Annie Brunet

Elaboration and use of nickel planar macrocyclic complex-based sensors for the direct electrochemical measurement of nitric oxide in biological media.

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.

Mitochondrial Arginase II Modulates Nitric-Oxide Synthesis through Nonfreely Exchangeable l-Arginine Pools in Human Endothelial Cells

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 Nω-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 μM. 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 μM. Extracellular l-arginine also dose-dependently increased NO release and arginase activity. When HUVECs were stimulated by thrombin in the presence of 100 μM 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.

Effects of chronic hypoxia and endurance training on muscle capillarity in rats.

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<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<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.

Lysophosphatidylcholine and 7-oxocholesterol modulate Ca2+ signals and inhibit the phosphorylation of endothelial NO synthase and cytosolic phospholipase A2.

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 microM) and LPC (20 microM) 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.

Superoxide release from interleukin-1β-stimulated human vascular cells: in situ electrochemical measurement

Release of superoxide anion by cultured vascular cells was investigated with the use of selective microelectrodes. Local concentration of superoxide anion (O2*-) was followed by differential pulse amperometry on a carbon microfiber at 0.1 V/SCE. The oxidation current allows O2*- detection in the 10(-8) M concentration range without interference of the other major oxygen species. Interleukin-1beta-stimulated O2*- 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 O2*- oxidation signal was suppressed in the presence of superoxide dismutase. Spontaneous O2*-release from unstimulated cells was undetectable. These results demonstrate that selective microelectrodes allow direct and real-time monitoring of local O2*- released from vascular endothelial as well as from smooth muscle cells submitted to an inflammatory stimulus.

Electrochemical sensing of nitric oxide for biological systems: methodological approach and new insights in examining interfering compounds.

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.

Effects of endurance training at high altitude on diaphragm muscle properties

The biochemical, histochemical, and structural changes induced by endurance training and long-term exposure to high altitude were studied in the diaphragm muscle of rats exposed to simulated altitude (HA: n = 16; Pb = 62 kPa, 463 Torr; 4000 m) and compared to animals maintained at sea-level (SL: n = 16). Half of the animals in each group were trained (T) by swimming for 12 weeks, the other half were kept sedentary (S). Except for a small decrease in type I fibres in the HA-S group (−7%, P<0.05), in favour of type IIab and type IIb fibres, neither high-altitude exposure nor endurance training had an overall effect on fibre type distribution. The mean fibre cross-sectional area was found to be unaffected by altitude and/or chronic exercise. Capillary density was shown to be increased by both high-altitude exposure (P<0.02) and training (P<0.001), whereas capillary growth, estimated by the capillary/fibre ratio, was unaffected in both cases. Following endurance training, a modest increase in citrate synthase was shown to occur to the same extent in the HA-T and SL-T groups (+15% and + 16% respectively, NS). Hexokinase increased following training (P<0.05) and high-altitude exposure (P<0.001). In normoxic and hypoxic animals, endurance training enhanced the ratio of the heart-specific lactate dehydrogenase isozyme LDH1 to total LDH activity (+59%, P<0.01; +92%, P<0.05 respectively). It may be hypothesized that the increased glucose phosphorylation capacity observed in diaphragm muscle contributes to the reduction of glycogen utilization during exercise.

Quinacrine increases endothelial nitric oxide release: role of superoxide anion.

The effect of acute quinacrine treatment on agonist-induced nitric oxide (NO) release was investigated in cultured human endothelial cells using electrochemical monitoring of the in situ NO concentration. Quinacrine dose-dependently increased NO release with an apparent EC50 of 0.2 microM and a maximal effect at 1 microM. Quinacrine did not modify the dependence of NO release on extracellular L-arginine. Acceleration or deceleration of O2- dismutation, which altered NO release in control cells, did not modify it in quinacrine-treated cells. Quinacrine did not modify NO amperometric signal or reaction with O2- produced by xanthine oxidation. In the presence of quinacrine, agonist-induced NO release became Mg2+ -independent and could not be attributed to an inhibition of phospholipase A2 activity. Quinacrine made NO release insensitive to Cu2+ chelation. The present study demonstrates that acute treatment by low quinacrine concentrations increases endothelial NO release, possibly through an inhibition of O2- production.

Analysis of agonist-evoked nitric oxide release from human endothelial cells: role of superoxide anion.

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.

Métaux de transition et production de monoxyde d’azote par les cellules endothéliales humaines

Abstract 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.