Modulation of the L-arginine/nitric oxide signalling pathway in vascular endothelial cells

Nitric oxide (NO) is synthesized from L-arginine, and in endothelial cells influx of L-arginine is mediated predominantly via Na+-independent cationic amino acid transporters. Constitutive, Ca2+-calmodulin-sensitive eNOS (endothelial nitric oxide synthase) metabolizes L-arginine to NO and L-citrulline. eNOS is present in membrane caveolae and the cytosol and requires tetrahydrobiopterin, NADPH, FAD and FMN as additional cofactors for its activity. Supply of L-arginine for NO synthesis appears to be derived from a membrane-associated compartment distinct from the bulk intracellular amino acid pool, e.g. near invaginations of the plasma membrane referred to as 'lipid rafts' or caveolae. Co-localization of eNOS and the cationic amino acid transport system y+ in caveolae in part explains the 'arginine paradox', related to the phenomenon that in certain disease states eNOS requires an extracellular supply of L-arginine despite having sufficient intracellular L-arginine concentrations. Vasoactive agonists normally elevate [Ca2+]i (intracellular calcium concentration) in endothelial cells, thus stimulating NO production, whereas fluid shear stress, 17beta-oestradiol and insulin cause phosphorylation of the serine/threonine protein kinase Akt/protein kinase B in a phosphoinositide 3-kinase-dependent manner and activation of eNOS at basal [Ca2+]i levels. Adenosine causes an acute activation of p42/p44 mitogen-activated protein kinase and NO release, with membrane hyperpolarization leading to increased system y+ activity in fetal endothelial cells. In addition to acute stimulatory actions of D-glucose and insulin on L-arginine transport and NO synthesis, gestational diabetes, intrauterine growth retardation and pre-eclampsia induce phenotypic changes in the fetal vasculature, resulting in alterations in the L-arginine/NO signalling pathway and regulation of [Ca2+]i. These alterations may have significant implications for long-term programming of the fetal cardiovascular system.     

Selective stimulation of L-arginine uptake contributes to shear stress-induced formation of nitric oxide

The aim of this study was to investigate the kinetics of L-arginine transport mechanisms and the role of extracellular L-arginine in nitric oxide formation during shear stress activation of endothelial cells. Porcine aortic endothelial cells were grown to confluence and were exposed to various amounts of shear stress for 40 min. Formation of nitric oxide was monitored by measuring elevation of endothelial cGMP. Activity of amino acid transport systems was determined by measuring the uptake of L-[3H]leucine (L system) and L-[3H]arginine (y+) under resting and shear stress condition. Shear stress-mediated nitric oxide formation critically depended on the presence of extracellular L-arginine, which increased shear stress-induced cGMP increases in a concentration dependent manner (EC50=123 microM). In addition, shear stress increased L-arginine uptake, while the transport capacity for neutral amino acids (L system) remained unchanged under shear stress conditions. Analysis of the kinetics of the uptake of L-arginine under resting and shear stress conditions indicate that shear stress increased velocity of the high affinity, low capacity transport (y+) without affecting affinity of this system. These data suggest that shear stress selectively activates uptake of L-arginine in endothelial cells and that the uptake of L-arginine might be important for shear stress-mediated nitric oxide formation.     

Cytoskeletal regulation of the L-arginine/NO pathway in pulmonary artery endothelial cells

We investigated possible involvement of the actin cytoskeleton in the regulation of the L-arginine/nitric oxide (NO) pathway in pulmonary artery endothelial cells (PAEC). We exposed cultured PAEC to swinholide A (Swinh), which severs actin microfilaments, or jasplakinolide (Jasp), which stabilizes actin filaments and promotes actin polymerization, or both. After treatment, the state of the actin cytoskeleton, L-arginine uptake mediated by the cationic amino acid transporter-1 (CAT-1), Ca(2+)/calmodulin-dependent (endothelial) NO synthase (eNOS) activity and content, and NO production were examined. Jasp (50-100 nM, 2 h treatment) induced a reversible activation of L-[(3)H]arginine uptake by PAEC, whereas Swinh (10-50 nM) decreased L-[(3)H]arginine uptake. The two drugs could abrogate the effect of each other on L-[(3)H]arginine uptake. The effects of both drugs on L-[(3)H]arginine transport were not related to changes in expression of CAT-1 transporters. Swinh (50 nM, 2 h) and Jasp (100 nM, 2 h) did not change eNOS activities and contents in PAEC. Detection of NO in PAEC by the fluorescent probe 4,5-diaminofluorescein diacetate showed that Swinh (50 nM) decreased and Jasp (100 nM) increased NO production by PAEC. The stimulatory effect of Jasp on NO production was dependent on the availability of extracellular L-arginine. Our results indicate that the state of actin microfilaments in PAEC regulates L-arginine transport and that this regulation can affect NO production by PAEC.     

Ethanol inhibits L-arginine uptake and enhances NO formation in human placenta.

The acute effects of ethanol (20-60 mM) on L-arginine uptake and nitric oxide (NO) formation was investigated in human placental cotyledons perfused at constant flow. Ethanol (40 mM) decreased L-[3H]arginine uptake from 27.6 +/- 2.3 to 15.8 +/- 1.3 per cent (P < 0.05) of the injected dose and significantly enhanced NO levels in the perfusate from 0.88 +/- 0.11 to 2.80 +/- 0.39 microM. Ethanol also elicited the constriction of placental vessels. The effects of ethanol (20-60 mM) on L-arginine uptake and endothelial NO synthase (eNOS) activity were also investigated in cultured human umbilical vein endothelial cells (HUVEC). After 60 min of ethanol (40 mM) exposure, basal L-[3H]arginine uptake (4.7 +/- 0.3 pmol/microg protein/min) was inhibited by 60 per cent (P < 0.05). Basal eNOS activity in HUVEC determined under "no flow" (static) conditions was significantly increased (approximately 1.8 fold) by 60 mM ethanol. These data are consistent with a stimulatory effect of ethanol on eNOS activity in both basal and flow-stimulated conditions, which may serve a protective role against its vasoconstrictive acute effect. While acute ethanol administration inhibits L-arginine uptake, the present results do not allow us to speculate on the effects of chronic ethanol exposure on NO formation in the fetoplacental unity.     

L-arginine uptake and release by cultured avian retinal cells: differential cellular localization in relation to nitric oxide synthase

The availability of L-arginine is of pivotal importance for the synthesis of nitric oxide, a signaling molecule in the CNS. Here we show the presence of a high-affinity L-arginine uptake system (Km of 4.4 +/- 0.5 microM and a Vmax of 26.0 +/- 0.9 fmol/well/min) in cultured chick retinal cells. Different compounds, such as N(G)-mono-methyl-L-arginine and L-lysine, were able to inhibit the uptake that was also inhibited 60-70% in the absence of sodium and/or calcium ions. No trans stimulation was observed when cells were preloaded with L-lysine. The data indicate that the L-arginine uptake in cultured retinal cells is partially mediated by the y+ system, but has a great contribution of the B(0,+) system. Autoradiographic studies revealed that the uptake is predominant in glial cells and can also be detected in neurons, whereas immunocytochemistry of nitric oxide synthase and L-citrulline showed that the enzyme is present in neurons and photoreceptors, but not in glial cells. L-[3H]Arginine is released from purified glial cultures incubated with high concentrations of potassium in the extracellular medium. Moreover, the amino acid released from preloaded glial cells was taken up by purified neuronal cultures. These results indicate that L-arginine released from glial cells is taken up by neurons and used as substrate for the synthesis of nitric oxide.     

Evidence for the pathophysiological role of endogenous methylarginines in regulation of endothelial NO production and vascular function

In endothelium, NO is derived from endothelial NO synthase (eNOS)-mediated L-arginine oxidation. Endogenous guanidinomethylated arginines (MAs), including asymmetric dimethylarginine (ADMA) and NG-methyl-L-arginine (L-NMMA), are released in cells upon protein degradation and are competitive inhibitors of eNOS. However, it is unknown whether intracellular MA concentrations reach levels sufficient to regulate endothelial NO production. Therefore, the dose-dependent effects of ADMA and L-NMMA on eNOS function were determined. Kinetic studies demonstrated that the Km for L-arginine is 3.14 microM with a Vmax of 0.14 micromol mg-1 min-1, whereas Ki values of 0.9 microM and 1.1 microM were determined for ADMA and L-NMMA, respectively. EPR studies of NO production from purified eNOS demonstrated that, with a physiological 100 microM level of L-arginine, MA levels of >10 microM were required for significant eNOS inhibition. Dose-dependent inhibition of NO formation in endothelial cells was observed with extracellular MA concentrations as low 5 microm. Similar effects were observed in isolated vessels where 5 microm ADMA inhibited vascular relaxation to acetylcholine. MA uptake studies demonstrated that ADMA and L-NMMA accumulate in endothelial cells with intracellular levels greatly exceeding extracellular concentrations. L-arginine/MA ratios were correlated with cellular NO production. Although normal physiological levels of MAs do not significantly inhibit NOS, a 3- to 9-fold increase, as reported under disease conditions, would exert prominent inhibition. Using a balloon model of vascular injury, approximately 4-fold increases in cellular MAs were observed, and these caused prominent impairment of vascular relaxation. Thus, MAs are critical mediators of vascular dysfunction following vascular injury.     

5-hydroxytryptamine evokes endothelial nitric oxide synthase activation in bovine aortic endothelial cell cultures.

Activation of endothelial nitric oxide synthase (eNOS) results in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells. The goal of this project was to address the possibility that 5-hydroxytryptamine (5-HT) stimulates eNOS activity in bovine aortic endothelial cell (BAEC) cultures. Here, we tested the hypothesis that 5-HT receptors mediate eNOS activation by measuring agonist-stimulated [3H]L-citrulline ([3H]L-Cit) formation in BAEC cultures. We found that 5-HT stimulated the conversion of [3H]L-arginine ([3H]L-Arg) to [3H]L-Cit, indicating eNOS activation. The high affinity 5-HT1B receptor agonist, 5-nonyloxytryptamine (5-NOT)-stimulated [3H]L-Cit turnover responses were concentration-(0.01 nM to 100 microM) and time-dependent. Maximal responses were observed within 10 min following agonist exposures. These responses were effectively blocked by the 5-HT1B receptor antagonist, isamoltane, the 5-HT1B/5-HT2 receptor antagonist, methiothepin, and the eNOS selective antagonists (0.01-10 microM): L-Nomega -monomethyl-L-arginine (L-NMMA) and L-N omega-iminoethyl-L-ornithine (L-NIO). Pretreatment of BAEC cultures with pertussis toxin (PTX; 1-100 ng/ml) for 16 hr resulted in significant inhibition of the agonist-stimulated eNOS activity, indicating the involvement of Gi proteins. These findings lend evidence of a 5-HT1B receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT. Further investigation is needed to determine the role of other vascular 5-HT receptors in the stimulation of eNOS activity.     

Intracellular L-arginine concentration does not determine NO production in endothelial cells: implications on the "L-arginine paradox"

We examined the relative contributory roles of extracellular vs. intracellular L-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of (15)N(4)-ARG, ARG, or L-arginine ethyl ester (ARG-EE) for 2h. To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, (15)N(4)-ARG, dimethylarginines, and L-citrulline by an LC-MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by(15)N-nitrite or estimated (15)N(3)-citrulline concentrations when (15)N(4)-ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced (15)N(4)-ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by (15)N-nitrite, total nitrite and (15)N(3)-citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, ARG can no longer gain access to the membrane-bound eNOS. These observations indicate that the "L-arginine paradox" should not consider intracellular ARG concentration as a reference point.     

D-glucose stimulation of L-arginine transport and nitric oxide synthesis results from activation of mitogen-activated protein kinases p42/44 and Smad2 requiring functional type II TGF-beta receptors in human umbilical vein endothelium

Elevated extracellular D-glucose increases transforming growth factor beta1 (TGF-beta1) release from human umbilical vein endothelium (HUVEC). TGF-beta1, via TGF-beta receptors I (TbetaRI) and TbetaRII, activates Smad2 and mitogen-activated protein kinases p44 and p42 (p42/44(mapk)). We studied whether D-glucose-stimulation of L-arginine transport and nitric oxide synthesis involves TGF-beta1 in primary cultures of HUVEC. TGF-beta1 release was higher ( approximately 1.6-fold) in 25 mM (high) compared with 5 mM (normal) D-glucose. TGF-beta1 increases L-arginine transport (half maximal effect approximately 1.6 ng/ml) in normal D-glucose, but did not alter high D-glucose-increased L-arginine transport. TGF-beta1 and high D-glucose increased hCAT-1 mRNA expression ( approximately 8-fold) and maximal transport velocity (V(max)), L-[(3)H]citrulline formation from L-[(3)H]arginine (index of NO synthesis) and endothelial NO synthase (eNOS) protein abundance, but did not alter eNOS phosphorylation. TGF-beta1 and high D-glucose increased p42/44(mapk) and Smad2 phosphorylation, an effect blocked by PD-98059 (MEK1/2 inhibitor). However, TGF-beta1 and high D-glucose were ineffective in cells expressing a truncated, negative dominant TbetaRII. High D-glucose increases L-arginine transport and eNOS expression following TbetaRII activation by TGF-beta1 involving p42/44(mapk) and Smad2 in HUVEC. Thus, TGF-beta1 could play a crucial role under conditions of hyperglycemia, such as gestational diabetes mellitus, which is associated with fetal endothelial dysfunction.     

Modulation of Neuronal Signal Transduction Systems by Extracellular ATP

The secretion of ATP by stimulated nerves is well documented. Following repetitive stimulation, extracellular ATP at the synapse can accumulate to levels estimated to be well over 100 microM. The present study examined the effects of extracellular ATP in the concentration range of 0.1-1.0 mM on second-messenger-generating systems in cultured neural cells of the clones NG108-15 and N1E-115. Cells in a medium mimicking the physiological extracellular environment were used to measure 45Ca2+ uptake, changes in free intracellular Ca2+ levels by the probes aequorin and Quin-2, de novo generation of cyclic GMP and cyclic AMP from intracellular GTP and ATP pools prelabeled with [3H]guanosine and [3H]adenine, respectively, and phosphoinositide metabolism in cells preloaded with [3H]inositol and assayed in the presence of LiCl. Extracellular ATP induced a concentration-dependent increase of 45Ca2+ uptake by intact cells, which was additive with the uptake induced by K+ depolarization. The increased uptake involved elevation of intracellular free Ca2+ ions, evidenced by measuring aequorin and Quin-2 signals. At the same concentration range (0.1-1.0 mM), extracellular ATP induced an increase in [3H]cyclic GMP formation, and a decrease in prostaglandin E1-stimulated [3H]cyclic AMP generation. In addition, extracellular ATP (1 mM) caused a large (15-fold) increase in [3H]inositol phosphates accumulation, and this effect was blocked by including La3+ ions in the assay medium. In parallel experiments, we found in NG108-15 cells surface protein phosphorylation activity that had an apparent Km for extracellular ATP at the same concentration required to produce half-maximal effects on Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo and in vitro evidence for ACh-stimulated L-arginine uptake

Whereas L-arginine is clearly recognized as the precursor for nitric oxide synthesis, and its entry into endothelial cells via system y(+) transport is established, few data exist regarding the acute regulation of this transport process. We specifically investigated the effect of ACh and isoprenaline (Iso) on L-arginine uptake in the human forearm and in cultured bovine aortic endothelial cells (BAEC). Sixteen healthy males were studied. During a steady-state intra-arterial infusion of [(3)H]L-arginine (100 nCi/min), the effects of ACh (9.25 and 37 microg/min), Iso (25-50 and 200 microg/min), and sodium nitroprusside (SNP) (1-2 and 8 microg/min) on forearm plasma flow (FPF), L-[(3)H]arginine uptake, and L-[(3)H]citrulline release were determined. In parallel experiments, the effects of ACh, Iso, and SNP on L-[(3)H]arginine uptake were studied in BAEC. L-Arginine uptake was inversely related to FPF (r = -0.50; P < 0.005). At a similar FPF (ACh 56.82 +/- 9.25, Iso 58.49 +/- 5.56, SNP 57.92 +/- 4.96 ml/min; P = ns), intra-arterial ACh significantly increased forearm uptake of L-[(3)H]arginine (54,655 +/- 8,018 dpm/min), compared with that observed with either Iso (40,517.23 +/- 6,841 dpm/min; P = 0.01) or SNP (36,816 +/- 4,650 dpm/min; P = 0.011). This was associated with increased ACh-induced L-[(3)H]citrulline release compared with Iso and SNP (P = 0.046). Similarly, in BAEC, ACh significantly increased L-[(3)H]arginine uptake compared with control, Iso, or SNP (ACh 12.0 x 10(7) +/- 1.83 x 10(7) vs. control 6.67 x 10(7) +/- 1.16 x 10(7) vs. Iso 7.35 x 10(7) +/- 1.63 x 10(7) vs. SNP 6.01 x 10(7) +/- 1.11 x 10(7) fmol.min(-1).mg(-1) at 300 micromol/l L-arginine; P = 0.043). Taken together, these data indicate that ACh stimulates L-arginine uptake in cultured endothelial cells and in human forearm circulation, indicating the potential for acute modulation of endothelial L-arginine uptake.     

Homocysteine induces endothelial dysfunction via inhibition of arginine transport.

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases. High levels of plasma homocysteine (HCY) increase oxidative stress and reduce endothelial-dependent relaxation. We determined whether hyperhomocysteinemia-induced endothelial dysfunction is mediated through inhibition of cellular transport of L-arginine. In endothelial cells, HCY had a biphasic effect on arginine transport. HCY treatment for 6 hr increased L-arginine uptake by 34%; however, uptake was decreased by 25% after 24 h. HCY caused membrane hyperpolarization during both 6 and 24 h incubation periods, indicating that the negative charge facilitating arginine uptake was maintained. HCY significantly reduced expression of cellular arginine transporter protein (CAT-1) after 24 h treatment; whereas endothelial nitric oxide synthase (eNOS) protein levels and basal eNOS activity were not altered. Nevertheless, nitric oxide (NO) formation was significantly decreased. The antioxidant ascorbic acid prevented the effect of HCY on arginine transport. HCY induced formation of the peroxynitrite biomarker nitrotyrosine, which was blocked by supplemental L-arginine. HCY treatment of aortic rings caused decreased vasorelaxation to acetylcholine, which was prevented by supplemental arginine. In conclusion, HCY decreased NO formation and induced endothelial dysfunction without altering protein level or basal activity of eNOS, but through decreases in function and protein expression of the CAT-1 transporter. Reduced arginine supply may lead to eNOS uncoupling and generation of superoxide, contributing to HCY-induced oxidative stress.     

Inhibition of protein synthesis by activation of NMDA receptors in cultured retinal cells: a new mechanism for the regulation of nitric oxide production

The synthesis of nitric oxide (NO) is limited by the intracellular availability of L-arginine. Here we show that stimulation of NMDA receptors promotes an increase of intracellular L-arginine which supports an increase in the production of NO. Although L-[3H]arginine uptake measured in cultured chick retina cells incubated in the presence of cycloheximide (CHX, a protein synthesis inhibitor) was inhibited approximately 75% at equilibrium, quantitative thin-layer chromatography analysis showed that free intracellular L-[3H]arginine was six times higher in CHX-treated than in control cultures. Extracellular L-[3H]citrulline levels increased threefold in CHX-treated groups, an effect blocked by NG-nitro-L-arginine, a NO synthase (NOS) inhibitor. NMDA promoted a 40% increase of free intracellular L-[3H]arginine in control cultures, an effect blocked by the NMDA antagonist 2-amino 5-phosphonovaleric acid. In parallel, NMDA promoted a reduction of 40-50% in the incorporation of 35[S]methionine or L-[3H]arginine into proteins. Western blot analysis revealed that NMDA stimulates the phosphorylation of eukaryotic elongation factor 2 (eEF2, a factor involved in protein translation), an effect inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801). In conclusion, we have shown that the stimulation of NMDA receptors promotes an inhibition of protein synthesis and a consequent increase of an intracellular L-arginine pool available for the synthesis of NO. This effect seems to be mediated by activation of eEF2 kinase, a calcium/calmodulin-dependent enzyme which specifically phosphorylates and blocks eEF2. The results raise the possibility that NMDA receptor activation stimulates two different calmodulin-dependent enzymes (eEF2 kinase and NOS) reinforcing local NO production by increasing precursor availability together with NOS catalytic activity.     

Characterization of two independent modes of action of ATP on human erythrocyte sugar transport

Intracellular ATP has been reported either to stimulate [Jacquez, J.A. (1983) Biochim. Biophys. Acta 727, 367-378] or to inhibit [Hebert, D. N., & Carruthers, A. (1986) J. Biol. Chem. 261, 10093-10099] human erythrocyte sugar transport. This current study provides a rational explanation for these divergent findings. Protein-mediated 3-O-methyl-alpha-D-glucopyranoside (3OMG) uptake by intact human red blood cells (lacking intracellular sugar) at ice temperature in isotonic KCl containing 2 mM MgCl2, 2 mM EGTA, and 5 mM Tris-HCl, pH 7.4 (KCl medium), is characterized by a Km(app) of 0.4 +/- 0.1 mM and a Vmax of 114 +/- 20 mumol L-1 min-1. Lysis of red cells in 40 volumes of EGTA-containing hypotonic medium and resealing in 10 volumes of KCl medium increase the Km(app) and Vmax for uptake to 7.1 +/- 1.8 mM and 841 +/- 191 mumol L-1 min-1, respectively. Addition of ATP (4 mM) to the resealing medium restores Michaelis and velocity constants for zero-trans 3OMG uptake to 0.42 +/- 0.11 mM and 110 +/- 15 mumol L-1 min-1, respectively. Addition of CaCl2 to extracellular KCl medium (calculated [Ca2+]o = 101 microM) reduces the Vmax for zero-trans 3OMG uptake in intact cells and ATP-containing ghosts by 79 +/- 4% and 61 +/- 9%, respectively. Intracellular Ca2+ (15 microM) reduces the Vmax for 3OMG uptake by ATP-containing ghosts by 38 +/- 12%. In nominally ATP-free ghosts, extracellular (101 microM) and intracellular (11 microM) Ca2+ reduce the Vmax for 3OMG uptake by 96 and 94%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium regulates estrogen increase in permeability of cultured CaSki epithelium by eNOS-dependent mechanism

Estrogen increasesbaseline transepithelial permeability across CaSki cultures andaugments the increase in permeability in response to hypertonicgradients. In estrogen-treated cells, lowering cytosolic calciumabrogated the hypertonicity-induced augmented increase in permeabilityand decreased baseline permeability to a greater degree than inestrogen-deprived cells. Steady-state levels of cytosolic calcium inestrogen-deprived cells were higher than in estrogen-treated cells.Increases in extracellular calcium increased cytosolic calcium more inestrogen-deprived cells than in estrogen-treated cells. However, inestrogen-treated cells, increasing cytosolic calcium was associatedwith greater increases in permeability in response to hypertonicgradients than in estrogen-deprived cells. Lowering cytosolic calciumblocked the estrogen-induced increase in nitric oxide (NO) release andin the in vitro conversion of L-[³H]arginineto L-[³H]citrulline. Treatment with estrogenupregulated mRNA of the NO synthase isoform endothelial nitric oxidesynthase (eNOS). These results indicate that cytosolic calcium mediatesthe responses to estrogen and suggest that the estrogen increase inpermeability and the augmented increase in permeability in response tohypertonicity involve an increase in NO synthesis by upregulation ofthe calcium-dependent eNOS.

     

Comparison of neuronal and endothelial isoforms of nitric oxide synthase in stably transfected HEK 293 cells

The neuronal and endothelial isoforms of nitric oxide (NO) synthase (nNOS and eNOS, respectively) both catalyze the production of NO but are regulated differently. Stably transfected HEK 293 cell lines containing nNOS, eNOS, and a soluble mutant of eNOS were therefore established to compare their activity in a common cellular environment. NOS activity was determined by measuring L-[3H]citrulline production in homogenates and intact cells, the conversion of oxyhemoglobin to methemoglobin, and the production of cGMP. The results indicate that nNOS is more active than eNOS, both in unstimulated as well as calcium-stimulated cells. Under basal conditions, the soluble mutant of eNOS appeared to be slightly more active than wild-type eNOS in terms of NO and cGMP formation, suggesting that membrane association may be crucial for inhibition of basal NO release but is not required for stimulation by Ca2+-mobilizing agents. The maximal activity of soluble guanylate cyclase was significantly reduced by transfection with wild-type eNOS due to downregulation of mRNA expression. These results demonstrate that nNOS and eNOS behave differently even in an identical cellular environment.     

Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate

Previous studies have shown that murine macrophages immunostimulated with interferon gamma and Escherichia coli lipopolysaccharide synthesize NO2-, NO3-, and citrulline from L-arginine by oxidation of one of the two chemically equivalent guanido nitrogens. The enzymatic activity for this very unusual reaction was found in the 100,000g supernatant isolated from activated RAW 264.7 cells and was totally absent in unstimulated cells. This activity requires NADPH and L-arginine and is enhanced by Mg2+. When the subcellular fraction containing the enzyme activity was incubated with L-arginine, NADPH, and Mg2+, the formation of nitric oxide was observed. Nitric oxide formation was dependent on the presence of L-arginine and NADPH and was inhibited by the NO2-/NO3- synthesis inhibitor NG-monomethyl-L-arginine. Furthermore, when incubated with L-[guanido-15N2]arginine, the nitric oxide was 15N-labeled. The results show that nitric oxide is an intermediate in the L-arginine to NO2-, NO3-, and citrulline pathway. L-Arginine is required for the activation of macrophages to the bactericidal/tumoricidal state and suggests that nitric oxide is serving as an intracellular signal for this activation process in a manner similar to that very recently observed in endothelial cells, where nitric oxide leads to vascular smooth muscle relaxation [Palmer, R. M. J., Ashton, D. S., & Moncada, S. (1988) Nature (London) 333, 664-666].     

Ascorbic acid blunts oxidant stress due to menadione in endothelial cells

Endothelial cells are exposed to potentially damaging reactive oxygen species generated both within the cells and in the bloodstream and underlying vessel wall. In this work, we studied the ability of ascorbic acid to protect cultured human-derived endothelial cells (EA.hy926) from oxidant stress generated by the redox cycling agent menadione. Menadione caused intracellular oxidation of dihydrofluorescein, which required the presence of D-glucose in the incubation medium, and was inhibited by intracellular ascorbate and desferrioxamine. At concentrations of 100 microM and higher, menadione depleted the cells of both GSH and ascorbate, and ascorbate loading partially prevented the decrease in GSH due to menadione. Menadione increased L-arginine uptake by the cells, but inhibited endothelial nitric oxide synthase, an effect that was prevented by acute loading with ascorbate. Ascorbate blunts menadione-induced oxidant stress in EA.hy926 cells, which may help to preserve nitric oxide synthase activity under conditions of excessive oxidant stress.     

Acute hypoxia increases intracellular L-arginine content in cultured porcine pulmonary artery endothelial cells

Exposure to hypoxia (0% O₂) for 4–24 h resulted in increased intracellular L-arginine content and increased activity of calpain, a calcium-dependent neutral cysteine protease, in pulmonary artery endothelial cells. Calpain-inhibitor I abolished the increased L-arginine content in hypoxic cells. When endothelial cell proteins were labeled with [³H]-L-arginine and the cells exposed to hypoxia, we observed an increase in free [³H]-L-arginine and a decrease in [³H]-L-arginine-labeled proteins. Once again, calpain-inhibitor I prevented the increases in free [³H]-L-arginine and the decreases in [³H]-L-arginine-labeled proteins in hypoxic cells. Hypoxia also inhibited the synthesis of L-arginine-containing proteins. Thus, the increase in intracellular L-arginine content in hypoxic pulmonary artery endothelial cells is caused by an increase in proteolysis secondary to calpain and a decrease in protein synthesis. These results indicate that hypoxia can modulate the availability of free intracellular L-arginine, the exclusive precursor of nitric oxide (NO) and the primary substrate of NO synthase, by affecting the synthesis and degradation of cellular proteins. © 1996 Wiley-Liss, Inc.     

Contrasting effects of thiol-modulating agents on endothelial NO bioactivity

The bioactivity of endothelium-derived nitric oxide(NO) is an important component of vascular homeostasis that issensitive to intracellular redox status. Because glutathione (GSH) is a major determinant of intracellular redox state, we sought to define itsrole in modulating endothelial NO bioactivity. In porcine aorticendothelial cells (PAECs), we depleted intracellular GSH (>70%) using1) buthionine-(S,R)-sulfoximine (BSO), whichinhibits GSH synthesis; 2) diamide, which oxidizes thiols;or 3) 1-chloro-2,4-dinitrobenzene (CDNB), which putativelydepletes GSH through glutathione S-transferase activity.Cellular GSH depletion with BSO had no effect on endothelial NObioactivity measured as A-23187-induced cGMP accumulation. In contrast,oxidation of intracellular thiols with diamide inhibited bothA-23187-induced cGMP accumulation and the cGMP response to exogenousNO. Diamide treatment of either PAECs, PAEC membrane fractions, orpurified endothelial nitric oxide synthase (eNOS) resulted insignificant inhibition (~75%) of eNOS catalytic activity measured asL-[³H]arginine-to-L-[³H]citrullineconversion. This effect appeared related to oxidation of eNOS thiols asit was completely reversed by dithiothreitol. Glutathione depletionwith CDNB inhibited A-23187-stimulated cGMP accumulation but not thecGMP response to exogenous NO. Rather, CDNB treatment impaired eNOScatalytic activity in intact PAECs, and this effect was reversed byexcess NADPH in isolated purified eNOS assays. Consistent with theseresults, we found spectral evidence that CDNB reacts with NADPH andrenders it inactive as a cofactor for either eNOS or glutathionereductase. Thus thiol-modulating agents exert pleiotropic effects onendothelial NO bioactivity, and these data may help to resolve a numberof conflicting previous studies linking GSH status with endothelialcell NO bioactivity.