Stimulation Of Calcium Efflux Research Articles

Lipid Domains at the Plasma Membrane of Red Blood Cells: Organization and Involvement in Deformation

Erythrocytes are highly deformable cells that go through capillaries eight times-narrower than their diameter to deliver oxygen throughout the body. This deformability is linked to erythrocytes specific features such as their biconcave shape, their highly regulated hemoglobin concentration and the strong anchorage of their membrane to the underlying cytoskeleton. Based on our recent evidence for submicrometric lipid domains upon labeling of cholesterol and polar lipids (sphingomyelin, phosphatidylcholine, ganglioside GM1) in erythrocyte outer plasma membrane leaflet, we here explore the mapping of lipid domains in erythrocytes at resting state and under deformation. Thanks to differential properties between cholesterol- and polar lipid-enriched domains (curvature association, lipid order, temperature dependence, cholesterol content dependence) and partial spatial dissociation, we suggest the coexistence of at least two types of lipid domains at the erythrocyte surface at resting state: those enriched in cholesterol and preferentially associated with high-curvature membranes and those mostly enriched in phosphatidylcholine and ganglioside GM1 and associated with low-curvature membranes. Biophysical properties of these domains are currently explored by atomic force microscopy and force correlation spectroscopy. Lipid domains abundance, composition and biophysical properties are differentially modulated by (i) impairment of erythrocyte deformability (energy depletion, modulation of anchorage to the cytoskeleton by pharmacological and genetic approaches), (ii) simulation of deformation (calcium channel activation, stretching in silicon chambers) and (iii) simulation of shape restoration after deformation (stimulation of calcium efflux). Indeed, high-curvature cholesterol-enriched domains gather under deformation while low-curvature domains become enriched in sphingomyelin and lose some of their ganglioside GM1 enrichment. We hypothesize that cholesterol-enriched domains can help to stabilize high curvature membranes during deformation while polar lipid-enriched domains, especially those enriched in sphingomyelin, are involved into calcium balance regulation during or after deformation. This hypothesis is under investigation.

Paclitaxel Induces Apoptosis in Breast Cancer Cells through Different Calcium—Regulating Mechanisms Depending on External Calcium Conditions

Previously, we reported that endoplasmic reticulum calcium stores were a direct target for paclitaxel initiation of apoptosis. Furthermore, the actions of paclitaxel attenuated Bcl-2 resistance to apoptosis through endoplasmic reticulum-mediated calcium release. To better understand the calcium-regulated mechanisms of paclitaxel-induced apoptosis in breast cancer cells, we investigated the role of extracellular calcium, specifically; whether influx of extracellular calcium contributed to and/or was necessary for paclitaxel-induced apoptosis. Our results demonstrated that paclitaxel induced extracellular calcium influx. This mobilization of extracellular calcium contributed to subsequent cytosolic calcium elevation differently, depending on dosage. Under normal extracellular calcium conditions, high dose paclitaxel induced apoptosis-promoting calcium influx, which did not occur in calcium-free conditions. In the absence of extracellular calcium an “Enhanced Calcium Efflux” mechanism in which high dose paclitaxel stimulated calcium efflux immediately, leading to dramatic cytosolic calcium decrease, was observed. In the absence of extracellular calcium, high dose paclitaxel’s stimulatory effects on capacitative calcium entry and apoptosis could not be completely restored. Thus, normal extracellular calcium concentrations are critical for high dose paclitaxel-induced apoptosis. In contrast, low dose paclitaxel mirrored controls, indicating that it occurs independent of extracellular calcium. Thus, extracellular calcium conditions only affect efficacy of high dose paclitaxel-induced apoptosis.

Platinum drugs and neurotoxicity: effects on intracellular calcium homeostasis

[Pt(O,O'-acac)(γ-acac)(DMS)] (PtAcacDMS) is a new platinum compound showing low reactivity with nucleobases and specific reactivity with sulfur ligands intracellularly. It induces apoptosis in breast cancer cells, but appears to be less neurotoxic to the developing cerebellum than cisplatin (cisPt). The aim of this study was to assess the neurotoxicity of platinum compounds on calcium homeostasis in the dentate gyrus and Cornu Ammonis regions of the hippocampal formation during rat postnatal development. Two intracellular calcium homeostasis systems were taken for measurement, calbindin, a calcium buffer protein, and a plasma membrane calcium ATPase (PMCA1). The platinum compounds showed different effects on these markers in the two areas. One day after injection (PD11), cisPt decreased calbindin immunoreactivity and PMCA1 labeling in both regions; at PD17, the downregulation of PMCA1 persisted. Instead, PtAcacDMS produced varying effects on calbindin immunoreactivity in the two regions at PD11 and PD17; but in all cases, the changes incurred in calbindin immunoreactivity were counterbalanced by changes produced in PMCA1 expression. In conclusion, PtAcacDMS seems to affect calcium homeostasis in the central nervous system differently than cisPt. Both the platinum compounds act early to alter the calbindin buffering system. However, the most important difference between cisPt and PtAcacDMS is that, in vivo, the latter acts early to stimulate calcium efflux from nerve cells as reflected by its effect on PMCA1. The rapid onset of an activated calcium pump appears to be essential to cope with the excessive intracellular calcium concentration stemming from the downregulation of calbindin which could damage neuron function and morphology.

Basic fibroblast growth factor (bFGF) regulation of the plasma membrane calcium ATPase (PMCA) as part of an anti-apoptotic mechanism of action

Basic fibroblast growth factor (bFGF) preserves the viability of at least 13 different cells, including epithelial, endothelial, smooth muscle and neuronal cells. In spite of this profound and rather universal effect on cell viability, detailed studies regarding the mechanism of bFGF’s action have not been conducted. Rather, most studies have simply shown that bFGF inhibits cells from undergoing programmed cell death (i.e. apoptosis). The most mechanistic studies to date have been conducted on either neurons or ovarian (granulosa) cells. These studies have shown that bFGF prevents apoptosis through both genomic and acute actions. Basic FGF’s acute actions involved the maintenance of normal levels of intracellular free calcium levels ([Ca 2+] i). In granulosa cells, bFGF maintained [Ca 2+] i through a protein kinase C delta (PKCδ)-dependent mechanism. Further, bFGF-activated PKCδ maintained [Ca 2+] i by stimulating calcium efflux. The ability of bFGF to stimulate calcium efflux involved the plasma membrane calcium ATPase (PMCA). Interestingly, bFGF-activated PKCδ appeared to regulate PMCA activity in part by promoting its membrane localization.

Basic Fibroblast Growth Factor Maintains Calcium Homeostasis and Granulosa Cell Viability by Stimulating Calcium Efflux via a PKCδ-Dependent Pathway

Previous studies have demonstrated that basic fibroblast growth factor prevents granulosa cell apoptosis. The following six observations provide insight into the mechanism by which basic fibroblast growth factor mediates its antiapoptotic action. First, loading granulosa cells with 1,2 bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, an intracellular calcium chelator, prevented apoptosis when granulosa cells were deprived of basic fibroblast growth factor. Second, treatment with thapsigargin, an agent known to increase intracellular free calcium, induced granulosa cell apoptosis even in the presence of basic fibroblast growth factor. Third, an activator of PKC mimicked, whereas PKC inhibitors blocked, basic fibroblast growth factor's antiapoptotic action. Fourth, continuous basic fibroblast growth factor exposure maintained relatively constant levels of intracellular free calcium, and a PKC inhibitor induced a sustained 2- to 3-fold increase in intracellular free calcium. Fifth, granulosa cells, as well as spontaneously immortalized granulosa cells, were shown to express PKC delta, -lambda, and -zeta. Finally, the PKC delta-specific inhibitor, rottlerin, blocked basic fibroblast growth factor's antiapoptotic action in granulosa cells and spontaneously immortalized granulosa cells. These studies suggest that basic fibroblast growth factor regulates intracellular free calcium through a PKC delta-dependent mechanism and that a sustained increase in intracellular free calcium is sufficient to induce and is required for granulosa cell apoptosis. Additional studies demonstrated that in spontaneously immortalized granulosa cells, basic fibroblast growth factor increased PKC delta activity by 60% within 2.5 min compared with serum-free control levels. Rottlerin attenuated basic fibroblast growth factor's ability to stimulate PKC delta activity and to maintain intracellular free calcium. Further, intracellular free calcium levels in spontaneously immortalized granulosa cells transfected with a PKC delta antibody in the presence of basic fibroblast growth factor were 2-fold higher than those spontaneously immortalized granulosa cells transfected with IgG. Similarly, transfecting spontaneously immortalized granulosa cells with a specific PKC delta-substrate increased intracellular free calcium compared with spontaneously immortalized granulosa cells transfected with a specific substrate for PKC epsilon. Moreover, basic fibroblast growth factor increased and rottlerin attenuated (45)Ca efflux by 50% compared with that in basic fibroblast growth factor-treated cells. Finally, an inhibitor of the plasma membrane calciumadenosine triphosphatase pump suppressed (45)Ca efflux, elevated intracellular free calcium, and induced apoptosis. Collectively, these studies demonstrate that basic fibroblast growth factor activates PKC delta, which, in turn, stimulates calcium efflux, accounting in part for basic fibroblast growth factor's ability to maintain calcium homeostasis and, ultimately, granulosa cell viability.

Parathyroid hormone(1-34) and parathyroid hormone-related protein(1-34) stimulate calcium release from human syncytiotrophoblast basal membranes via a common receptor.

The placental syncytiotrophoblast is the site for mineral and nutrient exchange across the maternal-fetal interface. It has been proposed that parathyroid hormone-related protein (PTHrP) is a key factor in the maintenance of a maternal-fetal calcium gradient. Using simultaneously prepared microvillous (maternal facing) and basal (fetal facing) syncytiotrophoblast membranes from term human placentae (n=8), we determined the relative contribution of PTH(1-34), PTHrP(1-34) and PTHrP(67-94) to the regulation of syncytiotrophoblast calcium efflux. The vesicles had correct right-side-out membrane orientation and specific markers validated the fractionation of microvillous and basal membrane vesicles. Calcium efflux was studied by preloading vesicles with calcium-45 in the presence of calcium and magnesium and then incubating the vesicles at 37 degrees C for 15 min with the peptides. In basal membranes, PTHrP(1-! 34) significantly stimulated calcium efflux at a dose of 12.5 nmol/l, whereas PTH(1-34)-stimulated efflux was significant at 50 nmol/l (P<0.05, ANOVA). This efflux was significantly reduced in the presence of the PTH/PTHrP receptor antagonist (PTHrP(7-34)). Midmolecule PTHrP(67-94) had no significant effect on basal membrane calcium efflux. PTH(1-34), PTHrP(1-34) or PTHrP(67-94) had no significant effects on MVM calcium efflux. This study, using the human syncytiotrophoblast in vitro membrane system, demonstrated that PTHrP(1-34) and PTH(1-34) stimulate calcium transport across the basal, but not microvillous, syncytiotrophoblast membrane vesicles, mediated via the PTH/PTHrP receptor.

Transmembrane Regulation of Intracellular Calcium by a Plasma Membrane Sodium/Calcium Exchanger in Mouse Ova1

Regulation of cytoplasmic free calcium concentration ([Ca2+)]i) is a key factor for maintenance of viability of cells, including oocytes. Indeed, during fertilization of an ovum, [Ca2+]i is known to undergo oscillations, but it is unknown how basal [Ca2+]i or calcium oscillations are regulated. In the present study we investigated the role of the plasma membrane in regulating [Ca2+]i of metaphase II-arrested mouse oocytes (ova). Ova were collected from B6C3F1 mice treated with eCG (10 IU) and hCG (5 IU), and intracellular calcium was determined by means of fura-2. Extracellular calcium flux across the zona pellucida was detected noninvasively by a calcium ion-selective, self-referencing microelectrode that was positioned by a computer-controlled micromanipulator. Under basal conditions ova exhibited a calcium net efflux of 20.6 +/- 5.2 fmol/cm2 per sec (n = 69). Treatment of ova with ethanol (7%) or thapsigargin (25 nM-2.5 microM) transiently increased intracellular calcium and stimulated calcium efflux that paralleled levels of [Ca2+]i. The presence of a Na+/Ca2+ exchanger was indicated by experiments employing both bepridil, an inhibitor of Na+/Ca2+ exchange, and sodium-depleted media. In the presence of bepridil, a net influx of calcium was revealed across the zona pellucida, which was reflected by an increase in the [Ca2+]i. In addition, replenishment of extracellular sodium to ova that had been incubated in sodium-depleted media induced a large calcium efflux, consistent with the actions of Na+/Ca2+ exchange. Sodium/calcium exchange in mouse ova may be an important mechanism that regulates [Ca2+]i.

Prostaglandins protect human intestinal cells against ethanol injury by stabilizing microtubules: role of protein kinase C and enhanced calcium efflux.

Prostaglandins (PG) protect gastrointestinal cells against damage induced by ethanol (EtOH) and other noxious agents, a process termed cytoprotection. The present study investigated the relationships between microtubule (MT) stability, protein kinase C (PKC) activation, and calcium efflux as a possible mechanism of PG's protective action using a human colonic cell line (Caco-2) exposed to known damaging concentrations of EtOH (7.5% and 10%). Preincubation of Caco-2 cells with 16,16-dimethyl-PGE2 (PG, 2.6 microM) significantly increased PKC activity in these cells. Pretreatment of Caco-2 cells with 50 microM OAG (a synthetic diacylglycerol and PKC activator) or 30 nM TPA (a direct PKC activator) prior to exposure to 7.5% or 10% EtOH for 5 min significantly reduced cell injury, as determined by trypan blue exclusion, and increased MT stability, as confirmed by confocal microscopy. Pretreatment of Caco-2 cells with 4 alpha-PDD (an inactive phorbol ester, 20 nM) failed to prevent cell injury and disruption of the MT cytoskeleton. Preincubation with staurosporine (a PKC inhibitor, 3 nM) abolished the protective effects of PG in cells exposed to 7.5% and 10% EtOH. Incubation of Caco-2 cells with A23187 (a Ca2+ ionophore), similar to 10% EtOH, caused a significant reduction in cell viability and MT stability. Preincubation with A23187 in combination with PG or OAG prior to subsequent exposure to EtOH significantly abolished the protective effects of PG or OAG pretreatment. Finally, pretreatment with OAG, TPA, or PG resulted in significant increases in calcium-45 efflux, which correlated with increased stability of the MT cytoskeleton. These data suggest that PG possesses direct protective effects against EtOH injury in Caco-2 cells and may act by stabilizing MT through the PKC signal transduction pathway and/or stimulation of calcium efflux from the cells.

Protein Kinase C Activation Stimulates Calcium Transport in Adrenal Zona Glomerulosa Cells

Adrenal zona glomerulosa (ZG) cells produce aldosterone in response to angiotensin II and extracellular potassium through different mechanisms which involve changes in cytosolic free calcium (Cai). Protein kinase C (PKC) activation is part of the angiotensin II signalling cascade but its effects on Caiare unknown. PKC activation with 1 μM phorbol 12-myristate 13-acetate (PMA) and 8 mM Kosignificantly increased the rate of calcium influx (P < 0.001). Both the PKC- and the Ko-induced calcium influx occurred via a nifedipine-sensitive pathway. When both were combined, PKC activation and 8 mM Kowere not additive over either agent alone. PKC activation and 8 mM Koalso stimulated calcium efflux (P < 0.01). When combined together PKC activation and 8 mM Kohad additive effects on calcium efflux (P < 0.05). PKC activation did not increase Cainor the exchangeable calcium pool in contrast to 8 mM Kowhich significantly increased both (P < 0.001). Thus, PKC activation in ZG cells induces a pattern of calcium transport characterized by accelerated calcium recycling across the cell membrane without increasing cell calcium content.

Adrenomedullin stimulates calcium efflux from adrenal chromaffin cells in culture: Possible involvement of an [formula omitted] exchange mechanism

The effect of adrenomedullin, a hypotensive peptide, on Ca 2+ efflux from cultured bovine adrenal chromaffin cells was examined. Adrenomedullin stimulated the efflux of 45Ca 2+ from the cells in a concentration-dependent manner (10 −7 M − 3 × 10 −6 M). Adrenomedullin did not increase the intracellular free Ca 2+ ([Ca 2+] i) level and catecholamine secretion. The adrenomedullin-stimulated 45Ca 2+ efflux was not inhibited by incubation with Ca 2+-free medium, but was inhibited by incubation with Na +-free medium. These results indicate that adrenomedullin stimulates extracellular Na +-dependent 45Ca 2+ efflux from cultured bovine adrenal chromaffin cells, probably through its stimulatory effect on membrane Na + Ca 2+ exchange.

Stimulation of Calcium Efflux from the Hagfish, Eptatretus cirrhatus, Gill Pouch by an Extract of Corpuscles of Stannius from an Eel (Anguilla dieffenbachii): Teleostei

A factor present in the homogenate of the corpuscles of Stannius from a teleost, the longfin eel Anguilla dieffenbachii, showed calcitropic effects in an agnathan, the hagfish Eptatretus cirrhatus. This factor greatly increased calcium efflux from an isolated perfused gill pouch preparation and had no effect on calcium influx. Hagfish regulated plasma total calcium at a concentration which was about 50% of that of the ambient seawater. Intact hagfish had rates of calcium influx and efflux which were similar to those recorded in teleosts. Hagfish are thus similar to teleosts to the extent that they can and do maintain a plasma calcium concentration which is quite different from that of the ambient medium. They also resemble teleosts in that transepithelial calcium fluxes across the hagfish gill pouches respond to extracts of Stannius corpuscles. Hagfish, however, are quite unlike teleosts in that the Stannius corpuscle extract stimulates calcium efflux rather than inhibiting calcium influx.

Solvent-Dependent Influences on Skeletal Muscle Sarcoplasmic Reticulum Calcium Uptake and Release

The organic cosolvents propylene glycol (PG) and polyethylene glycol 400 (PEG 400) have previously been shown to differ in their potential to cause muscle damage following im injection. PG was found to be more myotoxic than PEG 400, with indirect implications of the role of cytosolic calcium in mediating this damage. In the present study, the direct effects of these cosolvents were investigated on the sarcoplasmic reticulum (SR), the major intracellular muscle membrane that mobilizes calcium. The passive permeability of isolated SR microsomal vesicles to calcium was not affected by 5.3 and 10.5% (v/v) PG and PEG 400. At 10.5% (v/v), a concentration of the organic cosolvent that would not be unexpected at the injection site, PEG 400 stimulated calcium uptake by 40 and 140% in longitudinal tubular-derived and terminal cisternal-derived vesicles, respectively, without significantly altering the ATP hydrolytic activity of the calcium pump. The calcium pumping efficiency (Ca2+/ATP coupling ratio) was therefore also enhanced. On the other hand, 10.5% (v/v) PG did not significantly alter either calcium uptake or ATPase activity of the pump. PG stimulated calcium efflux from only the terminal cisternae vesicles via a pathway indicative of the ryanodine-sensitive calcium channel, as demonstrated by inhibition of PG-induced efflux by millimolar Mg2+. These results are consistent with multiple interactions of cosolvents with proteins in the membrane bilayer, with the distinction that the two cosolvents differentially influence the calcium pump and release channel, particularly at the terminal cisternae, where there is rapid change of calcium level during excitation-contraction coupling. These data provide further evidence for the role of calcium in mediating organic cosolvent-induced muscle damage. In addition, they provide a possible explanation for the myoprotective effect of PEG 400 (compared to PG) as a result of increased myoplasmic calcium removal and reduced calcium release.

Hormone-induced phosphorylation of the plasma membrane calcium pump in cultured aortic endothelial cells

The regulation of the plasma membrane Ca 2+ pump by hormones via phosphorylation in intact cells has not been clearly established. We now present evidence that the Ca 2+ pump is phosphorylated on both serine and threonine residues in unstimulated and stimulated cultured rat aortic endothelial cells. Among the stimuli tested, the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was most potent and increased the level of phosphorylation threefold, while the cAMP-dependent protein kinase activator 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) stimulated the phosphorylation 1.6-fold. Two-dimensional tryptic phosphopeptide maps of the Ca 2+ pump from unstimulated and CPT-cAMP-stimulated cells have identical patterns (five phosphopeptides) while PMA-stimulated cells have three additional phosphopeptides. Isoproterenol-, ATP-, angiotensin II-, and bradykinin-stimulated cells also have increased levels of Ca 2+ pump phosphorylation. Stimuli-induced phosphorylation of the Ca 2+ pump was rapid (5–10 min) and was concomitant with stimulated calcium efflux from the same cells. This is the first direct evidence that the plasma membrane Ca 2+ pump in intact cells is regulated by various hormones or agonists via cAMP-dependent protein kinase or protein kinase C phosphorylation.

Sodium inhibits hormone release and stimulates calcium efflux from isolated nerve endings of the rat neurohypophysis

1. We studied the effects of extracellular sodium on the secretion of vasopressin (VP) and oxytocin (OT) and the efflux of 45Ca from isolated, perfused nerve endings of the rat neurohypophysis (neurosecretosomes). 2. Upon removal of sodium from the perfusing medium, basal release of VP and OT increased by 3.95 +/- 0.23- and 3.71 +/- 0.22-fold, respectively, followed by a decline to about double the levels in normal (150 mM) sodium (P less than or equal to 0.1). 3. Compared to neurosecretosomes perfused in normal (150 mM) sodium, omission of sodium from the medium augmented ionomycin-induced VP and OT secretion by 66 +/- 5- and 20 +/- 3-fold, respectively, and A23187-induced secretion was increased 1.3 +/- 0.4- and 1.3 +/- 0.1-fold (P less than or equal to 0.01 for both ionophores). 4. The inhibition of ionomycin-induced secretion by sodium was concentration dependent (P less than or equal to 0.01 for sodium greater than or equal to 5 mM); the IC50 was about 10 mM sodium for both hormones, and the Hill slope was close to -1. 5. The rate of 45Ca efflux from neurosecretosomes showed 2.7 +/- 0.1-fold stimulation upon increasing sodium from 4.5 to 150 mM (P less than or equal to 0.01). 6. Our results suggest that sodium inhibits basal and stimulated secretion at the nerve terminal, possibly by reducing intraterminal calcium through sodium/calcium exchange.

Mechanism of amphotericin B stimulation of net calcium efflux from neonatal mouse calvariae

Amphotericin B is a polyene antifungal agent that binds to membrane sterols, creating aqueous pores that permit ion fluxes sufficient to cause cell lysis. It has also been shown to alter ion transport in mammalian cells, including proton secretion from renal tubular cells. The latter effect can lead to distal renal tubular acidosis in patients treated for systemic fungal infections. Based on the understanding that osteoclast-mediated bone resorption is dependent on proton secretion, we examined the effect of amphotericin B on calcium efflux from neonatal mouse calvariae in organ culture. Amphotericin B (5 micrograms/ml) stimulated net calcium efflux from calvariae within 24 h to a level almost as great as that produced by a maximally effective concentration of parathyroid hormone. The stimulated calcium efflux was completely inhibited by both 10 ng/ml salmon calcitonin, a physiologic inhibitor of osteoclast activity, and 4 x 10(-4) M acetazolamide, a specific inhibitor of carbonic anhydrase, the enzyme necessary for substantial proton generation by osteoclasts. These results indicate a direct effect of amphotericin B on bone in vitro to stimulate osteoclast-mediated calcium efflux.

Two distinct receptors for ATP can be distinguished in Swiss 3T6 mouse fibroblasts by their desensitization

The stimulation of calcium efflux from Swiss 3T6 mouse fibroblasts by extracellular ATP was studied. It was found that the cells could be desensitized to ATP by a previous exposure to the nucleotide, lending support to the theory that this is a receptor mediated process. Another ATP-receptor mediated process in Swiss 3T6 cells, that is also subject to desensitization, causes the permeabilization of the plasma membrane to nucleotides and other normally impermeant compounds [Gonzalez et al., J. Cell. Physiol. 139:109 (1989)]. Here we demonstrate that selective desensitization of the ATP-dependent calcium mobilization pathway can be achieved without affecting ATP-induced permeabilization. Data are presented in support of the existence of multiple ATP-receptors (purinoceptors) in Swiss 3T6 cells.

Verapamil: a novel probe of surfactant secretion from rat type II pneumocytes.

Surfactant from type II pneumocytes prevents the alveolar atelectasis found in both the neonatal and adult forms of respiratory distress syndrome. We have found that verapamil, a phenylalkene with calcium channel and alpha 1-receptor binding properties, has a multiphasic concentration effect on surfactant secretion from [3H]choline-labeled rat type II pneumocytes in culture. Verapamil (10(-8) M) caused a 24% stimulation of surfactant secretion, whereas an 8% inhibition was found at 10(-6) M and a 70% stimulation was found at 10(-4) M. Lactate dehydrogenase release occurred at 5 x 10(-4) M verapamil. Verapamil (10(-4) M) also produced a 100% increase in adenosine 3',5'-cyclic monophosphate (cAMP) in comparison with concentrations of less than or equal to 10(-6) M, an effect that could not be blocked by propranolol (10(-4) M). Verapamil (10(-6) M) increased the total formation of inositol phosphates (IP) by 23% in comparison with IP formation in control cells. Calcium influx was inhibited 15% by 10(-8) M verapamil and 37% by 10(-4) M verapamil. Calcium efflux was stimulated 44% by 10(-5) M verapamil. In combination with 50% effective concentrations (EC50) of terbutaline, phorbol ester, and ATP, the respective effects of verapamil (10(-4) M) on surfactant secretion were approximately additive. We conclude that verapamil has a novel multiphasic concentration effect on surfactant secretion, which appears to involve several signal transduction pathways including cAMP formation, IP formation, inhibition of calcium influx, and stimulation of calcium efflux.

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.

Age-related impairment in rat parotid cell α1-adrenergic action at the level of inositol trisphosphate responsiveness

α 1-Adrenergic-stimulated calcium efflux from rat parotid cell aggregates declines approx. 40% between 3 and 24 months of age, with the bulk of the reduction occurring between 12 and 24 months. Intracellular free calcium levels following α 1-adrenoceptor stimulation are also reduced about 40% between 3 and 24 months. No significant age differences in stimulation of inositol mono-, bis- or trisphosphate production are observed. However, the ability of inositol trisphosphate to directly stimulate calcium efflux is reduced by about 50% with increasing age. Concentrations of this inositol phosphate required for maximal calcium release do not change between 3 and 24 months. Differences in response are not due to a reduction in uptake of inositol trisphosphate into older cells, but suggest an age-related defect in the ability of inositol trisphosphate to liberate calcium from intracellular stores. Such dysfunction may be at least partially responsible for impaired α 1-adrenergic responsiveness during aging.

Stimulated calcium efflux from fura‐2‐loaded human platelets.

1. The reduction of cytoplasmic free calcium, [Ca2+]i following stimulation, has been investigated in fura-2-loaded human platelets in the presence of low extracellular calcium concentration. Thrombin produced a rapid rise in [Ca2+]i which then fell back to the basal level within 2 min. 2. Ionomycin produced a rapid elevation in [Ca2+]i which then declined to a plateau well above the basal calcium level. The addition of thrombin after ionomycin accelerated the decline in [Ca2+]i back towards basal levels, an action mimicked by phorbol myristate acetate (PMA). 3. Thrombin promoted the efflux of 45Ca2+ from cells co-loaded with fura-2 and the isotope. Ionomycin also promoted an efflux of 45Ca2+ which was increased by the subsequent addition of thrombin or PMA. These results confirm the ability of thrombin and PMA to stimulate Ca2+ removal from the cells. 4. The complete substitution of extracellular Na+ with N-methyl-D-glucamine (NMDG) did not alter the time course of the return of [Ca2+]i to basal following stimulation by thrombin, nor the ability of thrombin or PMA to promote Ca2+ efflux after elevation of [Ca2+]i by ionomycin. 5. The insensitivity to external Na+ suggests that the stimulated Ca2+ efflux is mediated by a Ca2+-ATPase rather than Na+-Ca2+ exchange. This pump does not appear to be activated by Ca2+-calmodulin since [Ca2+]i remains high when elevated by ionomycin. The ability of PMA to stimulate removal suggests that its known target, protein kinase C, can stimulate the Ca2+ pump. Forskolin, which stimulates adenylate cyclase, did not stimulate a fall in [Ca2+]i in the presence of ionomycin, indicating that cyclic AMP-dependent protein kinase does not stimulate Ca2+ extrusion.