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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 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.

Hypertension, which is characterized by multiple alterations in the structure and function of the cell membrane, is often associated with important metabolic abnormalities including those concerning lipid metabolism. Dyslipidemia accompanying essential hypertension consists of elevated plasma triglycerides, low HDL cholesterol, and increased levels of atherogenic LDL cholesterol particles. The altered membrane microviscosity seen in hypertensive subjects reflects the changes of membrane lipid composition resulting from intensive exchange between circulating and membrane lipids, as well as from abnormal cellular lipid synthesis and metabolism. The changes of membrane microviscosity are known to modulate the activity of proteins involved in ion transport, signal transduction, cell Ca2+ handling, intracellular pH regulation, etc. Alterations in plasma or membrane lipids are indeed closely associated with ion transport abnormalities as well as with impaired control of cytosolic Ca2+ and pH in various forms of hypertension in both humans and rats. Such lipid-dependent modifications of membrane properties in cells participating in the cardiovascular regulation might be a part of pathogenetic mechanisms responsible for chronic blood pressure elevation. Thus nutritional and pharmacologic interventions aiming to normalize abnormal lipid metabolism could be useful for amelioration of membrane abnormalities and lowering of high blood pressure. Future studies of functional membrane alterations in hypertension or dyslipidemia will therefore require the detailed determination of membrane lipid composition and the measurement of microviscosity in particular membrane domains.

Because arachidonate metabolites are potent mediators of inflammation, we have studied the effects of leukotriene B4 (LTB4) and the cysteinyl leukotrienes C4 and D4 (LTC4 and LTD4) on the release of nitric oxide (NO), in vitro, by human polymorphonuclear granulocytes (PMN). Two independent and highly sensitive real-time methods were used for these studies, ie, the NO-dependent oxidation of oxyhemoglobin (HbO2) to methemoglobin and a NO-sensitive microelectrode. When activated with LTB4, LTC4, or LTD4, but not with other lipoxygenase products such as 5S-HETE, 5-oxo-ETE or 5S, 12S-diHETE, PMN produced NO in a stimulus- and concentration-dependent manner. The rank order of potency was LTB4 = LTC4 > LTD4, corresponding to 232 +/- 50 pmol of NO/10(6) PMN for 100 nmol/L LTB4 after 30 minutes. The kinetic properties of the responses were similar for all three leukotrienes with a maximum response at 13 +/- 3 minutes. Cysteinyl leukotriene and LTB4 antagonists inhibited the agonist-induced NO production by 70%, and treatment with Bordetella pertussis toxin, or chelation of cytosolic Ca2+, [Ca2+]i, also efficiently inhibited this response. In contrast, treatment of PMN with cytochalasin B (5 microg/mL) enhanced the LTB4-induced NO formation by 86%. Thus, this is the first demonstration that the cysteinyl leukotrienes LTC4 and LTD4, as well as LTB4, activate NO release from human PMN by surface receptor, G-protein and [Ca2+]i-dependent mechanisms. This effect differs from activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, for which only LTB4 is an activator.

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, 7beta-hydroxycholesterol, 5alpha,6alpha-epoxycholesterol, 19-hydroxycholesterol (60 microg/mL), and alpha-lysophosphatidylcholine (10 microg/mL) were compared. Pretreatment of HUVECs with cholesterol, 5alpha,6alpha-epoxycholesterol, or 19-hydroxycholesterol did not alter histamine-activated NO production. In contrast, pretreatment with 7-ketocholesterol or 7beta-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.

This study was designed to assess whether platelet Ca2+ handling or membrane microviscosity could be considered as indexes of vascular tone, or could help to predict an increased risk of restenosis after coronary angioplasty. Vascular tone was quantified in 21 patients with stable angina by the vasodilator response to sin-1 intracoronary injection in the reference coronary segment and by the importance of the acute recoil after angioplasty in the narrowed segment. The degree of restenosis was quantified by coronary angiography 6 months later. Individual values of relative sin-1-induced changes in the reference coronary diameter were positively correlated with cytosolic Ca2+ concentration in unstimulated platelets, irrespective of the extracellular Ca2+ concentration (p < 0.01). This relationship was also observed with the thrombin-evoked Ca2+ changes, measured in the absence of a Ca2+ influx (p = 0.01). No relationship was found between sin-1-induced coronary changes and membrane microviscosity evaluated by TMA-DPH and DPH anisotropies or platelet volume, or between degree of acute recoil and platelet characteristics. In conclusion, platelet Ca2+ reflects the vasodilating efficacy in response to sin-1, but cannot help to predict restenosis after coronary angioplasty.

The causal relationships between cytosolic free-Ca2+ concentration ([Ca2+]i) increases and production of nitric oxide (NO) have been investigated mostly with indirect methods and remain unclear. Here we demonstrate, by direct real-time measurements of [NO] with a porphyrinic microsensor, that Ca2+ entry, but not an increase in [Ca2+]i, is required for triggering of NO production in human endothelial cells. Histamine, ranging from 0.1 to 100 microM, increased both NO production and [Ca2+]i when given in a single dose. However, histamine caused increased NO release but induced progressively smaller [Ca2+]i changes when cumulatively added. In the absence of a transmembrane Ca2+ gradient, no significant NO release was detectable, despite the marked Ca2+ peak induced by histamine. Inhibition of Ca2+ entry by SK&F 96365 abolished histamine-elicited NO production but only reduced the transient [Ca2+]i rise. The suppression of the sustained [Ca2+]i response under these two conditions suggests that NO release was closely associated with Ca2+ entry from the extracellular space. In addition, membrane depolarization, achieved by increasing the extracellular K+ concentration from 5 to 130 mM, reduced both the amplitude of histamine-induced sustained [Ca2+]i elevation and NO production. These results lead us to propose that the availability of numerous Ca2+ ions around the internal side of the plasma membrane would promote the association between nitric oxide synthase and calmodulin, thereby activating the enzyme.

1. Multiple cell membrane alterations have been described in humans and animals with various genetic forms of hypertension and/or dyslipidaemia. The aim of our study was to characterize membrane microviscosity, using two different fluorescent probes exploring either the outer membrane leaflet [trimethylamino-diphenylhexatriene (TMA-DPH)] or the lipid membrane core [diphenylhexatriene (DPH)], in platelets and erythrocytes of genetically hypertensive rats of the Prague hereditary hypertriglyceridaemic (HTG) strain. The relationships of membrane microviscosity to hypertension, hypertriglyceridaemia and cell calcium handling were also investigated. 2. Membrane microviscosity was similar in HTG and normotensive control Wistar rats when measured in platelets or erythrocyte ghosts incubated in Na(+)-containing medium. On the contrary, TMA-DPH fluorescence anisotropy was significantly reduced in HTG platelets incubated in Na(+)-free medium because external Na+ removal elicited a larger rise of TMA-DPH anisotropy in Wistar platelets. 3. Plasma triacylglycerols were associated positively with platelet TMA-DPH anisotropy and negatively with DPH anisotropy in both strains. The slopes of these relationships were reduced in HTG compared with Wistar rats. Platelet TMA-DPH anisotropy correlated positively and DPH anisotropy negatively with the cytosolic calcium concentration in unstimulated platelets, the slopes being almost identical in both strains. 4. Pulse pressure correlated negatively with TMA-DPH anisotropy and positively with DPH anisotropy found in erythrocyte ghosts. 5. The present results suggest that plasma triacylglycerols and cytosolic calcium are capable of modulating the membrane microviscosity in this new animal model of genetic hypertension associated with hypertriglyceridaemia.

To define better the relationships between lipid metabolism disturbances and platelet aggregation we have examined these parameters in hereditary hypertriglyceridemic and control Lewis rats. Hereditary hypertriglyceridemic rats are hypertensive and have high plasma triglycerides but not elevated plasma total cholesterol. In the present study, we have demonstrated that platelets from hereditary hypertriglyceridemic rats have lowered initial rate and maximal aggregation after stimulation with thrombin or ADP in comparison with controls. These two strains did not differ significantly in the inhibition of platelet aggregation by the thromboxane A2 receptor inhibitor, SQ 29 548. In hereditary hypertriglyceridemic rats, the thrombin response, as well as the contribution of the thromboxane A2-sensitive pathway, were positively associated with the plasma level of triglycerides. Similar trend was found in Lewis rats. However, the slopes of these relationships were reduced in hereditary hypertriglyceridemic rats. These alterations of the aggregatory responses in hereditary hypertriglyceridemic rats were independent of blood pressure and plasma cholesterol level. In conclusion, our results showed a clear-cut platelet hypoaggregability to both thrombin and ADP in hypertensive hypertriglyceridemic rats. This hypoaggregability was not due to an impaired function of the thromboxane A2 pathway but could be connected with disturbances of lipid metabolism.

The effects of the imidazole compound SK&F 96365 on Ca2+ movements and production of nitric oxide (NO) and von Willebrand factor (vWF) have been investigated in human endothelial cells. Changes in cytosolic Ca2+ concentration ([Ca2+]i) were measured with Fura-2. Real-time production of NO was monitored with a porphyrinic microsensor and the release of vWF with an enzyme-linked immunosorbent assay. Irrespective of the transmembrane Ca2+ gradient, 30 microM SK&F 96365 doubled [Ca2+]i suggesting a Ca2+ release from intracellular stores. The SK&F 96365-induced [Ca2+]i rise was not accompanied by detectable NO and vWF production, while 1 microM thapsigargin enhanced [Ca2+]i 2.5 times, doubled the secretion of vWF and increased the NO production to 10 +/- 4 nM (n = 5). Pretreatment with SK&F 96365 prevented thapsigargin from increasing [Ca2+]i, NO production and vWF secretion. To investigate the mechanism by which SK&F 96365 released Ca2+ from internal pools, its effect and that of thapsigargin on the ATP-dependent 45Ca2+ uptake into platelet membrane vesicles were compared. SK&F 96365 as thapsigargin, dose-dependently reduced the initial rate of 45Ca2+ uptake. In conclusion, we demonstrate that, in the absence of Ca2+ entry from the extracellular space, the [Ca2+]i increase elicited by SK&F 96365 or thapsigargin is not sufficient to initiate NO synthesis and vWF secretion. This confirms the important role of Ca2+ influx in endothelial secretion processes.

1. Nitric oxide (NO) donors inhibit platelet function and Ca2+ mobilization evoked by different agonists. This led us to investigate the direct effects of authentic NO on basal cytosolic Ca2+ concentration ([Ca2+]i) and on Ca2+ mobilization induced by thrombin or by two inhibitors of intracellular Ca(2+)-ATPases, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone (t-BuBHQ). 2. Cytosolic Ca2+ concentration was evaluated with Fura-2, in the absence of Ca2+ influx. Addition of 5 microM NO increased by 48% the basal cytosolic [Ca2+] of resting human platelets whereas a lower concentration (0.1 microM) did not induce significant modifications. This NO-induced Ca2+ increase was inversely correlated with the basal level of cytosolic [Ca2+]. 3. NO pretreatment for 30 to 120 s decreased by 42 to 57% the transient [Ca2+]i peak evoked by 0.10 u ml-1 thrombin and strongly attenuated the initial rate of [Ca2+]i rise induced by 1 microM thapsigargin or by 20 microM t-BuBHQ. The two components of the thapsigargin response, the Ca2+ release due to inhibition of Ca2+ pumps and the thromboxane A2-dependent self-amplification mechanism, were inhibited by NO. The observation that a subsequent stimulation was still capable of eliciting Ca2+ release suggests the presence of NO-insensitive Ca2+ stores. 4. These findings indicate that nitric oxide can modulate basal cytosolic [Ca2+] in unstimulated human platelets and decrease the Ca2+ mobilization from NO-sensitive internal stores evoked by stimulation of receptors or by Ca(2+)-ATPase inhibitors. This underlines the important role of nitric oxide in the modulation of platelet Ca2+ handling.