOS) is expressed when cells are induced or stimulated by proinflammatory cytokines and/or bacterial lipopolysaccharide (LPS). iNOS is a downstream gene of the NF-κB pathway. Our previous studies demonstrated that five Nfkb genes are expressed in mouse taste epithelium and taste organoids. However, it is unclear whether activation of the NF-κB pathway could induce iNOS gene expression and increase nitric oxide (NO) production in taste buds. In this study, we investigated the expression of iNOS mRNA and protein after LPS stimulation. Our results showed that a subset of taste bud cells and taste neurons express iNOS proteins after LPS stimulation. In addition, isolated mouse taste epithelium can release NO after exposure to LPS ex vivo. In taste behavioral tests, the NO donor nitroprusside enhanced mouse aversive responses to salty, bitter, and sour taste compounds. The enhanced aversive responses were especially strong for salty taste. In conclusion, our results suggest that iNOS and NO may play a role in the inflammation-associated taste disturbances.

Anidulafungin-Induced Suicidal Erythrocyte Death

Background/Aims: The novel antifungal drug Anidulafungin is used for the treatment of diverse fungal infections including candidiasis and aspergillosis. The traditional antifungal drug amphotericin B has previously been shown to trigger eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, ceramide, activated protein kinase C (PKC), casein kinase 1α or p38 kinase and activated caspases. Inhibitors of eryptosis include nitric oxide (NO). The present study explored, whether Anidulafungin induces eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was quantified by measuring haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Anidulafungin (1.5 - 6 µg/ml) significantly increased hemolysis and the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Anidulafungin (6 µg/ml) slightly, but significantly inceased Fluo3-fluorescence and the effect of Anidulafungin on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca²⁺. The effect of Anidulafungin on annexin-V-binding was further significantly blunted by the p38 kinase inhibitor SB203580 (2 µM) and NO donor nitroprusside (1 µM). An increase of extracellular K⁺ concentration significantly blunted the effect of Anidulafungin on cell volume but not on annexin-V-binding. Anidulafungin rather decreased DCFDA fluorescence and the effect of Anidulafungin on annexin-V-binding was not significantly blunted by the antioxidant N-acetylcysteine (1 mM). Moreover, the effect of Anidulafungin on annexin-V-binding was not paralleled by significant increase of ceramide abundance and was not significantly blunted by PKC inhibitor staurosporine (1 µM), casein kinase 1α inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM). Conclusions: Anidulafungin triggers hemolysis and eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to Ca²⁺ entry and activation of p38 kinase.

Change in vascular smooth muscle response to 5-HT due to short- or long-term endothelial denudation of the bovine digital vein

Several chronic progressive vascular diseases, such as laminitis, show vasocontractile dysfunction that might evolve into reperfusion injury and/or vessel structural remodelling, which may be traced back to aberrant endothelial function. In the present study, the vasomotor responses of bovine digital veins (BDVs) to 5-hydroxytryptamine (5-HT) were investigated in blood vessels, with and without endothelium present, and in samples deprived of endothelium before or after overnight incubation in tissue culture medium, to evaluate the effects of short- and long-term endothelial damage on vascular smooth muscle (VSM) reactivity. No significant effects were observed in the blood vessels tested immediately after the removal of endothelium. In contrast, a significant increase in VSM reactivity to 5-HT was seen in vessels incubated without endothelium. This long-term change in smooth muscle reactivity was prevented by exposure to the nitric oxide (NO) donor nitroprusside (P < 0.01), suggesting that the long-term lack of inhibitory control exerted by endothelium-derived NO is involved in increased VSM reactivity. The RhoA/ROCK pathway inhibitor fasudil reduced VSM hyper-contractility to ~65% (P < 0.001), the superoxide dismutase-mimetic tempol normalised the vascular response and the non-selective COX-inhibitor indomethacin exerted a moderate inhibitory effect (P < 0.05). Thus, over-activation of the RhoA/ROCK pathway and production of reactive oxygen species could account for VSM hyper-reactivity, triggered by long-term endothelium-deprivation in BDVs, suggesting that these biochemical mechanisms are potential targets for controlling the progressive vasocontractile dysfunction of digital veins in animals affected with laminitis.

Abstract 16720: Cytoglobin Regulates Cardiovascular Function and Vessel Tone by Controlling the Rate of Vascular Nitric Oxide Metabolism

Cytoglobin (Cygb) can effectively metabolize nitric oxide (NO), a potent vasodilator, in the presence of oxygen and reductants. Cygb in the vascular wall may affect cardiovascular functions by changing the rate of NO metabolism. In this study, we directly tested the vascular role of Cygb using Cygb knockout (Cygb-/-) mice. The mean blood pressure of Cygb-/- and C57BL/6 wild type (WT) mice was 65.3 ± 1.9 mmHg and 93.7 ± 1.5 mmHg, respectively (n=10). Using echocardiography, we observed that cardiac output (CO) was increased in Cygb-/- mice compared to WT with values of 29.8 ± 3.9 vs 17.7 ± 0.9 ml/min. The systemic vascular resistance (SVR) of Cygb-/- mice was decreased by ~60% vs that of WT mice (Fig. 1). Further, the inner diameter (id) of aorta of Cygb-/- mice was dilated compared to WT with values of 2.2 ± 0.1 mm vs 1.5 ± 0.05 mm (n=5), respectively. After treatment with the NO synthase inhibitor L-NAME, no difference in the aortic id remained between Cygb-/- (1.55 ± 0.03 mm) and WT (1.49 ± 0.02 mm) mice, indicating that the NO pathway is responsible for the difference in vascular inner diameters and tone. Myograph experiments show that the aortic vasodilation response of Cygb-/- mice is much more sensitive to acetylcholine (Ach) or the NO donor nitroprusside (SNP) (EC50 shifts from 13 nM and 2.9 nM (WT mice) to 0.33 nM and 0.16 nM (Cygb-/-) for Ach and SNP, respectively). Using NO electrodes to measure the rate of NO consumption by SMCs and quantitative imunoblotting to estimate Cygb content in RSMCs-AR and Cygb knockdown RSMCs, we observed that 90% of NO consumption by RSMCs-AR is caused by the intracellular Cygb. Our results indicate that Cygb deficiency in the vascular wall of Cygb-/- mice greatly reduces the rate of NO metabolism and increases vascular NO concentration, resulting in vasodilation, increase in vessel lumen diameter, and decrease in SVR. These results demonstrate that Cygb regulates cardiac function and vessel tone by controlling the rate of vascular NO metabolism.

Contribution of nitric oxide synthase isoforms to cholinergic vasodilation in murine retinal arterioles

Nitric oxide synthases (NOSs) are critically involved in regulation of ocular perfusion. However, the contribution of the individual NOS isoforms to vascular responses is unknown in the retina. Because some previous findings suggested an involvement of inducible nitric oxide synthase (iNOS) in the regulation of retinal vascular tone, a major goal of the present study was to examine the hypothesis that iNOS is involved in mediating cholinergic vasodilation responses of murine retinal arterioles. Another subject of this study was to test the contribution of the other two NOS isoforms, neuronal (nNOS) and endothelial NOS (eNOS), to cholinergic retinal arteriole responses. Expression of individual NOS isoforms was determined in murine retinal arterioles using real-time PCR. All three NOS isoforms were expressed in retinal arterioles. However, eNOS mRNA was found to be most, and iNOS mRNA least abundant. To examine the functional relevance of iNOS for mediating vascular responses, retinal vascular preparations from gene-targeted iNOS-deficient mice (iNOS−/−) and wild-type mice were studied in vitro. Changes in luminal vessel diameter in response to the thromboxane mimetic 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619), the endothelium-dependent vasodilator acetylcholine, and the nitric oxide donor nitroprusside were measured by video microscopy. To determine the contribution of individual NOS isoforms to cholinergic vasodilation responses, retinas from iNOS−/− and wild-type mice were incubated with Nω-nitro-l-arginine methyl ester (l-NAME), a non-isoform-selective inhibitor of NOS, 7-nitroindazole, a selective nNOS blocker and aminoguanidine, a selective iNOS inhibitor. U-46619 evoked concentration-dependent vasoconstriction that was similar in retinal arterioles from iNOS−/− and wild-type mice. In retinal arterioles preconstricted with U-46619, acetylcholine and nitroprusside produced dose-dependent dilation that did not differ between iNOS−/− and wild-type mice. Remarkably, in both genotypes, vasodilation to acetylcholine was negligible after incubation with l-NAME. In contrast, pharmacological inhibition of nNOS and iNOS had no effect on acetylcholine-induced vasodilation. These findings suggest that dilation of murine retinal arterioles to acetylcholine is mediated predominantly by eNOS.

Identification of the Muscarinic Acetylcholine Receptor Subtype Mediating Cholinergic Vasodilation in Murine Retinal Arterioles

To identify the muscarinic acetylcholine receptor subtype that mediates cholinergic vasodilation in murine retinal arterioles. Muscarinic receptor gene expression was determined in murine retinal arterioles using real-time PCR. To assess the functional relevance of muscarinic receptors for mediating vascular responses, retinal vascular preparations from muscarinic receptor-deficient mice were studied in vitro. Changes in luminal arteriole diameter in response to muscarinic and nonmuscarinic vasoactive substances were measured by video microscopy. Only mRNA for the M(3) receptor was detected in retinal arterioles. Thus, M(3) receptor-deficient mice (M3R(-/-)) and respective wild-type controls were used for functional studies. Acetylcholine concentration-dependently dilated retinal arterioles from wild-type mice. In contrast, vasodilation to acetylcholine was almost completely abolished in retinal arterioles from M3R(-/-) mice, whereas responses to the nitric oxide (NO) donor nitroprusside were retained. Carbachol, an acetylcholinesterase-resistant analog of acetylcholine, also evoked dilation in retinal arterioles from wild-type, but not from M3R(-/-), mice. Vasodilation responses from wild-type mice to acetylcholine were negligible after incubation with the non-subtype-selective muscarinic receptor blocker atropine or the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester, and were even reversed to contraction after endothelial damage with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. These findings provide evidence that endothelial M(3) receptors mediate cholinergic vasodilation in murine retinal arterioles via activation of NO synthase.

Participation of receptors of the NMDA type in regulation by glutamate of alimentary motor program of the freshwater mollusc Lymnaea stagnalis

A possible participation of receptors of the NMDA type in regulation by glutamate of the Lymnaea stagnalis feeding program was studied in electrophysiological experiments. The specific antagonist of receptors of the N-methyl-D-aspartate (NMDA) type MK-801 has been shown to turn off the endogenous generation of the standard three-phase rhythm or the two-phase rhythm. Stimulation of receptors of this type by their specific agonist, NMDA, on the contrary, increased frequency of the alimentary rhythm and transformed it into the two-phase one. All NMDA effects are eliminated by MK-801. Apart from action on generation of central feeding rhythms, ligands of receptors of the NMDA type changes the tonical level of depolarization and activity of the alimentary circuit motoneurons. MK-801 decreased the initial level of the motoneuron B4 activity and inhibited the excitatory effect both of NMDA and of glutamate itself. There are also obtained data in favor of that earlier reported effect of transformation of the inhibitory response of neurons B4 to glutamate into the excitatory one at action of nitric oxide (NO) donors can be mediated by the specific NO effect on the activity of receptors of the NMDA type. The blocker of NMDA receptors MK-801 has been shown to inhibit the effect of transformation of the response to glutamate. The NO donor nitroprusside enhanced essentially the NMDA excitatory action, while the NO acceptor oral PTIO decreased it. The results obtained with use of ODQ, the blocker of NO-sensitive guanylyl cyclase (GC), allow thinking that effect of NO on activity of the NMDA receptors of the pond snail feeding program can be realized through the metabolic pathway GC--cGMP. On the whole, the obtained results show the pond snail receptors of the NMDA type to participate in generation and rearragement of rhythmical alimentary programs in the tonical excitatory effect on the feeding program motoneurons in the NO-dependent transformation of the glutamate response.

Letter by Brown and Pretorius Regarding Article, “Effect of Sulfaphenazole on Tissue Plasminogen Activator Release in Normotensive Subjects and Hypertensive Patients”

HomeCirculationVol. 120, No. 19Letter by Brown and Pretorius Regarding Article, “Effect of Sulfaphenazole on Tissue Plasminogen Activator Release in Normotensive Subjects and Hypertensive Patients” Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBLetter by Brown and Pretorius Regarding Article, “Effect of Sulfaphenazole on Tissue Plasminogen Activator Release in Normotensive Subjects and Hypertensive Patients” Nancy J. Brown Mias Pretorius Nancy J. BrownNancy J. Brown Vanderbilt University School of Medicine, Division of Clinical Pharmacology, Department of Medicine, Nashville, TN Search for more papers by this author Mias PretoriusMias Pretorius Vanderbilt University School of Medicine, Department of Anesthesia, Nashville, TN Search for more papers by this author Originally published10 Nov 2009https://doi.org/10.1161/CIRCULATIONAHA.109.874743Circulation. 2009;120:e159To the Editor:We read with interest the article by Giannarelli et al regarding the effect of sulfaphenazole on tissue plasminogen activator release. 1 As always, the authors’ experimental design is elegant. However, it appears that an important error occurred in the calculation of net tissue plasminogen activator (t-PA) release.The authors report on the t-PA balance. On the basis of the units and Methods, this seems to be synonymous with net release, defined in the Methods as=(Cv−Ca)×[FBF×101−hct/100], where Cv and Ca are the venous and arterial concentrations. However, the units for concentrations provided in Table 2 are also expressed as ng · min−1×100 mL forearm tissue−1. Concentrations should be expressed as ng/mL. Did the authors multiply concentration by flow in Table 2, as well? If so, the presented data may disproportionately reflect changes in flow rather than in true net release.At the same time, the t-PA balance calculated during bradykinin seems to be 20- to 30-fold lower than that reported by others at comparable doses. The investigators gave bradykinin at a dose of 0.015 μg · 100 mL−1 · min−1. This dose would be equivalent to 225 ng/min in a man with a forearm volume of 1500 mL. Other investigators report a t-PA release of 20 to 80 ng · min−1 · 100 mL−1 at similar bradykinin doses.The effect of the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) on stimulated t-PA release is a bit confusing. L-NMMA decreased t-PA release, suggesting that NO increases t-PA release. If this were the case, the NO donor nitroprusside should also stimulate t-PA release. The authors report that sodium nitroprusside does not affect t-PA release. How do the authors reconcile these discrepant findings? DeSouza and colleagues suggest that NO decreases bradykinin-stimulated t-PA release.2 Because the authors infused sodium nitroprusside concurrently with L-NMMA, is it possible that the effect attributed to L-NMMA was really an effect of nitroprusside?Sources of FundingThis work was supported by National Institutes of Health grants HL065193, HL060906, and HL085740.DisclosuresDr Brown receives grant support from Forest Pharmaceuticals, which manufactures nebivolol. References 1 Giannarelli C, Virdis A, De Negri F, De NF, Magagna A, Duranti E, Salvetti A, Taddei S. Effect of sulfaphenazole on tissue plasminogen activator release in normotensive subjects and hypertensive patients. Circulation. 2009; 119: 1625–1633.LinkGoogle Scholar2 Smith DT, Hoetzer GL, Greiner JJ, Stauffer BL, DeSouza CA. Endothelial release of tissue-type plasminogen activator in the human forearm: role of nitric oxide. J Cardiovasc Pharmacol. 2003; 42: 311–314.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails November 10, 2009Vol 120, Issue 19 Advertisement Article InformationMetrics https://doi.org/10.1161/CIRCULATIONAHA.109.874743PMID: 19901200 Originally publishedNovember 10, 2009 PDF download Advertisement SubjectsClinical StudiesEndothelium/Vascular Type/Nitric OxideThrombosis

Modification of the effects of glutamate by nitric oxide (NO) in a pattern-generating network

Previous studies have shown that nitric oxide (NO) transforms the responses of various neurons to glutamate, though it remained unclear whether this mechanism is involved in the formation of behavior. We therefore studied the buccal generator of the feeding rhythm of the mollusk Lymnaea stagnalis (pond snail). In this organism, glutamate and NO are synthesized by defined neurons; glutamate is the neurotransmitter for the second phase of the standard triphasic feeding rhythm. Motoneuron B4 was used for monitoring. Studies using isolated CNS preparations showed that in some cases glutamate evoked hyperpolarization of B4 and terminated rhythmic network activity (n=17; group 1), while in other cases glutamate evoked depolarization of B4 and activation of a non-standard biphasic rhythm (n=12; group 2). In group 1, the NO donor nitroprusside lifted the inhibitory effect of glutamate (n=13), with transformation into an excitatory effect in nine cases. In group 2, the NO acceptor PTIO transformed the excitatory effect of glutamate into an inhibitory effect (n=7). These results provide evidence that: 1) the responses of the central generator of the buccal motor rhythm to glutamate depend on the NO level, and 2) this regulatory mechanism can modify feeding behavior.

Acute retinal ischemia inhibits endothelium‐dependent nitric oxide‐mediated dilation of retinal arterioles: role of endothelin‐1 and superoxide

Increases in endothelin‐1 (ET‐1) and oxidative stress are associated with retinal vascular disease. Herein, we assessed whether acute retinal ischemia increases local ET‐1 and superoxide (O2−), and alters nitric oxide (NO)‐mediated dilation of retinal arterioles. Retinal ischemia was established by elevating intraocular pressure (IOP) to 90 mmHg for 30 or 90 min via infusion of saline into the anterior chamber of the porcine eye. Following ischemia, retinal arterioles from control and ischemic eyes were isolated and pressurized without flow for in vitro study. Dilation to endothelium‐dependent NO‐mediated agonist bradykinin but not endothelium‐independent NO donor nitroprusside was significantly reduced in arterioles following only 90‐min ischemia. Dihydroethidium staining and ET‐1 ELISA revealed increase of O2− inarterioles and ET‐1 in the vitreous after ischemia. Pre‐ischemia intravitreal treatment with O2− scavenger TEMPOL or ET‐converting enzyme inhibitor phosphoramidon prevented elevation of O2− and preserved dilation to bradykinin. Phosphoramidon but not TEMPOL reduced vitreous ET‐1. Our data show that acute retinal ischemia inhibits endothelium‐dependent NO‐mediated dilation of retinal arterioles. The impairment is mediated by elevation of local ET‐l and subsequent NO inactivation via increased vascular O2−. Support: Retina Research Foundation and Scott &amp; White Research Foundation.

Hypotensive effect of hydroxylamine, an endogenous nitric oxide donor and SSAO inhibitor

The endogenous compound hydroxylamine relaxes vascular smooth muscle in vitro, apparently through conversion to the vasodilator factor nitric oxide, but its effect on blood pressure has not been characterized. We found that in the anesthetized rat the amine elicits dose-related hypotension when administered by continuous iv infusion. In experiments designed to explore the mechanism of this effect, hydroxylamine was compared with the nitric oxide donor nitroprusside and the direct-acting vasodilator hydralazine, using pretreatments known to modify diverse mechanisms of vasodilation. Hydroxylamine hypotension was enhanced by the SSAO inhibitor isoniazid and the SSAO substrate methylamine, a pattern shared by hydralazine. Responses were blocked by the guanylate cyclase inhibitor methylene blue and were increased by the nitric oxide synthase inhibitor L-NAME, a pattern shared by nitroprusside. It was concluded that hydroxylamine exerts hypotension partly through conversion to nitric oxide and partly by a "hydralazine-like" mechanism involving SSAO inhibition.

Effects of the Nitric Oxide Donor Nitroprusside on No-Reflow Phenomenon During Coronary Interventions for Acute Myocardial Infarction

We tested the effects of the nitric oxide donor nitroprusside as treatment for no reflow in 23 consecutive patients who underwent coronary angioplasty for acute myocardial infarction. No reflow was defined as a decrease of >/=1 Thrombolysis In Myocardial Infarction (TIMI) trial flow grade occurring after successful initial coronary recanalization. Nitroprusside induced a significant improvement in coronary flow, with an increase in TIMI flow grade from 1.5 +/- 0.8 to 2.9 +/- 0.3 (p <0.0001) and in TIMI frame count from 46 +/- 25 to 16 +/- 5 (p <0.0001). There were no significant adverse effects apart from transient hypotension. Intracoronary nitroprusside should be considered as a treatment of no reflow occurring in acute myocardial infarction.

Characterization of 5-HT7-receptor-mediated gastric relaxation in conscious dogs

We aimed to evaluate the gastric relaxant capacity of the 5-HT(1/7)-receptor agonist 5-carboxamidotryptamine (5-CT) in conscious dogs and to clarify the mechanism of action by use of selective antagonists, vagotomy, and in vitro experiments. A barostat enabled us to monitor the intragastric volume in response to different treatments (intravenously administered) before and after supradiaphragmatic vagotomy [results presented as the maximum volume change after treatment (mean; n = 5-11)]. In vitro experiments were performed with isolated muscle strips cut from four different stomach regions of the vagotomized dogs [results were fitted to the operational model of agonism to determine the efficacy parameter tau (n = 5)]. 5-CT (0.5-10 microg/kg) caused a dose-dependent gastric relaxation (29-267 ml) that was completely blocked by the selective 5-HT(7)-receptor antagonist SB-269970 (50 microg/kg). After vagotomy, the relaxation to 10 microg/kg 5-CT was significantly less pronounced (73 vs. 267 ml; P < 0.05) but still blocked by SB-269970, whereas the response to the nitric oxide donor nitroprusside was similar to that before vagotomy (178 vs. 218 ml). In vitro, 5-CT concentration dependently inhibited the PGF(2alpha)-contracted muscle strips before and after vagotomy. Although before and after vagotomy the response in every region was mediated by 5-HT(7) receptors (apparent affinity dissociation constant: SB-269970, 8.2-8.6 vs. 8.3-8.6, respectively), the response after vagotomy was less efficacious (log tau: 1.9 to 0.5 vs. 1.4 to -0.1). The results indicate that the 5-CT-induced proximal stomach relaxation in conscious dogs before and after vagotomy is mediated via 5-HT(7) receptors. The decreased efficacy of 5-CT in vitro after vagotomy is probably related to vagotomy-induced changes in receptor density or coupling efficiency and provides a possible explanation for the decreased in vivo response to 5-CT after vagotomy.

α‐Adrenoceptor constrictor responses and their modulation in slow‐twitch and fast‐twitch mouse skeletal muscle

Vasoconstrictor responses to sympathetic nerve stimulation and their sensitivity to metabolic modulation reportedly differ in fast-twitch and slow-twitch muscles, but the underlying mechanisms are not known. Both alpha(1)- and alpha(2)-adrenoceptors mediate these vascular responses in fast-twitch muscle, while their roles in slow-twitch muscle are less well defined. In this study, the phosphorylation of smooth muscle myosin regulatory light chain (smRLC) was measured as an index of vasoconstriction in slow-twitch soleus muscles and fast-twitch extensor digitorum longus (EDL) muscles isolated from C57BL/6J mice. In soleus muscles, incubation with phenylephrine (PE) or UK 14,304 to selectively activate alpha(1)- or alpha(2)-adrenoceptors resulted in concentration-dependent increases in smRLC phosphorylation. To evaluate metabolic modulation of these responses, vasodilator pathways previously implicated in such modulation in fast-twitch muscle were activated in soleus muscles by treatment with the nitric oxide (NO) donor nitroprusside or the ATP-sensitive potassium (K(ATP)) channel opener cromakalim. Both drugs inhibited responses to UK 14,304, but not to PE. The effect of nitroprusside to antagonize UK 14,304 responses was prevented by inhibition of guanylyl cyclase or by blockade of K(ATP) channels, but not by blockade of other potassium channels. Results were similar in EDL muscles. These data provide the first evidence for alpha(2)-adrenoceptor-mediated constriction in slow-twitch muscle, and show that it is sensitive to modulation by NO via a cGMP-dependent mechanism that requires K(ATP) channel activation. Based on the similar findings in soleus and EDL muscles, fibre type does not appear to determine the innate vascular response to alpha(1)- or alpha(2)-adrenoceptor activation.

NO and NO-independent mechanisms mediate ETBreceptor buffering of ET-1-induced renal vasoconstriction in the rat

Vascular endothelin (ET) type B (ET(B)) receptors exert dilator and constrictor actions in a complex interaction with ET(A) receptors. We aimed to clarify the presence and relative importance of nitric oxide (NO) and other mechanisms underlying the dilator effects of ET(B) receptors in rat kidneys. Complete inhibition of NO production with Nomega-nitro-L-arginine methyl ester (L-NAME, 25 mg/kg iv) enhanced the renal vasoconstriction elicited by ET-1 injected into the renal artery from -15 to -30%. Additional infusion of the NO donor nitroprusside (NP) into the renal artery did not reverse this effect (-29%) but effectively buffered ANG II-mediated vasoconstriction. Similarly, ET-1 responses were enhanced after a smaller intrarenal dose of L-NAME (-22 vs. -15%) and were unaffected by subsequent NP infusion (-21%). These results indicate that the responsiveness to ET-1 is buffered by ET(B) receptor-stimulated phasic release of NO, rather than its static mean level. Infusion of the ET(B) receptor antagonist BQ-788 into the renal artery further enhanced the ET-1 constrictor response to NP+L-NAME (-92 vs. -49%), revealing an NO-independent dilator component. In controls, vasoconstriction to ET-1 was unaffected by vehicle (-27 vs. -20%) and markedly enhanced by BQ-788 (-70%). The same pattern was observed when indomethacin (Indo) was used to inhibit cyclooxygenase (-20% for control, -22% with Indo, and -56% with ET(B) antagonist) or methylsulfonyl-6-(2-propargyloxyphenyl)-hexanamide (MS-PPOH) or miconazole+Indo was used to inhibit epoxygenase alone (-10% for control, -11% with MS-PPOH, and -35% with ET(B) antagonist) or in combination (-14% for control, -20% with Indo + miconazole, and -43% with ET(B) antagonist). We conclude that phasic release of NO, but not its static level, mediates part of the dilator effect of ET(B) receptors and that an NO-independent mechanism, distinct from prostanoids and epoxyeicosatetraenoic acids, perhaps ET(B) receptor clearance of ET-1, plays a major buffering role.

Nitroprusside Increases Gene Transfer Associated with Intracoronary Delivery of Adenovirus

Efficient gene transfer by vectors that can be easily delivered to target organs is desirable in clinical gene therapy. We tested the hypothesis that intracoronary infusion of the nitric oxide donor nitroprusside would increase the efficiency of adenovirus vector-mediated gene transfer to the heart. Intracoronary delivery of an adenovirus encoding murine adenylyl cyclase type VI (Ad.AC(VI)) was performed in adult pigs with and without simultaneous intracoronary infusion of nitroprusside. Animals were killed 12-14 days after Ad.AC(VI) delivery and myocardial adenylyl cyclase activity was measured. Addition of nitroprusside during intracoronary infusion of Ad.AC(VI) was associated with a 4-fold increase in cAMP-generating capacity in the left ventricle. Transgene expression was confirmed by immunoblotting. Intracoronary nitroprusside produced mild dose-dependent changes in blood pressure and heart rate during infusion. Intracoronary nitroprusside infusion is a safe and effective means to increase the extent of cardiac gene transfer with intracoronary delivery of adenovirus vectors.