Abnormalities in the regulation of blood platelet free cytosolic calcium in malignant hyperthermia. II. Pig platelets.

Since 1966 the domestic pig has served as the animal model in Malignant Hyperthermia (MH) research [1]. The use of genetically well-defined pigs rendered it possible to test the method for diagnosing MH-susceptibility of patients presented in the preceding paper. Thus, the effect of halothane on intracellular calcium movements was studied in Quin-2- and chlorotetracycline-loaded pig platelets. In 'Ca(2+)-free' suspensions the resting level of free cytosolic Ca2+ was about 60 nM. In contrast to the results with human platelets there were no significant differences between pig genotypes either in the absence or in the presence of external calcium. After addition of halothane, a mobilization of intracellular membrane-bound calcium can be observed. However, the calcium mobilization is not accompanied by a marked increase in fluorescence intensity of Quin-2-loaded platelets. Thus, in the absence of external calcium, halothane produces only a slight increase in free cytosolic Ca2+. Nevertheless, the calcium rises measured in platelets from affected animals were statistically significantly higher than those from normal subjects. However, in the presence of 1 mM external calcium, a rapid increase in free cytosolic calcium can be detected after halothane addition. This suggests that halothane causes a marked, dose-dependent increase in Ca2+ permeability of the plasma membrane. Compared to the control group, significantly enhanced calcium permeability was found, not only in homozygous positive pigs, but also in heterozygous animals.     

Erythropoietin induced transmembrane calcium influx in essential hypertension.

The effects of erythropoietin (EPO) on cytosolic free calcium concentration ([Ca2+]i) in platelets of 20 essential hypertensive patients (HT) and of 25 normotensive subjects (NT) were investigated using the fura2 technique. In resting platelets [Ca2+]i were not significantly higher in HT compared to NT (74.3 +/- 7.8 nM vs 59.8 +/- 7.0 nM, mean +/- SEM). Addition of EPO significantly increased [Ca2+]i in HT compared to NT (13.8 +/- 5.3 nM vs 0.9 +/- 1.9 nM, p less than 0.01). EPO increased the amount of calcium in intracellular stores. This was confirmed independently using thrombin-induced changes of [Ca2+]i in a calcium-free medium and using chlorotetracycline as a marker of stored calcium. After preincubation with EPO thrombin-induced changes of [Ca2+]i were significantly lower in HT compared to NT (306.1 +/- 30.0 nM vs 407.7 +/- 35.7 nM, p less than 0.05). In a calcium-free medium after preincubation with EPO thrombin-induced changes of [Ca2+]i were significantly lower in HT compared to NT (54.7 +/- 11.8 nM vs 100.9 +/- 10.5 nM, p less than 0.05) indicating lower storage capacity in HT. It is concluded that elevated response to EPO may provide a powerful tool to evaluate diagnosis and underlying pathophysiological mechanisms in essential hypertension.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

Modulation of cytoplasmic calcium in human platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine

The calcium-sensitive, fluorescent dye Quin 2 was used to quantitate changes in free intracellular calcium [( Ca2+]i) induced in platelets by the phospholipid platelet-activating factor 1-O-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine (AGEPC). The Ca2+]i of unstimulated platelets was 91 +/- 18 nM (mean +/- SD, n = 8), and treatment with 1 to 16 nM AGEPC increased [Ca2+]i in a dose-related manner, with 16 nM AGEPC increasing [Ca2+]i by 102 +/- 20 nM. [Ca2+]i was not increased by analogs of AGEPC which do not activate platelets including the lysophospholipid precursor of AGEPC, the optical isomer, and a C-2 benzoyl analog. The capacity of AGEPC to increase [Ca2+]i exceeded that required to induce maximal platelet aggregation. In four experiments, 100% platelet aggregation was induced by 4.5 +/- 2.4 nM AGEPC (mean +/- SD) and was associated with a submaximal increase in [Ca2+]i of 56 +/- 22 nM. Pretreatment of platelets with AGEPC rendered the platelets specifically unresponsive to repeat stimulation with AGEPC in terms of both platelet aggregation and increased [Ca2+]i, whereas the platelet response to thrombin was undiminished by pretreatment with AGEPC. In contrast, the platelet response to 0.5 microM calcium ionophore A23187 was undiminished by pretreatment with the same concentration of ionophore, suggesting that AGEPC does not activate platelets by an ionophore-like mechanism. IgG aggregates and AGEPC in combination activate platelets synergistically, as shown by the observation that a 1-min exposure of platelets to 60 micrograms/ml of IgG aggregates increased the platelet aggregation response to 2 nM AGEPC from 44 to 100%. In contrast, sequential exposure of platelets to IgG aggregates and AGEPC increased [Ca2+]i additively, suggesting that increased [Ca2+]i contributes to but does not fully mediate synergistic platelet activation by IgG aggregates and AGEPC. Quantitation of free intracellular calcium with the fluorescent dye Quin 2 is a highly sensitive technique for delineating the role of calcium in mediating platelet activation.     

Phosphoinositide signalling system in platelets of schizophrenic patients and the effect of neuroleptic therapy.

Alterations in the phosphoinositide signalling system have been proposed as a possible biological marker of schizophrenia. We studied the levels of inositol 1,4,5-trisphosphate (IP3), cytosolic Ca2+ concentrations ([Ca2+]i), and the incorporation of [32P]-orthophosphate into inositol phospholipids and phosphatidic acid (PA) in blood platelets of neuroleptic-treated schizophrenics in comparison with controls. The [Ca2+]i was significantly higher in platelets of one month neuroleptic-treated patients (155+/-5.8 nM) in comparison with controls (95+/-5.4 nM). Neuroleptic therapy decreased the [Ca2+]i, but even after long-term therapy it remained significantly higher (114+/-5.7 nM) than in controls. Differences were also found in the level of IP3 between controls (30+/-4.0 pmol/10(9) platelets), drug-free schizophrenics (52+/-9.0 pmol/10(9) platelets) and treated patients (50+/-6.0 pmol/10(9) platelets). The increased turnover of PA was observed in platelets of neuroleptic-treated schizophrenic patients. The study suggests that the regulation of calcium homeostasis and pathways involved in the phosphoinositide signalling system are altered in the platelets of schizophrenics. Neuroleptic therapy did not remove the observed changes in [Ca2+]i and IP3 levels.     

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.     

Na+/H+ exchange modulates Ca2+ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619

According to recent observations ADP stimulates platelets via activation of Na+/H+ exchange which increases cytosolic pH (pHi). This event initiates formation of thromboxane A2 (via phospholipase A2) and, thereafter, inositol 1,4,5-trisphosphate (via phospholipase C) which is known to mobilize Ca2+ from intracellular storage sites. We investigated changes in pHi and cytosolic free Ca2+, [Ca2+]i, activating platelets with ADP and the thromboxane mimetic U 46619. We found that ADP (5 microM) increased pHi from 7.15 +/- 0.08 to 7.35 +/- 0.04 (n = 8) in 2'-7'-bis-(carboxyethyl)-5,6-carboxyfluorescein-loaded platelets, whereas thromboxane A2 formation was inhibited by indomethacin. ADP also induced a dose-dependent Ca2+ mobilization in fura2-loaded platelets which again was not affected by indomethacin. [Ca2+]i increased by 54 +/- 10 nM (n = 8) at 1 microM and by 170 +/- 40 nM (n = 7) at 10 microM ADP above the resting value of 76 +/- 12 nM (n = 47). Inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA) reduced ADP-induced Ca2+ mobilization by more than 65% in indomethacin-treated platelets. This inhibition could be completely overcome by artificially raising pHi using either NH4Cl or the Na+/H+ ionophore monensin. We found that U 46619 increased pHi by 0.18 +/- 0.05 at 0.1 microM and by 0.29 +/- 0.07 (n = 7) at 1.0 microM above the resting value via an EIPA-sensitive mechanism. In conflict with the proposed role of the Na+/H+ exchange we found that U 46619 raised [Ca2+]i via a mechanism that for more than 50% depended on intact Na+/H+ exchange. Again, artificially elevating pHi restored U 46619-induced Ca2+ mobilization despite the presence of EIPA. Thus, our data show that Na+/H+ exchange is a common step in platelet activation by prostaglandin endoperoxides/thromboxane A2 and ADP and enhances Ca2+ mobilization independently of phospholipase A2 activity.     

Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs.

Using the rapid filtration technique to investigate Ca2+ movements across the sarcoplasmic reticulum (SR) membrane, we compare the initial phases of Ca2+ release and Ca2+ uptake in malignant hyperthermia susceptible (MHS) and normal (N) pig SR vesicles. Ca2+ release is measured from passively loaded SR vesicles. MHS SR vesicles present a 2-fold increase in the initial rate of calcium release induced by 0.3 microM Ca2+ (20.1 +/- 2.1 vs. 6.3 +/- 2.6 nmol mg-1 s-1). Maximal Ca2+ release is obtained with 3 microM Ca2+. At this optimal concentration, rate of Ca2+ efflux in absence of ATP is 55 and 25 nmol mg-1 s-1 for MHS and N SR, respectively. Ca(2+)-induced Ca2+ release is inhibited by Mg2+ in a dose-dependent manner for both MHS and N pig SR vesicles (K1/2 = 0.2 mM). Caffeine (5 mM) and halothane (0.01% v/v) increase the Ca2+ sensitivity of Ca(2+)-induced Ca2+ release. ATP (5 mM) strongly enhances the rate of Ca2+ efflux (to about 20-40-fold in both MHS and N pig SR vesicles). Furthermore, both types of vesicles do not differ in their high-affinity site for ryanodine (Kd = 12 nM and Bmax = 6 pmol/mg), lipid content, ATPase activity and initial rate of Ca2+ uptake (0.948 +/- 0.034 vs. 0.835 +/- 0.130 mumol mg-1 min-1 for MHS and N SR, respectively). Our results show that MH syndrome is associated to a higher rate of Ca2+ release in the earliest phase of the calcium efflux.     

Trypanosoma cruzi: mechanisms of intracellular calcium homeostasis.

Regulation of intracellular Ca2+ homeostasis was characterized in epimastigote forms of Trypanosoma cruzi using the fluorescence probe Fura-2. Despite an increase in extracellular Ca2+, [Ca2+]o, from 0 to 2 mM, cytosolic Ca2+, [Ca2+]i, increased only from 85 +/- 9 to 185 +/- 21 nM, indicating the presence of highly efficient mechanisms for maintaining [Ca2+]i. Exposure to monovalent Na+ (monensin)-, K+ (valinomycin, nigericin)-, and divalent Ca2+ (ionomycin)-specific ionophores, uncouplers of mitochondrial respiration (oligomycin), inhibitors of Na+/K(+)-ATPase (ouabain), and Ca(2+)-sensitive ATPase (orthovanadate) in 0 or 1 mM [Ca2+]o resulted in perturbations of [Ca2+]i, the patterns of which suggested both sequestration and extrusion mechanisms. Following equilibration in 1 mM [Ca2+]o, incubation with orthovanadate markedly increased [Ca2+]i, results which are compatible with an active uptake of [Ca2+]i by endoplasmic reticulum. In contrast, equilibration in 0 or 1 mM [Ca2+]o did not influence the relatively smaller increase in [Ca2+]i following incubation with oligomycin, suggesting a minor role for the mitochondrial compartment. In cells previously equilibrated in 1 mM [Ca2+]o, exposure to monensin or ouabain, conditions known to decrease the [Na+]o/[Na+]i gradient, upon which the Na+/Ca2+ exchange pathways are dependent, markedly increased [Ca2+]i. In a complementary manner, decreasing the extracellular Na+ gradient with Li+ increased [Ca2+]i in a dose-dependent manner. Finally, the calcium channel blockers verapamil and isradipine inhibited the uptake of Ca2+ by greater than 50%, whereas diltiazem, nifedipine, and nicardipine were ineffective. The results suggest that epimastigote forms of T. cruzi maintain [Ca2+]i by uptake, sequestration, and extrusion mechanisms, with properties common to eukaryotic organisms.     

Neural regulation of parvalbumin expression in mammalian skeletal muscle.

The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration-dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM-vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1-receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane-A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization.     

Cyclic AMP stimulates Ca(2+)-ATPase-mediated Ca2+ extrusion from human platelets.

The effect of cAMP on active Ca2+ extrusion across the plasma membrane of intact human platelets was studied using quin2, a fluorimetric indicator of free Ca2+ in the cytoplasmic compartment ([Ca2+]cyt). Elevations of cAMP were achieved by incubation with dibutyryl-cAMP or by forskolin, which was found to selectively elevate cAMP without affecting cGMP levels. Progress curves of Ca2+ extrusion from quin2-overloaded platelets were measured. The rate vs. [Ca2+]cyt characteristic was calculated as previously described (Johansson, J.S. and Haynes, D.H. (1988) J. Membr. Biol. 104, 147-163). Forskolin, at a maximally effective concentration of 10 microM, was shown to stimulate Ca2+ extrusion by increasing by a factor of 1.6 +/- 0.5 the Vm of a saturable component, previously identified with a Ca(2+)-Mg(2+)-ATPase located in the plasma membrane. Neither the Km (80 nM) or Hill coefficient (1.7 +/- 0.3) of the Ca(2+)-ATPase was affected. Forskolin had no effect on the linear, non-saturable component of extrusion (previously identified with a Na+/Ca2+ exchanger) over the [Ca2+]cyt range examined (50-1500 nM). Dibutyryl-cAMP (Bt2-cAMP, 1 mM) stimulated the Ca(2+)-Mg(2+)-ATPase component of Ca2+ extrusion by a factor of 2.0 +/- 0.6. Separate experiments showed that 10 microM forskolin reduces the resting [Ca2+]cyt from 112 nM to 96 nM. Mathematical analysis showed that this can be accounted for by the above-mentioned increase in Vm of the pump, countered by a 37-74% increase in the rate constant for passive Ca2+ leakage across the plasma membrane. The results suggest two mechanisms by which prostacyclin-induced elevation of cAMP inhibits platelet aggregation: (a) lowering of resting [Ca2+]cyt and (b) increasing the rate of Ca2+ extrusion after the initial influx or triggered release event.     

Measurement of sarcoplasmic reticulum Ca2+ content in intact amphibian skeletal muscle fibres with 4-chloro-m-cresol.

Single skeletal muscle fibres were isolated from the toad (Bufo marinus) and isometric force and myoplasmic free calcium concentration ([Ca2+]i) were measured. Brief applications of 4-chloro- m-cresol (4-CmC, 0.2-5 mM) elevated [Ca2+]i reversibly in a dose-dependent manner. The lowest concentration of 4-CmC which reliably gave maximal [Ca2+]i was 2 mM and it was, therefore, used for measurement of sarcoplasmic reticulum (SR) Ca2+ content. Tetanic stimulations (100 Hz) increased [Ca2+]i from a resting level of 105 +/- 47 nM (n = 10) to 1370 +/- 220 nM (n = 6). Application of 2 mM 4-CmC produced a contracture that was 54 +/- 16% (n = 6) of the tetanic force and elevated [Ca2+]i to a peak of 3520 +/- 540 nM (n = 8). Both force and [Ca2+]i levels (resting and tetanic) were restored after 10 min of washout of 4-CmC. In skinned muscle fibres, the myofibrillar Ca(2+)-sensitivity was not changed by 4-CmC, but maximal force was reduced to 74 +/- 10% (n = 4). The magnitude of the peak of the 4-CmC-induced Ca2+ transient was not significantly changed by removal of extracellular Ca2+ nor by inhibiting the SR Ca2+ pump with 2,5-di-tert-butylhydroquinone. Treatment of intact fibres with 30 mM caffeine produced a peak Ca2+ level that was indistinguishable from 2 mM 4-CmC. These results indicate that it is possible to measure the SR Ca2+ content in the same fibre with 4-CmC without loss of normal muscle function.     

Cytosolic alkalinization alone is not sufficient for Ca2+ mobilization, phosphatidic acid formation, and protein phosphorylation in human platelets

One of the earliest events following stimulation of human platelets with thrombin is a rise in the cytosolic pH, pHi, mediated by Na+/H+ exchange, and an increase in the cytosolic free Ca2+ concentration, [Ca2+]i. In the present study we investigated whether an increase in pHi alone, induced by the Na+/H+ ionophore monensin, is sufficient for platelet activation. Although monensin (20 microM) raised pHi from 7.10 +/- 0.05 (n = 21) to 7.72 +/- 0.17 (n = 13), neither Ca2+ influx nor mobilization were detectable upon this treatment in fura2-loaded platelets. In contrast, thrombin (0.05 U/ml) raised pHi to 7.31 +/- 0.10 (n = 10) and increased [Ca2+]i by more than 250 nM both in the presence and absence of extracellular Ca2+. Thrombin also caused the formation of phosphatidic acid and phosphorylation of the 20 kDa and 47 kDa proteins in platelets labeled with 32P. Monensin, however, induced none of these responses. It is concluded that an increase in pHi alone is not a sufficient trigger for platelet activation but enhances intracellular signal transduction in platelets stimulated by natural agonists.     

Histamine type I receptor occupancy increases endothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers

Considerable evidence suggests that Ca2+ modulates endothelial cell metabolic and morphologic responses to mediators of inflammation. We have used the fluorescent Ca2+ indicator, quin2, to monitor endothelial cell cytosolic free Ca2+, [Ca2+]i, in cultured human umbilical vein endothelial cells. Histamine stimulated an increase in [Ca2+]i from a resting level of 111 +/- 4 nM (mean +/- SEM, n = 10) to micromolar levels; maximal and half-maximal responses were elicited by 10(-4) M and 5 X 10(-6) M histamine, respectively. The rise in [Ca2+]i occurred with no detectable latency, attained peak values 15-30 s after addition of stimulus, and decayed to a sustained elevation of [Ca2+]i two- to threefold resting. H1 receptor specificity was demonstrated for the histamine-stimulated changes in [Ca2+]i. Experiments in Ca2+-free medium and in the presence of pyrilamine or the Ca2+ entry blockers Co2+ or Mn2+, indicated that Ca2+ mobilization from intracellular pools accounts for the initial rise, whereas influx of extracellular Ca2+ and continued H1 receptor occupancy are required for sustained elevation of [Ca2+]i. Ionomycin-sensitive intracellular Ca2+ stores were completely depleted by 4 min of exposure to 5 X 10(-6) M histamine. Verapamil or depolarization of endothelial cells in 120 mM K+ did not alter resting or histamine-stimulated [Ca2+]i, suggesting that histamine-elicited changes are not mediated by Ca2+ influx through voltage-gated channels. Endothelial cells grown on polycarbonate filters restricted the diffusion of a trypan blue-albumin complex; histamine (through an H1- selective effect) promoted trypan blue-albumin diffusion with a concentration dependency similar to that for the histamine-elicited rise in [Ca2+]i. Exposure of endothelial cells to histamine (10(-5) M) or ionomycin (10(-7) M) was associated with a decline in endothelial F- actin (relative F-actin content, 0.76 +/- 0.07 vs. 1.00 +/- 0.05; histamine vs. control, P less than 0.05; relative F-actin content, 0.72 +/- 0.06 vs. 1.00 +/- 0.05; ionomycin vs. control, P less than 0.01). The data support a role for cytosolic calcium in the regulation of endothelial shape change and vessel wall permeability in response to histamine.     

Unusual sensitivity of cytosolic free Ca2+ to changes in extracellular Ca2+ in rat C-cells

An essential function of C-cells is to monitor extracellular Ca2+ concentration ([Ca2+]e) and to respond to changes in [Ca2+]e by regulating hormone secretion. Using the calcitonin-secreting rat C-cell line rMTC 44-2, we have investigated a possible tight linkage between [Ca2+]e and cytosolic free Ca2+ ([Ca/+]i). We have demonstrated, using the Ca2+ indicator Quin 2, that the [Ca2+]i is particularly sensitive to changes in [Ca2+]e. Sequential increases in [Ca2+]e as small as 0.1 mM evoke clear elevations in [Ca2+]i. In contrast, other cell types tested did not alter their [Ca2+]i in response to increasing [Ca2+]e even to levels as high as 4.0 mM. Sequential 1.0 mM increments in [Ca2+]e caused the [Ca2+]i to rise from a base line of 357 +/- 20 nM Ca2+i at 1.0 mM Ca2+e to a maximum of 1066 +/- 149 nM Ca2+i at 5.0 mM Ca2+e. [Ca2+]e above 2.0 mM produced a biphasic response in [Ca2+]i consisting of an immediate (less than 5 s) spike followed by a decay to a new plateau. Treatment of rMTC 44-2 cells with either 50 mM K+ or 100 nM ionomycin at 1.0 mM Ca2+e caused an immediate spike in [Ca2+]i to micromolar levels. Pretreatment with EGTA or verapamil inhibited completely the increase in [Ca2+]i induced by 50 mM K+. However, pretreatment with EGTA only slightly attenuated the spike phase in [Ca2+]i produced by ionomycin, demonstrating that ionomycin released intracellular stores of calcium. We conclude that rMTC 44-2 cells regulate [Ca2+]i by monitoring small physiological changes in [Ca2+]e, the primary secretagogue for C-cells.     

Characterization of the effect of the adenosine agonist cyclohexyladenosine on platelet activating factor-induced increases in [Ca]i in human platelets in vitro

Previous studies have shown that adenosine agonists acting at A-2 receptors inhibit platelet aggregation. Since an increase in cytosolic Ca2+ concentration (delta [Ca2+]i) is closely associated with the time frame of platelet aggregation, we have examined the effect of adenosine receptor function on induced increases of [Ca2+]i by a potent platelet activator, platelet activating factor (PAF). We loaded washed platelets with Fura-2, then induced increases in [Ca2+]i with various concentrations of PAF, and then determined EC50 values (PAF concentration at half-maximal response) and values for maximal response of delta[Ca2+]i (max-delta[Ca2+]i). The EC50 for PAF-delta[Ca2+]i was 112 +/- 37 (SD) pM and the max-delta[Ca2+]i was 284 +/- 138 (SD) nM. Our results show that PAF-delta[Ca2+]i was inhibited in a non-competitive manner by the adenosine receptor agonist cyclohexyladenosine (CHA) with an IC50 of 14.9 microM. This inhibition was partially reversed by theophylline, an adenosine receptor antagonist, with an IC50 of 19 microM. Based on the results of these studies together with evidence from other research groups that platelets do not possess A-1 receptors, our results suggest that CHA inhibited PAF-delta[Ca2+]i in platelets through an activation of A-2 receptors.     

Increased cytosolic calcium. A signal for sulfonylurea-stimulated insulin release from beta cells

The mechanisms by which glyburide and tolbutamide signal insulin secretion were examined using a beta cell line (Hamster insulin-secreting tumor (HIT) cells). Insulin secretion was measured in static incubations, free cytosolic Ca2+ concentration ([Ca2+]i) was monitored in quin 2-loaded cells, and cAMP quantitated by radioimmunoassay. Insulin secretory dose-response curves utilizing static incubations fit a single binding site model and established that glyburide (ED50 = 112 +/- 18 nM) is a more potent secretagogue than tolbutamide (ED50 = 15 +/- 3 microM). Basal HIT cell [Ca2+]i was 76 +/- 7 nM (mean +/- S.E., n = 141) and increased in a dose-dependent manner with both glyburide and tolbutamide with ED50 values of 525 +/- 75 nM and 67 +/- 9 microM, respectively. The less active tolbutamide metabolite, carboxytolbutamide, had no effect on [Ca2+]i or insulin secretion. Chelation of extracellular Ca2+ with 4 mM EGTA completely inhibited the sulfonylurea-induced changes in [Ca2+]i and insulin release and established that the rise in [Ca2+]i came from an extracellular Ca2+ pool. The Ca2+ channel blocker, verapamil, inhibited glyburide- or tolbutamide-stimulated insulin release and the rise in [Ca2+]i at similar concentrations with IC50 values of 3 and 2.5 microM, respectively. At all concentrations tested, the sulfonylureas did not alter HIT cell cAMP content. These findings provide direct experimental evidence that glyburide and tolbutamide allow extracellular Ca2+ to enter the beta cell through verapamil-sensitive, voltage-dependent Ca2+ channels, causing a rise in [Ca2+]i which is the second messenger that stimulates insulin release.     

Increased internal calcium mobilization in platelets of patients with chronic renal failure.

Platelet free calcium concentrations ([Ca2+]i) were measured with Fura-2 to elucidate the intracellular calcium kinetics in patients with renal disease. There were no significant differences of the resting [Ca2+]i among the control subjects (C) (n = 12), patients with chronic glomerulonephritis (CGN) (n = 8), and patients with chronic renal failure (CRF) (n = 12). In all groups, platelets [Ca2+]i were significantly increased by agonists (thrombin, adenosine diphosphate) compared with their respective basal level. Thrombin-induced [Ca2+]i rise was significantly higher in CRF (840 +/- 265 nM) than in C (600 +/- 163) and CGN (562 +/- 137). Also adenosine diphosphate elicited similar responses. In the presence of calcium chelator in the incubation buffer, the elevation of [Ca2+]i after thrombin stimulation was statistically higher in CRF (469 +/- 85 nM) than in C (275 +/- 60) and CGN (301 +/- 41). These findings suggest that platelets of CRF were capable of increasing [Ca2+]i in response to agonists, through further mobilization of calcium from the intracellular pool rather than the elevation of transmembrane calcium influx.     

Effect of hyperglycemia on the changes of intracellular [Ca2+]i in heart myoblast

A rise in cytosolic free Ca2+ is the immediate trigger for contraction in heart muscle. In the present study, we investigated changes of intracellular Ca2+ increased by potassium chloride (KCl) and phenylephrine (PE) under hyperglycemia in rat heart myoblast H9c2 cells (BCRC 60096), respectively. We employed the fluorescent Ca2+-indicator, fura-2, and digital imaging microscopy to measure [Ca2+]i in H9c2 cells. Cells were cultured in hyperglycemic (30 mM glucose) Dulbecco's Modified Eagle's Medium. The variation of [Ca2+]i induced by KCI and PE in hyperglycemia was examined, respectively. Moreover, tiron, one of the antioxidants, was pretreated in hyperglycemia-treated H9c2 cells to measure the role of free radicals in the changes of intracellular [Ca2+]i. An influx in intracellular Ca2+ induced by KCl or PE was observed in a dose-dependent manner and reached the highest concentration of 434 +/- 42.3 nM and 443 +/- 42.8 nM (n = 24 cells), respectively. Moreover, this increase of intracellular [Ca2+]i induced by KCl or PE was markedly reduced in cells exposed to hyperglycemia (434 +/- 42.3 vs. 1.26 +/- 0.21 nM and 443 +/- 42.8 vs. 2.54 +/- 0.25 nM, n = 24 cells, P < 0.001, respectively). Similar changes were not observed in cells received mannitol showing same osmolarity. However, the reduction of intracellular [Ca2+]i induced by hyperglycemia was abolished significantly in the presence of tiron. Our results suggest that an increase of intracellular Ca2+ by KCl or PE in heart cell was markedly reduced by hyperglycemic treatment; mediation of free radicals in this action can be considered because it was reversed in the presence of tiron.     

Magnesium regulates intracellular free ionized calcium concentration and cell geometry in vascular smooth muscle cells.

Regulatory effects of extracellular magnesium ions ([Mg2+]o) on intracellular free ionized calcium ([Ca2+]i) were studied in cultured vascular smooth muscle cells (VSMCs) from rat aorta by use of the fluorescent indicator fura-2 and digital imaging microscopy. With normal Mg2+ (1.2 mM)-containing incubation media, [Ca2+]i in VSMCs was 93.6 +/- 7.93 nM with a heterogeneous cellular distribution. Lowering [Mg2+]o to 0 mM or 0.3 mM (the lowest physiological range) resulted in 5.8-fold (579.5 +/- 39.99 nM) and 3.5-fold (348.0 +/- 31.52 nM) increments of [Ca2+]i, respectively, without influencing the cellular distribution of [Ca2+]i. Surprisingly, [Mg2+]o withdrawal induced changes of cell geometry in many VSMCs, i.e., the cells rounded up. However, elevation of [Mg2+]o up to 4.8 mM only induced slight decrements of [Ca2+]i (mean = 72.0 +/- 4.55 nM). The large increment of [Ca2+]i induced by [Mg2+]o withdrawal was totally inhibited when [Ca2+]o was removed. The data suggest that: (1) [Mg2+]o regulates the level of [Ca2+]i in rat aortic smooth muscle cells, and (2) [Mg2+] acts as an important regulatory ion by modulating cell shapes in cultured VSMc and their metabolism to control vascular contractile activities.