Intracellular alkalinization leads to Ca2+ mobilization from agonist-sensitive pools in bovine aortic endothelial cells

Receptor-stimulated phosphoinositide turnover leads to activation of Na+/H+ exchange and subsequent intracellular alkalinization. To probe the effect of increased intracellular pH (pHi) on Ca2+ homeostasis in cultured bovine aortic endothelial cells (BAEC), we studied the effect of weak bases, ammonium chloride (NH4Cl) and methylamine (agents which increase pHi by direct passive diffusion), on resting and ATP (purinergic receptor agonist)-induced Ca2+ fluxes. Changes in cytosolic free Ca2+ ([Ca2+]i) or pHi were monitored in BAEC monolayers using the fluorescent dyes, fura-2 or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively. NH4Cl-induced, dose-dependent (5-20 mM) increases in [Ca2+]i (maximum change = 195 +/- 26 nM) which were temporally similar to the NH4Cl-induced pHi increases. Methylamine (20 mM) induced a more sustained pHi increase and also stimulated a prolonged [Ca2+]i increase. When BAEC were bathed in HCO3- buffer, removal of extracellular CO2/bicarbonate caused pHi to increase and also induced [Ca2+]i to increase transiently. Extracellular Ca2+ removal did not abolish the rapid NH4Cl-induced rise in [Ca2+]i, although the response was blunted and more transient. NH4Cl addition to BAEC cultures resulted in an increase in 45Ca efflux and decrease in total cell 45Ca content. BAEC treatment with ATP (100 microM) to deplete inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools completely blocked the NH4Cl (20 mM)-induced rise in [Ca2+]i. Likewise, prior NH4Cl addition partially inhibited ATP-induced increases in [Ca2+]i, as well as slowed the frequency of repetitive [Ca2+]i spikes in single endothelial cells due to agonist. NH4Cl augmented the rate of [Ca2+]i increase that occurs in response to the depletion of agonist-sensitive intracellular Ca2+ pools. However, the internal Ca2+ store remained depleted during the continued presence of NH4Cl, as indicated by a decreased [Ca2+]i response to ATP in Ca2(+)-free medium. Finally, NH4Cl exerted these actions without affecting basal or ATP-stimulated IP3 formation. These observations provide direct evidence that increased pHi leads to Ca2+ mobilization from an agonist-sensitive pool and impairs Ca2+ pool(s) refilling mechanisms without altering cellular IP3 levels.     

Effect of m-3M3FBS on Ca2+ movement in PC3 human prostate cancer cells

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in PC3 human prostate cancer cells is unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended PC3 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. M-3M3FBS at concentrations between 10-50 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 60% by removing extracellular Ca2+. M-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, 30 microM m-3M3FBS pretreatment greatly inhibited the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin or BHQ. Conversely, pretreatment with thapsigargin, BHQ or cyclopiazonic acid reduced the major part of m-3M3FBS-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not much alter m-3M3FBS-induced [Ca2+]i rise. Collectively, in PC3 cells, m-3M3FBS induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels.     

A slow sustained increase in cytosolic Ca2+ levels mediates stalk gene induction by differentiation inducing factor in Dictyostelium.

During Dictyostelium stalk cell differentiation, cells vacuolate, synthesize a cellulose cell wall and die. This process of programmed cell death is accompanied by expression of the prestalk gene ecmB and induced by the differentiation inducing factor DIF. Using cell lines expressing the recombinant Ca2+-sensitive photoprotein apoaequorin, we found that 100 nM DIF increases cytosolic Ca2+ ([Ca2+]i) levels from approximately 50 to 150 nM over a period of 8 h. The Ca2+-ATPase inhibitor 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ) induced a similar increase in [Ca2+]i levels and induced expression of the prestalk gene ecmB to the same level as DIF. The [Ca2+]i increases induced by DIF and BHQ showed similar kinetics and preceded ecmB gene expression by approximately 1-2 h. The Ca2+ chelator 1,2-bis(o-aminophenoxy)-ethane-N,N,N'N'-tetra-acetic acid (BAPTA) efficiently inhibited the BHQ-induced [Ca2+]i increase and blocked DIF-induced expression of the ecmB gene. These data indicate that the effects of DIF on stalk gene expression are mediated by a sustained increase in [Ca2-]i. Sustained [Ca2+]i elevation mediates many forms of programmed cell death in vertebrates. The Dictyostelium system may be the earliest example of how this mechanism developed during early eukaryote evolution.     

Effect of m-3m3FBS on Ca2+ handling and viability in OC2 human oral cancer cells

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells is unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. M-3M3FBS at concentrations between 10-60 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. M-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, 30 μM m-3M3FBS pretreatment inhibited the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, BHQ or cyclopiazonic acid partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. At concentrations between 5 and 100 μM m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 μM) induced apoptosis in a Ca2+-independent manner. Collectively, in OC2 cells, m-3M3FBS induced [Ca2+]i rise by causing inositol 1,4,5-trisphosphate-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive store-operated Ca2+ channels. M-3M3FBS also induced Ca2+-independent cell death and apoptosis.     

Effect of nortriptyline on Ca2+ handling in SIRC rabbit corneal epithelial cells

To explore the effect of nortriptyline, a tricyclic antidepressant, on cytosolic free Ca2+ concentrations ([Ca2+]i) in corneal epithelial cells, [Ca2+]i levels in suspended SIRC rabbit corneal epithelial cells were measured by using fura-2 as a Ca2+-sensitive fluorescent dye. Nortriptyline at concentrations between 20-200 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Nortriptyline-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and alteration of activity of protein kinase C. In Ca2+-free medium, 200 microM nortriptyline pretreatment greatly inhibited the rise of [Ca2+]i induced by the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ; another endoplasmic reticulum Ca2+ pump inhibitor) nearly abolished nortriptyline-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 decreased nortriptyline-induced [Ca2+]i rise by 75%. Taken together, nortriptyline induced [Ca2+]i rises in SIRC cells by causing phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels.     

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.     

Expression of active p21-activated kinase-1 induces Ca2+ flux modification with altered regulatory protein phosphorylation in cardiac myocytes

p21-Activated kinase-1 (Pak1) is a serine-threonine kinase that associates with and activates protein phosphatase 2A in adult ventricular myocytes and, thereby, induces increased Ca2+ sensitivity of skinned-fiber tension development mediated by dephosphorylation of myofilament proteins (Ke Y, Wang L, Pyle WG, de Tombe PP, Solaro RJ. Circ Res 94: 194-200, 2004). We test the hypothesis that activation of Pak1 also moderates cardiac contractility through regulation of intracellular Ca2+ fluxes. We found no difference in field-stimulated intracellular Ca2+ concentration ([Ca2+]i) transient amplitude and extent of cell shortening between myocytes expressing constitutively active Pak1 (CA-Pak1) and controls expressing LacZ; however, time to peak shortening was significantly faster and rate of [Ca2+]i decay and time of relengthening were slower. Neither caffeine-releasable sarcoplasmic reticulum (SR) Ca2+ content nor fractional release was different in CA-Pak1 myocytes compared with controls. Isoproterenol application revealed a significantly blunted increase in [Ca2+]i transient amplitude, as well as a slowed rate of [Ca2+]i decay, increased SR Ca2+ content, and increased cell shortening, in CA-Pak1 myocytes. We found no significant change in phospholamban phosphorylation at Ser16 or Thr17 in CA-Pak1 myocytes. Analysis of cardiac troponin I revealed a significant reduction in phosphorylated species that are primarily attributable to Ser(23/24) in CA-Pak1 myocytes. Nonstimulated, spontaneous SR Ca2+ release sparks were significantly smaller in amplitude in CA-Pak1 than LacZ myocytes. Propagation of spontaneous Ca2+ waves resulting from SR Ca2+ overload was significantly slower in CA-Pak1 myocytes. Our data indicate that CA-Pak1 expression has significant effects on ventricular myocyte contractility through altered myofilament Ca2+ sensitivity and modification of the [Ca2+]i transient.     

Overexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes

The functional consequences of overexpression of rat heart Na+/Ca2+ exchanger (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, cultured adult rat myocytes retained normal contractile function compared with freshly isolated myocytes for at least 48 h. Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h postinfection was not affected. When they were examined 48 h after infection, myocytes infected by adenovirus expressing both GFP and NCX1 had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+ concentration ([Ca2+]i) transients, and lower resting and diastolic [Ca2+]i when compared with myocytes infected by the adenovirus expressing GFP alone. The effects of NCX1 overexpression on sarcoplasmic reticulum (SR) Ca2+ content depended on extracellular Ca2+ concentration ([Ca2+]o), with a decrease at low [Ca2+]o and an increase at high [Ca2+]o. The half-times for [Ca2+]i transient decline were similar, suggesting little to no changes in SR Ca2+-ATPase activity. Western blots demonstrated a significant (P < or = 0.02) threefold increase in NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myocytes 48 h after infection by adenovirus expressing both GFP and NCX1 compared with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+]o resulted in enhanced Ca2+ influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and [Ca2+]i transient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting and diastolic [Ca2+]i.     

Thyroid status and beta-agonistic effects on cytosolic calcium concentrations in single rat cardiac myocytes activated by electrical stimulation or high-K+ depolarization.

The effects of the thyroid status on the cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were studied at rest and during contraction. The mean resting [Ca2+]i increased significantly from the hypothyroid (45 +/- 4 nM) through the euthyroid (69 +/- 12 nM) to the hyperthyroid condition (80 +/- 11 nM) at extracellular Ca2+ concentrations ([Ca2+]o) up to 2.5 mM. At [Ca2+]o above 2.5 mM the differences in [Ca2+]i between the groups became less. The amplitude of the Ca2+ transients became higher in all groups with increasing [Ca2+]o (1, 2.5 and 5 mM), and was highest at all [Ca2+]o in hyperthyroid myocytes. The beta-agonist isoprenaline elevated peak [Ca2+]i during contraction and increased the rate of the decay of the Ca2+ transients to a greater extent in hypothyroid myocytes than in hyperthyroid myocytes. Depolarization with high [K+]o induced a large but transient [Ca2+]i overshoot in hypothyroid myocytes, but not in hyperthyroid myocytes, before a new elevated steady-state [Ca2+]i was reached, which was not different between the groups. When isoprenaline was added to K+ o-depolarized myocytes after a steady state was reached, a significantly larger extra increase in [Ca2+]i was measured in the hypothyroid group (28%) compared with the hyperthyroid group (8%). It is concluded that in cardiac tissue exposed to increasing amounts of thyroid hormones (1) [Ca2+]i increases at rest and during contraction in cardiomyocytes and (2) interventions which favour Ca2+ entry into the cytosol [( Ca2+]o elevation, high [K+]o, beta-agonists) tend to have less impact on Ca2+ homoeostasis.     

High levels of cytosolic free calcium inhibit dephosphorylation of insulin receptor and glycogen synthase.

Treatment of adipocytes with depolarizing concentrations of K+ (40 mM) for 60 min increased [Ca2+]i from 158 +/- 28 nM to 328 +/- 38 nM. This significantly reduced (up to 80% inhibition) dephosphorylation of insulin receptor (IR), EGF receptor (EGF-R) and glycogen synthase (GS). The calcium channel blocker, nitrendipine (30 microM), or Ca2+ free medium completely prevented K(+)-induced inhibition of phosphoprotein phosphatase (PPTase). This effect of high [Ca2+]i was completely reversible when the cells were returned into the non-depolarizing medium. Trypsin treatment (4 micrograms/ml) of the membrane fraction containing inhibited PPTase activity, restored dephosphorylation activity to normal suggesting that elevated [Ca2+]i may inhibit PPTase by promoting its association with the inhibitors. These observations indicate that dephosphorylation of IR and GS can be regulated by [Ca2+]i.     

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators.

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.     

Modulation by external Ca2+ and nicardipine of Ca2+ influx and cytosolic concentration in human erythrocytes

Variations of Ca2+ influx (evaluated by the initial rate of 45Ca2+ uptake) and cytosolic free Ca2+ concentration ([Ca2+]i, measured with fura-2) were investigated in human erythrocytes. When external Ca2+ concentration ([Ca2+]o) rose from 1 to 2 mM, the initial rate of Ca2+ influx nearly doubled whereas [Ca2+]i increased only by 15%. Nicardipine dose-dependently decreased both initial rate of Ca2+ influx and [Ca2+]i (up to 53 and 18%. respectively at 10(-6) M). The less marked changes in [Ca2+]i than in Ca2+ influx indicate a partial adjustment of the Ca2+ extruding-pump activity to of Ca2+ influx. In vivo administration of nicardipine reduced [Ca2+]i only when its initial value exceeded 80 nM and prevented the rise in [Ca2+]i induced by the increase in [Ca2+]o. Our results indicate that nicardipine may reduce Ca2+ influx in human erythrocytes and participate in the control of [Ca2+]i when elevated.     

Basolateral store-operated Ca(2+)-entry in polarized human bronchial and colonic epithelial cells

Bronchial epithelial cells respond to extracellular nucleotides from the luminal and basolateral side activating Cl- secretion via [Ca2+]i increase. In this study we investigated the differences of apically (ap) and basolaterally (bl) stimulated [Ca2+]i signals in polarized human bronchial epithelial cells (16HBE14o-). Specifically we investigated the localization of 'capacitative Ca2+ entry' (CCE). 16HBE14o- cells grown on permeable filters were mounted into an Ussing chamber built for the simultaneous measurement of Fura-2 fluorescence and electrical properties. Application of ATP from both sides induced a rapid [Ca2+]i increase and subsequent sustained [Ca2+]i plateau due to transmembraneous Ca(2+)-influx. The use of different nucleotides revealed the following rank order or potency which was very similar for addition from the apical or basolateral side: UTP (EC50 ap: 4 microM, bl: 5 microM) > ATP (EC50 ap: 4 microM, bl: 10 microM) > ADP (n = 4-7 from both sides). 2-MeS-ATP, AMP, adenosine and beta gamma-methylene ATP were ineffective (n = 3 from both sides). The ATP- (ap and bl) induced Ca2+ influx was only abolished by removal of basolateral Ca2+. This was also true for receptor-independent activation of Ca(2+)-influx by intracellular Ca(2+)-store depletion with 2,5 Di-(tert-butyl)-1,4-benzohydroquinone (BHQ) (10 microM). Also in polarized T84 cells the basolateral carbachol and BHQ activated Ca2+ plateau was exclusively sensitive to removal of basolateral Ca2+. We propose that in all polarized epithelial cells the CCE entry pathway is located in the basolateral membrane. We furthermore suggest that Ca2+[i elevating agonists acting from the apical side of the epithelium lead to the opening of a basolateral CCE pathway.     

Glucose-induced increase in cytoplasmic pH in pancreatic beta-cells is mediated by Na+/H+ exchange, an effect not dependent on protein kinase C.

Glucose-induced changes in cytoplasmic pH (pHi) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Glucose, at concentrations above 3-5 mM, depolarized the beta-cell and increased pHi, cytoplasmic free Ca2+ ([Ca2+]i), and insulin release. This increase in pHi was dependent on the presence of extracellular Na+ and was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, a blocker of Na+/H+ exchange. Stimulation of protein kinase C with phorbol ester also induced an alkalinization. However, when protein kinase C activity was down-regulated, glucose stimulation still induced alkalinization. At 20 mM glucose, 10 mM NH4Cl induced a marked rise in pHi, paralleled by repolarization, inhibition of electrical activity, and decreases in both [Ca2+]i and insulin release. Reduction in [Ca2+]i was prevented by 200 microM tolbutamide, but not by 10 mM tetraethylammonium. At 4 mM glucose, NH4Cl induced a transient increase in insulin release, without changing [Ca2+]i. Exposure of beta-cells to 10 mM sodium acetate caused a persistent decrease in pHi, an effect paralleled by a small transient increase in [Ca2+]i. Acidification per se did not change the beta-cell sensitivity to glucose, not excluding that the activity of the ATP-regulated K+ channels may be modulated by changes in pHi.     

Effect of thyroid state on cytosolic free calcium in resting and electrically stimulated cardiac myocytes

The effects of the thyroid state on the cytosolic free Ca2+ concentration, [Ca2+]i, of resting and K+-depolarized cardiomyocytes were studied using the fluorescent Ca2+ indicator fura2. The mean resting [Ca2+]i in euthyroid myocytes (89 +/- 8 nM) was not significantly different from that in hyperthyroid myocytes (100 +/- 14 nM). The resting O2-consumption rate was identical for both groups when expressed per mg protein, but a 35% higher value was observed in the hyperthyroid group when expressed per cell on account of the cellular hypertrophy induced by thyroid hormone. Potassium induced depolarization (50 mM [K+]0) raised the level of [Ca2+]i by 50% in both groups. When ATP-coupled respiration was blocked with oligomycin, the 50 mM K+-induced rise in [Ca2+]i was accompanied in both groups by a 40% rise in glycolytic activity as inferred from measurement of lactate production. Ca2+-fluorescence transients were recorded from electrically stimulated myocytes of euthyroid, hyperthyroid and hypothyroid rats. The time taken to reach peak fluorescence (TPL) and that to 50% decay of peak fluorescence (RL0.5) decreased in the direction hypothyroid----hyperthyroid, indicating an increase in Ca2+ fluxes in the same direction. Isoproterenol (1 microM) enhanced the peak Ca2+ fluorescence in electrically stimulated hypothyroid and euthyroid myocytes but not in hyperthyroid myocytes. Both the TPL and RL0.5 were decreased by isoproterenol in euthyroid, but more so in hypothyroid myocytes. None of these parameters were influenced by isoproterenol in the hyperthyroid group. We conclude that (1) thyroid hormone increases neither the O2-consumption rate nor the level of [Ca2+]i of resting cardiomyocytes and (2) the effects of the beta-receptor-agonist isoproterenol on Ca2+ transients of electrically stimulated myocytes, are inversely related to the documented changes in beta-receptor density in heart tissue occurring with alterations in the thyroid state.     

Decreasing extracellular Na+ concentration triggers inositol polyphosphate production and Ca2+ mobilization

Removing extracellular Na+ (Na+o) evoked a large increase in cytosolic free Ca2+ concentration ([Ca2+]i in human skin fibroblasts. Decreasing [Na+]o from 120 to 14 mM caused the half-maximal peak increase in [Ca2+]i. Removing Na+o strongly stimulated 45Ca2+ efflux and decreased total cell Ca2+ by about 40%. Bradykinin caused changes in [Ca2+]i, total Ca2+, and 45Ca2+ fluxes similar to those evoked by removing Na+o. Prior stimulation of the cells with bradykinin prevented Na+o removal from increasing [Ca2+]i and vice versa. Na+o removal rapidly increased [3H]inositol polyphosphate production. Loading the cells with Na+ had no effect on the increase in 45Ca2+ efflux produced by Na+o removal. Therefore, decreasing [Na+]o probably stimulates a "receptor(s)" which is sensitive to extracellular, not intracellular, Na+. Removing Na+o also mobilized intracellular Ca2+ in smooth muscle and endothelial cells cultured from human umbilical and dog coronary arteries, respectively.     

Growth hormone promotes Ca(2+)-induced Ca2+ release in insulin-secreting cells by ryanodine receptor tyrosine phosphorylation

Elevation in cytoplasmic free Ca2+ concentration ([Ca2+]i) is a common mechanism in signaling events. An increased [Ca2+]i induced by GH, has been observed in relation to different cellular events. Little is known about the mechanism underlying the GH effect on Ca2+ handling. We have studied the molecular mechanisms underlying GH-induced rise in [Ca2+]i in BRIN-BD11 insulin-secreting cells. GH (500 ng/ml, 22 nm) induced a sustained increase in [Ca2+]i. The effect of GH on [Ca2+]i was prevented in the absence of extracellular Ca2+ and was inhibited by the ATP-sensitive K(+)-channel opener diazoxide and the voltage-dependent Ca(2+)-channel inhibitor nifedipine. However, GH failed to induce any changes in Ca2+ current and membrane potential, evaluated by patch-clamp recordings and by using voltage-sensitive dyes. When the intracellular Ca2+ pools had been depleted using the Ca(2+)-ATPase inhibitor thapsigargin, the effect of GH was inhibited. In addition, GH-stimulated rise in [Ca2+]i was completely abolished by ruthenium red, an inhibitor of mitochondrial Ca2+ transport, and caffeine. GH induced tyrosine phosphorylation of ryanodine receptors. The effect of GH on [Ca2+]i was completely blocked by the tyrosine kinase inhibitors genistein and lavendustin A. Interestingly, treatment of the cells with GH significantly enhanced K(+)-induced rise in [Ca2+]i. Hence, GH-stimulated rise in [Ca2+]i is dependent on extracellular Ca2+ and is mediated by Ca(2+)-induced Ca2+ release. This process is mediated by tyrosine phosphorylation of ryanodine receptors and may play a crucial role in physiological Ca2+ handling in insulin-secreting cells.     

Involvement of prostaglandins in histamine H1 receptor-operated Ca2+ entry in human gingival fibroblasts

In the absence of external Ca2+, 100 microM histamine evoked a transient increase in intracellular Ca2+ ([Ca2+]i), and subsequent addition of Ca2+ to the medium resulted in a sustained increase in [Ca2+]i in fura-2-loaded human gingival fibroblasts. These Ca2+ mobilizations are attributed to Ca2+ release from intracellular stores and Ca2+ entry, respectively. When the histamine H1 antagonist chlorpheniramine was added after the histamine-induced transient increase in [Ca2+]i, the Ca2+ entry induced by the addition of Ca2+ was inhibited. In the fibroblasts pretreated with cyclooxygenase inhibitors, indomethacin (1 microM) or aspirin (100 microM), histamine-induced Ca2+ entry was significantly inhibited, but not the transient [Ca2+]i increase. These results suggest that the histamine-induced Ca2+ entry requires the continuous binding of histamine to the H1 receptors and is regulated by prostaglandins, which are probably produced due to the H1 receptor activation.     

Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia on the intracellular Ca2+ concentration ([Ca2+]i) in rabbit pulmonary (PASMCs) and coronary arterial smooth muscle cells with fura-2. Perfusion of a glucose-free and hypoxic (PO2<50 mmHg) external solution increased [Ca2+]i in cultured as well as freshly isolated PASMCs. However it had no effect on [Ca2+]i in freshly isolated coronary arterial myocytes. In the absence of extracellular Ca2+, hypoxic stimulation elicited a transient [Ca2+]i increase in cultured PASMCs which was abolished by the simultaneous application of cyclopiazonic acid and ryanodine, suggesting the involvement of sarcoplasmic reticulum (SR) Ca2+ store. Pretreatment with the mitochondrial protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) enhanced the [Ca2+]i rise in response to hypoxia. A short application of caffeine gave a transient [Ca2+]i rise which was prolonged by CCCP. Decay of the caffeine-induced [Ca2+]i transients was significantly slowed by treatment of CCCP or rotenone. After full development of the hypoxia-induced [Ca2+]i rise, nifedipine did not decrease [Ca2+]i. These data suggest that the [Ca2+]i increase in response to hypoxia may be ascribed to both Ca2+ release from the SR and the subsequent activation of nifedipine-insensitive capacitative Ca2+ entry. Mitochondria appear to modulate hypoxia induced Ca2+ release from the SR.