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Studies of nitric oxide (NO) release from S-nitrosoglutathione (GSNO) decomposition by Cu2+ in the presence of reducing agents were performed using a nickel porphyrin and Nafion-coated microsensor in order to compare the efficiency of sodium hydrosulfite (Na2S2O4) and sodium borohydride (NaBH4) to that of the most abundant endogenous reducer, glutathione (GSH). When it was mixed to Cu(NO3)2 and added to equimolar concentration of GSNO, each reducing agent caused a NO release (measured in terms of oxidation current) but only NaBH4 induced a proportional rise if its concentration doubled and that of Cu2+ remained constant. For Na2S2O4, there was a mild increase and for GSH, no change. Furthermore, when Cu2+ concentrations ranging from 0.5 to 5 μM were mixed with 2 μM reducing agent and added to 2 μM GSNO, the NO oxidation current linearly increased with NaBH4 and was constant with Na2S2O4. Concerning GSH, Cu2+ dose-dependently increased the NO release from GSNO only if the Cu2+-to-reducer ratio was ≤1. However, GSH formed the catalytic species Cu+ even in excess of Cu2+ and GSNO as indicated by suppression of the Cu2+/GSH-induced NO release when the Cu+ chelator neocuproine was added to GSNO. This work shows that, among the 3 reducing agents, only NaBH4 allows Cu2+ to dose-dependently increase the NO release from GSNO for Cu2+-to-reducer ratios ranging from 0.25 to 2.5. Despite this good effectiveness, excess of NaBH4 compared to both Cu2+ and GSNO seems to be required for optimal NO release.

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

We report here on the appropriate analysis of some examples of interfering compounds that should be done to assess the specificity of the electrochemical sensing of nitric oxide in solution. To do so, we describe the design of a nickel porphyrin and Nafion(R)-coated carbon microfibre and discuss the methodological approach in examining interfering compounds.

1. Dichlorofluorescein oxidation and electrochemical monitoring of in situ nitric oxide (NO) release from cultured human endothelial cells reveals that agonists such as thrombin and histamine simultaneously stimulate transient superoxide production. 2. The duration of *NO release was increased only in the simultaneous presence of extracellular L-arginine and exogenous superoxide dismutase. In contrast, the inhibition of membrane reduced nicotinamide adenine dinucleotide (phosphate) oxidases, the major source of *O2- in endothelial cells, did not prolong *NO release, although extracellular L-arginine was also present. Comparison of these two experimental conditions suggested that H2O2 was involved in the extension of the *NO signal. 3. The present study demonstrates that, in the absence of external L-arginine, *O2- production does not constitute the major pathway controlling the duration of agonist-induced *NO signal. These results suggest that L-arginine and H2O2 act jointly to maintain nitric oxide synthase in an activated form.

The bioavailability of endothelial nitric oxide (NO) is regulated by transition metals but their mechanisms of action on NO synthesis and degradation are not clearly understood. Using differential pulse amperometry and NO microelectrodes, local NO concentration was measured at the surface of cultured human umbilical vein endothelial cells (HUVECs) stimulated by histamine or thrombin in the presence of transition metal chelators. The agonist-activated NO release required both extracellular Ca 2+ and transition metals. In the presence of 1 mM external Ca 2+ , a low concentration of EGTA (5 μM) inhibited by 40 % the NO release from stimulated HUVECs. In the presence of extracellular L-arginine, the inhibitory effect of EGTA was even more marked and, in its absence, it was suppressed by adding exogenous superoxide dismutase. The decrease in NO release induced by the copper chelators, cuprizone and DETC, suggests that extracellular traces of Cu 2+ could regulate NO availability.

Electrochemical monitoring with a porphyrinic microsensor of in situ nitric oxide production from cultured cells offers numerous advantages but requires cautious analysis, repeated calibration and accurate localisation of the electrode. It also had some limitations. We describe here some characteristics of this method, the stability of the electrode response during experiments and its application to nitric oxide production by constitutive and inducible nitric oxide synthases. Real-time measurements of NO concentration allow the study of the kinetics of NO production. This is illustrated by the time-course of NO release from cultured human endothelial cells. How NO production by inducible nitric oxide synthase in cultured smooth muscle cells obtained from human internal mammary artery can be evaluated by Nafion- and Ni-porphyrin-coated electrode is also described. The results thus obtained are compared to the cumulated NO 2 - production evaluated by the Griess method.

SummaryThe 3-morpholinosydnonimine (SIN-1) generates both nitric oxide (NO) and superoxide anion (O2−). It elicits dose-dependent vasodilation in vivo, in spite of the opposite effects of its breakdown products on vascular tone and platelet aggregation.This study was designed to investigate the influence of intravenous SIN-1 injection on platelet Ca2+ handling in patients undergoing coronary angiography. SIN-1 administration reduced cytosolic [Ca2+] in unstimulated platelets by decreasing Ca2+ influx. It attenuated Ca2+ mobilization from internal stores evoked by thrombin or thapsigargin. In vitro studies were used as an approach to investigate how simultaneous productions of NO and O2− from SIN-1 modify thrombin- or thapsigargin-induced platelet Ca2+ mobilization. Superoxide dismutase, the O2− scavenger, enhanced the capacity of SIN-1 to inhibit Ca2+ mobilization but catalase had no effect.This suggests that the effects of SIN-1 on platelet Ca2+ handling resemble those of NO, but are modulated by simultaneous O2− release, independently of H2O2 formation.

Abstract —Recent studies have demonstrated that, unlike cholesterol, cholesterol oxidized at position 7 can reduce the maximal endothelium-dependent relaxation of isolated rabbit aortas ( Circulation. 1997;95:723–731). The aim of the current study was to determine whether cholesterol oxides reduce the release of nitric oxide (NO) from human umbilical vein endothelial cells (HUVECs). The amount of NO released by histamine-stimulated HUVECs was determined by differential pulse amperometry using a nickel porphyrin– and Nafion-coated carbon microfiber electrode. The effects of cholesterol (preserved from oxidation by butylated hydroxytoluene), 7-ketocholesterol, 7β-hydroxycholesterol, 5α,6α-epoxycholesterol, 19-hydroxycholesterol (60 μg/mL), and α-lysophosphatidylcholine (10 μg/mL) were compared. Pretreatment of HUVECs with cholesterol, 5α,6α-epoxycholesterol, or 19-hydroxycholesterol did not alter histamine-activated NO production. In contrast, pretreatment with 7-ketocholesterol or 7β-hydroxycholesterol significantly decreased NO release. The inhibitory effect of 7-ketocholesterol was time and dose dependent and was maintained in the presence of l -arginine. In the absence of serum, lysophosphatidylcholine also reduced NO production. In ionomycin-stimulated cells, pretreatment with 7-ketocholesterol did not inhibit NO release. These results demonstrate that cholesterol derivatives oxidized at the 7 position, the main products of low density lipoprotein oxidation, reduce histamine-activated NO release in HUVECs. Such an inhibitory effect of cholesterol oxides may account, at least in part, for the ability of oxidized low density lipoprotein to reduce the endothelium-dependent relaxation of arteries.

As previously described for endothelin-3, platelet exposure to cyclic GMP-elevating agents such as sodium nitroprusside and M&B-22948 (2-o-propoxyphenyl-8-azapurin-6-one), a cGMP phosphodiesterase inhibitor, lowered Ca2+ mobilization in response to thrombin. Interestingly, when cGMP phosphodiesterases were blocked, endothelin-3 produced a dose-dependent cGMP accumulation (P < 0.001). Since endothelin-3 has been proposed to decrease the activity of Ca2+ accumulating pumps, we examined whether this latter effect could be mediated by a rise in cGMP content. Cyclic GMP decreased in a dose-dependent manner the initial rate and plateau value of the ATP-dependent 45Ca2+ uptake in platelet membrane vesicles (P = 0.006 for each). Furthermore, combined treatment with endothelin-3 and M&B-22948 or a moderate concentration of Na+-nitroprusside further reduced the thrombin-evoked Ca2+ discharge (P = 0.004 and 0.01, respectively), suggesting that endothelin-3 pre-exposure had reduced the amount of mobilizable Ca2+. We propose that the depletion of platelet Ca2+ stores and the reduction of Ca2+ release evoked by endothelin-3 could be due, at least in part, to the elevation of cGMP content and to a decrease in Ca2+ accumulating pump activity.

Skeletal muscle capillarity expressed as capillary density (CD), and number of capillaries per fibre (C/F), as well as the mean fibre cross-sectional area (FCSA), were determined in the extensor digitorum longus (EDL), plantaris (PLA) and soleus (SOL) muscles of four groups of eight rodents trained on a swimming exercise programme (T) or maintained sedentary (S), at sea level (SL) or at simulated altitude (HA), barometric pressure 61.7 kPa (463 torr) for 12 weeks. It was shown that both HA exposure and endurance training decreased body and skeletal muscles weights (P less than 0.001). However, neither HA exposure nor endurance training induce any variation in relative importance in the skeletal muscle mass. Altitude exposure and endurance training had increasing effects on CD in all muscles studied (P less than 0.001). This study confirms the fact that altitude exposure has no direct effect on capillary development. On the other hand, the capillary supply of the several slow- and fast-twitch skeletal muscles studied is increased by endurance training. This real enhancement in capillary network is ascertained by an increase in the C/F ratio (+7%, +26%, +16%, in PLA, EDL, and SOL muscles, respectively at sea level, and +19.5%, +30%, and +14% respectively at HA). These results indicate that the effects of chronic exercise on skeletal muscle capillarity estimated by the C/F ratio, are greater in an hypobaric environment than in a SL environment.

International audience; Reduced synthesis of nitric oxide (NO) contributes to the endothelial dysfunction and may be related to limited availability of L-arginine, the common substrate of constitutive nitric-oxide synthase (NOS) and cytosolic arginase I and mitochondrial arginase II. To determine whether arginases modulate the endothelial NO synthesis, we investigated the effects of the competitive arginase inhibitor N(omega)-hydroxy-nor-L-arginine (Nor-NOHA) on the activity of NOS, arginases, and L-arginine transporter and on NO release at surface of human umbilical vein endothelial cells (HUVECs). In unstimulated cells, Nor-NOHA dose-dependently reduced the arginase activity with maximal inhibition at 20 microM. When HUVECs were stimulated by thrombin without extracellular L-arginine, Nor-NOHA dose-dependently increased the NOS activity and the NO release with maximal effects at 20 microM. Extracellular L-arginine also dose-dependently increased NO release and arginase activity. When HUVECs were stimulated by thrombin in the presence of 100 microM L-arginine, NOS activity and NO release were similar in untreated and Nor-NOHA-treated cells. However, despite activation of L-arginine uptake, the inhibition of arginase activity by Nor-NOHA was still significant. The depletion of freely exchangeable L-arginine pools with extracellular L-lysine did not prevent Nor-NOHA from increasing the NO release. This indicates the presence of pools, which are accessible to NOS and arginase, but not exchangeable. Interestingly, the mitochondrial arginase II was constitutively expressed, whereas the cytosolic arginase I was barely detectable in HUVECs. These data suggest that endothelial NO synthesis depends on the activity of arginase II in mitochondria and l-arginine carriers in cell membrane.

International audience; Little is known about the effects of human free apolipoprotein A-I (Free-Apo A-I) and pre-beta-high density lipoprotein (pre-beta-HDL) on the endothelium function. In this study, we have investigated the effects of Free-Apo A-I and artificial pre-beta-HDL on endothelial NO synthase (eNOS) activity and on NO production by endothelial cells. Free-Apo A-I drastically inhibited NO production in human umbilical cord vein endothelial cells (HUVECs) and eNOS activity in bovine aortic endothelial cells (BAECs). Pre-beta-HDL and serum from human apolipoprotein A-I transgenic rabbits inhibited eNOS activity in BAECs but HDL3 did not. Free-Apo A-I displaced eNOS from BAEC plasma membrane towards intracellular pools without affecting eNOS activity and eNOS mass in BAEC crude homogenates. Free-Apo A-I and HDL3 did not decrease either caveolin bound to BAEC plasma membrane or caveola cholesterol content. As previously described, we showed that HDL3 directly induced endothelium-dependent relaxation of rings from rat aorta. We observed that pre-beta-HDL significantly decreased endothelium-dependent relaxation of rat aortic rings ex vivo.

The oxidation of plasma LDLs (low-density lipoproteins) is a key event in the pathogenesis of atherosclerosis. LPC (lysophosphatidylcholine) and oxysterols are major lipid constitutents of oxidized LDLs. In particular, 7-oxocholesterol has been found in plasma from cardiac patients and atherosclerotic plaque. In the present study, we investigated the ability of 7-oxocholesterol and LPC to regulate the activation of eNOS (endothelial nitric oxide synthase) and cPLA2 (cytosolic phospholipase A2) that synthesize two essential factors for vascular wall integrity, NO (nitric oxide) and arachidonic acid. In endothelial cells from human umbilical vein cords, both 7-oxocholesterol (150 µM) and LPC (20 µM) decreased histamine-induced NO release, but not the release activated by thapsigargin. The two lipids decreased NO release through a PI3K (phosphoinositide 3-kinase)-dependent pathway, and decreased eNOS phosphorylation. Their mechanisms of action were, however, different. The NO release reduction was dependent on superoxide anions in LPC-treated cells and not in 7-oxocholesterol-treated ones. The Ca2+ signals induced by histamine were abolished by LPC, but not by 7-oxocholesterol. The oxysterol also inhibited (i) the histamine- and thapsigargin-induced arachidonic acid release, and (ii) the phosphorylation of both cPLA2 and ERK1/2 (extracellular-signal-regulated kinases 1/2). The results show that 7-oxocholesterol inhibits eNOS and cPLA2 activation by altering a Ca2+-independent upstream step of PI3K and ERK1/2 cascades, whereas LPC desensitizes eNOS by interfering with receptor-activated signalling pathways. This suggests that 7-oxocholesterol and LPC generate signals which cross-talk with heterologous receptors, effects which could appear at early stage of atherosclerosis.

Intravenous gamma-immunoglobulin (i.v.Ig) is commonly used in the treatment of autoimmune and inflammatory vascular disorders to prevent thrombotic complications. The mechanism of action of i.v.Ig is, however, not yet elucidated. In view of this, we investigated the ability of i.v.Ig to modulate i) Ca(2+) signals of fura-2 loaded endothelial cells, and ii) the associated release of nitric oxide (NO) and von Willebrand factor (vWf). NO was measured either indirectly by radioimmunoassay of cGMP in unstimulated cells or directly by electrochemistry at the surface of stimulated endothelial cells from human umbilical cord veins (HUVEC). Short-term treatment of unstimulated HUVEC with intact i.v.Ig decreased the basal cytosolic Ca(2+) concentration by 20% while it activated the NO/cGMP synthesis. Following i.v.Ig treatment of HUVEC, the Ca(2+) liberation from internal stores and the vWf secretion induced by ATP, thrombin or histamine were significantly reduced by 38 and 60%, respectively. The effects on Ca(2+) signals were observed with intact i.v.Ig as well as with the F(ab')2 or the Fc fragments indicating that both portions are involved in the mechanism of action. The i.v.Ig treatment of HUVECs had no effect on the NO release induced by thrombin or histamine. By contrast, the i.v.Ig treatment increased the ATP-activated NO release by amplifying the Ser1177-eNOS phosphorylation. The i.v.Ig also activated the NO-dependent cGMP release in resting and collagen-stimulated platelets. Since NO is a potent inhibitor of platelet activation and vWF is a platelet adhesion cofactor, the beneficial effects of therapeutic i.v.Ig may lie in the inhibition of platelet adhesion to damaged endothelium.

Release of superoxide anion by cultured vascular cells was investigated with the use of selective microelectrodes. Local concentration of superoxide anion (O 2 •− ) was followed by differential pulse amperometry on a carbon microfiber at 0.1 V/SCE. The oxidation current allows O 2 •− detection in the 10 −8 M concentration range without interference of the other major oxygen species. Interleukin-1β-stimulated O 2 •− release that progressively increased to reach local concentrations at the cell membrane level of 76 ± 11 nm 40–60 min after stimulation in human cord vein endothelial cells, and 131 ± 18 nm 1–2 h after stimulation in internal mammary artery smooth muscle cells. In the two types of cells, the O 2 •− oxidation signal was suppressed in the presence of superoxide dismutase. Spontaneous O 2 •− release from unstimulated cells was undetectable. These results demonstrate that selective microelectrodes allow direct and real-time monitoring of local O 2 •− released from vascular endothelial as well as from smooth muscle cells submitted to an inflammatory stimulus.

We describe here the electrochemical detection of nitric oxide, NO, in biological systems by using chemically modified ultramicro carbon electrodes. In the first part of the paper, the different steps involved in the electrochemical preparation and characterization of the nickel-based sensor are described. This is illustrated by the use of nickel(II) tetrasulfonated phthalocyanine complex. The second part of the paper describes two examples of the direct electrochemical measurement of NO production in human blood platelets and endothelial cells from umbilical cord vein.

NO production in platelets has been followed by electrochemical detection. It was undetectable in unstimulated platelets and in thrombin or ADP-stimulated platelets, but dose-dependently stimulated by collagen. A production of 5 10(-19) mol/platelet was reached with 9 micrograms collagen. In collagen-stimulated platelets, preincubation with 1 mM L-Arg, D-Arg or L-NMMA increased by 77%, left unchanged or decreased by 63% NO production, respectively. NO production did not parallel cytosolic Ca2+ changes, although it decreased in low Ca2+ medium or when Ca2+ transients were attenuated by intracellular Ca2+ buffer. These results confirm that human platelets can generate NO. They demonstrate that cytosolic [Ca2+], although participating in the regulation of its synthesis, is not the messenger for NO synthase activation. Platelet NO production could become functionally important when collagen fibrils of the sub-endothelium are accessible.