Cyclic nucleotides in platelets of genetically hypertriglyceridemic and hypertensive rats. Thrombin and nitric oxide responses are unrelated to plasma triglyceride levels.

Prague hereditary hypertriglyceridemic (HTG) rats constitute a genetic model of hypertension associated with hyperlipidemia and insulin resistance. Various cell alterations, including changes in membrane dynamics, ion transport, and decreased platelet responses to thrombin have been observed in this strain. As hypertriglyceridemia appears to be associated with reduced endothelium-dependent vasodilation and platelet aggregation, we examined whether triglycerides could modulate cell responsiveness through changes in cyclic nucleotides in platelets of HTG rats. From the age of 6 weeks, these hypertensive animals were subjected for 10 weeks to interventions that modified circulating triglycerides levels (2.17+/-0.09 mmol/l), leading to their reduction (gemfibrozil treatment, 0.87+/-0.05 mmol/l) or elevation (high fructose intake, 3.23+/-0.07 mmol/l). Basal cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) contents were 15% and 48% lower in isolated platelets of HTG rats than in those of Lewis controls. cAMP level was further reduced in HTG rats subjected to high fructose intake. Irrespective of their plasma triglyceride levels, the thrombin-induced increase in platelet cGMP levels present in Lewis rats was absent in platelets of HTG rats. In contrast, no strain- or treatment-related differences were observed in the magnitude or kinetics of cGMP response to exogenous nitric oxide (NO). NO-induced cGMP and cAMP changes were associated in an opposite manner with trimethylamino-diphenylhexatriene (TMA-DPH) anisotropy, a biophysical parameter that reflects the microviscosity of the outer part of the cell membrane. Our results indicate that the attenuation of platelet responsiveness to thrombin in HTG rats represents a strain difference that cannot merely be due to a difference in plasma triglyceride levels. Platelet hyporesponsiveness to agonists such as thrombin in HTG rats cannot be explained by a change in levels of inhibitory cyclic nucleotides, since they were actually found to be low and not high.

[Transition metals and nitric oxide production in human endothelial cells]. / Métaux de transition et production de monoxyde d'azote par les cellules endothéliales humaines.

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 Ca2+ and transition metals. In the presence of 1 mM external Ca2+, a low concentration of EGTA (5 microM) 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 Cu2+ could regulate NO availability.

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. 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.

Chronic changes in plasma triglyceride levels do modify platelet membrane microviscosity in rats.

Lipid metabolism disorders were proposed to mediate numerous cell membrane alterations in various forms of hypertension. Elevated plasma triglycerides were found to be associated with changes in membrane structure and function related to altered microviscosity in particular domains of the cell membrane. The aim of our study was to determine if an abnormal triglyceride metabolism might play a causal role in these alterations of membrane dynamics. Using genetically hypertensive rats of the Prague hereditary hypertriglyceridemic (HTG) strain we investigated whether the elevation of circulating triglycerides induced by high fructose intake and/or their lowering by chronic gemfibrozil treatment (for 10 weeks starting at the age of 6 weeks) are followed by reciprocal changes in membrane microviscosity. Two different fluorescent probes exploring either the outer membrane leaflet (TMA-DPH anisotropy) or the membrane lipid core (DPH anisotropy) were used in platelets of HTG rats. DPH (diphenylhexatriene) fluorescence anisotropy was decreased in platelets of fructose-treated HTG animals with highly elevated plasma triglyceride levels, whereas it was increased in gemfibrozil-treated HTG rats in which triglyceride levels were almost normalized. On the contrary, TMA-DPH (trimethylamino-diphenylhexatriene) anisotropy was not substantially altered in platelets from HTG rats by the above modifications of circulating triglycerides. No changes of plasma cholesterol or blood pressure were associated with the triglyceride-dependent modifications of membrane core microviscosity. Our interventional study demonstrates a major causal role of circulating triglycerides in the control of the microviscosity of membrane lipid core.

Influence of SIN-1 on platelet Ca2+ handling in patients with suspected coronary artery disease: ex vivo and in vitro studies.

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.

Electrochemical detection of nitric oxide production in human polymorphonuclear neutrophil leukocytes.

The detection of nitric oxide (NO) release by human polymorphonuclear neutrophil leukocytes (PMNs) presents several difficulties, mainly due to concomitant production of O2- and H2O2, which could interfere with the measurements. A Nafion and nickel porphyrin-coated microelectrode was used to measure NO production in PMNs in vitro. It allowed detection of 6.3 +/- 1.9 nM NO in a PMN-containing system and was unaffected by added chemicals. Addition of the chemotactic oligopeptide f-met-leu-phe (fMLP; 100 nM) induced a NO release which reached a value of 71 +/- 30 pmol NO/10(6) PMN x ml(-1) 5 min after stimulation in the presence of SOD (150 U/ml). If SOD was omitted, the corresponding value was 36 +/- 20 pmol NO/10(6) PMN x ml(-1). Presence or absence of catalase did not alter the amount of NO measured. Addition of the NO-synthase inhibitor N(G)-monomethyl-L-arginine (LNMMA; 1 mM) reduced the current by 82 +/- 20%. These results agree with the rate of NO production in human PMNs when measured spectrophotometrically using the NO-dependent oxidation of oxyhaemoglobin to methaemoglobin. The NO production in human PMN was dependent on fMLP concentrations, but independent of cell-concentrations of 0.5-3.5 x 10(6)/ml. This paper shows that a electrochemical method, e.g. Nafion and porphyrin-coated microelectrode, is suitable for studies of NO release from stimulated human PMNs.

Superoxide release from interleukin-1B-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.

Membrane microviscosity, blood pressure and cytosolic pH in Dahl rats: the influence of plasma lipids.

OBJECTIVE: To determine the relationships between blood pressure, membrane microviscosity, plasma lipids and cytosolic pH in Dahl rats susceptible or resistant to salt hypertension. DESIGN AND METHODS: Blood pressure, plasma triglycerides and total cholesterol, platelet cytosolic pH (pHi) and the microviscosity of both outer membrane leaflet (TMA-DPH fluorescence anisotropy) and membrane lipid core (DPH fluorescence anisotropy) were studied in platelets and erythrocyte ghosts of Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) rats fed either a low-salt diet (0.3% NaCl) until the age of 9, 15 or 24 weeks or a high-salt diet (4% NaCl) for 5 or 10 weeks after weaning. RESULTS: At low salt intake, DPH but not TMA-DPH anisotropy increased with age in platelets of SS/Jr rats. Chronic high salt intake was accompanied by an increase of DPH anisotropy in platelets but not in erythrocyte ghosts of SS/Jr rats. Platelet DPH anisotropy correlated positively with blood pressure of salt-loaded SS/Jr rats. Chronic high salt intake also reduced pHi in platelets, the regulation of which seemed to be related to the changes in TMA-DPH anisotropy. This especially concerns the thrombin-induced pHi rise which was inversely related to basal pHi, plasma lipids and TMA-DPH anisotropy. Altered membrane lipid composition might be the underlying mechanism because both membrane microviscosity and platelet pHi regulation were reported to correlate significantly with plasma triglycerides and/or cholesterol. CONCLUSIONS: Platelets of salt hypertensive Dahl rats are characterized by an increased microviscosity of membrane lipid core which correlated positively with blood pressure. The major influence of plasma triglycerides on DPH anisotropy should be taken into consideration when investigating the links between membrane microviscosity and blood pressure. On the other hand, the changes in microviscosity of the outer membrane leaflet might be involved in pHi regulation (probably through control of the Na+/H+ exchanger).

Effect of pravastatin on erythrocyte rheological and biochemical properties in poorly controlled Type 2 diabetic patients.

AIMS: The rheological properties of erythrocytes are impaired in diabetes mellitus, especially because of changes in their membrane lipid composition. In hypercholesterolaemic patients, lowering plasma cholesterol is associated with an improvement of the erythrocyte rheological parameters. The aim of this study was to investigate the relationship between erythrocyte deformability, plasma lipids, lipid membrane composition and cytosolic cations in poorly controlled Type 2 diabetic patients and to test the effects of a cholesterol-lowering treatment on these parameters. METHODS: We compared 37 poorly controlled Type 2 diabetic patients with 26 controls. In 22 of the diabetic patients who showed an impairment in erythrocyte deformability (filtration index >10.5 on the Hanss' haemorheometer), a double-blind randomized trial compared the effect of the inhibitor of HMG CoA reductase pravastatin 20 mg per day for 4 months vs. placebo on the erythrocyte parameters. RESULTS: Compared with controls, diabetic patients had higher filtration index (FI), erythrocyte sodium and calcium contents and lower free cholesterol-phospholipids ratio in erythrocyte membranes. Erythrocyte sodium content correlated positively with the FI and the membrane free cholesterol-phospholipids ratio. In the pravastatin-treated group (11 patients), fibrinogen decreased significantly, FI reached a normal value (<10) in six patients. Four of the five other patients who still had abnormal FI after 4 months of treatment had either a high plasma triglycerides (> or =4.60 mmol/l) or a high plasma fibrinogen (> or =4 g/l) level at baseline. Only two of the 11 placebo-treated patients achieved a normal FI. CONCLUSION: These data suggest that in poorly controlled Type 2 diabetic patients there is a link between the chemical composition and the rheological properties of erythrocytes. Erythrocyte deformability may be improved by lowering plasma cholesterol with a statin.

Abnormalities of membrane function and lipid metabolism in hypertension: a review.

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.

Influence of hypercholesterolemia and endothelin-3 pre-treatment on the effects of shear forces on platelet aggregation and cyclic GMP content.

Shear forces induce platelet aggregation and stimulate the endothelial production of anti-aggregatory factors. Among them, endothelin-3 (ET-3) has been reported to reduce aggregation and to increase platelet cyclic GMP (cGMP) content. Since hypercholesterolemia modifies both platelet aggregability and endothelial function, we compared in 14 hypercholesterolemic and 15 normocholesterolemic subjects the influences of shear forces (240 and 650 s(-1)) on platelet aggregation and cGMP content, and their modulation by ET-3. Spontaneous maximal aggregation occurred earlier and at a greater extent in hypercholesterolemic than in normocholesterolemic subjects (63+/-2 vs 46+/-6% P < 0.01). Pre-treatment with ET-3 abolished the shear-induced facilitation of maximal aggregation in platelets of normocholesterolemic (from 70+/-2 to 52+2% at 240 s(-1) and from 73+/-1 to 59+/-2S at 650s(-1); P < 0.05) and hypercholesterolemic (from 78+/-3 to 64+/-2 at 240 s(-1) and from 78+/-2 to 66+/-3 at 650 s(-1); P < 0.05) subjects. cGMP content did not significantly differ between normocholesterolemic and hypercholesterolemic subjects (6.1+/-0.5 vs 6.9+/-0.7 pmol/10(9) platelets). It was reduced in platelets submitted to shear forces (P < 0.05). This shear-dependent reduction was suppressed by ET-3 pre-treatment. These results demonstrate that shear forces enhance platelet aggregation and diminish their cGMP content. ET-3 reduces the pro-aggregating effects of shear, suggesting a rise in cGMP content as a dynamic associated mechanism.

Activation of nitric oxide release and oxidative metabolism by leukotrienes B4, C4, and D4 in human polymorphonuclear leukocytes.

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.

Inhibition by cholesterol oxides of NO release from human vascular endothelial cells.

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.

Relations of platelet Ca2+ handling and membrane microviscosity to vascular tone and restenosis after angioplasty in human coronary artery.

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.

Nitric oxide production in human endothelial cells stimulated by histamine requires Ca2+ influx.

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.

Membrane microviscosity and plasma triacylglycerols in the rat.

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.

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.

Nitric oxide production by endothelial cells: comparison of three methods of quantification.

Vascular endothelial cells have been found to produce a relaxant mediator, identified as nitric oxide (NO) and implicated in numerous physiological functions. Subsequently, there has been an intensive search for accurate and specific detection methods to measure biological NO production. In the present study, we compared three approaches to evaluate NO production, based respectively on the Griess reaction (that quantifies nitrites and nitrates after their reduction), on the hemoglobin reaction (that quantifies oxyhemoglobin to methemoglobin transformation by NO), and on the electrochemical NO detection with a porphyrinic micro-probe. Comparison was made both under standard conditions and biological conditions, through calibration curves and measurements of histamine-induced NO production by cultured human endothelial cells and its modulation by L-arginine and N(omega)-monomethyl-L-arginine. We demonstrated that these three methods differ in terms of sensitivity and selectivity. The hemoglobin reaction and nitrate measurements suffer from a lack of specificity. Nitrite determination by the Griess reaction was hardly suitable for kinetic studies but it remains useful for the evaluation of basal NO production. The electrochemical technique, although it does not allow measurement of basal NO production, is the only one to exhibit great sensitivity and specificity and to allow instantaneous and non destructive measurements. This study brings up the potential hazards and pitfalls that may be associated with the various methods.

Platelet hypoaggregability in hereditary hypertriglyceridemic rats: relation to plasma triglycerides.

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.

Cytosolic pH and calcium in Dahl salt-sensitive and salt-resistant rats: the relationship to plasma lipids.

OBJECTIVE: To search for alterations of cytosolic pH and cell calcium handling in platelets and erythrocytes of Dahl rats susceptible and resistant to salt-induced hypertension. DESIGN AND METHODS: Blood pressure, plasma lipids, platelet cytosolic calcium concentration ([Ca2+]i) and pH (pHi) together with thrombin-induced changes in these parameters as well as erythrocyte [Ca2+]i and 45Ca influx were determined in Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) rats aged 9, 15 and 24 weeks, which were fed a low-salt diet (0.3% NaCl), and in animals fed high-salt diet (4% NaCl) for 5-10 weeks since weaning. RESULTS: With a low salt intake platelet pHi was lower in SS/Jr than it was in SR/Jr rats, whereas basal platelet [Ca2+]i was similar in rats of both strains. The difference in basal pHi between SS/Jr and SR/Jr rats increased progressively with age of animals. A high salt intake from youth did not influence platelet [Ca2+]i in rats of either strain but it caused an earlier decrease in pHi in SR/Jr than it did in SS/Jr rats. Thrombin stimulation induced similar elevations of pHi and [Ca2+]i in rats of both strains, irrespective of age, salt intake and response of blood pressure to salt intake. Erythrocyte 45Ca influx and [Ca2+]i were greater for SS/Jr rats but only the latter parameter was correlated positively to blood pressure. Both regulation of platelet pHi and erythrocyte Ca2+ handling were significantly related to plasma lipid levels. CONCLUSIONS: Platelets of SS/Jr rats fed a low-salt diet were characterized by a lower basal cytosolic pHi but unchanged [Ca2+]i relative to those of SR/Jr rats. Hypertension induced by high salt intake was associated with increased erythrocyte [Ca2+]i but not with elevation of platelet [Ca2+]i or alteration of response to stimulation with thrombin.