calcimycin and 5-nitro-2-(3-phenylpropylamino)benzoic-acid

We have characterized release of D-aspartate (D-Asp), a regulator of hormone synthesis and secretion, via a volume-sensitive organic anion channel (VSOC) in PC12 cells by studying its response to apoptotic stimuli. PC12 cells have been demonstrated to endogenously synthesize D-Asp. Apoptotic inducers, including staurosporin (STS), tumor necrosis factor (TNF)-alpha, H(2)O(2), and C2-ceramide, activate the release of D-Asp through a hypotonic stimulus-triggered mechanism. Putative blockers of the anion channel, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 4,4'-diisothiocyanostilbene-2,2'-sulphonic acid (DIDS), significantly inhibited stress-induced D-Asp release under hypotonic conditions following the application of apoptotic inducers. Hypotonic conditions are essential for activation by apoptotic inducers. Phorbol 12-mirystate 13-acetate and the Ca(2+) ionophore A23187 increased D-Asp efflux via the VSOC, implying the involvement of intracellular Ca(2+) in the activation of the D-Asp efflux. However, hypotonic stress and STS had no effect on the concentration of intracellular Ca(2+) in PC12 cells. Furthermore, an unknown EGTA-sensitive factor(s), other than Ca(2+), and peroxynitrite may play pivotal roles in STS-enhanced D-Asp release.

Topics: Animals; Apoptosis; Aspartic Acid; Calcimycin; Hydrogen Peroxide; Hypotonic Solutions; Ion Channels; Ionophores; Nitrobenzoates; PC12 Cells; Rats; Sphingosine; Staurosporine; Stereoisomerism; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; Uric Acid

Bone marrow mesenchymal stem cells (MSCs) are used as a cell source for cardiomyoplasty; however, the cellular electrophysiological properties are not fully understood. The present study was to investigate the functional ionic channels in undifferentiated mouse bone marrow MSCs using whole cell patch-voltage clamp technique, RT-PCR, and Western immunoblotting analysis. We found that three types of ionic currents were present in mouse MSCs, including a Ca(2+)-activated K(+) current (I(KCa)), an inwardly rectifying K(+) current (I(Kir)), and a chloride current (I(Cl)). I(Kir) was inhibited by Ba(2+), and I(KCa) was activated by the Ca(2+) ionophore A-23187 and inhibited by the intermediate-conductance I(KCa) channel blocker clotrimazole. I(Cl) was activated by hyposmotic (0.8 T) conditions and inhibited by the chloride channel blockers DIDS and NPPB. The corresponding ion channel genes and proteins, KCa3.1 for I(KCa), Kir2.1 for I(Kir), and Clcn3 for I(Cl), were confirmed by RT-PCR and Western immunoblotting analysis in mouse MSCs. These results demonstrate that three types of functional ion channel currents (i.e., I(Kir), I(KCa), and I(Cl)) are present in mouse bone marrow MSCs.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Barium Compounds; Blotting, Western; Bone Marrow Cells; Calcimycin; Cell Size; Cells, Cultured; Chloride Channels; Chlorides; Clotrimazole; Intermediate-Conductance Calcium-Activated Potassium Channels; Ionophores; Membrane Potentials; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Nitrobenzoates; Patch-Clamp Techniques; Potassium; Potassium Channel Blockers; Potassium Channels, Inwardly Rectifying; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Intracellular calcium ([Ca2+](i)), cell volume, membrane potential and currents were measured in neuroblastomaxglioma hybrid cells to gain insight into how [Ca2+](i) controls cell volume. [Ca2+](i) was increased by fluid shear stress, mechanical stimulation of the cells, the Ca2+ ionophore A23187, caffeine and thapsigargin. The increase in [Ca2+](i) induced by mechanical stimulation was decreased by about 50% by caffeine and abolished after incubation of the cells in a Ca2+-free solution. Mechanical stimulation by stirring the cell suspension induced cell shrinkage that was abolished by caffeine, but induced cell swelling in Ca2+-free solution. In the presence of caffeine, A23187 induced cell shrinkage whereas thapsigargin induced cell swelling. Both cell volume changes were inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid. The cells were hyperpolarized by fluid shear stress and A23187 and depolarized by caffeine, thapsigargin and intracellular EGTA. Under all these conditions, the membrane input resistance was decreased. Voltage-clamp experiments suggested that, in addition to an increased anionic current, fluid shear stress and A23187 increased a K+ current, whereas caffeine and intracellular Ca2+ chelation increased a non-selective cation current and thapsigargin increased both a K+ and a non-selective cation current. Taken together, these results suggest that, if cell volume is closely dependent on [Ca2+](i) and the activity of Cl- channels, its relative value is dependent on the ionic selectivity of co-activated channels and the membrane potential.

Topics: Brain Neoplasms; Caffeine; Calcimycin; Calcium; Cell Size; Chloride Channels; Electric Stimulation; Electrophysiology; Glioma; Humans; Hybrid Cells; Ion Channels; Ionophores; Membrane Potentials; Neuroblastoma; Nitrobenzoates; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Physical Stimulation; Thapsigargin; Tumor Cells, Cultured

We investigated the effects of the Cl- channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) on endothelin-1 (ET-1)-induced constriction of rat small pulmonary arteries (diameter 100-400 microm) in vitro, following endothelium removal. ET-1 (30 nM) induced a sustained constriction of rat pulmonary arteries in physiological salt solution. Arteries preconstricted with ET-1 were relaxed by niflumic acid (IC50: 35.8 microM) and NPPB (IC50: 21.1 microM) in a reversible and concentration-dependent manner. However, at concentrations known to block Ca++-activated Cl- channels, DIDS (

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Angiotensin II; Animals; Calcimycin; Calcium Channels; Chloride Channels; Endothelin-1; Glyburide; In Vitro Techniques; Male; Niflumic Acid; Nitrobenzoates; Peptides; Pulmonary Artery; Rats; Rats, Wistar; Uridine Triphosphate; Vasoconstriction; Vasodilation

1. Using a microelectrode technique, acetylcholine (ACh)-induced membrane potential changes were characterized using various types of inhibitors of K+ and Cl- channels in rabbit aortic valve endothelial cells (RAVEC). 2. ACh produced transient then sustained membrane hyperpolarizations. Withdrawal of ACh evoked a transient depolarization. 3. High K+ blocked and low K+ potentiated the two ACh-induced hyperpolarizations. Charybdotoxin (ChTX) attenuated the ACh-induced transient and sustained hyperpolarizations; apamin inhibited only the sustained hyperpolarization. In the combined presence of ChTX and apamin, ACh produced a depolarization. 4. In Ca2+-free solution or in the presence of Co2+ or Ni2+, ACh produced a transient hyperpolarization followed by a depolarization. In BAPTA-AM-treated cells, ACh produced only a depolarization. 5. A low concentration of A23187 attenuated the ACh-induced transient, but not the sustained, hyperpolarization. In the presence of cyclopiazonic acid, the hyperpolarization induced by ACh was maintained after ACh removal; this maintained hyperpolarization was blocked by Co2+. 6. Both NPPB and hypertonic solution inhibited the membrane depolarization seen after ACh washout. Bumetanide also attenuated this depolarization. 7. It is concluded that in RAVEC, ACh produces a two-component hyperpolarization followed by a depolarization. It is suggested that ACh-induced Ca2+ release from the storage sites causes a transient hyperpolarization due to activation of ChTX-sensitive K+ channels and that ACh-activated Ca2+ influx causes a sustained hyperpolarization by activating both ChTX- and apamin-sensitive K+ channels. Both volume-sensitive Cl- channels and the Na+-K+-Cl- cotransporter probably contribute to the ACh-induced depolarization.

Topics: Acetylcholine; Animals; Aortic Valve; Apamin; Bumetanide; Calcimycin; Calcium; Charybdotoxin; Chelating Agents; Chloride Channels; Cobalt; Diuretics; Dose-Response Relationship, Drug; Egtazic Acid; Endothelium; In Vitro Techniques; Indoles; Ionophores; Male; Membrane Potentials; Nickel; Nitrobenzoates; Potassium; Potassium Channel Blockers; Rabbits; Vasodilator Agents

Cl- secretion by pancreatic duct epithelial cells (PDEC) regulates cellular HCO3- secretion, an important component of the exocrine pancreas. In cystic fibrosis, for example, impaired function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel results in decreased pancreatic secretion and secondary pancreatic insufficiency. Studies of ion transport by PDEC have been hindered by the lack of a practical in vitro model. We have successfully cultured nontransformed dog PDEC on Vitrogen-coated permeable membranes overlying a feeder layer of myofibroblasts and report the characterization of Cl- channels in these cells. Cl- conductance, assessed through efflux of 125I from PDEC, was stimulated by agents acting via adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+. The Cl- conductances activated by cAMP and Ca2+ were distinct, since they were differentially inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and, to a lesser extent, by 5-nitro-2-(3-phenylpropylamino)benzoic acid and diphenylamine-2 carboxylate. Patch-clamp studies confirmed the presence of Cl- channels activated by cAMP and Ca2+, with differential inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The presence of CFTR Cl- channels in PDEC was confirmed by immunoblotting. These cultured PDEC are an optimal model for studies of pancreatic duct secretion.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Calcimycin; Calcium; Calcium Channel Blockers; Cells, Cultured; Chloride Channels; Colforsin; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Cytosol; Dogs; Epithelial Cells; Epithelium; Iodine; Nitrobenzoates; ortho-Aminobenzoates; Pancreatic Ducts; Patch-Clamp Techniques

The ability of a Cl-secreting epithelium to support net secretion of an anion other than a halide was investigated with 35SO4 flux measurements across the isolated, short-circuited rabbit distal colon. In most experiments, 36Cl fluxes were simultaneously measured to validate the secretory capacity of the tissues. Serosal addition of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 0.5 mM) stimulated a sustained net secretion of SO4 (about -3.0 nmol.cm-2.h-1 from a 0.20 mM solution) via an increase in the serosal-to-mucosal unidirectional flux, whereas Ca ionophore A-23187 (1 microM, serosal) produced a more transient stimulation of SO4 and Cl secretion. Net adenosine 3',5'-cyclic monophosphate (cAMP)-dependent SO4 and Cl secretion were strongly voltage sensitive, principally through the potential dependence of the serosal-to-mucosal fluxes, indicating an electrogenic transport process. Symmetrical replacement of either Na, K, or Cl inhibited cAMP-dependent SO4 secretion, whereas HCO3-free buffers had no effect on SO4 secretion. Serosal bumetanide (50 microM) or furosemide (100 microM) reduced DBcAMP-stimulated SO4 and Cl secretion, whereas serosal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (50 microM) blocked DBcAMP-induced SO4 secretion while enhancing net Cl secretion and short-circuit current. Mucosal 5-nitro-2-(3-phenylpropylamino)benzoic acid partially inhibited SO4 secretion and completely inhibited Cl secretion. It is concluded that secretagogue-stimulated SO4 secretion, like Cl secretion, may be an electrogenic process mediated by diffusive efflux through an apical anion conductance. Cellular accumulation of SO4 across the basolateral membrane appears to be achieved by a mechanism that is distinct from that employed by Cl.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Biological Transport, Active; Bucladesine; Calcimycin; Chloride Channels; Chlorides; Colon; Cross-Linking Reagents; Cyclic AMP; Electrolytes; Female; In Vitro Techniques; Intestinal Mucosa; Kinetics; Male; Nitrobenzoates; Rabbits; Stilbenes; Sulfates

Chloride secretion in primary cultures of cells originating from the secretory coil of human sweat glands was investigated by electron probe X-ray microanalysis. The total intracellular Cl concentration was lowered by muscarinic agonists (carbachol and acetylcholine), as well as by the calcium ionophore A23187. The muscarinic agonists also lowered the cellular K concentration. Cl- secretion induced by these agonists could be inhibited by the chloride channel blocker NPPB. After cAMP stimulation, the frequency distribution of the Cl concentration changed from Gaussian to bimodal, indicating that cAMP induces Cl- secretion only from a subpopulation of the cells. Also ATP stimulated Cl- secretion, indicating the presence of purinergic receptors. The results suggest that some of the cells in addition to Ca(2+)-regulated Cl- channels also possess cAMP-activated Cl- channels. Hence, the primary cultures still possess the Cl- transport mechanisms known to be present in intact glands. It can, however, not be excluded that some coil cells have acquired ductal characteristics during culture.

Topics: Adenosine Triphosphate; Adult; Calcimycin; Carbachol; Chloride Channels; Chlorides; Cyclic AMP; Electron Probe Microanalysis; Epithelium; Female; Humans; Ion Transport; Microscopy, Electron; Middle Aged; Nitrobenzoates; Potassium; Sodium; Sweat Glands

This report describes chloride and iodide effluxes across the basolateral membrane of porcine thyroid follicles reconstituted in culture. Basolateral chloride efflux is activated by thyrotropin (TSH). TSH (10 mU/ml) induces a twofold increase in the initial rate of chloride efflux. Forskolin (FSK, 5 microM) which increases intracellular cAMP also stimulates the initial rate of chloride efflux 3.5-fold, whereas an increase in the free cytosolic Ca2+ with the ionophore A23187 or thapsigargin, fails to mimic the TSH effect. The chloride channel blocker 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB) dose dependently inhibits chloride efflux rates with the maximal and half maximal effects observed for 100 microM and 30 microM, respectively. Basolateral chloride efflux rates are also inhibited in the presence of the organic anion transporter blocker probenecid (5 mM) or the Cl-/HCO3- exchanger blocker 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS, 250 microM), respectively, by 60% and 40%, whereas it is not affected by ClO4 (100 microM). The initial rate of iodide efflux is weakly activated (1.4-fold) by TSH (10 mU/ml). TSH effect could be reproduced by agents known to activate Ca(2+)-dependent processes as A23187, ionomycin (1 microM), phorbol 12-myristate 13-acetate (TPA, 0.1 microM) and epidermal growth factor (EGF, 0.1 microM) which increase the initial rate of iodide efflux from 1.2- to 1.8-fold, whereas FSK is without effect. The chloride channel blocker NPPB (500 microM) is required to significantly inhibit the initial rate of iodide efflux by 30%. The initial rate of iodide efflux is also reduced by 30% in the presence of SITS (250 microM) or probenecid (5 mM) whereas it is activated by ClO4 (100 microM). We conclude that basolateral chloride and iodide effluxes are both regulated by TSH, using two different transduction pathways. Chloride efflux regulation may involve a cAMP transduction signal, whereas the regulation of iodide efflux may involve a Ca2+ signal. Furthermore, as the sensitivities of chloride and iodide effluxes for the anion transporter blockers (especially NPPB) are different, it seems likely that chloride and iodide use two different transport pathways.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Antiporters; Calcimycin; Calcium; Cell Membrane; Cells, Cultured; Chloride Channels; Chlorides; Colforsin; Cyclic AMP; Iodides; Kinetics; Nitrobenzoates; Probenecid; Swine; Terpenes; Thapsigargin; Thyroid Gland; Thyrotropin

To determine how cell calcium ([Ca2+]c) regulates apical Cl- channels, we measured the rate of 125-Iodide (125I-) efflux to assay Cl- channel activity in intact cells and examined cell-free membrane patches from cultured canine tracheal epithelial cells. The Ca2+ elevating agonist bradykinin and the calcium ionophore A23187 increased 125I- efflux. This response did not require prostaglandin production. Under several conditions, changes in [Ca2+]c were temporally dissociated from changes in channel activation: a transient increase in [Ca2+]c caused a prolonged stimulation of 125I- efflux. Neither Cl- channel activation nor open-channel probability was affected by varying internal [Ca2+] in excised membrane patches. Adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca2(+)-dependent channel activation may be independent: cAMP-stimulated 125I- efflux did not require an increase in [Ca2+]c, Ca2(+)-stimulated efflux did not require an increase in cAMP, and simultaneous addition of A23187 and isoproterenol produced additive effects on 125I- efflux. The data suggest that an increase in [Ca2+]c activates Cl- channels, however, the effect of Ca2+ appears to be indirect, not involving a ligand-type interaction with the channel.

Topics: Animals; Bumetanide; Calcimycin; Calcium; Cells, Cultured; Chloride Channels; Chlorides; Cyclic AMP; Dogs; Epithelium; Indomethacin; Iodides; Ion Channels; Isoproterenol; Kinetics; Membrane Proteins; Muscle, Smooth; Nitrobenzoates; Potassium; Theophylline; Trachea; Valinomycin