Inter-individual variability in Ca2+ signalling in platelets from healthy volunteers: effects of aspirin and relationship with expression of endomembrane Ca2+-ATPases

Increased Ca2+ signal generation may lead to hyperactivity of platelets and contribute to thrombotic complications. Using fura-2-loaded platelets from 51 healthy volunteers, high variability was detected in the Ca2+ responses evoked by the receptor agonists, thrombin and collagen, and the inhibitor of sarco/endoplasmic reticulum Ca2+-ATPases (SERCA), thapsigargin (Tg). Oral intake of 500mg aspirin reduced the magnitude of the Ca2+ responses, and lowered the intra-individual coefficients of variance of the responses by 50%. However, the corresponding inter-individual variance coefficients were only a little influenced by aspirin intake, pointing to subject-dependent factors in Ca2+ handling that are unrelated to thromboxane formation. With each agonist, 6-9% of the subjects had platelets with relatively high Ca2+ responses (> mean + SD) both before and after aspirin intake. In 90% (9/10) of these cases the high responsiveness was confirmed in platelets obtained 6-12 months later. The Tg- but not thrombin-induced Ca2+ responses correlated inversely with the expression levels of SERCA PL/IM 430 (SERCA-3b) in platelets. After aspirin intake, the Ca2+ responses with collagen but not thrombin correlated inversely with SERCA-2b expression. These results suggest that, in the absence of potentiating effects of thromboxane, (i) the amount of PL/IM 430-recognizable SERCA may control the Ca2+ signal when SERCA-2b is specifically inhibited (with Tg), and (ii) the expression of SERCA-2b determine the collagen- but not the thrombin-evoked Ca2+ signal. Accordingly, limited Ca2+-pumping activity by low expression of one of the SERCA isoforms is likely to be one of the factors resulting in increased platelet activity towards collagen or thapsigargin but not thrombin.     

Cyclopiazonic acid and thapsigargin induce platelet aggregation resulting from Ca2+ influx through Ca2+ store-activated Ca2+-channels

The effects of cyclopiazonic acid and thapsigargin, selective inhibitors of the endoplasmic reticulum Ca2+-ATPase pump, on the platelet aggregation were investigated using washed rat platelets prepared by chromatography on Sepharose 2B columns. In Ca2+-free medium, cyclopiazonic acid and thapsigargin did not induce aggregation, but in the presence of 1 mM Ca2+, platelet aggregation was induced in a concentration-dependent manner. Cyclopiazonic acid- and thapsigargin-induced platelet aggregation was blocked by 1 mM Ni2+ but not by 100 microM indomethacin or 1 microM nifedipine. In aequorin-loaded platelets, cyclopiazonic acid and thapsigargin caused sustained elevation of the cytosolic Ca2+ concentration, an effect which was blocked by Ni2+, a non-selective Ca2+ channel blocker and SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl]-1H-imidazole hydrochloride), a putative receptor-operated Ca2+ channel antagonist. The above results indicated that both cyclopiazonic acid and thapsigargin induced platelet aggregation and elevation of cytosolic Ca2+ concentration, that extracellular Ca2+ was essential for cyclopiazonic acid- and thapsigargin-induced platelet aggregation, and that platelet aggregation may be associated with Ca2+ influx through Ca2+ store-activated Ca2+ channels.     

Endothelin receptor-mediated Ca2+ mobilization and contraction in bovine oviductal arteries: comparison with noradrenaline and potassium

1. The effects of endothelin-1 (ET-1) were studied in bovine oviductal arteries and compared to those of noradrenaline (NA) and high K+ (K+). The influence of endothelium, the receptor subtypes involved, and the mechanisms of Ca2+ mobilization were assessed. 2. ET-1 (0.1-300 nM) induced concentration-dependent contractions with a potency of 10(3) and 10(2) times higher than NA (0.1 microM-0.1 mM) and K+ (9.5-119 mM), respectively. Removal of endothelium or NG-nitro-L-arginine (L-NOARG, 0.1 mM) pretreatment did not affect responses to either ET-1 or K+, whereas the NA response was significantly increased. Indomethacin (1 microM) had no effect on either of these agonists. 3. The rank order of potency for the ET isopeptides was: ET-1 = ET-2 > ET-3. The ETA receptor-selective agonist, sarafotoxin 6c (S6c), had no effect. The ETA receptor-selective antagonist, BQ-123, showed a competitive antagonism on the ET-1 response (pA2 value of 6.58 +/- 0.01), whereas contractions to ET-3 were completely abolished by BQ-123 at 0.1 microM. 4. Concentration-response curves to both ET-1 and NA were shifted to the right and their maximum response reduced to approximately 56% and 65% of controls, respectively, under 30 min of incubation in Ca(2+)-free solution, whereas responses to K+ were almost abolished by this treatment. Contractions to both NA (30 microM) and ET-1 (30 nM) were maximally inhibited after 10 min of extracellular Ca2+ deprivation. 5. Contractions to ET-1 were more potently inhibited by nickel (Ni2+, 0.3 mM), whereas nifedipine (1 microM) and cadmium (Cd2+, 0.1 mM) induced only a slight effect. In contrast, opposite effects were found for both NA and K+. 6. Treatment with ryanodine (100 microM) and caffeine (10 mM) in Ca(2+)-free solution reduced the tension measured 5 min after NA (30 microM) and ET-1 (30 nM) addition, but the sustained response (tension at 25 min) remained unaffected. 7. Calphostin C (1 microM), a specific protein kinase C (PKC) inhibitor, reduced the maximum contractile response to ET-1 by about 50% without significantly affecting its pD2 value. 8. These results suggest that ET-1 acts in bovine oviductal arteries by directly activating a homogenous population of ETA receptors in smooth muscle, without endothelial modulation. Several Ca2+ activation mechanisms seem to be involved in the contractile action of the peptide, including: (1) extracellular Ca2+ entrance through Ni(2+)-sensitive and L-type Ca2+ channels; (2) intracellular Ca2+ release from a ryanodine-sensitive Ca2+ store; and (3) sensitization of the contractile machinery to Ca2+ via PKC.     

Activation of two types of Ca2+-permeable nonselective cation channel by endothelin-1 in A7r5 cells

1. In A7r5 cells loaded with the Ca2+ indicator fura-2, we examined the effect of a Ca2+ channel blocker SK&F 96365 on increases in intracellular free Ca2+ concentrations ([Ca2+]i) and Mn2+ quenching of fura-2 fluorescence by endothelin-1 (ET-1). Whole-cell patch-clamp was also performed. 2. Higher concentrations (> or = 10 nM) of ET-1 (higher [ET-1]) evoked a transient peak and a subsequent sustained elevation in [Ca2+]i: removal of extracellular Ca2+ abolished only the latter. A blocker of L-type voltage-operated Ca2+ channel (VOC) nifedipine at 1 microM reduced the sustained phase to about 50%, which was partially sensitive to SK&F 96365 (30 microM). 3. Lower [ET-1] (< or = 1 nM) evoked only a sustained elevation in [Ca2+]i which depends on extracellular Ca2+. The elevation was partly sensitive to nifedipine but not SK&F 96365. 4. In the presence of 1 microM nifedipine, higher [ET-1] increased the rate of Mn2+ quenching but lower [ET-1] had little effect. 5. In whole-cell recordings, both lower and higher [ET-1] induced inward currents at a holding potential of -60 mV with linear I-V relationships and reversal potentials close to 0 mV. The current at lower [ET-1] was resistant to SK&F 96365 but was abolished by replacement of Ca2+ in the bath solution with Mn2+. The current at higher [ET-1] was abolished by the replacement plus SK&F 96365. 6. In a bath solution containing only Ca2+ as a movable cation, ET-1 evoked currents: the current at lower [ET-1] was sensitive to Mn2+, whereas that at higher [ET-1] was partly sensitive to SK&F 96365. 7. These results indicate that in addition to VOC, ET-1 activates two types of Ca2+-permeable nonselective cation channel depending on its concentrations which differ in terms of sensitivity to SK&F 96365 and permeability to Mn2+.     

Alteration of Ca2+ fluxes in brain microsomes by K+ and Na+: modulation by sulfated polysaccharides and trifluoperazine

Rat brain microsomes accumulate Ca2+ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K+, Na+, or Li+. Both the Ca2+ uptake and the Ca(2+)-dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half-maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 micrograms/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca2+ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca2+ pump of brain microsomes. At low concentrations (20-80 microM) it stimulates the rate of Ca2+ influx, and at concentrations > 100 microM if inhibits both the Ca2+ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca(2+)-ATPase; for the Ca(2+)-ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration-dependent inhibition of Ca2+ uptake. Passive Ca2+ efflux from brain microsomes preloaded with Ca2+ is increased by trifluoperazine (50-150 microM), and this effect is potentiated by heparin (10 micrograms/ml), even in the presence of KCl. It is proposed that the Ca(2+)-ATPase isoforms from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.     

Differential effect of halothane and forskolin on platelet cytosolic Ca2+ mobilization and aggregation

BACKGROUND: Previous works have suggested that the impairment of platelet aggregation by halothane was partly related to a stimulation of cyclic adenosine monophosphate (cAMP) production, to an inhibitory effect on Ca2+ signaling, or both. Intracellular Ca2+ measurements therefore were undertaken, first to determine the critical steps in the platelet CaZ+ signaling cascade most likely to be affected by halothane or by an increase in cAMP production, and second to establish if the effect of halothane involves aggregation-related biochemical pathways triggered by an increase in internal Ca2+. METHODS: Human washed platelets were treated with halothane or forskolin for 5 min before application of either platelet-activating factor, thrombin, U46619, or thapsigargin. The cytosolic Ca2+ concentration ([Ca2+]i) was measured with the fluorescent Ca2+ indicator fura-2. Nephelometric measurements were also performed to assay the aggregation process. RESULTS: Our results indicate that pretreating platelets with halothane leads to a partial impairment of the [Ca2+]i increase induced either by U46619, thrombin, or platelet-activating factor, but this had no significant effect on the [Ca2+]i response triggered by thapsigargin. In addition, our results show that halothane inhibits platelet aggregation triggered by U46619, but not by thapsigargin. Conversely, forskolin completely inhibited the [Ca2+]i response to U46619 and thapsigargin and prevented platelet aggregation induced by both agonists. CONCLUSIONS: These results suggest that halothane and cAMP exert their effects on platelet aggregation and Ca2+ signaling through different mechanisms, and that halothane cannot impair platelet aggregation independently of phospholipase C stimulation.     

Ca2+ in the dense tubules: a model of platelet Ca2+ load

In this work, we explored the relationship between the freely exchangeable Ca2+ (FECa2+) in the dense tubules (DT) and the sarco(endo)plasmic reticulum (SER) Ca2+-ATPase (SERCA) in circulating human platelets and examined the relationship between blood pressure (BP) and these platelet parameters. Studying platelets from 32 healthy men, we showed that the maximal reaction velocity (Vmax) of the SERCA significantly correlated with FECa2+ in the DT and with the protein expressions of SERCA 2 and 3. BP positively correlated with both the Vmax of the SERCA (r=.462, P=.010) and the FECa2+ sequestered in the DT (r=.492, P=.005). The relationships between these platelet Ca2+ parameters and BP were in part confounded by increased levels of serum triglycerides and diminished HDL cholesterol with a higher BP. No correlation was observed between the resting cytosolic Ca2+ and BP. Collectively, these findings indicate that (1) an increase in the cellular Ca2+ load in platelets is expressed by a higher activity of the SERCA and an increase in the expressions of SERCA 2 and 3 proteins, coupled with an increase in the FECa2+ in the DT, and (2) a higher BP is associated with an increase in platelet Ca2+ load in human beings, expressed by a rise in the FECa2+ in the DT and the upregulation of SERCA activity.     

Ca2+ release from Ca2+ stores, particularly from ryanodine-sensitive Ca2+ stores, is required for the induction of LTD in cultured cerebellar Purkinje cells

1. Primary-cultured cerebellar Purkinje cells (PCs) from mouse embryos were whole cell voltage clamped, and L-glutamate (Glu) was applied iontophoretically to the dendrite. Long-term depression (LTD) of Glu-evoked currents was induced through the conjunction of repeated depolarizations and Glu applications. 2. Thapsigargin, a specific inhibitor of Ca(2+)-ATPase on the endoplasmic reticulum, and ryanodine and ruthenium red, inhibitors of the ryanodine receptor, blocked the induction of LTD. 3. Thapsigargin and ryanodine alone did not affect influx of Ca2+ through voltage-gated Ca2+ channels and inward currents evoked by Glu applications. 4. Our results suggest that Ca2+ release from internal stores, particularly from ryanodine-sensitive stores, is necessary for the induction of LTD in cultured PCs.     

Pharmacological characterization of endothelin-induced contraction in the guinea-pig oesophageal muscularis mucosae

1. In the oesophageal muscularis mucosae, we examined the effects of endothelin-1 (ET-1), endothelin-2 (ET-2), endothelin-3 (ET-3) and sarafotoxin S6c (SX6c) as agonists, and FR139317, BQ-123 and RES-701-1 as endothelin receptor antagonists. 2. All of the endothelins produced tonic contractions which were frequently superimposed on rhythmic motility in a concentration-dependent manner. The order of potency (-log EC50) was ET-1 (8.61)=SX6c (8.65)>ET-2 (8.40)>ET-3 (8.18). 3. FR139317 (1-3 microM) and BQ-123 (1 microM) caused parallel rightward shifts of the concentration-response curve to ET-1, but at higher concentrations caused no further shift. RES-701-1 (3 microM) caused a rightward shift of the concentration-response curve to ET-1, while RES-701-1 (10 microM) had no additional effect. RES-701-1 (0.1-1 microM) concentration-dependently caused a rightward shift of the concentration-response curve to SX6c. The contraction to ET-1 (10 nM) in preparations desensitized to the actions of SX6c was greatly inhibited by pretreatment with FR139317 (10 microM). 4. Modulation of the Ca2+ concentration in the Krebs solution caused the concentration-response curve to ET-1 or SX6c to shift to the right and downward as external Ca2+ concentrations decreased. Verapamil (30 microM) abolished rhythmic motility induced by ET-1 or SX6c. Ni2+ (0.1 mM) weakly inhibited ET-1- or SX6c-induced tonic contraction. SK&F 96365 (60 microM) completely inhibited ET-1-induced contractions. 5. We conclude that there are two types of ET-receptors, excitatory ET(A)- and ET(B)-receptors in the oesophageal muscularis mucosae. These receptors mediate tonic contractions predominantly by opening receptor-operated Ca2+ channels (ROCs) and partly by opening T-type Ca2+ channels, and mediate rhythmic motility by opening L-type Ca2+ channels.     

Endothelin-1 inhibits L-type Ca2+ current enhanced by isoprenaline in rat atrial myocytes

Endothelin-1 (ET-1) was shown to exert direct cardiac effects by complex signaling pathways and to interact with neurotransmitter regulation of cardiac activity. The effect of ET-1 was investigated on the beta-adrenergic stimulation of cardiac L-type Ca2+ current (ICaL) on isolated rat atrial myocytes by using the patch-clamp technique. ET-1 (5 x 10(-8) M) reversed the increase in ICaL induced by isoprenaline (10(-6) M) but had no effect on basal ICaL and on (-) Bay K 8644-increased ICaL (10(-6) M); so ET-1 might exert an effect only when the Ca2+ channels are phosphorylated. The antiadrenergic action of ET-1, blocked by BQ-123 (10(-6) M) and unaffected by IRL 1038 (3.5 x 10(-8) M) should be mediated by ET-A receptors. The inhibitory action of ET-1 was still observed when ICaL was previously increased by forskolin (3 x 10(-6) M), 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP; 200 microM), or cAMP (100 microM) in presence of isobutyl methyl xanthine (IBMX; 10(-6) M), suggesting that the antiadrenergic action of ET-1 on ICaL was exerted independent of the cAMP-dependent phosphorylation pathway. ET-1 is known to be an activator of phosphoinositide hydrolysis, resulting in an increased production of IP3 and diacylglycerol (DAG). A Ca(2+)-dependent inhibition of ICaL consequently to an elevation of the intracellular Ca2+ pool via IP3 might be excluded in the action of ET-1, because of the presence of EGTA in the intrapipette medium. ET-1 reversed the isoprenaline-induced increase in ICaL in the presence of protein kinase C inhibitor [PKC(19-31); 100 microM), making unlikely the involvement of a DAG-dependent activation of PKC. Therefore the antiadrenergic action of ET-1 might also be independent on the phosphoinositide pathway.     

Thrombin-induced thromboxane synthesis by human platelets. Properties of anion binding exosite I-independent receptor

These studies have examined the effects of thrombin-related agonists in stimulating thromboxane production by human platelets. The results presented show that (1) the maximal response elicited by thrombin receptor agonist peptide (TRAP) stimulation was 40% to 50% of that seen with thrombin or the thrombin mutant Thrombin Quick I; (2) pretreatment of platelets with prostaglandin E1 or genistein resulted in differential inhibition of thromboxane production in response to TRAP compared with either enzyme agonist; (3) an antibody to the thrombin receptor cleavage site that inhibits increases in intracellular [Ca2+] only partially reduced thromboxane production in response to 5 nmol+L thrombin and 15 nmol/L Thrombin Quick I; (4) preincubation with 20 mumol/L TRAP resulted in desensitization to further stimulation by 100 mumol/L TRAP, but not by 100 nmol/L thrombin; and (5) the response to thrombin after TRAP desensitization was completely inhibited by the tyrosine kinase inhibitor genistein and was independent of an intracellular [Ca2+] flux, The cumulative results may be explained by the existence of two proteolytically activated receptors that result in thromboxane production in response to thrombin. One is the thrombin receptor/substrate, PAR-1. Stimulation through the second receptor/substrate depends on a genistein-sensitive step, is independent of an intracellular Ca2+ flux, and is initiated by a thrombin-activated receptor that does not depend on interaction with anion-binding exosite I, as previously indicated by the relative activity of Thrombin Quick I in stimulating platelet aggregation and thromboxane production. The proposed second thrombin receptor on platelets represents an additional member of the class of proteolytically activated receptors.     

Sensitivity of G-protein involved in endothelin-1-induced Ca2+ influx to pertussis toxin in porcine endothelial cells in situ

1. We designed a new method to determine quantitatively the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ, using front-surface fluorometry and fura-2-loaded porcine aortic valvular strips. Using this method, we investigated the characteristics of the G-protein involved in endothelin-1 (ET-1)-induced changes in [Ca2+]i of endothelial cells in situ. 2. Endothelial cells were identified by specific uptake of acetylated-low density lipoprotein labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). Double staining with DiI-Ac-LDL and fura-2 showed that the valvular strip was covered with a monolayer of endothelial cells and that the cellular component which contributed to the fura-2 fluorescence, [Ca2+]i signal, was exclusively endothelial cells. 3. ET-1 (10(-7) M) induced an elevation of [Ca2+]i consisting of two components: the first was a rapid and transient elevation to reach a peak, followed by a second, sustained elevation (the second phase). The first phase was composed of extracellular Ca(2+)-independent and -dependent components, while the second phase was exclusively extracellular Ca(2+)-dependent. The extracellular Ca(2+)-independent component of the first phase was due to the release of Ca2+ from intracellular storage sites. The second phase and part of the first phase of [Ca2+]i elevation were attributed to the influx of extracellular Ca2+. The Ca2+ influx component was completely inhibited by 10(-3) M Ni2+ but was not affected by 10(-5) M diltiazem. 4. Pertussis toxin (IAP) markedly inhibited the extracellular Ca2+-dependent elevation of [Ca2+]j, but had no effect on the extracellular Ca2+-independent elevation of [Ca2+], caused by ET-1 (10-7M).5. Bradykinin (10-7 M) or ATP (10- 5M) elevated [Ca2+]i and these responses also consisted of extracellular Ca2+-independent and extracellular Ca2+-dependent components. IAP had no effect on either component of the [Ca2+]i elevation induced by bradykinin or ATP.6. From these findings we conclude that, in porcine endotheliel cells in situ, ET-1 elevates [Ca2+]i as are result of a Ca2+ influx component from the extracellular space and release of intracelluarly stored Ca2+ .The Ca2+ influx is regulated by an IAP-sensitive G-protein, while the release of Ca2+ from the intracellular store is not.     

Evidence for mitochondrial Ca(2+)-induced Ca2+ release in permeabilised endothelial cells

Generally most intracellular Ca2+ is stored in the endoplasmic reticulum (ER) and mitochondria. Recently a mitochondrial Ca(2+)-induced Ca2+ release (mCICR) mechanism, unconnected with ryanodine receptors (RyR's), has been shown in tumour cells. The existence of a mitochondrial Ca2+ release mechanism in BAE cells was investigated using saponin-permeabilised BAE cells. When buffered intracellular solution were 'stepped' from 10 nM to 10 microM free Ca2+, the mitochondrial inhibitors CN (2 mM), FCCP (1 microM), and RR (20 microM) significantly reduced total CICR by approximately 25%. The ER Ca(2+)-ATPase inhibitor thapsigargin (100 nM) had no effect. Furthermore, cyclosporin A (200 nM), an inhibitor of the mitochondrial permeability transition pore (PTP), abolished total CICR. Therefore, the novel ryanodine-caffeine insensitive CICR mechanism previously reported in BAE cells involves mitochondrial Ca2 release. It is proposed that in BAE cells, mCICR occurs via the mitochondrial PTP and may be physiologically important in endothelial cell Ca2+ signalling.     

Nitric oxide inhibits capacitative cation influx in human platelets by promoting sarcoplasmic/endoplasmic reticulum Ca2+-ATPase-dependent refilling of Ca2+ stores

Nitric oxide (NO) is a potent inhibitor of thrombin-induced increase in cytoplasmic free Ca2+ concentration and aggregation in platelets, but the precise mechanism of this inhibition is unclear. To measure Ca2+/Mn2+ influx in intact platelets and to monitor Ca2+ uptake into the stores in permeabilized platelets, fura-2 was used. In intact platelets, maximal capacitative Ca2+ and Mn2+ influx developed rapidly (within 30 s) after fast release of Ca2+ from the stores with thrombin (0.5 U/mL) or slowly (within 5 to 10 minutes) following passive Ca2+ leak caused by inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) with 30 micromol/L 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ). NO (1 micromol/L) inhibited capacitative Ca2+ and Mn2+ influx independently of the time after thrombin application. In contrast, the effect of NO on BHQ-induced Ca2+ and Mn2+ influx was observed only during the first few minutes after BHQ application and completely disappeared when capacitative cation influx reached its maximum. In Ca2+-free medium, NO reduced the peak Ca2+ rise caused by thrombin and significantly promoted Ca2+ back-sequestration into the stores. Both effects disappeared in the presence of BHQ. Inhibition of guanylate cyclase with H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (10 micromol/L) attenuated but did not prevent the effects of NO on cytoplasmic free Ca2+ concentration. Inhibition of Ca2+ uptake by mitochondria did not change the effects of NO. In permeabilized platelets, NO accelerated back-sequestration of Ca2+ into the stores after inositol-1,4,5-trisphosphate-induced Ca2+ release or after addition of Ca2+ (1 micromol/L) in the absence of inositol-1,4,5-trisphosphate. The effect of NO depended on the initial rate of Ca2+ uptake and on the concentration of ATP and was abolished by BHQ, indicating the direct involvement of SERCA. These data strongly support the hypothesis that NO inhibits store-operated cation influx in human platelets indirectly via acceleration of SERCA-dependent refilling of Ca2+ stores.     

Thromboxane A2 synthase inhibition and thromboxane A2 receptor blockade by 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) in rat platelets

The effects of 2-[(4-acetylphenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15), a synthetic 1,4-naphthoquinone derivative, on platelet activity and its mechanism of action were investigated. NQ-Y15 caused a concentration-dependent inhibition of the aggregation induced by thrombin, collagen, arachidonic acid (AA), and A23187. The IC50 values of NQ-Y15 on thrombin (0.1 U/mL)-, collagen (10 microg/mL)-, AA (50 microM)-, and A23187 (2 microM)-induced aggregation were 36.2 +/- 1.5, 6.7 +/- 0.7, 35.4 +/- 1.7, and 93.1 +/- 1.4 microM, respectively. NQ-Y15 also inhibited thrombin-, collagen-, AA-, and A23187-stimulated serotonin secretion in a concentration-dependent manner. However, a high concentration (100 microM) of NQ-Y15 showed no significant inhibitory effect on ADP-induced primary aggregation, which is independent of thromboxane A2 (TXA2) production in rat platelets. In fura-2-loaded platelets, the elevation of intracellular free calcium concentration stimulated by AA, thrombin, and 4-bromo-A23187 was inhibited by NQ-Y15 in a concentration-dependent manner. The formation of TXA2 caused by AA, thrombin, and collagen was inhibited significantly by NQ-Y15. NQ-Y15 inhibited TXA2 synthase in intact rat platelets, since this agent reduced the conversion of prostaglandin (PG) H2 to TXA2. Similarly, NQ-Y15 selectively inhibited the TXA2 synthase activity in human platelet microsomes, whereas it had no effect on activity of phospholipase A2, cyclooxygenase, and PGI2 synthase in vitro. NQ-Y15 inhibited platelet aggregation induced by the endoperoxide analogue U46619 in human platelets, indicating TXA2 receptor antagonism, possibly of a competitive nature. These results suggest that the antiplatelet effect of NQ-Y15 is due to a combination of TXA2 synthase inhibition with TXA2 receptor blockade, and that it may be useful as an antithrombotic agent.     

Characterization of the intracellular and the plasma membrane Ca2+-ATPases in fractionated pig brain membranes using calcium pump inhibitors

The Ca2+-ATPase activity of isolated membranes and purified plasma membrane ATPase from pig brain was measured in the presence of specific inhibitors. The inhibition of the enzymatic activity by vanadate presents a lower affinity in microsomes than in the synaptic plasma membrane vesicles, showing K0.5 of 0.4 and 0.2 microM, respectively. The purified enzyme showed a higher sensitivity to vanadate with a K0.5 of 0.10 microM. Thapsigargin (Tg) and 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) were stronger inhibitors of the Ca2+-ATPase activity in microsomes than in the synaptic membrane vesicles. The activity of the purified enzyme was not affected by Tg and only partially by BHQ. Cyclopiazonic acid inhibited the enzymatic activity in all fractions, being more sensitive in microsomes. The microsome preparation incorporated 32P from [gamma-32P]ATP into two main proteins that appear at approx 110,000 and 140,000. According to the inhibition pattern, the lower phosphorylated band was identified as the sarco(endo)plasmic reticulum Ca2+-ATPase, being in a higher percentage than the upper band. Synaptic membrane vesicles also incorporated radioactive 32P into two protein bands. The 140,000 protein (upper band) shows the typical behavior of the purified plasma membrane Ca2+-ATPase, being more abundant in this preparation than the organellar Ca2+-pump (lower band). This study highlights the heterogeneous nature of the Ca2+-ATPase activity measured in brain membrane fractions.     

Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles

Microparticles are released during platelet activation in vitro and have been detected in vivo in syndromes of platelet activation. They have been reported to express both pro- and anticoagulant activities. Nevertheless, their functional significance has remained unresolved. To address the mechanism(s) of cellular activation by platelet microparticles, we examined their effects on platelets and endothelial cells. Activation of human platelets by diverse stimuli (thrombin, 0.1 U/ml; collagen, 4 microg/ml; and the calcium ionophore A23187, 1 microM) results in shedding of microparticles. Pretreatment of these particles, but not membrane fractions from resting platelets, with (s)PLA2 evokes a dose-dependent increase in platelet aggregation, intracellular [Ca2+] movement, and inositol phosphate formation. These effects localize to the arachidonic acid fraction of the microparticles and are mimicked by arachidonic acid isolated from them. However, platelet activation requires prior metabolism of microparticle arachidonic acid to thromboxane A2. Thus, pretreatment of platelets with the cyclooxygenase (COX) inhibitor, indomethacin (20 microM), the thromboxane antagonist SQ29,548 (1 microM), or the protein kinase C inhibitor GF109203X (5 microM) prevents platelet activation by microparticles. However, platelet microparticles fail to evoke an inositol phosphate response directly, via either of the cloned thromboxane receptor isoforms stably expressed in human embryonic kidney (HEK) 293 cells. Prelabeling platelets with [2H(8)] arachidonate was used to demonstrate platelet metabolism of the microparticle-derived substrate to thromboxane. Platelet microparticles can also induce expression of COX-2 and prostacyclin (PGI2) production, but not expression of COX-1, in human endothelial cells. These effects are prevented by pretreatment with actinomycin D (12 microM) or cycloheximide (5 microg/ml). Expression of COX-2 is again induced by the microparticle arachidonate fraction, which it may then use to synthesize PGI2. Both PGE2 and iloprost, a stable PGI2 analog, evoke human umbilical vein endothelial cell COX-2 expression, albeit with kinetics that differ from the response to platelet microparticles. These studies indicate a novel mechanism of transcellular lipid metabolism whereby platelet activation may be amplified or modulated by concentrated delivery of arachidonic acid to adjacent platelets and endothelial cells.     

Difference of (Ca2+)i movements in platelets stimulated by thrombin and TRAP: the involvement of alpha(IIb)beta3-mediated TXA2 synthesis

This study investigated the difference of [Ca2+]i movement in platelets in response to thrombin and TRAP. The involvement of alpha(IIb)beta3 in this signaling was also studied. Stimulation of platelets with thrombin at 0.03 U/ml caused platelet aggregation and a two-peak increase in [Ca2+]i. The second peak of [Ca2+]i, but not the first peak was abolished by the inhibition of platelet aggregation with alpha(IIb)beta3 antagonists or by scavenging endogenous ADP with apyrase. A cyclooxygenase inhibitor, aspirin, and a TXA2 receptor antagonist, BM 13505, also abolished the second peak of [Ca2+]i but not the first peak, although these regents did not inhibit aggregation. Under the same assay conditions, measurement of TXB2 demonstrated that alpha(IIb)beta3 antagonists and aspirin almost completely inhibited the production of TXB2. In contrast to thrombin-stimulation, TRAP caused only a single peak of [Ca2+]i even in the presence of platelet aggregation, and a high level of [Ca2+]i increase was needed for the induction of platelet aggregation. The inhibition of aggregation with alpha(IIb)beta3 antagonists had no effect on [Ca2+]i change and TXB2 production induced by TRAP. Inhibition studies using anti-GPIb antibodies suggested that GPIb may be involved in the thrombin response, but not in the TRAP. Our findings suggest that low dose thrombin causes a different [Ca2+]i response and TXA2 producing signal from TRAP. Endogenous ADP release and fibrinogen binding to alpha(IIb)beta3 are responsible for the synthesis of TXA2 which results in the induction of the second peak of [Ca2+]i in low thrombin- but not TRAP-stimulated platelets.     

Pathway for Ca2+ influx into cells by trichosporin-B-VIa, an alpha-aminoisobutyric acid-containing peptide, from the fungus Trichoderma polysporum

Trichosporin (TS) -B-VIa, a fungal alpha-aminoisobutyric acid (Aib) -containing peptide consisting of 19 amino acid residues and a phenylalaninol, produced both 45Ca2+ influx into bovine adrenal chromaffin cells and catecholamine secretion from the cells. The secretion induced by TS-B-VIa at lower concentrations (2-5 microM) was completely dependent on the external Ca2+, while that induced by TS-B-VIa at higher concentrations (10-30 microM) was partly independent of the Ca2+. The concentration-response curves (2-5 microM) for the TS-B-VIa-induced Ca2+ influx and secretion correlated well. The TS-B-VIa (at 5 microM) -induced secretion was not antagonized by diltiazem, a blocker of L-type voltage-sensitive Ca2+ channels. The treatment of fura-2-loaded C6 glioma cells with TS-B-VIa (2-5 microM) led to an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner but the stimulatory effects of TS-B-VIa on [Ca2+]i were only slightly observed in Ca(2+)-free medium, indicating that TS-B-VIa causes Ca2+ influx from the external medium into the C6 cells. The TS-B-VIa-induced increase in [Ca2+]i in the C6 cells was not antagonized by diltiazem and by SK&F 96365, a novel blocker of receptor-mediated Ca2+ entry. High K+ increased neither [Ca2+]1 in the C6 cells nor Mn2+ influx into the cells, while TS-B-VIa increased Mn2+ influx. Also in other non-excitable cells, bovine platelets, similar results were obtained. These results strongly suggest that the mechanism of Ca2+ influx by TS-B-VIa at the lower concentrations is distinct from the event of Ca2+ influx through receptor-operated or L-type voltage-sensitive Ca2+ channels in both excitable cells (the chrornaffin cells) and non-excitable cells (the C6 cells and the platelets) and that TS-B-VIa per se may form Ca(2+)-permeable ion channels in biological membranes. On the other hand, the peptide at the higher concentrations seems to damage cell membranes.     

The structure and interactions of Ca(2+)-ATPase

Electron crystallographic studies on membrane crystals of Ca(2+)-ATPase reveal different patterns of ATPase-ATPase interactions depending on enzyme conformation. Physiologically relevant changes in Ca2+ concentration and membrane potential affect these interactions. Ca2+ induced difference FTIR spectra of Ca(2+)-ATPase triggered by photolysis of caged Ca2+ are consistent with changes in secondary structure and carboxylate groups upon Ca2+ binding; the changes are reversed during ATP hydrolysis suggesting that a phosphorylated enzyme form of low Ca2+ affinity is the dominant intermediate during Ca2+ transport. A two-channel model of Ca2+ translocation is proposed involving the membrane-spanning helices M2-M5 and M4, M5, M6 and M8 respectively, with separate but interacting Ca2+ binding sites.