sq-23377 and fenamic-acid

In many epithelial tissues, the Cl(-) efflux via Ca(2+)-activated Cl(-) channels (Cl(Ca)) play a key role for the fluid secretion. To elucidate the mechanism of prostatic fluid secretion, the properties of whole-cell chloride conductance were investigated.. Rat prostate secretory epithelial cells (RPSECs) were isolated by collagenase treatment, and were used for the whole-cell voltage clamp. Both extra- and intracellular monovalent cations were replaced by N-methyl-D-glucamate to record the Cl(-) current selectively.. A bath application of Ca(2+)-ionophore, ionomycin (0.2 micro M), increased the membrane conductance with outwardly rectifying voltage-dependence. On step-like depolarization from -60 to +80 mV (500 msec), the ionomycin-induced current showed slowly activating kinetics, a known property of Cl(Ca) current (I(Cl(Ca))) of other tissues. The relative permeability of Cl(Ca) to various anions was calculated from the reversal potentials measured under a total replacement of extracellular Cl(-) with various anions, and the relative order of permeability was SCN(-)>I(-)>Br(-)>Cl(-)>>gluconate. The amplitude of I(Cl(Ca)) was decreased by various anion channel blockers: niflumic acid (100 micro M), DPC (100 micro M), DIDS (1 mM), and NPPB (200 micro M).. RPSECs have Cl(Ca) that may provide Cl(-) efflux pathways for the exocrine secretions of the prostate.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anions; Calcium; Chloride Channels; Electric Conductivity; Epithelial Cells; Ionomycin; Ionophores; Male; Niflumic Acid; Nitrobenzoates; ortho-Aminobenzoates; Patch-Clamp Techniques; Permeability; Prostate; Rats; Rats, Sprague-Dawley

Sections of posterior intestine of the euryhaline killifish Fundulus heteroclitus adapted to sea water were stimulated by the calcium ionophore ionomycin (1 micromol l(-1)) in combination with agents to elevate intracellular cyclic AMP levels, 0.5 mmol l(-1) dibutyryl-cyclic AMP (db-cAMP) with 0.1 mmol l(-1) 3-isobutyl-1-methylxanthine (IBMX). Intestinal bag preparations from recently fed animals (but not from overnight unfed animals) changed from fluid absorption (+18.9+/-8.30 microl cm(-2) h(-1), N=8) in the untreated control period to net fluid secretion after stimulation (-7.43+/-1.30 microl cm(-2) h(-1), N=8, P<0.01; means +/- S.E.M.), indicative of the capacity of teleost intestine to undergo secretion. Posterior intestinal pieces mounted in vitro in Ussing-style membrane chambers showed net Cl(-) uptake (+2.245+/-0.633 microequiv cm(-2) h(-1), N=7) that turned to net secretion following stimulation by ionomycin + db-cAMP + IBMX (-3.809+/-1.22 microequiv cm(-2) h(-1), N=7, P<0.01). Mucosal application of the anion channel blocker 1 mmol l(-1) diphenylamine-2-carboxylate (DPC) after ionomycin + db-cAMP + IBMX treatment significantly reduced serosal-to-mucosal unidirectional Cl(-) flux (P<0.001), net Cl(-) flux (P<0.05), short-circuit current (I(sc), P<0.001) and tissue conductance (G(t), P<0.001), while 0.1 mmol l(-1) 4,4'-diisothiocyano-2,2'-stilbene-disulphonic acid (DIDS, a blocker of anion exchange) was without effect. Stimulation by db-cAMP + IBMX (no ionomycin) significantly increased unidirectional fluxes, I(sc) and G(t) but did not produce net Cl(-) secretion. Ionomycin alone produced a transient increase in I(sc) but had no effect on G(t) and caused no significant changes in unidirectional or net Cl(-) fluxes. Addition of db-cAMP + IBMX after ionomycin treatment produced net secretion of Cl(-) and large increases in unidirectional fluxes and G(t). Cystic fibrosis transmembrane conductance regulator (CFTR) was immunocytochemically localized with a monoclonal mouse antibody to the carboxy terminus and found to be present in the cytoplasm and basolateral membranes of all enterocytes and in the brush-border membrane of some cells, whereas NKCC immunofluorescence, demonstrating the presence of the Na(+)/K(+)/2Cl(-) cotransporter, was present in the cytoplasm and brush-border membrane. We conclude that the teleost intestine is capable of salt and fluid secretion only if intracellular Ca(2+) and cyclic AMP pathways are stimulated together and that

Topics: 1-Methyl-3-isobutylxanthine; Animals; Body Fluids; Bucladesine; Calcium; Calcium Channel Blockers; Chlorides; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Electric Conductivity; Female; Fluorescent Antibody Technique; Fundulidae; Immunohistochemistry; Intestinal Mucosa; Intestines; Ionomycin; Male; ortho-Aminobenzoates; Sodium Chloride; Sodium-Potassium-Chloride Symporters

We have developed a cell culture of guinea pig gallbladder epithelial cells with which to study ion transport. When grown on permeable supports, the cultured epithelia developed a transepithelial resistance (R(t)) of approximately 500 Omega. cm(2). The epithelial cell origin of the cell culture was further confirmed by immunocytochemical localization of cytokeratin. Ionomycin and forskolin increased transepithelial voltage and short-circuit current (I(sc)) and decreased R(t). The response to ionomycin was transient, whereas that to forskolin was sustained. Both were attenuated by replacement of Cl(-) and/or HCO(3)(-). Mucosal addition of the anion transport inhibitors DIDS or diphenylamine-2-carboxylic acid (DPC) blocked the response to ionomycin. The response to forskolin was blocked by DPC but not by DIDS. Ionomycin, but not forskolin, increased intracellular Ca(2+) concentration in fura 2-loaded cells. PGE(2), histamine, vasoactive intestinal polypeptide, and secretin elicited a sustained increase in I(sc). Responses to ATP and CCK were transient. Thus cultured guinea pig gallbladder epithelia display the range of responses observed in the native tissue and are an appropriate model for studies of ion transport in gallbladder and intestinal epithelia.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Animals; Anions; Biological Transport; Calcium; Calcium Channel Blockers; Cells, Cultured; Cholecystokinin; Colforsin; Cyclic AMP; Dinoprostone; Epithelial Cells; Gallbladder; Gastrointestinal Agents; Guinea Pigs; Histamine; Ionomycin; Ionophores; Male; ortho-Aminobenzoates; Secretin; Vasoactive Intestinal Peptide

Secretion of salt and water by the epithelial cells that line pancreatic ducts depends on activation of apical membrane Cl- conductance. In the present study, we characterized two types of Cl- conductances present in the apical cell membrane of bovine pancreatic duct epithelial cells. Primary cultures of bovine main pancreatic duct epithelium and an immortalized cell line (BPD1) derived from primary cultures were used. Elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) or Ca2+ in intact monolayers of duct epithelium induced sustained anion secretion. Agonist-induced changes in plasma membrane Cl- permeability were accessed by 36 Cl- efflux, whole cell current recording, and measurements of transepithelial Cl- current across permeabilized epithelial monolayers. Elevation of intracellular cAMP elicited a sustained increase in Cl- permeability, whereas elevation of intracellular Ca2+ induced only a transient increase in Cl- permeability. Ca(2+)- but not cAMP-induced increases in Cl- permeability were abolished by preincubation of cells with the Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl) ester (BAPTA-AM). N-phenylanthranilic acid (DPC; 1 mM) and glibenclamide (100 microM), but not 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 500 microM), inhibited the cAMP-induced increase in Cl- permeability. In contrast, DPC and DIDS, but not glibenclamide, inhibited the Ca(2+)-induced increase in Cl- permeability. We conclude from these experiments that bovine pancreatic duct epithelial cells express at least two types of Cl- channels, cAMP and Ca2+ activated, in the apical cell membrane. Because the Ca(2+)-activated increase in Cl- permeability is transient, the extent to which this pathway contributes to sustained anion secretion by the ductal epithelium remains to be determined.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Animals; Anions; Calcium; Cattle; Cell Line; Cells, Cultured; Chelating Agents; Chloride Channels; Chlorides; Colforsin; Cyclic AMP; Egtazic Acid; Epithelial Cells; Epithelium; Glyburide; Ionomycin; Kinetics; Membrane Potentials; ortho-Aminobenzoates; Pancreatic Ducts; Theophylline

We developed a convenient flux assay that permits simultaneous measurement of Cl and K conductance pathways in Cl-secreting epithelial cells. Monolayers of the colonic tumor cell line T84 were preloaded with 125I and 86Rb, and isotope effluxes were monitored by a sample-replace procedure. The adenosine 3',5'-cyclic monophosphate (cAMP)-mediated agonists forskolin and prostaglandin E2 increased I efflux with little effect on Rb efflux, whereas the Ca-mediated agonists ionomycin, A23187, and carbachol increased both I and Rb effluxes. Simultaneous determinations of I and Cl or Rb and K effluxes indicated that I and Rb provide good measures of the effluxes of Cl and K, respectively. Forskolin- and ionomycin-stimulated I effluxes were inhibited by the Cl-channel blockers diphenylamine-2-dicarboxylate (DPC), 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB), and 2-[cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H- inden-5-yl)oxy]acetic acid (IAA-94) and by high external K. The Rb efflux evoked by ionomycin was inhibited by the K-channel blockers Ba and charybdotoxin. These findings suggest that I and Rb effluxes provide qualitative estimates of agonist-stimulated Cl and K conductance pathways. Thus this method can provide a simple and relatively inexpensive screening assay for Cl and K conductances in cultured cells to assess the effects of agonist, blockers, or genetic manipulations.

Topics: Barium; Bumetanide; Carbachol; Cell Line; Charybdotoxin; Chloride Channels; Chlorides; Colforsin; Colonic Neoplasms; Dinoprostone; Epithelium; Humans; Iodides; Iodine Radioisotopes; Ion Channels; Ionomycin; Kinetics; Mannitol; Membrane Proteins; ortho-Aminobenzoates; Potassium Channels; Radioisotope Dilution Technique; Rubidium; Rubidium Radioisotopes; Scorpion Venoms; Sodium; Tumor Cells, Cultured