Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin

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.     

GSH depletion, K-Cl cotransport, and regulatory volume decrease in high-K/high-GSH dog red blood cells

Thiol reagents activateK-Cl cotransport (K-Cl COT), the Cl-dependent and Na-independentouabain-resistant K flux, in red blood cells (RBCs) of several species,upon depletion of cellular glutathione (GSH). K-Cl COT isphysiologically active in high potassium (HK), high GSH (HG) dog RBCs.In this unique model, we studied whether the same inverse relationshipexists between GSH levels and K-Cl COT activity found in other species.The effects of GSH depletion by three different chemical reactions[nitrite (NO₂)-mediated oxidation, diazene dicarboxylicacid bis-N,N-dimethylamide (diamide)-induceddithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB)] were tested on K-Cl COT and regulatory volume decrease (RVD).After 85% GSH depletion, all three interventions stimulated K-Cl COThalf-maximally with the following order of potency: diamide > NO₂ > CDNB. Repletion of GSH reversed K-Cl COTstimulation by 50%. Cl-dependent RVD accompanied K-Cl COT activationby NO₂ and diamide. K-Cl COT activation at concentrationratios of oxidant/GSH greater than unity was irreversible, suggestingeither nitrosothiolation, heterodithiol formation, or GST-mediateddinitrophenylation of protein thiols. The data support the hypothesisthat an intact redox system, rather than the absolute GSH levels,protects K-Cl COT activity and cell volume regulation from thiol modification.

     

eNOS translocation but not eNOS phosphorylation is dependent on intracellular Ca2+ in human atrial myocardium

In endothelial cells, two ways of endothelial nitric oxide (NO) synthase (eNOS) activation are known: 1) translocation and 2) Akt-dependent phosphorylation of the enzyme at Ser¹¹⁷⁷ (Ser¹¹⁷⁷ eNOS). We have recently shown that agonist-induced Ser¹¹⁷⁷ eNOS phosphorylation also occurs in human myocardium (10). In this study, we investigated the Ca²⁺ dependency of these two mechanisms in human atrium. Therefore, atrial tissue was obtained from patients who underwent coronary artery bypass operations. In immunohistochemical experiments, the translocated form of eNOS and phosphorylated Ser¹¹⁷⁷ eNOS were labeled using specific antibodies. eNOS translocation was measured in the absence and presence of the Ca²⁺ chelator BAPTA before and after application of BRL 37344 (BRL), a ₃-adrenoceptor agonist that increases eNOS activity (34). In the absence of BAPTA, BRL time dependently increased the staining intensity of translocated eNOS, whereas in the presence of BAPTA, this effect was blunted. In contrast, BRL clearly increased the staining of phosphorylated Ser¹¹⁷⁷ eNOS even in the presence of BAPTA. This observation was confirmed using Western blot analysis. Using the NO-sensitive dye diaminofluorescein, we have demonstrated that BRL induced a strong NO release. This effect was completely abolished in the presence of BAPTA but was unaffected by LY-292004, an inhibitor of phosphatidylinositol 3-kinase activity and eNOS phosphorylation. Although Ca²⁺ dependent, neither the translocation of eNOS nor NO release was changed by the adenylate cyclase activator forskolin. In conclusion, 1) in human atrial myocardium, BRL-induced eNOS translocation but not Ser¹¹⁷⁷ eNOS phosphorylation is dependent on intracellular Ca²⁺. 2) In atrial myocardium, eNOS-translocation and not Ser¹¹⁷⁷ eNOS phosphorylation is responsible for generating the main amount of NO. 3) Although Ca²⁺ dependent, eNOS translocation and NO release could not be mimicked by adenylate cyclase activation as a mediator of -adrenergic stimulation. ₃-adrenoceptor; BRL 37344; cardiomyocyte; heart; Ca²⁺ regulation     

Endothelial response to stress from exogenous Zn2+ resembles that of NO-mediated nitrosative stress, and is protected by MT-1 overexpression

While nitric oxide (NO)-mediated biological interactions have been intensively studied, the underlying mechanisms of nitrosative stress with resulting pathology remain unclear. Previous studies have demonstrated that NO exposure increases free zinc ions (Zn²⁺) within cells. However, the resulting effects on endothelial cell survival have not been adequately resolved. Thus the purpose of this study was to investigate the role of altered zinc homeostasis on endothelial cell survival. Initially, we confirmed the previously observed significant increase in free Zn²⁺ with a subsequent induction of apoptosis in our pulmonary artery endothelial cells (PAECs) exposed to the NO donor N-[2-aminoethyl]-N-[2-hydroxy-2-nitrosohydrazino]-1,2-ethylenediamine. However, NO has many effects upon cell function and we wanted to specifically evaluate the effects mediated by zinc. To accomplish this we utilized the direct addition of zinc chloride (ZnCl₂) to PAEC. We observed that Zn²⁺-exposed PAECs exhibited a dose-dependent increase in superoxide (O₂^–·) generation that was localized to the mitochondria. Furthermore, we found Zn²⁺-exposed PAECs exhibited a significant reduction in mitochondrial membrane potential, loss of cardiolipin from the inner leaflet, caspase activation, and significant increases in TdT-mediated dUTP nick end labeling-positive cells. Furthermore, using an adenoviral construct for the overexpression of the Zn²⁺-binding protein, metallothionein-1 (MT-1), we found either MT-1 overexpression or coincubation with a Zn²⁺-selective chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene-diamide, in PAECs significantly protected the mitochondria from both NO and Zn²⁺-mediated disruption and induction of apoptosis and cell death. In summary, our results indicate that a loss of Zn²⁺ homeostasis produces mitochondrial dysfunction, increased oxidative stress, and apoptotic cell death. We propose that regulation of Zn²⁺ levels may represent a potential therapeutic target for disease associated with both nitrosative and oxidative stress. reactive nitrogen species; apoptosis mitochondrial dysfunction     

Hydrogen peroxide restrains endothelium-derived nitric oxide bioactivity -- role for iron-dependent oxidative stress

Vascular diseases are characterized by impairment of endothelial-derived nitric oxide (NO) bioactivity and increased vascular levels of hydrogen peroxide (H(2)O(2)). Here we examined the implications of H(2)O(2) for agonist-stimulated endothelial NO bioactivity in rabbit aortic rings and cultured porcine aortic endothelial cells (PAEC). Vessels pre-treated with H(2)O(2) exhibited impaired endothelial-dependent relaxation induced by acetylcholine or calcium ionophore. In contrast, H(2)O(2) had no effect on endothelium-independent relaxation induced by a NO donor, indicating a defect in endothelium-derived NO. This defect was not related to eNOS catalytic activity; treatment of PAEC with H(2)O(2) enhanced agonist-stimulated eNOS activity indicated by increased eNOS phosphorylation at Ser-1177 and de-phosphorylation at Thr-495 and enhanced conversion of [(3)H]-L-arginine to [(3)H]-L-citrulline that was prevented by inhibitors of Src and phosphatidylinositol-3 kinases. Despite activating eNOS, H(2)O(2) impaired endothelial NO bioactivity indicated by attenuation of the increase in intracellular cGMP in PAEC stimulated with calcium ionophore or NO. The decrease in cGMP was not due to impaired guanylyl cyclase as H(2)O(2) treatment increased cGMP accumulation in response to BAY 41-2272, a NO-independent activator of soluble guanylyl cyclase. At concentrations that impaired endothelial NO bioactivity H(2)O(2) increased intracellular oxidative stress and size of the labile iron pool in PAEC. The increase in oxidative stress was prevented by the free radical scavenger's tempol or tiron and the iron chelator desferrioxamine and these antioxidants reversed the H(2)O(2)-induced impairment of NO bioactivity in PAEC. This study shows that despite promoting eNOS activity, H(2)O(2) impairs endothelial NO bioactivity by promoting oxidative inactivation of synthesized NO. The study highlights another way in which oxidative stress may impair NO bioactivity during vascular disease.     

Lack of endothelial nitric oxide synthase decreases cardiomyocyte proliferation and delays cardiac maturation

We recently demonstrated that deficiency in endothelial nitric oxide synthase (eNOS) results in congenital septal defects and postnatal heart failure. The aim of this study was to investigate the role of eNOS in cardiomyocyte proliferation and maturation during postnatal development. Cultured eNOS knockout (eNOS^–/–) cardiomyocytes displayed fewer cells and lower bromodeoxyuridine (BrdU) incorporation in vitro compared with wild-type (WT) cardiomyocytes (P < 0.05). Treatment with the nitric oxide (NO) donor diethylenetriamine NONOate increased BrdU incorporation and cell counts in eNOS^–/– cardiomyocytes (P < 0.05). Inhibition of nitric oxide synthase activity using N^G-nitro-L-arginine methyl ester decreased the level of BrdU incorporation and cell counts in WT cardiomyocytes (P < 0.05). Vascular endothelial growth factor (VEGF) increased the level of BrdU incorporation in cultured WT cardiomyocytes in a dose- and time-dependent manner (P < 0.05). Conversely, VEGF did not alter BrdU incorporation in eNOS^–/– cardiomyocytes (P = not significant). Furthermore, deficiency in eNOS significantly decreased BrdU labeling indexes in neonatal hearts in vivo. Although WT hearts displayed a rapid decrease in atrial natriuretic peptide (ANP) expression in the first week of neonatal life, ANP expression in eNOS^–/– hearts remain elevated. Our study demonstrated that NO production from eNOS is necessary for postnatal cardiomyocyte proliferation and maturation, suggesting that eNOS plays an important role during postnatal heart development. proliferation; heart development     

Stimulation of cystine uptake by nitric oxide: regulation of endothelial cell glutathione levels

Nitric oxide (NO)is known to produce some of its biological activity throughmodification of cellular thiols. Return of cellular thiols to theirbasal state requires the activity of the GSH redox cycle, suggestingimportant interactions between NO signaling and regulation of cellularredox status. Because continuous exposure to NO may lead to adaptiveresponses in cellular redox systems, we investigated the effects of NOon cellular GSH levels in vascular endothelial cells. Acute exposure (1 h) of cells to >1 mMS-nitroso-N-acetyl-penicillamine (SNAP) led to depletion of GSH. On the other hand, chronic exposure tolower concentrations of SNAP (1 mM) led to a progressive increase incytosolic GSH, reaching fourfold above basal by 16 h. The mechanism mayinvolve an increase in GSH biosynthesis through effects on biosyntheticenzymes or through increased supply of cysteine, the limitingsubstrate. In this regard, we report that chronic exposure to SNAP ledto a concentration-dependent increase in cystine uptake over a timecourse similar to that seen for elevation of GSH. The effect of SNAP oncystine uptake was inhibitable by either cycloheximide or actinomycinD, suggesting a requirement for both RNA and protein synthesis.Furthermore, uptake was Na⁺independent and was blocked by extracellular glutamate. Extracellular glutamate also blocked SNAP-mediated elevation of cytosolic GSH. Finally, in a coculture model, NO produced by cytokine-pretreated RAW264.7 cells increased both GSH levels and cystine uptake in naiveendothelial cells. These findings strongly suggest that NO leads toadaptive induction of thex_c amino acidtransport system, increased cystine uptake, and elevation ofintracellular GSH levels.     

Redox regulation of the signaling pathways leading to eNOS phosphorylation

Oxidative stress mediates positive and negative effects on physiological processes. Recent reports show that H(2)O(2) induces phosphorylation and activation of endothelial nitric oxide synthase (eNOS) through an Akt-phosphorylation-dependent pathway. In this study, we assessed activation of eNOS and Akt by determining their phosphorylation status. Whereas moderate levels of H(2)O(2) (100 microM) activated the Akt/eNOS pathway, higher levels (500 microM) did not, suggesting differential effects by differing levels of oxidative stress. We then found that two pro-oxidants with activity on sulfhydryl groups, 1-chloro-2,4-dinitrobenzene (CDNB) and diethyl maleate (DEM), blocked the phosphorylation events induced by 100 microM H(2)O(2). GSH was not a target thiol in this system because buthionine sulfoximine did not inhibit this phosphorylation. However, down-regulation of cell membrane surface and intracellular free thiols was associated with the inhibition of phosphorylation, suggesting that oxidation of non-GSH thiols inhibits the H(2)O(2)-induced phosphorylation of eNOS and Akt. DTT reversed the inhibitory effects of CDNB and DEM on Akt phosphorylation and concomitantly restored cell surface thiol levels more efficiently than it restored intracellular thiols, suggesting a more prominent role for the former. Similarly, DEM and CDNB inhibited TNF-alpha-induced Akt and eNOS phosphorylation, suggesting that thiol modification is involved in eNOS inductive pathways. Our findings suggest that eNOS activation is exquisitely sensitive to regulation by redox and that cell surface thiols, other than glutathione, regulate signal transduction leading to phosphorylation of Akt and eNOS.     

Plasma membrane-associated endothelial nitric oxide synthase and activity in aging rat aortic vascular endothelia markedly decline with age

The mechanisms leading to the age-related loss of endothelial nitric oxide (NO) and NO-dependent vasodilation remain largely unknown. Freshly isolated endothelium from young (6 months) and old (36 months) F344xBrN rats were analyzed for endothelial nitric oxide synthase (eNOS) protein, its subcellular distribution, and association with regulatory proteins. Results show that both vessel ring vasoreactivity and A23187-induced eNOS activity in isolated endothelial cells significantly (p < or = 0.05) declined with age. Levels of cGMP, a reliable marker for NO bioactivity also declined significantly (p < or = 0.01). However, no change in overall eNOS protein was evident. Subcellular fractionation studies revealed an age-related loss in active, plasma membrane-bound eNOS relative to eNOS in the Golgi/cytosol of the endothelium. Plasma membrane-associated eNOS in aged endothelium was also less complexed with the activating proteins Hsp90 and Akt and more associated with to caveolin-1, which inhibits eNOS activity. These results suggest that age-dependent loss of NO may be partly caused by differences in eNOS subcellular distribution and its association with inhibitory proteins.     

Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N-monomethyl-L-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation. vascular biology; atherosclerosis; mouse models     

Apocynin improves oxygenation and increases eNOS in persistent pulmonary hypertension of the newborn

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.     

Regional differences in endothelial function in horse lungs: possible role in blood flow distribution?

We investigated regional differences of in vitroresponses of pulmonary arteries (6-mm OD) from the dorsocaudal (top)and cranioventral (bottom) lung regions to endothelium-dependentvasodilators (methacholine, bradykinin, and calcium ionophore A-23187).Methacholine relaxed endothelium-intact top vessels; however, in bottomvessels, a small relaxation preceded a profound contraction. In topvessels, removal of endothelial cells converted relaxation tocontraction, and in bottom vessels it abolished relaxation and enhancedcontraction. Bradykinin and A-23187 were more potent and caused greaterendothelium-mediated relaxation in top than in bottom arteries. Theendothelium-independent vasodilator sodium nitroprusside caused similarrelaxations in all rings.N-nitro-L-arginine andN^G-monomethyl-L-arginine andmethylene blue abolished relaxation of top and bottom arteries tomethacholine; meclofenamate had little effect. We conclude thatregional differences in endothelium-mediated relaxation are caused bydifferences in the magnitude of the endothelial release of nitricoxide. Similar differences in endothelium-dependent flow-mediatedvasodilation and endothelial nitric oxide release may result inpreferential perfusion of caudodorsal lung regions.

     

Chronic intrauterine pulmonary hypertension increases endothelial cell Rho kinase activity and impairs angiogenesis in vitro

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by endothelial dysfunction and decreased vascular growth. The role of Rho kinase activity in modulating endothelial function and regulating angiogenesis during normal lung development and in PPHN is unknown. We hypothesized that PPHN increases Rho kinase activity in fetal pulmonary artery endothelial cells (PAECs) and impairs angiogenesis in vitro. Proximal PAECs were harvested from fetal sheep with partial ligation of the ductus arteriosus in utero (PPHN) and age-matched controls. Rho kinase activity was measured by RhoA, Rho GTP, and phosphorylated MYPT-1 protein content. The effects of Rho kinase activity on angiogenesis, endothelial nitric oxide (NO) synthase (eNOS) protein expression, and NO production were determined in normal and PPHN PAECs. Angiogenesis was assessed by tube formation in vitro with/without Y-27632 (a Rho kinase inhibitor) and calpeptin (a Rho kinase activator) in the presence/absence of N-nitro-l-arginine (l-NA, an NOS inhibitor). RhoA, Rho GTP, and phosphorylated MYPT-1 protein were increased in PPHN PAECs. Tube formation was reduced 29% in PPHN PAECs (P < 0.001) and increased with Y-27632 treatment in normal and PPHN PAECs, with PPHN PAECs achieving levels similar to those of normal PAECs. l-NA inhibited the Y-27632-induced increase in tube formation in normal, but not PPHN, PAECs. Calpeptin reduced tube formation in normal and PPHN PAECs. eNOS expression was reduced 42% in PPHN PAECs (P < 0.01). Y-27632 increased eNOS protein and NO production in normal and PPHN PAECs. Calpeptin decreased eNOS protein only in normal PAECs but reduced NO production in normal and PPHN PAECs. We conclude that Rho kinase activity is increased in PPHN PAECs and impairs angiogenesis and downregulates eNOS protein and NO production in vitro.     

Sodium-Coupled Neutral Amino Acid Transporter 1 (SNAT1) Modulates L-Citrulline Transport and Nitric Oxide (NO) Signaling in Piglet Pulmonary Arterial Endothelial Cells

Rationale

There is evidence that impairments in nitric oxide (NO) signaling contribute to chronic hypoxia-induced pulmonary hypertension. The L-arginine-NO precursor, L-citrulline, has been shown to ameliorate pulmonary hypertension. Sodium-coupled neutral amino acid transporters (SNATs) are involved in the transport of L-citrulline into pulmonary arterial endothelial cells (PAECs). The functional link between the SNATs, L-citrulline, and NO signaling has not yet been explored.

Objective

We tested the hypothesis that changes in SNAT1 expression and transport function regulate NO production by modulating eNOS coupling in newborn piglet PAECs.

Methods and Results

A silencing RNA (siRNA) technique was used to assess the contribution of SNAT1 to NO production and eNOS coupling (eNOS dimer-to-monomer ratios) in PAECs from newborn piglets cultured under normoxic and hypoxic conditions in the presence and absence of L-citrulline. SNAT1 siRNA reduced basal NO production in normoxic PAECs and prevented L-citrulline-induced elevations in NO production in both normoxic and hypoxic PAECs. SNAT1 siRNA reduced basal eNOS dimer-to-monomer ratios in normoxic PAECs and prevented L-citrulline-induced increases in eNOS dimer-to-monomer ratios in hypoxic PAECs.

Conclusions

SNAT1 mediated L-citrulline transport modulates eNOS coupling and thus regulates NO production in hypoxic PAECs from newborn piglets. Strategies that increase SNAT1-mediated transport and supply of L-citrulline may serve as novel therapeutic approaches to enhance NO production in patients with pulmonary vascular disease.     

Regulation of intracellular Ca2+ release in corpus cavernosum smooth muscle: synergism between nitric oxide and cGMP

Tonic contraction of corpus cavernosum smooth muscle cells (SMCs) maintains the flaccid state of the penis, and relaxation is initiated by nitric oxide (NO), leading to erection. Our aim was to investigate the effect of NO on the smooth muscle cellular response to adrenergic stimulation in corpus cavernosum. Fura-2 fluorescence was used to record intracellular Ca²⁺ concentration ([Ca²⁺]_i) from freshly isolated SMCs from rat and human. Phenylephrine (PE) transiently elevated [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺, indicating release from intracellular stores. Whereas the NO donor S-nitroso-N-acetylpenicillamine (SNAP) with sildenafil citrate (SIL) caused no change in basal [Ca²⁺]_i, the PE-induced rise of [Ca²⁺]_i was reversibly inhibited by 27 ± 7% (n = 21, P < 0.005) in rat and by 55 ± 15% (n = 9, P < 0.01) in human SMCs. SNAP and SIL also reduced the contractile response to PE. To investigate the mechanism, we applied mediators alone or in combination. The soluble guanylyl cyclase inhibitor ODQ reduced the effect of SNAP and SIL. SIL, cGMP analogs, and NO donors without SIL did not reduce the PE-induced rise of [Ca²⁺]_i. However, the combination of 8-bromo-cGMP with SNAP reduced the Ca²⁺ peak by 42 ± 9% (n = 22, P < 0.01). Our results demonstrate that NO and cGMP act synergistically to reduce Ca²⁺ release from intracellular stores. Reduction of intracellular Ca²⁺ release may contribute to relaxation of the corpus cavernosum, leading to erection. calcium stores; nitric oxide; sildenafil citrate; inositol 1,4,5-trisphosphate receptor     

Lipoic acid and vitamin C potentiate nitric oxide synthesis in human aortic endothelial cells independently of cellular glutathione status

Vitamin C and thiol agents improve vasomotor function. To determine whether these compounds directly affect endothelial function, nitric oxide (NO) synthesis was measured in human aortic endothelial cells treated with ascorbic acid or the thiol modulating agents lipoic acid or L-2-oxothiazolidine-4-carboxylic acid (OTC). A dose-dependent increase in A23187-stimulated NO synthesis and elevated cGMP levels were observed in all cases except for OTC. Cellular GSH levels were not significantly increased, and the GSH/GSSG ratio was not significantly affected by treatment of the cells with lipoic acid, OTC, or ascorbic acid. Thus, vitamin C and lipoic acid potentiate endothelial NO synthesis and bioactivity by mechanisms that appear to be independent of cellular GSH levels and redox environment.     

Peroxisome proliferator-activated receptor-gamma ligands regulate endothelial membrane superoxide production

Recently, we demonstrated that the peroxisome proliferator-activated receptor- (PPAR-) ligands, either 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂) or ciglitazone, increased endothelial nitric oxide (·NO) release without altering endothelial nitric oxide synthase (eNOS) expression (4). However, the precise molecular mechanisms of PPAR--stimulated endothelial·NO release remain to be defined. Superoxide anion radical (O₂^–·) combines with ·NO to decrease·NO bioavailability. NADPH oxidase, which produces O₂^–·, and Cu/Zn-superoxide dismutase (Cu/Zn-SOD), which degrades O₂^–·, thereby contribute to regulation of endothelial cell·NO metabolism. Therefore, we examined the ability of PPAR- ligands to modulate endothelial O₂^–· metabolism through alterations in the expression and activity of NADPH oxidase or Cu/Zn-SOD. Treatment with 10 µM 15d-PGJ₂ or ciglitazone for 24 h decreased human umbilical vein endothelial cell (HUVEC) membrane NADPH-dependent O₂^–· production detected with electron spin resonance spectroscopy. Treatment with 15d-PGJ₂ or ciglitazone also reduced relative mRNA levels of the NADPH oxidase subunits, nox-1, gp91^phox (nox-2), and nox-4, as measured using real-time PCR analysis. Concordantly, Western blot analysis demonstrated that 15d-PGJ₂ or ciglitazone decreased nox-2 and nox-4 protein expression. PPAR- ligands also stimulated both activity and expression of Cu/Zn-SOD in HUVEC. These data suggest that in addition to any direct effects on endothelial·NO production, PPAR- ligands enhance endothelial·NO bioavailability, in part by altering endothelial O₂^–· metabolism through suppression of NADPH oxidase and induction of Cu/Zn-SOD. These findings further elucidate the molecular mechanisms by which PPAR- ligands directly alter vascular endothelial function. reduced nicotinamide adenine dinucleotide phosphate oxidase; copper/zinc superoxide dismutase; nitric oxide; endothelial cells     

Nitric oxide relaxes human myometrium by a cGMP-independent mechanism

The role of intracellular guanosine 3',5'-cyclicmonophosphate concentration([cGMP]_i) in nitricoxide (NO)-mediated relaxations in the uterus has become controversial.We found the NO donor S-nitroso-L-cysteine(CysNO) to potently (IC₅₀ = 30 nM)inhibit spontaneous contractions in the nonpregnant human myometrium. CysNO treatment increased[cGMP]_i significantly(P < 0.001), and this increase wasblocked by the guanylyl cyclase inhibitors methylene blue (10 µM) orLY-83583 (1 µM); however, pretreatment with these guanylyl cyclaseinhibitors failed to block CysNO-mediated relaxations. IntracellularcAMP concentrations were not altered by treatment of tissues with 10 µM CysNO. Incubation with the cGMP analogs 8-bromo-cGMP or-phenyl-1,N²-etheno-cGMPdid not significantly affect spontaneous contractility. Pretreatment oftissues with charybdotoxin [a calcium-dependent potassium channel(BK) blocker] completely reversed CysNO-induced relaxations. Weconclude that NO is a potent inhibitor of spontaneous contractileactivity in the nonpregnant human uterus and that, although guanylylcyclase and BK activities are increased by NO, increases in[cGMP]_i are notrequired for NO-induced relaxations in this tissue.

     

Endothelium-dependent vasorelaxation is inhibited by in vivo depletion of vascular thiol levels: Role of endothelial nitric oxide synthase

Thiols like glutathione may serve as reducing co-factors in the production of nitric oxide (NO) and protect NO from inactivation by radical oxygen species. Depletion of thiol compounds reduces NO-mediated vascular effects in vitro and in vivo. The mechanisms underlying these actions are not clear, but may involve decreased synthesis of NO and/or increased degradation of NO. This study investigates the effect of glutathione depletion on the response to NO-mediated vasodilation induced by acetylcholine (Ach, 10 μg/kg), endothelial NO synthase (eNOS) activity and potential markers of vascular superoxide anion (O^·-₂) production in conscious chronically catheterized rats. Thiol depletion induced by buthionine sulfoximine (BSO, 1 g ip within 24 h) decreased the hypotensive effect of Ach by 30% (MAP reduction before BSO 27 ± 3 mmHg, 19 ± 3 mmHg after BSO, (mean ± SEM), p < .05n = 8). The impaired effect of Ach was associated with a significant reduction in eNOS activity (control: 7.7 ± 0.8, BSO: 3.9 ± 0.4 pmol/min/mg protein (p < .05), n = 6). In contrast, neither NADH/NADPH driven membrane-associated oxidases nor lucigenin reductase activity were significantly (p < .05) affected by BSO (BSO: 4415 ± 123, control: 4105 ± 455 counts/mg, n = 6) in rat aorta. It is concluded that in vivo thiol depletion results in endothelial dysfunction and a reduced receptor-mediated vascular relaxation. This effect is caused by reduced endothelial NO formation.     

Effects of NO donors and synthase agonists on endothelial cell uptake of L-Arg and superoxide production

It is commonly believed thatthe activity of NO synthase (NOS) solely controls NO production fromits substrates, L-Arg and O₂. The Michaelis-Menten constant(K_m) of NOS forL-Arg is in the micromolarrange; cellular levels of L-Argare much higher. However, evidence strongly suggests that cellularsupply of L-Arg may becomelimiting and lead to reduced NO and increased superoxide anion(O₂·) formation, promotingcardiovascular dysfunction. Uptake ofL-Arg into cells occursprimarily (~85%) through the actions of aNa⁺-independent, carrier-mediatedtransporter (system y⁺). We haveexamined the effects of NOS agonists (substance P, bradykinin, and ACh)and NO donors(S-nitroso-N-acetyl-penicillamine and dipropylenetriamine NONOate) on transport ofL-Arg into bovine aorticendothelial cells (BAEC). Our results demonstrate that NOS agonistsincrease y⁺ transporter activity.A rapidly acting NO donor initially increases L-Arg uptake; however, afterlonger exposure, L-Arg uptake is suppressed. Exposure of BAEC withoutL-Arg to substance P and aCa²⁺ ionophore (A-23187) increasedO₂· formation, which was blockedwith concurrent presence ofL-Arg or the NOS antagonistN-nitro-L-arginine methyl ester.We conclude that factors including NO itself controly⁺ transport function and theproduction of NO and O₂·.