Comparison of maitotoxin with thromboxane A2 in rabbit platelet activation.

Maitotoxin (MTX), a Ca2+ channel-activating marine toxin, caused shape change followed by aggregation in rabbit platelets, like U46619, a thromboxane A2 analogue. Although both drugs failed to cause aggregation in the absence of external Ca2+, U46619, but not maitotoxin, elicited shape change in the absence of external Ca2+. The observations of platelets with a scanning electron microscope showed that both drugs caused contraction of platelets and extension of pseudopodia (shape change) followed by aggregation with a clot in the presence of Ca2+. It is noteworthy that long term exposure to MTX caused the lysis of platelets in the presence of Ca2+. While U46619 transiently increased the internal Ca2+ concentration ([Ca2+]i), maitotoxin slowly but irreversibly increased [Ca2+]i in an external Ca2(+)-dependent manner. MTX-induced phosphoinositide hydrolysis was totally dependent on the presence of external Ca2+, but U46619-induced phosphoinositide hydrolysis was still observed in the absence of external Ca2+. MTX-induced phosphoinositide hydrolysis was partly inhibited by SK&F96365, a voltage-independent Ca2+ channel antagonist, or by genistein, a tyrosine kinase inhibitor. MTX caused phosphorylation of tyrosine residues of several proteins, like U46619. Thus, MTX is similar to U46619 in functions of Ca2+ mobilization, phosphoinositide hydrolysis and tyrosine phosphorylation, but MTX-induced actions are strictly dependent on the presence of external Ca2+.     

Platelet aggregation caused by a partially purified jellyfish toxin from Carybdea rastonii

A partially purified toxin (pCrTX) was obtained from the tentacles of the jellyfish, Carybdea rastonii. When pCrTX (3 X 10(-8) - 3 X 10(-7) g/ml) was added to citrated platelet-rich plasma, aggregation was produced in a concentration-dependent manner. Scanning electron microscopic examination revealed that both pCrTX and collagen produced aggregates of platelets possessing many pseudopods. The concentration which produced 50% aggregation for pCrTX was 1.8 X 10(-7) g/ml, as compared to 2.3 X 10(-6) g/ml for collagen. The pCrTX-induced aggregation was only slightly inhibited by indomethacin and quinacrine in concentrations sufficient to inhibit arachidonic acid- and collagen-induced aggregation. pCrTX was less active in washed platelets suspended in Ca2+ free medium, whereas the pCrTX-induced aggregation was significantly augmented in the presence of Ca2+. The augmentation of aggregation by Ca2+ was only slightly attenuated by pretreatment with 100 microM verapamil. pCrTX significantly increased the concentration of cytoplasmic free Ca2+ ([Ca2+]i) and depolarized the platelet membrane in concentrations that produced aggregation. The increase in [Ca2+]i caused by pCrTX was little affected by verapamil. The depolarization by pCrTX was unchanged in the presence or absence of Ca2+, or by sodium or potassium transport inhibitors. The movement of 22Na+ into platelets was significantly increased by pCrTX. This increase in the movement of 22N+ into platelets was unaffected by tetrodotoxin. On the other hand, pCrTX-induced aggregation, depolarization and the increase in [Ca2+]i were all significantly attenuated in low Na+ medium. These results suggest that pCrTX causes a massive depolarization by increasing cation permeability indiscriminately and this generalized depolarization permits an inward movement of calcium down an electrochemical gradient which, in turn triggers platelet aggregation.     

The mechanism of action of maitotoxin in relation to Ca2+ movements in guinea-pig and rat cardiac muscles.

Maitotoxin (MTX), the most potent marine toxin known, produced a dose-dependent positive inotropic effect on guinea-pig isolated left atria and rat ventricle strips at concentrations of 0.1 ng to 4 ng ml-1. MTX (4 ng ml-1) also exhibited a positive chronotropic effect on guinea-pig right atria. The MTX-induced inotropic effect was almost abolished by Co2+ or verapamil, but was little affected by propranolol, reserpine or tetrodotoxin. The tissue Ca content of guinea-pig left atria was increased by MTX (2-30 ng ml-1) in a dose-dependent manner, and this increase was markedly inhibited by Co2+ or verapamil. Furthermore, on the rat isolated cardiac myocytes MTX (0.01-10 ng ml-1) caused an arrhythmogenic effect which was followed by their transformation into irreversibly rounded cells. The effects of MTX on the isolated cells were inhibited by verapamil or Ca2+-free solution. These results suggest that the excitatory effects of MTX on heart muscle are caused by a direct action on the cardiac muscle membrane mainly due to an increase in Ca2+ permeability.     

Apparent efficacy of kappa-opioid receptor ligands on serum prolactin levels in rhesus monkeys

ADP produces a series of responses in rabbit platelets such as shape changes, aggregation and intracellular Ca2+ mobilization. In human platelets, the P2X1 receptor mediates a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) upon stimulation with ADP. We investigated whether this phenomenon is also present in rabbit platelets. We found that the P2X1 receptor-mediated response was absent because there was (1) no elevation of [Ca2+]i in response to alpha,beta-methylene-ATP, a selective P2X1 receptor agonist, in fura-2-loaded platelets; (2) no change in the ADP-induced [Ca2+]i increase and platelet aggregation after P2X1 receptor desensitization with alpha,beta-methylene-ATP; (3) complete inhibition of the ADP-induced [Ca2+]i elevation by the P2Y1 receptor specific antagonist, A3'P5'PS, with a similar ID50 value both in the presence and absence of external Ca2+. These results indicate that ADP-induced [Ca2+]i elevation is mainly mediated by P2Y1 receptors in rabbit platelets. We conclude that the P2X1 receptor does not play a significant role in the ADP-induced platelet shape changes and aggregation.     

Maitotoxin effects are blocked by SK&F 96365, an inhibitor of receptor-mediated calcium entry.

The dinoflagellate toxin maitotoxin (MTX) elicited a sustained increase of [Ca2+]i in C6 glioma cells. This response was inhibited by SK&F 96365, a blocker of receptor-mediated calcium entry. In C6 cells, endothelin-1 elicited a rapid but transient increase in [Ca2+]i, followed by a smaller sustained increase. SK&F 96365 inhibited the sustained increase in [Ca2+]i. In both C6 glioma cells and RIN insulinoma cells, MTX elicited a marked influx of 45Ca2+. SK&F 96365 inhibited MTX-induced 45Ca2+ influx by 95% at 30 microM. The L-type calcium channel blocker nifedipine, even at 10 microM, inhibited MTX-induced calcium uptake by only 20% in RIN cells and by only 10% in C6 cells. MTX elicited calcium-dependent phosphoinositide breakdown in both C6 and RIN cells. In both cell lines, the MTX-induced phosphoinositide breakdown was inhibited by 90% by SK&F 96365 at 30 microM. Endothelin-1 and carbamylcholine elicited phosphoinositide breakdown in C6 cells and RIN cells, respectively. The stimulations were unaffected by the presence of SK&F 96365 up to 100 microM. In RIN insulinoma cells, MTX elicited calcium-dependent release of insulin. SK&F 96365 at 30 microM inhibited MTX-induced insulin release by 75%, whereas nifedipine, even at 30 microM, inhibited release by only 10%. The blockade of MTX-induced responses by SK&F 96365 indicates that MTX increases intracellular calcium by interacting directly with a calcium-entry system that is similar, in its sensitivity to SK&F 96365, to the calcium-entry system activated by receptors that elicit phosphoinositide breakdown. Activation of phospholipase C and hormone release by MTX also are blocked by SK&F 96365 and, thus, may be secondary to the activation of such a calcium-entry system.     

Caffeine-evoked, calcium-sensitive membrane currents in rabbit aortic endothelial cells.

1. Single cell photometry and whole-cell patch clamp recording were used to study caffeine-induced intracellular Ca2+ signals and membrane currents, respectively, in endothelial cells freshly dissociated from rabbit aorta. 2. Caffeine (5 mM) evoked a transient increase in [Ca2+]i in fura-2-loaded endothelial cells. Pretreatment of cells with 10 microM ryanodine did not alter resting [Ca2+]i but irreversibly inhibited the caffeine-induced rise in [Ca2+]i. The caffeine-induced increase in [Ca2+]i was not attenuated by the removal of extracellular Ca2+ and did not stimulate the rate of Mn2+ quench of fura-2 fluorescence. 3. Bath application of caffeine evoked a dose- and voltage-dependent outward current. The rate of onset and amplitude of the caffeine-evoked outward current increased with higher caffeine concentrations and membrane depolarization. The relationship between caffeine-evoked current amplitude and membrane potential was non linear, suggesting that the channels underlying the current are voltage-sensitive. 4. In the absence of extracellular Ca2+, the amplitude of the caffeine-evoked outward current was reduced by approximately 50% but the duration of the current was prolonged compared to that observed in the presence of external Ca2+. Ca(2+)-free external solutions produced an unexpected increase in both the frequency and amplitude of spontaneous transient outward currents (STOCs). 5. Inclusion of heparin (10 micrograms ml-1) in the patch pipette abolished the acetylcholine (ACh)-induced outward current but failed to inhibit either STOCs or the caffeine-evoked outward current in native endothelial cells. In the absence of extracellular Ca2+, heparin did not affect either STOCs or the caffeine-induced outward current.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual effects of mastoparan on intracellular free Ca2+ concentrations in human astrocytoma cells.

1. The effect of mastoparan, a wasp venom toxin, on intracellular free Ca2+ concentration ([Ca2+]i) was examined in human astrocytoma cells. Mastoparan inhibited [Ca2+]i induced by carbachol (100 microM) in a concentration-dependent manner in the absence of extracellular Ca2+, consistent with our previous results showing that mastoparan inhibits phosphoinositide hydrolysis in human astrocytoma cells. 2. In contrast, mastoparan itself increased [Ca2+]i and augmented carbachol-induced increase in the [Ca2+]i in the presence of extracellular Ca2+, suggesting that mastoparan elicited Ca2+ influx from the extracellular medium. The increase appeared to be maximum at extracellular Ca2+ concentrations of 0.1-0.2 mM. The higher concentrations of extracellular Ca2+ depressed the influx. 3. Pertussis toxin did not affect mastoparan-induced inhibition of [Ca2+]i in the absence of extracellular Ca2+, consistent with the previous results that pertussis toxin did not affect mastoparan-induced inhibition of phosphoinositide hydrolysis. 4. Pertussis toxin augmented mastoparan-induced increase in [Ca2+]i in the presence of extracellular Ca2+, suggesting that pertussis toxin substrate(s) seems to be inhibitory for Ca2+ influx induced by mastoparan. 5. Verapamil, nifedipine and diltiazem (each 10 microM), L-type Ca2+ antagonists, did not affect mastoparan-induced Ca2+ influx. However, verapamil (10 microM) slightly inhibited the increase in [Ca2+]i induced by carbachol in the presence of mastoparan. 6. The results obtained in the present study indicate that mastoparan has two opposite effects on [Ca2+]i in human astrocytoma cells and possibly has at least two sites of action.     

Activation of rabbit platelets by Ca2+ influx and thromboxane A2 release in an external Ca(2+)-dependent manner by zooxanthellatoxin-A, a novel polyol.

1. Zooxanthellatoxin-A (ZT-A), a novel polyhydroxylated long chain compound, isolated from a symbiotic marine alga Simbiodinium sp., caused aggregation in rabbit washed platelets in a concentration-dependent manner (1-4 microM), accompanied by an increase in cytosolic Ca2+ concentration ([Ca2+]i). 2. ZT-A did not cause platelet aggregation or increase [Ca2+]i in a Ca(2+)-free solution, and Cd2+ (0.1-1 mM), Co2+ (1-10 mM) and Mn2+ (1-10 mM) inhibited ZT-A-induced aggregation. SK&F96365 (1-100 microM), a receptor operated Ca2+ channel antagonist, and mefenamic acid (0.1-10 microM), a non-specific divalent cation channel antagonist, inhibited platelet aggregation and the increase in [Ca2+]i induced by ZT-A. 3. Indomethacin (0.1-10 microM), a cyclo-oxygenase inhibitor, and SQ-29548 (0.1-10 microM), a thromboxane A2 (TXA2) receptor antagonist, inhibited platelet aggregation and the increase in [Ca2+]i induced by ZT-A. 4. Methysergide (0.01-1 microM), a 5-HT2 receptor antagonist, inhibited ZT-A-induced platelet aggregation but did not affect the increase in [Ca2+]i induced by ZT-A. 5. Tetrodotoxin (1 microM), a Na+ channel blocker and chlorpheniramine (1 microM), a H1-histamine receptor antagonist, neither affected ZT-A-induced platelet aggregation nor the increase in [Ca2+]i induced by ZT-A. 6. Genistein (1-100 microM), a protein tyrosine kinase inhibitor, and staurosporine (0.01-1 microM), a protein kinase C inhibitor, also inhibited ZT-A-induced platelet aggregation. 7. The present results suggest that ZT-A elicits Ca(2+)-influx from platelet plasma membranes. The resulting increase in [Ca2+]i subsequently stimulates the secondary release of TXA2 from platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca-dependent arrhythmogenic effects of maitotoxin, the most potent marine toxin known, on isolated rat cardiac muscle cells

Maitotoxin (MTX) (0.1-10 ng/ml) caused an increase in the degree and the rate of contraction and subsequent arrhythmogenic actions in rat myocardial cells. These effects of MTX were inhibited by verapamil and were abolished by Ca2+-free medium. The MTX-induced effects on cardiac myocytes were enhanced by increasing the Ca2+ concentration. These results suggest that MTX elevates the permeability of cardiac muscle membrane to Ca2+ to increase the intracellular Ca2+ concentration, and thus induces excitatory effects.     

Activation of human blood platelets by arginine-vasopressin. Role of bivalent cations

Arginine-vasopressin caused platelet activation, i.e., a shape change reaction and a rise in intracellular free Ca2+ ([Ca2+]i) only in the presence of certain bivalent cations. The EC50 of arginine-vasopressin (concentration causing half-maximal shape change) decreased with rising concentrations of Mn2+, Mg2+, or Ca2+ in the medium, but was at least an order higher with Ca2+ than with Mn2+ or Mg2+. The EC50 of the active bivalent cations (concentrations enabling 100 nM arginine-vasopressin to exert half-maximal shape change and rise in [Ca2+]i) varied with the individual cations, being by far the highest for Ca2+. The KD of [3H]arginine-vasopressin binding to platelet membranes and intact platelets markedly decreased when extracellular Mg2+ or Mn2+ were present, and the KD values were inversely related to the concentration of the cations. Ca2+ also lowered the KD values; however, the effect was less marked than that of Mg2+ or Mn2+ and, in physiological conditions, significant only in intact platelets. Vasopressin-1 antagonists counteracted arginine-vasopressin binding and the shape change reaction and [Ca2+]i rise induced by arginine-vasopressin. In the presence of Mn2+ in the medium, administration of arginine-vasopressin led to quenching of the intracellular fluorescence of 2-methyl-6-methoxy-8-nitroquinoline-loaded platelets, possibly due to influx of Mn2+. In conclusion, the dependency of the arginine-vasopressin-induced platelet activation on bivalent cations is at least partly due to an enhancement by these cations of the affinity of the vasopressin-1 receptor for arginine-vasopressin. Thereby, under physiological conditions, Mg2+ seems to be of primary importance. Other mechanisms may be involved, too, e.g., an enhancement by arginine-vasopressin of the influx of bivalent cations into the platelets.     

Influence of authentic nitric oxide on basal cytosolic [Ca2+] and Ca2+ release from internal stores in human platelets.

1. Nitric oxide (NO) donors inhibit platelet function and Ca2+ mobilization evoked by different agonists. This led us to investigate the direct effects of authentic NO on basal cytosolic Ca2+ concentration ([Ca2+]i) and on Ca2+ mobilization induced by thrombin or by two inhibitors of intracellular Ca(2+)-ATPases, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone (t-BuBHQ). 2. Cytosolic Ca2+ concentration was evaluated with Fura-2, in the absence of Ca2+ influx. Addition of 5 microM NO increased by 48% the basal cytosolic [Ca2+] of resting human platelets whereas a lower concentration (0.1 microM) did not induce significant modifications. This NO-induced Ca2+ increase was inversely correlated with the basal level of cytosolic [Ca2+]. 3. NO pretreatment for 30 to 120 s decreased by 42 to 57% the transient [Ca2+]i peak evoked by 0.10 u ml-1 thrombin and strongly attenuated the initial rate of [Ca2+]i rise induced by 1 microM thapsigargin or by 20 microM t-BuBHQ. The two components of the thapsigargin response, the Ca2+ release due to inhibition of Ca2+ pumps and the thromboxane A2-dependent self-amplification mechanism, were inhibited by NO. The observation that a subsequent stimulation was still capable of eliciting Ca2+ release suggests the presence of NO-insensitive Ca2+ stores. 4. These findings indicate that nitric oxide can modulate basal cytosolic [Ca2+] in unstimulated human platelets and decrease the Ca2+ mobilization from NO-sensitive internal stores evoked by stimulation of receptors or by Ca(2+)-ATPase inhibitors. This underlines the important role of nitric oxide in the modulation of platelet Ca2+ handling.     

In vitro inhibition by endothelins of thrombin-induced aggregation and Ca2+ mobilization in human platelets.

1. The in vitro effects of endothelins (ET-1 and ET-3) on human platelets were investigated by measurement of the aggregatory responses of washed platelets to thrombin and by the determination of cytosolic pH (pHi) and free Ca2+ concentration ([Ca2+]i) determined with the fluorescent indicators, BCECF and Fura-2. 2. ET-1 and ET-3 at concentrations ranging from 10(-10) to 5 x 10(-7) M, did not promote platelet aggregation but inhibited in a dose-dependent manner the aggregation induced by 0.05 u ml-1 thrombin (P less than 0.002 and less than 0.001, respectively) with maximal effects reached at 10(-8) M (17 +/- 3 and 15 +/- 2%, n = 11, P = 0.002 for each). 3. Even at 5 x 10(-7) M, ET-1 and ET-3 did not cause a measurable change in basal [Ca2+]i and pHi. When tested in combination with thrombin, 5 x 10(-7) M ET-1 and ET-3 decreased the transient peak of [Ca2+]i by 17 +/- 7 and 28 +/- 7% (n = 7 and 11, P = 0.03 and P = 0.002). No effect on pHi variations was detected. In the virtual absence of external Ca2+, 5 x 10(-7) M ET-3 inhibited the peak of [Ca2+]i by 18 +/- 6% (n = 6, P = 0.02). 4. The anti-aggregating agents, prostacyclin (PGI2, 10(-8)-10(-7) M) and nitroprusside (NP, 10 ng-50 micrograms l-1) also induced a dose-dependent inhibition of the thrombin-induced [Ca2+]i peak (P = 0.001 for each).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tamoxifen-induced Ca2+ mobilization in bladder female transitional carcinoma cells

This study examined the effect of tamoxifen, an anti-breast cancer drug, on Ca2+ handling in bladder female transitional cancer cells. Changes in cytosolic free Ca2+ levels were recorded by using the Ca2+-sensitive dye fura-2. In a dose-dependent manner, tamoxifen induced intracellular free Ca2+ concentrations ([Ca2+]i) increases between 5 and 20 microM with an EC50 of 10 microM. External Ca2+ removal reduced the response by 60+/-6%. Addition of 3 mM Ca2+ caused a [Ca2+]i increase after pretreatment with 10 microM tamoxifen in Ca2+-free medium. In Ca2+-free medium, pretreatment with 10 microM tamoxifen abolished the [Ca2+]i increase induced by 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 1 microM thapsigargin prevented tamoxifen from releasing more Ca2+. Inhibition of phospholipase C-dependent inositol 1,4,5-tris-phosphate formation with 2 microM U73122 did not alter 10 microM tamoxifen-induced Ca2+ release. The [Ca2+]i increase induced by 5 microM tamoxifen was not altered by 10 microM La3+, nifedipine, verapamil, and diltiazem. Collectively, it was found that tamoxifen increased [Ca2+]i in bladder cancer cells by releasing Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity, and by inducing Ca2+ entry from external medium.     

槲皮素单硫酸酯钠盐对凝血酶诱导的猪血小板聚集的抑制作用

研究槲皮素单硫酸酯钠盐对凝血酶诱导的猪血小板聚集的抑制作用。方法：用比浊法测定血小板聚集,Ｆｕｒａ ２－ＡＭ荧光法检测胞浆游离钙浓度（「Ｃａ＾２＋）ｉ」。用组蛋白ⅢＳ,「γ＾３２Ｐ」ＡＴＰ与蛋白激酶Ｃ酶液一起温育的方法测定ＰＫＣ的活性。用ＳＤＳ－ＰＡＧＥ分离骨架蛋白。     

Mechanisms of AM404-induced [Ca(2+)](i) rise and death in human osteosarcoma cells

The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca2+ levels ([Ca2+]i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations > or = 5 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 60 microM. The Ca2+ signal was reduced partly by removing extracellular Ca2+. AM404 induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was sensitive to La3+, Ni2+, nifedipine and verapamil. In Ca2+-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), AM404-induced [Ca2+]i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca2+]i rise. At concentrations between 10 and 200 microM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 microM AM404 was partly reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via L-type Ca2+ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis.     

血小板释放的ADP通过稳定细胞内钙浓度而稳定PAF诱导的兔血小板 …

AIM: To examine whether platelet-released adenosine diphosphate (ADP) would contribute to the stabilization of rabbit platelet aggregation induced by platelet activating factor (PAF). METHODS: Rabbit platelet aggregation induced by PAF was measured turbimetrically. ADP release from rabbit platelets stimulated by PAF was determined by HPLC. Intracellular Ca2+ was measured using Ca(2+)-sensitive fluorescent indicator Fura 2-AM. RESULTS: PAF > or = 1 nmol.L-1 induced full platelet aggregation, which did not deaggregate over 5 min after aggregation reached peak. Platelet aggregation was deaggregated in a concentration-dependent manner by subsequent addition of ADP scavenger ATP-diphosphohydrolase (apyrase) at 5-100 mg.L-1. PAF 3 nmol.L-1 stimulated release of ADP (29% vs 6% of control), and elicited a rapid rise in intracellular calcium ([Ca2+]i) which peaked at approximately 15 s. Then the [Ca2+]i gradually decayed from 585 +/- 80 nmol.L-1 within 100 s to a low level (364 +/- 82 nmol.L-1). Apyrase 100 mg.L-1, added 2 min after PAF, reduced [Ca2+]i to a lower level (171 +/- 29 nmol.L-1). CONCLUSION: Platelet-released ADP stabilizes PAF-induced rabbit platelet aggregation by stabilizing [Ca2+]i at elevated level.     

Characterization of fMet-Leu-Phe receptor-mediated Ca2+ influx across the plasma membrane of human neutrophils

N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe) stimulation of human neutrophils leads to a rapid increase of the cytosolic free Ca2+ concentration, [Ca2+]i, which is significantly reduced by removal of extracellular calcium. In the present study we show that fMet-Leu-Phe-induced [Ca2+]i increases are, in part, mediated by an increase of the plasma membrane permeability to Ca2+. This conclusion is based on the following evidence. In the presence of extracellular calcium, addition of La3+ reduced the fMet-Leu-Phe-induced [Ca2+]i increase to approximately the same level as that observed in the absence of extracellular calcium. A net increase of the plasma membrane permeability for Mn2+ could be observed after fMet-Leu-Phe stimulation, as revealed by intracellular quenching of the quin2 signal. The influx of Mn2+, like that of Ca2+, was inhibited by La3+ and was more pronounced in the absence of extracellular Ca2+, suggesting competition for the same pathway. Temporal dissociation of intracellular Ca2+ release from stores and Ca2+ influx from the medium could be demonstrated by readdition of calcium to cells stimulated in the absence of this cation. This second [Ca2+]i increase could be abolished either by giving the specific chemotactic peptide receptor antagonist, BOC-Met-Leu-Phe, or Co2+. We could also show that the fMet-Leu-Phe-dependent Ca2+ influx was not due to the activation of voltage-dependent calcium channels since depolarization either by K+ or gramicidin D did not affect the resting [Ca2+]i, nor did it affect a subsequent [Ca2+]i increase induced by fMet-Leu-Phe. Furthermore, nifedipine and verapamil, at concentrations known to block classical voltage-dependent calcium channels, had no significant effects on the Ca2+ influx induced by fMet-Leu-Phe. We suggest that fMet-Leu-Phe promotes influx of Ca2+ ions across the plasma membrane of human neutrophils by opening of receptor-dependent calcium channels.     

Inhibition by Zn2+ of uridine 5'-triphosphate-induced Ca(2+)-influx but not Ca(2+)-mobilization in rat phaeochromocytoma cells.

1. Uridine 5''-triphosphate (UTP)-evoked increase in intracellular Ca2+ concentration ([Ca]i) and release of dopamine were investigated in rat phaeochromocytoma PC12 cells. UTP (1-100 microM) evoked an increase in [Ca]i in a concentration-dependent manner. This response was decreased to about 30% by extracellular Ca(2+)-depletion, but not abolished. This [Ca]i rise was mimicked by 100 microM ATP but not by 100 microM 2-methyl-thio-ATP or alpha,beta-methylene-ATP in the absence of external Ca2+, suggesting that the response was mediated by P2U purinoceptors, a subclass of P2-purinoceptors. 2. The UTP-evoked [Ca]i rise consisted of two components; a transient and a sustained one. When external Ca2+ was removed, the sustained component was abolished while the transient component was decreased by about 70% but did not disappear. These results suggest that UTP induces Ca(2+)-mobilization and, subsequently, Ca(2+)-influx. 3. The UTP-evoked increase in [Ca]i was not affected by Cd2+ (100 and 300 microM) or nicardipine (30 microM), inhibitors of voltage-gated calcium channels, but was significantly inhibited by Zn2+ (10-300 microM) in the presence of external Ca2+. Zn2+, however, did not affect the Ca2+ response to UTP in the absence of external Ca2+. 4. UTP (30 microM-1 mM) evoked the release of dopamine from the cells in a concentration-dependent manner. This dopamine release was abolished by Ca(2+)-depletion or Zn2+ but not by Cd2+ or nicardipine. 5. Taken together, the data demonstrate that UTP stimulates P2U-purinoceptors and induces a rise in [Ca]i both by Ca(2+)-mobilization and Ca(2+)-influx in PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of maitotoxin-induced elevation of the cytosolic free calcium level in rat cerebrocortical synaptosomes

The present study was conducted to elucidate the mechanism of the maitotoxin (MTX)-induced increase in intrasynaptosomal free calcium level ([Ca2+]i). The MTX (1 ng/ml)-induced increase in [Ca2+]i was partially inhibited by the omission of extracellular Ca2+ (Ca2+e) or the addition of verapamil, but not by adding nifedipine, omega-agatoxin IVA, omega-conotoxin GVIA and omega-conotoxin MVIIC. An increase in [Ca2+]i in the absence of Ca2+e was sensitive to procaine, TMB-8, genistein and verapamil, but not to ryanodine and U-73122. These results may suggest that MTX increases [Ca2+]i by stimulating Ca2+ entry through voltage-independent nonselective cation channels and Ca2+ release from stores through a phospholipase C-gamma1-mediated pathway in rat cerebrocortical synaptosomes.     

Thromboxane A2-mediated shape change: independent of Gq-phospholipase C--Ca2+ pathway in rabbit platelets.

1. Thromboxane A2 (TXA2) receptor-mediated signal transduction was investigated in washed rabbit platelets to clarify the mechanisms of induction of shape change and aggregation. 2. The TXA2 agonist, U46619 (1 nM to 10 microM) caused shape change and aggregation in a concentration-dependent manner. A forty-times higher concentration of U46619 was needed for aggregation (EC50 of 0.58 microM) than shape change (EC50 of 0.013 microM). The aggregation occurred only when external 1 mM Ca2+ was present, but the shape change could occur in the absence of Ca2+. 3. SQ29548 at 30 nM and GR32191B at 0.3 microM (TXA2 receptor antagonists) competitively inhibited U46619-induced shape change and aggregation with similar potency, showing that both aggregation and shape change induced by U46619 were TXA2 receptor-mediated events. However, ONO NT-126 at 1 nM, another TXA2 receptor antagonist, inhibited U46619-induced aggregation much more potently than the shape change, suggesting the possible existence of TXA2 receptor subtypes. 4. ONO NT-126 (2 nM to 3 microM) by itself caused a shape change without aggregation in a concentration-dependent manner, independent of external Ca2+. Therefore, ONO NT-126 is a partial agonist at the TXA2 receptor in rabbit platelets. 5. U46619 (10 nM to 10 microM) increased internal Ca2+ concentration ([Ca2+]i) and activated phosphoinositide (PI) hydrolysis in a concentration-dependent manner with a similar concentration-dependency. 6. U46619 (3 nM to 10 microM) also activated GTPase concentration-dependently in the membranes derived from platelets. U46619-induced activation of GTPase was partly inhibited by treatment of membranes with QL, an antibody against Gq/11. 7. The EC50 values of U46619 in Ca2+ mobilization (0.15 microM), PI hydrolysis (0.20 microM) and increase in GTPase activity (0.12 microM) were similar, but different from the EC50 value in shape change (0.013 microM), suggesting that activation of TXA2 receptors might cause shape change via an unknown mechanism. 8. U46619-induced shape change was unaffected by W-7 (30 microM), a calmodulin antagonist or ML-7 (30 microM), a myosin light-chain kinase inhibitor, indicating that an increase in [Ca2+]i might not be involved in the shape change. In fact, U46619 (10 nM) could cause shape change without affecting [Ca2+]i level, determined by simultaneous recordings. 9. [3H]-SQ29548 and [3H]-U46619 bound to platelets at a single site with a Kd value of 14.88 nM and Bmax of 106.1 fmol/10(8) platelets and a Kd value of 129.8 nM and Bmax of 170.4 fmol/10(8) platelets, respectively. The inhibitory constant Ki value for U46619 as an inhibitor of 3H-ligand binding was similar to the EC50 value of U46619 in GTPase activity, phosphoinositide hydrolysis and Ca2+ mobilization, but significantly different (P < 0.001 by Student''s t test) from the effect on shape change. 10. Neither U46619 nor ONO NT-126 affected the adenosine 3'',5''-cyclic monophosphate (cyclic AMP) level in the presence or absence of external Ca2+ and/or isobutyl methylxanthine. 11. The results indicate that TXA2 receptor stimulation causes phospholipase C activation and increase in [Ca2+]i via a G protein of the Gq/11 family leading to aggregation in the presence of external Ca2+, and that shape change induced by TXA2 receptor stimulation might occur without involvement of the Gq-phospholipase C-Ca2+ pathway.