cyclic-gmp and sapropterin

The regulation of myocardial function by constitutive nitric oxide synthases (NOS) is important for the maintenance of myocardial Ca(2+) homeostasis, relaxation and distensibility, and protection from arrhythmia and abnormal stress stimuli. However, sustained insults such as diabetes, hypertension, hemodynamic overload, and atrial fibrillation lead to dysfunctional NOS activity with superoxide produced instead of NO and worse pathophysiology.. Major strides in understanding the role of normal and abnormal constitutive NOS in the heart have revealed molecular targets by which NO modulates myocyte function and morphology, the role and nature of post-translational modifications of NOS, and factors controlling nitroso-redox balance. Localized and differential signaling from NOS1 (neuronal) versus NOS3 (endothelial) isoforms are being identified, as are methods to restore NOS function in heart disease.. Abnormal NOS signaling plays a key role in many cardiac disorders, while targeted modulation may potentially reverse this pathogenic source of oxidative stress.. Improvements in the clinical translation of potent modulators of NOS function/dysfunction may ultimately provide a powerful new treatment for many hearts diseases that are fueled by nitroso-redox imbalance.

Topics: Animals; Arginase; Autocrine Communication; Biopterins; Calcium Signaling; Cyclic GMP; Diabetes Mellitus; Disease Progression; Enzyme Activation; Enzyme Induction; Heart Diseases; Heart Failure; Humans; Hypertension; Myocardium; Myocytes, Cardiac; Nitric Oxide; Nitric Oxide Synthase; Paracrine Communication; Protein Processing, Post-Translational; Protein Structure, Tertiary; Protein Transport; Signal Transduction; Superoxides

Left ventricular hypertrophy (LVH) is regarded as a complication common to a number of cardiovascular diseases, including hypertension, myocardial infarction and ischaemia associated with coronary artery disease. Initially LVH is a compensatory mechanism, but in the long term cardiac hypertrophy predisposes individuals to heart failure, myocardial infarction and sudden death. Alteration of the nitric oxide (NO) pathway is believed to play an important role in the haemodynamically overloaded heart and pathological cardiac remodelling. Although re-establishment of the physiological NO pathway could be considered an important therapeutic target, the use of conventional nitrates is limited in the clinical setting by the development of tissue resistance and tolerance and by the shortage of large-scale clinical trials unequivocally confirming the beneficial impact of NO donors on cardiovascular morbidity and mortality. The aim of this review is to present current therapeutic options for dealing with changes in the L-arginine-NO pathway. The most promising therapeutic approach is represented by a new neutral sugar organic nitrate, LA-419, the thiol group of which seems to protect NO from degradation, thereby increasing its bioavailability. In a model of aortic stenosis-induced pressure overload, LA-419 has been found to restore the complete NO signalling cascade and reduce left ventricular remodelling, but without restoring the original pressure gradient, indicating a possible direct antiproliferative effect. Future studies are needed to confirm this therapeutic benefit in other animal models of hypertension and in the clinical setting.

Topics: Arginine; Biopterins; Cardiomegaly; Cyclic GMP; Humans; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase

Topics: Arginine; Biopterins; Calcium; Calmodulin; Cyclic GMP; Guanylate Cyclase; Humans; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Phosphorylation; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Transcription, Genetic

The potential transient vanilloid receptor type 1 (TRPV1) plays important functional roles in the vascular system. In the present study, we explored the role of the TRPV1 in the production of nitric oxide (NO), biopterines (BH4 and BH2), cyclic guanosine monophosphate (cGMP), malondialdehyde (MDA), phosphodiesterase-3 (PDE-3), total antioxidant capacity (TAC), and calcitonin gene-related peptide (CGRP) in the rat aorta. Wistar rats were divided into four groups: (1) control, (2) capsaicin (CS, 20 mg/kg), (3) capsazepine (CZ, 24 mg/kg), and (4) CZ + CS. Treatments were applied daily for 4 days before removing the thoracic aortas for testing of aortic tissue and endothelial cells. TRPV1 activation produced increases in BH4 14%, cGMP 25%, NO 29%, and TAC 59.2% in comparison to the controls. BH2 and MDA increased with CZ. CGRP shows a tendency to decrease with CZ. The analysis by immunocytochemistry confirmed that the TRPV1 is present in aortic endothelial cells. Aortic endothelial cells were obtained from healthy rats and cultured to directly explore the effects of CS and CZ. The activation of the TRPV1 (CS 30 

Topics: Animals; Aorta; Biopterins; Capsaicin; Cyclic GMP; Endothelial Cells; Male; Malondialdehyde; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Rats; Rats, Wistar; TRPV Cation Channels

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases (CVDs). Stachydrine (STA) is an active component in Chinese motherwort Leonurus heterophyllus sweet, which has been widely used for gynecological and cardiovascular disorders. This study is aimed to examine the effects of STA on homocysteine (Hcy)-induced endothelial dysfunction.. The effects of STA on vascular relaxation in rat thoracic aortas (TA), mesenteric arteries (MA) and renal arteries (RA) were measured by using Multi Myograph System. The levels of nitric oxide (NO), tetrahydrobiopterin (BH4) and guanosine 3', 5' cyclic monophosphate (cGMP) were determined. Endothelial nitric oxide synthase (eNOS) dimers and monomers were assayed by using Western blotting. GTP cyclohydrolase 1 (GTPCH1) and dihydrofolate reductase (DHFR) expressions were measured by using quantitative reverse transcriptase-PCR (qRT-PCR) and Western blotting.. STA effectively blocked Hcy-induced impairment of endothelium-dependent vasorelaxation in rat TA, MA and RA. STA-elicited arterial relaxations were reduced by NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) or the NO-sensitive guanylyl cyclase inhibitor 1H- [1, 2, 4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), but not by inducible iNOS inhibitor 1400 W nor the nonselective COX inhibitor indomethacin. Hcy caused eNOS uncoupling and decreases in NO, cGMP and BH4, which were attenuated by STA. Moreover, STA prevented decreases of GTPCH1 and DHFR levels in Hcy-treated BAECs.. We demonstrated that STA effectively reversed the Hcy-induced endothelial dysfunction and prevented eNOS uncoupling by increasing the expression of GTPCH1 and DHFR. These results revealed a novel mechanism by which STA exerts its beneficial vascular effects.

Topics: Animals; Aorta, Thoracic; Biopterins; Cattle; Cell Line; Cyclic GMP; Endothelial Cells; Endothelium, Vascular; Homocysteine; Male; Mesenteric Arteries; Nitric Oxide; Nitric Oxide Synthase Type III; Proline; Rats, Sprague-Dawley; Renal Artery; Vasodilation

Guanosine triphosphate cyclohydrolase/tetrahydrobiopterin (GTPCH)/(BH4) pathway has been proved to regulate the function of endothelial progenitor cells (EPCs) in deoxycorticosterone acetate-salt hypertensive mice, indicating that GTPCH/BH4 pathway may be an important repair target for hypertension-related endothelial injury. Shear stress is an important nonpharmacologic strategy to modulate the function of EPCs. Here, we investigated the effects of laminar shear stress on the GTPCH/BH4 pathway and endothelial repair capacity of circulating EPCs in hypertension.. Laminar shear stress was loaded on the human EPCs from hypertensive patients and normotensive patients. The in-vitro function, in-vivo reendothelialization capacity and GTPCH/BH4 pathway of human EPCs were evaluated.. Both in-vitro function and reendothelialization capacity of EPCs were lower in hypertensive patients than that in normotensive patients. The GTPCH/BH4 pathway of EPCs was downregulated in hypertensive patients. Shear stress increased in-vitro function and reendothelialization capacity of EPCs from hypertensive patients and normotensive patients. Furthermore, shear stress upregulated the expression of GTPCH I and levels of BH4, nitric oxide, and cGMP of EPCs, and reduced thrombospondin-1 expression. With treatment of GTPCH knockdown or nitroarginine methyl ester inhibition, shear stress-induced increased levels of BH4, nitric oxide and cGMP of EPCs was suppressed. When GTPCH/BH4 pathway of EPCs was blocked, the effects of shear stress on in-vitro function and reendothelialization capacity of EPCs were inhibited.. The study demonstrates for the first time that shear stress-induced upregulation of the GTPCH/BH4 pathway ameliorates hypertension-related decline in endothelial repair capacity of EPCs. These findings provide novel nonpharmacologic therapeutic approach for hypertension-related endothelial repair.

Topics: Animals; Biopterins; Cells, Cultured; Cyclic GMP; Down-Regulation; Endothelial Progenitor Cells; Gene Knockdown Techniques; GTP Cyclohydrolase; Human Umbilical Vein Endothelial Cells; Humans; Hypertension; Male; Metabolic Networks and Pathways; Mice; Middle Aged; NG-Nitroarginine Methyl Ester; Nitric Oxide; Stress, Mechanical; Thrombospondin 1; Up-Regulation

Tetrahydrobiopterin (BH4) is a critical determinant of nitric oxide (NO) production by nitric oxide synthase (NOS) in the vascular endothelium and its biosynthesis is regulated by the enzymatic activity of GTP-cyclohydrolase I (GTPCH I). The present study was designed to determine the effects of endothelium-targeted overexpression of GTPCH I (eGCH-Tg) on murine cerebral vascular function. Endothelium targeted over-expression of GTPCH I was associated with a significant increase in levels of BH4, as well as its oxidized product, 7,8-dihydrobiopterin (7,8-BH2) in cerebral microvessels. Importantly, ratio of BH4 to 7,8-BH2, indicative of BH4 available for eNOS activation, was significantly increased in eGCH-Tg mice. However, expression of endothelial NOS, levels of nitrate/nitrite--indicative of NO production--remained unchanged between cerebral microvessels of wild-type and eGCH-Tg mice. Furthermore, increased BH4 biosynthesis neither affected production of superoxide anion nor expression of antioxidant proteins. Moreover, endothelium-specific GTPCH I overexpression did not alter intracellular levels of cGMP, reflective of NO signaling in cerebral microvessels. The obtained results suggest that, despite a significant increase in BH4 bioavailability, generation of endothelial NO in cerebral microvessels remained unchanged in eGCH-Tg mice. We conclude that under physiological conditions the levels of BH4 in the cerebral microvessels are optimal for activation of endothelial NOS and NO/cGMP signaling.

Topics: Analysis of Variance; Animals; Biopterins; Catecholamines; Cerebral Arteries; Cyclic GMP; Endothelium, Vascular; Gene Expression Regulation; GTP Cyclohydrolase; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microvessels; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Superoxides

Peroxisome proliferator-activated receptor (PPAR)-δ is a nuclear hormone receptor that is mainly involved in lipid metabolism. Recent studies have suggested that PPAR-δ agonists exert vascular protective effects. The present study was designed to characterize vascular function in mice with genetic inactivation of PPAR-δ in the endothelium. Mice with vascular endothelial cell-specific deletion of the PPAR-δ gene (ePPARδ(-/-) mice) were generated using loxP/Cre technology. ePPARδ(-/-) mice were normotensive and did not display any sign of metabolic syndrome. Endothelium-dependent relaxations to ACh and endothelium-independent relaxations to the nitric oxide (NO) donor diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate were both significantly impaired in the aorta and carotid arteries of ePPARδ(-/-) mice (P < 0.05). In ePPARδ(-/-) mouse aortas, phosphorylation of endothelial NO synthase at Ser(1177) was significantly decreased (P < 0.05). However, basal levels of cGMP were unexpectedly increased (P < 0.05). Enzymatic activity of GTP-cyclohydrolase I and tetrahydrobiopterin levels were also enhanced in ePPARδ(-/-) mice (P < 0.05). Most notably, endothelium-specific deletion of the PPAR-δ gene significantly decreased protein expressions of catalase and glutathione peroxidase 1 and resulted in increased levels of H2O2 in the aorta (P < 0.05). In contrast, superoxide anion production was unaltered. Moreover, treatment with catalase prevented the endothelial dysfunction and elevation of cGMP detected in aortas of ePPARδ(-/-) mice. The findings suggest that increased levels of cGMP caused by H2O2 impair vasodilator reactivity to endogenous and exogenous NO. We speculate that chronic elevation of H2O2 predisposes PPAR-δ-deficient arteries to oxidative stress and vascular dysfunction.

Topics: Animals; Antioxidants; Aorta; Biopterins; Carotid Artery, Common; Catalase; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Glutathione Peroxidase; Glutathione Peroxidase GPX1; GTP Cyclohydrolase; Hydrogen Peroxide; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Phosphorylation; PPAR gamma; Superoxides; Vasodilation; Vasodilator Agents

(6R)-5,6,7,8-Tetrahydro-L-biopterin (BH4) availability regulates nitric oxide and superoxide formation by endothelial nitric oxide synthase (eNOS). At low BH4 or low BH4 to 7,8-dihydrobiopterin (BH2) ratios the enzyme becomes uncoupled and generates superoxide at the expense of NO. We studied the effects of exogenously added BH2 on intracellular BH4/BH2 ratios and eNOS activity in different types of endothelial cells. Incubation of porcine aortic endothelial cells with BH2 increased BH4/BH2 ratios from 8.4 (controls) and 0.5 (BH4-depleted cells) up to ~20, demonstrating efficient reduction of BH2. Uncoupled eNOS activity observed in BH4-depleted cells was prevented by preincubation with BH2. Recycling of BH4 was much less efficient in human endothelial cells isolated from umbilical veins or derived from dermal microvessels (HMEC-1 cells), which exhibited eNOS uncoupling and low BH4/BH2 ratios under basal conditions and responded to exogenous BH2 with only moderate increases in BH4/BH2 ratios. The kinetics of dihydrofolate reductase-catalyzed BH4 recycling in endothelial cytosols showed that the apparent BH2 affinity of the enzyme was 50- to 300-fold higher in porcine than in human cell preparations. Thus, the differential regulation of eNOS uncoupling in different types of endothelial cells may be explained by striking differences in the apparent BH2 affinity of dihydrofolate reductase.

Topics: Animals; Aorta; Biopterins; Cell Line; Cells, Cultured; Cyclic GMP; Dermis; Endothelium, Vascular; Human Umbilical Vein Endothelial Cells; Humans; Microvessels; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidation-Reduction; Reactive Oxygen Species; Superoxides; Sus scrofa; Tetrahydrofolate Dehydrogenase

The cGMP/protein kinase G (PKG) pathway is involved in the cardioprotective effects of postconditioning (PoCo). Although PKG signaling in PoCo has been proposed to depend on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt cascade, recent data bring into question a causal role of reperfusion injury signaling kinase (RISK) in PoCo protection. We hypothesized that PoCo increases PKG activity by reducing oxidative stress-induced endothelial nitric oxide synthase (NOS) uncoupling at the onset of reperfusion.. Isolated rat hearts were submitted to 40 minutes of ischemia and reperfusion with and without a PoCo protocol. PoCo reduced infarct size by 48% and cGMP depletion. Blockade of cGMP synthesis (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) and inhibition of PKG (KT5823) or NOS (l-NAME) abolished protection, but inhibition of PI3K/Akt cascade (LY294002) did not (n=5 to 7 per group). Phosphorylation of the RISK pathway was higher in PoCo hearts. However, this difference is due to increased cell death in control hearts because in hearts reperfused with the contractile inhibitor blebbistatin, a drug effective in preventing cell death at the onset of reperfusion, RISK phosphorylation increased during reperfusion without differences between control and PoCo groups. In these hearts, PoCo reduced the production of superoxide (O2(-)) and protein nitrotyrosylation and increased nitrate/nitrite levels in parallel with a significant decrease in the oxidation of tetrahydrobiopterin (BH4) and in the monomeric form of endothelial NOS.. These results demonstrate that PoCo activates the cGMP/PKG pathway via a mechanism independent of the PI3K/Akt cascade and dependent on the reduction of O2(-) production at the onset of reperfusion, resulting in attenuated oxidation of BH4 and reduced NOS uncoupling.

Topics: Animals; Biopterins; Cell Death; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Enzyme Activation; Enzyme Inhibitors; Ischemic Postconditioning; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Nitrates; Nitric Oxide Synthase Type III; Nitrites; Oxidative Stress; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Superoxides; Time Factors; Tyrosine

NADPH oxidase is a major source of superoxide anions in the pulmonary arteries (PA). We previously reported that intratracheal SOD improves oxygenation and restores endothelial nitric oxide (NO) synthase (eNOS) function in lambs with persistent pulmonary hypertension of the newborn (PPHN). In this study, we determined the effects of the NADPH oxidase inhibitor apocynin on oxygenation, reactive oxygen species (ROS) levels, and NO signaling in PPHN lambs. PPHN was induced in lambs by antenatal ligation of the ductus arteriosus 9 days prior to delivery. Lambs were treated with vehicle or apocynin (3 mg/kg intratracheally) at birth and then ventilated with 100% O(2) for 24 h. A significant improvement in oxygenation was observed in apocynin-treated lambs after 24 h of ventilation. Contractility of isolated fifth-generation PA to norepinephrine was attenuated in apocynin-treated lambs. PA constrictions to NO synthase (NOS) inhibition with N-nitro-l-arginine were blunted in PPHN lambs; apocynin restored contractility to N-nitro-l-arginine, suggesting increased NOS activity. Intratracheal apocynin also enhanced PA relaxations to the eNOS activator A-23187 and to the NO donor S-nitrosyl-N-acetyl-penicillamine. Apocynin decreased the interaction between NADPH oxidase subunits p22(phox) and p47(phox) and decreased the expression of Nox2 and p22(phox) in ventilated PPHN lungs. These findings were associated with decreased superoxide and 3-nitrotyrosine levels in the PA of apocynin-treated PPHN lambs. eNOS protein expression, endothelial NO levels, and tetrahydrobiopterin-to-dihydrobiopterin ratios were significantly increased in PA from apocynin-treated lambs, although cGMP levels did not significantly increase and phosphodiesterase-5 activity did not significantly decrease. NADPH oxidase inhibition with apocynin may improve oxygenation, in part, by attenuating ROS-mediated vasoconstriction and by increasing NOS activity.

Topics: Acetophenones; Animals; Animals, Newborn; Biopterins; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Endothelium, Vascular; Hypertension, Pulmonary; Lung; NADPH Oxidases; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Norepinephrine; Pulmonary Artery; Reactive Oxygen Species; Sheep; Superoxides; Tyrosine; Vasoconstriction; Vasodilation

Tetrahydrobiopterin (BH4) is a major endogenous vasoprotective agent that improves endothelial function by increasing nitric oxide (NO) synthesis and scavenging of superoxide and peroxynitrite. Therefore, administration of BH4 is considered a promising therapy for cardiovascular diseases associated with endothelial dysfunction and oxidative stress. Here we report on a novel function of BH4 that might contribute to the beneficial vascular effects of the pteridine. Treatment of cultured porcine aortic endothelial cells with nitroglycerin (GTN) or 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) resulted in heme oxidation of soluble guanylate cyclase (sGC), as evident from diminished NO-induced cGMP accumulation that was paralleled by increased cGMP response to a heme- and NO-independent activator of soluble guanylate cyclase [4-([(4-carboxybutyl)[2-(5-fluoro-2-([4'-(trifluoromethyl)biphenyl-4-yl]methoxy)phenyl)ethyl]amino]methyl)benzoic acid (BAY 60-2770)]. Whereas scavenging of superoxide and/or peroxynitrite with superoxide dismutase, tiron, Mn(III)tetrakis(4-benzoic acid)porphyrin, and urate had no protective effects, supplementation of the cells with BH4, either by application of BH4 directly or of its precursors dihydrobiopterin or sepiapterin, completely prevented the inhibition of NO-induced cGMP accumulation by GTN and ODQ. Tetrahydroneopterin had the same effect, and virtually identical results were obtained with RFL-6 fibroblasts, suggesting that our observation reflects a general feature of tetrahydropteridines that is unrelated to NO synthase function and not limited to endothelial cells. Protection of sGC against oxidative inactivation may contribute to the known beneficial effects of BH4 in cardiovascular disorders associated with oxidative stress.

Topics: Animals; Aorta; Biopterins; Cardiovascular Diseases; Cells, Cultured; Cyclic GMP; Endothelial Cells; Fibroblasts; Guanylate Cyclase; Heme; Nitric Oxide; Nitric Oxide Synthase; Nitroglycerin; Oxidation-Reduction; Oxidative Stress; Peroxynitrous Acid; Pterins; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Superoxides; Swine

Cerebral blood flow (CBF) is known to be dysregulated in persons with human immunodeficiency virus 1 (HIV-1), for uncertain reasons. This is an important issue because impaired vasoreactivity has been associated with increased risk of ischemic stroke, elevated overall cardiovascular risk and cognitive impairment.. To test whether dysregulation of CBF might be due to virally-induced neuroinflammation, we used a well-defined animal model (GFAP-driven, doxycycline-inducible HIV-1 Tat transgenic (Tat-tg) mice). We then exposed the mice to a brief hypercapnic stimulus, and assessed cerebrovascular reactivity by measuring 1) changes in cerebral blood flow, using laser Doppler flowmetry and 2) changes in vascular dilation, using in vivo two-photon imaging.. Exposure to brief hypercapnia revealed an underlying cerebrovascular pathology in Tat-tg mice. In control animals, brief hypercapnia induced a brisk increase in cortical flow (20.8% above baseline) and vascular dilation, as measured by laser Doppler flowmetry and in vivo two-photon microscopy. These responses were significantly attenuated in Tat-tg mice (11.6% above baseline), but cortical microvascular morphology and capillary density were unaltered, suggesting that the functional pathology was not secondary to vascular remodeling. To examine the mechanistic basis for the diminished cerebrovascular response to brief hypercapnia, Tat-tg mice were treated with 1) gisadenafil, a phosphodiesterase 5 (PDE5) inhibitor and 2) tetrahydrobiopterin (BH4). Gisadenafil largely restored the normal increase in cortical flow following hypercapnia in Tat-tg mice (17.5% above baseline), whereas BH4 had little effect. Gisadenafil also restored the dilation of small (<25 μm) arterioles following hypercapnia (19.1% versus 20.6% diameter increase in control and Tat-tg plus gisadenafil, respectively), although it failed to restore full dilation of larger (>25 μm) vessels.. Taken together, these data show that HIV-associated neuroinflammation can cause cerebrovascular pathology through effects on cyclic guanosine monophosphate (cGMP) metabolism and possibly on PDE5 metabolism.

Topics: Animals; Arterioles; Biopterins; Blood Circulation Time; Carbon Dioxide; Cardiovascular System; Cerebral Cortex; Cerebrovascular Circulation; Chlorocebus aethiops; COS Cells; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Disease Models, Animal; Encephalitis; Gene Expression Regulation, Enzymologic; HIV Infections; Humans; Lectins; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitric Oxide; tat Gene Products, Human Immunodeficiency Virus; Time Factors; Vasodilation

The thromboxane receptor (TPr) and multiple TPr ligands, including thromboxane A(2) (TxA(2)) and prostaglandin H(2), are elevated during vascular and atherothrombotic diseases. How TPr stimulation causes vascular injury remains poorly defined. This study was conducted to investigate the mechanism by which TPr stimulation leads to vascular injury.. Exposure of bovine aortic endothelial cells to either [1S-(1α,2β(5Z),3α(1E,3R),4α]-7-[3-(3-hydroxy-4-(d'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1] heptan-2-yl]-5'-heptenoic acid (IBOP) or U46619, 2 structurally related TxA(2) mimetics, for 24 hours markedly increased the release of superoxide anions (O(2)(·-)) and peroxynitrite (ONOO(-)) but reduced cyclic GMP, an index of nitric oxide bioactivity. IBOP also significantly suppressed activity of endothelial nitric oxide synthase (eNOS), increased enzyme-inactive eNOS monomers, and reduced levels of tetrahydrobiopterin, an essential eNOS cofactor. IBOP- and U46619-induced increases in O(2)(·-) were accompanied by the membrane translocation of the p67(phox) subunit of NAD(P)H oxidase. Pharmacological or genetic inhibition of either NAD(P)H oxidase or TPr abolished IBOP-induced O(2)(·-) formation. Furthermore, TPr activation significantly increased protein kinase C-ζ (PKC-ζ) in membrane fractions and PKC-ζ phosphorylation at Thr410. Consistently, PKC-ζ inhibition abolished TPr activation-induced membrane translocation of p67(phox) and O(2)(·-) production. Finally, exposure of isolated mouse aortae to IBOP markedly increased O(2)(·-) in wild-type but not in those from gp91(phox) knockout mice.. We conclude that TPr activation via PKC-ζ-mediated NAD(P)H oxidase activation increases both O(2)(·-) and ONOO(-), resulting in eNOS uncoupling in endothelial cells.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Apoptosis; Biopterins; Bridged Bicyclo Compounds, Heterocyclic; Cattle; Cell Survival; Cells, Cultured; Cyclic GMP; Cytochrome P-450 Enzyme System; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; Fatty Acids, Unsaturated; Intramolecular Oxidoreductases; Mice; Mice, Inbred C57BL; Mice, Knockout; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Peroxynitrous Acid; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein Transport; Receptors, Immunologic; Receptors, Thromboxane A2, Prostaglandin H2; RNA Interference; Signal Transduction; Superoxides; Time Factors; Tyrosine

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.

Topics: Angiotensin II; Animals; Antihypertensive Agents; Biopterins; Cyclic GMP; GTP Cyclohydrolase; Hydralazine; Hydrochlorothiazide; Hypertension; Male; Mesenteric Arteries; Nitric Oxide; Nitric Oxide Synthase Type III; Rats; Rats, Sprague-Dawley; Reserpine; Signal Transduction

The diabetic heart is known to be susceptible to ischemia/reperfusion (I/R) injury by increased oxidative stress. Although oxidative stress upregulates inducible nitric oxide (iNOS), the role of iNOS in I/R injury in the diabetic heart has been poorly understood. Because iNOS-derived nitric oxide (NO) plays a crucial role in cardioprotection against I/R injury, we hypothesized that inhibition of iNOS uncoupling would restore tolerance to I/R injury in the diabetic heart. The present study demonstrated that iNOS-derived superoxide generation was reduced, and that the NO bioavailability was increased, by treatment with the NOS-cofactor, tetrahydrobiopterin (BH4), before I/R in the hearts isolated from diabetic rats. This was associated with a reduction of infarct size and improvement of left ventricular (LV) function after I/R. The cardioprotective effect of BH4 was abrogated by treatment with a thiol reducing agent dithiothreitol (DTT), but not a NO-sensitive guanylyl cyclase inhibitor ODQ, suggesting that iNOS-derived NO-mediated cardioprotection occurs through protein S-nitrosylation but not cGMP-dependent signaling in the diabetic heart. Indeed, protein S-nitrosylation was increased by treatment with BH4 in the diabetic heart and was inhibited by DTT. These results suggest that the inhibition of iNOS uncoupling unmasks tolerance to I/R injury through enhanced protein S-nitrosylation in the diabetic rat heart.

Topics: Animals; Biopterins; Cyclic GMP; Diabetes Complications; Diabetes Mellitus, Experimental; Dithiothreitol; Imines; Male; Myocardial Reperfusion Injury; Nitric Oxide; Nitric Oxide Synthase Type II; Oxadiazoles; Oxidative Stress; Quinoxalines; Rats; Rats, Sprague-Dawley; Superoxides; Tiopronin; Tyrosine; Up-Regulation; Ventricular Function, Left

Tetrahydrobiopterin (BH4) is an important cofactor of endogenous nitric oxide synthesis. In the present preclinical study, we investigated the effects of BH4 on cardiac and pulmonary function during early reperfusion in an experimental model of cardioplegic arrest and extracorporal circulation.. Twelve anesthetized dogs underwent hypothermic cardiopulmonary bypass. After 60 min of hypothermic cardiac arrest, reperfusion was started after application of either saline vehicle (control, n = 6), or BH4 (n = 6). Left-ventricular end-systolic pressure volume relationship (E(es)) was measured by a combined pressure-volume conductance catheter at baseline and after 60 min of reperfusion. Left anterior descending (LAD) coronary (CBF) and pulmonary blood flow (PBF), endothelium-dependent vasodilatation to acetylcholine (ACh), endothelium-independent vasodilatation to sodium nitroprusside (SNP) and alveolo-arterial O₂ gradient were determined.. The administration of BH4 led to a significantly better recovery of E(es) (given as percent of baseline: 85 ± 22 vs 46 ± 15%, p<0.05). CBF was also significantly higher in the BH4 group (38 ± 5 vs 22 ± 5 ml min⁻¹, p<0.05). While the vasodilatatory response to SNP was similar in both groups, injection of ACh resulted in a significantly higher increase in CBF (64 ± 12 vs 25 ± 12%, p < 0.05) and PBF (49 ± 15 vs 36 ± 14%, p<0.05) in the BH4-treated animals. Alveolo-arterial O₂ gradient was significantly lower after BH4 supplementation (80 ± 15 vs 49 ± 14 mm Hg, p < 0.05).. Application of BH4 improves myocardial, endothelial and pulmonary function after cardiopulmonary bypass with hypothermic cardiac arrest. The observed protective effects indicate that BH4 could be a novel therapeutic option in the treatment of ischemia/reperfusion injury.

Topics: Animals; Biopterins; Cardiopulmonary Bypass; Coronary Circulation; Cyclic GMP; Disease Models, Animal; Dogs; Drug Evaluation, Preclinical; Endothelium, Vascular; Heart Arrest, Induced; Hemodynamics; Myocardial Reperfusion Injury; Oxygen; Postoperative Care; Vasodilation; Ventricular Function, Left

In the present study, we used the hph-1 mouse, which displays GTP-cyclohydrolase I (GTPCH I) deficiency, to test the hypothesis that loss of tetrahydrobiopterin (BH(4)) in conduit and small arteries activates compensatory mechanisms designed to protect vascular wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and BH(4) levels were reduced in the aortas and small mesenteric arteries of hph-1 mice. However, the BH(4)-to-7,8-dihydrobiopterin ratio was significantly reduced only in hph-1 aortas. Furthermore, superoxide anion and 3-nitrotyrosine production were significantly enhanced in aortas but not in small mesenteric arteries of hph-1 mice. In contrast to the aorta, protein expression of copper- and zinc-containing superoxide dismutase (CuZnSOD) was significantly increased in small mesenteric arteries of hph-1 mice. Protein expression of catalase was increased in both aortas and small mesenteric arteries of hph-1 mice. Further analysis of endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) signaling demonstrated that protein expression of phosphorylated Ser(1177)-eNOS as well as basal cGMP levels and hydrogen peroxide was increased in hph-1 aortas. Increased production of hydrogen peroxide in hph-1 mice aortas appears to be the most likely mechanism responsible for phosphorylation of eNOS and elevation of cGMP. In contrast, upregulation of CuZnSOD and catalase in resistance arteries is sufficient to protect vascular tissue from increased production of reactive oxygen species generated by uncoupling of eNOS. The results of our study suggest that anatomical origin determines the ability of vessel wall to cope with oxidative stress induced by uncoupling of eNOS.

Topics: Animals; Antioxidants; Aorta; Biopterins; Catalase; Cyclic GMP; GTP Cyclohydrolase; Hydrogen Peroxide; Male; Mesenteric Arteries; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase Type III; Oxidative Stress; Phosphorylation; Serine; Superoxide Dismutase; Superoxides; Tyrosine

The aquaporin (AQP) water channel is expected to play a decisive role of hyponatremia and water retention in cirrhotic patients. Despite the importance of the water channel, however, previous findings vary widely when it concerns AQP2 of the kidneys in subjects with cirrhosis. The purpose of this study was to investigate the expression of AQP2 in the distal renal tubule in cirrhosis, and the presence of the nitric oxide-AQP2 signaling pathway as a possible vasopressin-aquaporin-independent pathway. Sixty male Wister rats were assigned to six groups: (1) control; (2) TAA (thioacetamide); (3) TAA with nitric oxide donor; (4) TAA with nitric oxide inhibitor; (5) TAA with HMG CoA reductase inhibitor; (6) TAA with tetrahydrobiopterin. Immunohistochemical staining for AQP2, real-time polymerase chain reaction (PCR) for AQP2 and 3, citrulline assay, and renal cGMP concentration were measured. The AQP2-positivity of cirrhotic rats were higher than the controls (P < 0.05). The AQP2-positivity decreased in the nitric oxide donor group, but the proportion rose back up when the subjects were injected with the nitric oxide inhibitor (P < 0.05). The expression of AQP2 and AQP3 mRNA was also found to show an increase in the cirrhotic group as compared with the normal controls (P < 0.05). The cirrhotic group administered with nitric oxide donor showed a significant decline in the expression of the mRNA. The control group's cGMP concentration was lower than that of the cirrhotic group (P < 0.05), but a comparison of the two groups injected with nitric oxide modulators, such as statin and BH4, did not show significant differences in the cGMP concentration level. The expression of AQP2 of the kidneys increased in the cirrhotic rats. AQP2 had relations to the activity changes of nitric oxide synthetase.

Topics: Analysis of Variance; Animals; Aquaporin 2; Biopterins; Cyclic GMP; Disease Models, Animal; Immunoenzyme Techniques; Isosorbide Dinitrate; Kidney; Liver Cirrhosis; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Random Allocation; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Simvastatin; Statistics, Nonparametric; Thioacetamide

The requirement of endothelial NO synthase (NOS3) calcium to produce NO is well described, although the effect of NO on intracellular calcium levels [Ca(2+)](i) is still confusing. Therefore, NO and [Ca(2+)](i) cross-talk were studied in parallel in endothelial cells possessing a functional or a dysfunctional NO pathway.. Dysfunctional porcine endothelial cells were obtained either in vitro by successive passages or in vivo from regenerated endothelium 1 month after coronary angioplasty. Activity of NOS3 was characterized by conversion of arginine to citrulline, BH(4) intracellular availability, cGMP, and superoxide anion production. Imaging of the Ca(2+) indicator FURA 2-AM was recorded and sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) pump activity was analysed by (45)Ca(2+) uptake into cells. In endothelial cells with a functional NO pathway, NOS3 inhibition increased [Ca(2+)](i) and, conversely, an NO donor decreased it. In aged cells with an uncoupled NOS3 as shown by the reduced BH(4) level, the increase in superoxide anion and the lower production of cGMP and the decrease in NO bioavailability were linearly correlated with the increase in basal [Ca(2+)](i). Moreover, when stimulated by bradykinin, the calcium response was reduced while its decay was slowed down. These effects on the calcium signalling were abolished in calcium-free buffer and were similarly induced by SERCA inhibitors. In aged cells, NO improved the reduced SERCA activity and tended to normalize the agonist calcium response.. In control endothelial cells, NO exerts a negative feedback on cytosolic Ca(2+) homeostasis. In aged cells, uncoupled NOS3 produced NO that was insufficient to control the [Ca(2+)](i). Consequently, under resting conditions, SERCA activity decreased and [Ca(2+)](i) increased. These alterations were reversible as exogenous NO, in a cGMP-independent way, refilled intracellular calcium stores, reduced calcium influx, and improved the agonist-evoked calcium response. Therefore, prevention of the decrease in NO in dysfunctional endothelium would normalize the calcium-dependent functions.

Topics: Animals; Arginine; Biopterins; Bradykinin; Calcium; Cells, Cultured; Cellular Senescence; Citrulline; Cyclic GMP; Endothelial Cells; Enzyme Inhibitors; Homeostasis; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Superoxides; Swine; Time Factors

Tetrahydrobiopterin is an essential cofactor for NOS enzymes to synthesize NO. It has been suggested that reduced intrahepatic tetrahydrobiopterin decreases intrahepatic NO and contributes to increase hepatic vascular resistance and portal pressure in cirrhosis. The main aim of the study was to evaluate the effect of tetrahydrobiopterin supplementation in portal pressure in CCl4 cirrhotic rats.. Cirrhotic rats received vehicle or tetrahydrobiopterin (10mg/kg/day i.p.) for 3 days. Hepatic and systemic hemodynamics and hepatic tetrahydrobiopterin, NOS activity and cGMP levels were measured. In addition, hepatic and systemic hemodynamics were evaluated in normal rats in which tetrahydrobiopterin deficiency was induced by administrating 2,4-diamino-6-hydroxy-pyrimidine (DAHP) for 8h.. In cirrhotic rats, tetrahydrobiopterin administration increased liver NOS activity and cGMP levels and markedly and significantly reduced portal pressure. Amelioration of portal hypertension was associated with a normalization of arterial pressure. In normal rats DAHP decreased hepatic tetrahydrobiopterin and NOS activity and increased hepatic vascular tone. These effects of DAHP administration were corrected by tetrahydrobiopterin supplementation.. The present study shows that tetrahydrobiopterin markedly reduces portal hypertension and improves systemic hemodynamics in cirrhotic rats. These data support the concept that tetrahydrobiopterin supplementation may represent a new therapeutic strategy for portal hypertension.

Topics: Animals; Biopterins; Carbon Tetrachloride; Cyclic GMP; Enzyme Inhibitors; Hypertension, Portal; Hypoxanthines; Liver; Liver Cirrhosis; Male; Nitric Oxide Synthase; Rats; Rats, Wistar; Splanchnic Circulation

Endothelium-dependent relaxation in conduit vessels is mediated largely by nitric oxide (NO), produced by the enzyme endothelial nitric oxide synthase (eNOS) in the presence of the cofactor tetrahydrobiopterin (BH4) and mediated through a cGMP-dependent downstream signalling cascade. Endothelial NOS regulates blood pressure in vivo, and impaired endothelial NO bioactivity in vascular disease states may contribute to systemic hypertension. In the absence of sufficient levels of the cofactor BH4, NO becomes uncoupled from arginine oxidation and eNOS produces superoxide rather than NO. The enzymatic uncoupling of eNOS is an important feature of vascular disease states associated with increased oxidative stress. However, whether eNOS coupling, rather than overall eNOS activity, has specific effects on endothelium-dependent vasorelaxation in vitro, or on blood pressure regulation in vivo, remains unclear. In this study, we evaluate the relationships between blood pressure and endothelial function in models of eNOS uncoupling, using mice with endothelium-targeted transgenic eNOS overexpression (eNOS-Tg), in comparison with littermates in which eNOS coupling was rescued by additional endothelium-targeted overexpression of GTP cyclohydrolase 1 (eNOS/GCH-Tg) to increase endothelial BH4 levels. Despite the previously characterized differences in eNOS-dependent superoxide production between these animals, we find that blood pressure is equally reduced in both genotypes, compared with wild-type animals. Furthermore, both eNOS-Tg and eNOS/GCH-Tg mice exhibit similarly impaired endothelium-dependent vasorelaxation. We show that reduced vasorelaxation responses result from desensitization of cGMP-mediated signalling and are associated with increased NO production rather than changes in superoxide production.

Topics: Animals; Aorta, Thoracic; Biopterins; Blood Pressure; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; GTP Cyclohydrolase; Mice; Mice, Inbred C57BL; Mice, Transgenic; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Ribavirin is a broad-spectrum antiviral drug that is used to treat hepatitis C virus (HCV)-infected patients. The virological response after ribavirin treatment appears to be insufficient to fully explain ribavirin-induced beneficial effects. Angiogenesis plays a pathogenic role in HCV-induced liver damage. Here, we investigated the influence of therapeutic ribavirin concentrations on angiogenesis. Ribavirin inhibited endothelial cell tube formation in vitro and vessel formation in the chick chorioallantoic membrane assay in vivo. Ribavirin inhibits inosine monophosphate dehydrogenase, which causes depletion of cellular GTP and in turn reduction of cellular tetrahydrobiopterin levels. The availability of tetrahydrobiopterin limits NO production by endothelial NO synthase. Ribavirin reduced levels of tetrahydrobiopterin (as revealed by HPLC), NO (as revealed by electron spin resonance spectroscopy), and cGMP (as revealed by RIA) in endothelial cells. Addition of tetrahydrobiopterin or NO prevented ribavirin-induced tube formation inhibition. In conclusion, angiogenesis inhibition by ribavirin has not been described before. This inhibition may contribute to ribavirin-induced pharmacological effects including adverse events.

Topics: Angiogenesis Inhibitors; Animals; Biopterins; Cell Proliferation; Cells, Cultured; Chickens; Chromatography, High Pressure Liquid; Cyclic GMP; Electron Spin Resonance Spectroscopy; Endothelium, Vascular; Humans; Neovascularization, Pathologic; Nitric Oxide; Nitric Oxide Synthase Type III; Radioimmunoassay; Ribavirin

C-reactive protein (CRP), a cardiovascular risk marker, induces endothelial dysfunction. We have previously shown that CRP decreases endothelial nitric oxide synthase (eNOS) expression and bioactivity in human aortic endothelial cells (HAECs). In this study, we examined the mechanisms by which CRP decreases eNOS activity in HAECs. To this end, we explored different strategies such as availability of tetrahydrobiopterin (BH4)-a critical cofactor for eNOS, superoxide (O(2)(-)) production resulting in uncoupling of eNOS and phosphorylation/dephosphorylation of eNOS. CRP treatment significantly decreased levels of BH4 thereby promoting eNOS uncoupling. Pretreatment with sepiapterin, a BH4 precursor, prevented CRP-mediated effects on BH(4) levels, superoxide production as well as eNOS activity. The gene expression and enzymatic activity of GTPCH1, the first enzyme in the de novo biosynthesis of BH(4), were significantly inhibited by CRP. Importantly, GTPCH1 is known to be regulated by cAMP-mediated pathway. In the present study, CRP-mediated inhibition of GTPCH1 activity was reversed by pretreatment with cAMP analogues. Furthermore, CRP-induced O(2)(-) production was reversed by pharmacologic inhibition and siRNAs to p47 phox and p22 phox. Additionally, CRP treatment significantly decreased the eNOS dimer: monomer ratio confirming CRP-mediated eNOS uncoupling. The pretreatment of cells with NO synthase inhibitor (N-nitro-l-arginine methyl ester [l-NAME]) also prevented CRP-mediated O(2)(-) production further strengthening CRP-mediated eNOS uncoupling. Additionally, CRP decreased eNOS phosphorylation at Ser1177 as well as increased phosphorylation at Thr495. CRP appears to mediate these effects through the Fcgamma receptors, CD32 and CD64. To conclude, CRP uncouples eNOS resulting in increased superoxide production, decreased NO production and altered eNOS phosphorylation.

Topics: Biopterins; Blotting, Western; C-Reactive Protein; Cells, Cultured; Cyclic GMP; Dimerization; Electron Transport; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; GTP Cyclohydrolase; Humans; Models, Biological; NADPH Oxidases; Neutralization Tests; Nitric Oxide Synthase Type III; Pterins; RNA, Messenger; Time Factors; Uncoupling Agents; Vascular Endothelial Growth Factor A

Shear stress, imposed on the vascular endothelium by circulating blood, critically sustains vascular synthesis of nitric oxide (NO). Endothelial NO synthase (eNOS) activity is determined by heat shock protein 90 (HSP90), caveolin-1, and the cofactor tetrahydrobiopterin (BH4). To determine whether increased blood flow concomitantly upregulates eNOS and GTP cyclohydrolase I (GTPCH I, the rate-limiting enzyme in BH4 biosynthesis), an aortocaval fistula model in the rat was employed wherein aortic blood flow is enhanced proximal but decreased distal to the fistula. Eight weeks after the creation of the aortocaval fistula, the proximal and distal aortic segments were harvested; sham-operated rats served as controls. Vasomotor function was assessed by isometric force recording. Expression of eNOS, HSP90, caveolin-1, Akt, phosphorylated eNOS (eNOS-Ser1177), and GTPCH I were determined by Western blot analysis. Biosynthesis of BH4 and GTPCH-I activity was examined by HPLC. In the aortic segments exposed to increased flow, contractions to KCl and phenylephrine were reduced, whereas endothelium-dependent relaxations were not affected compared with sham-operated or aortic segments with reduced blood flow. Expression of eNOS, caveolin-1, phosphorylated Akt, and eNOS-Ser1177 was enhanced in aortas exposed to increased blood flow. High flow augmented levels of cGMP and BH4 and increased expression of GTPCH I. In aggregate, these findings provide the first demonstration in vivo that coordinated vascular upregulation of eNOS, and GTPCH I accompanies increased blood flow. This induction of GTPCH I increases BH4 production, thereby optimizing the generation of NO by eNOS and thus the adaptive, vasorelaxant response required in sustaining increased blood flow.

Topics: Animals; Aorta; Aortic Diseases; Arteriovenous Fistula; Biopterins; Blotting, Western; Cyclic GMP; GTP Cyclohydrolase; Hemodynamics; Nitric Oxide Synthase Type III; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Up-Regulation; Vasomotor System; Vena Cava, Inferior

This study examined whether intravenous administration of tetrahydrobiopterin (BH4) reduces myocardial infarct size following ischemia/reperfusion (I/R) in rats, and the mechanisms of its protective effect were also investigated. Rats were subjected to 30 minutes of ischemia by ligation of the left coronary artery and 2 hours of reperfusion. The infarct size was determined as a percentage of the area at risk by triphenyltetrazolium staining. Intravenous administration of BH4 (0.01 mg/kg-1 mg/kg) significantly reduced the myocardial infarct size. Nitrite plus nitrate (NOx) and cGMP levels in the hearts were significantly increased by the treatment with BH4, and the infarct size-limiting effect of BH4 was abolished by the co-administration of NG-nitro-L-arginine methyl ester, a specific inhibitor of nitric oxide synthase, or 5-hydroxydecanoic acid, a specific inhibitor of mitochondrial ATP-sensitive potassium channel (mitoKATP channel). These findings suggest that BH4 has a cardioprotective effect against I/R in vivo, and its protective effect appeared to be involved in the opening of mitoKATP channels through increased nitric oxide production.

Topics: Animals; Anti-Arrhythmia Agents; Arginine; Biopterins; Cyclic GMP; Decanoic Acids; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydroxy Acids; Male; Malondialdehyde; Myocardial Infarction; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitrites; Potassium Channels; Rats; Rats, Sprague-Dawley

We hypothesized that cardiopulmonary bypass induces a selective alteration of the coronary arterial endothelial cell signal transduction which could be explained by a state of depletion and/or decreased activity of endogenous tetrahydrobiopterin (BH(4)). The aim of this study was to assess the effects of cardiopulmonary bypass and BH(4) on the endothelial function of epicardial coronary arteries in a swine model of cardiopulmonary bypass.. Swine underwent 90 min of cardiopulmonary bypass alone (N=19) or in association with a brief cardioplegic arrest with (N = 6) or without (N = 5) in vivo BH(4) administration, followed by a 60-min period following weaning from cardiopulmonary bypass and were compared to a control group (N = 7). Endothelium-dependent relaxations of epicardial coronary artery rings were studied using standard organ chamber experiments in the presence or absence of in vitro BH(4) or superoxide dismutase (SOD) and catalase.. Cardiopulmonary bypass caused a statistically significant reduction of endothelium-dependent relaxations to serotonin (p < 0.0001), bradykinin (p < 0.001), UK14304 (p < 0.0001) and calcium ionophore (p < 0.01) in epicardial porcine coronary arteries. In vitro and in vivo BH(4) supplementation improved endothelium-dependent relaxations to serotonin and bradykinin, which were left unchanged by SOD-catalase administration. Cardiopulmonary bypass was associated with a decrease in nitric oxide availability (p = 0.002) and increased oxidative stress (p < 0.001), which were both restored by in vivo BH(4) administration (p < 0.001).. Treatment with BH(4) improves the endothelial dysfunction of porcine epicardial coronary arteries, restores nitric oxide availability and reduces the oxidative stress associated with cardiopulmonary bypass.

Topics: Animals; Biopterins; Bradykinin; Cardiopulmonary Bypass; Coronary Vessels; Cyclic GMP; Dose-Response Relationship, Drug; Endothelial Cells; Endothelium, Vascular; Models, Animal; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Pericardium; Serotonin; Serotonin Agents; Signal Transduction; Superoxide Dismutase; Swine; Vasodilator Agents

Increased production of reactive oxygen species and loss of endothelial nitric oxide (NO) bioactivity are key features of vascular disease states such as atherosclerosis. Tetrahydrobiopterin (BH4) is a required cofactor for NO synthesis by endothelial nitric oxide synthase (eNOS); pharmacologic studies suggest that reduced BH4 availability may be an important mediator of endothelial dysfunction in atherosclerosis. We aimed to investigate the importance of endothelial BH4 availability in atherosclerosis using a transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, GTP-cyclohydrolase I (GTPCH).. Transgenic mice were crossed into an ApoE knockout (ApoE-KO) background and fed a high-fat diet for 16 weeks. Compared with ApoE-KO controls, transgenic mice (ApoE-KO/GCH-Tg) had higher aortic BH4 levels, reduced endothelial superoxide production and eNOS uncoupling, increased cGMP levels, and preserved NO-mediated endothelium dependent vasorelaxations. Furthermore, aortic root atherosclerotic plaque was significantly reduced in ApoE-KO/GCH-Tg mice compared with ApoE-KO controls.. These findings indicate that BH4 availability is a critical determinant of eNOS regulation in atherosclerosis and is a rational therapeutic target to restore NO-mediated endothelial function and reduce disease progression.

Topics: Animals; Aorta; Aortic Diseases; Apolipoproteins E; Arteriosclerosis; Biopterins; Coenzymes; Crosses, Genetic; Cyclic GMP; Diet, Atherogenic; Endothelium, Vascular; GTP Cyclohydrolase; Humans; Hyperlipoproteinemia Type II; Hyperlipoproteinemia Type IV; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nitric Oxide; Organ Specificity; Receptor, TIE-2; Recombinant Fusion Proteins; Superoxides; Vasodilation

Salt-sensitive hypertension is associated with impaired NO/cGMP signaling. We hypothesized that increased superoxide production by NADPH oxidase and altered endothelial NO synthase (NOS3) phosphorylation determine endothelial dysfunction in hypertension. Experiments tested if NO/cGMP signaling and NOS3 serine phosphorylation are decreased and NADPH oxidase activity is increased in mesenteric arteries from deoxycorticosterone acetate (DOCA)-salt rats compared with arteries from placebo rats. Concentration response curves to phenylephrine were performed in mesenteric arteries in the presence and absence of Nomega-nitro-L-arginine (LNA) and antioxidants to determine the influence of basal NO and superoxide production on vascular tone. LNA increased phenylephrine sensitivity in arteries from placebo, but not DOCA-salt rats, regardless of antioxidant treatment. To determine basal cGMP production, mesenteric arteries were incubated with 3-isobutyl-1-methylxanthine in the presence or absence of LNA, sodium nitroprusside (SNP), antioxidants, or tetrahydrobiopterin. NOS-dependent cGMP production was reduced in arteries from DOCA-salt rats compared with arteries from placebo rats and was not restored by acute treatment with antioxidants or tetrahydrobiopterin. SNP-induced cGMP production was similar between groups as was NADPH oxidase activity, measured by lucigenin chemiluminescence, in mesenteric arteries. Expression and phosphorylation of NOS3 were examined by Western blotting. Phosphorylation of NOS3 was decreased in arteries from DOCA-salt rats compared with placebo at serine residues 1179 and 635. These findings indicate that diminished NO/cGMP signaling in mesenteric arteries from DOCA-salt rats is caused by reduced phosphorylation of NOS3 at serine 1179 and serine 635, rather than NO scavenging by superoxide.

Topics: Adrenergic alpha-Agonists; Animals; Antioxidants; Biopterins; Codon; Cyclic GMP; Desoxycorticosterone; Hypertension; Male; Mesenteric Arteries; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroarginine; Nitroprusside; Phenylephrine; Phosphorylation; Phosphoserine; Polyethylene Glycols; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Signal Transduction; Sodium Chloride, Dietary; Superoxide Dismutase

Nitric oxide (NO), a central regulator of vascular tone and homeostasis, is generated upon activation of endothelial NO synthase (eNOS), which is mediated by an increase of intracellular calcium and/or by eNOS phosphorylation. A reduction of NO bioavailability leads to endothelial dysfunction that has been shown to be improved by alpha-tocopherol in certain conditions. The underlying mechanisms, however, are not completely clarified. The present study was performed to investigate whether alpha-tocopherol is able to affect endothelial NO synthesis. The formation of NO was measured in human umbilical vein endothelial cells using citrulline (coproduct) and cGMP (product of the NO-activated soluble guanylate cyclase) as indicator molecules. alpha-Tocopherol (10-200 microM, 24 hr) increased ionomycin-induced citrulline and cGMP formation in intact cells in a concentration-dependent manner. In parallel, ionomycin-stimulated phosphorylation of eNOS at serine 1177, known to support enzyme activation, was increased by alpha-tocopherol, suggesting that this was the mechanism responsible for enhanced NO formation. The effect of alpha-tocopherol was dependent on its hydrophobic structure because it was mimicked by gamma-tocopherol but not by trolox, a hydrophilic derivative of alpha-tocopherol. Coincubation with ascorbic acid (100 microM, 24 hr) amplified the effects of alpha-tocopherol on eNOS phosphorylation and NO formation, which is possibly related to the regeneration of oxidized alpha-tocopherol by ascorbate. Our data suggest that vasoprotective effects of alpha-tocopherol in vivo may be related to an increase of NO formation. The effect of alpha-tocopherol seems to be dependent on tissue saturation with ascorbic acid, and both vitamins may act synergistically to provide optimal conditions for endothelial NO formation.

Topics: alpha-Tocopherol; Biopterins; Cells, Cultured; Citrulline; Cyclic GMP; Endothelium, Vascular; Humans; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Phosphorylation; Serine; Umbilical Veins

In cultured endothelial cells, the antioxidant, L-ascorbic acid (vitamin C), increases nitric oxide synthase (NOS) enzyme activity via chemical stabilization of tetrahydrobiopterin. Our objective was to determine the effect of vitamin C on NOS function and tetrahydrobiopterin metabolism in vivo. Twenty-six to twenty-eight weeks of diet supplementation with vitamin C (1%/kg chow) significantly increased circulating levels of vitamin C in wild-type (C57BL/6J) and apolipoprotein E (apoE)--deficient mice. Measurements of NOS enzymatic activity in aortas of apoE-deficient mice indicated a significant increase in total NOS activity. However, this increase was mainly due to high activity of inducible NOS, whereas eNOS activity was reduced. Significantly higher tetrahydrobiopterin levels were detected in aortas of apoE-deficient mice. Long-term treatment with vitamin C restored endothelial NOS activity in aortas of apoE-deficient mice, but did not affect activity of inducible NOS. In addition, 7,8-dihydrobiopterin levels, an oxidized form of tetrahydrobiopterin, were decreased and vascular endothelial function of aortas was significantly improved in apoE-deficient mice. Interestingly, vitamin C also increased tetrahydrobiopterin and NOS activity in aortas of C57BL/6J mice. In contrast, long-term treatment with vitamin E (2000 U/kg chow) did not affect vascular NOS activity or metabolism of tetrahydrobiopterin. In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of tetrahydrobiopterin and restoration of eNOS enzymatic activity.

Topics: Animals; Aorta; Apolipoproteins E; Arteriosclerosis; Ascorbic Acid; Biopterins; Cyclic AMP; Cyclic GMP; Dietary Supplements; Disease Models, Animal; Endothelium, Vascular; Enzyme Activation; In Vitro Techniques; Lipids; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Superoxides; Time; Tyrosine; Vasomotor System; Vitamin E

In cultured rat vascular smooth muscle cells (VSMC), inducible nitric oxide synthase (iNOS) expression evoked by interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha was greatly enhanced in hypoxia (2% O(2)), compared to in normoxia. In contrast, iNOS induction by interferon-gamma, lipopolysaccharide or their combination was barely influenced by hypoxia. These results indicate that iNOS induction is regulated by hypoxia in different manners, depending on the stimuli in VSMC. Nitric oxide (NO) production in response to stimulation with interferon-gamma plus lipopolysaccharide was significantly decreased in hypoxia, due to a decrease in the concentration of O(2) as a substrate. In contrast, the level of NO production in hypoxia was almost the same as that in normoxia when the cells were stimulated by IL-1beta. In addition, cGMP increased in response to IL-1beta in hypoxia to a level comparable to that in normoxia. Thus, it seems that the IL-1beta-induced expression of iNOS is up-regulated in hypoxia to compensate for a decrease in the enzyme activity due to the lower availability of O(2) as a substrate, and consequently a sufficient amount of NO is produced to elevate cGMP to an adequate level. In addition, the IL-1beta-induced synthesis of tetrahydrobiopterin, a cofactor for iNOS, was also greatly stimulated by hypoxia in VSMC.

Topics: Animals; Biopterins; Cell Hypoxia; Cells, Cultured; Cyclic GMP; Cytokines; Interferon-gamma; Interleukin-1; Lipopolysaccharides; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxygen; Rats; RNA, Messenger; Tumor Necrosis Factor-alpha

We have recently found evidence for impairment of nitric oxide (NO) formation and induction of oxidative stress in residents of an endemic area of chronic arsenic poisoning in Inner Mongolia, China. To investigate the underlying mechanisms responsible for these phenomena, a subchronic animal experiment was conducted using male New Zealand White rabbits. After 18 weeks of continuous exposure of rabbits to 5 mg/l of arsenate in drinking water, a significant decrease in systemic NO production occurred, as shown by significantly reduced plasma NO metabolites levels (76% of control) and a tendency towards decreased serum cGMP levels (81.4% of control). On the other hand, increased oxidative stress, as shown by significantly increased urinary hydrogen peroxide (H(2)O(2)) (120% of control), was observed in arsenate-exposed rabbits. In additional experiments measuring aortic tension, the addition of either the calcium ionophore A23187 or acethylcholine (ACh) induced a transient vasoconstriction of aortic rings prepared from arsenate-exposed rabbits, but not in those prepared from control animals. This calcium-dependent contractility action observed in aorta rings from arsenate-exposed rabbits was markedly attenuated by the superoxide (O2(.-)) scavenging enzyme Cu, Zn-SOD, as well as diphenyleneiodonium (DPI) or N(G)-nitro-L-arginine methyl ester (L-NAME), which are inhibitors for nitric oxide synthase (NOS). However, the cyclooxygenase inhibitor indomethacin or the xanthine oxidase blocker allopurinol had no effect on this vasoconstriction. These results suggest that arsenate-mediated reduction of systemic NO may be associated with the enzymatic uncoupling reaction of NOS with a subsequent enhancement of reactive oxygen species such as O2(.-), an endothelium-derived vasoconstricting factor. Furthermore, hepatic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)), a cofactor for NOS, were markedly reduced in arsenate-exposed rabbits to 62% of control, while no significant change occurred in cardiac L-arginine levels. These results suggest that prolonged exposure of rabbits to oral arsenate may impair the bioavailability of BH(4) in endothelial cells and, as a consequence, disrupt the balance between NO and O2(.-) produced from endothelial NOS, such that enhanced free radicals are produced at the expense of NO.

Topics: Acetylcholine; Administration, Oral; Allopurinol; Animals; Aorta; Arsenates; Biopterins; Calcimycin; Cyclic GMP; Cyclooxygenase Inhibitors; Endothelium, Vascular; Enzyme Inhibitors; Hydrogen Peroxide; Indomethacin; Ionophores; Liver; Male; New Zealand; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Onium Compounds; Oxidative Stress; Rabbits; Superoxide Dismutase; Superoxides; Teratogens; Vasodilator Agents; Water; Xanthine Oxidase

Tetrahydrobiopterin (BH4) acts as an essential cofactor for the enzymatic activity of nitric oxide (NO) synthases. Biosynthesis of the cofactor BH4 starts from GTP and requires 3 enzymatic steps, which include GTP cyclohydrolase I (GCH I) catalysis of the first and rate-limiting step. In this study we examined the effects of cGMP on GCH I activity in human umbilical vein endothelial cells under inflammatory conditions. Exogenous application of the cGMP analogue 8-bromo-cGMP markedly inhibited GCH I activity in the short term, whereas an cAMP analogue had no effect on GCH I activity under the same condition. NO donors, NOR3 and sodium nitroprusside, elevated the intracellular cGMP level and reduced GCH I activity in the short term. This inhibition of GCH I activity was obliterated in the presence of an NO trapper carboxy-PTIO. NO donors had no effect on GCH I mRNA expression in the short term. Moreover, cycloheximide did not alter the inhibition by NO donors of GCH I activity. These findings suggest that stimulation of the cGMP signaling cascade down-regulates GCH I activity through post translational modification of the GCH I enzyme.

Topics: Biopterins; Cyclic GMP; Dose-Response Relationship, Drug; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; GTP Cyclohydrolase; Humans; Umbilical Veins

Tetrahydrobiopterin (BH4) is an essential co-factor for nitric oxide synthases (NOS). The aim of the present work was to study whether BH4 deficiency affects the vulnerability of neurones in primary culture to hypoxia. Intracellular BH4 levels were depleted by pre-incubating neurones with 5 mm 2,4-diamino-6-hydroxypyrimidine (DAHP) for 18 h, after which cells were exposed for 1 h to normoxic or hypoxic conditions. Our results showed that whereas neurones were resistant to hypoxia-induced cellular damage, BH4 deficiency in neurones led to oxidative stress, mitochondrial depolarization, ATP depletion and necrosis after 1 h of hypoxia. Indeed, hypoxia specifically inhibited mitochondrial complex IV activity in BH4-deficient neurones. All these effects were counteracted when neuronal BH4 levels were restored by incubating cells with exogenous BH4 during the hypoxic period. Moreover, hypoxia-induced damage in BH4-deficient neurones was prevented when Nomega-nitro-l-arginine monomethyl ester (NAME), haemoglobin or superoxide dismutase plus catalase were present during the hypoxic period, suggesting that peroxynitrite might be involved in the process. In fact, BH4 deficiency elicited neuronal NO dysfunction, resulting in an increase in peroxynitrite generation by cells, as shown by the enhancement in tyrosine nitration; this was prevented by supplements of BH4, NAME, haemoglobin or superoxide dismutase plus catalase during hypoxia. Our results suggest that BH4 deficiency converts neuronal NOS into an efficient peroxynitrite synthase, which is responsible for the increase in neuronal vulnerability to hypoxia-induced mitochondrial damage and necrosis.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Biopterins; Cell Death; Cell Hypoxia; Cells, Cultured; Cyclic GMP; Enzyme Inhibitors; GTP Cyclohydrolase; Hypoxanthines; Hypoxia, Brain; Mitochondria; Neurons; Nitric Oxide Synthase; Oxidative Stress; Peroxynitrous Acid; Rats; Rats, Wistar

Nitric oxide (NO) regulates the biological activity of many enzymes and other functional proteins as well as gene expression. In this study, we tested whether pretreatment with NO regulates NO production in response to cytokines in cultured rat hepatocytes. Hepatocytes were recovered in fresh medium for 24 h following pretreatment with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and stimulated to express the inducible NO synthase (iNOS) with interleukin-1beta and interferon-gamma or transfected with the human iNOS gene. NO pretreatment resulted in a significant increase in NO production without changing iNOS expression for both conditions. This effect, which did not occur in macrophages and smooth muscle cells, was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO(2)(-), or NO(3)(-) did not increase the cytokine-induced NO production. SNAP pretreatment increased cytosolic iNOS activity measured only in the absence of exogenous tetrahydrobiopterin (BH(4)). SNAP pretreatment suppressed the level of GTP cyclohydrolase I (GTPCHI) feedback regulatory protein (GFRP) and increased GTPCHI activity without changing GTPCHI protein level. SNAP pretreatment also increased total cellular levels of biopterin and active iNOS dimer. These results suggest that SNAP pretreatment increased NO production from iNOS by elevating cellular BH(4) levels and promoting iNOS subunit dimerization through the suppression of GFRP levels and subsequent activation of GTPCHI.

Topics: Animals; Biopterins; Blotting, Northern; Blotting, Western; Cells, Cultured; Cyclic GMP; Cytokines; Dimerization; Dose-Response Relationship, Drug; Enzyme Activation; GTP Cyclohydrolase; Hepatocytes; Humans; Male; Muscle, Smooth; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Penicillamine; Protein Binding; Rats; Rats, Sprague-Dawley; Time Factors; Transfection

Tetrahydrobiopterin(BH4) serves as an essential cofactor for the biosynthesis of nitric oxide (NO). BH4 is de novo synthesized from GTP and GTP cyclohydrolase I(GCH I) is the rate-limiting enzyme in the biosynthesis of BH4. Under inflammatory conditions, it is reported that endothelial cells release large amount of BH4. In this study, we examined the regulation mechanism of the biosynthesis of BH4 in human umbilical vein endothelial cells(HUVEC). Prostacyclin and forskolin, reagents of stimulation of cAMP signaling cascade, reduced cytokine induced biosynthesis of BH4 through the inhibition of expression of GCH I mRNA. On the other hand, stimulations of NO-cGMP signaling pathway inhibited GCH I activities through the post translational modification of GCH I enzyme. Both two signaling cascade lead to vasodilation. It is suggested that the biosynthesis of BH4 can be regulated by negative feed back regulation systems between endothelium and smooth muscle cells to prevent over stimulated vasodilation.

Topics: Biopterins; Cells, Cultured; Cyclic AMP; Cyclic GMP; Endothelium, Vascular; Epoprostenol; Feedback, Physiological; GTP Cyclohydrolase; Humans; Inflammation; Muscle, Smooth, Vascular; Nitric Oxide; Protein Processing, Post-Translational; Signal Transduction; Umbilical Veins; Vasodilation

Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time- and concentration-dependent fashion without affecting NO synthase (NOS) expression or l-arginine uptake. The present study investigates if the underlying mechanism is related to the NOS cofactor tetrahydrobiopterin. Pretreatment of human umbilical vein endothelial cells with ascorbate (1 microm to 1 mm, 24 h) led to an up to 3-fold increase of intracellular tetrahydrobiopterin levels that was concentration-dependent and saturable at 100 microm. Accordingly, the effect of ascorbic acid on Ca(2+)-dependent formation of citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) was abolished when intracellular tetrahydrobiopterin levels were increased by coincubation of endothelial cells with sepiapterin (0.001-100 microm, 24 h). In contrast, ascorbic acid did not modify the pterin affinity of endothelial NOS, which was measured in assays with purified tetrahydrobiopterin-free enzyme. The ascorbate-induced increase of endothelial tetrahydrobiopterin was not due to an enhanced synthesis of the compound. Neither the mRNA expression of the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I, nor the activities of either GTP cyclohydrolase I or 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the de novo synthesis pathway, were altered by ascorbate. Our data demonstrate that ascorbic acid leads to a chemical stabilization of tetrahydrobiopterin. This was evident as an increase in the half-life of tetrahydrobiopterin in aqueous solution. Furthermore, the increase of tetrahydrobiopterin levels in intact endothelial cells coincubated with cytokines and ascorbate was associated with a decrease of more oxidized biopterin derivatives (7,8-dihydrobiopterin and biopterin) in cells and cell supernatants. The present study suggests that saturated ascorbic acid levels in endothelial cells are necessary to protect tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis.

Topics: Ascorbic Acid; Biopterins; Cells, Cultured; Citrulline; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; GTP Cyclohydrolase; Humans; Hypoxanthines; Nitric Oxide; Nitric Oxide Synthase; Phosphorus-Oxygen Lyases; Pteridines; Pterins; RNA, Messenger; Solutions; Umbilical Cord

The retina of newborn rats consists of the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL) containing amacrine cells and the neuroblastic layer (NBL). In retinal explants, the GCL enters cell death after sectioning of the optic nerve, whereas there is almost no cell death in the NBL. When protein synthesis is inhibited with anisomycin, cell death is blocked in the GCL and induced in the NBL. We tested the roles of nitric oxide (NO) on cell death in the retina in vitro. Either L-arginine, the substrate for NO synthase or the NO donor S:-nitroso-acetylpenicillamine (SNAP) blocked cell death induced by anisomycin in the NBL, but had no effect in the GCL. Sepiapterin, a precursor of the nitric oxide synthase (NOS)-cofactor tetrahydrobiopterin also had a protective effect against anisomycin. The use of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble form of guanylyl cyclase, showed that anti-apoptotic effect of SNAP is partially mediated by cGMP generated by activation of guanylyl cyclase. NADPH-diaphorase histochemistry stained cells only in the GCL and INL. Thus, the degenerative effect of anisomycin is observed within the NBL, whereas the localization of NOS is restricted to the GCL and INL. The protective effect of both the NO substrate and cofactor upon cell death induced by anisomycin in the NBL, indicates that NO produced by amacrine and ganglion cells is a paracrine modulator of cell death within the retinal tissue.

Topics: Animals; Animals, Newborn; Anisomycin; Arginine; Biopterins; Cell Death; Cells, Cultured; Cyclic GMP; Guanylate Cyclase; In Vitro Techniques; NADPH Dehydrogenase; Neuroprotective Agents; Nitric Oxide; Paracrine Communication; Penicillamine; Pteridines; Pterins; Rats; Rats, Inbred Strains; Retina; Retinal Ganglion Cells; S-Nitroso-N-Acetylpenicillamine

Hypochlorous acid/hypochlorite, generated by the myeloperoxidase/H(2)O(2)/halide system of activated phagocytes, has been shown to oxidize/modify low density lipoprotein (LDL) in vitro and may be involved in the formation of atherogenic lipoproteins in vivo. Accordingly, hypochlorite-modified (lipo)proteins have been detected in human atherosclerotic lesions where they colocalize with macrophages and endothelial cells. The present study investigates the influence of hypochlorite-modified LDL on endothelial synthesis of nitric oxide (NO) measured as formation of citrulline (coproduct of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) upon cell stimulation with thrombin or ionomycin. Pretreatment of human umbilical vein endothelial cells with hypochlorite-modified LDL led to a time- and concentration-dependent inhibition of agonist-induced citrulline and cGMP synthesis compared with preincubation of cells with native LDL. This inhibition was neither due to a decreased expression of endothelial NO synthase (eNOS) nor to a deficiency of its cofactor tetrahydrobiopterin. Likewise, the uptake of l-arginine, the substrate of eNOS, into the cells was not affected. Hypochlorite-modified LDL caused remarkable changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the Golgi location without altering myristoylation or palmitoylation of the enzyme. In contrast, cyclodextrin known to deplete plasma membrane of cholesterol and to disrupt caveolae induced only a disappearance of eNOS from the plasma membrane that was not associated with decreased agonist-induced citrulline and cGMP formation. The present findings suggest that mislocalization of NOS accounts for the reduced NO formation in human umbilical vein endothelial cells treated with hypochlorite-modified LDL and point to an important role of Golgi-located NOS in these processes. We conclude that inhibition of NO synthesis by hypochlorite-modified LDL may be an important mechanism in the development of endothelial dysfunction and early pathogenesis of atherosclerosis.

Topics: Antioxidants; Arginine; Biopterins; Blotting, Western; Cell Membrane; Cells, Cultured; Centrifugation, Density Gradient; Citrulline; Cyclic GMP; Cyclodextrins; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Endothelium, Vascular; Golgi Apparatus; Humans; Hypochlorous Acid; Immunoblotting; Immunohistochemistry; Ionomycin; Ionophores; Lipoproteins, LDL; Microscopy, Fluorescence; Myristic Acids; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Palmitic Acids; Precipitin Tests; RNA, Messenger; Subcellular Fractions; Sucrose; Time Factors; Umbilical Veins

In intrapulmonary arteries cultured under hypoxic conditions (5% oxygen) for 7 days, endothelium-dependent relaxation and cGMP accumulation induced by substance P were decreased as compared to those of a normoxic control (20% oxygen). In rabbit mesenteric arteries exposed to chronic hypoxia, however, endothelial dysfunction was not observed. Furthermore, in endothelium-denuded pulmonary arteries exposed to hypoxia, neither relaxation nor cGMP accumulation due to sodium nitroprusside differed from those of the normoxic control. Hypoxia did not change the mRNA expression of endothelial NO synthase (eNOS), the protein expression of eNOS or the eNOS regulatory protein caveolin-1 as assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) or whole-mount immunostaining. Morphological study revealed atrophy of endothelial cells and condensation of the eNOS protein in many cells. These results suggest that chronic hypoxia impaired NO-mediated arterial relaxation without changing either the eNOS protein expression or the NO-sensitivity of smooth muscle cells in pulmonary arteries. Changes in cell structure and organization may be involved in endothelial dysfunction.

Topics: Animals; Arginine; Biopterins; Caveolin 1; Caveolins; Cyclic GMP; Dinoprost; Dose-Response Relationship, Drug; Endothelium, Vascular; Hypoxia; Ionomycin; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroprusside; Organ Culture Techniques; Pulmonary Artery; Rabbits; RNA, Messenger; Substance P; Superoxide Dismutase; Vasoconstriction; Vasodilation; Vasodilator Agents

Substitution of the nitric oxide- (NO-) pathway improves early graft function following lung transplantation. We previously demonstrated that 8-Br-cGMP (second messenger of NO) to the flush solution and tetrahydrobiopterin (BH4, coenzyme of NO synthase) given as additive during reperfusion improve post-transplant graft function. In the present study, the combined treatment with 8-Br-cGMP and BH4 was evaluated.. Unilateral left lung transplantation was performed in weight matched outbred pigs (24-31 kg). In group I, grafts were preserved for 30 h (n=5). 8-Br-cGMP (1mg/kg) was added to the flush solution (Perfadex, 1.5l, 1 degrees C) and BH4 (10mg/kg/h) was given to the recipient for 5h after reperfusion. In group II, lungs were transplanted after a preservation time of 30 h (n=3) and prostaglandin E(1) (250 g) was given into the pulmonary artery (PA) prior to flush. In all recipients 1h after reperfusion the contralateral right PA and bronchus were ligated to assess graft function only. Survival time after reperfusion, extravascular lung water index (EVLWI), hemodynamic variables, and gas exchange (PaO(2)) were assessed during a 12h observation period.. All recipients in group I survived the 12h assessment, whereas none of the group II animals survived more than 4h after reperfusion with a rapid increase of EVLWI up to 24.8+/-6.7 ml/kg. In contrast, in group I EVLWI reached up to 8.9+/-1.5 ml/kg and returned to nearly normal levels at 12h (6.1+/-0.8 ml/kg). In two animals of group I the gas exchange deteriorated slightly. The other three animals showed normal arterial oxygenation over the entire observation time.. Our data indicate that the combined substitution of the NO pathway during preservation and reperfusion reduces ischemia/reperfusion injury substantially and that this treatment even allows lung transplantation after 30 h preservation in this model.

Topics: Animals; Biopterins; Cell Movement; Coenzymes; Cyclic GMP; Extravascular Lung Water; Graft Survival; Hemodynamics; Infusions, Intravenous; Lipid Peroxidation; Lung; Lung Transplantation; Neutrophils; Nitric Oxide Synthase; Organ Preservation; Organ Preservation Solutions; Peroxidase; Pulmonary Gas Exchange; Reperfusion Injury; Swine; Thiobarbituric Acid Reactive Substances; Time Factors

We investigated the effects of anaesthesia on dynamic nitric oxide production, concentrations of tetrahydrobiopterin and the accumulation of cyclic GMP (cGMP) in the rat central nervous system (CNS). Rats were assigned to anaesthesia with halothane, isoflurane, pentobarbital, diazepam, ketamine or xenon (n=6 per group). After 30 min, [14C]L-arginine (i.v.) was given and, after a further 60 min of anaesthesia, rats were killed and exposed immediately to focused microwave radiation. After removal of the brain and spinal cord, nitric oxide production from radiolabelled arginine (and hence nitric oxide synthase activity during anaesthesia) was measured as [14C]L-citrulline by scintillation counting. cGMP was determined by enzyme immunoassay and tetrahydrobiopterin by fluorescence HPLC, in brain regions and the spinal cord. Nitric oxide synthase activity was similar in all brain regions but was lower in the spinal cord, and was unaffected by anaesthesia. cGMP was similar in all areas of the CNS and was significantly decreased in rats anaesthetized with halothane. Isoflurane produced similar effects. In contrast, ketamine and xenon anaesthesia increased cGMP in the spinal cord, brainstem and hippocampus. Diazepam and pentobarbital had no effect. Tetrahydrobiopterin concentrations were similar in all areas of the CNS and were increased in the cortex and hippocampus after anaesthesia. We have shown profound differential effects of anaesthesia on the nitric oxide pathway in the rat CNS.

Topics: Adjuvants, Anesthesia; Anesthetics, Dissociative; Anesthetics, General; Anesthetics, Inhalation; Animals; Biopterins; Brain; Cyclic GMP; Female; Nitric Oxide; Nitric Oxide Synthase; Rats; Spinal Cord

Platelet-derived nitric oxide inhibits platelet aggregation via constitutive NO synthase (NOS). Tetrahydrobiopterin (BH(4)), a cofactor of NOS, augments NO formation, whereas its deficiency decreases NO bioactivity and increases superoxide generation by NOS. The roles of intraplatelet BH(4) in platelet aggregation and thrombus formation, however, are unknown. Accordingly, we investigated whether intraplatelet BH(4) is involved in regulating cyclic flow variations (CFVs) and platelet aggregation in a canine model with stenosed and endothelium-injured coronary arteries that mimics acute coronary syndromes in humans.. After developing CFVs, dogs received saline or BH(4) (10 or 30 mg/kg) intravenously. Intraplatelet BH(4) and cGMP levels were decreased and intraplatelet nitrotyrosine production was increased during CFVs. ADP- and U46619-induced ex vivo platelet aggregation and platelet P-selectin expression were augmented during CFVs. BH(4) administration restored intraplatelet BH(4) and cGMP levels and decreased intraplatelet nitrotyrosine production, resulting in reduced CFVs and inhibited ex vivo platelet aggregation and platelet P-selectin expression. CFVs again developed after N(G)-monomethyl-L-arginine, an inhibitor of NOS, in BH(4)-treated dogs. Ex vivo platelet NOS activity at baseline, during CFVs, and after BH(4) administration did not differ.. Intraplatelet BH(4) may play an important role in regulating thrombus formation by modulating platelet-derived nitric oxide and superoxide generation by platelet NOS.

Topics: Animals; Biopterins; Blood Platelets; Coronary Thrombosis; Cyclic GMP; Dogs; Enzyme Inhibitors; Hemodynamics; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; P-Selectin; Platelet Aggregation; Superoxides; Tyrosine

Ascorbic acid enhances NO bioactivity in patients with vascular disease through unclear mechanism(s). We investigated the role of intracellular ascorbic acid in endothelium-derived NO bioactivity. Incubation of porcine aortic endothelial cells (PAECs) with ascorbic acid produced time- and dose-dependent intracellular ascorbic acid accumulation that enhanced NO bioactivity by 70% measured as A23187-induced cGMP accumulation. This effect was due to enhanced NO production because ascorbate stimulated both PAEC nitrogen oxide (NO(2)(-) + NO(3)(-)) production and l-arginine to l-citrulline conversion by 59 and 72%, respectively, without altering the cGMP response to authentic NO. Ascorbic acid also stimulated the catalytic activity of eNOS derived from either PAEC membrane fractions or baculovirus-infected Sf9 cells. Ascorbic acid enhanced bovine eNOS V(max) by approximately 50% without altering the K(m) for l-arginine. The effect of ascorbate was tetrahydrobiopterin (BH(4))-dependent, because ascorbate was ineffective with BH(4) concentrations >10 microm or in PAECs treated with sepiapterin to increase intracellular BH(4). The effect of ascorbic acid was also specific because A23187-stimulated cGMP accumulation in PAECs was insensitive to intracellular glutathione manipulation and only ascorbic acid, not glutathione, increased the intracellular concentration of BH(4). These data suggest that ascorbic acid enhances NO bioactivity in a BH(4)-dependent manner by increasing intracellular BH(4) content.

Topics: Animals; Aorta; Arginine; Ascorbic Acid; Atrial Natriuretic Factor; Biopterins; Calcimycin; Cattle; Cell Line; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Kinetics; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroprusside; Pteridines; Pterins; Recombinant Proteins; Spodoptera; Swine; Transfection

Nocardia sp. strain NRRL 5646 contains a nitric oxide synthase (NOS) enzyme system capable of generating nitric oxide (NO) from arginine and arginine-containing peptides. To explain possible roles of the NOS system in this bacterium, guanylate cyclase (GC) and tetrahydrobiopterin (H(4)B) biosynthetic enzymes were identified in cell extracts and in culture media. Cell extracts contained GC activity, as measured by the conversion of GTP to cyclic guanosine-3',5'-monophosphate (cGMP) at 9.56 pmol of cGMP h(-1) mg of protein(-1). Concentrations of extracellular cGMP in culture media were significantly increased, from average control levels of 45 pmol cGMP liter(-1) to a maximum of 315 pmol liter(-1), in response to additions of GTP, L-arginine, H(4)B, and sodium nitroprusside to growing Nocardia cultures. On the other hand, the NOS inhibitor N(G)-nitro-L-arginine and the GC inhibitor 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one both dramatically decreased extracellular cGMP levels. Activities for GTP-cyclohydrase-1, 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase, enzymes essential for H(4)B biosynthesis, were present in Nocardia culture extracts at 77.5 pmol of neopterin and 45.8 pmol of biopterin h(-1) mg of protein(-1), respectively. In Nocardia spp., as in mammals, GTP is a key intermediate in H(4)B biosynthesis, and GTP is converted to cGMP by a GC enzyme system that is activated by NO.

Topics: Alcohol Oxidoreductases; Biopterins; Cyclic GMP; GTP Cyclohydrolase; Guanylate Cyclase; Neopterin; Nocardia

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Biopterins; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme Inhibitors; Humans; Isoenzymes; Male; Nitric Oxide; Nitric Oxide Synthase; Phosphoric Diester Hydrolases; Piperazines; Protein Conformation; Purines; Signal Transduction; Sildenafil Citrate; Sulfones

Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether L-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1-100 microM ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 microM) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 microM and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor molecule L-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate L-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid.

Topics: Antioxidants; Ascorbic Acid; Biopterins; Calcium; Cells, Cultured; Citrulline; Cyclic GMP; Drug Synergism; Endothelium, Vascular; Enzyme Induction; Free Radicals; Humans; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III

Exogenous nitric oxide reduces ischemia-reperfusion injury after solid organ transplantation. Tetrahydrobiopterin, an essential cofactor for nitric oxide synthases, may restore impaired endothelium-dependent nitric oxide synthesis. We evaluated whether tetrahydrobiopterin administration to the recipient attenuates lung reperfusion injury after transplantation in swine.. Unilateral left lung transplantation was performed in 15 weight-matched pigs (24-31 kg). Donor lungs were flushed with 1.5 L cold (1 degrees C) low-potassium-dextran solution and preserved for 20 hours. Group I animals served as controls. Group II and III animals were treated with a bolus of tetrahydrobiopterin (20 mg/kg). In addition, in group III a continuous infusion of tetrahydrobiopterin (10 mg/kg per hour over 5 hours) was given. One hour after reperfusion, the recipient right lung was occluded. Cyclic guanosine monophosphate levels were measured in the pulmonary venous and central venous blood. Extravascular lung water index, hemodynamic variables, lipid peroxidation, and neutrophil migration to the allograft were assessed.. In group III a significant reduction of extravascular lung water was noted in comparison with the controls (P =.0047). Lipid peroxidation in lung allograft tissue was significantly reduced in group II (P =.0021) and group III ( P =. 0077) in comparison with group I. Pulmonary venous levels of cyclic guanosine monophosphate increased up to 23 +/- 1 pmol/mL at 5 hours in group II and up to 40 +/- 1 pmol/mL in group III (group I, 4.1 +/- 0.5 pmol/mL [I vs III]; P <.001), whereas central venous levels of cyclic guanosine monophosphate were unchanged in all groups.. Tetrahydrobiopterin administration during lung allograft reperfusion may reduce posttransplantation lung edema and oxygen-derived free radical injury in the graft. This effect is mediated by local enhancement of the nitric oxide/cyclic guanosine monophosphate pathway.

Topics: Animals; Antioxidants; Biopterins; Cryopreservation; Cyclic GMP; Dextrans; Disease Models, Animal; Edema; Endothelium, Vascular; Extravascular Lung Water; Free Radical Scavengers; Hemodynamics; Infusions, Intravenous; Injections, Intravenous; Lipid Peroxidation; Lung Diseases; Lung Transplantation; Neutrophil Infiltration; Nitric Oxide; Nitric Oxide Synthase; Plasma Substitutes; Potassium; Pulmonary Veins; Reactive Oxygen Species; Reperfusion Injury; Swine

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.

Topics: Animals; Biopterins; Brain; Cerebellum; Cyclic GMP; Dystonic Disorders; GTP Cyclohydrolase; Mice; Mice, Neurologic Mutants; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Prosencephalon; Second Messenger Systems; Serotonin; Stimulation, Chemical

Constitutive nitric oxide synthase (NOS) is expressed in rat adenohypophysis and clonal GH3 cells. The mechanisms of action of nitric oxide (NO) to inhibit hormone secretion and the possible role of (6R)-5, 6, 7, 8-tetrahydro-L-biopterin (THB) in the action of endogenous NO were studied in GH3 cells. Inhibiting NOS with N(G)-nitro-L-arginine or trapping NO with oxyhemoglobin enhanced both the basal and TRH-stimulated rat GH release. Sodium nitroprusside did not further decrease either the basal or the TRH-stimulated GH secretion, suggesting that endogenous NO exerted the maximal inhibitory effect. Inhibition of de novo synthesis of THB increased GH secretion. A cyclic guanosine-monophosphate (cGMP) antagonist did not increase the basal GH secretion but enhanced TRH-induced GH release. These findings suggest that endogenous NO plays an inhibitory role on basal GH release and TRH-stimulated hormone release from GH3 cells in an autocrine or paracrine fashion, at least partly, through a cGMP-dependent pathway. It is also suggested that endogenous THB plays a role in NO production and subsequent inhibition of hormone secretion in GH3 cells.

Topics: Animals; Biopterins; Cell Line; Cyclic GMP; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Growth Hormone; Nitric Oxide; Oxyhemoglobins; Rats; Thionucleotides; Thyrotropin-Releasing Hormone

We assessed the role of .NO in recombinant adenovirus-mediated gene transfer both in vitro and in vivo. NIH3T3 fibroblasts, stably transfected with the human inducible nitric oxide synthase, but lacking tetrahydrobiopterin (NIH3T3/iNOS [inducibile nitric oxide synthase]), were infected with replication-deficient adenovirus (E1-deleted), containing either the luciferase or the Lac Z reporter genes (AdCMV-Luc and AdCMV-Lac Z; 1-10 plaque forming units [pfu]/cell). Incubation of infected cells with sepiapterin (50 microM), a precursor of tetrahydrobiopterin, progressively increased nitrate/nitrite levels in the medium and decreased both luciferase and beta-galactosidase protein expression to approximately 60% of their corresponding control values, 24 h later. NIH3T3/iNOS cells had normal ATP (adenosine 5'-triphosphate) levels and did not release LDH(lactic dehydrogenase) into the medium. Pretreatment of these cells with N(G)-monomethyl-L-arginine (L-NMMA; 1 mM), an inhibitor of iNOS, prevented the sepiapterin-mediated induction of .NO and restored gene transfer to baseline values. Incubation of NIH3T3/iNOS with 8-bromo-cGMP (400 microM) in the absence of sepiapterin, or exposure of AdCMV-Luc to large concentrations of .NO, did not alter the efficacy of gene transfer. .NO produced by NIH3T3/iNOS cells also suppressed beta-galactosidase expression in NIH3T3 cocultured cells stably transfected with beta-galactosidase gene, suggesting .NO inhibited gene expression at either the transriptional or posttranscriptional levels. To investigate the effects of inhaled .NO on gene transfer in vivo, CD1 mice received an intratracheal instillation of AdCMV-Luc (4 x 10(9) pfu in 80 microl of saline) and exposed to .NO (25 ppm in room air) for 72 h. At that time, no significant degree of lung inflammation was detected by histological examination. However, lung luciferase activity decreased by 53% as compared with air breathing controls (P < 0.05; n > or = 8). We concluded that overproduction of .NO decreases the efficiency of adenovirus-mediated gene transfer in lung cells in the absence of cytotoxicity or inflammation.

Topics: 3T3 Cells; Adenoviruses, Human; Animals; Antioxidants; beta-Galactosidase; Biopterins; Cell Survival; Cyclic GMP; Enzyme Inhibitors; Gene Transfer Techniques; Genes, Reporter; Genetic Vectors; Humans; Luciferases; Lung; Mice; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; omega-N-Methylarginine; Pteridines; Pterins; Transfection

1. Basal electrogenic Cl- secretion, measured as the short-circuit current (Isc), was variable in ileum removed from tetrahydrobiopterin (BH4)-deficient hph-1 mice and wild-type controls in vitro, although values were not significantly different. 2. The basal nitrite release and mucosal cyclic guanosine 3',5'-monophosphate (cyclic GMP) production were similar in control and BH4-deficient ileum. 3. Mucosally added Escherichia coli heat-stable toxin (STa, 55 ng ml-1) increased the nitrite release, cyclic GMP levels and the Isc in control ileum, but its secretory actions were reduced in BH4-deficient ileum. 4. L-Arginine (1 mM) increased the nitrite release, cyclic GMP production and the Isc in control ileum, but the actions were reduced in BH4-deficient ileum. 5. Serosal carbachol (1 mM) stimulated maximum short-circuit currents of similar magnitude in both control and BH4-deficient ileum, whilst nitrite release and cyclic GMP production were minimal. 6. E. coli STa and L-arginine increased electrogenic Cl- secretion across intact mouse ileum in vitro by releasing nitric oxide and elevating mucosal cyclic GMP. The inhibition of these processes in the hph-1 mouse ileum suggests that BH4 may be a target for the modulation of electrogenic transport, and highlight the complexity of the interactions between nitric oxide and cyclic GMP in the gut.

Topics: Animals; Antioxidants; Arginine; Biopterins; Carbachol; Chloride Channels; Cholinergic Agonists; Cyclic GMP; Ileum; In Vitro Techniques; Intestinal Mucosa; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Muscle, Smooth; Nitric Oxide; Nitrites; Time Factors

Smooth muscle cells (SMCs) play a key role in the pathogenesis of vascular diseases. The objectives of this study were to determine whether transfer of recombinant endothelial nitric oxide synthase (eNOS) gene to porcine coronary artery smooth muscle cell (CSMCs) would result in expression of a functional enzyme and to assess the effect of expression of eNOS on cell proliferation. CSMCs were transduced in vitro with adenoviral vectors encoding cDNA for eNOS (AdeNOS) and beta-galactosidase (Ad beta Gal). In contrast to Ad beta Gal- or sham-transduced cells, CSMCs transduced with AdeNOS stained positive with the NADPH-diaphorase stain, acquired calcium-dependent NOS activity (measured by the conversion of [3H]L-arginine to [3H]L-citrulline), had increasing cyclic 3',5' cGMP levels with increasing concentrations of the vector, and produced increased amounts of nitrite. cGMP production by AdeNOS-transduced cells was augmented by increasing intracellular levels of the eNOS cofactor tetrahydrobiopterin. CSMCs transduced with AdeNOS showed diminished serum-stimulated DNA synthesis as measured by thymidine uptake. Cell proliferation was diminished in AdeNOS-transduced CSMCs as assessed by cell counts 3 and 6 days after serum stimulation of quiescent CSMCs. The present study demonstrates that adenovirus-mediated gene transfer of eNOS to CSMCs results in the expression of a functional enzyme whose activity can be augmented by increasing intracellular levels of tetrahydrobiopterin. Expression of recombinant eNOS in CSMCs results in inhibition of serum-stimulated DNA synthesis and cell proliferation. These findings imply that eNOS gene transfer to SMCs may be a unique mode of increasing local NO production in the arterial wall.

Topics: Adenoviridae; Animals; Biopterins; Cattle; Cell Division; Cells, Cultured; Coronary Vessels; Cyclic GMP; DNA Replication; Enzyme Induction; Genes, Reporter; Genetic Vectors; GTP Cyclohydrolase; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Pteridines; Pterins; Recombinant Fusion Proteins; Superoxide Dismutase; Swine; Transfection

Smoking impairs the endothelium-dependent relaxation of arteries and veins, with the maximum relaxation in response to the calcium ionophore A23187 of saphenous vein rings being reduced from 53 +/- 4% in nonsmokers to 27 +/- 5% in smokers. We have investigated whether this endothelial dysfunction was attributable to altered activity or concentration of nitric oxide synthase (NOS). The concentration of NOS in saphenous vein endothelium, determined by Western blotting and immunohistochemistry, was not different in nonsmokers and smokers. Nitrite production from vein strips stimulated with A23187 was higher in nonsmokers (median 23.6 nmol.cm-2.h-1) than smokers (median 3.3 nmol.cm-2.h-1), P=.001, this difference being abolished when vein strips were preincubated in the presence of NG-monomethyl-L-arginine. Organ chamber studies to monitor the endothelium-dependent relaxation of vein rings in response to A23187 showed that preincubation of rings from smokers with either L-arginine (3mmol/L) or superoxide dismutase (250 U/mL) did not improve the maximum relaxation. In contrast, preincubation of vein rings from smokers with 20 micromol/L tetrahydrobiopterin increased the maximum relaxation from 27 +/- 5% to 51 +/- 6%, P=.01. Preincubation of vein from smokers with tetrahydrobiopterin also significantly increased nitrite and cGMP production in response to stimulation with A23187. The impaired endothelium-dependent relaxation of saphenous vein rings from smokers appears to be caused by a reduction in the activity of endothelial NOS that is attributable to an inadequate supply of the coenzyme tetrahydrobiopterin.

Topics: Adult; Biopterins; Blotting, Western; Calcimycin; Cyclic GMP; Endothelins; Endothelium, Vascular; Female; Humans; Immunohistochemistry; Ionophores; Male; Microscopy, Electron, Scanning; Middle Aged; Nitric Oxide Synthase; Nitrites; Organ Culture Techniques; Saphenous Vein; Smoking; Vasodilation

Tetrahydrobiopterin is an essential cofactor required for activation of NO synthase. However, in intact arteries, the exact role of tetrahydrobiopterin in the regulation of NO synthase activity is not fully understood. The present study was designed to determine the effect of increasing intracellular tetrahydrobiopterin levels on endothelial function in isolated canine middle cerebral arteries. The arterial segments were incubated in MEM for 24 hours at 37 degrees C in the presence or absence of a tetrahydrobiopterin precursor, sepiapterin (10(-4) mol/L), and/or superoxide dismutase (150 U/mL). The rings were suspended for isometric tension recording. Tetrahydrobiopterin levels were assayed by high-performance liquid chromatography. Production of cGMP was measured by radioimmunoassay. Incubation with sepiapterin markedly increased intracellular tetrahydrobiopterin levels. In sepiapterin-treated arteries, endothelium-dependent relaxations to calcium ionophore A23187 and intracellular cGMP levels were significantly reduced. Superoxide dismutase alone did not affect either relaxation to A23187 or production of cGMP. However, when arteries were incubated with superoxide dismutase plus sepiapterin, endothelium-dependent relaxations to A23187, as well as cGMP production, were significantly augmented. The augmentation of cGMP was observed in rings with (but not without) endothelium. Incubation of arteries in calcium-free medium almost abolished the synergistic effect of tetrahydrobiopterin and superoxide dismutase on cGMP production. These results demonstrate that increased availability of tetrahydrobiopterin may activate endothelial NO synthase. This effect appears to be critically dependent on the presence of superoxide dismutase.

Topics: Animals; Anions; Biopterins; Calcium; Cerebral Arteries; Culture Media; Cyclic GMP; Dogs; Endothelium, Vascular; In Vitro Techniques; Pteridines; Pterins; Superoxides; Vasodilation

In this study, the effect of tetrahydrobiopterin deficiency on the nitric oxide/cGMP pathway has been investigated in cerebellar slices derived from the hph-1 mouse. This animal displays a partial deficiency of tetrahydrobiopterin. Basal levels of cGMP were significantly reduced (-29.5%) in the hph-1 mouse cerebellum compared to controls. Following kainate stimulation (500 microM) cGMP levels increased in both control and hph-1 preparations but were again significantly lower (-29.1%) in the hph-1 mouse. Exposure of slices to the nitric oxide donors, S-nitroso-N-acetylpenicillamine and S-nitroso-glutathione, revealed no difference in cGMP accumulation between the two groups. These findings suggest that the cerebellar nitric oxide/cGMP pathway may be impaired in partial tetrahydrobiopterin deficiency states due to diminished nitric oxide formation.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Antioxidants; Biopterins; Cerebellum; Cyclic GMP; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutathione; Kainic Acid; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Mutant Strains; Mutation; Nitric Oxide; Nitroarginine; Nitroso Compounds; Organ Culture Techniques; Oxyhemoglobins; Penicillamine; Platelet Aggregation Inhibitors; S-Nitroso-N-Acetylpenicillamine; S-Nitrosoglutathione

The L-arginine/nitric oxide pathway plays a key role in the regulation of arterial tone. Biosynthesis of nitric oxide requires activation of nitric oxide synthase in the presence of tetrahydrobiopterin as a cofactor. Biochemical studies demonstrated that activation of purified nitric oxide synthase at suboptimal concentrations of tetrahydrobiopterin leads to production of hydrogen peroxide. The present experiments were designed to determine whether in coronary arteries inhibition of tetrahydrobiopterin synthesis may favor nitric oxide synthase-catalyzed production of hydrogen peroxide.. Primary branches of canine left anterior descending artery were incubated for 6 hours in minimum essential medium in the presence or in the absence of the tetrahydrobiopterin synthesis inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP; 10(-2) mol/L). Arterial rings were suspended for isometric tension recording. Production of cGMP was measured by radioimmunoassay. Experiments were performed in the presence of indomethacin (10(-5) mol/L). During contractions to the thromboxane A2/prostaglandin H2 receptor agonist U46619 (10(-7) mol/L), calcium ionophore A23187 (10(-9) to 10(-6) mol/L) caused endothelium-dependent relaxations. A nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (3 x 10(-4) mol/L), significantly inhibited these relaxations. In DAHP-treated arteries, relaxations to A23187 and its stimulating effect on cGMP production were significantly reduced in the presence of catalase (1200 U/mL). By contrast, catalase did not exert any effect in rings incubated in the absence of DAHP. Furthermore, the inhibitory effect of catalase on A23187-induced relaxations was abolished when coronary arteries were incubated in the presence of DAHP plus a liposoluble analogue of tetrahydrobiopterin, 6-methyltetrahydropterin (10(-4) mol/L).. The present study suggests that hydrogen peroxide may be a mediator of endothelium-dependent relaxations in coronary arteries depleted of tetrahydrobiopterin. This initially compensatory response, triggered by a dysfunctional nitric oxide synthase, may represent an important mechanism underlying oxidative vascular injury.

Topics: Amino Acid Oxidoreductases; Animals; Biopterins; Calcimycin; Coronary Vessels; Cyclic GMP; Dogs; Endothelium, Vascular; Hydrogen Peroxide; Hypoxanthines; Muscle Relaxation; Nitric Oxide Synthase

Hepatocyte plating density is known to affect cell function. Human and rat hepatocytes have been shown to express the inducible nitric oxide synthase (INOS) in response to cytokines plus lipopolysaccharide (LPS). The following studies were performed to determine the effects of hepatocyte plating density on the regulation of INOS. Rat hepatocytes were plated at densities from 10(4) to 20 x 10(4) hepatocytes/cm2 and stimulated with a combination of LPS, interferon-gamma, interleukin-1, and tumor necrosis factor. We found that NO2- plus NO3- released from stimulated hepatocytes declines with increasing hepatocyte density. Similar effects were seen for 3',5'-cyclic monophosphate release into supernatants and in the amount of nonheme iron-nitrosyl signals measured by electron paramagnetic resonance spectroscopy. Limitations of substrate (L-arginine) and 5,6,7,8-tetrahydrobiopterin were excluded as cause of the reduced nitric oxide generation at higher densities. Although mRNA levels for INOS were not influenced when measured at 24 h, there was a marked reduction in INOS enzyme activity and INOS protein detectable by Western blotting at higher cell density. Total protein synthesis decreased as hepatocyte density increased in both nonstimulated and stimulated hepatocytes at higher cell densities. These data suggest that reduced INOS translation may account for the density-dependent reduction in INOS activity in cultured hepatocytes. The importance of this phenomenon remains to be determined in vivo but has important implications for the in vitro study of INOS expression.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Biopterins; Cell Count; Cells, Cultured; Cyclic GMP; Cytokines; Humans; Lipopolysaccharides; Liver; Male; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Rats; Rats, Sprague-Dawley; RNA, Messenger

Stimulation of nitric oxide (NO) synthase in endothelial cells by Ca2+ influx leads to increased intracellular levels of cGMP. NO synthase from various sources is known to use tetrahydrobiopterin, flavins, and NADPH as cofactors. We studied the effect of interferon-gamma, tumor necrosis factor-alpha, and lipopolysaccharide on tetrahydrobiopterin biosynthetic activities in human umbilical vein endothelial cells (HUVEC). These stimuli led to an up to 40-fold increase of GTP cyclohydrolase I (EC 3.5.4.16) activity and to increased accumulation of neopterin and tetrahydrobiopterin in HUVEC. Further enzyme activities of tetrahydrobiopterin biosynthesis, i.e. 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase (EC 1.1.1.153), remained unchanged. NO synthase activity in protein fractions from homogenates of cells treated with interferon-gamma plus tumor necrosis factor-alpha was not influenced as compared with untreated controls. However, interferon-gamma alone or in combination with tumor necrosis factor-alpha significantly increased intracellular cGMP formation in intact HUVEC by 50 and 80%, respectively. These stimuli increased intracellular tetrahydrobiopterin concentrations up to 14-fold. NO-triggered cGMP formation was similarly increased by incubation of otherwise untreated cells with sepiapterin, leading to elevated intracellular tetrahydrobiopterin levels. Thus, cytokines indirectly stimulate the activity of constitutive NO synthase in HUVEC by upregulating production of the cofactor tetrahydrobiopterin.

Topics: Amino Acid Oxidoreductases; Biopterins; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; GTP Cyclohydrolase; Humans; Hypoxanthines; Interferon-gamma; Lipopolysaccharides; Neopterin; Nitric Oxide; Nitric Oxide Synthase; Pteridines; Pterins; Tumor Necrosis Factor-alpha; Umbilical Veins

Studies with cell homogenates and cultures had indicated earlier that nitric oxide (NO) synthase is dependent on tetrahydrobiopterin, which apparently functions as a cofactor for the enzyme. The present results showed a vasodilator response to tetrahydrobiopterin in precontracted aorta, which is attained via an increase of the intracellular cyclic GMP level. Furthermore, L-Ng-nitro-arginine-methyl ester inhibits the tetrahydrobiopterin-induced vasodilation, showing the involvement of NO synthase.

Topics: Animals; Aorta, Thoracic; Arginine; Biopterins; Cyclic GMP; In Vitro Techniques; Methoxamine; Muscle Contraction; NG-Nitroarginine Methyl Ester; Radioimmunoassay; Rats; Vasodilation

We investigated the mechanisms by which cytokines lead to a diminished responsiveness of vascular smooth muscle to vasoconstrictors. The attenuation of noradrenaline-induced contraction by 6 to 24 h incubations with the cytokines, tumor necrosis factor and interleukin-1, in endothelium-denuded rabbit aorta was associated with an increase in intracellular cyclic GMP level. This increase was abolished by the stereoselective inhibitor of nitric oxide-synthase, NG-nitro-L-arginine and by cycloheximide. Formation of nitric oxide was detected in the cytosol of cytokine-treated native and cultured smooth muscle cells by activation of purified soluble guanylate cyclase, and depended on tetrahydrobiopterin, but not on Ca2(+)-calmodulin. The results indicate that cytokines induce a nitric oxide-synthase of the macrophage-type in vascular smooth muscle.

Topics: Amino Acid Oxidoreductases; Animals; Aorta, Thoracic; Arginine; Biopterins; Cells, Cultured; Cyclic GMP; Cytokines; Enzyme Induction; In Vitro Techniques; Interferon-gamma; Interleukin-1; Muscle, Smooth, Vascular; NADP; Nitric Oxide; Nitric Oxide Synthase; Rabbits; Tumor Necrosis Factor-alpha

L-Arginine-derived nitric oxide acts as an inter- and intracellular signal molecule with cytosolic guanylyl cyclase as the effector system. Two NO synthase isoenzymes are postulated: a cytokine-inducible enzyme in macrophages and a constitutive, Ca2(+)-regulated enzyme in various other cells. An NO synthase was isolated from porcine cerebellum by ammonium sulfate precipitation and affinity chromatography on 2',5'-ADP-Sepharose. The enzyme was identified as an NO synthase with a specific NO-chemiluminescence method and with purified cytosolic guanylyl cyclase as an NO-sensitive detection system. The purified NO synthase was, besides Ca2+/calmodulin and NADPH, largely dependent on tetrahydrobiopterin as a cofactor.

Topics: Amino Acid Oxidoreductases; Animals; Biopterins; Calcium; Calmodulin; Cerebellum; Citrulline; Cyclic GMP; Guanylate Cyclase; Molecular Weight; Nitric Oxide Synthase; Swine