ATP mediates flow-induced NO production in thick ascending limbs

Mechanical stimulation caused by increasing flow induces nucleotide release from many cells. Luminal flow and extracellular ATP stimulate production of nitric oxide (NO) in thick ascending limbs. However, the factors that mediate flow-induced NO production are unknown. We hypothesized that luminal flow stimulates thick ascending limb NO production via ATP. We measured NO in isolated, perfused rat thick ascending limbs using the fluorescent dye DAF FM. The rate of increase in dye fluorescence reflects NO accumulation. Increasing luminal flow from 0 to 20 nl/min stimulated NO production from 17 ± 16 to 130 ± 37 arbitrary units (AU)/min (P < 0.02). Increasing flow from 0 to 20 nl/min raised ATP release from 4 ± 1 to 21 ± 6 AU/min (P < 0.04). Hexokinase (10 U/ml) plus glucose, which consumes ATP, completely prevented the measured increase in ATP. Luminal flow did not increase NO production in the presence of luminal and basolateral hexokinase (10 U/ml). When flow was increased with the ATPase apyrase in both luminal and basolateral solutions (5 U/ml), NO levels did not change significantly. The P2 receptor antagonist suramin (300 μmol/l) reduced flow-induced NO production by 83 ± 25% (P < 0.03) when added to both and basolateral sides. Luminal hexokinase decreased flow-induced NO production from 205.6 ± 85.6 to 36.6 ± 118.6 AU/min (P < 0.02). Basolateral hexokinase also reduced flow-induced NO production. The P2X receptor-selective antagonist NF023 (200 μmol/l) prevented flow-induced NO production when added to the basolateral side but not the luminal side. We conclude that ATP mediates flow-induced NO production in the thick ascending limb likely via activation of P2Y receptors in the luminal and P2X receptors in the basolateral membrane.     

Genetic engineering of Escherichia coli for production of tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is an essential cofactor for various enzymes in mammals. In vivo, it is synthesized from GTP via the three-step pathway of GTP cyclohydrolase I (GCHI), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SPR). BH4 is a medicine used to treat atypical hyperphenylalaninemia. It is currently synthesized by chemical means, which consists of many steps, and requires costly materials and complicated procedures. To explore an alternative microbial method for BH4 production, we utilized recombinant DNA technology to construct recombinant Escherichia coli (E. coli) strains carrying genes expressing GCHI, PTPS and SPR enzymes. These strains successfully produced BH4, which was detected as dihydrobiopterin and biopterin, oxidation products of BH4. In order to increase BH4 productivity we made further improvements. First, to increase the de novo GTP supply, an 8-azaguanine resistant mutant was isolated and an additional guaBA operon was introduced. Second, to augment the activity of GCHI, the folE gene from E. coli was replaced by the mtrA gene from Bacillus subtilis. These modifications provided us with a strain showing significantly higher productivity, up to 4.0 g of biopterin/L of culture broth. The results suggest the possibility of commercial BH4 production by our method.     

NAD(P)H oxidase-derived peroxide mediates elevated basal and impaired flow-induced NO production in SHR mesenteric arteries in vivo

Nitric oxide (NO) and reactive oxygen species (ROS) have fundamentally important roles in the regulation of vascular tone and remodeling. Although arterial disease and endothelial dysfunction alter NO and ROS levels to impact vasodilation and vascular structure, direct measurements of these reactive species under in vivo conditions with flow alterations are unavailable. In this study, in vivo measurements of NO and H2O2 were made on mesenteric arteries to determine whether antioxidant therapies could restore normal NO production in spontaneously hypertensive rats (SHR). Flow was altered from approximately 50-200% of control in anesthetized Wistar-Kyoto rats (WKY) and SHR by selective placement of microvascular clamps on adjacent arteries while NO and H2O2 were directly measured with microelectrodes. Relative to WKY, SHR had significantly increased baseline NO and H2O2 concentrations (2,572 +/- 241 vs. 1,059 +/- 160 nM, P < 0.01; and 26 +/- 7 vs. 7 +/- 1 microM, P < 0.05, respectively). With flow elevation, H2O2 but not NO increased in SHR; NO but not H2O2 was elevated in WKY. Apocynin and polyethylene-glycolated catalase decreased baseline SHR NO and H2O2 to WKY levels and restored flow-mediated NO production. Suppression of NAD(P)H oxidase with gp91ds-tat decreased SHR H2O2 to WKY levels. Addition of topical H2O2 to increase peroxide to the basal concentration measured in SHR elevated WKY NO to levels observed in SHR. The results support the hypothesis that increased vascular peroxide in SHR is primarily derived from NAD(P)H oxidase and increases NO concentration to levels that cannot be further elevated with increased flow. Short-term and even acute administration of antioxidants are able to restore normal flow-mediated NO signaling in young SHR.     

Gender-specific compensation for the lack of NO in the mediation of flow-induced arteriolar dilation

Flow-induced dilation of gracilis muscle arterioles was examined in both genders of control rats and rats chronically treated with N(omega)-nitro-L-arginine methyl ester (L-NAME). After L-NAME treatment (4 wk), systolic blood pressure was significantly increased compared with control, whereas the plasma concentration of nitrate/nitrite was significantly reduced. Isolated and pressurized arterioles dilated significantly in response to increases in flow (0-25 microl/min). Flow-induced dilation was comparable in arterioles of control and L-NAME-treated rats but was significantly greater in female than in male rats. L-NAME + indomethacin, which abolished flow-induced dilation in arterioles of male control rats, inhibited the dilation by only ~75% in female control rats. The residual portion of the response was eliminated by additional administration of miconazole, an inhibitor of cytochrome P-450. Indomethacin did not affect the dilation in female L-NAME-treated rats but completely inhibited the response in male L-NAME-treated rats. The indomethacin-insensitive, flow-induced dilation in female L-NAME-treated arterioles was abolished by miconazole, 6-(2-proparglyoxyphenyl)hexanoic acid, or charybdotoxin. Thus an augmented release of endothelial prostaglandins accounts for the preserved flow-induced dilation in arterioles of male rats, whereas a metabolite of cytochrome P-450 is responsible for the maintenance of flow-induced dilation in female rats, suggesting important differences in the adaptation of the endothelium of arterioles from male and female rats to the lack of nitric oxide (NO) synthesis.     

autoregulatory role of endothelium-derived nitric oxide (NO) on Lipopolysaccharide-induced vascular inducible NO synthase expression and function

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is responsible for sepsis-induced hypotension and plays a major contributory role in the ensuing multiorgan failure. The present study aimed to elucidate the role of endothelial NO in lipopolysaccharide (LPS)-induced iNOS expression, in isolated rat aortic rings. Exposure to LPS (1 mug/ml, 5 h) resulted in a reversal of phenylephrine precontracted tone in aortic rings (70.7 +/- 3.2%). This relaxation was associated with iNOS expression and NF-kappaB activation. Positive immunoreactivity for iNOS protein was localized in medial and adventitial layers of LPS-treated aortic rings. Removal of the endothelium rendered aortic rings resistant to LPS-induced relaxation (8.9 +/- 4.5%). Western blotting of these rings demonstrated an absence of iNOS expression. However, treatment of endothelium-denuded rings with the NO donor, diethylamine-NONOate (0.1 mum), restored LPS-induced relaxation (61.6 +/- 6.6%) and iNOS expression to levels comparable with arteries with intact endothelium. Blockade of endothelial NOS (eNOS) activation using geldanamycin and radicicol, inhibitors of heat shock protein 90, in endothelium-intact arteries suppressed both LPS-induced relaxation and LPS-induced iNOS expression (9.0 +/- 8.0% and 2.0 +/- 6.2%, respectively). Moreover, LPS treatment (12.5 mg/kg, intravenous, 15 h) of wild-type mice resulted in profound elevation of plasma [NO(x)] measurements that were reduced by approximately 50% in eNOS knock-out animals. Furthermore, LPS-induced changes in vascular reactivity and iNOS expression evident in wild-type tissues were profoundly suppressed in tissues taken from eNOS knockout animals. Together, these data suggest that eNOS-derived NO, in part via activation of NF-kappaB, regulates iNOS-induction by LPS. This study provides the first demonstration of a proinflammatory role of vascular eNOS in sepsis.     

Homocysteine induces oxidative stress by uncoupling of NO synthase activity through reduction of tetrahydrobiopterin

Hyperhomocysteinemia is a risk factor for cardiovascular diseases that induces endothelial dysfunction. Here, we examine the participation of endothelial NO synthase (eNOS) in the homocysteine-induced alterations of NO/O(2)(-) balance in endothelial cells from human umbilical cord vein. When cells were treated for 24 h, homocysteine dose-dependently inhibited thrombin-activated NO release without altering eNOS phosphorylation and independently of the endogenous NOS inhibitor, asymmetric dimethylarginine. The inhibitory effect of homocysteine on NO release was associated with increased production of reactive nitrogen and oxygen species (RNS/ROS) independent of extracellular superoxide anion (O(2)(-)) and was suppressed by the NOS inhibitor L-NAME. In unstimulated cells, L-NAME markedly decreased RNS/ROS formation and the ethidium red fluorescence induced by homocysteine. This eNOS-dependent O(2)(-) synthesis was associated with reduced intracellular levels of both total biopterins (-45%) and tetrahydrobiopterin (-80%) and increased release of 7,8-dihydrobiopterin and biopterin in the extracellular medium (+40%). In addition, homocysteine suppressed the activating effect of sepiapterin on NO release, but not that of ascorbate. The results show that the oxidative stress and inhibition of NO release induced by homocysteine depend on eNOS uncoupling due to reduction of intracellular tetrahydrobiopterin availability.     

Evidence against tetrahydrobiopterin depletion of vascular tissue exposed to nitric oxide/superoxide or nitroglycerin

Several cardiovascular disorders, including atherosclerosis and tolerance to the antianginal drug nitroglycerin (GTN), may be associated with the generation of superoxide anions, which react with nitric oxide (NO) to yield peroxynitrite. According to a widely held view, oxidation of tetrahydrobiopterin (BH₄) by peroxynitrite causes uncoupling of endothelial NO synthase (eNOS), resulting in reduced NO bioavailability and endothelial dysfunction under conditions of oxidative stress. In this study we determined the levels of reduced biopterins and endothelial function in cultured cells exposed to peroxynitrite and GTN as well as in blood vessels isolated from GTN-tolerant guinea pigs and rats. BH₄ was rapidly oxidized by peroxynitrite and 3-morpholino sydnonimine (SIN-1) in buffer, but this was prevented by glutathione and not observed in endothelial cells exposed to SIN-1 or GTN. Prolonged treatment of the cells with 0.1 mM GTN caused slow N^G-nitro-l-arginine-sensitive formation of reactive oxygen species without affecting eNOS activity. Endothelial function and BH₄/BH₂ levels were identical in blood vessels of control and GTN-tolerant animals. Our results suggest that peroxynitrite-triggered BH₄ oxidation does not occur in endothelial cells or GTN-exposed blood vessels. GTN seems to trigger minor eNOS uncoupling that is unrelated to BH₄ depletion and without observable consequence on eNOS function.     

Synthetic melanin suppresses production of proinflammatory cytokines

An overproduction of proinflammatory cytokines mediates the damaging sequelae of inflammation in pathologic conditions such as rheumatoid arthritis, graft-vs-host reaction, cachexia, and sepsis syndrome. We examined the cytokine regulatory activity of synthetic melanin, exemplified by biosynthetic l-glycine-l-tyrosine-based polymer (ME-1) and chemosynthetic dihydroxyphenylalanine-based polymer (MC-1). At nontoxic concentrations, both compounds effectively (>/=60%) and reversibly suppressed the production of tumor necrosis factor (TNF), even when applied after stimulation of human peripheral blood monocytes with lipopolysaccharide (LPS). The inhibitory activity of melanin was selective with regard to cytokine response but not inducer- or cell-type-specific. In addition to TNF, melanin inhibited production of interleukin (IL)-1beta, IL-6, and IL-10 but not granulocyte-macrophage colony-stimulating factor by the LPS-stimulated monocytes. Melanin was equally effective in inhibiting production of TNF by monocytes stimulated with the purified protein derivative of Mycobacterium tuberculosis and production of IL-6 by IL-1alpha-stimulated human fibroblasts and endothelial cells. Northern blot analysis, mRNA stability determination, immunoprecipitation studies on metabolically labeled intracellular TNF, and pulse chase experiments revealed that melanin reduced efficiency of mRNA translation. The finding that melanin arrests ongoing cytokine synthesis suggests that this compound may be useful as an adjunct therapy for conditions showing involvement of proinflammatory cytokines.     

Endothelin-3 stimulates production of endothelium-derived nitric oxide via phosphoinositide breakdown.

Cultured bovine endothelial cells (EC) have specific receptors for endothelin (ET)-3 functionally coupled to phosphoinositide breakdown. We studied whether ET-3 stimulates synthesis of nitric oxide (NO), an endothelium-derived relaxing factor that activates soluble guanylate cyclase in EC, and whether the ET-3-induced NO formation involves G-proteins. ET-3 dose-dependently stimulated production of intracellular cGMP in EC, of which effects were abolished by pretreatment with NG-monomethyl L-arginine, an inhibitor of NO synthesis, and methylene blue, an inhibitor of soluble guanylate cyclase. The stimulatory effects of ET-3 on cGMP production, inositol trisphosphate formation and increase in cytosolic free Ca2+ concentration were similarly blocked by pretreatment with pertussis toxin (PTX). These data suggest that ET-3 induces synthesis of NO mediated by phosphoinositide breakdown via PTX-sensitive G-protein in EC.     

Antihypertensive therapy increases tetrahydrobiopterin levels and NO/cGMP signaling in small arteries of angiotensin II-infused hypertensive rats

We previously reported that small mesenteric arteries from hypertensive rats have increased NOS-derived H(2)O(2) and reduced NO/cGMP signaling. We hypothesized that antihypertensive therapy lowers blood pressure through a tetrahydrobiopterin (BH(4))-dependent mechanism restoring NO/cGMP signaling and endothelial NOS (NOS3; eNOS) phosphorylation in small arteries. To test this hypothesis, small mesenteric arteries from normotensive rats (NORM), angiotensin II-infused rats (ANG), ANG rats with triple therapy (reserperine, hydrochlorothiazide, and hydralazine), or ANG rats with oral BH(4) therapy were studied. Both triple therapy and oral BH(4) therapy attenuated the rise in systolic blood pressure in ANG rats and restored NO/cGMP signaling in small arteries similarly. Triple therapy significantly increased vascular BH(4) levels and BH(4)-to-BH(2) ratio similar to ANG rats with BH(4) supplementation. Furthermore, triple therapy (but not oral BH(4) therapy) significantly increased GTP cyclohydrolase I (GTPCH I) activity in small arteries without a change in expression. NOS3 phosphorylation at Ser1177 was reduced in small arteries from ANG compared with NORM, while NOS3 phosphorylation at Ser633 and Thr495 were similar in ANG and NORM. NOS3 phosphorylation at Ser1177 was restored with triple therapy or oral BH(4) in ANG rats. In conclusion, antihypertensive therapy regulates NO/cGMP signaling in small arteries through increasing BH(4) levels and NOS3 phosphorylation at Ser1177.     

Stimulation of the brain NO/cyclic GMP pathway by peripheral administration of tetrahydrobiopterin in the hph-1 mouse

Mutations in GTP-cyclohydrolase I (GTP-CH) have been identified as causing a range of inborn errors of metabolism, including dopa-responsive dystonia. GTP-CH catalyses the first step in the biosynthesis of tetrahydrobiopterin (BH4), a cofactor necessary for the synthesis of catecholamines and serotonin. Current therapy based on monoamine neurotransmitter replacement may be only partially successful in correcting the neurological deficits. The reason might be that BH4 is also a cofactor for nitric oxide synthase. Using a strain of mutant GTP-CH-deficient (hph-1) mice, we demonstrate that in addition to impaired monoamine metabolism, BH4 deficiency is also associated with diminished nitric oxide synthesis in the brain (as evaluated by measuring the levels of cyclic GMP), when compared with wild-type animals. We have found a decline in the levels of BH4 with age in all animals, but no gender-related differences. We found a strong association between the levels of BH4 and cyclic GMP in hph-1 mice but not in wild-type animals. We also demonstrate that acute peripheral administration of BH4 (100 micromol/kg s.c.) in hph-1 mice significantly elevated the brain BH4 concentration and subsequently cyclic GMP levels in cerebellum, with peaks at 2 and 3 h, respectively. We suggest that BH4 administration should be considered in BH4 deficiency states in addition to monoamine replacement therapy.     

L-Arginine and tetrahydrobiopterin supported nitric oxide production is crucial for the microbicidal activity of neutrophils

Recent report from this lab has shown role of Rac2 in the translocation of inducible nitric oxide synthase (iNOS) to the phagosomal compartment of polymorphonuclear leukocytes (PMNs) following phagocytosis of beads. This study was undertaken to further assess the status and role of tetrahydrobiopterin (BH₄), a redox-sensitive cofactor, L-arginine, and the substrate of nitric oxide synthase (NOS) in sustained nitric oxide (˙NO) production in killing of phagocytosed microbes (Escherichia coli) by human PMNs. Time-dependent study revealed consistent NO and reactive oxygen species (ROS) production in the PMNs following phagocytosis of beads. In addition, levels of L-arginine and BH₄ were maintained or increased simultaneously to support the enzymatic activity of NOS in the bead activated PMNs. Moreover, translocation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunits along with iNOS was reconfirmed in the isolated phagosomes. We demonstrate that increase in the level of NO was supported by L-arginine and BH₄ to kill E. coli, by using PMNs from NOS2^?/? mice, human PMNs treated with biopterin inhibitor, N-acetyl serotonin (NAS), or by suspending human PMNs in L-arginine deficient medium. Altogether, this study demonstrates that following phagocytosis, sustained. NO production in the PMNs was well-maintained by redox sensitive cofactor, BH₄ and substrate, and L-arginine to enable microbial killing. Further results suggest NO production in the human PMNs, along with ROS and myeloperoxidase (MPO) is important to execute antimicrobial activity.     

Chemical inhibition of bacterial protein tyrosine phosphatase suppresses capsule production

Capsule polysaccharide is a major virulence factor for a wide range of bacterial pathogens, including Streptococcus pneumoniae. The biosynthesis of Wzy-dependent capsules in both gram-negative and -positive bacteria is regulated by a system involving a protein tyrosine phosphatase (PTP) and a protein tyrosine kinase. However, how the system functions is still controversial. In Streptococcus pneumoniae, a major human pathogen, the system is present in all but 2 of the 93 serotypes found to date. In order to study this regulation further, we performed a screen to find inhibitors of the phosphatase, CpsB. This led to the observation that a recently discovered marine sponge metabolite, fascioquinol E, inhibited CpsB phosphatase activity both in vitro and in vivo at concentrations that did not affect the growth of the bacteria. This inhibition resulted in decreased capsule synthesis in D39 and Type 1 S. pneumoniae. Furthermore, concentrations of Fascioquinol E that inhibited capsule also lead to increased attachment of pneumococci to a macrophage cell line, suggesting that this compound would inhibit the virulence of the pathogen. Interestingly, this compound also inhibited the phosphatase activity of the structurally unrelated gram-negative PTP, Wzb, which belongs to separate family of protein tyrosine phosphatases. Furthermore, incubation with Klebsiella pneumoniae, which contains a homologous phosphatase, resulted in decreased capsule synthesis. Taken together, these data provide evidence that PTPs are critical for Wzy-dependent capsule production across a spectrum of bacteria, and as such represents a valuable new molecular target for the development of anti-virulence antibacterials.     

Chronic blockade of NO synthase paradoxically increases islet NO production and modulates islet hormone release

Islet production of nitric oxide (NO) and CO in relation to islet hormone secretion was investigated in mice given the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) in their drinking water. In these mice, the total islet NO production was paradoxically increased, reflecting induction of inducible NOS (iNOS) in background of reduced activity and immunoreactivity of constitutive NOS (cNOS). Unexpectedly, normal mice fasted for 24 h also displayed iNOS activity, which was further increased in L-NAME-drinking mice. Glucose-stimulated insulin secretion in vitro and in vivo was increased in fasted but unaffected in fed mice after L-NAME drinking. Glucagon secretion was increased in vitro. Control islets incubated with different NOS inhibitors at 20 mM glucose displayed increased insulin release and decreased cNOS activity. These NOS inhibitors potentiated glucose-stimulated insulin release also from islets of L-NAME-drinking mice. In contrast, glucagon release was suppressed. In islets from L-NAME-drinking mice, cyclic nucleotides were upregulated, and forskolin-stimulated hormone release, CO production, and heme oxygenase (HO)-2 expression increased. In conclusion, chronic NOS blockade evoked iNOS-derived NO production in pancreatic islets and elicited compensatory mechanisms against the inhibitory action of NO on glucose-stimulated insulin release by inducing upregulation of the islet cAMP and HO-CO systems.     

The role of hyperhomocysteinemia in nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation.

Hyperhomocysteinemia (HHcy) is associated with impaired endothelial-dependent vasodilatation and increased risk of atherosclerosis and thrombosis. Here, we summarize some of our previous work on the effect of HHcy on pathways involved in endothelium-dependent vasodilatation, and present new data concerning the endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation. We showed that the 894 G>T single-nucleotide polymorphism in the human endothelial nitric oxide synthase gene (eNOS) increased the risk of recurrent venous thrombosis in individuals with elevated homocysteine levels, indicating that the pathophysiological mechanism in HHcy involves impaired NO-mediated vasodilatation. In addition, the EDHF-mediated vasodilatation of the renal artery was disturbed in diet-induced hyperhomocysteinemic rats. Interestingly, we demonstrated that pretreatment of rats with periodate-oxidized adenosine (Adox), which is an inhibitor of S-adenosylhomocysteine hydrolase, prevented the methionine-induced rise in plasma total Hcy (tHcy) levels but not the inhibition of the EDHF pathway. Furthermore, we demonstrated that S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (AdoMet) levels were increased in the kidneys of diet-induced HHcy rats, resulting in a decreased AdoMet:AdoHcy ratio. In addition, we demonstrated that mRNA expression of Connexin 40, which is one of the structural subunits of gap-junctions, was down-regulated in endothelial cells of HHcy rats, and correlated with elevated AdoHcy levels in kidney of these rats. These finding suggest a key role for AdoHcy in relation to decreased Cx40 mRNA expression and impaired EDHF-mediated vasodilatation of HHcy rats.