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 1,117 records · Page 62Linked to original sources
PubMed1995

Vasopressin stimulation of Ca2+ mobilization, two bivalent cation entry pathways and Ca2+ efflux in A7r5 rat smooth muscle cells.

1. Arg8-vasopressin (AVP)-regulated Ca2+ transport were investigated in fura-2-loaded A7r5 cells using both single cell and population measurements. 2. AVP evokes an initial concentration-dependent rise in cytosolic free Ca2+ concentration ([Ca2+ ]i) to a peak which is independent of extracellular Ca2+, and a sustained Ca2+ signal that results from a balance between stimulation of Ca2+ entry and efflux. 3. Depletion of intracellular Ca2+ stores with thapsigargin, ionomycin, or prior treatment with AVP in Ca2(+)-free medium activates 'capacitative' entry of Ca2+, Ba2+ or Mn2+. Capacitative Mn2+ entry is inhibited by refilling stores with Ca2+; neither Sr2+ nor Ba2+ substitute for Ca2+ to give this effect. 4. In cells with empty stores, AVP stimulates further bivalent cation entry, and the effect persists when extracellular Na+ is replaced by N-methyl-D-glucamine or under depolarizing condition (extracellular KCl concentration ([KCl]o), 135 mM). This effect of AVP is not therefore merely a consequence of AVP causing membrane hyperpolarization or stimulation of Na(+)-Ca2+ exchange, but results from opening of a bivalent cation influx pathway. 5. Several lines of evidence indicate that AVP-stimulated bivalent cation entry is not a consequence of more complete emptying of the intracellular stores and consequent further activation of the capacitative pathway. AVP stimulates Ba2+ entry when the intracellular Ca2+ stores have been both emptied by ionomycin and prevented from refilling by thapsigargin. Mn2+ permeates the capacitative pathway, but AVP does not further increase Mn2+ entry, confirming that AVP does not further activate the capacitative pathway and that the two pathways differ in their permeability to Mn2+. When the extracellular [Sr2+] is low, empty stores do not stimulate detectable Sr2+ entry, but addition of AVP causes substantial Sr2+ entry. 6. A decrease in [Ca2+]i occurs when 50 nM AVP is added during a sustained elevation of [Ca2+]i evoked by thapsigargin. Since AVP does not inhibit the capacitative pathway, this result suggests that AVP stimulates Ca2+ extrusion. 7. We conclude that stimulation of Ca2+ mobilization, two modes of bivalent cation entry, and Ca2+ efflux all contribute to the complex concentration-dependent effects of AVP in A7r5 smooth muscle cells.

Animals↗
PubMed1996

Role of calbindin-D9k in buffering cytosolic free Ca2+ ions in pig duodenal enterocytes.

1. The aim of the present study was to test whether the vitamin D-dependent Ca(2+)-binding protein calbindin-D9k could function as an important cytosolic Ca2+ buffer in duodenal enterocytes while facilitating transepithelial active transport of Ca2+ ions. For the investigations we used dual-wavelength, fluorescence ratio imaging, with fura-2 as the Ca(2+)-sensitive dye, to measure changes in cytosolic concentrations of free Ca2+ ions ([Ca2+]i) in isolated pig duodenal enterocytes affected by different cytosolic calbindin-D9k concentrations. 2. Epithelial cells were obtained from weaned piglets with normal calbindin-D9k concentrations (con-piglets), from piglets with low calbindin-D9k levels due to inherited calcitriol deficiency caused by defective renal 25-hydroxycholecalciferol D3-1 alpha-hydroxylase activity (def-piglets), and from piglets with reconstituted calbindin-D9k concentrations, i.e. def-animals treated with high doses of vitamin D3 which elevated plasma calcitriol levels by extrarenal production (def-D3-piglets). Basal levels of [Ca2+]i ranged between 170 and 205 nM and did not differ significantly between the groups. 3. After addition of 5 mM theophylline, the [Ca2+]i in enterocytes from con-piglets doubled during the 10 min incubation. This effect, however, was three times higher in enterocytes from def-piglets compared with those from con-piglets. Similar results were obtained after 4 min incubation of enterocytes from con- and def-piglets in the presence of 1 microM ionomycin. In preparations from def-D3-piglets, ionomycin-induced increases in [Ca2+]i were significantly lower compared with enterocytes from def-piglets and were not different from the control values. 4. From the results, substantial support is given for the hypothesis that one of the major functions of mucosal calbindin-D9k is the effective buffering of Ca2+ ions.

Animals↗
PubMed1995

Human peripheral blood CD4+ and CD8+ T cells express Th1-like cytokine mRNA and proteins following in vitro stimulation with heat-inactivated Brucella abortus.

Defining the pattern of lymphokine production associated with Brucella abortus is critical for advancing the development of B. abortus as a vaccine carrier. In the present study we investigated the ability of heat-inactivated B. abortus or lipopolysaccharide from B. abortus to induce lymphokine production from purified human T cells in vitro. Gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5 induction was assayed by mRNA-specific PCR and by enzyme-linked immunosorbent assay and bioassay for protein production. Following depletion of monocytes and B cells, B. abortus increased IFN-gamma and IL-2 mRNA expression in purified T cells compared with expression in unstimulated cells. In contrast, no IL-5 mRNA expression and only transient low-level IL-4 mRNA expression and no IL-4 protein secretion were detected. Phytohemagglutinin or phorbol myristate acetate plus ionomycin induced mRNA and protein for all these cytokines. Similar results were obtained with LPS purified from B. abortus. Removal of NK cells did not reduce lymphokine production, and enriched NK cells did not express IFN-gamma mRNA or secrete IFN-gamma protein in response to B. abortus, indicating that NK cells were not the responding population. Both CD4+ and CD8+ populations produced IFN-gamma and IL-2 in response to B. abortus. Preincubation of resting T cells with B. abortus or LPS from B. abortus for 7 days induced their differentiation into Th1-like cells as judged by their subsequent lymphokine response to phorbol myristate acetate plus ionomycin. These results suggest that B. abortus can induce differentiation of Th0 into Th1-type cells.

Base Sequence↗
PubMed2002

Effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta and RANTES mRNA expression in guinea pig cells exposed to attenuated and virulent mycobacteria.

The effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta (IL-1 beta) or regulated-upon-activation, normally T-cell-expressed and -secreted chemokine (RANTES) mRNA expression in guinea pig spleen cells stimulated with concanavalin A, lipopolysaccharide (LPS), phorbol myristate acetate (PMA) plus ionomycin, or purified protein derivative (PPD) was studied in vitro. Similarly, peritoneal exudate cell-derived macrophages from naïve and BCG-vaccinated guinea pigs were infected with M. bovis BCG, Mycobacterium avium, the attenuated Mycobacterium tuberculosis H37Ra strain, or virulent strains H37Rv and Erdman of M. tuberculosis. Total RNA was subjected to Northern blot analysis using probes generated from guinea pig IL-1 beta or RANTES cDNA. Although IL-1 beta and RANTES mRNA could be detected in the spleen cells from naïve animals stimulated with LPS or PMA plus ionomycin, the levels were significantly enhanced after BCG vaccination. mRNA expression was also elevated in macrophages infected with live mycobacteria after BCG vaccination. However, macrophages infected with the virulent H37Rv strain of M. tuberculosis showed 75 to 90% reductions in IL-1 beta expression and 25 to 60% reductions in RANTES mRNA expression compared with macrophages infected with the attenuated H37Ra strain. The IL-1 beta mRNA levels peaked as soon as 1 h after PPD stimulation and 4 h after M. tuberculosis H37Rv infection of macrophages. In contrast, RANTES mRNA expression was delayed until 48 h after infection. These results indicate that molecular mediators produced in response to various stimuli associated with protective immunity against mycobacteria are upregulated after BCG vaccination; however, a significantly weaker response was observed with virulent M. tuberculosis. These initial studies indicate that BCG vaccination has a positive effect on IL-1 beta and RANTES mRNA expression by host cells in a highly relevant animal tuberculosis model.

Animals↗
PubMed2002

Regulation of intracellular calcium in N1E-115 neuroblastoma cells: the role of Na(+)/Ca(2+) exchange.

In fura 2-loaded N1E-115 cells, regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) following a Ca(2+) load induced by 1 microM thapsigargin and 10 microM carbonylcyanide p-trifluoromethyoxyphenylhydrazone (FCCP) was Na(+) dependent and inhibited by 5 mM Ni(2+). In cells with normal intracellular Na(+) concentration ([Na(+)](i)), removal of bath Na(+), which should result in reversal of Na(+)/Ca(2+) exchange, did not increase [Ca(2+)](i) unless cell Ca(2+) buffer capacity was reduced. When N1E-115 cells were Na(+) loaded using 100 microM veratridine and 4 microg/ml scorpion venom, the rate of the reverse mode of the Na(+)/Ca(2+) exchanger was apparently enhanced, since an approximately 4- to 6-fold increase in [Ca(2+)](i) occurred despite normal cell Ca(2+) buffering. In SBFI-loaded cells, we were able to demonstrate forward operation of the Na(+)/Ca(2+) exchanger (net efflux of Ca(2+)) by observing increases (approximately 6 mM) in [Na(+)](i). These Ni(2+) (5 mM)-inhibited increases in [Na(+)](i) could only be observed when a continuous ionomycin-induced influx of Ca(2+) occurred. The voltage-sensitive dye bis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used to measure changes in membrane potential. Ionomycin (1 microM) depolarized N1E-115 cells (approximately 25 mV). This depolarization was Na(+) dependent and blocked by 5 mM Ni(2+) and 250-500 microM benzamil. These data provide evidence for the presence of an electrogenic Na(+)/Ca(2+) exchanger that is capable of regulating [Ca(2+)](i) after release of Ca(2+) from cell stores.

Animals↗
PubMed2002

The calcium-dependent chloride conductance mediator pCLCA1.

The regulatory behavior, inhibitor sensitivity, and properties of the whole cell chloride conductance observed in cells expressing the cDNA coding for a chloride conductance mediator isoform of the CLCA gene family, pCLCA1, have been studied. Common C-kinase consensus phosphorylation sites between pCLCA1 and the closely related human isoform hCLCA1 are consistent with a role for calcium in channel activation. Both channels are activated rapidly on exposure to the calcium ionophore ionomycin. Direct involvement of calcium in the activation of pCLCA1 was supported by the finding that treatment with the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM reduced the rate of chloride efflux from NIH/3T3 cells expressing the pCLCA1 channel. No combination of A-kinase activators used was effective in activating chloride efflux via this channel despite the presence of a unique strong A-kinase consensus site in pCLCA1. Notable differences of pCLCA1 from the reported properties of CLCA family members include the failure of phorbol 12-myristate 13-acetate to activate chloride efflux in cells expressing pCLCA1 and a lack of inhibition of chloride efflux from these cells after treatment with DIDS or dithiothreitol. However, selected inhibitors of anionic conductance inhibited pCLCA1-dependent anion efflux. The electrogenic nature of the ionomycin-dependent efflux of chloride from cells expressing pCLCA1 was confirmed by detection of outwardly rectifying chloride current and inhibition of this current by chloride conductance inhibitors in a whole cell patch-clamp study.

3T3 Cells↗
PubMed1987

Effects of bradykinin and angiotensin II on intracellular Ca2+ dynamics in endothelial cells.

The purpose of this study was to investigate the effects of angiotensin II and bradykinin on intracellular Ca2+ dynamics in cultured endothelial cells. We used the "second-generation" fluorescent Ca2+ indicator fura-2, in conjunction with dual-wavelength fluorescence spectroscopy, in cultured adherent pulmonary arterial endothelial cells. Angiotensin II (up to 2 microM) had no consistent effect on intracellular Ca2+ levels. In contrast, bradykinin (10 nM) elicited a transient increase of cytosolic free Ca2+, from the resting value of 37 +/- 5 to 647 +/- 123 nM, followed by a decline to a steady-state value of 113 +/- 14 nM, which was significantly higher than the resting Ca2+ levels. Bradykinin's Ca-stimulatory effect was dose dependent, having a half-maximally effective concentration of approximately 1 nM and a maximally effective concentration of 10 nM. A B1-receptor agonist, Des-Arg9-bradykinin, was much less effective than bradykinin as modulator of cytosolic Ca2+. Moreover, a B1-receptor antagonist, Des-Arg9, [Leu8]-bradykinin, did not significantly affect the increase of cytosolic Ca2+ elicited by bradykinin. On the other hand, the bradykinin-elicited increase of Ca2+ was almost completely inhibited by a novel B2-receptor antagonist, D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin. Bradykinin increased cytosolic free Ca2+ levels in cells maintained in Ca2+-deficient extracellular medium, suggesting that the peptide mobilized Ca2+ from intracellular stores. However, the absence of extra-cellular Ca2+ resulted in an 80-90% attenuation of the transient Ca2+ response, whereas the posttransient steady-state response was completely absent. These findings are consistent with the notion that the bradykinin-elicited transient Ca2+ response is dependent on both extra- and intracellular Ca2+ and that the posttransient steady-state response is entirely dependent on extracellular Ca2+. Endothelial cells were responsive to a second dose of bradykinin after a 10-min interim period of incubation in the absence of the peptide hormone. The absence of extracellular Ca2+ during the interim period, or the pretreatment of cells with ionomycin in the absence of extracellular Ca2+, prevented the response of the cells to a second dose of bradykinin. Bradykinin- or ionomycin-desensitized cells could be resensitized by a brief incubation period in Ca2+-replete medium. The results are consistent with the notions that cellular resensitization requires the replenishment of intracellular Ca2+ and that bradykinin, but not angiotensin II, modulates intracellular Ca2+ dynamics in endothelial cells by interacting with a B2-type receptor.

Angiotensin II↗
PubMed1988

Cyclic nucleotide-dependent protein kinase inhibition by H-8: effects on ion transport.

We explored the potential role of cyclic nucleotide-dependent protein phosphorylation in regulating ion transport across flounder intestinal mucosa by studying the effects of N-[2(methylamino)-ethyl]-s-isoquinolinesulfonamide (H-8), a selective inhibitor of cyclic nucleotide-dependent protein kinase in vitro. Addition of H-8 reversed the inhibitory effects of 8-bromoguanosine 3',5'-cyclic-monophosphate (8-BrcGMP), 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), atriopeptin III (AP III), and vasoactive intestinal peptide (VIP) on the short-circuit current (Isc) and transepithelial potential difference (PD). Flux measurements established that these changes in Isc and PD directly reflected changes in Na and Cl absorption by the intestine. H-8 was unable, however, to reverse the inhibitory effects on Isc and PD of the Ca ionophore ionomycin and of substance P at dosages exceeding those needed to reverse the effects of AP III, VIP, and the cyclic nucleotides. We conclude that 1) H-8 (100 microM or less) does not exert toxic effects, 2) exogenously added cyclic nucleotide analogues inhibit ion transport through activation of cyclic nucleotide-dependent kinases resulting in protein phosphorylation, 3) activation of these kinases is an essential intermediate step in the inhibitory action of AP III and VIP on ion transport, and 4) the Ca ionophore ionomycin and substance P appear to inhibit ion transport by a mechanism that is independent of cyclic nucleotide-dependent protein phosphorylation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Activation of an apical Cl- conductance by Ca2+ ionophores in cystic fibrosis airway epithelia.

Cystic fibrosis (CF) airway epithelia express a defect in adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of apical membrane Cl- channels. Recent patch-clamp studies have raised the possibility that Ca2+ -dependent mechanisms for the activation of Cl- secretion may be preserved in CF airway epithelia. To determine 1) whether intact normal (N1) and CF airway epithelia exhibit a Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanisms initiate Cl- secretion via activation of an apical membrane Cl- conductance (GCl-), nasal epithelia from N1 and CF subjects were cultured on collagen membranes, and responses to isoproterenol or Ca2- ionophores [A23187 10(-6) M; ionomycin (10(-5)M)] were measured with transepithelial and intracellular techniques. Isoproterenol induced activation of an apical membrane GCl- in N1 cultures but was ineffective in CF. In contrast, in both N1 and CF amiloride-pretreated cultures, A23187 induced an increase in the equivalent short-circuit current that was associated with an activation of an apical membrane Gc1- and was bumetanide inhibitable. A23187 addition during superfusion of the lumen with a low Cl- (3 mM) solution reduced intracellular Cl- activity of CF cells. A Ca2+ ionophore of different selectivity properties, ionomycin, was also an effective Cl- secretagogue in both N1 and CF cultures. We conclude that 1) the A23187 induced Cl- secretion via activation of an apical GCl- in N1 human nasal epithelium, and 2) in contrast to an isoproterenol-dependent path, a Ca2+ -dependent path for GCl- activation is preserved in CF epithelia.

Adolescent↗
PubMed1989

Multiple calcium-mediated effector mechanisms regulate chloride secretory responses in T84-cells.

Free cytosolic Ca2+ [( Ca2+]i) has been implicated as a second messenger mediating the ion transport effects of carbachol, histamine, taurodeoxycholate, ionomycin, and 4-bromo-A23187 (4-BrA23187) in T84-cells. In this study, we correlated short-circuit current (Isc, reflective of Cl- secretion) and [Ca2+]i responses in T84-cell monolayers stimulated by these agents to evaluate the role of [Ca2+]i in Cl- secretory responses. Time-course studies showed that the duration of [Ca2+]i and Isc responses did not correlate with one another. Isc responses were more prolonged than [Ca2+]i responses with carbachol and histamine (both derived [Ca2+]i partly from intracellular sources), less prolonged than [Ca2+]i with taurodeoxycholate, and continued to increase after [Ca2+]i stabilized with ionomycin and 4-BrA23187. Isc and [Ca2+]i responses to histamine and carbachol were additive. A comparison of the magnitude of [Ca2+]i and Isc responses in cells stimulated by different agonists showed that the change in [Ca2+]i accompanying equivalent Isc responses varied greatly, suggesting that secretagogues vary in their dependency on [Ca2+]i. These findings suggest the existence of multiple [Ca2+]i-mediated effector mechanisms or the existence of multiple mediators that augment or attenuate the action of [Ca2+]i.

Calcium↗
PubMed1991

CaMKII mediates stimulation of chloride conductance by calcium in T84 cells.

We used the secretory colonic cell line T84 to study the regulatory pathways controlling the Ca-stimulated Cl conductance [GCl(Ca)]. Under whole cell patch clamp, basal (unstimulated) current levels averaged 73 +/- 9 pA/20 pF (n = 93) and increased to 600 +/- 100 pA/20 pF (n = 53; at +100 mV) on exposure to 1-2 microM ionomycin. Bath application of the calmodulin (CaM) antagonists trifluoperazine, calmidazolium, or sphingosine (50 microM) reversibly inhibited GCl(Ca), whereas the protein kinase C antagonists H7 and phloretin (50 microM) were without effect. This suggests that increases in intracellular Ca stimulate GCl(Ca) via a CaM-dependent process rather than activating Cl channels directly. To assess the involvement of protein kinases in the Ca-dependent stimulation of Cl conductance, we employed pseudosubstrate peptide inhibitors of protein kinase C (PKC) and the Ca/CaM-dependent protein kinase II (CaMKII). Cellular concentrations of inhibitors during whole cell recording were estimated to be 4-20 times the inhibitory constant values for kinase inhibition observed in vitro. Pipette solutions containing the PKC peptide inhibitor PKC-(19-36) (7.5 microM) had no effect on GCl(Ca). In contrast, stimulation of GCl(Ca) by ionomycin was abolished when pipette solutions contained 10 microM CaMKII peptide inhibitor CaMKII-(273-302). The truncated peptide CaMKII-(284-302) (20 microM) lacks the CaMKII inhibitory domain and did not affect GCl(Ca). These data suggest that CaM, acting through the multifunctional CaMKII, mediates the Ca-dependent stimulation of Cl conductance in colonic secretory cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Carbachol-stimulated phosphorylation of a 170-kDa endogenous protein in avian salt gland cells.

The effect of cholinergic stimulation of cellular protein phosphorylation was studied using an intact cell preparation isolated from the avian salt gland. Isolated cells were allowed to incorporate 32Pi into the cellular ATP pool and then challenged with compounds known to induce ion secretion in this tissue. Addition of carbachol resulted in a time- and concentration-dependent (EC50 = 500 nM) increase in 32Pi content of a 170-kDa protein (pp170). The stimulated phosphorylation could be blocked by the inclusion of atropine (100 microM). Subcellular fractionation studies localized pp170 to the plasma membrane fraction of the tissue. The integral nature of this protein was demonstrated by detergent-solubilization experiments with Triton X-100. The possibility that carbachol stimulates phosphorylation of pp170 via activation of protein kinase C (PKC) was investigated. Incubating salt gland cells with 4 beta-phorbol 12-myristate 13-acetate (PMA; 1 microM) or carbachol (100 microM) resulted in a translocation of soluble PKC from the cytosol to a plasma membrane fraction. Addition of either PMA (1 microM) or ionomycin (1 microM) alone did not enhance phosphorylation of pp170. A 4.5-fold increase in the phosphorylation state of pp170 was only observed when PMA and ionomycin were added concurrently. Preincubation of salt gland cells with PKC inhibitors H-7 (50 microM) or staurosporine (10 microM) inhibited the carbachol-stimulated phosphorylation of pp170. These findings suggest that carbachol mediates its secretomimetic effects via activation of PKC and that pp170 may represent a novel integral membrane PKC substrate protein.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Extracellular Na+ dependency of free cytosolic Ca2+ regulation in aortic vascular smooth muscle cells.

This study examined contribution of Na(+)-dependent processes to the regulation of free cytosolic calcium (Ca2+i) in cultured vascular smooth muscle cells (VSMC) using fura-2. Removal of Na+ from superfusate (replacement with choline) resulted in an increment of Ca2+i that was greatly augmented by pretreatment with ouabain. Under both conditions, Ca2+i increase was followed by partial recovery to a new steady state that was still significantly higher than that seen before removal of external Na+ (Na+o). In ouabain-pretreated cells lowering of Na+o caused progressive increases in Ca2+i. Addition of NiCl2, a Na(+)-Ca2+ exchange inhibitor, completely blocked the increase in Ca2+i produced by removal of Na+o, indicating that the Na(+)-Ca2+ antiporter was responsible for observed Ca2+i changes. Ca2+i increase produced by reduction of Na+o was also seen after depletion of inositol trisphosphate-sensitive Ca2+ stores with repeated pulses of angiotensin II or after blockade of sarcoplasmatic reticulum Ca2+ release with TMB-8 but was not observed in the absence of external Ca2+. These observations indicate that the source of Ca2+i increase in response to changes in the transmembrane Na+ gradient is largely external, and potentiation of the Ca2+i surge by ouabain suggests Ca2+ influx via the Na(+)-Ca2+ exchanger operating in the reverse mode. The relative contribution of a Na(+)-dependent and -independent component of Ca2+i recovery was investigated by superfusing cells with ionomycin in a Na(+)-free medium and later adding Na+ to the medium. This Ca2+ ionophore increased Ca2+i to a peak, and this was followed by a rapid but partial recovery to a new steady state. Readdition of varying amounts of Na+ to the superfusate, in the continued presence of ionomycin, resulted in concentration-related decline in Ca2+i, thereby uncovering a substantial contribution of a Na(+)-dependent mechanism of Ca2+i regulation. Decline of Ca2+i produced by readdition of Na+ was blocked by addition of NiCl2 to the superfusate. Our findings thereby provide evidence for Ca2+i regulation in VSMC via a Na(+)-dependent mechanism, consistent with a Na(+)-Ca2+ exchanger, which acts as a Ca2+ efflux mechanism when Ca2+i is elevated. Na(+)-Ca2+ exchanger acts as a Ca2+ influx mechanism when intracellular Na+ is elevated by prior exposure to ouabain.

Animals↗
PubMed1992

Calcium-activated phosphatidylcholine-specific phospholipase C and D in MDCK epithelial cells.

Calcium ionophore exposure generates diglycerides (DAG) from phosphatidylcholine (PC) hydrolysis in Madin-Darby canine kidney (MDCK) epithelial cells. This study compares calcium ionophore-activated PC hydrolysis with the previously described phorbol ester-stimulated PC hydrolysis pathway using MDCK cells labeled with [14C]-linoleic acid. Lipid species were measured using thin-layer chromatography. DAG resulted in part from PC hydrolysis because DAG increased in cells labeled with [palmitoyl-2-14C]phosphatidylcholine. Neither protein kinase C (PKC) inhibitors nor PKC depletion affected the ionomycin (IONO)-induced increase in DAG. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the increased DAG after IONO but not after phorbol 12,13-dibutyrate (PDBu) exposure. The EGTA effect was reversed by adding excess calcium but was not reversed by adding excess Mg2+. IONO exposure also increased phosphatidic acid (PA) production. The PA was produced by phospholipase D (PLD) because phosphatidylethanol was produced when IONO was added to the cells in the presence of ethanol. Although increasing concentrations of ethanol resulted in progressively less PA, it had no effect on increased DAG after IONO exposure at any time point tested. These data are consistent with both increased phospholipase C (PLC) and increased PLD activity following ionomycin. In contrast to IONO exposure, ethanol completely prevented the increase in DAG after PDBu exposure, consistent with DAG produced by PLD activation. These results demonstrate that calcium activates both PC-specific PLC and PLD in MDCK cells and that the calcium-activated pathway is independent of the previously described PKC activation pathways.

Animals↗
PubMed1993

Absence of endonuclease activation during acute cell death in renal proximal tubules.

The role of endonuclease and poly(ADP-ribose) polymerase activation in various types of cell injuries and death to rabbit renal proximal tubule suspensions was examined. Proximal tubules were exposed to the mitochondrial inhibitor antimycin A (0.1 microM), the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, 1 microM), the calcium ionophore ionomycin (5 microM), or the oxidant t-butyl hydroperoxide (TBHP, 0.5 mM) in the absence or presence of the endonuclease inhibitor aurintricarboxylic acid or the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. Lactate dehydrogenase (LDH) release was used as a marker of cell death and analysis of genomic DNA for internucleosomal cleavage was used as a marker of endonuclease activation. Aurintricarboxylic acid and 3-aminobenzamide had no effect on the proximal tubule LDH release produced by 1 h exposure to antimycin A, FCCP, or ionomycin, or 2 h exposure to TBHP. Furthermore, there was no evidence of DNA fragmentation with any compound prior to or after cell death began. As a positive control, proximal tubules exposed to digitonin in the absence of metabolic substrates resulted in the chelator-inhibitable fragmentation of DNA, indicating that the endonuclease is present in proximal tubules. These results show that endonuclease activation did not occur prior to or after cell death began. Furthermore, these results suggest that endonuclease and poly(ADP-ribose) polymerase activation do not play a role in this model of acute renal proximal tubule cell injury and death induced by agents that cause oxidative stress, mitochondrial dysfunction, or increases in cytosolic free calcium.

Animals↗
PubMed1994

Anomalies in ion transport in CF mouse tracheal epithelium.

The cystic fibrosis (CF) mouse trachea has become a model for gene transfer. To characterize ion transport properties of tracheal epithelium from normal and CF mice, tracheas were excised, mounted in Ussing chambers, and basal properties and responses to pharmacological agents and/or ion substitution protocols measured. No difference in basal short-circuit (Isc) was observed between normal (29.1 +/- 3.8 muA/cm2, n = 21) and CF (34.7 +/- 4.5 muA/cm2, n = 16) tracheas. The relative contribution of Na+ transport to basal Isc was small (30-40%). Ionomycin stimulated large increases in Isc in both normal and CF murine tracheas [change in Isc (delta Isc) with ionomycin: 30.5 +/- 8.8 muA/cm2, n = 11, normal; 27.3 +/- 6.7 muA/cm2, n = 6, CF]. Unexpectedly, forskolin increased Isc in both CF and normal amiloride-pretreated tracheas (delta Isc: 10.5 +/- 2.1 muA/cm2, n = 21, normal; 13 +/- 2.3 muA/cm2, n = 16, CF). Forskolin was observed to increase intracellular Ca2+ in both normal and CF tracheal cells, suggesting this as a mechanism to induce Cl- secretion. These similarities in ion transport, in part reflecting the dominance of Ca(2+)-regulated Cl- conductance, suggest that the murine trachea is not an ideal target for assessment of CF correction by gene transfer.

Animals↗
PubMed1996

Caffeine- and carbachol-induced Cl- and cation currents in single opossum esophageal circular muscle cells.

Cl- and cation currents may play important roles in esophageal smooth muscle membrane potential changes and contraction. We studied Ca2+ release-activated cell-shortening and membrane currents in single cells freshly dispersed from the circular muscle of the opossum esophagus using the standard patch-clamp whole cell recording method. Caffeine (10-20 microM) and carbachol (10-100 microM) shortened the single smooth muscle cells by releasing intracellular Ca2+. At a holding potential of 0 mV, spontaneous transient outward currents STOCs, representing spontaneous Ca(2+)-activated K+ currents) were recorded. Caffeine, carbachol, or ionomycin evoked large outward currents (up to 1,650 pA) and subsequently abolished STOCs. At a holding potential of -50 mV in K(+)-containing solutions, an outward current in response to the agonists was observed; in some cells, the outward current followed an inward current. In K(+)-free solutions, the agonists induced only an inward current whose reversal potential was shifted by alteration of the anion gradient but not by that of the cation. With a low-Cl- pipette solution (Cl- substituted by glucuronate or glutamate), the inward currents were dependent mainly on the external cation gradient. This cation channel was permeable to Ba2+. Inclusion of 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the pipette solution abolished all these currents. These data suggest that in the opossum esophageal circular muscle 1) Ca2+ released from the intracellular stores by caffeine and carbachol is sufficient to induce single smooth muscle cell contraction and 2) the caffeine-, carbachol-, and ionomycin-induced membrane currents consist of Ca(2+)-activated K+, Cl-, and cation conductances.

Animals↗
PubMed1997

Mechanisms of electrolyte transport across the endometrium. II. Regulation by GRP and substance P.

The purpose of this study was to investigate the regulation of electrolyte transport across the porcine endometrium by gastrin-releasing peptide (GRP) and substance P (SP). Luminal addition of GRP, neuromedin B (NMB), SP, or neurokinin A(NKA) to mucosal tissues mounted in Ussing chambers produced a multiphasic change in short-circuit current (Isc) characterized by an initial rapid increase and subsequent decrease in current. A similar response was obtained after addition of ionomycin or thapsigargin to the tissues. The Isc response to the peptides or Ca ionophore was inhibited by pretreatment of the tissues with luminal amiloride or benzamil. GRP and SP were more potent [50% effective concentration (EC50) of 3 nM] than NMB or NKA (EC50 values of 46 and 26 nM, respectively) in producing the decrease in Isc. Pretreatment with the GRP receptor antagonist 3-Phe-His-Trp-Ala-Val-D-Ala-His-D-Pro-psi Phe-NH2 blocked the Isc response to GRP and NMB but not to SP or NKA, whereas the NMB receptor antagonist D-Nal-[Cys-Try-D-Trp-Orn-Val-Cys]-Nal-NH2 was ineffective in inhibiting the Isc response to any of the peptides. In contrast, pretreatment of the tissue with the nonpeptide SP receptor antagonist CP-99,994 blocked the Isc response to SP and NKA but not to GRP or NMB. Experiments with amphotericin B-permeabilized tissues showed that GRP, SP, ionomycin, and thapsigargin increased current through an outwardly rectifying K conductance located on the apical membrane of the cells. The K-to-Na selectivity ratio of this conductance was calculated to be 2.5:1. These experiments showed that GRP and SP, acting through different receptors, produced an increase in K efflux through a Ca-dependent K conductance present in the apical membrane of surface endometrial epithelial cells. In addition, immunohistochemistry data showed that GRP-like immunoreactivity was localized to surface and glandular epithelial cells, whereas GRP receptor antibody labeling was observed in both epithelial and stromal cells. These results suggest that GRP functions as both an autocrine and paracrine regulatory peptide in the endometrium.

Amiloride↗