Cytoplasmic free calcium concentration in porcine platelets: Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation

Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1·10^?7 M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thromin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.     

Erythropoietin increases cytosolic free calcium concentration and thrombin induced changes in cytosolic free calcium in platelets from spontaneously hypertensive rats

Using fura-2 cytosolic free calcium concentrations were measured in intact washed platelets from 9 spontaneously hypertensive rats (SHR) and from 9 age-matched normotensive Wistar-Kyoto rats (WKY). In resting platelets cytosolic free calcium concentration was significantly higher in SHR than in WKY (171.8 +/- 64.4 nM vs 93.1 +/- 59.0 nM, p less than 0.05). After preincubation with erythropoietin cytosolic free calcium concentration was significantly higher in SHR than in WKY (197.5 +/- 83.2 vs 93.0 +/- 60.1, p less than 0.01). Using platelets from SHR erythropoietin increased mean resting cytosolic free calcium concentration by 14.9% (p less than 0.05) and mean thrombin induced changes of cytosolic free calcium by 58.3% (p less than 0.01). In contrast, erythropoietin caused no significant increase in the resting calcium concentration or in thrombin induced changes of cytosolic free calcium in platelets from WKY. It is concluded that erythropoietin is involved in the pathogenesis of hypertension by elevating cytosolic free calcium concentration.     

Neurons respond to hyposmotic conditions by an increase in intracellular free calcium

The effect of hyposmotic conditions on the concentration of intracellular free calcium ([Ca²⁺]_i) was studied in cultured cerebellar granule cells and cerebral cortical neurons after loading of the cells with the fluorescent Ca²⁺ chelator Fluo-3. It was found that in both types of neurons exposure to media with a decrease in osmolarity of 20 to 50% of the osmolarity in the isosmotic medium (320 mOsm) led to a dose dependent increase in [Ca²⁺]_i with a time course showing the highest value at the earliest measured time point, i.e. 40 s after exposure to the hyposmotic media and a subsequent decline towards the basal level during the following 320 s. The response in the cortical neurons was larger than in the granule cells but both types of neurons exhibited a similar increase in [Ca²⁺]_i after expoxure to 50 mM K⁺ which was of the same magnitude as the increase in [Ca²⁺]_i observed in the cortical neurons exposed for 40 s to a medium with a 50% reduction in osmolarity. In both types of neurons the blocker of voltage gated Ca²⁺ channels verapamil had no effect on the hyposmolarity induced increase in [Ca²⁺]_i. On the contrary, this increase in [Ca²⁺]_i was dependent upon external calcium and could be inhibited partly or completely by the inorganic blockers of Ca²⁺ channels Mg²⁺ and La³⁺. Dantrolene which prevents release of Ca²⁺ from internal stores had no effect. The results show that exposure of neurons to hyposmotic conditions leading to swelling results in a large increase in free intracellular Ca²⁺ which represents an influx of Ca²⁺ rather than a release of Ca²⁺ from internal, dantrolene sensitive stores.     

Low-density lipoproteins increase intracellular calcium in aequorin-loaded platelets

Low-density lipoproteins activate isolated human platelets. The mechanism of this activation is unknown, but may involve increased phosphoinositide turnover. We have examined the effect of low-density lipoproteins on intracellular calcium concentrations in platelets loaded with the photoprotein aequorin. The lipoproteins induced concentration-dependent increases in intracellular calcium, associated with shape change and aggregation. These responses could be partially inhibited by the removal of extracellular calcium and by pre-incubation with acetylsalicylic acid. They were also antagonised by agents which increase cellular concentrations of cyclic adenosine and guanosine monophosphates. It is not clear whether the platelet-lipoprotein interaction involves a 'classical' lipoprotein receptor.     

整合素介导小鼠卵内钙离子增加

为了研究整合素是否作为跨膜信号传递受体介导小鼠卵[Ca^2 ]i的变化并探讨其机制。本实验采用甘-精-甘-天冬-丝-脯(GLY-ARG-GLY-ASP-SER-PRO,RGD肽)、纤连蛋A(fibronectin,Fn)及抗整合素α6、β1的单克隆抗体作用于负载了钙探针Fluo-3／AM的去透明带小鼠卵,用激光共聚焦显微镜检测小鼠卵的荧光强度以反映卵[Ca^2 ];用无钙液替代有钙液、或用酪氨酸激酶抑制剂或蛋白激酶C的抑制剂预先作用于卵,然后再观察RGD肽所致卵[Ca^2 ]i的变化。结果显示整合素配体RGD肽或Fn作用于去透明带小鼠卵可引起卵[Ca^2 ]i增加,增加的程度与精子作用相似;去除培养液中的Ca^2 后,再用RGD肽、Fn作用仍可引起卵[Ca^2 ]i增加：用功能性的抗小鼠整合素α6、β1的单克隆抗体也可引起不同程度的卵[Ca^2]i增加,尤其以抗小鼠整合素α6、β1单克隆抗体的作用明显;用酪氨酸激酶抑制剂预先作用于鼠卵,RGD肽或精子作用都不再引起卵[Ca^2 ]i增加;蛋白激酶C抑制剂预先作用鼠卵,RGD肽及Fn也不再引起卵[Ca^2 ]i增加。实验证明。小鼠卵膜整合素与其配体结合可使卵内贮存钙离子释放,引起卵[Ca^2 ]i增加这一卵激活的早期事件;整合素介导小鼠卵激活需要酪氨酸激酶信号转导途径的参与;蛋白激酶C也参与了整合素介导的卵激活。     

Regulation of cytosolic free calcium concentration by intrasynaptic mitochondria.

By the use of digitonin permeabilized presynaptic nerve terminals (synaptosomes), we have found that intrasynaptic mitochondria, when studied "in situ," i.e., surrounded by their cytosolic environment, are able to buffer calcium in a range of calcium concentrations close to those usually present in the cytosol of resting synaptosomes. Adenine nucleotides and polyamines, which are usually lost during isolation of mitochondria, greatly improve the calcium-sequestering activity of mitochondria in permeabilized synaptosomes. The hypothesis that the mitochondria contributes to calcium homeostasis at low resting cytosolic free calcium concentration ([Ca2+]i) in synaptosomes has been tested; it has been found that in fact this is the case. Intrasynaptic mitochondria actively accumulates calcium at [Ca2+]i around 10(-7) M, and this activity is necessary for the regulation of [Ca2+]i. When compared with other membrane-limited calcium pools, it was found that depending on external concentration the calcium pool mobilized from mitochondria is similar or even greater than the IP3- or caffeine-sensitive calcium pools. In summary, the results presented argue in favor of a more prominent role of mitochondria in regulating [Ca2+]i in presynaptic nerve terminals, a role that should be reconsidered for other cellular types in light of the present evidence.     

Response of a human megakaryocytic cell line to thrombin: increase in intracellular free calcium and mitogen release.

The CHRF-288-11 cell line has been previously shown to exhibit properties consistent with a megakaryocytic origin. The response of these cells to thrombin has now been investigated. Thrombin treatment of CHRF-288-11 cells results in both an increase in intracellular free calcium levels and secretion of mitogenic activity and beta-thromboglobulin. Cell viability is not affected. The mitogenic activity released from the cells is due primarily to the presence of basic fibroblast growth factor. Immunohistochemical data indicate a packaging of basic fibroblast growth factor into granular structures. Trypsin and phorbol 12-myristate 13-acetate also initiate release of mitogenic activity from this cell line, whereas under non-stirred conditions collagen and ADP do not. Through measurements of intracellular calcium levels it was determined that thrombin pretreatment of cells ablates a further response to thrombin, but does not block an increase in intracellular calcium levels due to trypsin. This suggests that these two agonists may act through different mechanisms. The thrombin-induced release reaction is inhibited almost completely by the reagents hirudin and dipyridamole, and only partially by indomethacin. These data indicate that the CHRF-288-11 cell line should provide an excellent model system in which to study the packaging of factors into granules which undergo regulated release.     

Contribution of intracellular calcium stores to an increase in cytosolic calcium concentration induced by Mannheimia haemolytica leukotoxin

The contribution of intracellular calcium stores to Mannheimia haemolytica leukotoxin (LKT)-induced increase in cytosolic calcium concentration was studied by pharmacologically inhibiting transport of calcium across the plasma and endoplasmic reticulum membranes of bovine neutrophils exposed to LKT. Active intracellular storage of calcium by sarcoplasmic/endoplasmic reticulum calcium ATPase, influx of extracellular calcium across the plasma membrane, and release of stored calcium via inositol triphosphate receptors and ryanodine-sensitive calcium channels were inhibited using thapsigargin, lanthanum chloride, xestospongin C, and magnesium chloride, respectively. Pre-incubation with thapsigargin attenuated the increase in cytosolic calcium concentration produced by LKT, thus confirming the involvement of intracellular calcium stores. Inhibitory effects of lanthanum chloride, xestospongin C, and magnesium chloride indicated that the increase in cytosolic calcium concentration induced by LKT resulted from both influx of calcium across the plasma membrane and release of calcium from intracellular stores.     

Irregular spiking in free calcium concentration in single, human platelets. Regulation by modulation of the inositol trisphosphate receptors.

Fluorescence ratio imaging indicates that immobilized, aspirin-treated platelets, loaded with Fura-2, respond to inositol 1,4,5-trisphosphate- (InsP3)-generating agonists such as thrombin by high-frequency, irregular rises in cytosolic [Ca2+]i with spikes that vary in peak level and peak-to-peak interval. This differs from the regular [Ca2+]i oscillations observed in other, larger cells. We found that the thiol-reactive compounds thimerosal (10 microm) and U73122 (10 microm) evoked similar irregular Ca2+ responses in platelets, but in this case in the absence of InsP3 generation. Thrombin-induced spiking was acutely abolished by inhibiting phospholipase C or elevating intracellular cAMP levels, while spiking with sulfhydryl reagents was only partially blocked by cAMP elevation. Confocal laser scanning microscopy using fluo-3-loaded platelets indicated that, with all agonists or conditions, the irregular spikes were almost instantaneously raised in various regions within a single platelet. When using saponin-permeabilized platelets, we found that InsP3-induced Ca2+ release from stores was stimulated by modest Ca2+ concentrations, pointing to a mechanism of InsP3-dependent Ca2+-induced Ca2+ release (CICR). This process was completely inhibitable by heparin. The Ca2+ release by InsP3, but not the CICR sensor, was negatively regulated by cAMP elevation. Thimerosal treatment did not release Ca2+ from intracellular stores, but markedly potentiated the stimulatory effect of InsP3. In contrast, U73122 caused a heparin/cAMP-insensitive Ca2+ leak from stores that differed from those used by InsP3. Taken together, these results demonstrate that InsP3 receptor channels play a crucial role in the irregular, spiking Ca2+ signal of intact platelets, even when induced by agents such as thimerosal or U73122 which do not stimulate InsP3 formation. The irregular Ca2+ release events appear to be subjected to extensive regulation by: (a) InsP3 level, (b) the potentiating effect of elevated Ca2+ on InsP3 action via CICR, (c) InsP3 channel sensitization by sulfhydryl (thimerosal) modification, (d) InsP3 channel-independent Ca2+ leak with U73122, and (e) down-regulation via cAMP elevation. The observation that individual Ca2+ peaks were generated in various parts of a platelet at similar intervals and amplitudes points to effective cooperation of the various stores in the Ca2+-release process.     

Limits to the early increase in free cytoplasmic calcium concentration during the mitogenic stimulation of lymphocytes.

Three aspects of the calcium hypothesis we have proposed previously [Metcalfe, Pozzan, Smith & Hesketh (1980) Biochem. Soc. Symp. 45, 1-26] for the control of mitogenic stimulation of lymphocytes are examined in studies on the mitogenic action of the Ca2+ ionophore A23187 and its effect on cap formation. (1) Pig lymphocytes that were mitogenically stimulated by continuous incubation with 3H-labelled A23187 for 48 h contained between 3 and 15 amol of ionophore per cell. Lymphocytes exposed to 3H-labelled A23187 for 2h before washing the cells and resuspending them in ionophore-free medium were only stimulated mitogenically at 48h if the residual ionophore associated with the cells after washing was in the concentration range 3-15 amol per cell. When the cells were washed repeatedly after 2h incubation with ionophore to reduce the cell-associated ionophore below the critical concentration range, no mitogenic stimulation occurred as a result of short-term exposure to any ionophore concentration. Re-addition of ionophore to within the indicated range of cell-associated concentrations restored mitogenic stimulation at 48h. We conclude that large, short-term Ca2+ fluxes into the cells induced by the ionophore cannot generate a mitogenic signal that commits the cells to enter the cell cycle. (2) Further experiments with the ionophore showed that detectable mitogenic stimulation at 48h required a minimum of 3h exposure to optimal ionophore concentrations, and that maximal stimulation required at least 20h exposure. This is consistent with the view that a prolonged increase in the free cytoplasmic calcium concentration is required to stimulate the maximum proportion of the cells into the cell cycle. (3) Mouse splenic lymphocytes treated for short periods with very high ionophore concentrations (30 microM) in the presence of various external Ca2+ concentrations showed significant inhibition of cap formation of surface immunoglobulin receptors in the range 1-10 microM-Ca2+ in normal or depolarizing medium. We conclude that mitogens at optimal concentrations for the stimulation of lymphocytes do not cause any early increase in the free cytoplasmic Ca2+ concentration above 10 microM.     

Gonadotropin-releasing hormone stimulation of pituitary gonadotrope cells produces an increase in intracellular calcium

Gonadotropin-releasing hormone (GnRH) stimulates pituitary gonadotrope cells to release luteinizing hormone (LH). Previous studies have indicated a role for Ca+2 in this process; however, the present study provides the first measurements of an increased intracellular Ca+2 concentration. Pituitary cell cultures enriched for gonadotropes were loaded with quin 2, a fluorescent Ca+2-sensitive molecule. Subsequent addition of GnRH to these cells produced a rapid (within 10 sec) increase in fluorescence (indicating an increase in intracellular free Ca+2). In contrast, two GnRH analogs, des1 GnRH (a very low-affinity binder to the GnRH receptor) and Ac[D-pCl-Phe1,2] DTrp3 DLys6 DAla10-GnRH (a pure GnRH antagonist) produced no such Ca+2 change, thus showing a correlation between increased intracellular Ca+2 and LH release. A functional relationship between increased Ca+2 and LH release was suggested by experiments in which LH release was inhibited from cells loaded with high levels of intracellular quin 2 (in order to chelate intracellular Ca+2). Since this inhibition was completely reversed by addition of the Ca+2 ionophore A23187, quin 2 was not toxic at the concentrations used and apparently inhibited LH release by buffering intracellular Ca+2. The results presented here are consistent with the hypothesis that GnRH-stimulated LH release is mediated by increased intracellular Ca+2 and support the notion that the rate-limiting step in GnRH-stimulated LH release is distal to Ca+2 mobilization.     

Regulation of intracellular calcium concentration by nanosecond pulsed electric fields

Changes in [Ca²⁺]_i response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca²⁺]_i was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca²⁺]_i response was due to the release of Ca²⁺ from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca²⁺]_i was due to both internal release and to influx across the plasma membrane. Spontaneous changes in [Ca²⁺]_i exhibited a more gradual increase over several seconds. The initial, pulse-induced [Ca²⁺]_i response initiates at the poles of the cell with respect to electrode placement and co-localizes with the endoplasmic reticulum. The results suggest that nsPEFs target both the plasma membrane and subcellular membranes and that one of the mechanisms for Ca²⁺ release may be due to nanopore formation in the endoplasmic reticulum.     

Effects of dihydropyridines and inorganic calcium blockers on aggregation and on intracellular free calcium in platelets.

[Ca2+]i increase is necessary in physiological platelet activity, particularly aggregation and release. The increase of [Ca2+]i observed during platelet activation depends in part on Ca2+ influx from the extracellular medium. The participation of voltage-operated Ca2+ channels as a pathway for Ca2+ entry is controversial. In the present study we have attempted to reinvestigate this problem by measuring aggregation and [Ca2+]i changes in platelets activated by ADP or thrombin and incubated with organic or inorganic blockers of calcium channels. The main findings of the present paper can be summarized as follows: (i) Ni2+, Co2+ and Mn2+, well known inorganic blockers of Ca2+ channels, inhibited platelet aggregation induced by ADP or thrombin in a dose-dependent manner, Ni2+ being the most effective agent. (ii) Thrombin induced a rise in free [Ca2+]i in platelets incubated both in 1 mmol/l Ca(2+)-containing medium and in nominally Ca(2+)-free medium; the rise of free [Ca2+]i was in the first case up to 370 +/- 31 nmol/l and in the second case up to 242 +/- 26 nmol/l, indicating that this observed difference was due to Ca2+ entry from the extracellular medium. Co2+ and Ni2+ abolished that difference by inhibiting Ca2+ influx. (iii) Nisoldipine, nitrendipine and nimodipine (10-50 nmol/l) inhibited in a dose-dependent manner platelet aggregation induced by either ADP or thrombin in platelets incubated in normal-Ca2+ normal-K+ medium, also, aggregation was inhibited to a similar extent in platelets incubated in normal-Ca2+ high-K+ medium. (iv) Nisoldipine--the most effective dihydropyridine to inhibit platelet aggregation--also inhibited Ca2+ influx in platelets incubated in normal-Ca2+ medium, either in normal-K+ or high-K+ media. Our data support the existence of voltage-operated, dihydropyridine-sensitive calcium channels (L-type) and a physiological role for them in platelet function.     

Direct measurement of the increase in intracellular free calcium ion concentration in response to the action of complement.

1. The effect of rabbit anti-(pigeon erythrocyte) antibodies plus human complement on the concentration of intracellular free Ca2+ in sealed pigeon erythrocyte ''ghosts'' was investigated with the photoprotein obelin. 2. The addition of human serum, as a source of complement, to ''ghosts'' coated with antibody caused a rapid increase in intracellular free Ca2+ after a lag of 20-40 s, as detected by an increase in obelin luminescence. 3. The increase in obelin luminescence could not be explained by release of obelin into the medium. It was also Ca2+-dependent in that extracellular EGTA abolished the effect and intracellular EGTA inhibited it and required the complete terminal complex (C56789). No effect was seen with C5678. 4. The concentration of intracellular free Ca2+ before addition of complement was approx. 0.3 microM. This increased to a maximum of 5-30 microM after complement addition and then remained constant for at least 1-2 min. 5. Antibody plus complement induced a rapid increase in 42K+ efflux and an inhibition of cyclic AMP formation. 6. When partially purified complement components (C5b-9) were used in ''reactive lysis'' it was possible to inhibit the release of macromolecules from pigeon erythrocyte ''ghosts'' by extracellular EGTA. 7. It was concluded that the increase in intracellular free Ca2+ concentration caused by anti-cell antibody plus complement occurred before cell lysis and may be involved in the mechanism of complement-induced cell injury.     

Synergistic stimulation of thromboxane biosynthesis by calcium ionophore and phorbol ester or thrombin in human platelets

Phorbol ester PMA and low concentrations of calcium ionophore A-23187, which given separately have minimal effect in stimulating thromboxane synthesis in human platelets, showed marked synergism when given simultaneously. A similar synergism can be also demonstrated between thrombin or collagen and low concentrations of A-23187 but not of PMA. Simultaneous addition of thrombin and PMA results in less synthesis of thromboxane than that of thrombin alone. These studies suggest that protein kinase C activation by agonists may not only induce but also regulate thromboxane synthesis in human platelets.     

Direct demonstration of increased intracellular concentration of free calcium in rabbit and human neutrophils following stimulation by chemotactic factor

An increase in the level of intracellular free calcium concentration in rabbit and human neutrophils stimulated by chemotactic factors has been demonstrated directly using the calcium-sensitive fluorescent probe quin-2. Addition of f-Met-Leu-Phe (10(-9) M), C5a (3 x 10(-9) M) or leukotriene B4 (6 x 10(-8) M) to the neutrophils induces a rapid increase in the intracellular concentration of free calcium that reaches a maximum value 15 seconds following stimulation. At concentrations of f-Met-Leu-Phe less than 10(-8) M the enhancement is dose dependent with an ED50 of 8 x 10(-11) M and is significantly reduced in the presence of EGTA in the suspending medium.     

Melatonin elevates intracellular free calcium in human platelets by inositol 1,4,5-trisphosphate independent mechanism

Several studies have indicated the existence of direct effects of melatonin on platelets. Here we show that, melatonin at high concentration is capable of significantly raising platelet intracellular calcium even in the absence of an agonist. The effect of melatonin on platelets was abolished by luzindole, a melatonin receptor blocker, and rotenone, while it was unaffected by cell-permeable antagonists of either inositol 1,4,5-trisphosphate (IP(3)) receptor, phospholipase C (PLC), or bafilomycin A1, which discharges acidic calcium stores. Melatonin-induced manganese entry provided evidence for activation of bivalent cation entry. Thus, our data suggest that melatonin evoked the elevation of platelet intracellular calcium through depletion of mitochondrial Ca(2+) stores and store-operated calcium entry (SOCE), while the action was independent of the PLC-IP(3) axis.     

Synthetic diacylglycerols trigger an increase of intracellular free calcium in promyelocytic HL60 cells

Phosphoinositide turnover is known to play an important role in intracellular free calcium homeostasis through the inositol trisphophate-mediated release of calcium from intracellular stores. We find that the other product of phosphoinositide turnover, 1,2-diacylglycerol, elicits an increase in intracellular free calcium in HL60 cells which is due, at least in part, to release of calcium from intracellular stores. This effect is specific for calcium, since intracellular sodium and potassium levels and cellular volume were unaffected. Concomitant with the intracellular calcium increase, we find an increase in cellular inositol trisphosphate levels, suggesting that the effect of diacylglycerol on calcium may be mediated by inositol trisphosphate. Diacylglycerols also stimulate calcium efflux. This stimulation is not simply due to the increase in intracellular calcium. These effects appear not to be mediated through stimulation of a phorbol ester-activatable protein kinase C (Ca2+/phospholipid-dependent enzyme) since phorbol esters do not elicit an increase in cytoplasmic free calcium or an increase in calcium efflux.