Cellular Ca2+ ATPase activity in diabetes mellitus

Basal and maximal Ca2+ ATPase activity was studied in erythrocytes of 29 healthy controls, 15 patients with insulin-dependent diabetes mellitus (IDDM) and 22 patients with non-insulin-dependent diabetes mellitus (NIDDM). Basal and maximal Ca2+ ATPase activity was significantly decreased in insulin-dependent diabetes mellitus (8.4 +/- 0.5 and 22.5 +/- 1.1 pmol/10(6) RBC/min) and non-insulin-dependent diabetes mellitus (7.3 +/- 1.0 and 18.6 +/- 1.8 pmol/10(6) RBC/min) compared to healthy controls (9.3 +/- 1.0 and 24.6 +/- 1.1 pmol/10(6) RBC/min). Maximal Ca2+ ATPase activity showed a significant correlation to systolic blood pressure in both insulin-dependent diabetes mellitus and non-insulin-dependent diabetes mellitus. There was no significant correlation of maximal Ca2+ ATPase activity to fasting serum glucose concentration and to HbA1 levels. Maximal Ca2+ ATPase activity was significantly correlated to creatinine clearance in non-insulin-dependent diabetes mellitus, but not in insulin-dependent diabetes mellitus. It is concluded that a decreased cellular Ca2+ ATPase activity may predispose to the development of hypertension in diabetes mellitus.     

Increase in functional Ca2+ channels in cerebral smooth muscle with renal hypertension

The hypothesis that availability of functional Ca2+ channels in vascular smooth muscle is augmented in hypertension was tested in basilar artery cells from Wistar rats exhibiting stable systolic blood pressure (BPsys) for 2 to 11 weeks after partial renal artery ligation (Goldblatt 2-kidney 1-clip [2K1C] model). Cells were freshly isolated and patch-clamped using a nystatin-perforated patch method. BPsys ranged from 110 to 280 mm Hg and correlated with normalized kidney mass. Macroscopic current-voltage curves were fit to a Boltzmann function to obtain maximum conductance (gmax), steepness and midpoint potential for the voltage dependence of activation (k and E1/2, respectively), and extrapolated reversal potential for the chord conductance (Erev). Linear regression of normalized conductance (ng(max)=g(max)/cell capacitance) versus BPsys for 103 cells indicated a strong relationship, with a slope of 0.0019 nS x pF(-1) x mm Hg(-1) (P<0.0001). Similar analysis of data from 35 other cells exposed to 500 nmol/L Bay K 8644 gave a slope of 0.0041 nS x pF(-1) x mm Hg(-1) (P=0.001). Voltage-dependent parameters, k, E1/2, and Erev, were not significantly related to BPsys. Single-channel measurements in cell-attached patches revealed that the number of channels in 32 patches was significantly related to BPsys (P=0.0024) but that slope conductance, open dwell times at 0 mV, and distribution between 2 open states were not. Finally, in a subgroup of 61 cells from animals made hypertensive (180 mm Hg相似文献   

Sarcoplasmic reticular Ca2+ pump ATPase activity in congestive heart failure due to myocardial infarction

OBJECTIVE: Earlier studies have shown a depression in the sarcoplasmic reticular (SR) Ca2+ uptake and gene expression in Ca2+ pump ATPase protein in congestive heart failure subsequent to myocardial infarction. It is the objective of this study to understand further the mechanisms of depressed SR Ca2+ pump activity in the failing heart. METHODS: Heart failure in rats was induced by occluding the left coronary artery for 16 weeks and the viable left ventricle was processed for the isolation of SR membranes. Sham-operated animals were used as control. The characteristics of SR Ca2+ pump ATPase in the presence of different concentrations of K+, Ca2+ and ATP were examined and the purity of these membranes was monitored by determining the marker enzyme activities. In addition to measuring changes in cyclic adenosine monophosphate (cAMP) protein kinase and Ca(2+)-calmodulin induced phosphorylation, alterations in SR phospholipid composition as well as sulfhydryl (SH) group content were investigated. RESULTS: Ca(2+)-stimulated ATPase activity, unlike Mg(2+)-ATPase activity, was depressed in the left ventricular SR from failing hearts as compared to control. The decrease in Ca(2+)-stimulated ATPase activity was seen at different concentrations of Ca2+, K+ and ATP but no changes in the affinities of the enzyme for Ca2+ and ATP were evident. The SR Ca(2+)-stimulated ATPase activities in the presence of both cAMP-dependent protein kinase and Ca(2+)-calmodulin were markedly decreased in the failing hearts when compared to control preparations. Furthermore, the 32P incorporation in the presence of cAMP-dependent protein kinase or Ca(2+)-calmodulin was also reduced in the experimental heart SR membranes. The phospholipid composition of the SR membranes from the failing heart was markedly altered. No changes in SH-group or the degree of cross contamination with other membranes were apparent in the failing heart SR. CONCLUSIONS: These results suggest that abnormalities in membrane phospholipid composition and phosphorylation of the enzyme may partly explain the observed depression in SR Ca2+ pump ATPase activity in heart failure following myocardial infarction.     

Characterization of dimethylguanosine, phenylethylamine, and phenylacetic acid as inhibitors of Ca2+ ATPase in end-stage renal failure

The activity of the plasma membrane Ca2+ ATPase of chronic renal failure patients is decreased by circulating inhibitors yet to be characterized. In this study, inhibitors of Ca2+ ATPase were isolated from ultrafiltrate of patients with end-stage renal failure. They were identified as dimethylguanosine, phenylethylamine, and phenylacetic acid by chromatography and mass spectrometry. Ca2+ ATPase activity was measured spectrophotometrically as the difference in hydrolysis of ATP in the presence and absence of Ca2+ with different concentrations of ATP and the isolated substances. All of the identified compounds are sufficiently lipophilic to penetrate the blood-brain barrier and to accumulate in cerebral tissue. The inhibitory effects of these agents were additive. The apparent K(m) values for ATP and Ca2+ were not altered by these substances, suggesting a noncompetitive mechanism of inhibition. In plasma of healthy subjects, the substances were not detectable. The Ca2+ ATPase inhibitors identified may play a role in the pathophysiology of end-stage renal failure and, potentially, in monitoring toxic effects on cellular Ca2+ metabolism in renal failure.     

Phencyclidine block of Ca2+ ATPase in rat heart sarcoplasmic reticulum

Phencyclidine hydrochloride (PCP) also known as Angel Dust is a very potent psychotomimetic drug of abuse. Besides its central nervous system (CNS) effects PCP produces a number of adverse effects in a variety of tissues including the cardiovascular system. Since PCP is known to alter the cellular calcium homeostasis the present studies were initiated to determine the changes in cardiac Ca2+ ATPase activity in rats treated with PCP. For in vitro studies the cardiac sarcoplasmic reticulum (SR) fractions prepared from normal rats were incubated with 25, 50 and 100 microM PCP and the enzyme activities were estimated. Whereas, for in vivo studies the cardiac SR fractions prepared from rats treated with PCP (10 mg/kg body wt. single dose, intra-peritoneally (i.p.)) and sacrificed at different time intervals were used. PCP reduced the Ca2+ ATPase activity significantly both in vitro and in vivo. A 50% inhibition of the enzyme activity was obtained with 100 microM PCP in vitro. A significant reduction of SR Ca2+ ATPase was also evident as early as 1 h after treatment of rats with PCP. The reduction of Ca2+ ATPase activity in SR was irreversible even at 12 h after treatment. The in vitro kinetic studies revealed that PCP was found to be a competitive inhibitor of Ca2+ ATPase with respect to the substrate, ATP, and non-competitive with respect to Ca2+ activation. These results indicate that PCP alters the myocardial Ca2+ homeostasis by inhibiting the Ca2+ ATPase in cardiac SR in rats. Inhibition of SR Ca2+ ATPase may result in the impairment of contraction and relaxation coupling processes in the myocardium.     

Sarcoplasmic reticulum Ca2+ ATPase promoter activity during endothelin-1 induced hypertrophy of cultured rat cardiomyocytes

Proliferation in mammalian cells is controlled primarily in the G1-phase of the cell cycle through the action of the G1 cyclin-dependent kinases, CDK4 and CDK2. To explore the mechanism of cellular response to extrinsic factors, specific loss of function mutations were generated in two negative regulators of G1 progression, p21 and pRB. Individually, these mutations were shown to have significant effects in G1 regulation, and when combined, Rb and p21 mutations caused more profound defects in G1. Moreover, cells deficient for pRB and p21 were uniquely capable of anchorage-independent growth. In contrast, combined absence of pRB and p21 function was not sufficient to overcome contact inhibition of growth nor for tumor formation in nude mice. Finally, animals with the genotype Rb+/-;p21(-/-) succumbed to tumors more rapidly than Rb+/- mice, suggesting that in certain contexts mutations in these two cell cycle regulators can cooperate in tumor development.     

Intracellular signaling through long-range linked functions in the Ca2+ transport ATPase

The Ca2+ transport ATPases of intracellular membranes exhibit an intracellular long-range functional linkage which is the basic mechanistic device for Ca2+ transport through ATP utilization. The functional linkage operates between a phosphorylation (catalytic) domain located in the extramembranous region, and a Ca2+ binding domain located in the membrane bound region of the enzyme. The two domains are separated by a distance of approximately 50 A, and are both affected by binding of a single molecule of the highly specific inhibitor, thapsigargin, to the enzyme. Functional and structural features are here described to explain the long-range linkage through the protein structure.     

Ca2+ translocation across sarcoplasmic reticulum ATPase randomizes the two transported ions

Cytoplasmic Ca2+ dissociation is sequential, and the Ca2+ ions bound to the nonphosphorylated ATPase are commonly represented as superimposed on each other, so that the superficial Ca2+ is freely exchangeable from the cytoplasm, whereas the deeper Ca2+ is not. Under conditions where ADP-sensitive phosphoenzyme accumulates (leaky vesicles, 5 degrees C, pH 8, 300 mM K+), luminal Ca2+ dissociation is sequential as well, so that the representation of two superimposed Ca2+ ions still holds on the phosphoenzyme, with the superficial Ca2+ facing the lumen freely exchangeable and the deeper Ca2+ blocked by the superficial Ca2+. Under the same conditions, we have investigated whether a prebuilt Ca2+ order is maintained during membrane translocation. Starting from a prebuilt order on the cytoplasmic side, we showed that the Ca2+ ions cannot be identified after translocation to the luminal side. The same result was obtained starting from a prebuilt order on the luminal side and following the luminal to cytoplasmic translocation. We conclude that the two Ca2+ ions are mixed during ATP-induced phosphorylation as well as during ADP-induced dephosphorylation.     

PLA2 activity regulates Ca2+ storage-dependent cellular proliferation

The objective of this study is to determine the role of arachidonic acid (AA) in cell proliferation by inhibiting AA synthetic enzyme phospholipase A2 (PLA2) and to determine its involvement in the role of the second messenger intracellular calcium (Ca2+). Methods used to determine the effects on proliferation of cell cultures of primary meningioma and astrocytoma U373-MG included treatment with micromolar concentrations of PLA2 inhibitors 4-bromophenacylbromide and quinacrine. Effects of these drugs on proliferation were further investigated by the application of concentrations that inhibit growth by 50% while antagonizing these agents with AA replacement. Free cytosolic Ca2+ was measured with the use of fluorescent dye Fura-2 during PLA2 agonist/antagonist studies. These Ca2+ measurements were performed in the absence of extracellular Ca2+ to identify the contribution of intracellular Ca2+ sources. PLA2 inhibition resulted in decreased growth of cultured astrocytoma and meningioma cells in a dose-dependent manner in the micromolar range. This inhibitory effect was antagonized by the addition of AA. PLA2 inhibition caused an elevation of basal-cytosolic-free [Ca2+] while depleting internal Ca2+ stores. These Ca2+ changes were also antagonized by the addition of AA. In conclusion, these results demonstrate that AA, a PLA2 enzyme product, is involved in regulating the growth rate of these cell types. The PLA2 pathway also regulates the maintenance of the internal Ca2+ stores. Ca2+ is known to be a growth-related intracellular second messenger. These results suggest that the growth regulatory functions of AA are mediated by Ca2+-dependent mechanisms.     

Mechanism of nitric oxide-induced vasodilatation: refilling of intracellular stores by sarcoplasmic reticulum Ca2+ ATPase and inhibition of store-operated Ca2+ influx

The precise mechanisms by which nitric oxide (NO) decreases free [Ca2+]i, inhibits Ca2+ influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca2+]i were resistant to nifedipine, suggesting Ca2+ entry through non-L-type Ca2+ channels. Relaxations to NO were inhibited by thapsigargin (TG) or cyclopiazonic acid (CPA) indicating the involvement of sarcoplasmic reticulum ATPase (SERCA). Studies of the effect of NO on [Ca2+]i and the rate of Mn2+ influx with fura-2 fluorometry in rabbit aortic smooth muscle cells in primary culture were designed to test how SERCA is involved in mediating the response to NO. When cells were stimulated with angiotensin II (AII), NO accelerated the removal of Ca2+ from the cytoplasm, decreased [Ca2+]i, and inhibited Ca2+ and Mn2+ influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na+/Ca2+exchanger or the plasma membrane Ca2+ ATPase had no influence on the ability of NO to decrease [Ca2+]i. NO maximally decreased [Ca2+]i within 5 s, whereas significant inhibition of AII-induced Ca2+ and Mn2+ influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca2+]o and functional SERCA, suggesting that during the delay before NO inhibits Ca2+ influx, the influx of Ca2+ and the uptake into intracellular stores are required. In the absence of [Ca2+]o, NO diminished the AII-induced [Ca2+]i transient by a SERCA-dependent mechanism and increased the amount of Ca2+ in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca2+]i caused by NO in vascular smooth muscle is accounted for by the uptake of Ca2+ by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca2+ influx through non-L-type Ca2+ conducting ion channels and that this maintains the decrease in [Ca2+]i and NO-induced relaxation.     

Region-specific activity of the plasma membrane Ca2+ pump and delayed activation of Ca2+ entry characterize the polarized, agonist-evoked Ca2+ signals in exocrine cells

The initial release of Ca2+ from the intracellular Ca2+ stores is followed by a second phase during which the agonist-dependent Ca2+ response becomes sensitive to the extracellular Ca2+, indicating the involvement of the plasma membrane (PM) Ca2+ transport systems. The time course of activation of these transport systems, which consist of both Ca2+ extrusion and Ca2+ entry pathways, is not well established. To investigate the participation of these processes during the agonist-evoked Ca2+ response, isolated pancreatic acinar cells were exposed to maximal concentrations of an inositol 1,4,5-trisphosphate-mobilizing agonist (acetylcholine, 10 microM) in different experimental conditions. Following the increase of [Ca2+]i, there was an almost immediate activation of the PM Ca2+ extrusion system, and maximal activity was reached within less than 2s. The rate of Ca2+ extrusion was dependent on the level of [Ca2+]i, with a steep activation at values just above the resting [Ca2+]i and reached a plateau value at 700 nM Ca2+. In contrast, the PM Ca2+ entry pathway was activated with a much slower time course. There was also a delay of 3-4 s between the maximal effective depletion of the intracellular Ca2+ stores and the activation of this entry pathway. By use of digital imaging data, the PM Ca2+ transport systems were also analyzed independently in two regions of the cells, the lumenal and the basal poles. With respect to the activation of the Ca2+ entry pathways, no significant difference existed between these two regions. In contrast, the PM Ca2+ pump displayed a different pattern of activity in these regions. In the basal pole, the pump activity was more sensitive to changes of [Ca2+]i and had a higher maximal activity. Also, in the lumenal pole, the pump became saturated at values of [Ca2+]i around 700 nM, whereas at the basal pole [Ca2+]i had a biphasic effect on the pump activity, and higher [Ca2+]i inhibited the pump. It is argued that these differences in sensitivity to the levels of [Ca2+]i and the different relationship between [Ca2+]i and the rate of extrusion at the two functional poles of the pancreatic acinar cells indicate that the plasma membrane Ca2+ ATPase might play an important role in the polarization of the Ca2+ response.     

Ca2+ transport by reconstituted synaptosomal ATPase is associated with H+ countertransport and net charge displacement

The synaptosomal plasma membrane Ca2+-ATPase (PMCA) purified from pig brain was reconstituted with liposomes prepared by reverse phase evaporation at a lipid to protein ratio of 150/1 (w/w). ATP-dependent Ca2+ uptake and H+ ejection by the reconstituted proteoliposomes were demonstrated by following light absorption and fluorescence changes undergone by arsenazo III and 8-hydroxy-1,3, 6-pyrene trisulfonate, respectively. Ca2+ uptake was increased up to 2-3-fold by the H+ ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, consistent with relief of an inhibitory transmembrane pH gradient (i.e. lumenal alkalinization) generated by H+ countertransport. The stoichiometric ratio of Ca2+/H+ countertransport was 1.0/0.6, and the ATP/Ca2+ coupling stoichiometry was 1/1 at 25 degrees C. The electrogenic character of the Ca2+/H+ countertransport was demonstrated by measuring light absorption changes undergone by oxonol VI. It was shown that a 20 mV steady state potential (positive on the lumenal side) was formed as a consequence of net charge transfer associated with the 1/1 Ca2+/H+ countertransport. Calmodulin stimulated ATPase activity, Ca2+ uptake, and H+ ejection, demonstrating that these parameters are linked by the same mechanism of PMCA regulation.     

The Ca2+ transport mechanisms of mitochondria and Ca2+ uptake from physiological-type Ca2+ transients

Mitochondria contain a sophisticated system for transporting Ca2+. The existence of a uniporter and of both Na+-dependent and -independent efflux mechanisms has been known for years. Recently, a new mechanism, called the RaM, which seems adapted for sequestering Ca2+ from physiological transients or pulses has been discovered. The RaM shows a conductivity at the beginning of a Ca2+ pulse that is much higher than the conductivity of the uniporter. This conductivity decreases very rapidly following the increase in [Ca2+] outside the mitochondria. This decrease in the Ca2+ conductivity of the RaM is associated with binding of Ca2+ to an external regulatory site. When liver mitochondria are exposed to a sequence of pulses, uptake of labeled Ca2+ via the RaM appears additive between pulses. Ruthenium red inhibits the RaM in liver mitochondria but much larger amounts are required than for inhibition of the mitochondrial Ca2+ uniporter. Spermine, ATP and GTP increase Ca2+ uptake via the RaM. Maximum uptake via the RaM from a single Ca2+ pulse in the physiological range has been observed to be approximately 7 nmole/mg protein, suggesting that Ca2+ uptake via the RaM and uniporter from physiological pulses may be sufficient to activate the Ca2+-sensitive metabolic reactions in the mitochondrial matrix which increase the rate of ATP production. RaM-mediated Ca2+ uptake has also been observed in heart mitochondria. Evidence for Ca2+ uptake into the mitochondria in a variety of tissues described in the literature is reviewed for evidence of participation of the RaM in this uptake. Possible ways in which the differences in transport via the RaM and the uniporter may be used to differentiate between metabolic and apoptotic signaling are discussed.     

The effect of minoxidil on blood pressure and plasma renin activity in patients with essential and renal hypertension

The effect of the new vasodilator, minoxidil, on blood pressure and plasma renin activity was studied in 21 hypertensive patients: 12 patients with essential and 9 with renal hypertension. The average maximum dosage of minoxidil was 27.9 +/- 6.0 mg/day (M +/- SD). Average duration of treatment was 84.5 days. During the observation period the average systolic blood pressure fell from 195 +/- 18 to 159 +/- 7 mm Hg (M +/- SD), and the mean diastolic blood pressure fell from 120 +/- 8.3 to 92.5 +/- 8 mm Hg (p less than 0.01). These patients had been treated earlier with other antihypertensive agents, such as reserpine, saluretics, hydralazine, alpha-methyldopa, and clonidine, without any significant reduction in blood pressure. Before treatment, plasma renin activity after resting was 59 +/- 6.4 ng/ml/16 h (M +/- SE) and after saluretics and orthostasis 89 +/- 12.7 ng/ml/16 h. After treatment, the decline in renin value after resting was statistically significant: 42.7 +/- 3.3 ng/ml/16 h (p less than 0.05), and the stimulated renin had fallen to 70 +/- 3.4 ng/ml/16 h (p greater than 0.1). A comparison of the renin stimulation values of patients with renal hypertension also revealed a significant reduction (p less than 0.01). Side effects which appeared at a daily dose of 15 to 30 mg consisted mainly of tachycardia and fluid retention and could be controlled by the administration of propranolol and chlorthalidone. In 5 women and in 1 man was observed a cosmetically disturbing, reversible hypertrichosis. Orthostatic hypotension was observed in one patient. Minoxidil is an effective antihypertensive agent. However, because of its side effects, it generally must be administered with beta-receptor blocking agents and saluretics. It is possible that its blood pressure lowering effect is due, at least in part, to a suppression of the plasma renin activity.     

Does essential hypertension cause progressive renal disease?

In the distant past, terminal renal failure occurred mainly as a result of malignant hypertension. The introduction of effective antihypertensive therapy has made malignant hypertension rare, and researchers have stopped focusing on the kidney's role in their hypertension research. However, recent long-term observational studies have documented an impressive relationship between hypertension and impaired renal function in patients without primary chronic renal disease; elderly and African American individuals with hypertension have the worst prognoses. The hallmark of hypertensive renal injury is thought to be a progressive increase in intrarenal vascular resistance, which may precede changes in renal structure. Because we lack evidence from renal biopsy studies, it is unclear whether an increase in albumin (protein) excretion correlates with these disturbances of renal function and structure. Nevertheless, because urinary excretion of albumin in patients with essential hypertension is related to the risk of cardiovascular complications, its measurement provides important clinical information.     

Targeted disruption of the Ca2+ channel beta3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons

In comparison to the well characterized role of the principal subunit of voltage-gated Ca2+ channels, the pore-forming, antagonist-binding alpha1 subunit, considerably less is understood about how beta subunits contribute to neuronal Ca2+ channel function. We studied the role of the Ca2+ channel beta3 subunit, the major Ca2+ channel beta subunit in neurons, by using a gene-targeting strategy. The beta3 deficient (beta3-/-) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic beta3-/- neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence, analogous to the "reluctant" and "willing" states reported for N-type channels. The absence of the beta3 subunit was associated with a hyperpolarizing shift of the "reluctant" component of activation. Norepinephrine inhibited wt and beta3-/- neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca2+ channels. The reduction in the expression of N-type Ca2+ channels in the beta3-/- mice may be expected to impair Ca2+ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.     

Blood pressure and renin activity in essential hypertension under pindolol

20 essential hypertension patients with diastolic blood pressure of 100-140 mm Hg were treated with increasing doses (15-45 mg/day by mouth) of pindolol for 14 weeks after an initial placebo period of 5 weeks. Systolic and diastolic blood pressure decreased significantly with as little as 15 mg of pindolol (p less than 0.001). No further changes in systolic and diastolic blood pressure were observed when the doses of pindolol were increased. Plasma renin activity (PRA) determined by radioimmunoassay did not change under increasing doses of pindolol. The blood pressure changes did not correlate with initial PRA or with individual changes in PRA under increasing doses with individual changes in PRA under increasing doses of pindolol. These results do not afford evidence for a renin-dependent hypotensive effect of pindolol.     

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.