Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Ionomycin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 415 records · Page 23Linked to original sources
PubMed2004

Dual role of sumoylation in the nuclear localization and transcriptional activation of NFAT1.

The nuclear import of nuclear factor of activated T cells (NFAT) transcription factors is critical for regulating NFAT activity. Here we demonstrate that the sumoylation of NFAT1 defines a novel mechanism of the nuclear anchorage and transcriptional activation downstream from the known mechanism of calcineurin-mediated dephosphorylation and nuclear import. We show that Lys(684) and Lys(897) of NFAT1 can be sumoylated. The sumoylation at Lys(684) is required for NFAT1 transcriptional activity and subsequent sumoylation of Lys(897), whereas the sumoylation of Lys(897) is only required for nuclear anchorage. Because Lys(897) of NFAT1 is not conserved among other members of the NFAT family, we propose that sumoylation of Lys(897) may provide a mechanism for NFAT1 isotype-specific regulation of nuclear anchorage and transcriptional activation. Furthermore, we found that treatment with both ionomycin and phorbol 12-myristate 13-acetate ensured efficient nuclear anchorage with the recruitment of NFAT1 into the SUMO-1 bodies, whereas treatment with ionomycin alone induced nuclear translocation of NFAT1 but not recruitment into the SUMO-1 bodies. Our results suggest that the recruitment of NFAT1 into SUMO-1 bodies may be required for the progressive transcriptional activity of NFAT1 upon co-stimulation with ionomycin and phorbol 12-myristate 13-acetate, whereas anergic transcription stimulated by ionomycin alone may occur without recruitment into the SUMO-1 bodies.

Animals↗
PubMed2005

Overexpression of cytosolic group IVA phospholipase A2 protects cells from Ca2+-dependent death.

The calcium ionophore ionomycin induces apoptosis-like events in the human embryonic kidney cell line at early times. Plasma membrane blebbing, mitochondrial depolarization, externalization of phosphatidylserine, and nuclear permeability changes can all be observed within 15 min of treatment. However, there is no activation of caspases or chromatin condensation. Expression of a fusion protein containing the enhanced green fluorescent protein (EGFP) and human cytosolic Group IVA phospholipase A(2)alpha (EGFP-cPLA(2)alpha) in these cells prevents ionomycin-induced phosphatidylserine externalization and death. Cells expressing the cPLA(2)alpha mutant D43N, which does not bind calcium, retain their susceptibility to ionomycin-induced cell death. Both nonexpressing and EGFP-D43N-cPLA(2)alpha-expressing human embryonic kidney cells can be spared from ionomycin-induced cell death by pretreating them with exogenous arachidonic acid. Moreover, during calcium overload, mitochondrial depolarization is significantly lower in the EGFP-cPLA(2)alpha-expressing cells than in cells expressing normal amounts of cPLA(2)alpha. These results suggest that early cell death events promoted by an overload of calcium can be prevented by the presence of high levels of arachidonic acid.

Animals↗
PubMed1988

Immunocytochemical evidence for translocation of protein kinase C in human megakaryoblastic leukemic cells: synergistic effects of Ca2+ and activators of protein kinase C on the plasma membrane association.

Immunological analysis using monoclonal antibodies against subspecies of protein kinase C revealed the predominant expression of the isozyme, type II, in human megakaryoblastic leukemic cells. We investigated the effects of phorbol diester 12-O-tetradecanoyl phorbol-13-acetate (TPA), the Ca2+ ionophore ionomycin and synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) on the immunocytochemical localization of protein kinase C in these cells. Indirect immunofluorescence techniques revealed the enzyme to be located in a diffuse cytosolic pattern, in the intact cells. When the cells were exposed to 100 nM TPA, the immunofluorescent staining was translocated from the cytoplasm to the plasma membrane. The translocation was protracted and staining on the membrane decreased in parallel with the Ca2+, phospholipid-dependent protein kinase activity. Treatment of the cells with 500 nM ionomycin caused an apparent translocation comparable with that seen with TPA, however, this translocation was transient and most of the cytosolic staining was within 60 min. We also found that 30 micrograms/ml OAG did not have significant effects on distribution of the staining, but rather acted synergistically on the translocation with the suboptimal concentration of 100 nM ionomycin. A similar synergism was also observed with 10 nM TPA and 100 nM ionomycin. These results obtained in situ provide evidence that intracellular Ca2+ and diacylglycerol regulate membrane binding of the enzyme in vivo.

Anti-Bacterial Agents↗
PubMed1992

Transmembrane-mediated changes in [Ca2+] are involved in the signaling pathway leading to macrophage cytocidal differentiation: implications of localized changes in intracellular [Ca2+] and of interferon priming on Ca2+ utilization.

Macrophage cytocidal activation requires the sequential impingement on the macrophage of a priming stimulus (interferon [IFN] alpha, beta, or gamma) and a triggering stimulus (such as polyinosinic acid:polycytidylic acid [poly [I:C]] or bacterial lipopolysaccharide). The mechanism of progression from the IFN-primed state to the cytocidal state is poorly understood. By quantifying the level of expression of a gene product (complement component factor B [Bf]) associated with cytocidal activation and through the use of phenotypically distinct populations of macrophages (unprimed and IFN-primed), we have investigated the functional necessity of changes in intracellular concentration of free calcium ions ([Ca2+]i) in signaling the transition from the primed to the cytocidal state. Elevating the [Ca2+]i by incubation of unprimed macrophages with the calcium ionophore, ionomycin, failed to induce the expression of Bf. By contrast, Bf was expressed at high levels when IFN-primed macrophages were exposed to ionomycin, suggesting that priming induced within the macrophages the capacity to respond to a nonspecific change in [Ca2+]i. Quantification of the [Ca2+]i in response to exposure to ionomycin revealed an initial transient elevation, followed by a secondary sustained component. No differences in these changes were observed between unprimed and IFN-primed macrophages. We therefore questioned if changes in [Ca2+]i were also implicated in the transition between the primed and the cytocidal state using the ligand, poly [I:C]. In contrast to ionomycin, incubation of IFN-primed macrophages with poly [I:C] did not sustain measurable increases in [Ca2+]i, yet fully stimulated the transition from the IFN primed to the cytocidal state. However, incubation of IFN-primed macrophages with poly [I:C] in the presence of 1) a Ca2+/ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffer calculated to clamp the extracellular concentration of free calcium ions to a value approximately equal to the resting [Ca2+]i; 2) the calcium channel blocker verapamil; or 3) the intracellular Ca2+ antagonists (W-7, W-13, and TMB-8) substantially inhibited the induction of Bf. Collectively, these data support the following conclusions. First, that changes in [Ca2+]i comprise an important element in the induction of progression from the IFN-primed to the cytocidal state. Second, the failure to detect global changes in [Ca2+]i in response to the ligand, poly [I:C], suggests that changes in [Ca2+]i or Ca2+ movement may occur in either a spatially restricted or in an asynchronous cyclical fashion and are not detected by population fluorescence measurements. Third, the source of the relevant Ca2+ is extracellular. Fourth, our findings suggest that priming influences macrophage functional responses at a locus that is distal to the changes in [Ca2+]i, thereby potentially allowing signaling processes to be utilized to initiate different cellular responses.

Animals↗
PubMed1991

Effects of anti-immunoglobulin antibodies, interleukin-4 and second messenger agonists on B cells from neonatal mice.

Crosslinking of surface Ig receptors on mature B cells with mitogenic anti-Ig antibodies stimulates phosphoinositide breakdown with subsequent activation of protein kinase C (PKC) and elevation of [Ca2+]i leading to B-cell activation. This response can be mimicked using second messenger agonists such as phorbol 12,13-dibutyrate (PDB) plus a Ca2+ ionophore. Furthermore, interleukin-4 (IL-4) synergizes with sub-mitogenic concentrations of anti-Ig (or with PDB) to activate adult B cells. In contrast anti-Ig does not activate neonatal B cells but rather desensitizes or kills them. The nature of the signals involved in these effects on neonatal B cells is poorly understood. Here we have investigated the proliferative responses of small, resting B cells from 1-, 4-, 8-, and 12-week-old mice stimulated with combinations of anti-Ig, PDB, ionomycin and IL-4. We find that B cells from 8- and 12-week-old mice show an adult pattern of reactivity. B cells from 4-week-old mice respond to high doses of anti-Ig, anti-Ig + IL-4 and also to PDB + ionomycin + IL-4, but not to PDB + ionomycin alone. Neonatal (1-week-old) B cells respond only to the combination of PDB, ionomycin and IL-4. Most strikingly, pre-incubation of neonatal cells with anti-Ig completely abrogates this response, whilst IL-4 renders them refractory to such anti-Ig mediated inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Calcium-ionophore-induced formation of platelet-activating factor and leukotrienes by horse eosinophils: a comparative study.

Horse eosinophils preincubated with 3H-labelled acetate and stimulated with the Ca2+ ionophores ionomycin or A23187 form a radioactive compound, which we have shown to be 1-O-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine (platelet-activating factor). We could detect no 1-O-acyl-2-[3H]acetyl-glycero-3-phosphocholine in the radioactive fraction. The formation of platelet-activating factor was strongly correlated to the generation of leukotriene C4, the main arachidonate metabolite in horse eosinophils, suggesting that platelet-activating factor and leukotriene C4 have a common precursor pool (1-O-alkyl-2-arachidonyl-glycero-3-phosphocholine) and a common regulation of synthesis. Even though both ionomycin and A23187 are described as Ca2+ ionophores, they have a series of significantly different effects on the eosinophils with respect to formation of platelet-activating factor and leukotriene C4. While A23187 induces an asymptotic maximum of mediator formation at concentrations higher than 20 microM, ionomycin expressed a narrow optimum at 2 microM. The effects of exogenous pH on the release of mediators also differ strongly between the two ionophores: for A23187 the effects are the same for both mediators but when ionomycin is used as stimulant, generation of platelet-activating factor and leukotriene C4 are affected differently.

Animals↗
PubMed1992

Differential sensitivities of the prostacyclin and nitric oxide biosynthetic pathways to cytosolic calcium in bovine aortic endothelial cells.

1. Bovine aortic endothelial cells were cultured in vitro, and shown to release both prostacyclin (PGI2; Kact = 24.1 nM) and endothelium-derived relaxing factor (EDRF, NO; Kact = 0.7 nM) in a concentration-dependent manner when exposed to bradykinin. 2. The bradykinin-dependent release of PGI2 (but not EDRF) was inhibited by 1 microM isoprenaline or 5 microM forskolin, and the inhibitory effect of isoprenaline could be reversed by the beta 2-adrenoceptor antagonist, ICI 118551. In contrast, isoprenaline had no capacity to inhibit PGI2 release stimulated by exogenous arachidonic acid. 3. Exposure of cells to bradykinin increased the cytosolic concentration of Ca2+ ions ([Ca2+]i; Kact = 4.8 nM), and the effect was inhibited by both 1 microM isoprenaline and 5 microM forskolin. 4. In similar experiments, exposure of cells to ionomycin also increased [Ca2+]i and the values of [Ca2+]i were calibrated in terms of the ionomycin concentration. In subsequent experiments involving exposure of endothelial cells to selected concentrations of ionomycin, it was possible to show that the biosynthesis of NO was triggered at ionomycin concentrations about one tenth of the required for PGI2 biosynthesis and that these corresponded to a [Ca2+]i threshold of 350 nM for PGI2 release while that for EDRF release was less than 200 nM. 5. These differences in Ca2+ ion sensitivity explain the selective inhibition of bradykinin-stimulated PGI2 biosynthesis (to the exclusion of NO biosynthesis) by isoprenaline or forskolin, both of which attenuate bradykinin-dependent increases in [Ca2+]i.

Adrenergic beta-Antagonists↗
PubMed1993

Bradykinin-induced release of PGI2 from aortic endothelial cell lines: responses mediated selectively by Ca2+ ions or a staurosporine-sensitive kinase.

1. Bradykinin (100 nM) triggers release of nitric oxide and prostacyclin from both AG07680A and AG04762 bovine cultured aortic endothelial cells. The exposure of these cells to bradykinin is in each case associated with a striking rise in intracellular calcium ion concentration. 2. Exposure of AG07680A cells to 250 nM ionomycin was followed also by a significant release of prostacyclin, whereas 250 nM ionomycin had no capacity to stimulate release of prostacyclin from AG04762 cells. 3. There was a similar concentration-dependent increase in intracellular calcium ion concentration on exposure of AG07680A and AG04762 cells to ionomycin. 4. Exposure of AG04762 cells for 10 min to staurosporine produced a concentration-dependent inhibition (IC50 = 107 +/- 14 nM) in bradykinin-stimulated prostacyclin release. There was no similar inhibitory effect of staurosporine in AG07680A cells. 5. Bradykinin (10 nM) triggered release of nitric oxide from both AG07680A and AG04762 cells, and the effect was not inhibited by 500 nM staurosporine. There was a similar ionomycin-dependent release of nitric oxide from both cell types. 6. These results identify a common pathway for bradykinin-dependent nitric oxide release from both AG07680A and AG04762 cells, involving increases in intracellular calcium ion concentration. In contrast, the bradykinin-dependent release of prostacyclin may involve one of two pathways (involving an increase in intracellular calcium or activation of a staurosporine-sensitive kinase), and the two pathways are selectively exploited in AG07680A and AG04762 cells, respectively.

6-Ketoprostaglandin F1 alpha↗
PubMed1996

Inhibition of a store-operated Ca2+ entry pathway in human endothelial cells by the isoquinoline derivative LOE 908.

1. The novel cation channel blocker, LOE 908, was tested for its effects on Ca2+ entry and membrane currents activated by depletion of intracellular Ca2+ stores in human endothelial cells. 2. LOE 908 inhibited store-operated Ca2+ entry induced by direct depletion of Ca2+ stores with 100 nM thapsigargin or 100 nM ionomycin with an EC50 of 2 microM and 4 microM, respectively. 3. LOE 908 did not affect thapsigargin- or ionomycin-induced Ca2+ release from intracellular stores up to concentrations of 3 microM. 4. LOE 908 reversibly suppressed thapsigargin- as well as ionomycin-induced whole-cell membrane currents. 5. The LOE 908-sensitive membrane conductance corresponded to a cation permeability of 5.5 and 6.9 fold selectivity for Ca2+ over K+ in the presence of thapsigargin and ionomycin, respectively. 6. Our results suggest that the isoquinoline, LOE 908 is a novel, potent inhibitor of the store-operated (capacitive) Ca2+ entry pathway in endothelial cells.

Acetamides↗
PubMed1988

Effect of staphylococcal alpha-toxin on intracellular Ca2+ in polymorphonuclear leukocytes.

Staphylococcal alpha-toxin, a channel-forming protein, stimulates leukotriene B4 formation in rabbit polymorphonuclear leukocytes (PMN) (N. Suttorp, W. Seeger, J. Zucker-Reimann, L. Roka, and S. Bhakdi, Infect. Immun. 55:104-110, 1987). The concept was advanced that transmembrane toxin pores act as Ca2+ gates allowing passive Ca2+ influx into the cell, thus initiating stimulus response coupling. A critical step in this hypothesis is the demonstration of an increase in the cytosolic free Ca2+ concentration [( Ca2+]i). [Ca2+]i and membrane-associated Ca2+ were therefore monitored in quin-2- or chlorotetracycline-loaded PMN exposed to alpha-toxin. The effects of the Ca2+ ionophore ionomycin and the chemotactic tripeptide formylmethionyl-leucylphenylalanine (fMLP) were studied in parallel. All stimuli increased [Ca2+]i in dose- and time-dependent manner. In the presence of an EDTA excess there was a decrease of [Ca2+]i due to an efflux of Ca2+ in alpha-toxin- and ionomycin-treated cells, while addition of fMLP still induced an increase of [Ca2+]i. In the presence of verapamil, a Ca2+ channel blocker, [Ca2+]i was reduced after stimulation with fMLP but not with alpha-toxin or ionomycin. Addition of fMLP and ionomycin but not of alpha-toxin to PMN resulted in a rapid and substantial mobilization of membrane-associated Ca2+. The collective data demonstrate that exposure of PMN to staphylococcal alpha-toxin results in an increase in [Ca2+]i which is due to an influx of extracellular Ca2+ and not to a mobilization of intracellularly stored Ca2+. The concept of initiating stimulus response coupling by Ca2+ influx through transmembrane pores may be generally applicable to other channel-forming cytolysins.

Animals↗
PubMed1986

Role of prostaglandin release in the response of tight epithelia to Ca2+ ionophores.

We have studied the effects of the Ca2+ ionophores A23187 and ionomycin on ion transport across amphibian skin and urinary bladder. Both A23187 and ionomycin stimulated transepithelial Na+ transport across the skin. Ionomycin also markedly increased the conductance of an amiloride-insensitive pathway. Both ionophores markedly stimulated the release of prostaglandin E2 (PGE2) into the solution bathing the serosal surface of the skin. Addition of indomethacin to the serosal bathing solution of the skin blocked both the stimulation of short-circuit current (Isc) and the release of prostaglandin caused by the ionophores. Acetylsalicylic acid also blocked the ionomycin-induced stimulation of Isc. These results suggest that the stimulation of Na+ transport caused by Ca2+ ionophores is mediated by the release of a product of the cyclooxygenase pathway, very likely PGE2. Ca2+ ionophores also stimulated the release of PGE2 in urinary bladders; however, they generally depressed Isc. Since the effect on Isc caused by the addition of exogenous PGE2 was different in urinary bladders than in skins, we suggest that at least part of the difference in the action of ionophores is due to the difference in the sensitivity of these epithelia to PGE2. Our results suggest that the heterogeneity of effects that Ca2+ ionophores cause in the physiological parameters of tight epithelia are not always the direct result of increased cytoplasmic Ca2+ but that they may be mediated by other tissue responses triggered by the addition of the ionophores.

Animals↗
PubMed1991

Regulation of calcium-activated potassium efflux by neurotensin and other agents in HT-29 cells.

Neurotensin receptors have been shown previously to be coupled to polyphosphoinositide turnover and intracellular Ca2+ ([Ca2+]i) mobilization in HT-29 colonic epithelial cells (Bozou et al. Biochem. J. 264: 871, 1989; Turner et al. J. Pharmacol. Exp. Ther. 253: 1049, 1990). In this study, neurotensin was found to enhance dramatically the Ba2(+)- and tetraethylammonium chloride-sensitive K(+)-efflux rate (measured with 86Rb+) in the presence of ouabain and bumetanide, with basal efflux increasing 4.5 +/- 0.5-fold with 10 nM neurotensin. The K(+)-efflux rate, which was partially dependent on the extracellular Ca2+ concentration, was also increased by carbachol and ATP, two other [Ca2+]i-mobilizing agonists in HT-29 cells, as well as by the Ca2+ ionophores ionomycin and A23187, suggesting that the efflux was through Ca2(+)-activated K+ channels. Pretreatment of cells with neurotensin, carbachol, or ATP desensitized subsequent neurotensin-stimulated efflux by 82, 57, and 63%, respectively, confirming our previous results which indicated homologous and heterologous desensitization of the neurotensin receptor-signal transduction pathway. Pretreatment of cells with the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and mezerein did not affect [Ca2+]i mobilization or K+ efflux directly but desensitized neurotensin-stimulated efflux by greater than 80%. Pretreatment (2 h) with PMA also decreased K+ efflux in response to ionomycin by 59%, although ionomycin-induced [Ca2+]i mobilization was not inhibited. Downregulation of protein kinase C by overnight pretreatment with PMA resulted in recovery of ionomycin-stimulated efflux. These results suggest that agonist-stimulated Ca2(+)-activated K+ channels in HT-29 cells are regulated at multiple steps in the signal transduction pathway.

Adenocarcinoma↗
PubMed1993

Properties of cAMP-dependent and Ca(2+)-dependent whole cell Cl- conductances in rat epididymal cells.

Single rat epididymal cell studied under whole cell patch-clamp condition responded to 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) (500 microM) and to ionomycin (1 microM) by an increase in whole cell conductance. A major part of the stimulated current was carried by Cl-, although a small part was due to nonselective cation current. After elimination of the cation current component by using impermeant cation, the cells revealed different Cl- conductance properties in response to adenosine 3',5'-cyclic monophosphate (cAMP) and ionomycin. The cAMP-stimulated Cl- conductance was independent of time and voltage and showed a linear current-voltage relationship. The anion permselectivity was NO3- > Br- > Cl- approximately I- >> SO(4)2-. The ionomycin-stimulated Cl- conductance showed marked time and voltage dependency. In contrast to the cAMP-induced anion permselectivity, the ionomycin-induced anion permselectivity was I- > Br- approximately NO3- > Cl- >> SO(4)2-. These results indicate that the epididymal epithelial cells exhibit different Cl- conductances sensitive to cAMP and Ca2+. The cAMP-activated conductance has properties resembling the type associated with the cystic fibrosis transmembrane conductance regulator found in cystic fibrosis-affected epithelia. This finding supports the notion that the epididymis is a cystic fibrosis epithelium.

Animals↗
PubMed1993

Regulation of K secretion across the porcine gallbladder epithelium.

Porcine gallbladder epithelium from the neck and the fundus of the organ was stripped of serosal muscle and mounted in Ussing chambers to investigate the mechanisms of K secretion. The sensitivity to K channel blockers and regulation by norepinephrine (NE), adenosine 3',5'-cyclic monophosphate (cAMP), and increases in intracellular Ca concentration ([Ca]) were studied. The porcine gallbladder secretes K (approximately 0.8 mu eq/cm2.h) under basal conditions. Mucosal tetraethylammonium (TEA) produced a concentration-dependent increase in short-circuit current (Isc) and inhibited the unidirectional serosal-to-mucosal 86Rb flux JsmRb, resulting in a > 60% reduction in net Rb secretion. In contrast, serosal Ba produced a concentration-dependent decrease in Isc and stimulated JsmRb, resulting in a > 200% increase in net Rb secretion. NE inhibited JsmRb and net Rb secretion in both regions. In the fundic region the mucosal-to-serosal Rb flux (JmsRb) was also significantly increased, suggesting that active K absorption was activated. Exogenous cAMP increased JsmRb and net Rb secretion by > 85% in both regions. This increase in net Rb secretion was blocked by mucosal TEA but unaffected by NE. The Ca ionophore ionomycin also increased JsmRb and net Rb secretion and reduced the Isc by approximately 50%. Neither mucosal TEA nor Ba blocked changes in steady-state Rb secretion induced by ionomycin. Although both serosal Ba and ionomycin produced significant reductions in Isc, the effects of Ba were blocked by ionomycin pretreatment. These findings indicate that basal K secretion occurs through TEA-sensitive apical K channels and is regulated by intracellular cAMP. NE likely reduces K secretion by decreasing intracellular concentration of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Multiple modes of regulation of airway epithelial chloride secretion by extracellular ATP.

Cultured normal and cystic fibrosis (CF) airway epithelia were exposed to 5'-(N-ethylcarboxamido)-adenosine (NECA), ATP, or ionomycin. NECA activated a sustained, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-insensitive Cl- secretory response in normal but not CF, consistent with stimulation of the CF transmembrane conductance regulator (CFTR). In normal and CF, ionomycin or ATP induced Cl- secretion with an initial peak that was inhibited > 50% by DIDS, but in normals there was a prolonged current that was not inhibited by DIDS. The ATP and ionomycin responses in CF were of greater magnitude, and the prolonged phase was inhibited by DIDS. Although we expected ATP to regulate Cl- conductance through intracellular Ca2+ activity, ATP further stimulated Cl- secretion in tissues pretreated to maximally elevate intracellular Ca2+ activity. ATP also activated whole cell Cl- currents in cells dialyzed with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Thus ATP and ionomycin regulate a Cl- conductance that is distinct from CFTR, but the regulation by ATP is not tightly coupled to intracellular Ca2+ activity. Alternatively, ATP regulates separate Ca(2+)-sensitive and Ca(2+)-insensitive Cl- conductances. Furthermore, extracellular ATP activates DIDS-resistant Cl- secretion in normal but not CF cultured epithelia, consistent with activation of CFTR by extracellular ATP.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Characterization of volume-sensitive taurine- and Cl(-)-permeable channels.

Volume-sensitive Cl- channels [ICl(vol)] were studied using taurine efflux and patch-clamp experiments in 9HTEo- human tracheal cells. Cells were stimulated with the Ca(2+)- elevating agents ATP and ionomycin in isotonic medium or in hypotonic solutions. ATP (100 microM) or ionomycin (1 microM) and hypotonic shock produced a synergic effect. Indeed, the resulting taurine efflux was much higher than the sum of the single effects elicited by ATP, ionomycin, or hypotonic medium. The taurine release elicited by hypotonic shock and the potentiation by ATP and ionomycin were markedly inhibited by using a Ca(2+)-free extracellular medium and by incubating the cells with the membrane-permeable 1,2-bis(2-amino- phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester chelating agent. Patch-clamp experiments confirmed the role of Ca2+ on ICl(vol) channels. Swelling-induced taurine efflux was inhibited by reactive blue 2, suramin, and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid. Patch-clamp experiments demonstrated that these compounds shift the voltage-dependent inactivation of ICl(vol) channels toward more negative values. This study indicates that the sensitivity of ICl(vol) to cell volume changes is modulated by intracellular Ca2+ and that purinergic receptor antagonists represent a new class of CI- channel blockers.

Adenosine Triphosphate↗
PubMed1989

Carbachol-induced protein phosphorylation in parietal cells: regulation by [Ca2+]i.

1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), an intracellular calcium [( Ca2+]i) chelator, was used to investigate the role of [Ca2+]i in acid secretory activity and protein phosphorylation in parietal cells from rabbit. Chelation of extracellular calcium [( Ca2+]o) with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not prevent the initial carbachol-induced elevation of [Ca2+]i as measured with the fluorescent Ca2+ probe, fura-2, and only partially inhibited [14C]-aminopyrine (AP) accumulation, an indirect indicator of acid secretory activity. [Ca2+]i chelation with BAPTA/AM eliminated carbachol-stimulated increases in [Ca2+]i and AP accumulation but only transiently reduced histamine stimulation of AP accumulation. Carbachol increased phosphorylation of a 36-kDa, pI approximately 7 protein (pp36) and transient phosphorylation of a 28-kDa, pI approximately 5 protein (pp28), whereas histamine increased phosphorylation of 40-kDa, pI approximately 6.5 (pp40) and 27-kDa, pI approximately 6.2 (pp27) proteins. Phosphorylation of pp36 and pp28 were mimicked by the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore, ionomycin, respectively. Two other phosphoproteins with molecular weights of 66,000 and pIs of 5.7 and 5.9 were also phosphorylated in response to TPA and carbachol. Chelation of [Ca2+]i and [Ca2+]o blocked carbachol-induced phosphorylation of pp28 and pp36 and ionomycin phosphorylation of pp28 but not TPA-stimulated phosphorylation of pp36 or the two pp66s or histamine-stimulated phosphorylation of pp27 or pp40. Chelation of [Ca2+]i alone did not block increases in [Ca2+]i or phosphorylation of pp28 in response to ionomycin. Both pp28 and pp36 were localized in both microsomal and cytosolic fractions of cells, which suggests involvement in cytoskeleton-membrane interactions. These phosphoproteins could be common elements of Ca2+-dependent stimulus-secretion coupling as similar proteins were phosphorylated by carbachol and cholecystokinin (CCK) in chief cells. Based on data with TPA and ionomycin, both protein kinase C and an as yet unidentified Ca2+-dependent protein kinase(s) appear to be activated upon stimulation with cholinergic agonists and CCK.

Aminopyrine↗
PubMed1996

Ca(2+)-activated C1- channels in a human biliary cell line: regulation by Ca2+/calmodulin-dependent protein kinase.

Biliary epithelial cells contribute to bile formation through absorption and secretion of fluid and electrolytes. Recent studies indicate that membrane Cl- permeability is regulated in part by increases in intracellular Ca2+ concentration. The purpose of these studies was to evaluate the effects of intracellular Ca2+ on channel activity, using the human Mz-ChA-1 cholangiocarcinoma cell line as a model, and to assess the possible roles of Ca(2+)-dependent kinases in channel regulation. Exposure to ionomycin (1 microM) activated ion channels in the cell-attached configuration in 63 of 74 attempts, increasing open probability (NPo) from 0 to 0.26 +/- 0.15 (n = 17). Multiple channels were present in each patch, and the effects of ionomycin were reversed by subsequent addition of ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'- tetraacetic acid (2 mM) to the bath. With Cl(-)-containing solutions, channels had a slope conductance of 14 +/- 4 pS (n = 11), and the mean open time was estimated to be 5.3 +/- 1.8 ms. These channels were anion selective, and currents were carried by efflux of Cl- at the resting potential. Exposure to the Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist calmidazolium (100 microM) decreased NPo in ionomycin-stimulated cells to 0.02 +/- 0.06 (n = 19). The protein kinase C antagonist chelerythrine (50 microM) was without effect. In parallel studies in subconfluent cell monolayers, CaMKII antagonists were also potent inhibitors of ionomycin-stimulated 125I efflux. These findings indicate that Ca(2+)-dependent increases in membrane Cl- permeability are related in part to opening of 14.pS anion channels through a mechanism that depends on both Ca2+ and CaMKII. These channels represent a potential target for pharmacological modulation of biliary cell transport and function.

Bile Ducts↗