Production of NAADP and its role in Ca2+ mobilization associated with lysosomes in coronary arterial myocytes

The present study was designed to determine the production of nicotinic acid adenine dinucleotide phosphate (NAADP) and its role associated with lysosomes in mediating endothelin-1 (ET-1)-induced vasoconstriction in coronary arteries. HPLC assay showed that NAADP was produced in coronary arterial smooth muscle cells (CASMCs) via endogenous ADP-ribosyl cyclase. Fluorescence microscopic analysis of intracellular Ca2+ concentration ([Ca2+]i) in CASMCs revealed that exogenous 100 nM NAADP increased [Ca2+]i by 711 +/- 47 nM. Lipid bilayer experiments, however, demonstrated that NAADP did not directly activate ryanodine (Rya) receptor Ca2+ release channels on the sarcoplasmic reticulum. In CASMCs pretreated with 100 nM bafilomycin A1 (Baf), an inhibitor of lysosomal Ca2+ release and vacuolar proton pump function, NAADP-induced [Ca2+]i increase was significantly abolished. Moreover, ET-1 significantly increased NAADP formation in CASMCs and resulted in the rise of [Ca2+]i in these cells with a large increase in global Ca2+ level of 1,815 +/- 84 nM. Interestingly, before this large Ca2+ increase, a small Ca2+ spike with an increase in [Ca2+]i of 529 +/- 32 nM was observed. In the presence of Baf (100 nM), this ET-1-induced two-phase [Ca2+]i response was completely abolished, whereas Rya (50 microM) only markedly blocked the ET-1-induced large global Ca2+ increase. Functional studies showed that 100 nM Baf significantly attenuated ET-1-induced maximal constriction from 82.26 +/- 4.42% to 51.80 +/- 4.36%. Our results suggest that a lysosome-mediated Ca2+ regulatory mechanism via NAADP contributes to ET-1-induced Ca2+ mobilization in CASMCs and consequent vasoconstriction of coronary arteries.     

Effect of t-butyl hydroperoxide on Ca2+ movement in PC12 pheochromocytoma cells

The effect of the oxidant t-butyl hydroperoxide on intracellular free levels of Ca2+ ([Ca2+]i) in PC12 pheochromocytoma cells was examined by using fura-2 as a fluorescent dye. t-Butyl hydroperoxide induced an increase in [Ca2+]i in a concentration-dependent fashion between 50-250 microM with an EC50 of 100 microM. The [Ca2+]i signal consisted of a slow rise and a sustained phase. The response was decreased by 65% by removal of extracellular Ca2+. In Ca(2+)-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) abolished 150 microM t-butyl hydroperoxide-induced [Ca2+]i increase, and conversely, pretreatment with t-butyl hydroperoxide abrogated thapsigargin-induced [Ca2+]i increase. The 150 microM t-butyl hydroperoxide-induced [Ca2+]i increase in Ca2+ medium was reduced by 42 +/- 5% by pretreatment with 0.1 microM nicardipine but not by 10 microM verapamil, nifedipine, nimodipine or diltiazem, or by 50 microM La3+ or Ni2+. Pretreatment with 10 microM t-butyl hydroperoxide for 40 min did not affect 10 microM ATP-induced [Ca2+]i increase. Together, the results show that t-butyl hydroperoxide induced significant [Ca2+]i increase in PC12 cells by causing store Ca2+ release from the thapsigargin-sensitive endoplasmic reticulum pool in an inositol 1,4,5-trisphosphate-independent manner and by inducing Ca2+ influx via a nicardipine-sensitive pathway.     

Intracellular Ca2+ mobilization in immature and more mature U937 induced to differentiate by dimethyl sulfoxide or phorbol myristate acetate

Intracellular Ca2+ mobilization in U937 cells was studied. Stimulation of immature U937 cells with leukotriene B4 (LTB4) increased intracellular Ca2+ levels, whereas stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) failed to increase intracellular Ca2+ levels. U937 cells cultured with 1.5% dimethyl sulfoxide (DMSO) for 4 days (DMSO-U937 cells) responded to LTB4 and possessed the ability to respond to fMLP. U937 cells cultured with 1 ng/ml phorbol myristate acetate (PMA) for 4 days (PMA-U937 cells) lost the ability to respond to LTB4, although they responded to fMLP. Treatment of DMSO-U937 cells with 100 ng/ml PMA for 3 min suppressed intracellular Ca2+ increase induced by LTB4 and fMLP. The fMLP-induced Ca2+ rise in PMA-U937 cells was not suppressed by a further treatment with 100 ng/ml PMA. DMSO-U937 cells responded to inositol 1,4,5-trisphosphate (IP3), indicating that IP3 functions as a messenger of intracellular Ca2+ mobilization from endoplasmic reticulum in U937. The magnitude and duration of the rise in Ca2+ induced by IP3 in DMSO-U937 cells treated with 100 ng/ml PMA for 3 min were similar to those of the controls. When DMSO-U937 cells were Ca2+-depleted, addition of Ca2+ resulted in a transient overshoot of Ca2+ influx. However, the transient overshoot was not observed, when PMA-U937 cells were tested. These results indicate that Ca2+ efflux in PMA-U937 cells is increased by an activated exit pump, which may be directly or indirectly related to the functional state of PMA-U937 cells.     

Electrophysiological study of the action mechanism of somatoliberin (GH-RH) on hypophyseal GH3 tumor cells]

We have investigated the electrical response of patched GH3 cells to Growth-Hormone Releasing-Hormone (GH-RH). GH-RH (100 nM) enhanced firing frequency of action potentials. This is accompanied by membrane depolarization (5-10 mV) and conductance increase. Voltage clamp studies reveal that GH-RH potentiates calcium inward currents and a calcium-dependent chloride current; transient outward current is diminished. These changes in membrane conductance account for the cytosolic free calcium rise shown by Indo-1 fluorescence measurements.     

Muscarinic receptor-mediated increase in cytoplasmic free Ca2+ in isolated bovine adrenal medullary cells. Effects of TMB-8 and phorbol ester TPA

The change in cytoplasmic free calcium, [Ca2+]i in isolated bovine adrenal medullary cells during stimulation by acetylcholine (ACh) in Ca2+-free incubation medium was measured using the fluorescent Ca2+ indicator quin2. ACh (1-100 microM) caused an increase in [Ca2+]i by mobilization of Ca2+ from the intracellular pool. Nicotine (10 microM) did not increase [Ca2+]i in the absence of extracellular Ca2+. Pretreatment of the cells with atropine (10 microM) completely inhibited ACh-induced increase in [Ca2+]i, whereas pretreatment with hexamethonium (100 microM) did not. The intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), inhibited ACh-induced increase in [Ca2+]i. The activator of protein kinase C 12-O-tetradecanoylphorbol-13-acetate (TPA), but not its 'inactive' analog 4 alpha-phorbol-12,13-didecanoate (PDD), also inhibited ACh-induced increase in [Ca2+]i. These findings suggest that in bovine adrenal medullary cells, stimulation of muscarinic ACh receptor causes an increase in [Ca2+]i by mobilizing Ca2+ from the intracellular pool and that protein kinase C is involved in 'termination' or 'down regulation' of this response.     

Extracellular calcium-dependent regulation of transmembrane calcium fluxes in murine keratinocytes.

Because the level of extracellular Ca2+ is an important stimulus for differentiation of epidermal cells in vitro, we characterized the extracellular Ca(2+)-dependent transmembrane Ca2+ fluxes in BALB/MK mouse keratinocytes. Increasing levels of extracellular Ca2+, ranging from 0.07 to 1.87 mM, stimulated the rate of 45Ca2+ uptake into these cells 10- to 70-fold and doubled the rate of 45Ca2+ efflux. The divalent cations, Ni2+ and Co2+, were able to block the influx of Ca2+, but dihydropyridines and verapamil were not. Furthermore, 10 to 100 microM of the trivalent cation La3+ induced a dose-dependent 2- to 100-fold increase of Ca2+ uptake, independently of the level of extracellular Ca2+. These observations suggest that keratinocytes possess a cell-surface "Ca(2+)-receptor," activation of which stimulates the influx of 45Ca2+ through a type of voltage-independent, receptor-operated Ca2+ channels. Epidermal growth factor induced an accumulation of 45Ca2+ of a much smaller magnitude than elevations of the level of extracellular Ca2+, without a detectable increase of Ca2+ efflux. Thus, the divergent cellular responses of keratinocytes to EGF and extracellular Ca2+ may be due, in part, to the distinct changes in transmembrane Ca2+ fluxes that these two stimuli generate. Treatment of cells with type beta transforming growth factor led to a gradual 6-fold increase of the Ca(2+)-activated rate of Ca2+ uptake over a period of 4 hours, but reduced the Ca2+ efflux by approximately 50% within 10 minutes. Thus, type beta transforming growth factor apparently stimulates Ca2+ influx indirectly, but may control the differentiation of keratinocytes by direct inhibition of Ca2+ efflux pumps.     

Neopterin inhibits ATP-induced calcium release in alveolar epithelial cells in vitro

BACKGROUND: Serum neopterin concentrations rise during activation of the cellular immune system. It is suggested that neopterin interacts with cellular redox mechanisms. This induces oxidative stress, which inhibits intracellular Ca2+ transients in various cell types. In type II alveolar epithelial cells, Ca2+ increase is considered involved in the exocytosis of surfactants. This exocytosis is disturbed during inflammation. AIMS: To clarify whether neopterin affects adenosine triphosphate (ATP)-induced Ca2+ transients in an alveolar epithelial cell line (L2). METHODS: Ca2+ transients were detected as fura-2 fluorescence by an image analysis system. RESULTS: Cells were exposed for 100 sec to ATP (1 microM, repeated four times). The first application of ATP induced an increase of the fluorescence ratio by approximately 100%, while the following stimulations resulted in smaller transients. In a second set of experiments, L2 cells were exposed to ATP or ATP + neopterin (100 nM), alternately. Simultaneous application of neopterin inhibited Ca2+ transients almost completely. CONCLUSIONS: Inhibition of Ca2+ transients by neopterin may lead to suppressed exocytosis of surfactant proteins in alveolar epithelial cells. This might contribute to the deterioration of pulmonary functions in the course of inflammatory processes.     

Ca2+-activated Na+ fluxes in human red cells. Amiloride sensitivity

The effect of Ca2+ on the ouabain- and bumetanide-resistant Na+ fluxes in intact red cells was studied at relatively constant internal Ca2+, membrane potential, and cell volume. The red cell calcium concentration was modified using the ionophore A23187. In fresh red cells, the Na+ influx and efflux (1.2 +/- 0.13 and 0.26 +/- 0.07 mmol/liter cells x h, respectively) were not affected by amiloride (1 mM). When external Ca2+ was raised from 0 to 150 microM, in the presence of A23187, both the Na+ influx and efflux were stimulated (about 3.5-fold). The Ca2+-activated Na+ efflux and influx had an apparent Km for activation by Ca2+o of about 25 microM. The Ca2+-dependent Na+ transport was inhibited 30-60% by amiloride (ID50 = 17.3 +/- 8 microM). Amiloride, however, had no effect on the Ca2+-dependent K+ influx. The amiloride-sensitive (AS) transport pathway was a linear function of the Na+o concentration in the range from 0 to 75 mM. The Ca2+i activation seems to depend on the metabolic integrity of red cells. 1) It does not take place in ATP-depleted red cells; 2) ATP-repletion of ATP-depleted red cells fully restored AS Na influx; and 3) ATP-enrichment (ATP-red cells) enhanced the AS Na influx by about 100%. The Ca2+-activated AS Na+ influx was not affected by either DIDS or trifluoperazine. The present results indicate that in human erythrocytes an increase in internal Ca2+ activates on otherwise silent AS Na+-transport system, which is dependent on the metabolic integrity of the red cells.     

Participation of transient-type Ca2+ channels in the sustained increase of Ca2+ level in GH3 cells

Participation of two types of Ca2+ channels (T- and L-types) in the sustained increase of cytosolic-free Ca2+ concentration [( Ca2+]i) was studied in thyrotropin-releasing hormone (TRH)-stimulated clonal GH3 pituitary cells. The effects of Ca2+ channel blockers were analyzed by measuring Ca2+ channel current and [Ca2+]i, using whole-cell voltage-clamp and Fura-2 fluorometry, respectively. Phenytoin (100 microM) and Ni2+ (100 microM) selectively blocked T-type Ca2+ channels and suppressed the TRH-induced sustained [Ca2+]i increase in single cells. Synthetic omega-conotoxin (omega-CgTX, 2 microM) preferentially blocked L-type Ca2+ channels, but it did not suppress the TRH-induced sustained [Ca2+]i increase. The present results suggest that the sustained elevations of [Ca2+]i triggered by TRH may be mediated by T-type Ca2+ channels in GH3 cells.     

Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells

We have investigated the effects of extracellular and intracellular Ca deficits and of pharmacologic agents thought to inhibit Ca influx or intracellular Ca mobilization on vasopressin-evoked changes of cytosolic Ca2+ levels and PG synthesis in cultured rat mesenteric arterial vascular smooth muscle cells. Vasopressin rapidly increased cytosolic Ca2+ as well as PG synthesis. The increase of cytosolic Ca2+ and the rate of PG synthesis were both maximal within the first minute of incubation. An extracellular Ca deficit of short duration partially inhibited both vasopressin-evoked PG synthesis and the increase of cytosolic Ca2+ by 40 to 60%. Two procedures which deplete cells of some of their intracellular Ca, namely a 30 min incubation in EGTA-supplemented, Ca-lacking media, or a 1 min incubation with ionophore A23187 in Ca-deficient media, decreased PG synthesis by 65% to 100%. The addition of extracellular Ca to Ca-depleted cells restored the ability of vasopressin to stimulate PG synthesis. Two Ca channel antagonists, nifedipine or cinnarizine, had no effect on either vasopressin-evoked PG synthesis or increased cytosolic Ca2+, whereas TMB-8 (10 microM), a putative inhibitor of intracellular Ca mobilization, decreased PG synthesis by 75% by inhibiting acylhydrolase as well as cyclo-oxygenase activities, but had no effect on basal or vasopressin-evoked increase of cytosolic Ca2+, documenting that its inhibitory effect was not a consequence of decreased cytosolic Ca2+. These results demonstrate that decreased cellular Ca levels are associated with decreased cytosolic Ca2+ levels and PG synthesis, and support the hypothesis of a link between, on the one hand, cellular Ca and/or cytosolic Ca2+ and on the other hand, PG synthesis.     

Calcium dependence of the thrombin-induced increase in endothelial albumin permeability

We examined whether the increase in endothelial albumin permeability induced by alpha-thrombin is dependent on extracellular Ca2+ influx. Permeability of 125I-albumin across confluent monolayers of cultured bovine pulmonary artery endothelial cells was measured before and after the addition of 0.1 microM alpha-thrombin. In the presence of normal extracellular Ca2+ concentration ([Ca2+]o, 1000 microM), alpha-thrombin produced a 175 +/- 10% increase in 125I-albumin permeability. At lower [Ca2+]o (100, 10, 1, or less than 1 microM), alpha-thrombin caused a 140% increase in permeability (P less than 0.005). LaCl3 (1 mM), which competes for Ca2+ entry, blunted 38% of the increase in permeability. Preloading endothelial monolayers with quin2 to buffer cytosolic Ca2+ (Cai2+) produced a dose-dependent inhibition of the increase in 125I-albumin permeability. Preincubation with nifedipine or verapamil was ineffective in reducing the thrombin-induced permeability increase. A 60 mM K+ isosmotic solution did not alter base-line endothelial permeability. alpha-Thrombin increased [Ca2+]i in a dose-dependent manner and the 45Ca2+ influx rate. Extracellular medium containing 60 mM K+ did not increase 45Ca2+ influx, and nifedipine did not block the rise in 45Ca2+ influx caused by alpha-thrombin. Ca2+ flux into endothelial cells induced by alpha-thrombin does not occur through voltage-sensitive channels but may involve receptor-operated channels. In conclusion, the increase in endothelial albumin permeability caused by alpha-thrombin is dependent on Ca2+ influx and intracellular Ca2+ mobilization.     

Regulation of the mitochondrial matrix volume in vivo and in vitro. The role of calcium.

The ability of alpha-adrenergic agonists and vasopressin to increase the mitochondrial volume in hepatocytes is dependent on the presence of extracellular Ca2+. Addition of Ca2+ to hormone-treated cells incubated in the absence of Ca2+ initiates mitochondrial swelling. In the presence of extracellular Ca2+, A23187 (7.5 microM) induces mitochondrial swelling and stimulates gluconeogenesis from L-lactate. Isolated liver mitochondria incubated in KCl medium in the presence of 2.5 mM-phosphate undergo energy-dependent swelling, which is associated with electrogenic K+ uptake and reaches an equilibrium when the volume has increased to about 1.3-1.5 microliter/mg of protein. This K+-dependent swelling is stimulated by the presence of 0.3-1.0 microM-Ca2+, leading to an increase in matrix volume at equilibrium that is dependent on [Ca2+]. Ca2+-activated K+-dependent swelling requires phosphate and shows a strong preference for K+ over Na+, Li+ or choline. It is not associated with either uncoupling of mitochondria or any non-specific permeability changes and cannot be produced by Ba2+, Mn2+ or Sr2+. Ca2+-activated K+-dependent swelling is not prevented by any known inhibitors of plasma-membrane ion-transport systems, nor by inhibitors of mitochondrial phospholipase A2. Swelling is inhibited by 65% and 35% by 1 mM-ATP and 100 microM-quinine respectively. The effect of Ca2+ is blocked by Ruthenium Red (5 micrograms/ml) at low [Ca2+]. Spermine (0.25 mM) enhanced the swelling seen on addition of Ca2+, correlating with its ability to increase Ca2+ uptake into the mitochondria as measured by using Arsenazo-III. Mitochondria derived from rats treated with glucagon showed less swelling than did control mitochondria. In the presence of Ruthenium Red and higher [Ca2+], the mitochondria from hormone-treated animals showed greater swelling than did control mitochondria. These data imply that an increase in intramitochondrial [Ca2+] can increase the electrogenic flux of K+ into mitochondria by an unknown mechanism and thereby cause swelling. It is proposed that this is the mechanism by which alpha-agonists and vasopressin cause an increase in mitochondrial volume in situ.     

Sphingosine stimulates calcium mobilization in rat parotid acinar cells

In fura-2-loaded parotid acinar cells, 50-200 microM sphingosine induced an increase in cytosolic Ca2+ ([Ca2+]i). When extracellular Ca2+ was chelated by EGTA, 50 microM sphingosine failed to increase [Ca2+]i, but 100 or 200 microM sphingosine induced a slight and transient increase in [Ca2+]i. The addition of LaCl3 to the medium resulted in the same effect as chelation of extracellular Ca2+. When cells were incubated in low Ca2+ medium containing sphingosine, and extracellular Ca2+ was subsequently added, a rapid increase in [Ca2+]i depending on the concentration of sphingosine was shown. In low Ca2+ medium, a slight increase in [Ca2+]i induced by high concentrations of sphingosine was not shown after the transient increase in [Ca2+]i elicited by methacholine. Inhibitors of protein kinase C, H-7 and K252a, did not mimic the effect of sphingosine on [Ca2+]i. These results suggest that sphingosine stimulates Ca(2+)-influx and further stimulates the release of Ca2+ from agonist-sensitive intracellular pools by a mechanism that is independent of protein kinase C.     

Involvement of ion channels in the induction of proenkephalin A gene expression by nicotine and cAMP in bovine chromaffin cells

The involvement of Na+ and Ca2+ channels in the stimulatory effect of nicotine and cAMP upon proenkephalin A mRNA (mRNA ENK) levels in primary cultures of bovine adrenal chromaffin cells was analyzed. Nicotine (10 microM) caused about a 2-3-fold increase in mRNA ENK which was abolished by the nicotinic receptor antagonist tubocurarine (4 X 10(-7) M), inhibited by the Ca2+ channel antagonist nifedipine (100 nM) abolished by the Ca2+ channel blocker D600 (10 microM), and augmented by the Ca2+ channel agonist BayK 8644 (100 nM). In contrast, blockade of the Na+ channel by tetrodotoxin (1 microM) did not modulate the nicotine-induced increase in mRNA ENK. Incubation of the cells with forskolin (25 microM) and 8-bromo-cAMP (1 mM) also resulted in an increase in mRNA ENK levels that was inhibited by the Ca2+ channel blocker verapamil (50 microM) and nifedipine (100 nM), whereas it was enhanced by BayK 8644 (100 nM). In addition, the effect of forskolin and 8-bromo-cAMP was decreased by the Na+ channel blocker tetrodotoxin (1 microM). These results suggest that the induction of proenkephalin A gene expression by cAMP and nicotine involves the modulation of ion channels. It appears that changes in Ca2+ flux are involved in mediating this induction. The dihydropyridines nifedipine and BayK 8644 and the Ca2+ channel blockers verapamil and D600 all modulate 45Ca uptake. In addition, we show that incubation of the cells with A23187 (10(-7) M), a Ca2+ ionophore, resulted in an increase in mRNA ENK, indicating that changes in intracellular Ca2+ levels may indeed modulate proenkephalin A gene expression. Although it appears that an elevation of mRNA ENK upon nicotinic receptor activation occurs rapidly (an increase could be detected after 2 h incubation), the findings that the rise in mRNA ENK could be abolished by the Ca2+ channel blocker D600 but not affected by tetrodotoxin (1 microM), and that agents such as KCl (20 mM) and veratridine (5 microM) that increase mRNA ENK by activation of voltage-dependent Ca2+ channels do not result in an increase in intracellular cAMP, provide no evidence for a major role of the adenylate cyclase system in the inducing effect of nicotine upon proenkephalin A gene expression.     

Effect of hypothyroidism on the cytosolic free Ca2+ concentration in rat hepatocytes during rest and following stimulation by noradrenaline or vasopressin

The mean resting concentration of cytosolic free Ca2+ [( Ca2+]i) in parenchymal liver cells, as determined with the intracellular Ca2+ indicator quin2, was lowered by about 30% in hypothyroidism (0.17 microM vs. 0.27 microM in normal cells). The [Ca2+]i level in hypothyroid cells at 10 s following stimulation by noradrenaline (1 microM) was about 64% lower than in normal cells (0.33 microM vs. 1.0 microM). The response to noradrenaline in hypothyroid cells was slower in onset (significant at 5 s vs. 3 s in euthyroid cells), and the maximum of the initial [Ca2+]i increase was reached later (14 s vs. 8 s in normal cells). In hypothyroid hepatocytes the initial increase was followed by a slow but prolonged secondary increase in [Ca2+]i. With vasopressin similar results were found. Chelation of extracellular Ca2+ with EGTA immediately prior to stimulation had no effect on the initial [Ca2+]i increase. Treatment with T3 in vivo (0.5 micrograms/100 g body weight daily during 3 days) completely restored the basal and stimulated [Ca2+]i in hypothyroid cells. The half-maximally effective dose of noradrenaline was the same in euthyroid and hypothyroid liver cells (1.8 X 10(-7) M). Hypothyroidism had no significant effect on the number of alpha 1-receptors determined by [3H]prazosin labeling in crude homogenate fractions, while the Kd for [3H]prazosin was 21% lower than in the euthyroid group. These results show that thyroid hormone has a general stimulating effect on intracellular Ca2+ mobilization by Ca2+-mobilizing hormones, probably at a site distal to the binding of the agonist to its receptor. The results also support our idea that thyroid hormone may control metabolism during rest and activation, at least partially, by altering Ca2+ homeostasis.     

Influence of inhibitors on increase in intracellular free calcium and proliferation induced by platelet-activating factor in bovine oviductal cells.

Oviductal endosalpingeal cells were isolated mechanically from heifers and cultured until there was 100% confluency. The cells were loaded with the Ca(2+)-sensitive fluorochrome, fura-2/acetoxymethylester, and cytosolic free calcium ([Ca2+]i) was monitored by spectrofluorimetry. Platelet-activating factor, at a concentration of 30 nmol l-1, induced an intracellular Ca2+ increase in cultured bovine oviductal cells, mainly via influx from the extracellular space. In fura-2-loaded oviductal cells, different Ca2+ channel blockers were investigated to characterize the pathways responsible for the Ca2+ influx. The negative effects of Ni(2+)-, La(3+)-activated K+ channel blockers, such as apamin and charybdotoxin, and Ca2+ channel blockers, such as dotarizine, on the platelet-activating factor-induced [Ca2+]i increase indicate the minor participation of the voltage-gated Ca2+ channels. TMB-8 and flufenamic acid blocked the platelet-activating factor-induced Ca2+ increase directly on non-selective cationic channels or acted via a Ca2+ release-triggered Ca2+ influx. Platelet-activating factor, at concentrations of 1.25 mumol l-1 and 2.5 mumol l-1, significantly stimulated the proliferation and depolarization of oviductal cells, but 10 mumol l-1 significantly decreased both parameters and exerted a cytotoxic effect on cells. After incubation with TMB-8 or flufenamic acid, the cell proliferation was inhibited in a concentration-dependent manner, with IC50 values of 26.57 mumol l-1 and 95.29 mumol l-1, respectively. The depolarization was significantly inhibited at 50 mumol l-1 for both TMB-8 and flufenamic acid. The results of the present study may contribute to further understanding of the mechanism behind the actions of platelet-activating factor on oviductal cells.     

Hormonal and environmental regulation of epithelial calcium channel in gill of rainbow trout (Oncorhynchus mykiss)

We indirectly tested the idea that the epithelial Ca2+ channel (ECaC) of the trout gill is regulated in an appropriate manner to adjust rates of Ca2+ uptake. This was accomplished by assessing the levels of gill ECaC mRNA and protein in fish exposed to treatments known to increase or decrease Ca2+ uptake capacity. Exposure of trout to soft water ([Ca2+]=20-30 nmol/l) for 5 days (a treatment known to increase Ca2+ uptake capacity) caused a significant increase in ECaC mRNA levels and an increase in ECaC protein expression. The inducement of hypercalcemia by infusing fish with CaCl2 (a treatment known to reduce Ca2+ uptake) was associated with a significant decrease in ECaC mRNA levels, yet protein levels were unaltered. ECaC mRNA and protein expression were increased in fish treated with the hypercalcemic hormone cortisol. Finally, exposure of trout to 48 h of hypercapnia (approximately 7.5 mmHg, a treatment known to increase Ca2+ uptake capacity) elicited an approximately 100-fold increase in the levels of ECaC mRNA and a significant increase in protein expression. Immunocytochemical analysis of the gills from hypercapnic fish suggested a marked increase in the apical expression of ECaC on pavement cells and a subpopulation of mitochondria-rich cells. The results of this study provide evidence that Ca2+ uptake rates are, in part, regulated by the numbers of apical membrane Ca2+ channels that, in turn, modulate the inward flux of Ca2+ into gill epithelial cells.     

Ca2+ dependence of Ca2+ release from intracellular stores of intact and permeabilized Ehrlich ascites tumour cells

Effects of Ca2+ ions on the mobilization of Ca2+ from intracellular stores of intact and permeabilized (15 microM digitonin) Ehrlich ascites tumour cells (EATC) have been compared. For permeabilized cells, the dependences of the initial rate and amplitude of Ca2+ mobilization evoked by the addition of 100 nM inositol 1,4,5-trisphosphate (IP3) on preexisting [Ca2+] were bell-shaped within a [Ca2+] range 10(-7)-10(-6) M with the maxima at [Ca2+] = 166 nM. In intact cells, different concentrations of free cytosolic Ca2+ ([Ca2+]i) were produced using low (up to 0.005%) concentrations of digitonin which selectively increased the permeability of the plasma membrane. Stimulation of cells by exogenous ATP at [Ca2+]i = 10(-8)-10(-6) M resulted in Ca2+ mobilization the rate and amplitude of which were maximal at 102-115 nM Ca2+. The experimental Ca2+ dependences were fit by a model which includes channel opening upon Ca2+ binding and transition to the inactive states upon Ca2+ binding to the closed and open channel forms. Three inactivation types (including two particular cases) demonstrate a slight priority of inhibitory binding of Ca2+ only to the open channel, but predict markedly different parameter values. We conclude that an increase in [Ca2+] can stimulate IP3-induced mobilization, but in intact EATC, deviations of [Ca2+]i from the resting level (about 100 nM) attenuate responses to the agonist stimulation.     

Role of calcium and calcium-activated proteases in CYP2E1-dependent toxicity in HEPG2 cells.

The objective of this work was to investigate whether CYP2E1- and oxidative stress-dependent toxicity in HepG2 cells is mediated by an increase of cytosolic Ca2+ and activation of Ca2+-modulated processes. HepG2 cells expressing CYP2E1 (E47 cells) or control cells not expressing CYP2E1 (C34 cells) were preloaded with arachidonic acid (AA, up to 10 microm) and, after washing, incubated with iron-nitrilotriacetic acid (up to 100 microm) for variable periods (up to 12 h). Toxicity was greater in E47 cells than in C34 cells at all times and combinations of iron/AA tested. Cytosolic calcium increased with incubation time in both cell lines, but the increase was higher in E47 cells than in C34 cells. The rise in calcium was an early event and preceded the developing toxicity. Toxicity in E47 cells and the increase in Ca2+ were inhibited by omission of Ca2+ from the extracellular medium, and toxicity was restored by reincorporation of Ca2+. An inhibitor of Ca2+ release from intracellular stores did not prevent the toxicity or the increase in Ca2+, reflecting a role for the influx of extracellular Ca2+ in the toxicity. Reactive oxygen production was similar in media with or without calcium, indicating that calcium was not modulating CYP2E1-dependent oxidative stress. Toxicity, lipid peroxidation, and the increase of Ca2+ in E47 cells exposed to iron-AA were inhibited by alpha-tocopherol. E47 cells (but not C34 cells) exposed to iron-AA showed increased calpain activity in situ (40-fold). The toxicity in E47 cells mirrored calpain activation and was inhibited by calpeptin, suggesting that calpain activation plays a causal role in toxicity. These results suggest that CYP2E1-dependent toxicity in this model depends on the activation of lipid peroxidation, followed by an increased influx of extracellular Ca2+ and activation of Ca2+-dependent proteases.     

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.