valinomycin and fenamic-acid

In isolated rhesus eccrine clear cells, regulatory volume decrease (RVD) occurs after osmotic swelling. RVD was completely inhibited by 1 mM quinidine, 200 nM charybdotoxin, 1 mM diphenylamine-2-carboxylic acid (DPC), or 0.1 mM 4-nitro-2(3-phenylpropyl-amino)benzoate. RVD was also inhibited in Ca(2+)-free medium by vinblastine (antimicrotubular agent), N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide (W-7), or 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Valinomycin reversed quinidine- and DIDS-induced inhibition of RVD but not the inhibition caused by Ca(2+)-free medium, DPC, vinblastine, or W-7. The cytosolic free Ca2+ concentration, as determined by the fura 2 method, increased from 220 nM in the control to 435 nM during RVD. Activation of both K+ and Cl-currents was also directly demonstrated with the whole cell current-voltage clamp method. DIDS inhibited swelling-induced K+, but not Cl-, currents and depolarized the membrane potential during RVD, further supporting the notion that DIDS inhibited swelling-activated K+, but not Cl-, pathways. We conclude that the observed RVD is mediated by the activation of conductive Ca(2+)-dependent K+ and Cl- pathways.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biopsy; Calcium; Calmodulin; Cells, Cultured; Charybdotoxin; Chlorides; Eccrine Glands; Egtazic Acid; Homeostasis; Hypotonic Solutions; Macaca mulatta; Membrane Potentials; Nitrobenzoates; ortho-Aminobenzoates; Osmolar Concentration; Potassium; Scorpion Venoms; Skin; Sulfonamides; Valinomycin; Vinblastine

The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is highly enriched in Na+/K+ ATPase and has been shown by electrophysiological methods to be highly conductive to Cl-. In order to study the Cl- conductive pathways, membrane vesicles were isolated from the TALH-containing region of the porcine kidney, the red outer medulla, and Cl- channel activity was determined by a 36Cl uptake assay where the uptake of the radioactive tracer is driven by the membrane potential (positive inside) generated by an outward Cl- gradient. The accumulation of 36Cl- inside the vesicles was found to be dependent on the intravesicular Cl- concentration and was abolished by clamping the membrane potential with valinomycin. The latter finding indicated the involvement of conductive pathways. Cl- channel activity was also observed using a fluorescent potential-sensitive carbocyanine dye, which detected a diffusion potential induced by an imposed inward Cl- gradient. The anion selectivity of the channels was Cl- greater than NO3- = I- much greater than gluconate. Among the Cl- transport inhibitors tested, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPAB), 4,4'-diisothiocyano-stilbene-2,2'-disulfonate (DIDS), and diphenylamine-2-carboxylate (DPC) showed IC50 of 110, 200 and 550 microM, respectively. Inhibition of 36Cl uptake by NPPAB and two other structural analogues was fully reversible, whereas that by DIDS was not. The nonreactive analogue of DIDS, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), was considerably less inhibitory than DIDS (25% inhibition at 200 microM). The irreversible inhibition by DIDS was prevented by NPPAB, whereas DPC was ineffective, consistent with its low inhibitory potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anions; Benzothiazoles; Carbocyanines; Chlorides; Fluorescent Dyes; Ion Channels; Kidney Medulla; Membrane Potentials; Membranes; ortho-Aminobenzoates; Swine; Valinomycin

Chloride transport across human placental microvillous vesicle membrane was investigated using the fluorescent probe SPQ (6-methoxy-N[3-sulfopropyl]quinolinium). Chloride influx (JCl) was calculated from the initial rate of quenching of intravesicular SPQ fluorescence by chloride. JCl measured by SPQ fluorescence was not significantly different from JCl measured by uptake of 36Cl; SPQ did not affect measurements of JCl.JCl was increased 51% by a 58-mV membrane potential (internal positive). Voltage-stimulated JCl showed a saturable dependence on chloride concentration with a dissociation constant (Kd) of 18 +/- 5 mM and was inhibited by diphenylamine-2-carboxylate with an apparent inhibitory constant of 0.13 +/- 0.03 mM. The activation energy calculated for voltage-stimulated JCl was 4.6 +/- 0.6 kcal/mol. JCl was also stimulated by a reduction in the external pH from 7.0 to 5.5 (internal pH = 7.0). pH-stimulated chloride influx was increased by trans-HCO3 (25 mM) and was inhibited by dihydro-4,4'-diisothiocyano-2,2'-disulfonic stilbene. Uptake of 36Cl into microvillous vesicles was stimulated by trans-Cl. pH-stimulated JCl showed a saturable dependence on chloride with a Kd of 38 +/- 6 mM but was not affected by membrane potential. No evidence was found for Na- or K-coupled chloride cotransport. These findings demonstrate the presence of a saturable chloride conductance and an electroneutral chloride-bicarbonate exchanger in the placental microvillous membrane.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Bicarbonates; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Chlorides; Female; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Kinetics; Mathematics; Microvilli; Models, Theoretical; ortho-Aminobenzoates; Placenta; Potassium; Pregnancy; Quinolinium Compounds; Spectrometry, Fluorescence; Valinomycin

This report describes a Cl- transport pathway in confluent monolayer cultures of the T84 human colonic carcinoma cell line which is: 1) activated by vasoactive intestinal polypeptide, or other agents which induce or mimic cAMP; 2) independent of extracellular Na+ or K+; 3) refractory to inhibition by 0.1 mM bumetanide and 1 mM 4-acetamido-4'-isothiocyanostilbene-2,-2'-disulfonic acid; 4) competitively inhibited by NO3-, I-, SCN-, and Br-; 5) inhibited in a noncompetitive-complex manner by the putative Cl- channel-blocking agent, N-phenylanthranilic acid; and 6) localized to the apical membrane of confluent monolayers. This Cl- transport system is, therefore, distinct from the bumetanide-sensitive, basolateral membrane-localized, Na+, K+, Cl- cotransport system previously described in these cells (Dharmsathaphorn, K., Mandel, K., Masui, H., and McRoberts, J.A. (1985) J. Clin. Invest. 75, 462-471). Kinetic studies revealed that Cl- transport by this pathway fit simple Michaelis-Menten kinetics with an apparent Km for Cl- of about 6 mM. Activation by vasoactive intestinal polypeptide increased the Vmax but did not alter the apparent Km. We discuss the possibility that this transport system is a Cl- channel which is intimately involved in hormonally mediated, electrogenic Cl- secretion across T84 cell monolayers.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anthracenes; Biological Transport, Active; Bromides; Bucladesine; Bumetanide; Calcimycin; Cell Line; Cell Membrane; Chlorides; Colon; Cyclic AMP; Dose-Response Relationship, Drug; Epithelium; Humans; Hydrogen-Ion Concentration; Kinetics; ortho-Aminobenzoates; Sodium; Valinomycin; Vasoactive Intestinal Peptide