Pro-atherogenic effect of interleukin-4 in endothelial cells: modulation of oxidative stress, nitric oxide and monocyte chemoattractant protein-1 expression.

BACKGROUND: Although considered as an anti-inflammatory cytokine, interleukin-4 (IL-4) has been shown to be pro-atherogenic in mice models of atherosclerosis. OBJECTIVES: In order to elucidate this paradox, we have investigated the effects of IL-4 on characteristic atherogenic parameters in human umbilical vein endothelial cells (HUVECs): production of reactive oxygen species, expression of monocyte chemoattractant protein-1 (MCP-1) and nitric oxide (NO) bioavailability. RESULTS: Incubation of HUVECs with IL-4 resulted in an increased production of reactive oxygen species and extracellular O(2)(-)(*) measured using fluorogenic probes and Cytochrome c that was inhibited by superoxide dismutase or gp91ds-tat, a selective NADPH oxidase inhibitor. The latter also inhibited IL-4 induced over-expression of MCP-1 mRNA measured by classical and real time RT-PCR. Incubation of HUVECs with IL-4 reduced thrombin-induced NO release, detected by electrochemistry, an effect which was reversed by incubation with superoxide dismutase. Both production of reactive oxygen species and MCP-1 mRNA over-expression induced by IL-4 were fully inhibited by selective inhibitors of phosphatidyl inositol 3-kinase. CONCLUSION: The data demonstrate that IL-4 up-regulates the expression of MCP-1 and decreases NO bioavailability through activation of NADPH oxidase in endothelial cells. These results are in favor of a pro-inflammatory and pro-atherogenic effect of IL-4 in vascular tissues.

Fluorescence measurements of free Ca2+ concentration in human erythrocytes using the Ca2+-indicator fura-2.

We report here the use of the fluorescent Ca2+-chelator fura-2 to directly measure free Ca2+ concentration within intact human erythrocytes and the influence of viscosity on the fluorescence of this probe. The bright fluorescence of fura-2 has permitted the use of low concentrations of indicator and cells, thus minimizing the screening effect and the intrinsic fluorescence of haemoglobin. Erythrocytes (10(8) cells/ml) were loaded with 0.5 microM fura-2AM then diluted at 10(7) cells per ml for measurements. The extracellular signal was suppressed by addition of manganese ions just before recording spectra. Under these conditions, a blood sample of 100 microliter was sufficient for analysis. To study the influence of viscosity on fura-2 fluorescence, gelatin and polyvinylpyrrolidone at various concentrations were added to a physiological buffer to perform fura-2-Ca fluorescence standard curves. Fluorescence intensities and the apparent affinity constant for Ca2+ were modified by viscosity. When intra-erythrocytic viscosity was simulated with 21 g/l polyvinylpyrrolidone to obtain a mean viscosity of 14 mPa.s similar to that observed in human erythrocytes, the mean value of free Ca2+ concentration measured in erythrocytes from healthy subjects was 78 +/- 16 nM (mean +/- S.D., n = 29).

SK&F 96365 inhibits intracellular Ca2+ pumps and raises cytosolic Ca2+ concentration without production of nitric oxide and von Willebrand factor.

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.

Control of the erythrocyte free Ca2+ concentration in essential hypertension.

Since Ca2+ ions seem to directly participate in the control of erythrocyte membrane structure and deformability and because cell Ca2+ metabolism has been repeatedly proposed to be modified in hypertension, the intracellular calcium ion concentration ([Ca2+]i) was investigated in red blood cells from hypertensive and normotensive subjects. [Ca2+]i was measured by using the fluorescent Ca2+ chelator fura-2. Red blood cell [Ca2+]i was increased in hypertensive compared with normotensive subjects in the whole population and further increased when hypertensive were compared with age-matched normotensive subjects. An inverse relation between age and [Ca2+]i was observed when calculated with blood pressure adjusted. In hypertensive patients, high [Ca2+]i values were associated with a reduced erythrocyte deformability. The initial rate of 45Ca2+ uptake did not differ between the two blood pressure groups. Similarly, when the extracellular Ca2+ concentration was elevated from 1 to 2 mmol/l, [Ca2+]i increased by 16 +/- 4% (p less than 0.03) in red blood cells from both groups, thus maintaining a significant difference between hypertensive and normotensive subjects. Under these conditions, the addition of 10(-7) mol/l nicardipine, a dihydropyridine Ca2+ antagonist, decreased [Ca2+]i by 15 +/- 4% (p less than 0.05) and 7 +/- 5% in erythrocytes from hypertensive and normotensive subjects, respectively, thereby reducing the difference in [Ca2+]i observed between these two groups. This nicardipine effect was positively correlated to the initial [Ca2+]i. In the presence of 5 mumol/l W7, a calmodulin antagonist, [Ca2+]i increased significantly only in erythrocytes from hypertensive patients (26 +/- 6%, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Direct characterization of the Na+/H+ exchanger in human platelets.

The kinetic properties of the Na+/H+ exchanger in human platelets were investigated by direct measurements of pHi as detected with the fluorescent dye, BCECF. In acid-loaded cells, the antiporter displayed a hyperbolic dependence regarding external Na+ with an apparent Km of 38 +/- 4 mM (pHo 7.2 at 25 degrees C) whereas its pHi-dependent activation between 7.3 to 6.4 did not obey a Michaelian model. External acidification from 7.7 to 6.5 decreased significantly the initial rate of Na(+)-dependent H+ efflux. The amiloride derivative, ethylisopropylamiloride blocked this exchanger and exerted a non-competitive inhibition with respect to Na+o (Ki = 17 nM). The cation selectivity of the external site of the antiporter was Na+ greater than Li+ greater than K+ and choline. These results indicate that the BCECF technique allows to evaluate the main features of the Na+/H+ exchanger in human platelets, which possesses kinetic properties similar to those reported in other cell types.

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.

Further investigation of platelet cytosolic alkalinization in essential hypertension.

OBJECTIVE: To verify that platelet cytosolic pH is altered in essential hypertension and to investigate the mechanisms involved. METHODS: Cytosolic pH was determined in unstimulated platelets by the fluorescent indicator 2,7-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF). Membrane microviscosity was evaluated by the fluorescence anisotropies of diphenylhexatriene (DPH) and its cationic derivative trimethylamino-diphenylhexatriene (TMA-DPH). RESULTS: The cytosolic alkalinization previously observed in platelets from untreated hypertensive patients was confirmed. The buffering capacity appeared unaltered and the cytosolic pH was not modified by 50 mumol/l N-5-ethylisopropylamiloride, a specific inhibitor of the Na(+)-H+ exchange. Exposure to external Na(+)-free media produced an intracellular acidification that was similar in hypertensive and normotensive donors and maintained the cytosolic pH difference between the two groups. In the two blood pressure groups platelet cytosolic pH varied inversely with the steady-state anisotropy of TMA-DPH but not with that of DPH. Experimentally induced acidification of the cytosol by Na+ removal with or without nigericin treatment was accompanied by rises in TMA-DPH anisotropy. CONCLUSIONS: This study of platelet intracellular pH in essential hypertension confirms cytosolic alkalinization and demonstrates its association with changes in the dynamic properties of the platelet plasma membrane.

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.

In vitro inhibition by endothelins of thrombin-induced aggregation and Ca2+ mobilization in human platelets.

1. The in vitro effects of endothelins (ET-1 and ET-3) on human platelets were investigated by measurement of the aggregatory responses of washed platelets to thrombin and by the determination of cytosolic pH (pHi) and free Ca2+ concentration ([Ca2+]i) determined with the fluorescent indicators, BCECF and Fura-2. 2. ET-1 and ET-3 at concentrations ranging from 10(-10) to 5 x 10(-7) M, did not promote platelet aggregation but inhibited in a dose-dependent manner the aggregation induced by 0.05 u ml-1 thrombin (P less than 0.002 and less than 0.001, respectively) with maximal effects reached at 10(-8) M (17 +/- 3 and 15 +/- 2%, n = 11, P = 0.002 for each). 3. Even at 5 x 10(-7) M, ET-1 and ET-3 did not cause a measurable change in basal [Ca2+]i and pHi. When tested in combination with thrombin, 5 x 10(-7) M ET-1 and ET-3 decreased the transient peak of [Ca2+]i by 17 +/- 7 and 28 +/- 7% (n = 7 and 11, P = 0.03 and P = 0.002). No effect on pHi variations was detected. In the virtual absence of external Ca2+, 5 x 10(-7) M ET-3 inhibited the peak of [Ca2+]i by 18 +/- 6% (n = 6, P = 0.02). 4. The anti-aggregating agents, prostacyclin (PGI2, 10(-8)-10(-7) M) and nitroprusside (NP, 10 ng-50 micrograms l-1) also induced a dose-dependent inhibition of the thrombin-induced [Ca2+]i peak (P = 0.001 for each).A combination of 10-9M PGI2 and 1O ng P' NP augmented the inhibitory effect of each drug(PGI2 alone 52 +/-11, plus NP 90 +/- 2; NP alone 26 +/- 4, plus PGI2 69 +/- 5% inhibition of [Ca2 ], peak, n = 6 for each, P <0.01 and P <0.001, respectively). Platelet preincubation with 5 x 10-7M ET-3 increased by 34+/-11% (n = 6, P = 0.0 14) the inhibitory effect of NP 1O ng without a significant influence on the PGI2 effect.5. In conclusion, endothelins ET-1 and ET-3 can reduce in vitro the aggregating response of human platelets to thrombin by a mechanism that is probably due to decrease Ca2+ mobilization.

Refilling state of internal Ca2+ stores is not the only intracellular signal stimulating Ca2+ influx in human endothelial cells.

To further analyse the role of the refilling state of internal Ca2+ pools in the stimulation of Ca2+ influx in human endothelial cells, we investigated the combined effect of thapsigargin (TG) and histamine on cytosolic Ca2+ concentration ([Ca2+]i) and inositol polyphosphate production. At normal extracellular Ca2+ levels, TG induced a progressive and sustained elevation in [Ca2+]i which was dose-dependently prevented by pretreatment with 1-10 microM histamine. Similarly, pretreatment with 0.1 and 1 microM TG suppressed histamine-induced Ca2+ transients partially and totally, respectively. TG pretreatment did not alter the inositol triphosphate (IP3) level liberated by histamine, but modified IP3 metabolism by decreasing inositol biphosphate (IP2) and increasing inositol monophosphate (IP1) contents. In the absence of Ca2+ influx, 1 microM TG only induced a small transient increase in [Ca2+]i whereas the Ca2+ mobilization evoked by 10 microM histamine was unchanged. In both cases, the absence of any additional effect of either TG, histamine or 2 microM ionomycin indicated the complete depletion of Ca2+ stores. The re-establishment of the transmembrane Ca2+ gradient induced a transient rise in [Ca2+]i. Its amplitude differed between histamine- and TG-treated cells. It was imposed by cell pretreatment and was selectively affected by changes in the membrane potential. At 5 mM external K+, the transient rise in [Ca2+]i was more marked in histamine- than in TG-stimulated cells; this difference was suppressed by TG pretreatment. The presence of 130 mM external K+ increased Ca2+ entry in TG-treated cells but reduced it in histamine-stimulated cells. These results indicate that the refilling state of internal Ca2+ stores does not constitute the single regulator of Ca2+ influx. TG and histamine seem to activate Ca2+ influx through distinct but interdependent pathways regulated by membrane potential.

In vivo and in vitro effects of isradipine on cytosolic Ca2+ concentration in erythrocytes from spontaneously hypertensive rats.

Cytosolic Ca2+ concentration ([Ca2+]i) was investigated in erythrocytes from spontaneously hypertensive rats (SHR) and their normotensive controls (WKY), after an acute treatment with the Ca2+ antagonist isradipine. Blood samples were obtained from conscious rats and [Ca2+]i measured with fura-2. The [Ca2+]i was higher in SHR than in WKY erythrocytes (P < .05). Isradipine administration had no effect on WKY [Ca2+]i, but reduced that of SHR to WKY levels after 1 h. In vitro, isradipine dose-dependently decreased [Ca2+]i only in SHR (P = .006). The reduction by isradipine of the elevated [Ca2+]i in SHR suggests the presence of a greater dihydropyridine-sensitive Ca2+ influx in the SHR erythrocyte.

Cytosolic pH in cultured cardiac myocytes and fibroblasts from newborn spontaneously hypertensive rats.

Newborn spontaneously hypertensive rats (SHR) develop cardiac hypertrophy before a rise in blood pressure. Cytosolic pH (pHi) has been discovered to modulate cell growth and proliferation; therefore, we have investigated pHi in myocytes and fibroblasts from 3- to 4-day-old SHR and normotensive Wistar (W) and Wistar-Kyoto controls (WKY). The ratio of heart to body weight was higher in SHR than in W and WKY (7.56 +/- 0.10 v 6.21 +/- 0.10 and 5.98 +/- 0.14 mg/g in 10, 5, and 7 groups of 20 to 40 animals; P less than .001 for both). Cytosolic pH, determined with the fluorescent probe BCECF, was measured from the sixth to the eighth day in culture on confluent cells. The mean pHi was higher in myocytes from SHR than in those from W or WKY rats (7.19 +/- 0.03, N = 30, v 7.09 +/- 0.03 and 7.11 +/- 0.02, N = 25 and 30; P = .008 and .024, respectively). In contrast, pHi was similar in fibroblasts from the three strains (7.21 +/- 0.03, 7.18 +/- 0.03, and 7.19 +/- 0.02, N = 15, 15, and 14, in SHR, W, and WKY rats, respectively). External acidification induced similar decreases in pHi from SHR and WKY myocytes, maintaining higher pHi values in SHR myocytes along the entire external pH (pHo) range studied. The inhibition of Na+/H+ exchange by the amiloride derivative, ethylisopropylamiloride, decreased the steady-state pHi of myocytes independently of the initial pHi values. This study demonstrated a cytosolic alkalinization in contractile cardiac cells from SHR before a significant rise in blood pressure and in the absence of hemodynamic influences and specific plasma factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Isradipine affects histamine-induced cytosolic Ca2+ movements in human endothelial cells.

Although endothelial actions of dihydropyridines remain controversial, isradipine has been observed to exert anti-atherosclerotic actions in which endothelium could be involved. This study was designed to investigate the direct effects of isradipine on cytosolic Ca2+ concentration in cultured human umbilical vein endothelial cells. Isradipine (from 10 nM to 1 microM) had no effect on unstimulated cells but dose-dependently decreased both the transient [Ca2+]i peak and the sustained increase induced by histamine. Its maximal effects were reached at 0.1 microM. In the absence of Ca2+ influx or in depolarized cells, 1 microM isradipine still significantly decreased the transient [Ca2+]i peak (by 23 +/- 8% and 42 +/- 11%). Ca2+ influx induced by re-establishment of transmembrane Ca2+ gradient was also inhibited by isradipine, as was that induced by 1 microM thapsigargin. These results demonstrate that isradipine is able to reduce both Ca2+ release from internal stores and the consequent Ca2+ entry in stimulated human endothelial cells.

Stimulation by nifedipine of calcium transport by cardiac sarcolemmal vesicles from spontaneously hypertensive rats.

The effects of the calcium antagonist nifedipine on the binding and ATP-dependent accumulation of calcium by cardiac plasma membranes from spontaneously hypertensive rats (SHR) and from their normotensive controls (WKY) were studied at free calcium concentrations of 2 X 10(-8) M and 4 X 10(-7) M corresponding to very high affinity and high affinity binding sites respectively. Nifedipine did not significantly modify calcium binding to either class of sites in SHR or WKY membranes. In contrast, in a free calcium concentration of 2 X 10(-8) M, nifedipine enhanced ATP-dependent Ca2+ transport. The concentration of nifedipine required for significant stimulation was smaller in SHR than in WKY membranes (10(-7) M and 10(-5) M respectively). Another calcium antagonist, D600, did not modify ATP-dependent calcium accumulation by SHR or WKY vesicles. These results raise the question of the mechanism of action of nifedipine on the calcium pump and confirm the presence of abnormalities in cardiac plasma membranes from young SHR, rendering them more sensitive than WKY membranes to the calcium antagonist nifedipine.

Na(+)- and Ca(2+)-dependent pH regulation in unstimulated human platelets.

The influence of transmembrane Na+ and Ca2+ gradients on cytosolic pH (pHi) and free Ca2+ concentration ([Ca2+]i) have been examined in unstimulated human platelets with the aid of BCECF and Fura-2 fluorescent dyes. The removal of external Na+ (Na+o) acidified the cytosol in a pHo-dependent manner which was insensitive to EIPA and DIDS, the inhibitors of the Na+/H+ exchanger and bicarbonate transporters. Na+o removal also increased [Ca2+]i by 17 +/- 5%, but the amplitude of the concomitant acidification was independent on Ca2+ influx or cytosolic Ca2+ concentration. In contrast, in the presence of 145mM Na+o, a rise in external Ca2+ concentration from 1 to 2mM increased [Ca2+]i by 38 +/- 11% and acidified the cytosol by 0.16 +/- 0.04 pH units. These results indicated that, in resting human platelets, the transmembrane Na+ gradient is a major determinant of pHi. Two Na(+)-dependent processes have been found: one is triggered by an external acidification and the other activated by a rise in Ca2+ influx or cytosolic concentration.

Calmodulin abolishes the changes in Ca2+ binding and transport by heart sarcolemmal membranes of spontaneously hypertensive rats.

Both Ca2+ transport and binding properties of heart sarcolemmal membranes are altered in spontaneously hypertensive rats (SHR) when compared to their normotensive controls (WKY). The effects of calmodulin on these two processes were studied at free calcium concentrations presumed to be the physiological levels in the cytosol. At a calcium concentration of 2.10(-8)M, calmodulin did not significantly modify either binding or ATP-dependent accumulation of calcium by membranes of both origins. In contrast, at a free calcium concentration of 4.10(-7)M, calmodulin enhanced the calcium binding to SHR membranes and the ATP-dependent calcium transport by SHR and WKY membranes. Differences in calcium binding and ATP-dependent accumulation between the two substrains were suppressed in presence of calmodulin. These data demonstrate that modifications in calcium handling by SHR cardiac plasma membranes might be due to altered intracellular content or function of calmodulin in SHR.

Alterations of membrane properties in erythrocytes of salt hypertensive Sabra rats.

This study was designed to investigate the effects of a hypertensive stimulus, high salt intake, in hypertension-prone (SBH) and -resistant (SBN) Sabra rats on erythrocyte Na+ content (Na+i), Ca2+ influx and cytosolic Ca2+ concentration ([Ca2+]i). The relationships of these parameters to plasma lipids, circulating digoxin-like immunoreactivity and membrane microviscosity, determined by the fluorescence anisotropy of trimethylamino-diphenylhexatriene (TMA-DPH) and diphenylhexatriene (DPH), were also evaluated. Erythrocytes of SBH rats were characterized by increased [Ca2+]i, unchanged Ca2+ influx and reduced Na+i. There were no significant differences in the plasma digoxin-like immunoreactivity between the two strains. High-salt intake decreased membrane microviscosity (DPH anisotropy) in SBH rats but did not alter the above parameters. Erythrocyte [Ca2+]i correlated positively with diastolic blood pressure and negatively with erythrocyte Na+i. Membrane dynamics evaluated by the two fluorescent probes did not correlate with [Ca2+]i, Ca2+ influx or Na+i whereas DPH anisotropy was inversely related to blood pressure. These relationships were independent of plasma cholesterol or triglycerides. It can be concluded that 1) similarly to earlier observations in essential hypertension and spontaneously hypertensive rats, erythrocyte [Ca2+]i correlates positively with blood pressure in salt-dependent hypertension, and 2) increased erythrocyte Na+ content need not be a hallmark of hypertension.

Erythrocyte Ca2+ handling in the spontaneously hypertensive rat, effect of vanadate ions.

Cytosolic Ca2+ concentration ([Ca2+]i) and 45Ca2+ influx were investigated in erythrocytes from conscious spontaneously hypertensive rats (SHR) and their normotensive controls Wistar-Kyoto (WKY). [Ca2+]i was evaluated with fura-2 and intra- and extra-cellular calibration parameters were compared. Irrespective of the calibration parameters used, erythrocyte [Ca2+]i was always significantly higher in SHR than in WKY and Wistar rats (by 25 and 40%, p < 0.01 and 0.001). A rise of the external Ca2+ concentration from 1 to 2 mmol/l increased less [Ca2+]i in SHR than in WKY erythrocytes (17 vs 37%, p < 0.01). SHR erythrocytes incorporated more 45Ca2+ than those from WKY, with an initial rate of 45Ca2+ uptake higher by 57% than that of WKY erythrocytes (p < 0.05). Vanadate ions, after corrections of their quenching effect on red cell and fura-2 fluorescence signals, increased [Ca2+]i by 19% in WKY erythrocytes (p = 0.05), but did not modify the SHR values. They also increased 45Ca2+ accumulation and the initial rate of 45Ca2+ influx in WKY erythrocytes only (p < 0.01). This study indicates that, when compared to WKY rats, erythrocytes from SHR are characterized by higher [Ca2+]i values, higher initial rate of Ca2+ influx and low sensitivity to vanadate ions.

Cell calcium handling and intracellular pH regulation in hereditary hypertriglyceridemic rats: reduced platelet response to thrombin stimulation.

Multiple cell membrane alterations have been described in humans and animals with various genetic forms of hypertension and/or dyslipidemia. The aim of our study was to characterize some properties of platelets and/or erythrocytes (cytosolic calcium handling, intracellular pH regulation and thrombin responsiveness) in a new model of genetic hypertension associated with hyperlipidemia-Prague hereditary hypertriglyceridemic (HTG) rats. There were no differences in basal cytosolic Ca2+ values in platelets or erythrocytes of HTG rats and control Wistar rats. Ca2+ influx into erythrocytes was also similar in HTG and control rats. In both strains Ca2+ influx correlated positively with plasma triglycerides. The slope of this relationship was less steep in HTG than in Wistar rats. Cytosolic Ca2+ response to thrombin stimulation was smaller in HTG platelets, which were also characterized by a major reduction of thrombin-induced Mn2+ entry through receptor-operated Ca2+ channels. Platelets of HTG rats had the same basal intracellular pHi values and similar buffering capacity as control rats but their pHi response to thrombin stimulation was substantially reduced. It can be concluded that reduced responsiveness to thrombin stimulation is a major alteration found in platelets of hypertensive hereditary hypertriglyceridemic rats.

Changes in platelet free Ca2+ concentration after chronic digoxin treatment.

Cell Na+ and Ca2+ concentrations control each other by various mechanisms. In excitable cells from various origins, Ca2+ extrusion from the cell and its entry are dependent for a large part on the activity of the Na+, Ca2+-countertransport system. Cytosolic free Ca2+ concentration is also controlled by the Na+-H+ exchange activity. To analyze the changes in cytosolic Ca2+ concentration accompanying the reduction of the membrane Na+ gradient, cytosolic free Ca2+ concentration ([Ca2+]i) was measured by fluorescent dyes in platelets and erythrocytes from healthy subjects, before and during digoxin treatment (0.25 mg/day for 6 days). [Ca2+]i was increased in platelets from 169 +/- 30 to 321 +/- 61 nmol/l (n = 7, P less than 0.02) and unchanged in erythrocytes (121 +/- 6 and 104 +/- 7 nmol/l). This increase in platelet [Ca2+]i was not accompanied by a change in serotonin content (5.43 +/- 0.67 vs 5.49 +/- 0.61 10(-7) mol per 10(11) cells) and could not be reproduced by in vitro addition of 10(-4) mol/l ouabain (198 +/- 33 vs 186 +/- 73 nmol/l). The enhanced [Ca2+]i in platelets is thus not a short-term consequence of a reduced membrane Na+ gradient, but reflects either the overload of intracellular Ca2+ stores or an enhanced in vivo stimulation by hormones or neurotransmitters.