Increased expression of plasma membrane Ca(2+)ATPase 4b in platelets from hypertensives: a new sign of abnormal thrombopoiesis?

Platelet Ca(2+) homeostasis is controlled by a multi-Ca(2+)ATPase system including two PMCA (plasma membrane Ca(2+)ATPase) and seven SERCA (sarco/endoplasmic reticulum Ca(2+)ATPase) isoforms. Previous studies have shown similar platelet Ca(2+) abnormalities in diabetic and hypertensive patients, including an increase in intracellular [Ca(2+)](I), a possible modulation of PMCA activity and increased PMCA tyrosine phosphorylation. Very recently, we found that platelets from diabetic patients also exhibited increased PMCA4b expression. In the present study we looked for further similarities between diabetic and hypertensive patients. We first confirmed a decrease in Ca(2+)ATPase activity (mean 55 + 7%) in mixed platelet membranes isolated from 10 patients with hypertension compared with those from 10 healthy controls. In addition, the decreased Ca(2+)ATPase activity correlated with the DBP of the different patients, as expected for PMCA activity. Second, we performed a pilot study of six hypertensives to examine their expressions of PMCA and SERCA mRNA and proteins. Like the diabetic patients, 100% of hypertensives were found to present a major increase in PMCA4b expression (mean value of 218 +/- 21%). We thus determined that platelets from diabetic and hypertensive patients showed similar increased PMCA4b isoform. Since increased PMCA4b expression was recently found to be associated with a perturbation of megakaryocytopoiesis, these findings may also point to an abnormality in platelet maturation in hypertension.     

Increased expression of plasma membrane Ca2+ATPase 4b in platelets from hypertensives: A new sign of abnormal thrombopoiesis?

Platelet Ca²⁺ homeostasis is controlled by a multi-Ca²⁺ATPase system including two PMCA (plasma membrane Ca²⁺ATPase) and seven SERCA (sarco/endoplasmic reticulum Ca²⁺ATPase) isoforms. Previous studies have shown similar platelet Ca²⁺ abnormalities in diabetic and hypertensive patients, including an increase in intracellular [Ca²⁺]_I, a possible modulation of PMCA activity and increased PMCA tyrosine phosphorylation. Very recently, we found that platelets from diabetic patients also exhibited increased PMCA4b expression. In the present study we looked for further similarities between diabetic and hypertensive patients. We first confirmed a decrease in Ca²⁺ATPase activity (mean 55?+?7%) in mixed platelet membranes isolated from 10 patients with hypertension compared with those from 10 healthy controls. In addition, the decreased Ca²⁺ATPase activity correlated with the DBP of the different patients, as expected for PMCA activity. Second, we performed a pilot study of six hypertensives to examine their expressions of PMCA and SERCA mRNA and proteins. Like the diabetic patients, 100% of hypertensives were found to present a major increase in PMCA4b expression (mean value of 218?±?21%). We thus determined that platelets from diabetic and hypertensive patients showed similar increased PMCA4b isoform. Since increased PMCA4b expression was recently found to be associated with a perturbation of megakaryocytopoiesis, these findings may also point to an abnormality in platelet maturation in hypertension.     

Dominant role of mitochondria in calcium homeostasis of single rat pituitary corticotropes

The rise in cytosolic free Ca2+ concentration ([Ca2+]i) is the major trigger for secretion of ACTH from pituitary corticotropes. To better understand the shaping of the Ca2+ signal in corticotropes, we investigated the mechanisms regulating the depolarization-triggered Ca2+ signal using patch-clamp techniques and indo-1 fluorometry. The rate of cytosolic Ca2+ clearance was unaffected by inhibitors of Na+/Ca2+ exchanger or plasma membrane Ca2+-ATPase (PMCA), slightly slowed by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, but dramatically slowed by mitochondrial uncouplers or inhibitor of mitochondrial uniporter. Measurements with rhod-2 revealed that depolarization-triggered increase in mitochondrial Ca2+ concentration. Thus, mitochondria have a dominant role in cytosolic Ca2+ clearance. Using the Mn2+ quench technique, we found the presence of a continuous basal Ca2+ influx in corticotropes. This basal Ca2+ influx was balanced by the combined actions of mitochondrial uniporter and PMCA and SERCA pumps. Inhibition of the mitochondrial uniporter or PMCA or SERCA pumps elevated basal [Ca2+]i. Using membrane capacitance measurement, we found that the change in the shape of the depolarization-triggered Ca2+ signal after mitochondrial inhibition was associated with enhancement of the exocytotic response. Thus, mitochondria have a dominant role in the regulation of Ca2+ signal and exocytosis in corticotropes.     

Role of tyrosine phosphorylation in regulation of inositol 1,4,5-trisphosphate-mediated Ca 2+ release in human platelets

Thrombin activation of human platelets is accompanied by tyrosine phosphorylation of many cellular proteins, as well as changes in the cytosolic Ca 2+ concentration. Consequently, we assessed tyrosine phosphorylation of the platelet inositol 1,4,5-trisphosphate receptor (IP 3 R) to determine if phosphorylation modulates Ca 2+ release from internal stores. Stimulation of platelets with thrombin resulted in tyrosine phosphorylation of the type 1 IP 3 R, which peaked at 3 min followed by dephosphorylation to the basal level by 10 min. Thrombin also increased the extent of IP 3 -mediated Ca 2+ release 1.6-fold in crude platelet membranes isolated from thrombin-stimulated (3 min) platelets. Since we have previously shown that the plasma membrane Ca 2+ -ATPase (PMCA) is also phosphorylated on tyrosine residues during thrombin-stimulated platelet activation, resulting in inhibition of the pump (Dean et al ., J Biol Chem 1997; 272: 15113-9), the present results suggest that early in thrombin-stimulated platelet activation, tyrosine phosphorylation of PMCA and IP 3 R results in transient enhancement in platelet cytosolic Ca 2+ , but that this enhancement is attenuated by subsequent dephosphorylation.     

Role of tyrosine phosphorylation in regulation of inositol 1,4,5-trisphosphate-mediated Ca2+ release in human platelets

Thrombin activation of human platelets is accompanied by tyrosine phosphorylation of many cellular proteins, as well as changes in the cytosolic Ca2+ concentration. Consequently, we assessed tyrosine phosphorylation of the platelet inositol 1,4,5-trisphosphate receptor (IP(3)R) to determine if phosphorylation modulates Ca2+ release from internal stores. Stimulation of platelets with thrombin resulted in tyrosine phosphorylation of the type 1 IP(3)R, which peaked at 3 min followed by dephosphorylation to the basal level by 10 min. Thrombin also increased the extent of IP(3)R-mediated Ca2+ release 1.6-fold in crude platelet membranes isolated from thrombin-stimulated (3 min) platelets. Since we have previously shown that the plasma membrane Ca2+-ATPase (PMCA) is also phosphorylated on tyrosine residues during thrombin-stimulated platelet activation, resulting in inhibition of the pump (Dean et al., J Biol Chem 1997; 272: 15113-9), the present results suggest that early in thrombin-stimulated platelet activation, tyrosine phosphorylation of PMCA and IP(3)R results in transient enhancement in platelet cytosolic Ca2+, but that this enhancement is attenuated by subsequent dephosphorylation.     

The platelet Ca2+ transport ATPase system

The Ca2+ signal accompanying cell function involves the activities of plasma membrane Ca2+ transport ATPases (PMCA) which transport Ca2+ ions out of the cell and those of sarco/endoplasmic reticulum Ca2+ transport ATPases (SERCA), which pump Ca2+ ions into intracellular Ca2+ pools. Although a platelet Ca2+ transport ATPase was described three decades ago, for a long time it remained poorly understood in terms of its cellular localization and identity. By integrating data obtained during recent years, including newly available information in the literature for the PMCAs and aspects of our work concerning the SERCAs, the present review will show how the overall view of the platelet Ca2+ATPase system has to be modified due to the presence of a number of Ca2+ATPases in these cells. These Ca2+ATPases include a typical 144 kDa PMCA protein, although its molecular identity still remains to be established, expressed together with a multi-SERCA system constituted by the ubiquitous 100 kDa SERCA 2b isoform, the 97 kDa SERCA 3 isoform and a new 97 kDa SERCA isoform recognized by the monoclonal antibody termed PL/IM 430 which also remains to be identified. The new paradigm of the platelet multi-Ca2+ATPase system will be discussed including: (i) the problems solved, as it has now become possible to reconciliate previous contradictory observations and (ii) those which still remain due to the fact that the platelet Ca2+ATPase system is more complex than previously assumed. Finally, to put this complexity of the platelet Ca2+ transport ATPase system into perspective, the biological significance of the multi-SERCA system in the context of Ca2+ signalling will be tentatively discussed in an attempt to produce a model of the organization of the intracellular Ca2+ pools in platelets.     

Endogenously generated reactive oxygen species reduce PMCA activity in platelets from patients with non-insulin-dependent diabetes mellitus

Intracellular Ca2+ homeostasis in platelets of patients with non-insulin-dependent diabetes mellitus (NIDDM) has been reported to be altered, leading to an increased adhesiveness and spontaneous aggregation. Among the disturbed Ca2+ mechanism in platelets from NIDDM subjects, a reduced Ca2+ extrusion by the plasma membrane Ca2+-ATPase (PMCA) is especially relevant, maintaining an elevated cytosolic free Ca2+ concentration that results in platelet hypersensitivity. Here we show that treatment of platelets from NIDDM patients with 300 U/mL catalase or 5 mM D-mannitol, which prevent H2O2- and hydroxyl radicals-mediated oxidative stress, respectively, increases Ca2+ extrusion after treatment with thapsigargin (TG) plus ionomycin (Iono). In contrast, 1 mM trolox, a scavenger of ONOO-, did not alter TG + Iono-induced response. Catalase and D-mannitol reversed the enhanced tyrosine phosphorylation of PMCA induced by TG + Iono in NIDDM patients. These findings open up new horizon for the development of therapeutic strategies to palliate cardiovascular disorders in NIDDM.     

Contribution of thromboxane and endomembrane Ca2+-ATPases to variability in Ca2+ signalling of platelets from healthy volunteers

Inter-individual variability in Ca2+ signal generation was studied in platelets from 15 healthy volunteers. The possible involvement of variation in thromboxane A production and variation in sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) was investigated by using platelets isolated before and after intake of 500 mg aspirin, and by measuring the expression levels of two main SERCA isoforms (SERCA-2b and PL/IM 430-recognizable SERCA). Considerable difference in Ca2+ responses were detected after platelet stimulation with thrombin, collagen or the SERCA-2b inhibitor, thapsigargin (TG), with inter-individual coefficients of variance of 22-43% in the absence and 15-41% in the presence of aspirin. Differences in thromboxane A2 generation and SERCA expression contributed to this variability in various ways. In the absence of aspirin, the amount of formed thromboxane A2 partially explains the level of the Ca2+ response induced by TG. On the other hand, in the absence of thromboxane-dependent effects, the expression levels of SERCA-2b and SERCA PL/IM 430 were inversely related to the responses evoked by collagen and TG, respectively. None of these factors were related to the level of the thrombin-evoked Ca2+ signal.     

Contribution of thromboxane and endomembrane Ca2+-ATPases to variability in Ca2+ signalling of platelets from healthy volunteers

Inter-individual variability in Ca2+ signal generation was studied in platelets from 15 healthy volunteers. The possible involvement of variation in thromboxane A production and variation in sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) was investigated by using platelets isolated before and after intake of 500 mg aspirin, and by measuring the expression levels of two main SERCA isoforms (SERCA-2b and PL/IM 430-recognizable SERCA). Considerable difference in Ca2+ responses were detected after platelet stimulation with thrombin, collagen or the SERCA-2b inhibitor, thapsigargin (TG), with inter-individual coefficients of variance of 22-43% in the absence and 15-41% in the presence of aspirin. Differences in thromboxane A2 generation and SERCA expression contributed to this variability in various ways. In the absence of aspirin, the amount of formed thromboxane A2 partially explains the level of the Ca2+ response induced by TG. On the other hand, in the absence of thromboxane-dependent effects, the expression levels of SERCA-2b and SERCA PL/IM 430 were inversely related to the responses evoked by collagen and TG, respectively. None of these factors were related to the level of the thrombin-evoked Ca2+ signal.     

Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ATPase system in human platelets

We recently identified a multi-SERCA (sarco/endoplasmic reticulum Ca²⁺ ATPase) system in haemopoietic cells comprising the SERCA 2b, SERCA 3 and a new monoclonal anti-Ca²⁺ ATPase antibody (PL/IM 430) recognizable SERCA isoforms. We have now investigated the subcellular localization of these enzymes in human platelets by Western blotting of subcellular membrane fractions and by immunoelectron microscopy. We precisely defined the recognition specificity of the polyclonal anti-SERCA 2b, anti-SERCA 3, anti-SERCA 1 antibodies as well as of the monoclonal antibody PL/IM 430 by testing their recognition of the tryptic fragments of the SERCA isoforms. The analysis of fragmented membranes enriched in plasma membrane and intracellular membrane components by Western blotting showed that the SERCA 2b and the SERCA 3 isoforms were found in both the plasma membrane and the intracellular membrane fractions, whereas the PL/IM 430 recognizable SERCA isoform was restricted to membranes associated with the plasma membrane fraction. The immunoelectron microscopical study of the SERCA isoforms in resting platelets showed that: (i) the SERCA 2b isoform was expressed in membranes associated with the plasma membrane and open canalicular system, some α-granules and in unidentified membranes; (ii) the SERCA 3 isoform was found associated with plasma and intracellular membranes; and (iii) the PL/IM 430 recognizable SERCA isoform was observed only in structures associated with the cytoplasmic face of the plasma membranes, as confirmed by flow cytometry. Finally, since the PL/IM 430 antibody was raised against intracellular membranes, we looked for a potential membrane redistribution during the isolation procedure used for the preparation of the immunizing membranes. Neuraminidase treatment indeed induced a translocation of the PL/IM 430 recognizable SERCA isoform from plasma to intracellular membranes. Thus, the multi-SERCA system in platelets: (i) is distributed over different platelet membranes, (ii) presents a sub-compartmental organization with some overlapping, and (iii) is partly associated with motile membranes, reflecting an unrecognized level of complexity of Ca²⁺ stores in these cells.     

Regulation of plasma membrane Ca2+-ATPase in human platelets by calpain

The plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca(2+) in resting human platelets by extruding Ca(2+) from the cytoplasm across the plasma membrane. Since PMCA is the main agent of Ca(2+) efflux in platelets, it is a key point for regulation of platelet Ca(2+) metabolism. PMCA has been shown to be an excellent substrate for the Ca(2+)-activated cysteine protease calpain, a major platelet protein that is turned on during platelet activation. The objectives of the present work were to determine if PMCA is degraded during thrombin- and collagen-mediated platelet activation, and if calpain is responsible. The kinetics of PMCA degradation during platelet activation were analysed using SDS polyacrylamide gel electrophoresis and immunoblotting. The role of calpain was tested using the calpain inhibitors calpeptin and ALLN. Platelet activation mediated by both collagen and thrombin resulted in degradation of 60% of platelet PMCA within 18 minutes. Calpeptin and ALLN significantly inhibited the rate and extent of PMCA degradation. We conclude that calpain-mediated degradation of PMCA during platelet activation likely contributes significantly to Ca(2+) regulation and, therefore, to platelet function.     

How many Ca(2)+ATPase isoforms are expressed in a cell type? A growing family of membrane proteins illustrated by studies in platelets

Ca(2+) signaling plays a key role in normal and abnormal platelet functions. Understanding platelet Ca(2+) signaling requires the knowledge of proteins involved in this process. Among these proteins are Ca(2+)ATPases or Ca(2+) pumps that deplete the cytosol of Ca(2+) ions. Here, we will particularly focus on two Ca(2+) pump families: the plasma membrane Ca(2+)ATPases (PMCAs) that extrude cytosolic Ca(2+) towards the extracellular medium and the sarco/endoplasmic reticulum Ca(2+)ATPases (SERCAs) that pump Ca(2+) into the endoplasmic reticulum (ER). In the present review, we will summarize data on platelet Ca(2+)ATPases including their identification and biogenesis. First of all, we will present the Ca(2+)ATPase genes and their isoforms expressed in platelets. We will especially focus on a member of the SERCA family, SERCA3, recently found to give rise to a number of species-specific isoforms. Next, we will describe the differences in Ca(2+)ATPase patterns observed in human and rat platelets. Last, we will analyze how the expression of Ca(2+)ATPase isoforms changes during megakaryocytic maturation and show that megakaryocytopoiesis is associated with a profound reorganization of the expression and/or activity of Ca(2+)ATPases. Taken together, these data provide new aspects of investigations to better understand normal and abnormal platelet Ca(2+) signaling.     

TSH/cAMP up-regulate sarco/endoplasmic reticulum Ca2+-ATPases expression and activity in PC Cl3 thyroid cells

OBJECTIVE: We recently reported that the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2b is the SERCA form preferentially expressed in rat thyroid. Moreover, SERCA2b expression dramatically decreases in virally transformed, highly tumorigenic, PC Cl3 thyroid cells. These results suggest that, in the thyroid, SERCA2b, in addition to its housekeeping role, is linked to differentiation and is a regulated gene. We therefore sought to study the effect of TSH, the main regulator of thyroid function, on SERCA2b expression and activity. METHODS: PC Cl3 cells were hormone starved in low-serum medium and stimulated for long (48 h) or short (1, 2 and 4 h) times. SERCA2b expression and activity were evaluated by Northern and Western blots, Ca2+-ATPase activity and Ca2+ store content. RESULTS: In PC Cl3 cells, SERCA2b mRNA and protein were induced twofold by a 48-h long treatment with TSH. Long-term elevation (48 h) of intracellular cAMP levels, by forskolin or 8-Br-cAMP, had similar effects on SERCA2b mRNA and protein. We also measured Ca2+-ATPase activity and Ca2+ store content. Both long (48 h) and short (0.5-1 h) treatments with TSH, forskolin or 8-Br-cAMP induced a marked increase of SERCA2b activity. This effect was completely abolished by H89, a specific inhibitor of cAMP-dependent protein kinase A (PKA). TSH and 8-Br-cAMP increased Ca2+ store content after both long (48 h) and short (1-2 h) treatments. CONCLUSIONS: These data suggested that TSH/cAMP acts as an important regulator of both SERCA2b expression and activity in the thyroid system, through PKA activation.     

Ca2+ uptake by the sarcoplasmic reticulum in ventricular myocytes of the SERCA2b/b mouse is impaired at higher Ca2+ loads only

SERCA2a is the cardiac-specific isoform of Ca2+-ATPase of the sarcoplasmic reticulum (SR). A reduction of SERCA2a has been implicated in the contractile dysfunction of heart failure, and partial knockout of the SERCA2 gene (Atp2a2+/- mice) reiterated many of the features of heart failure. Yet, mice with a mutation of Atp2a2, resulting in full suppression of the SERCA2a isoform and expression of the SERCA2b isoform only (SERCA2b/b), showed only moderate functional impairment, despite a reduction by 40% of the SERCA2 protein levels. We examined in more detail the Ca2+ handling in isolated cardiac myocytes from SERCA2b/b. At 0.25 Hz stimulation, the amplitude of the [Ca2+]i transients, SR Ca2+ content, diastolic [Ca2+]i, and density of ICaL were comparable between WT and SERCA2b/b. However, the decline of [Ca2+]i was slower (t1/2 154+/-7 versus 131+/-5 ms; P<0.05). Reducing the amplitude of the [Ca2+]i transient (eg, SR depletion), removed the differences in [Ca2+]i decline. In contrast, increasing the Ca2+ load revealed pronounced reduction of SR Ca2+ uptake at high [Ca2+]i. There was no increase in Na+-Ca2+ exchange protein or function. Theoretical modeling indicated that in the SERCA2b/b mouse, the higher Ca2+ affinity of SERCA2b partially compensates for the 40% reduction of SERCA expression. The lack of SR depletion in the SERCA2b/b may also be related to the absence of upregulation of Na+-Ca2+ exchange. We conclude that for SERCA isoforms with increased affinity for Ca2+, a reduced expression level is better tolerated as Ca2+ uptake and storage are impaired only at higher Ca2+ loads.     

Fiberoptic bronchoscopy induces platelet activation

The plasma membrane Ca²⁺-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca²⁺ in resting human platelets by extruding Ca²⁺ from the cytoplasm across the plasma membrane. Since PMCA is the main agent of Ca²⁺ efflux in platelets, it is a key point for regulation of platelet Ca²⁺ metabolism. PMCA has been shown to be an excellent substrate for the Ca²⁺-activated cysteine protease calpain, a major platelet protein that is turned on during platelet activation. The objectives of the present work were to determine if PMCA is degraded during thrombin- and collagen-mediated platelet activation, and if calpain is responsible. The kinetics of PMCA degradation during platelet activation were analysed using SDS polyacrylamide gel electrophoresis and immunoblotting. The role of calpain was tested using the calpain inhibitors calpeptin and ALLN. Platelet activation mediated by both collagen and thrombin resulted in degradation of 60% of platelet PMCA within 18 minutes. Calpeptin and ALLN significantly inhibited the rate and extent of PMCA degradation. We conclude that calpain-mediated degradation of PMCA during platelet activation likely contributes significantly to Ca²⁺ regulation and, therefore, to platelet function.     

Plasma membrane calcium ATPase overexpression in arterial smooth muscle increases vasomotor responsiveness and blood pressure

In vascular smooth muscle cells (SMCs), several mechanisms act in concert to regulate the intracellular calcium concentration [Ca2+]i, which may in turn affect vascular tone. One such mechanism is the extrusion of Ca2+ by the plasma membrane calcium ATPase (PMCA). To address, in particular, the role of the neuronal nitric oxide synthase (nNOS)-associating isoform PMCA4b in regulating vascular tone, a doxycycline-responsive transgene for human PMCA4b was overexpressed in arterial SMCs of mice. Overexpression of hPMCA4b resulted in a 2-fold increase in total aortic PMCA4 protein expression and significant real-time RT-PCR-documented differences in the levels of endogenous mouse PMCA1, PMCA4, SERCA2, and IP3R1 gene expression in arterial SMCs. Whereas no significant difference in basal [Ca2+]i or Ca2+ sensitivity was observed in vascular SMCs or mesenteric arteries, respectively, from hPMCA4b-overexpressing versus control mice, hPMCA4b-overexpressing mice revealed a reduced set-point and increased extent of myogenic response and heightened sensitivity to vasoconstrictors. Treatment of arteries with an nNOS inhibitor resulted in a reduced set-point and increased extent of the myogenic response in control but not hPMCA4b-overexpressing mice. Moreover, aortic SMCs from hPMCA4b-overexpressing mice exhibited reduced levels of cGMP under both basal and phenylephrine-stimulated conditions. These changes were associated with significant doxycycline-reversible elevations in blood pressure. Taken together, these data show that overexpression of hPMCA4b in arterial SMCs increases vascular reactivity and blood pressure, an effect that may be mediated in part by negative regulation of nNOS.     

Impaired megakaryocytopoiesis in type 2B von Willebrand disease with severe thrombocytopenia

In type 2B von Willebrand disease, there is spontaneous binding of mutated von Willebrand factor (VWF) multimers to platelets. Here we report a family in which severe thrombocytopenia may also be linked to abnormal megakaryocytopoiesis. A heterozygous mutation in the VWF A1 domain gave a R1308P substitution in an interactive site for glycoprotein Ibalpha (GPIbalpha). Electron microscopy showed clusters of platelets in close contact. Binding of antibodies to the GPIbalpha N-terminal domain was decreased, whereas GPIX and GPV were normally detected. In Western blotting (WB), GPIbalpha, alphaIIb, and beta3 were normally present. Proteins involved in Ca(2+) homeostasis were analyzed by quantitating platelet mRNA or by WB. Plasma membrane Ca(2+) ATPase (PMCA)-4b and type III inositol trisphosphate receptor (InsP(3)-R3) were selectively increased. The presence of degradation products of polyadenosine diphosphate (ADP)-ribose polymerase protein (PARP) suggested ongoing caspase-3 activity. These were findings typical of immature normal megakaryocytes cultured from peripheral blood CD34(+) cells with TPO. Significantly, megakaryocytes from the patients in culture produced self-associated and interwoven proplatelets. Immunolocalization showed VWF not only associated with platelets, but already on the megakaryocyte surface and within internal channels. In this family, type 2B VWD is clearly associated with abnormal platelet production.     

Dominant role of sarcoendoplasmic reticulum Ca2+-ATPase pump in Ca2+ homeostasis and exocytosis in rat pancreatic beta-cells

The exocytosis of insulin-containing granules from pancreatic beta-cells is tightly regulated by changes in cytosolic Ca2+ concentration ([Ca2+]i). We investigated the role of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pump, Na+/Ca2+ exchanger, and plasma membrane Ca2+-ATPase pump in the Ca2+ dynamics of single rat pancreatic beta-cells. When the membrane potential was voltage clamped at -70 mV (in 3 mm glucose at approximately 22 or 35 C), SERCA pump inhibition dramatically slowed (approximately 4-fold) cytosolic Ca2+ clearance and caused a sustained rise in basal [Ca2+]i via the activation of capacitative Ca2+ entry. SERCA pump inhibition increased ( approximately 1.8-fold) the amplitude of the depolarization-triggered Ca2+ transient at approximately 22 C. Inhibition of the Na+/Ca2+ exchanger or plasma membrane Ca2+-ATPase pump had only minor effects on Ca2+ dynamics. Simultaneous measurement of [Ca2+]i and exocytosis (with capacitance measurement) revealed that SERCA pump inhibition increased the magnitude of depolarization-triggered exocytosis. This enhancement in exocytosis was not due to the slowing of the cytosolic Ca2+ clearance but was closely correlated to the increase in the peak of the depolarization-triggered Ca2+ transient. When compared at similar [Ca2+]i with controls, the rise in basal [Ca2+]i during SERCA pump inhibition did not cause any enhancement in the magnitude of the ensuing depolarization-triggered exocytosis. Therefore, we conclude that in rat pancreatic beta-cells, the rapid uptake of Ca2+ by SERCA pump limits the peak amplitude of depolarization-triggered [Ca2+]i rise and thus controls the amount of insulin secretion.     

Influence of low density lipoprotein from insulin-dependent diabetic patients on platelet functions.

In the present work we studied in vitro the action of low density lipoproteins (LDL) isolated from normolipemic insulin-dependent diabetic (IDDM) patients on transmembrane cation transport, nitric oxide synthase (NOS) activity, and aggregating response to stimuli of platelets from healthy subjects to elucidate whether the modified interaction between circulating lipoproteins and cells might be one of the pathogenetic mechanisms of the increased platelet activation in IDDM. LDL were obtained by discontinuous gradient ultracentrifugation from 15 IDDM out-patients and 15 sex- and age-matched healthy subjects and used for incubation experiments with control platelets. Lipid composition and hydroperoxide concentrations were studied in LDL. Platelet aggregation responses to ADP, NOS activity, cytosolic Ca2+ concentrations, and platelet membrane Na+/K+-adenosine triphosphatase (Na+/K+-ATPase) and Ca2+-ATPase activities were measured after incubation. IDDM LDL showed an increased lysophosphatidylcholine content compared with that of control LDL. IDDM LDL significantly increased the platelet aggregating response to ADP, cytosolic Ca2+ concentrations, and plasma membrane Ca2+-ATPase activity and significantly reduced NOS activity and platelet membrane Na+/K+-ATPase activity compared with those of platelets incubated in buffer or cells incubated with control LDL. The effects exerted by IDDM LDL on platelet suspensions from healthy subjects mimic the alterations observed in platelets from diabetic subjects in basal conditions. Both the decreased activity of NOS and the higher cytoplasmic concentrations of Ca2+ might cause increased platelet activation, as observed in IDDM. In conclusion, the present study suggests a new mechanism with a potential role in the early development of atherosclerosis in diabetic patients, i.e. an altered interaction between circulating lipoproteins and platelets.