Immunopathological manifestations in Kabuki syndrome: a registry study of 177 individuals.

PURPOSE: Kabuki syndrome (KS) (OMIM 147920 and 300867) is a rare genetic disorder characterized by specific facial features, intellectual disability, and various malformations. Immunopathological manifestations seem prevalent and increase the morbimortality. To assess the frequency and severity of the manifestations, we measured the prevalence of immunopathological manifestations as well as genotype-phenotype correlations in KS individuals from a registry. METHODS: Data were for 177 KS individuals with KDM6A or KMT2D pathogenic variants. Questionnaires to clinicians were used to assess the presence of immunodeficiency and autoimmune diseases both on a clinical and biological basis. RESULTS: Overall, 44.1% (78/177) and 58.2% (46/79) of KS individuals exhibited infection susceptibility and hypogammaglobulinemia, respectively; 13.6% (24/177) had autoimmune disease (AID; 25.6% [11/43] in adults), 5.6% (10/177) with ≥2 AID manifestations. The most frequent AID manifestations were immune thrombocytopenic purpura (7.3% [13/177]) and autoimmune hemolytic anemia (4.0% [7/177]). Among nonhematological manifestations, vitiligo was frequent. Immune thrombocytopenic purpura was frequent with missense versus other types of variants (p = 0.027). CONCLUSION: The high prevalence of immunopathological manifestations in KS demonstrates the importance of systematic screening and efficient preventive management of these treatable and sometimes life-threatening conditions.

Inflammatory and oxidative stress biomarkers in alkaptonuria: data from the DevelopAKUre project.

OBJECTIVE: The aim of this work was to assess baseline serum levels of established biomarkers related to inflammation and oxidative stress in samples from alkaptonuric subjects enrolled in SONIA1 (n = 40) and SONIA2 (n = 138) clinical trials (DevelopAKUre project). METHODS: Baseline serum levels of Serum Amyloid A (SAA), IL-6, IL-1ß, TNFα, CRP, cathepsin D (CATD), IL-1ra, and MMP-3 were determined through commercial ELISA assays. Chitotriosidase activity was assessed through a fluorimetric method. Advanced Oxidation Protein Products (AOPP) were determined by spectrophotometry. Thiols, S-thiolated proteins and Protein Thiolation Index (PTI) were determined by spectrophotometry and HPLC. Patients' quality of life was assessed through validated questionnaires. RESULTS: We found that SAA serum levels were significantly increased compared to reference threshold in 57.5% and 86% of SONIA1 and SONIA2 samples, respectively. Similarly, chitotriosidase activity was above the reference threshold in half of SONIA2 samples, whereas CRP levels were increased only in a minority of samples. CATD, IL-1ß, IL-6, TNFα, MMP-3, AOPP, thiols, S-thiolated protein and PTI showed no statistically significant differences from control population. We provided evidence that alkaptonuric patients presenting with significantly higher SAA, chitotriosidase activity and PTI reported more often a decreased quality of life. This suggests that worsening of symptoms in alkaptonuria (AKU) is paralleled by increased inflammation and oxidative stress, which might play a role in disease progression. CONCLUSIONS: Monitoring of SAA may be suggested in AKU to evaluate inflammation. Though further evidence is needed, SAA, chitotriosidase activity and PTI might be proposed as disease activity markers in AKU.

Ketone Bodies as a Possible Adjuvant to Ketogenic Diet in PDHc Deficiency but Not in GLUT1 Deficiency.

OBJECTIVE: Ketogenic diet is the first line therapy for neurological symptoms associated with pyruvate dehydrogenase deficiency (PDHD) and intractable seizures in a number of disorders, including GLUT1 deficiency syndrome (GLUT1-DS). Because high-fat diet raises serious compliance issues, we investigated if oral L,D-3-hydroxybutyrate administration could be as effective as ketogenic diet in PDHD and GLUT1-DS. METHODS: We designed a partial or total progressive substitution of KD with L,D-3-hydroxybutyrate in three GLUT1-DS and two PDHD patients. RESULTS: In GLUT1-DS patients, we observed clinical deterioration including increased frequency of seizures and myoclonus. In parallel, ketone bodies in CSF decreased after introducing 3-hydroxybutyrate. By contrast, two patients with PDHD showed clinical improvement as dystonic crises and fatigability decreased under basal metabolic conditions. In one of the two PDHD children, 3-hydroxybutyrate has largely replaced the ketogenic diet, with the latter that is mostly resumed only during febrile illness. Positive direct effects on energy metabolism in PDHD patients were suggested by negative correlation between ketonemia and lactatemia (r 2 = 0.59). Moreover, in cultured PDHc-deficient fibroblasts, the increase of CO2 production after 14C-labeled 3-hydroxybutyrate supplementation was consistent with improved Krebs cycle activity. However, except in one patient, ketonemia tended to be lower with 3-hydroxybutyrate administration compared to ketogenic diet. CONCLUSION: 3-hydroxybutyrate may be an adjuvant treatment to ketogenic diet in PDHD but not in GLUT1-DS under basal metabolic conditions. Nevertheless, ketogenic diet is still necessary in PDHD patients during febrile illness.

Perinatal and infantile hypophosphatasia: clinical features and treatment.

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.

Biochemical and functional alterations associated with hypercholesterolemia in platelets from hypertensive patients.

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.

Increased platelet cytosolic free calcium concentration in essential hypertension.

Cytosolic free calcium concentration [Ca2+] was studied in platelets of hypertensive patients with the use of the fluorescent indicator Quin 2/AM. Cytosolic free Ca2+ was significantly higher in platelets of hypertensive patients than in those of normotensive subjects (241 +/- 9 versus 192 +/- 7 nmol/l, n = 58 and 57, respectively P less than 0.001). When all 115 subjects were included, there was a significant correlation between cytosolic free Ca2+ and systolic or diastolic blood pressure (r = 0.262, P less than 0.0025 and r = 0.251, P less than 0.0025, respectively). Intracellular Quin 2 concentration was measured to evaluate the formaldehyde production (a product of Quin 2/AM hydrolysis which has been described as reducing the adenosine triphosphate (ATP) production). The Quin 2 concentrations in platelets of the two groups of subjects were observed to be similar (0.41 +/- 0.03 versus 0.38 +/- 0.03 mmol/l, n = 8 and 7 for hypertensives and normotensives, respectively). The effects of prostaglandin E1 (PGE1), an adenylate cyclase stimulator, on cytosolic free Ca2+ were studied. The presence of 10(-7) mol/l PGE1 lowered the Ca2+ in platelets of hypertensive patients only, suppressing the difference between the two groups.

Platelet cytosolic free Ca2+ concentration and serotonin (5-HT) content in essential hypertension.

Cytosolic free [Ca2+] and serotonin (5-HT) content were measured in platelets from 22 untreated hypertensive patients and 16 normotensive subjects. In hypertensive patients, cytosolic free [Ca2+] was significantly higher (239 +/- 13 nmol/l versus 186 +/- 7 nmol/l, n = 22 and 16, P less than 0.01) and 5-HT content was significantly lower than those measured in cells from control subjects (3.22 +/- 0.26 versus 4.99 +/- 0.38 X 10(-7) mol/10(11) cells, n = 22 and 16, P less than 0.001). These two parameters were closely correlated (r = -0.565, n = 38, P less than 0.001). These two concomitant changes in platelet characteristics might result from a common cause, such as cell membrane alterations. As ritanserine, a specific 5-HT2 receptor antagonist, did not modify the cytosolic free [Ca2+], a higher stimulation of 5-HT2 receptors is not likely to be responsible for the enhanced free Ca2+ levels.

Platelet cyclic AMP in essential hypertension.

Various abnormalities in platelet metabolism, including increased sensitivity to several aggregating agents, have been described in essential hypertension. Platelet response is controlled by Ca2+ and cyclic AMP-dependent mechanisms (stimulatory and inhibitory, respectively) which oppose one another. In the present study, the cyclic AMP contents of unstimulated platelets were measured by radio-immunoassay and observed to be lower in hypertensive than in normotensive subjects, either in the basal state or after prostaglandin E1 (PGE1) stimulation. In the presence of 7-bromo-1,5,dihydro-3,6-dimethylimidazo [2,1-b] quinazolin-2(3H)-one (Ro 15-2041), a specific inhibitor of phosphodiesterase, the increases in cyclic AMP content were similar in platelets from both groups, indicating that this enzyme was not responsible for the alterations in cyclic AMP metabolism observed in hypertension. Low external Ca2+ reduced basal and PGE1-stimulated cyclic AMP content in both normotensive and hypertensive groups but cyclic AMP levels remained lower in hypertensive patients than in normotensive subjects, indicating that Ca2+ influx is not responsible for this altered metabolism of cyclic AMP in hypertension. These data suggest that the reduced platelet cAMP content may participate in the hyperreactivity to various aggregating agents previously reported to accompany essential hypertension.

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.

Platelet cytosolic calcium concentration, plasma lipids and hypertension.

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.

Inhibition of Ca2+ uptake into A7r5 vascular smooth muscle cells by farnesol: lack of effect on membrane fluidity and Ca2+-ATPase activities.

BACKGROUND: Previous studies have shown that farnesol, a 15-carbon nonsterol derivative of mevalonic acid, inhibits vasoconstriction. Because of its lipophilic properties, we hypothesized that farnesol increased membrane dynamics, thus reducing uptake of Ca2+ and contraction. OBJECTIVE: To characterize the effect of farnesol on cell membrane fluidity. DESIGN: The study was conducted using A7r5 cells, a rat aortic vascular smooth muscle cell line. Inhibition of Ca2+ uptake by farnesol was first established in these cells. Then, the effect of farnesol on membrane dynamics was determined. Finally, to ascertain that activation of Ca2+ extrusion and reuptake processes by farnesol did not occur, Ca2+-ATPase activity was examined. METHODS: Membrane fluidity in cell homogenates was estimated using two fluorescent dyes (1,6-diphenyl-1,3,5-hexatriene) and (1-[-(trimethylamino)-phenyl]-6-phenyl-1,3,5-hexatriene). Ca2+ uptake was determined by monitoring the changes in cytosolic Ca2+ concentration ([Ca2+]i) in fura-2-loaded cells after addition of KCI. Ca2+-ATPase activity was measured in 100000 x g cell fractions. RESULTS: Farnesol reduced KCI-induced (Ca2+]i transients significantly (P < 0.001), but did not modify membrane dynamic properties [0.214+/-0.007 versus 0.218+/-0.007 (n = 10) and 0.142+/-0.002 versus 0.146+/-0.003 (n = 5) for 1 -[-(trimethylamino)-phenyl]-6-phenyl-1,3,5-hexatriene and 1,6-diphenyl-1,3,5-hexatriene anisotropies, respectively; NS]. Administration of up to 30 micromol/l farnesol did not affect Ca2+-ATPase activity. CONCLUSION: Farnesol inhibits KCI-dependent rise of [Ca2+]i in A7r5 cells. This effect of farnesol is not related to a global change in plasma membrane lipid organization or to activation of Ca2+ pumps. Other mechanisms such as direct inhibition of voltage-dependent Ca2+ channels could therefore explain the biologic action of farnesol in the vascular tissue.

Plasma lipids and platelet membrane fluidity in essential hypertension.

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.

Platelet aggregation and in vivo shear forces.

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.

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.

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.

Influence of authentic nitric oxide on basal cytosolic [Ca2+] and Ca2+ release from internal stores in human platelets.

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.

Platelet and erythrocyte membrane microviscosity in Lyon hypertensive rats.

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.

Plasma digitalis-like activity and cytosolic Ca2+ in essential hypertension.

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.

Acute membrane effects of trimetazidine in human platelets.

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.

Regulation of the dynamic properties of platelet plasma membrane by intracellular sodium ions.

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.