Synergistic effect and possible mechanisms of tumor necrosis factor and cisplatin cytotoxicity under moderate hyperthermia against gastric cancer cells

Photosensitization induces intracellular free calcium changes ([Ca2+]i) in some eukaryotic cell systems which either contribute to or protect against cell inactivation. We have investigated whether or not similar changes can be induced in prokaryotes. The skin bacterium Propionibacterium acnes was sensitized using protoporphyrin IX (PP IX) or 5-aminolevulinic acid (ALA). Exogenous ALA resulted in either a preferential accumulation of protoporphyrin (ALA-PP) or of coproporphyrin and/or uroporphyrin (ALA-CP/UP) in P. acnes. For PP IX or ALA-PP sensitization, exposure to broad-band red light resulted in an increase in [Ca2+]i. For ALA-PP sensitization, this increase was transient and [Ca2+]i returned to basal levels within 5-10 min after irradiation. However, the elevated [Ca2+]i levels obtained after PP IX sensitization were maintained for at least 1 h after irradiation. In both cases, the reduction in the external calcium concentration led to an enhancement in the cell survival, indicating that induced [Ca2+]i changes may participate in photoinactivation. Sensitization by hydrophilic coproporphyrin and/or uroporphyrin (ALA-CP/UP) did not affect the [Ca2+]i levels, but higher levels of cell inactivation were obtained. It therefore appears that damage to membrane-associated components is at least partly responsible for [Ca2+]i alterations after photosensitization.     

Opposing actions of hepatocyte growth factor and basic fibroblast growth factor on cell contact, intracellular free calcium levels, and rat ovarian surface epithelial cell viability

Previous studies demonstrated that cell-to-cell contact stimulates a tyrosine phosphorylation signal transduction pathway that prevents rat ovarian surface epithelial (ROSE) cells from undergoing apoptosis. Hepatocyte growth factor (HGF), also know as scatter factor (SF), is expressed by ovarian stromal and thecal cells and has been shown to reduce cell contact in nonovarian tissues. The present studies were designed to determine whether HGF/SF promotes ROSE cells to dissociate and subsequently become apoptotic. Because an increase in intracellular free calcium ([Ca2+]i) is often an early event in the apoptotic cascade, the effects of HGF/SF on [Ca2+]i levels were also assessed. ROSE cells were cultured in serum-free medium with HGF/SF, basic fibroblast growth factor (bFGF), thapsigargin, Bay K, actinomycin D, cycloheximide, and/or BAPTA depending on the experimental design. Cell contact was assayed by time-lapse photography; [Ca2+]i levels were measured with Fluo-3, and apoptosis was assessed by in situ DNA staining. HGF/SF decreased cell contact within 1 h, increased [Ca2+]i levels by 3 h, and induced apoptosis by 6 h of culture. bFGF inhibited these HGF/SF-induced responses. The increase in [Ca2+]i appears to represent a point in the apoptotic cascade that commits ROSE cells to die. This concept is based on the observations that: 1) in the presence of the calcium chelator BAPTA, HGF/SF decreased cell contact but did not increase [Ca2+]i or apoptosis; 2) bFGF blocked HGF/SF-induced increase in [Ca2+]i; 3) bFGF did not attenuate HGF/SF's apoptotic action if exposed to cells after the increase in [Ca2+]i; and 4) RNA and protein synthesis were required for HGF/SF to increase [Ca2+]i, whereas the thapsigargin- and Bay K-induced increase in [Ca2+]i and apoptosis were independent of RNA/protein synthesis. These observations indicate that the components of the apoptotic cascade distal to the increase in [Ca2+]i are present within ROSE cells and are activated by a sustained elevation of [Ca2+]i. The present studies also show that when ROSE cells establish contact with 3T3 cells that express N-cadherin, [Ca2+]i levels are maintained at low basal levels. In contrast, cell contact with 3T3 cells that do not express N-cadherin results in elevated [Ca2+]i levels. Similarly, a synthetic N-cadherin peptide, which inhibits homophilic N-cadherin binding, increases [Ca2+]i levels. Taken together, these data indicate that homophilic N-cadherin binding between adhering cells plays an important role in maintaining calcium homeostasis. Further, these data support the concept that HGF/SF's ability to promote the dissociation of ROSE cells accounts in part for its ability to increase [Ca2+]i levels.     

The regulation of membrane 125I- and 86Rb+ permeability in a virally transformed cell line (NCL-SG3) derived from the human sweat gland epithelium

We have explored the factors that may regulate membrane permeability in a cell line (NCL-SG3) derived from the human sweat gland epithelium. Ionomycin increased the rate of 125I-efflux from preloaded cells and this action appeared to be due to an increase in intracellular free calcium ([Ca2+]i). The ionomycin-evoked increase in 125I- efflux was reduced in cells that were exposed either to barium or to valinomycin in the presence of a high concentration of external potassium. It thus appears that a fraction of the ionomycin-evoked increase in 125I- efflux is due to the activation of potassium channels and experiments using 86Rb+ also suggested that ionomycin increased the rate of potassium efflux, an effect which was totally abolished by barium. Blockade of Na(+)-K(+)-2Cl- cotransport and of Cl- -HCO3- exchange reduced the basal rate of 125I- efflux and the ionomycin-evoked increase in 125I-efflux from control cells and from cells depolarized by valinomycin. These transport systems thus contribute to anion efflux, although [Ca2+]i-dependent chloride channels also appear to be present. Acetylcholine increases [Ca2+]i in the secretory cells of human sweat glands, but this neurotransmitter did not increase [Ca2+]i in NCL-SG3 cells and so membrane permeability was not under cholinergic control. Adrenaline did not increase [Ca2+]i, but this hormone did evoke cyclic-3',5'-adenosine monophosphate (cyclic AMP) production. However, membrane permeability was not under adrenergic control, as the cells did not appear to express functional, cyclic AMP-dependent anion channels. This may be because they were not fully differentiated under the culture conditions. ATP consistently evoked a dose-dependent increase in anion efflux that appeared to be mediated by [Ca2+]i. The increase in [Ca2+]i was initiated by the release of calcium from a limited internal store and was subsequently sustained by calcium influx. UTP and ADP also increased [Ca2+]i, whereas adenosine, AMP and alpha,beta-methylene ATP were without effect. These data thus suggest that a subclass of type 2 purine receptor, which is functionally coupled to phosphoinositidase C, is present in these cells.     

Integrin alphavbeta5 participates in the binding of photoreceptor rod outer segments during phagocytosis by cultured human retinal pigment epithelium

Because glycolysis is thought to be important for maintenance of cellular ion homeostasis, the aim of the present study was to examine the role of glycolysis in the control of cytosolic calcium ([Ca2+]i) and cell shortening during conditions of increased calcium influx. Thus, [Ca2+]i and unloaded cell shortening were measured in fura-2/AM loaded rat ventricular myocytes. All cells were superfused with Tyrode's solution containing glucose and pyruvate (to preserve oxidative metabolism), and glycolysis was inhibited by iodoacetate (IAA, 100 microM). Calcium influx was increased, secondary to an increase in intracellular sodium, by addition of veratrine (1 microgram/ml), or directly by either elevating [Ca2+]o from 2 to 5 mM or by exposing the cells to isoproterenol (1 to 100 nm). Veratrine exposure caused a time-dependent increase in both diastolic and systolic [Ca2+]i that resulted in cellular calcium overload and hypercontraction. The rate of increase in [Ca2+]i was more rapid in IAA-treated than in untreated myocytes, leading to a 13+/-3 v 5+/-2% increase (P<0.05) in diastolic [Ca2+]i after 5 min of exposure. The corresponding increases in systolic [Ca2+]i were 43+/-6 and 24+/-5% (P<0.05). Elevated [Ca2+]o resulted in increased [Ca2+]i transient amplitudes and cell shortening. These responses were each attenuated by inhibiting glycolysis, so that the increase was 38+/-5 v 68+/-9% ([Ca2+]i transient amplitude, P<0.05) and 41+/-11 v 91+/-18% (cell shortening, P<0.05). Inhibition of glycolysis did not, however, affect the increase in calcium transient or cell shortening during addition of isoproterenol. We conclude that glycolysis plays an essential role in the maintenance of intracellular calcium homeostasis during severe calcium overload. Glycolysis was also essential for signalling the inotropic effect that accompanied elevation in extracellular calcium, while the changes in intracellular calcium following administration of isoproterenol were not influenced by glycolysis in the present model.     

Hydrogen peroxide induces intracellular calcium overload by activation of a non-selective cation channel in an insulin-secreting cell line

Fura-2 fluorescence was used to investigate the effects of H2O2 on [Ca2+]i in the insulin-secreting cell line CRI-G1. H2O2 (1-10 mM) caused a biphasic increase in free [Ca2+]i, an initial rise observed within 3 min and a second, much larger rise following a 30-min exposure. Extracellular calcium removal blocked the late, but not the initial, rise in [Ca2+]i. Thapsigargin did not affect either response to H2O2, but activated capacitive calcium entry, an action abolished by 10 microM La3+. Simultaneous recordings of membrane potential and [Ca2+]i demonstrated the same biphasic [Ca2+]i response to H2O2 and showed that the late increase in [Ca2+]i coincided temporally with cell membrane potential collapse. Buffering Ca2+i to low nanomolar levels prevented both phases of increased [Ca2+]i and the H2O2-induced depolarization. The H2O2-induced late rise in [Ca2+]i was prevented by extracellular application of 100 microM La3+. La3+ (100 microM) inhibited the H2O2-induced cation current and NAD-activated cation (NSNAD) channel activity in these cells. H2O2 increased the NAD/NADH ratio in intact CRI-G1 cells, consistent with increased cellular [NAD]. These data suggest that H2O2 increases [NAD], which, coupled with increased [Ca2+]i, activates NSNAD channels, causing unregulated Ca2+ entry and consequent cell death.     

Regulation of the intracellular concentration of free calcium ions in pinealocytes of the rainbow trout and the rat

Together with cAMP, calcium ions play an important role in the regulation of melatonin synthesis in the pineal organ of all vertebrate species, irrespective of the conspicuous phylogenetic transformation of the melatonin-producing cell, the pinealocyte. Here we address the question how the intracellular concentration of free calcium ions [Ca2+]i is regulated in directly light-sensitive trout pinealocytes and in rat pinealocytes which have lost the direct light sensitivity and respond to norepinephrine. Isolated pinealocytes identified by the S-antigen immunoreaction were investigated by means of the fura-2 technique, image analysis and patch clamp recordings. Approximately 30% of the trout pinealocytes exhibited spontaneous [Ca2+]i oscillations that were not affected by light or dark adaptation of the cells. Removal of extracellular Ca2+ or application of 10 microM nifedipine caused a reversible breakdown of the [Ca2+]i oscillations. Treatments with 60 mM KCl and nifedipine suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca2+]i in both oscillating and nonoscillating trout pinealocytes. Experiments with thapsigargin (2 microM) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role in the regulation of [Ca2+]i remains elusive. Norepinephrine had no apparent effect on [Ca2+]i in any trout pinealocyte. In rat pinealocytes, [Ca2+]i did not show spontaneous oscillations. Norepinephrine evoked a dramatic biphasic rise in [Ca2+]i in more than 95% of the cells via stimulation of alpha1-adrenergic receptors. The response reflects a combination of calcium mobilization from intracellular, thapsigargin-sensitive calcium stores and an increased calcium influx. Voltage-gated calcium channels of the L-type are present in the rat pinealocyte membrane, but they are not involved in the norepinephrine-induced calcium response. These channels, however, mediate the increase in calcium influx which is observed in virtually all rat pinealocytes upon stimulation with acetylcholine or nicotine. The results show that the mechanisms which regulate [Ca2+]i in pinealocytes are complex and differ considerably between poikilothermic and mammalian species.     

Role of intracellular calcium ([Ca2+]i) and tyrosine phosphorylation in adhesion of cultured vascular smooth muscle cells to fibrinogen

OBJECTIVE: Fibrinogen is an independent risk factor for cardiovascular disease. This study has investigated the role of intracellular Ca2+ ([Ca2+]i) and tyrosine phosphorylation in the attachment of human and rat-derived cultured vascular smooth muscle cells to fibrinogen. METHODS: Cells were cultured from human saphenous vein segments (HVSMC) and from an established rat aortic cell line (A7r5). [Ca2+]i was measured using fura-2 and adhesion was studied using pre-coated 96 well polystyrene plates. RESULTS: Fibrinogen increased [Ca2+]i in both cell types. In A7r5 cells fibrinogen-induced increases in [Ca2+]i were partially inhibited by a peptide containing the amino acid sequence Arg-Gly-Asp (RGD) which interferes with binding to integrins. In contrast RGD increased [Ca2+]i in HVSMC, but did not inhibit responses to fibrinogen. Ni2+, an inorganic calcium channel blocker largely abolished the rise in [Ca2+]i, but blockers of voltage-operated calcium channels failed to affect [Ca2+]i responses to fibrinogen in either cell type. Genistein, an inhibitor of tyrosine kinase inhibited fibrinogen-induced rises in [Ca2+]i, while daidzein, an inactive analogue, was without effect. Adhesion of cells to fibrinogen was concentration- and time-dependent. Cell adhesion to fibrinogen was partially inhibited by RGD peptide in both cell types. Adhesion of cell to fibrinogen was inhibited by chelation of [Ca2+]i with BAPTA-AM, inhibition of Ca2+ entry by Ni2+, and inhibition of tyrosine kinases by genistein, but heparin had no effect on adhesion. CONCLUSIONS: Vascular smooth muscle cells attach to fibrinogen in part through RGD-type interactions. Activation of tyrosine kinase(s) and a subsequent rise in [Ca2+]i appear to be important signals mediating the response to fibrinogen.     

Dose-dependent effects of chloroquine on secretory granule formation in the melanotroph

Intracellular calcium ([Ca2+]i) and hydrogen ion concentrations (pHi) are important regulators of cell function. Those ions also may interact and it is important, therefore, to measure their concentrations simultaneously. In the present studies we used a system developed for that purpose, a fluorescent emission ratio technique for simultaneous analysis of calcium (Indo-1) and pH (SNARF-1) in single cells at video rates, and determined if arginine vasopressin (AVP, 12.5 mumol/l) evoked [Ca2+]i and pHi signals interact in MDCK cells. We also employed a simple system for analysing the side specific (basolateral or apical) application of agonist to polarized cell layers on permeable membranes. AVP is found to evoke simultaneous changes in both pHi and [Ca2+]i. Basolateral application induced transient acidification, followed by partial recovery, and a [Ca2+]i transient with kinetic pattern similar to that of the pHi. Apical application also caused a mirror image pHi and [Ca2+]i pattern but of smaller magnitude (no peak). Selective removal of extracellular calcium ([Ca2+]e) or sodium ([Na+]e) dissociated the pHi and [Ca2+]i responses in both cases. Na+e removal abolished the pHi changes, but not the [Ca2+]i transients. [Ca2+]e removal abolished the [Ca2+]i changes and reduced, but did not abolish, the pHi responses. Thus, AVP induces pHi changes which are modified by calcium while calcium signalling is not modified by changes in pHi.     

Rat Sertoli cell calcium response to basement membrane and follicle-stimulating hormone

Sertoli cells cultured on basement membrane substrates differentiate morphologically into polarized cells and exhibit an enhanced responsiveness to FSH. The signal transduction mechanisms by which the extracellular matrix induces changes in the morphology and function of Sertoli cells are not known. Since calcium has been implicated in mediating changes in cytoskeletal assembly and organization, we investigated to see if basement membrane can modulate cytosolic free calcium concentrations during the process of adhesion and spreading of Sertoli cells. A direct quantification of the intracellular free cytosolic calcium concentration [Ca2+]i in freshly isolated immature rat Sertoli cells plated on laminin was performed by digital imaging microscopy using the fluorescent probe Fura-2 AM. [Ca2+]i levels rose by 1.5-2-fold within 1 h after plating on laminin, suggesting that calcium may be involved in adhesion and spreading of the cells on basement membrane. Furthermore, the possibility that matrix influences [Ca2+]i levels upon stimulation with FSH was examined by adding FSH directly to the cells spreading on laminin. A dramatic decrease in [Ca2+]i was observed compared to the level in untreated cells. Similarly, a significant decrease in [Ca2+]i in response to FSH was observed in cells already spread on laminin or Matrigel. Addition of dibutyryl cAMP did not significantly alter the basal calcium levels. Long-term exposure of Sertoli cells cultured on either laminin or Matrigel to FSH was studied by incubating the cells with 45CaCl2 in the presence or absence of FSH for 24 h. FSH induced a decrease or no change in 45Ca concentration in cells cultured on basement membrane. Addition of dibutyryl cAMP, instead of FSH, did not alter the basal 45Ca concentrations. In cells cultured on the peptides derived from laminin (RGD and SIKVAV), FSH increased the uptake of 45Ca significantly, whereas on YIGSR, also a laminin-derived peptide, it did not have any effect. Thus, basement membrane induces an early increase in [Ca2+]i in cultured Sertoli cells during spreading, and FSH appears to significantly decrease [Ca2+]i levels.     

Atrial natriuretic peptide modulates the effect of angiotensin II on the concentration of free calcium in the cytosol of Mandin-Darby canine kidney cells

The effect of angiotensin II (ANG II) and atrial natriuretic peptide (ANP) on intracellular free calcium concentration [Ca2+]i was investigated in Mandin-Darby canine kidney (MDCK) cells in culture. Changes in [Ca2+]i were monitored fluorometrically with the Ca(2+)-sensitive probe fura-2/AM at 37 degrees C using a Perkin-Elmer LS-5 spectrofluorimeter (excitation 340/380 nm, slit 3 nm; emission 520 nm, slit 10 nm). MDCK cells exhibited a mean baseline [Ca2+]i of 98 +/- 10 nM. The addition of increasing concentrations of ANG II (1 pM to 1 microM) to the cell suspension led to a progressive increase in [Ca2+]i to 2-3 times basal levels. In contrast, addition of 1 microM ANP to the cell suspension led to a very rapid 60% decrease in [Ca2+]i. The addition of 1 pM to 1 microM ANG II immediately after 1 microM ANP caused an increase in [Ca2+]i which never exceeded the basal level in the absence of ANP. The data indicate that ANG II increases cell [Ca2+]i, as expected, and provide the new observation that ANP reduces [Ca2+]i in these cells. Furthermore, ANP reduces the increase in [Ca2+]i elicited by ANG II, thus modulating the effect of ANG II on [Ca2+]i.     

Insulin exocytosis and glucose-mediated increase in cytoplasmic free Ca2+ concentration in the pancreatic beta-cell are independent of cyclic ADP-ribose

Stimulation of pancreatic beta-cells by glucose gives rise to an increase in the cytoplasmic free calcium concentration ([Ca2+]i) and exocytosis of insulin. Cyclic adenosine 5'-diphosphate ribose (cADPR), a metabolite of beta-NAD+, has been reported to increase [Ca2+]i in pancreatic beta-cells by releasing Ca2+ from inositol 1,4,5-trisphosphate-insensitive intracellular stores. In the present study, we have examined the role of cADPR in glucose-mediated increases in [Ca2+]i and insulin exocytosis. Dispersed ob/ob mouse beta-cell aggregates were either pressure microinjected with fura-2 salt or loaded with fura-2 acetoxymethyl ester, and [Ca2+]i was monitored by microfluorimetry. Microinjection of beta-NAD+ into fura-2-loaded beta-cells did not increase [Ca2+]i nor did it alter the cells' subsequent [Ca2+]i response to glucose. Cells microinjected with the cADPR antagonist 8NH2-cADPR increased [Ca2+]i in response to glucose equally well as those injected with cADPR. Finally, the ability of cADPR to promote exocytosis of insulin in electropermeabilized beta-cells was investigated. cADPR on its own did not increase insulin secretion nor did it potentiate Ca2+-induced insulin secretion. We conclude that cADPR neither plays a significant role in glucose-mediated increases in [Ca2+]i nor interacts directly with the molecular mechanisms regulating exocytosis of insulin in normal pancreatic beta-cells.     

An immunosuppressive agent, FTY720, increases intracellular concentration of calcium ion and induces apoptosis in HL-60

We previously reported that FTY720 is an efficient inducer of apoptosis in lymphocytes and cultured cell lines. In the present study, HL-60 human promyerocytoma cells also induced apoptosis through in vitro treatment with the drug, demonstrating extensive DNA fragmentation 6 hr after incubation. The major target of FTY720 was the common signalling pathway of apoptosis, since a rapid (< 1 min) increase in the intracellular Ca2+ concentration ([Ca2+]i) was found in the cells treated with the drug. Calcium chelation in the culture medium with EGTA did not affect the [Ca2+]i mobilization. A phospholipase C inhibitor, U73122, inhibited the increase in [Ca2+]i as well as the fragmentation of the nuclear DNA, whereas U73343, a non-effective analogue of U73122, had little effect. These results suggest that FTY720-induced apoptosis is mediated through an activation of phospholipase C and the subsequent release of Ca2+ from intracellular calcium pools. In addition, the treatment of HL-60 with pertussis toxin (PTX) did not inhibit Ca2+ mobilization or apoptosis, suggesting that the activation of phospholipase C is independent of PTX-sensitive G-proteins.     

Different mechanisms are involved in intracellular calcium increase by insulin-like growth factors 1 and 2 in articular chondrocytes: voltage-gated calcium channels, and/or phospholipase C coupled to a pertussis-sensitive G-protein

This study describes the mechanisms involved in the IGF-1 and IGF-2-induced increases in intracellular calcium concentration [Ca2+]i in cultured chondrocytes and the involvement of type 1 IGF receptors. It shows that IGF-1, IGF-2, and insulin increased the cytosolic free calcium concentration [Ca2+]i in a dose-dependent manner, with a plateau from 25 to 100 ng/ml for both IGF-1 and IGF-2 and from 1 to 2 micrograms/ml for insulin. The effect of IGF-1 was twice as great as the one of IGF-2, and the effect of insulin was 40% lower than IGF-1 effect. Two different mechanisms are involved in the intracellular [Ca2+]i increase. 1) IGF-1 and insulin but not IGF-2 involved a Ca2+ influx through voltage-gated calcium channels: pretreatment of the cells by EGTA and verapamil diminished the IGF-1 or insulin-induced [Ca2+]i but did not block the effect of IGF-2. 2) IGF-1, IGF-2, and insulin also induced a Ca2+ mobilization from the endoplasmic reticulum: phospholipase C (PLC) inhibitors, neomycin, or U-73122 partially blocked the intracellular [Ca2+]i increase induced by IGF-1 and insulin and totally inhibited the effect of IGF-2. This Ca2+ mobilization was pertussis toxin (PTX) dependent, suggesting an activation of a PLC coupled to a PTX-sensitive G-protein. Lastly, preincubation of the cells with IGF1 receptor antibodies diminished the IGF-1-induced Ca2+ spike and totally abolished the Ca2+ influx, but did not modify the effect of IGF-2. These results suggest that IGF-1 action on Ca2+ influx involves the IGF1 receptor, while part of IGF-1 and all of IGF-2 Ca2+ mobilization do not implicate this receptor.     

Identification of a novel adhesin-like glycoprotein from Mycoplasma hyopneumoniae

A Ca2+-activated (ICl,Ca) and a swelling-activated anion current (ICl,vol) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying currents were activated by an increase in the free intracellular calcium concentration ([Ca2+]i), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular Cl- concentration. ICl,Ca current density increased with increasing [Ca2+]i, and this current was abolished by lowering [Ca2+]i to <1 nm using 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA). In contrast, activation of ICl,vol did not require an increase in [Ca2+]i. The kinetics of ICl,Ca and ICl,vol were different: at depolarized potentials, ICl,Ca as activated in a [Ca2+]i- and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, ICl,vol exhibited time- and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials. The deactivation of ICl, vol was dependent on the extracellular Mg2+ concentration. The anion permeability sequence for both currents was I- > Cl- > gluconate. ICl,Ca was inhibited by niflumic acid (100 micron), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 micron) and 4, 4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS, 100 micron), niflumic acid being the most potent inhibitor. In contrast, ICl,vol was unaffected by niflumic acid (100 micron), but abolished by tamoxifen (10 micron). Thus, in Ehrlich cells, separate chloride currents, ICl,Ca and ICl,vol, are activated by an increase in [Ca2+]i and by cell swelling, respectively.     

Na+/Ca2+ exchanger modulates kainate-triggered Ca2+ signaling in Bergmann glial cells in situ

The role of sodium-calcium exchanger in calcium homeostasis in Bergmann glial cells in situ was investigated by monitoring cytoplasmic calcium ([Ca2+]i) and sodium ([Na+]i) concentrations. The [Ca2+]i and [Na+]i transients were measured either separately by using fluorescent indicators fura-2 and SBFI, respectively, or simultaneously using the indicators fluo-3 and SBFI. Since the removal of extracellular Na+ induced a relatively small (approximately 50 nM) elevation of [Ca2+]i, the Na+/Ca2+ exchanger seems to play a minor role in regulation of resting [Ca2+]i. In contrast, kainate-triggered [Ca2+]i increase was significantly suppressed by lowering of the extracellular Na+ concentration ([Na+]o). In addition, manipulations with [Na+]o dramatically affected the recovery of the kainate-induced [Ca2+]i transients. Simultaneous recordings of [Ca2+]i and [Na+]i revealed that kainate-evoked [Ca2+]i transients were accompanied with an increase in [Na+]i. Moreover, kainate induced significantly larger [Ca2+]i and smaller [Na+]i transients under current-clamp conditions as compared to those recorded when the membrane voltage was clamped at -70 mV. The above results demonstrate that the Na(+)-Ca2+ exchanger is operative in Bergmann glial cells in situ and is able to modulate dynamically the amplitude and kinetics of [Ca2+]i signals associated with an activation of ionotropic glutamate receptors.     

Ca2+ signalling by endothelin receptors in rat and human cultured airway smooth muscle cells

1. The aim of the current study was to characterize the ET receptor subtypes in cultured airway smooth muscle cells derived from rat trachea and human bronchus using radioligand binding techniques and to investigate the coupling of ET receptors to intracellular calcium signalling mechanisms using endothelin receptor-selective agonists (sarafotoxin S6c) and antagonists (BQ-123, BQ-788) and digital image fluorescence microscopy. 2. Confluent rat airway smooth muscle cells in culture possessed a mixed ET receptor population (30% ETA : 70% ETB), with a density of approximately 3400+/-280 ETA and 8000+/-610 ETB receptors/cell (n = 3 experiments). The density of ETB, but not ETA receptors increased substantially in serum-containing medium. However, a 2-day period of serum deprivation, which inhibited cellular growth, substantially reduced ETB receptor density such that the ET receptor subtype proportions were approximately equal (55% ETA; 45% ETB) and similar to those previously observed in intact rat tracheal smooth muscle. 3. Challenge of rat airway smooth muscle cells in culture with endothelin- 1 elicited a concentration-dependent biphasic increase in [Ca2+]i (EC50: 16 nM), that comprised an initial transient peak [Ca2+]i increase (typically 350 nM) followed by a modest sustained component. The endothelin-1-induced biphasic [Ca2+]i increase was primarily due to ETA receptor activation, although a modest and inconsistent ETB response was observed. The ETA-mediated [Ca2+]i increase was due primarily to the mobilization of IP3-sensitive and to a lesser extent ryanodine-sensitive intracellular calcium stores. In contrast, ETB receptor activation was exclusively coupled to extracellular calcium influx. 4. Somewhat surprisingly, human airway smooth muscle cells in culture contained a homogeneous population of ETA receptors at a density of 6100+/-800 receptors cell(-1) (n = 3 experiments). Serum deprivation was without effect on either ET receptor subtype proportion or ETA receptor density. Challenge of human airway smooth muscle cells with endothelin-1 provoked a concentration-dependent increase in [Ca2+]i (EC50: 15 nM), with a peak [Ca2+]i increase to greater than 700 nM. Furthermore, the ETA-mediated calcium response in these human airway smooth muscle cells in culture was entirely dependent upon the mobilization of calcium from intracellular stores. 5. In summary, rat cultured tracheal airway smooth muscle cells contained both ETA and ETB receptors. ETA receptors, the numbers of which remained constant during cell growth, were linked to the release of Ca2+ from intracellular stores and a strong rise in [Ca2+]i in the majority of airway smooth muscle cells. In stark contrast, the numbers of ETB receptors increased significantly during cell growth, an effect that was diminished substantially by incubation in serum-free medium. Moreover, despite the greater number of ETB receptors, their activation in a small number of airway smooth muscle cells produced only a weak rise in [Ca2+]i, which appeared to be attributable to the influx of extracellular Ca2+. In contrast, the populations of ET receptors and their linkage to [Ca2+]i were markedly different in the human cultured airway smooth muscle cells used in the current study compared to that previously observed in intact human isolated bronchial smooth muscle.     

Oscillatory Cl- current induced by angiotensin II in rat pulmonary arterial myocytes: Ca2+ dependence and physiological implication

We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca2+]i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca2+]i. In cells clamped at -60 mV, ANG II (10 microM) or ATP (100 microM) induced an oscillatory inward current. Caffeine (5 mM) induced only one transient inward current. In control conditions, the reversal potential (Erev) of these currents was close to the equilibrium potential for Cl- ions (Ecl = -2.1 mV) and was shifted towards more positive values in low-Cl- solutions. Niflumic acid (10-50 microM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca2+]i by indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca2+]i whereas the caffeine-induced current was accompanied by only one transient increase in [Ca2+]i. Niflumic acid (25 microM) had no effect on agonist-induced [Ca2+]i responses, whereas thapsigargin (1 microM) abolished both membrane current and the [Ca2+]i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca2+]i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 microM) or nifedipine (1 microM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca2+]i and that both oscillatory phenomena are primarily IP3-mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca2+ channels. The resulting Ca2+ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade.     

Requirement for Ca2+ mobilization and increased prostaglandin production for maximal decidualization in rats and the involvement of angiotensin II

Studies were performed to determine whether the inhibition of the decidual cell reaction induced by intrauterine infusion of the angiotensin converting enzyme inhibitor enalaprilat in rats is reversed by activation of Ca2+ influx. Influx of Ca2+ was shown to be stimulated by angiotensin II in endometrial cells in this study. Ovariectomized, adult female rats were sensitized for the decidual cell reaction with steroid treatments. For experiments in vivo, intrauterine infusions of enalaprilat alone, or in combination with the Ca2+ ionophore A23187, a synthetic diacylglycerol, and dioctanoyl-sn-glycerol (diC8), and PGE2 were initiated on the day of uterine sensitivity. Enalaprilat inhibited the increases in uterine PG concentrations and uterine weight that occur following infusion of the vehicle. Concurrent infusion of A23187 partially, but not completely, reversed the inhibition of uterine weight increase; diC8 did not affect the inhibition of enalaprilat. A23187 did not reverse the effects of enalaprilat on uterine PG concentrations. Concurrent infusion of A23187 and PGE2 fully reversed the inhibitory effect of enalaprilat on uterine weight. For experiments in vitro, endometrial stromal and epithelial cells were obtained from uteri on the day of sensitivity and maintained in suspension. Cytosolic free calcium concentration ([Ca2+]i) was monitored in cell suspensions by fluorescence spectrophotometry using the Ca(2+)-sensitive probe, indo-1. Angiotensin II induced a transient increase in [Ca2+]i of endometrial stromal cell suspensions, but not of epithelial cells; PGE2 did not increase [Ca2+]i in stromal or epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric study of mitochondrial dysfunction after AMPA receptor activation

The effect of AMPA-receptor stimulation on MMP and on the concentration of intracellular calcium ([Ca2+]i) was studied in dissociated CGC from rat pups, by flow cytometry. In the presence of cyclothiazide, AMPA induced a sodium-independent decrease in MMP up to 30.7+/-2.5%. This effect was antagonized by CNQX and NBQX. Mepacrine and dibucaine reversed the effect of AMPA on MMP, suggesting that it is mediated by a release of arachidonic acid. AMPA alone induced a slight (about 7%) increase in [Ca2+]i. In the presence of cyclothiazide, AMPA induced a concentration-dependent [Ca2+]i increase up to 29.10+/-2.10% that was not reversed by flunarizine. This increase was similar to that observed in a Na+-free medium, and was antagonized by CNQX and NBQX, but not by MK-801. Mitochondria play a key role in the modulation of [Ca2+]i since a significant [Ca2+]i increase was found in the presence of FCCP. On the other hand, the dantrolene-sensitive calcium pools do not participate in the [Ca2+]i increase induced by stimulation of AMPA receptors. It is concluded that when AMPA-receptor desensitization is blocked, a decrease in MMP and an increase in [Ca2+]i occurs, which could be additional events to potentiate neuronal cell death induced by glutamate.