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Hypophosphatasia (HPP) is a rare hereditary disease characterized by defective skeletal mineralization, and with a broad severity spectrum. The perinatal forms, lethal and non-lethal, are associated with severe neonatal respiratory distress, potential seizures, hypotrophy and marked hypotonia. The diagnosis is rapidly suggested by a combination of typical radiological signs, hypercalcemia, hyperphosphatemia and low alkaline phosphatase (ALP) activity. In the infantile form, the clinical signs appear before the age of six months, but the patients usually have no or very mild signs at birth. The diagnosis should be considered in the event of early deformation of the pectus, feeding difficulties, hypotonia, frequent respiratory tract infections, hypercalcemia, and even early constitution of craniosynostosis. Radiological signs may be less obvious characterized by irregular metaphyses and generalized hypomineralization. Management is initially symptomatic, and adjusted to the symptoms. Care should be provided by a multidisciplinary team, in close collaboration with Reference Centers experts for the disease. Currently, recombinant enzyme replacement therapy (ERT) is under development for the severe form of HPP. The course of the disease, depending on the degree of severity and the various types of management, requires long-term evaluation through joint prospective follow-up to assess the long-term outcomes of these patients. Multidisciplinary follow up is needed to identify the medical and socio-economic outcomes of children and adults affected by HPP., (© 2017 Elsevier Masson SAS. All rights reserved.)

Recent studies have suggested that an oral dose of acetylcysteine could play a prophylactic role in the prevention of nephrotoxicity from iodine contrast media in patients affected by chronic renal failure. Between June 2001 and September 2002 we selected 120 patients with a basal plasma creatinine level greater than 1.36 mg/dl investigated by coronary angiography. The treatment group included 60 patients who received 600 mg of acetylcysteine in the morning and evening before the day of the examination together with intravenous saline hydration. The control group patients received hydration alone. The clinical characteristics of the groups were comparable as well as the basal plasma creatinine level: 2.01+/-1.1 mg/dl in the acetylcysteine group and 1.81+/-0.69 in the control group. The plasma creatinine level was measured 24 and 48 hours after coronary angiography. The respective changes in plasma creatinine level at 24 and 48 hours were 0.12+/-0.29 and 0.02+/-0.29 mg/dl in the acetylcysteine group and 0.06+/-0.29 and 0.07+/-0.43 mg/dl in the control group (NS). Acute renal failure caused by the contrast medium, defined by an increase of 25% in the plasma creatinine level compared to the basal value, occurred in 3 patients from the acetylcysteine group and 2 patients from the control group. The only predictive factor for acute renal failure was the quantity of contrast medium (316+/-141 vs 173+/-115 ml, p<0.05). In conclusion, acute renal failure caused by contrast medium is rare in sufficiently hydrated patients with moderate chronic renal failure when a low dose of contrast medium is used. Our study does not confirm a prophylactic effect of acetylcysteine in the prevention of nephrotoxicity from contrast media following coronary angiography in patients with moderate chronic renal failure.

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.

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.

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.

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.

Objective: To study the relationship between high blood pressure and hyperlipidaemia and the cytosolic calcium concentration in unstimulated platelets, focusing on the effects of an alteration in membrane dynamics., Materials and Methods: Basal cytosolic calcium concentrations were determined in the presence and the absence of a significant calcium influx in platelets of 47 untreated hypertensive patients and 26 normotensive subjects. Membrane microviscosity was investigated by fluorescence depolarization of diphenylhexatriene and trimethylaminodiphenylhexatriene. To study the influence of plasma factors, unstimulated platelets were loaded in the presence of plasma with Quin-2, which forms a relatively strong intracellular calcium buffer. The cytosolic calcium concentration was then determined at two extracellular calcium concentrations (1 mmol/l and in the absence of a Ca2+ influx)., Results: Irrespective of the external calcium concentration, the cytosolic calcium concentration increased significantly with diastolic blood pressure (P = 0.026 in the presence and P = 0.003 in the absence of Ca2+ influx) and with plasma triacylglycerols (P = 0.03 and 0.001, respectively). Multiple regression analysis indicated that the cytosolic Ca+ concentration was independently related to these two factors [Ca2+ = 35 + (18.6 +/- 4.6). In triacylglycerols (mmol/l) + (0.45 +/- 0.15) mmHg diastolic blood pressure; P < 0.001]. The relationship between the cytosolic calcium concentration and diphenylhexatriene or trimethylaminodiphenylhexatriene anisotropies was not independent of blood pressure and plasma triacylglycerol levels., Conclusions: The present results confirm the link between blood pressure and the platelet cytosolic calcium concentration and indicate that plasma triacylglycerols directly or indirectly modulate the ex vivo efficacy of platelet calcium storage and/or extrusion mechanisms. They could facilitate cell stimulation.

This study was designed to evaluate the relationships between platelet cytosolic Ca2+ concentration ([Ca2+]i) and plasma lipids in patients with primary hypercholesterolemia. In a double-blind, placebo-controlled trial, we determined platelet [Ca2+]i in the presence and virtual absence of extracellular Ca2+ and the effects of prolonged treatment with pravastatin, a selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Platelet [Ca2+]i and membrane microviscosity were determined in 22 normotensive hypercholesterolemic men. Platelet [Ca2+]i was observed to vary with in vivo plasma lipid characteristics: in untreated patients, [Ca2+]i determined at low extracellular Ca2+ concentration was significantly associated with plasma triacylglycerols (P = .008) and with the total cholesterol to HDL cholesterol ratio (P = .044). Triacylglycerol levels also correlated inversely with the external Ca(2+)-dependent [Ca2+]i rise. Pravastatin treatment reduced plasma total cholesterol (-20 +/- 3%), LDL cholesterol (-30 +/- 3%), triacylglycerols (-17 +/- 6%), and apoB levels (-25 +/- 4%) and simultaneously decreased platelet [Ca2+]i measured in a low-Ca2+ medium by 14 +/- 6% (P = .03). However, [Ca2+]i values remained positively correlated with the total cholesterol to HDL cholesterol ratio (P = .04). Prvastatin treatment did not induce marked changes in membrane microviscosity, although the changes in trimethylaminodiphenylhexatriene anisotropy were inversely correlated with those of HDL cholesterol. These results indicate that plasma lipids can modulate cytosolic Ca2+ in platelets by affecting Ca2+ transport pathways that are dependent and independent of Ca2+ influx.

1. To investigate the possibility that arterial hypertension is associated with changes in the physicochemical properties of cell membranes, we have studied the effects of dietary salt loading on platelet membrane microviscosity in hypertension-prone and -resistant Sabra rats. 2. Sixteen hypertension-prone and 14 hypertension-resistant Sabra rats were submitted to either a low-salt (0.25% NaCl) or a high-salt (4% NaCl) diet for 3-4 weeks. Platelet membrane anisotropy was determined, in the presence and absence of extracellular Na+, using two fluorescent probes, diphenylhexatriene and trimethylamino-diphenylhexatriene, inserted in different areas of the cell membranes. 3. A decrease in diphenylhexatriene anisotropy was demonstrated when platelets of hypertension-prone (but not hypertension-resistant) Sabra rats were suspended in a Na(+)-free medium. This alteration in membrane dynamic properties is localized within the hydrophobic core of the platelet membranes and is independent of salt intake. It reflects an abnormal fluidizing effect of extracellular Na+ removal. 4. Platelets of hypertension-prone and hypertension-resistant Sabra rats did not differ significantly in trimethylamino-diphenylhexatriene fluorescence anisotropy, irrespective of the incubation media used. Extracellular Na+ removal caused an increase in trimethylamino-diphenylhexatriene fluorescence anisotropy in all groups, the change being greatest in salt-loaded rats. 5. This study indicates that platelet membrane microviscosity is specifically altered in the hypertension-prone Sabra rat irrespective of salt intake. This raises the question of the relation of this inherited defect with the susceptibility of this strain to dietary salt loading.

The altered membrane microviscosity demonstrated in various cells of spontaneously hypertensive rats (SHR) and essential hypertensive (EH) patients has been proposed to play an important role in the pathogenesis of genetic forms of hypertension. The aim of this study was to evaluate possible changes of membrane microviscosity in platelets and red cell ghosts of Lyon hypertensive (LH) and normotensive (LN) rats. Both erythrocyte and platelet membranes of LH rats had a clear tendency to reduced DPH fluorescence anisotropy reflecting the decreased core membrane microviscosity. On the other hand, there were no changes in TMA-DPH fluorescence anisotropy that characterizes the dynamic properties of the outer membrane leaflet. DPH, but not TMA-DPH, anisotropy correlated negatively with blood pressure. This was true for both red cell ghosts and platelets. Membrane microviscosity had no significant relationship to plasma cholesterol or triglycerides. In platelets, TMA-DPH anisotropy correlated positively with cytosolic free calcium concentration ([Ca2+]i). A similar trend was observed in erythrocytes. In contrast, DPH anisotropy had an inverse relationship to platelet [Ca2+]i. It can be concluded that the alterations of membrane microviscosity seen in LH rats are completely different from those reported in SHR animals and that surface and core membrane microviscosity differ in their relationship to blood pressure and [Ca2+]i.

Haemodynamic shear forces have been reported to exert direct and indirect effects on platelet reactivity. In vitro, they activate platelets leading to spontaneous or facilitated aggregation. In vivo, they stimulate the production of endothelium-derived anti-aggregatory agents. This study was designed to evaluate in hypertensive patients, before and after antihypertensive treatment, the possible role of these haemodynamic forces, determined at the brachial artery level on the ex vivo platelet aggregatory response to ADP and collagen. Platelet reactivity, evaluated by EC50 for ADP and collagen, was found to be related to blood velocity, shear rate and shear stress (p < 0.01 for each). These inverse correlations of platelet aggregation with stress levels did not depend on age, body mass index, mean blood pressure, serum cholesterol and triglycerides or haematocrit. They were also independent of platelet cytosolic Ca2+ and cyclic AMP. The changes in shear forces and in aggregatory responses to ADP and collagen induced by nitrendipine treatment for 6 months remained negatively correlated, confirming the relationships existing between haemodynamic shear forces and platelet reactivity. These results indicate that the shear antiaggregant effects, likely mediated by flow-dependent endothelium-derived factors, prevail over its direct platelet aggregating effects.

The mechanisms by which trimetazidine (1-[2,3,4-trimethoxybenzyl]-piperazine) exerts its cytoprotective action have not been identified. This study was designed to investigate in human platelets and erythrocyte ghosts a possible perturbation of membrane dynamics by trimetazidine. Its effects on the steady-state anisotropies of two fluorescent probes, trimethylamino-diphenyl-hexatriene (TMA-DPH) and diphenylhexatriene (DPH) were compared. The effects on the aggregatory responses to collagen and ADP, and on platelet cAMP content were also investigated. In platelets, trimetazidine dose-dependently raised TMA-DPH anisotropy but not that of DPH. It reduced cAMP content (in the presence of Ro 15-2041, a phosphodiesterase inhibitor) and the aggregation responses to collagen and ADP. This suggests that trimetazidine decreases the 'fluidity' of the outer part of the plasma membrane, the adenylyl cyclase activity and some steps involved in platelet activation. In erythrocyte ghosts, the fluorescence anisotropy of TMA-DPH was not modified by trimetazidine. The membrane effects reported here could participate in the protection of cell metabolism afforded by a long-term treatment with trimetazidine.

Essential hypertension is often associated with high levels of plasma cholesterol or triglycerides. The relationships between plasma lipids and platelet lipids, membrane fluidity and functions in untreated hypertensive patients were investigated by measuring the fluorescence anisotropies of two fluorescent dyes (DPH and its cationic derivative, TMA-DPH, with different subcellular localization), cytosolic Ca2+ and pH, cyclic AMP content and aggregation to ADP and collagen. Hypercholesterolemia was found to be accompanied by a rise in platelet cholesterol content without changes in TMA-DPH or DPH anisotropies whereas hypertriglyceridemia was associated with a decreased cholesterol to phospholipid molar ratio, a decreased DPH anisotropy and a tendency of the cytosol to alkalinize. These results point out the differences between the effects of an acute cholesterol load and those of chronic hypercholesterolemia on platelet membrane microviscosity and aggregation. They demonstrate a strong association between plasma triglyceride levels and platelet membrane structure.

Our previous experiments in human and rat platelets demonstrated that the absence of extracellular Na+ increased the fluorescence anisotropy of TMA-DPH (trimethylamino-diphenylhexatriene, probe preferentially incorporated into the outer leaflet of the plasma membrane). Here we investigated further the in vitro effects of Na+ ions on membrane dynamic properties. Na(+)-dependent changes were reversible and they required about 10-20 min to be induced. They were specifically located in the TMA-DPH environment because they were not observed with diphenylhexatriene (probe non-selectively incorporated into all hydrophobic domains of the cell). To evaluate the possible influence of the intracellular Na+, the effects of sodium replenishment, monensin, ouabain and thrombin on TMA-DPH anisotropy were measured. A rise in intracellular Na+ above the physiological level was associated with unchanged or slightly decreased TMA-DPH anisotropy whereas its decrease was accompanied by a pronounced rise in TMA-DPH anisotropy. Our data indicate that the changes in intracellular Na+ concentration, rather than those in extracellular Na+ concentration, are responsible for the alterations in platelet membrane fluidity probed by TMA-DPH.

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.

Hypercholesterolemia and hypertension are two of the major risk factors associated with increased atherosclerotic vascular disease. An abnormal platelet function is one of the mechanisms proposed to participate in atherogenesis. This study was undertaken to find out whether hypercholesterolemia in hypertensive patients can change platelet lipid composition and reactivity. Twenty-nine untreated hypertensive patients were distributed into 3 age, body mass index and blood pressure-matched groups according to their plasma cholesterol levels (normal, borderline or elevated, group NC, BC and HC respectively). Their platelet lipid composition, cytosolic Ca2+ concentration, cyclic AMP content and aggregating response to ADP and collagen were determined. Platelet from group HC patients were characterized by reduced cyclic AMP content (evaluated in the presence and absence of a platelet phosphodiesterase inhibitor) and aggregating responses to ADP and collagen, increased palmitic acid content and decreased arachidonic, eicosapentaenoic and docosatetraenoic and pentaenoic acid content, resulting in a lowered polyunsaturated to saturated fatty acid ratio (P less than 0.001). In contrast, platelet cytosolic Ca2+ concentration, DPH steady-state anisotropy and cholesterol to phospholipid molar ratio were not significantly changed. This indicates that hypercholesterolemia is accompanied in hypertensive patients by marked changes in platelet fatty acid composition, cyclic AMP content and response to aggregating agents. These changes, which clearly differ from those induced by in vitro cholesterol loading, could reflect not only the balance between LDL and HDL stimulation but also an adaptation to hemodynamic perturbations.

1. The metabolism of blood platelets, taken as an accessible model of excitable cells, has been reported to be altered in hypertension. Most of the identified alterations concern the functions of various plasma membrane constituents. 2. A possible modification of membrane microviscosity was investigated by 1,6-diphenyl-1,3,5-hexatriene and 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene fluorescence depolarization. In order to determine whether or not the membrane structures probed by these indicators were related to platelet physiological functions, the cytosolic free Ca2+ concentration was determined in parallel. 3. At physiological temperature, the fluorescence anisotropy of 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene was decreased in untreated hypertensive patients (0.276 +/- 0.002 versus 0.288 +/- 0.002, n = 23 and 22, P less than 0.001), indicating a lowered microviscosity at the lipid-water interface of cell membrane. It correlated inversely with blood pressure (P less than 0.001) and cytosolic free Ca2+ concentration (P less than 0.030). On the contrary, 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy was observed to vary with sex but not with blood pressure. 4. These results suggest that structural membrane modifications may participate in the various functional abnormalities observed in platelets from hypertensive patients.

Na+ ions, which can play a pathogenic role in the development of high blood pressure, have been reported to regulate membrane enzymatic activities, receptor-ligand interaction and coupling of G-protein receptors to their effectors. This study was designed to investigate the in vitro effects of Na+ ions on membrane dynamic properties. The fluorescence anisotropy values of TMA-DPH (trimethylamino-diphenylhexatriene, probe selectively incorporated into the outer leaflet of the plasma membrane) was evaluated in platelets and erythrocytes of sodium-dependent hypertension-prone and -resistant rats of the Sabra Strain. Whereas no difference was observed between the 2 strains, TMA-DPH anisotropy was found to be strongly influenced in platelets by external Na+ ions. In the absence of external Na+, TMA-DPH anisotropy increased in human and rat platelets. In contrast, Na+ ions did not affect the anisotropy when the probe was inserted into erythrocyte ghosts. This indicates that Na+ ions can acutely regulate order parameter and microviscosity of platelet plasma membrane in the regions explored by the probe.

1. Blood cells and vascular endothelial cells are subjected to a wide range of haemodynamically generated shear stress forces. In vitro, membrane stretching or shear stress have been observed to activate ion channels and cell metabolism and to facilitate erythrocyte and platelet aggregation. 2. The present study was designed to evaluate the participation of shear stresses in the control of apparent platelet cytosolic free Ca2+ concentration in hypertensive patients. 3. Shear conditions and platelet cytosolic free Ca2+ concentration in vitro were studied after a dynamic perturbation induced by 3 months of double-blind treatment with one of two beta-antagonists, carteolol and atenolol. Brachial artery wall shear rate and stress were estimated by means of a pulsed Doppler velocimeter, and blood viscosity was measured by a co-axial viscometer at a shear rate of 96 s-1. Platelet cytosolic free Ca2+ concentration was simultaneously measured by using the Quin-2 fluorescent chelator. The direct effect of atenolol and carteolol on platelet cytosolic free Ca2+ concentration in vitro was also measured after addition of the beta-blockers to platelet-rich plasma. 4. Atenolol and carteolol decreased blood pressure similarly but their effects on shear rate (P less than 0.02), shear stress (P less than 0.01) and platelet cytosolic free Ca2+ concentration (P less than 0.05) differed after 3 months of therapy. In contrast, neither of the drugs significantly altered platelet cytosolic free Ca2+ concentration, in vitro per se. 5. In the overall population, strong positive correlations existed not only between changes in platelet cytosolic free Ca2+ concentration and those in shear rate (r = 0.81, P less than 0.001) and shear stress (r = 0.83, P less than 0.001), but also between their absolute values, suggesting a possible haemodynamic shear-dependent modulation of transmembrane Ca2+ transport.

Digitalis compounds are known to increase Ca2+ influx in various cells. As platelet cytosolic free [Ca2+] and plasma digitalis-like activity have been reported independently to be higher in some untreated hypertensive patients than in normotensive subjects, we have investigated a possible relationship between these two parameters. Platelet cytosolic free [Ca2+], determined using Quin-2, the capacity of plasma extracts to inhibit renal Na+, K(+)-ATPase activity and ouabain binding on human erythrocytes were measured in parallel in 25 untreated hypertensive patients and 11 normotensive subjects. Enhanced values for all 3 parameters were observed in the same hypertensive patient. Platelet cytosolic free [Ca2+] was positively correlated to the plasma digitalis-like activity, which was evaluated by the inhibition of Na+, K(+)-ATPase activity and ouabain binding (r = 0.430, P = .010 and r = 0.448, P = .006, respectively). These relationships were independent of age and blood pressure. These results indicate that endogenous digitalis-like compounds may participate in the control of cytosolic free [Ca2+], in agreement with their proposed hypertensive role.

Inhibition of the Na(+)-K+ pump by digitalis compounds has been reported to increase intracellular Na+ and Ca2+ concentrations and to stimulate Na(+)-H+ exchange. The activity of endogenous digitalis-like compounds, proposed to promote natriuresis and to raise blood pressure, has been found to be increased in volume expansion and hypertension. The enhanced cytosolic [Ca2+] present in platelets from hypertensive patients may thus originate from inhibition of the Na(+)-K+ pump by endogenous inhibitors, enhanced mobilization of internal Ca2+ stores due to phospholipase C activation and/or structural membrane defects. In unstimulated platelets from essential hypertensives, the increase in [Ca2+]i depends on external Ca2+, thereby underlining the importance of Ca2+ influx. The observation that [Ca2+]i was also enhanced in erythrocytes (p = 0.03) demonstrates that intracellular stores are not required for this rise. Plasma digitalis-like activity was positively correlated with platelet [Ca2+]i (inhibition of renal Na+,K(+)-ATPase, competition with ouabain binding, p less than 0.01). Platelet [Ca2+]i also rose during chronic digoxin administration (p less than 0.02) but not after acute in vitro ouabain treatment. The alkalinisation of platelet cytosol (p = 0.005) also agrees with the stimulation of the Na(+)-H(+)-exchange. In conclusion, these results are compatible with a participation of endogenous Na(+)-K+ pump inhibitors in the control of cytoplasmic [Ca2+] and cell excitability.

It has been proposed that plasma cholesterol, a major risk factor for atherosclerosis, may modify cellular Ca2+. In particular, higher membrane cholesterol contents induce higher Ca2+ influx and decrease the activity of the Ca2+ pumps. Cellular Ca2+ may also control the number of accessible low density lipoprotein receptors. We investigated the question of whether plasma cholesterol influences cellular Ca2+ metabolism, by analysing the cytosolic free Ca2+ concentration [( Ca2+]i) in unstimulated platelets from 61 untreated hypertensive patients [systolic and diastolic arterial pressure: 157 +/- 3/95 +/- 2 mmHg (mean +/- s.e.m.), respectively, age 41.8 +/- 1.7 years, body mass index 24.7 +/- 0.6 kg/m2]. The subjects' plasma total cholesterol (5.5 +/- 0.5 mmol/l) and platelet [Ca2+]i concentration (228 +/- 7 nmol/l) were positively correlated (r = 0.375, P less than 0.003). This correlation persisted at constant age, arterial pressure or body mass index. Platelet [Ca2+]i tended to increase with plasma low-density lipoprotein concentration (n = 21, P = 0.01), and to decrease with the ratio of high-density lipoprotein cholesterol to total cholesterol (n = 21, P = 0.08). The observation that in normocholesterolaemic hypertensive patients [Ca2+]i concentration in unstimulated platelets was correlated with plasma cholesterol suggests that cell activation may be modulated by membrane fluidity or that cholesterol metabolism is influenced by cell Ca2+.

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.

Cytosolic free Ca2+ concentrations were measured in platelets from hypertensive and normotensive humans and rats with the use of the fluorescent indicator Quin-2/AM. Without external Ca2+ added, no difference was observed between platelets of hypertensive patients and those normotensive subjects or between platelets of spontaneously hypertensive rats and those of the normotensive Wistar Kyoto and regular Wistar rats. In the presence of 0.5-1 mM external Ca2+, the cytosolic free Ca2+ concentrations were higher both in patients with essential hypertension and rats with genetic hypertension than in their respective normotensive controls. These results suggest that primary hypertension is accompanied by a disequilibrium between cellular Ca2+ influx, storage and extrusion. Such a characteristic if present in other excitable cells and in particular in vascular smooth muscle cells may play a major role in the rise of peripheral resistances.

Intraplatelet free [Ca2+] was studied in human primary hypertension using the fluorescent indicator Quin-2. The cytosolic free [Ca2+] was observed to increase in human platelets as a function of the external [Ca2+]. In the absence of added external calcium, no difference in [Ca2+]i was observed between platelets of hypertensive patients and normotensive subjects (101 +/- 5 nM vs 89 +/- 5 nM, n = 27 and 33). In the presence of 1 nM Ca2+ in the external medium, [Ca2+]i was significantly higher in hypertensive patients than those of normotensive subjects (254 +/- 16 nM vs 202 +/- 10 nM, n = 27 and 34, p less than 0.01). Since an increase in cytosolic free Ca2+ concentration has been proposed to stem from the enhanced intracellular Na+ concentration due to the Na+ pump inhibition, the effects on [Ca2+]i of ouabain and removal of external K+ ions were investigated. No effect of 10(-4)M ouabain was observed whereas [Ca2+]i rose from 198 +/- 8 to 273 +/- 29 (n = 6) in absence of external K+ ions. The increase of intracellular free [Ca2+] in platelets of essential hypertensives observed only in the presence of external Ca2+ thus reflects a structural membrane alteration leading to an increased influx and/or a limited extrusion capacity. Its relationship with cell Na+ handling remains unclear.

Studies in humans have shown that chronic elevation of blood pressure induces early alterations in the large arteries, consisting mainly of increased diameter, increased pulse wave velocity, and decreased arterial compliance. Acute reduction of blood pressure, per se, does not necessarily correct these alterations homogeneously. For instance, cadralazine reduced brachial artery diameter and urapidil decreased pulse wave velocity, without accompanying changes in forearm arterial compliance. In contrast, acebutolol, isosorbide dinitrate, nicardipine, and nitrendipine, for the same acute decrease in blood pressure, improved forearm arterial compliance by a concomitant decrease in pulse wave velocity and increase in arterial diameter. To determine whether or not these vascular effects persist with long-term acebutolol therapy, nine hypertensive patients were treated for a period of 3 months. Forearm hemodynamic and cytosolic free calcium concentrations in platelets were simultaneously assessed before and after treatment. Compared with placebo baseline values, chronic acebutolol therapy significantly decreased mean arterial pressure (p less than 0.01), pulse wave velocity (p less than 0.01), and platelet free calcium concentration (p less than 0.05); forearm arterial compliance was increased (p less than 0.01), but brachial artery diameter did not change. Platelet calcium concentration correlated closely with pulse wave velocity even at constant mean arterial pressure. These findings suggest a relaxant effect of acebutolol on the smooth musculature of large arteries, which is independent of changes in blood pressure and arterial diameter, and possibly mediated by changes in cytosolic calcium levels.

A new procedure was applied to rainbow trout for collecting cerebrospinal fluid (CSF). CSF was withdrawn continuously from the third ventricle at a flow rate of 0.7 microliter/min for up to 6 hr. The 5-HIAA concentrations in trout CSF are temperature-dependent and decrease exponentially after pargyline injection. The computed half-life of 5-HIAA production in CSF is 78 min at 15 degrees C. 5-HIAA concentrations in CSF are considered to reflect serotonin (5-HT) metabolism. The 5-HIAA content in the CSF of trout held in freshwater for several weeks is significantly higher than in trout held in either 1.6 or 3.0% saltwater while sodium content only exhibits a very slight change in the CSF of trout held in 3.0% saltwater. It is hypothesized that 5-HT could participate in the neurally-mediated adaptation to various osmotic conditions.

1. Plasma renin activity (PRA) and platelet cytosolic free calcium concentration ([Ca2+]i) were simultaneously determined in 18 untreated essential hypertensive subjects and 17 normotensive controls. A significant positive correlation was found between [Ca2+]i and PRA (slope = 42 nmol/l/ng/ml/h) in these 35 subjects. 2. Two determinations more than one week apart in nine subjects confirmed the parallel fluctuations of [Ca2+]i and PRA. A strict sodium restriction produced a progressive PRA elevation associated with a parallel rise in [Ca2+]i in one subject. 3. These results are consistent with the hypothesis that angiotensin II causes a concentration-dependent calcium mobilization.