Phospholipase A2 activity in platelets of patients with uremia

Platelets of patients with uremia develop a defective platelet function and have a decreased production of thromboxane B2 (TxB2). Activated platelets generate thromboxane from free arachidonate that is previously released from the membrane phospholipids (PLs) by phospholipases. Phospholipase A2 (PLA2) release up to 70% of the arachidonate in normal platelets, and to date, the activity of this enzyme in uremia is unknown. This work studied the PLA2 activity in the platelets of nine uremic patients and nine healthy volunteers. Washed platelets were labelled with [(14)C]arachidonic acid and activated with calcium ionophore A-23187 (4 microgr/ml). Lipids were resolved by TLC and identified by autoradiography. The distribution of [(14)C]arachidonic acid in the five major platelet phospholipids was found to be normal. Uremic platelets released more radioactivity than normal platelets (19.0 +/- 5.2% versus 11.3 +/- 1.6%, P = 0.001). The production of both, radioactive thromboxane B2 and hydroxyheptadecatrienoic acid was normal (2.6 +/- 1.2% and 3.5 +/- 1.6% of total radioactivity respectively), but the formation of the lipoxygenase metabolite hydroxyeicosatetraenoic acid was increased with respect to the controls (12.9 +/- 4.6% vs 7.0 +/- 1.3% of total radioactivity, P = 0002). In conclusion, platelets of patients with uremia have an increased activity of phospholipase A2 and produce increased amounts of hydroxyeicosatetraenoic acid, an inhibitor of the platelet function.     

Reduced plasma membrane Ca2+-ATPase function in platelets from patients with non-insulin-dependent diabetes mellitus

We clearly show that plasma membrane Ca2+ ATPase (PMCA) activity is lower in platelets from patients with non-insulin-dependent diabetes mellitus (NIDDM) than in those from healthy controls. The lower activity is likely due to reduced PMCA expression and increased tyrosine phosphorylation. These findings provide an explanation for the cellular ionic defects occurring in insulin resistant conditions.     

Ca2+ leakage rate from agonist-sensitive intracellular pools is altered in platelets from patients with type 2 diabetes

Platelets from patients with type 2 diabetes show abnormalities in intracellular Ca(2+) homeostasis that are involved in platelet hyperaggregability and the development of thrombotic complications. Different Ca(2+) transport mechanisms have been reported to be altered in platelets from patients with type 2 diabetes, including the sarcoendoplasmic and plasma membrane Ca(2+)-ATPases, plasma membrane Ca(2+) channels, or the Na(+)/Ca(2+) exchanger. Here, we have investigated whether passive Ca(2+) leak from the stores is altered in platelets from patients with type 2 diabetes. Resting cytosolic Ca(2+) concentration ([Ca(2+)](i)) was found to be greater in platelets from patients with type 2 diabetes than in healthy controls. In a Ca(2+)-free medium, platelet stimulation with thrombin or ADP evokes a rapid and transient increase in [Ca(2+)](i) that was found to be greater in patients with diabetes than in healthy controls. Sequential or combined inhibition of Ca(2+) extrusion and Ca(2+) sequestration into the stores reduced the difference between the responses to agonists in patients with diabetes and healthy controls, although agonist-induced Ca(2+) efflux from the stores was still significantly greater in patients with diabetes. Ca(2+) leak from the dense tubular system or the acidic stores, induced by a low concentration of thapsigargin or 2,5-di-(t-butyl)-1,4-hydroquinone (TBHQ), respectively, was clearly greater in patients with diabetes than in controls, and was not significantly modified by treatment with 2-APB. These findings indicate that passive Ca(2+) leakage rate from the intracellular stores in platelets is significantly enhanced in patients with type 2 diabetes mellitus and this might explain the increased resting [Ca(2+)](i).     

Normalizing effect of Ca2+ ionophore on cytoplasmic Ca2+ and parathyroid hormone release of dispersed parathyroid cells from patients with hyperparathyroidism

The effects of the Ca2+ ionophore A23187 on parathyroid hormone (PTH) secretion and cytoplasmic free Ca2+ concentration (Ca2+i) were measured at different extracellular Ca2+ concentrations using dispersed cells from patients with hyperparathyroidism (HPT). The addition of a low concentration of the Ca2+ ionophore to quin2-loaded cell preparations resulted in the apparent normalization of calcium-regulated Ca2+i. At all extracellular calcium concentrations Ca2+i reached significantly higher values in the presence of the ionophore and the dose-response relationship was shifted to the left. Under similar conditions calcium-regulated PTH release was correspondingly corrected with an increased suppressibility and left-shifted dose-response relationship. The data render strong support for a disturbed regulation of Ca2+i as a major factor in the pathophysiology of HPT.     

Mitochondrial Ca2+ uptake contributes to buffering cytoplasmic Ca2+ peaks in cardiomyocytes

Mitochondrial ability of shaping Ca(2+) signals has been demonstrated in a large number of cell types, but it is still debated in heart cells. Here, we take advantage of the molecular identification of the mitochondrial Ca(2+) uniporter (MCU) and of unique targeted Ca(2+) probes to directly address this issue. We demonstrate that, during spontaneous Ca(2+) pacing, Ca(2+) peaks on the outer mitochondrial membrane (OMM) are much greater than in the cytoplasm because of a large number of Ca(2+) hot spots generated on the OMM surface. Cytoplasmic Ca(2+) peaks are reduced or enhanced by MCU overexpression and siRNA silencing, respectively; the opposite occurs within the mitochondrial matrix. Accordingly, the extent of contraction is reduced by overexpression of MCU and augmented by its down-regulation. Modulation of MCU levels does not affect the ATP content of the cardiomyocytes. Thus, in neonatal cardiac myocytes, mitochondria significantly contribute to buffering the amplitude of systolic Ca(2+) rises.     

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.     

Increased Ca2+ influx into platelets induced by thromboxane A2 analog in patients with ischemic heart disease

We compared platelet aggregation and intracellular free calcium concentrations [( Ca2+]i) following stimulation with STA2, an analog of thromboxane A2 between patients with ischemic heart disease (IHD) showing significant stenosis in coronary angiograms and controls. In the presence of extracellular calcium, platelet aggregation and intracellular Ca2+ increase were enhanced by STA2 stimulation in a dose-dependent fashion and were higher in the IHD patients than in the controls. However, in the absence of extracellular calcium, no difference in intracellular Ca2+ increase was observed due to its total dependence on release from intracellular stores. These results suggest that thromboxane A2 increases platelet intracellular Ca2+-inducing aggregation, and this increase and aggregation, which is enhanced by thromboxane A2 in IHD patients, is due to promotion of Ca2+ influx by thromboxane A2. IHD patients appear to have an abnormality in the platelet membrane which may explain such thromboxane A2-dependent increased permeability to Ca2+.     

The relationship between Ca2+-ATPase and freely exchangeable Ca2+ in the dense tubules: A study in platelets from women

The main aims of this work were to examine in women: the relationship between the freely exchangeable Ca²⁺ (FECa²⁺) in the dense tubules and the activity of the sarco(endo)plasmic reticulum (SER) Ca²⁺-ATPase (SERCA) in platelets, and the relationship of these parameters with blood pressure and serum lipoproteins. Platelets from 14 white and 13 black women in good health were studied. The FECa²⁺ was measured as the ionomycin-evoked Ca²⁺ release (in the presence of thapsigargin) in Ca²⁺-free medium. SERCA activity was measured as the thapsigargin sensitive, Ca²⁺ dependent and ouabain resistant, ATP hydrolyses in platelet membranes. Relative expressions of SERCA 2 and 3 isoforms and Ras-related protein (Rap) 1 in platelet membranes were determined by Western immunoblots. Highly significant correlations were observed for FECa²⁺ in the dense tubules with: 1) the maximal reaction velocity (V_max) of the SERCA (r = 0.592, P = .0014), and 2) Rap1 (r = 0.551, P = .0035). In addition, negative correlations were observed between FECa²⁺ in the dense tubules and age. No correlations were observed for these variables with blood pressure or serum lipoproteins. We conclude the FECa²⁺ and the V_max of the SERCA are reliable indicators of Ca²⁺ load in platelets from women. However, in women, unlike previous observations in men, these platelet parameters are not correlated with blood pressure and serum lipoproteins.     

Coupled Ca2+/H+ transport by cytoplasmic buffers regulates local Ca2+ and H+ ion signaling

Ca²⁺ signaling regulates cell function. This is subject to modulation by H⁺ ions that are universal end-products of metabolism. Due to slow diffusion and common buffers, changes in cytoplasmic [Ca²⁺] ([Ca²⁺]_i) or [H⁺] ([H⁺]_i) can become compartmentalized, leading potentially to complex spatial Ca²⁺/H⁺ coupling. This was studied by fluorescence imaging of cardiac myocytes. An increase in [H⁺]_i, produced by superfusion of acetate (salt of membrane-permeant weak acid), evoked a [Ca²⁺]_i rise, independent of sarcolemmal Ca²⁺ influx or release from mitochondria, sarcoplasmic reticulum, or acidic stores. Photolytic H⁺ uncaging from 2-nitrobenzaldehyde also raised [Ca²⁺]_i, and the yield was reduced following inhibition of glycolysis or mitochondrial respiration. H⁺ uncaging into buffer mixtures in vitro demonstrated that Ca²⁺ unloading from proteins, histidyl dipeptides (HDPs; e.g., carnosine), and ATP can underlie the H⁺-evoked [Ca²⁺]_i rise. Raising [H⁺]_i tonically at one end of a myocyte evoked a local [Ca²⁺]_i rise in the acidic microdomain, which did not dissipate. The result is consistent with uphill Ca²⁺ transport into the acidic zone via Ca²⁺/H⁺ exchange on diffusible HDPs and ATP molecules, energized by the [H⁺]_i gradient. Ca²⁺ recruitment to a localized acid microdomain was greatly reduced during intracellular Mg²⁺ overload or by ATP depletion, maneuvers that reduce the Ca²⁺-carrying capacity of HDPs. Cytoplasmic HDPs and ATP underlie spatial Ca²⁺/H⁺ coupling in the cardiac myocyte by providing ion exchange and transport on common buffer sites. Given the abundance of cellular HDPs and ATP, spatial Ca²⁺/H⁺ coupling is likely to be of general importance in cell signaling.Most cells are exquisitely responsive to calcium (Ca²⁺) (1) and hydrogen (H⁺) ions (i.e., pH) (2). In cardiac myocytes, Ca²⁺ ions trigger contraction and control growth and development (3), whereas H⁺ ions, which are generated or consumed metabolically, are potent modulators of essentially all biological processes (4). By acting on Ca²⁺-handling proteins directly or via other molecules, H⁺ ions exert both inhibitory and excitatory effects on Ca²⁺ signaling. For example, in the ventricular myocyte, H⁺ ions can reduce Ca²⁺ release from sarcoplasmic reticulum (SR) stores, through inhibition of the SR Ca²⁺ ATPase (SERCA) pump and ryanodine receptor (RyR) Ca²⁺ channels (5, 6). In contrast, H⁺ ions can enhance SR Ca²⁺ release by stimulating sarcolemmal Na⁺/H⁺ exchange (NHE), which raises intracellular [Na⁺] and reduces the driving force for Ca²⁺ extrusion on Na⁺/Ca²⁺ exchange (NCX), leading to cellular retention of Ca²⁺ (7, 8). Ca²⁺ signaling is thus subservient to pH.Cytoplasmic Ca²⁺ and H⁺ ions bind avidly to buffer molecules, such that <1% of all Ca²⁺ ions and <0.001% of all H⁺ ions are free. Some of these buffers bind H⁺ and Ca²⁺ ions competitively, and this has been proposed to be one mechanism underlying cytoplasmic Ca²⁺/H⁺ coupling (9). Reversible binding to buffers greatly reduces the effective mobility of Ca²⁺ and H⁺ ions in cytoplasm (10, 11) and can allow for highly compartmentalized ionic microdomains, and hence a spatially heterogeneous regulation of cell function. In cardiac myocytes under resting (diastolic) conditions, the cytoplasm-averaged concentration of free [Ca²⁺] ([Ca²⁺]_i) and [H⁺] ([H⁺]_i) ions is kept near 10⁻⁷ M by membrane transporter proteins. Thus, [H⁺]_i is regulated by the balance of flux among acid-extruding and acid-loading transporter proteins at the sarcolemma [e.g., NHE and Cl⁻/OH⁻ (CHE) exchangers, respectively] (4). Similarly, the activity of SERCA and NCX proteins returns [Ca²⁺]_i to its diastolic level after evoked signaling events (3, 12). Despite these regulatory mechanisms, cytoplasmic gradients of [H⁺]_i and [Ca²⁺]_i do occur in myocytes and are an important part of their physiology. Gradients arise from local differences in transmembrane fluxes that alter [H⁺]_i or [Ca²⁺]_i. For example, spatial [H⁺]_i gradients are produced when NHE transporters, expressed mainly at the intercalated disk region, are activated (4, 13) or when membrane-permeant weak acids, such as CO₂, are presented locally (14). Similarly, release of Ca²⁺ through a cluster of RyR channels in the SR produces [Ca²⁺]_i nonuniformity in the form of Ca²⁺ sparks (15). Given the propensity of cytoplasm to develop ionic gradients, it is important to understand their underlying mechanism and functional role.The present work demonstrates a distinct form of spatial interaction between Ca²⁺ and H⁺ ions. We show that cytoplasmic [H⁺] gradients can produce stable [Ca²⁺]_i gradients, and vice versa, and that this interaction is mediated by low-molecular-weight (mobile) buffers with affinity for both ions. We demonstrate that the diffusive counterflux of H⁺ and Ca²⁺ bound to these buffers comprises a cytoplasmic Ca²⁺/H⁺ exchanger. This acts like a “pump” without a membrane, which can, for instance, recruit Ca²⁺ to acidic cellular microdomains. Cytoplasmic Ca²⁺/H⁺ exchange adds a spatial paradigm to our understanding of Ca²⁺ and H⁺ ion signaling.     

Dysregulated intracellular Ca2+ stores and Ca2+ signaling in synovial fluid T lymphocytes from patients with chronic inflammatory arthritis

OBJECTIVE: Peripheral blood (PB) T cells from rheumatoid arthritis (RA) patients proliferate poorly to mitogen, a change that is related to decreased intracellular Ca2+ ([Ca2+]i) signaling after T cell receptor (TCR) stimulation. We hypothesized that this was, in part, due to the effect of mediators of inflammation and predicted that greater changes in [Ca2+]i signaling would be seen in synovial fluid (SF) T cells. We also examined the mechanisms underlying the altered [Ca2+]i signals. METHODS: Paired PB and SF T cells from patients with chronic inflammatory arthritis were stimulated with mitogen to assess the magnitude of the [Ca2+]i signal in cell populations by fluorometry, the pattern of the [Ca2+]i signal in individual cells in a single-cell ion-imaging system, and the spatial distribution of Ca2+ within intracellular organelles. RESULTS: There was a significantly smaller [Ca2+]i signal after phytohemagglutinin protein stimulation of SF T cells (peak rise in [Ca2+]i signal PB versus SF 200 nM versus 180 nM; P < 0.05). In single SF T cells, a change in the pattern of the [Ca2+]i signal and a reduction in the number of responding cells was seen. These changes were a magnification of those seen in RA PB compared with control PB T cells. The contribution of Ca2+ release from intracellular stores to the final [Ca2+]i signal in PB and SF T cells was equal, but there was a significant increase in the Ca2+ remaining in the endoplasmic reticulum (ER) in SF T cells after TCR activation (PB versus SF 6 nM versus 19 nM; P < 0.05). Non-ER Ca2+ stores were not similarly affected. CONCLUSION: We found abnormalities in the magnitude, pattern, and spatial distribution of [Ca2+]i signaling in T cells from SF of patients with chronic inflammatory arthritis. A reduction in the number of responding SF T cells may partly explain some of our observations. However, we propose that the observed redistribution of SF Ca2+ stores may underlie the altered [Ca2+]i signaling, thus making these cells hyporesponsive to mitogen. The inflammatory environment of the joint and the late stage of differentiation of SF T cells are both likely to contribute to these changes in [Ca2+]i signaling, resulting in aberrant T cell function and promotion of disease chronicity.     

Intracellular free Ca2+ in platelets of essential hypertensive patients. Lack of correlation with clinical and laboratory data

Platelet intracellular free Ca2+, [Ca2+]i, was found to be higher in 64 hypertensive patients than in 65 age- and sex-matched normotensive controls (142.7 +/- 3.8 nmol/l vs 126.6 +/- 2.4 nmol/l, M +/- SEM, respectively; P less than 0.001). No differences were observed in hypertensive patients in relation to age, duration of hypertension or severity of retinopathy, but a slight correlation was observed (r = 0.275, P less than 0.05) between platelet [Ca2+]i and systolic blood pressure. No correlations were found between platelet [Ca2+]i and plasma cholesterol, triglycerides, aldosterone or renin activity. These data appear to support the hypothesis that increased platelet [Ca2+]i in hypertensive patients is more likely to be related to a primary cellular abnormality than to activation by extrinsic humoral or vascular factors.     

Abnormal Ca2+ handling and increased Mg2+ permeability in platelets of hypertensive rats.

In order to test the hypothesis that intracellular Na+ accumulation and cellular Mg2+ deficiency may be involved in the abnormalities in Ca2+ handling and reactivity in spontaneously hypertensive rats (SHR) platelets, the metabolism of Na+, Ca2+ and Mg2+ was determined in fluorescent dye loaded platelets from 15 SHR and 15 Wistar-Kyoto rats (WKY) at 12 weeks of age. Mg2+ leak was estimated as the Mg2+ influx induced by an increase in extracellular [Mg2+] (from 1 to 5 mmol/l) and Mg2+/Na+ exchange activity was estimated as the Mg2+ influx induced by a decrease in extracellular [Na+] (from 140 to 50 mmol/l). Cellular metabolism of the fluorescent dye was similar in the two groups. Mean platelet [Ca2+]i was significantly increased under basal and thrombin (0.1 U/ml)-stimulated conditions in SHR compared to WKY, both in the presence and absence of extracellular Ca2+. Mean Ca2+ discharge capacity was similar between the two groups. There was no difference in mean [Na+]i between the two groups. Basal [Mg2+]i was also increased in SHR platelets. Mg2+ leak was higher in SHR than in WKY, while Mg2+/Na+ exchange activity was similar in the two groups. There was no difference in serum Mg2+ concentration between SHR and WKY. These data suggest that abnormal Ca2+ handling is accompanied by elevation in [Mg2+]i via increased permeability of platelet cell membranes to Mg2+ in SHR without any alteration in [Na+]i, and do not support the Mg2+ deficiency hypothesis in genetically hypertensive rats.     

Oleic acid glucose-independently stimulates glucagon secretion by increasing cytoplasmic Ca2+ via endoplasmic reticulum Ca2+ release and Ca2+ influx in the rat islet alpha-cells

The effect of long-chain free fatty acids on glucagon secretion from islet alpha-cells has been a controversial issue. This study examined direct effects of oleic acid (OA) on glucagon release from rat pancreatic islets and on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in single alpha-cells by fura-2 fluorescence imaging. OA at 30 microM increased glucagon release from isolated islets in the presence of low (2.8 mM) and elevated (8.3 mM) glucose concentrations. OA at 6-10 microm concentration-dependently increased [Ca(2+)](i) in alpha-cells, irrespective of glucose concentrations (1.4, 2.8, and 8.3 mM). OA at 10 mum increased [Ca(2+)](i) in 90% of alpha-cells. OA-induced [Ca(2+)](i) increases were strongly inhibited by the endoplasmic reticulum Ca(2+)-pump inhibitors cyclopiazonic acid and thapsigargin and by 2-aminoethoxydiphenyl borate, the blocker of both inositol 1,4,5-trisphosphate receptors and store-operated Ca(2+) channels. Furthermore, the amplitude, but not incidence, of OA-induced [Ca(2+)](i) increases was reduced substantially by Ca(2+)-free conditions and mildly by an L-type Ca(2+) channel blocker, nitrendipine, and an ATP-sensitive K(+) channel activator, diazoxide. OA-induced glucagon release was also inhibited mildly by nitrendipine and strongly by 2-aminoethoxydiphenyl borate. These results indicate that OA glucose-independently stimulates glucagon release by increasing [Ca(2+)](i) in rat pancreatic alpha-cells and that the [Ca(2+)](i) increase is triggered by Ca(2+) release from endoplasmic reticulum and amplified by Ca(2+) influx possibly via store-operated channels and via voltage-dependent L-type Ca(2+) channels. The glucose-independent action of OA to stimulate glucagon release from alpha-cells may operate under hypoglycemic conditions when plasma free fatty acids levels are elevated, possibly playing a role in maintaining glucose metabolism.     

Glycoprotein IIb-IIIa complex and Ca2+ influx into stimulated platelets

Changes in intracellular Ca2+ concentrations [( Ca2+]i) in platelets stimulated with aggregating agents were measured with the fluorescent indicator dye quin 2. Ca2+ influx, but not intracellular mobilization, in response to adenosine diphosphate (ADP), platelet aggregating factor (PAF-acether), and sodium arachidonate was significantly inhibited by monoclonal antibodies against the glycoprotein (GP) IIb-IIIa complex; inhibition of thrombin-stimulated influx was inhibited to a lesser extent and reached statistical significance only at thrombin concentrations of 0.1 U/mL and below. Anti-GP Ib and HLA-ABC monoclonal antibodies had no effect on Ca2+ influx in response to any agonist. Thrombasthenic platelets gave normal [Ca2+]i responses to ADP and thrombin, which were not inhibited by an anti-GP IIb-IIIa antibody. It is suggested that Ca2+ influx in response to weak agonists occurs predominantly via a channel closely adjacent to the GP IIb-IIIa complex, but that higher concentrations of thrombin and A23187 also stimulate influx via another pathway.     

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.     

Simultaneous detection of changes in cytoplasmic Ca(2+), aminophospholipid exposure and micro-vesiculation in activated platelets

We have used flow cytometry to compare the temporal relationship between cytoplasmic Ca(2+)-fluxes and micro-vesiculation during platelet activation. Changes in fluorescence of the Ca(2+)-dye, fluo-3, and in forward light scatter as a measure of the decrease in platelet size that accompanies micro-vesiculation, were assessed simultaneously. In other experiments, changes in Ca(2+) levels and aminophospholipid exposure were assessed using fura-red, which is a long wavelength range indicator, and FITC-annexin V. Results obtained using the ionophore A23 187 and the ATPase inhibitor, thapsigargin, showed that micro-vesiculation is a relatively late event compared with intracellular Ca(2+) elevation. The relatively slow binding kinetics of annexin V prevented the establishment of a temporal relationship between increases in intracellular Ca(2+) and aminophospholipid exposure. Nevertheless, the combined use of fura-red and annexin V highlighted the heterogeneous response seen on some occasions with thapsigargin and always with a thrombin plus collagen mixture, and confirmed that individual platelets that bound annexin V were also those with elevated intracellular Ca(2+) levels.     

Sarcoplasmic reticulum Ca2+ refilling controls recovery from Ca2+-induced Ca2+ release refractoriness in heart muscle

In cardiac muscle Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is initiated by Ca2+ influx via L-type Ca2+ channels. At present, the mechanisms underlying termination of SR Ca2+ release, which are required to ensure stable excitation-contraction coupling cycles, are not precisely known. However, the same mechanism leading to refractoriness of SR Ca2+ release could also be responsible for the termination of CICR. To examine the refractoriness of SR Ca2+ release, we analyzed Na+-Ca2+ exchange currents reflecting cytosolic Ca2+ signals induced by UV-laser flash-photolysis of caged Ca2+. Pairs of UV flashes were applied at various intervals to examine the time course of recovery from CICR refractoriness. In cardiomyocytes isolated from guinea-pigs and mice, beta-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by approximately 2-fold. Application of cyclopiazonic acid at moderate concentrations (<10 micromol/L) slowed down recovery from refractoriness in a dose-dependent manner. Compared with cells from wild-type littermates, those from phospholamban knockout (PLB-KO) mice exhibited almost 5-fold accelerated recovery from refractoriness. Our results suggest that SR Ca2+ refilling mediated by the SR Ca2+-pump corresponds to the rate-limiting step for recovery from CICR refractoriness. Thus, the Ca2+ sensitivity of CICR appears to be regulated by SR Ca2+ content, possibly resulting from a change in the steady-state Ca2+ sensitivity and in the gating kinetics of the SR Ca2+ release channels (ryanodine receptors). During Ca2+ release, the concomitant reduction in Ca2+ sensitivity of the ryanodine receptors might also underlie Ca2+ spark termination by deactivation.     

Pancreatic beta-cells from obese-hyperglycemic mice are characterized by excessive firing of cytoplasmic Ca2+ transients

Pancreatic β-cells from obese-hyperglycemic (ob/ob) mice are widely used for studying the mechanisms of insulin release, including its regulation by the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). In this study, we compared changes of [Ca²⁺]_i in single β-cells isolated from ob/ob mice with those from lean mice using dual-wavelength microfluorometry and the indicator fura-2. There were no differences in the frequency, amplitude, and half-width of the slow oscillations induced by glucose. Most β-cells from the obese mice responded to 10 mM caffeine with transformation of the oscillations into sustained elevation of [Ca²⁺]_i, a process counteracted by ryanodine. The β-cells from the obese mice were characterized by ample generation of [Ca²⁺]_i transients, which increased in number in the presence of glucagon. The transients became less frequent when leptin was added at a concentration as low as 1 nM. It is suggested that the excessive firing of [Ca²⁺]_i transients in the ob/ob mice is owing to the absence of leptin and is mediated by activation of the phospholipase C signaling pathway.