Ethanol Inhibits Store‐Operated Ca²⁺ Entry of Platelets

Abstract

Moderate drinking is known to prevent atherosclerotic diseases (Marmot et al. 1981). In particular, the incidence of coronary heart disease is negatively correlated with the degree of ethanol consumption (Kagan et al. 1981). The beneficial effect of ethanol on atherosclerosis is in part explained by its action on lipid metabolism: drinking increases serum levels of high-density lipoprotein but decreases those of low-density lipoprotein (Kagan etal. 1981; Marmot et al. 1981; Constant 1997; Rimm et al. 1999). Drinking also prevents thrombus formation by modulating blood coagulation-fibrinolysis balance (Rimm et al. 1999): drinking reduces blood fibrinogen concentration and increases the activity of plasminogen activator in blood. The inhibitory effect of ethanol on platelet aggregation is also a well-known mechanism involved in ethanol action against thrombosis (Constant 1997). Moreover, some polyphenol compounds, which are mainly involved in the beneficial actions of red wine on cardiovascular diseases (French paradox), have been known to inhibit platelet functions via promoting nitric oxide production by the vascular endothelium (Soleas et al. 1997). The major inhibitory effects of ethanol in vitro on platelet activation are generally observed in the order of 100 mM, and ethanol in vitro was reported to inhibit platelet aggregation in response to collagen, thrombin and Ca2+ ionophore, but not that in response to arachidonate (Fenn & Littleton 1983). Thus, it is thought as a mechanism of this inhibitory action of ethanol on platelet aggregation that ethanol attenuates phospholipase A2 in the plasma membrane, resulting in decreased production of arachidonate metabolites such as thromboxane A2, a strong inducer of platelet aggregation. Ethanol was also reported to directly inhibit membrane-associated phospholipase A2 isolated from rat platelets (Stubbs & Rubin 1992). However, ethanol effects on other signal transduction pathways of platelet aggregation, including cellular Ca2+ homeostasis, are still obscure. In non-excitable cells such as platelets, transplasmalemmal Ca2+ entry is not dependent on membrane potential but has recently been proposed to be linked with depletion of intracellular Ca2+ stores (Putney 1993). This mechanism is called store-operated Ca2+ entry or capacitative Ca2+ entry. Thus, the purpose of the present study was to investigate the effects of ethanol in vitro on store-operated Ca2+ entry and the subsequent aggregation of platelets. Blood was obtained from healthy donors who had not been given any drug for at least 10 days before the experiments. The blood (18 ml) was rapidly transfered to a plastic tube containing 2 ml of an anticoagulant, d-phenylalanyl-prolyl-arginine chloromethyl ketone dihydrochloride (final concentration of 10 μM), and mixed. The blood was then centrifuged at 150×g for 10 min., and the supernatant was obtained as platelet-rich plasma. Platelet-rich plasma was then mixed with 40 ml of Ca2+-free Tyrode solution that had been buffered by HEPES (pH 7.4) and contained 1 mM EGTA, and the mixture was centrifuged at 150 x g for 10 min. After the supernatant obtained had been further centrifuged at 400×g for 5 min., the pellet obtained was suspended with 40 ml of the above Tyrode-HEPES solution and then again centrifuged at 400×g for 5 min. The pellet obtained was suspended again with 2 ml of the Ca2+-free Tyrode-HEPES solution, and the suspension was used for the experiments. Intracellular Ca2+ was ascertained using a fluorescent Ca2+ indicator, fura-2, and fluorescence measurements were carried out using a dual-wavelength spectrofluorometer (CAF-110 Intracellular Ion Analyzer, JASCO Corporation, Tokyo, Japan). The level of intracellular free-Ca2+ ([Ca2+]i) was calculated using the formula described by Grynkiewicz et al. (1985). Platelet aggregation was evaluated using an aggregometer (NBS HEMA TRACER 801, Niko Bioscience, Inc., Hachiouji, Japan), which measures increases in light transmission. The light transmission through a platelet suspension without any treatment and that through the buffer alone were considered as 0 and 1, respectively, and the relative level of maximum aggregation after Ca2+ addition was calculated. The data were presented as means±S.E., and statistical analysis was done using analysis of variance and subsequent Scheffé F-test. P values less than 0.05 were regarded as significant. Fig. 1A shows representative charts of changes in [Ca2+]i. In nominally Ca2+-free solution, thapsigargin (1 μM), a Ca2+-ATPase inhibitor, induced a small increase in [Ca2+]i. The depletion of Ca2+ stores was confirmed by this increase in [Ca2+]i. Then Ca2+ was added extracellularly, resulting in a large increase in [Ca2+]i (store-operated Ca2+ influx). The addition of ethanol (170 mM) to the platelet suspension at 2 min. after thapsigargin stimulation did not affect the level of [Ca2+]i. In the ethanol-treated platelets, the extracellular Ca2+-induced increase in [Ca2+]i after thapsigargin stimulation (store-operated Ca2+ influx) was lower than that in the vehicle-treated control (fig. 1A). As shown in fig. 1B, ethanol (85–340 mM) concentration-dependently inhibited store-operated Ca2+ influx. In the presence of SKF-96365 1-[β-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride, 100 μM), an antagonist of receptor-operated Ca2+ channels, store-operated Ca2+influx, calculated by subtracting the basal [Ca2+]i level, was strongly inhibited [202.0±21.5 nM (control) versus 80.5±3.5 nM (SKF-96365-pretreated), P<0.05]. In our preliminary experiment, lanthanum (1 mM) abolished the extracellular Ca2+-induced increase in [Ca2+]i after thapsigargin stimulation in a solution not containing Ca2+ (data not shown). Thus, this increase in [Ca2+]i is completely dependent on the transplasmalemmal Ca2+ entry. On the other hand, addition of Ca2+ to the platelet suspension in a nominally Ca2+-free medium not containing thapsigargin caused a slight increase in [Ca2+]i(leak Ca2+influx), which was much smaller than the [Ca2+]i increase induced by store-operated Ca2+ influx. This leak Ca2+ influx, which is expressed as the increase in [Ca2+]i after addition of Ca2+, was not affected by neither ethanol (85–340 mM) nor SKF-96365 (100 μM) [63.2±4.2 nM (control), 62.2±2.9 nM (ethanol 85 mM), 62.7±1.5 nM (ethanol 170 mM), 69.3±4.1 nM (ethanol 340 mM), 64.2±4.1 nM (SKF-96365)]. Representative charts of changes in [Ca2+]i. Washed platelets were incubated in nominally Ca2+-free solution at 37°. After stabilization, the platelets were stimulated with thapsigargin (1 μM). At 2 min. after thapsigargin stimulation, ethanol (EtOH: 170 mM) or a vehicle (Control) was added to the platelet suspension. After further incubation for 2 min., CaCl2 (0.5 mM) was added to the suspension. B. Effects of pretreatment with different concentrations of ethanol (EtOH) on store-operated Ca2+ influx of platelets. The experimental protocol was the same as that described above. Asterisks denote significant differences compared with the values of the control pretreated with a vehicle (n=6). Experiments of platelet aggregation were performed under the same experimental protocol as that for [Ca2+]i measurement. In nominally Ca2+-free solution, thapsigargin did not affect the basal level of light transmission of washed platelet suspension (data not shown). Then, the addition of Ca2+ elicited platelet aggregation, which was concentration-dependently attenuated by pretreatment with ethanol (85–340 mM) (fig. 2A, B). This external Ca2+-induced platelet aggregation after Ca2+ store depletion was abolished in the presence of SKF-96365 (100μM) [0.378±0.020 (control) versus 0.035±0.008 (SKF-96365-pretreated), P<0.05]. A. Representative charts of platelet aggregation. Washed platelets were incubated in nominally Ca2+-free solution at 37°. After stabilization, the platelets were stimulated with thapsigargin (1μM). At 2 min. after thapsigargin stimulation, ethanol (EtOH: 85, 170, 340 mM) or a vehicle (Control) was added to the platelet suspension. After further incubation for 2 min., CaCl2 (0.5 mM) was added to the suspension at time 0. B. Effects of pretreatment with different concentrations of ethanol (EtOH) on platelet aggregation caused by store-operated Ca2+ influx. The experimental protocol was the same as that described above. Asterisks denote significant differences compared with the values of the control pretreated with a vehicle (n=6). It has been shown that Ca2+ store depletion, induced by Ca2+-ATPase inhibitors such as thapsigargin, causes transplasmalemmal Ca2+ influx of platelets (Authi et al. 1993). Since this Ca2+ entry is sensitive to antagonists of receptor-operated Ca2+ channels such as SKF-96365, this mechanism is thought also to be involved in agonist-induced transmembraneous Ca2+ entry that is linked with Ca2+-store depletion due to 1,4,5-inositol trisphosphate. Moreover, the previous study showed that the maximally effective concentration (1 μM) of thapsigargin released 77% of the 45Ca2+ accumulated in non-mitochondrial intracellular Ca2+ stores of human platelets and that addition of exogenous inositol trisphosphate together with thapsigargin resulted in a non-additive 45Ca2+ release which was the same as with thapsigargin alone (Authi et al. 1993). These findings suggest that thapsigargin treatment induces a sufficient depletion of intracellular Ca2+ stores and that thapsigargin releases Ca2+ from a pool that totally overlaps the inositol trisphosphate-sensitive pool. The present study clearly demonstrated that both store-operated Ca2+ influx and the subsequent aggregation of platelets are inhibited by ethanol in vitro. Moreover, the degree of ethanol inhibition of platelet aggregation was not larger than that of Ca2+ influx (1 A, 2). Therefore, the inhibitory effect of ethanol on platelet aggregation under the present experimental conditions may be due mainly to attenuation of this Ca2+ entry pathway. Furthermore, the fact that activation of phospholipase A2 requires Ca2+-calmodulin suggests that ethanol-induced phospholipase A2 inhibition, which was previously reported to be a mechanism of ethanol inhibition of platelet aggregation (Fenn & Littleton 1983; Stubbs & Rubin 1992), may be due to at least in part a decrease in [Ca2+]i resulting from suppression of agonist-induced Ca2+ entry linked with Ca2+-store depletion. It is well known that ethanol inhibits the Ca2+ entry through L-type or N-type Ca2+ channels in various kinds of cells including neuronal cells (Walter & Messing 1999). However, it has not been determined whether ethanol affects on the Ca2+ entry through voltage-independent Ca2+ channels in non-excitable cells such as platelets. Ca2+ release-activated Ca2+ entry is thought to be a major pathway for Ca2+ entry after receptor stimulation in many kinds of non-excitable cells (Putney 1993). To our knowledge, this is the first study showing that ethanol inhibits store-operated Ca2+ influx, resulting in attenuation of platelet aggregation. Although it was not determined how store-operated Ca2+ entry is modulated by ethanol in the present study, it is likely that ethanol directly acts on store-operated Ca2+ channels, since ethanol has an affinity with the plasma membrane and inhibits other types (L-type and N-type) of Ca2+ channels in neuronal cells (Walter & Messing 1999). Further studies are also needed to determine whether ethanol perturbs the coupling of plasmalemmal Ca2+ channels and Ca2+ stores, although this coupling mechanism has still not been clarified in detail.