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The role of cystic fibrosis transmembrane conductance regulator (CFTR) in the control of Cl(-) currents was studied in mouse kidney. Whole cell clamp was used to analyze Cl(-) currents in primary cultures of proximal and distal convoluted and cortical collecting tubules from wild-type (WT) and cftr knockout (KO) mice. In WT mice, forskolin activated a linear Cl(-) current only in distal convoluted and cortical collecting tubule cells. This current was not recorded in KO mice. In both mice, Ca(2+)-dependent Cl(-) currents were recorded in all segments. In WT mice, volume-sensitive Cl(-) currents were implicated in regulatory volume decrease during hypotonicity. In KO mice, regulatory volume decrease and swelling-activated Cl(-) current were impaired but were restored by adenosine perfusion. Extracellular ATP also restored swelling-activated Cl(-) currents. The effect of ATP or adenosine was blocked by 8-cyclopentyl-1,3-diproxylxanthine. The ecto-ATPase inhibitor ARL-67156 inhibited the effect of hypotonicity and ATP. Finally, in KO mice, volume-sensitive Cl(-) currents are potentially functional, but the absence of CFTR precludes their activation by extracellular nucleosides. This observation strengthens the hypothesis that CFTR is a modulator of ATP release in epithelia.

Cl(-) currents induced by cell swelling were characterized in an immortalized cell line (DC1) derived from rabbit distal bright convoluted tubule by the whole cell patch-clamp techniques and by (125)I(-) efflux experiments. Exposure of cells to a hypotonic shock induced outwardly rectifying Cl(-) currents that could be blocked by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM DIDS, and by 1 mM diphenylamine-2-carboxylate. (125)I(-) efflux experiments showed that exposure of the monolayer to a hypotonic medium increased (125)I(-) loss. Preincubation of cells with LaCl(3) or GdCl(3) prevented the development of the response. The addition of 10 microM adenosine to the bath medium activated outwardly rectifying whole cell currents similar to those recorded after hypotonic shock. This conductance was inhibited by the A(1)-receptor antagonist 8-cyclopentyl-1,3-diproxylxanthine (DPCPX), LaCl(3), or GdCl(3) and was activated by GTPgammaS. The selective A(1)-receptor agonist N(6)-cyclopentyladenosine (CPA) mimicked the effect of hypotonicity on (125)I(-) efflux. The CPA-induced increase of (125)I(-) efflux was inhibited by DPCPX and external application of LaCl(3) or GdCl(3). Adenosine also enhanced Mn(2+) influx across the apical membrane. Overall, the data show that DC1 cells possess swelling- and adenosine-activated Cl(-) conductances that share identical characteristics. The activation of both conductances involved Ca(2+) entry into the cell, probably via mechanosensitive Ca(2+) channels. The effects of adenosine are mediated via A(1) receptors that could mediate the purinergic regulation of the volume-sensitive Cl(-) conductance.

We characterized Cl(-) conductance activated by extracellular ATP in an immortalized cell line derived from rabbit distal bright convoluted tubule (DC1). (125)I(-) efflux experiments showed that ATP increased (125)I(-) loss with an EC(50) = 3 microM. Diphenylamine-2-carboxylate (10(-3) M) and NPPB (10(-4) M) abolished the (125)I(-) efflux. Preincubation with 10 microM 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester or 10(-7) M thapsigargin inhibited the effect of ATP. Ionomycin (2 microM) increased (125)I(-) efflux with a time course similar to that of extracellular ATP, suggesting that the response is dependent on the intracellular Ca(2+) concentration ([Ca(2+)](i)). The ATP agonist potency order was ATP >/= UTP > ATPgammaS. Suramin (500 microM) inhibited the ATP-induced (125)I(-) efflux, consistent with P2 purinoceptors. (125)I(-) effluxes from cells grown on permeable filters suggest that ATP induced an apical efflux that was mediated via apical P2 receptors. Whole cell experiments showed that ATP (100 microM) activated outwardly rectifying Cl(-) currents in the presence of 8-cyclopentyl-1,3-dipropylxanthine, excluding the involvement of P1 receptors. Ionomycin activated Cl(-) currents similar to those developed with ATP. These results demonstrate the presence of a purinergic regulatory mechanism involving ATP, apical P2Y2 receptors, and Ca(2+) mobilization for apical Cl(-) conductance in a distal tubule cell line.

Cl- conductances were studied in an immortalized cell line (DC1) derived from rabbit distal bright convoluted tubule (DCTb). The DC1 clone was obtained after transfection of primary cultures of DCTb with pSV3 neo. RT-PCR experiments showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in the DC1 cell line. Using the whole cell patch-clamp technique, we recorded a linear Cl- conductance activated by forskolin (FK). This conductance was insensitive to DIDS and corresponded to a CFTR-like channel conductance. Fluorescence experiments with 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ) showed that FK induced an increase in Cl- efflux and influx in DC1 cells similar to that observed in cultured DCTb cells. 125I- efflux experiments performed on DC1 cells grown on collagen-coated filters showed that exposure of the monolayer to FK led to an increased 125I- loss through the apical membrane only. The addition of 10 microM adenosine activated a linear conductance identical to that recorded with FK and corresponding to the CFTR-like conductance. This conductance was also activated by 5'-(N-ethylcarboxamido)adenosine and CGS-21680 and inhibited in the presence of 8-cyclopentyl-1, 3-diproxylxanthine (DPCPX). This Cl- conductance could also be activated by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The addition of protein kinase A (PKA) inhibitor to the pipette solution inhibited the development of the current activated by CGS-21680. Finally, 125I- efflux showed that adenosine induced an apical efflux mediated through basolateral A2 receptors. Overall, the data show that the DC1 cell line expressed an apical CFTR Cl- conductance that could be activated by adenosine via A2A receptors located in the basolateral membrane and involving G protein and PKA pathways.

Cl- conductances were studied in cultured rabbit proximal convoluted tubule (PCT) epithelial cells and compared with those measured in cultured distal bright convoluted tubule (DCTb) epithelial cells. Using the whole cell patch-clamp technique, three types of Cl- conductances were identified in DCTb cultured cells. These consisted of volume-sensitive, Ca2+-activated, and forskolin-activated Cl- currents. In PCT cultured cells, only volume-sensitive and Ca2+-activated Cl- currents were recorded. The characteristics of Ca2+-activated currents in PCT cells closely resembled those in DCTb cells. Volume-sensitive Cl- currents could be elicited both in PCT and in DCTb cells by hypotonic stress. The pharmacological profile of this conductance was established for both cell types. Forskolin activated a linear Cl- current in DCTb cells but not in PCT cells. This conductance was insensitive to DIDS and corresponds to cystic fibrosis transmembrane conductance regulator (CFTR)-like channels. Quantitative measurements of SPQ fluorescence showed that only the apical membrane of DCTb cells possessed a Cl- pathway that was sensitive to forskolin. RT-PCR experiments showed the presence of CFTR mRNA in both cultures, whereas immunostaining experiments revealed the expression of CFTR in DCTb cells only. The physiological role of the different types of channels is discussed.

Ionic currents induced by cell swelling were characterized in primary cultures of rabbit distal bright convoluted tubule (DCTb) by the whole cell patch-clamp technique. Cl- currents were produced spontaneously by whole cell recording with an isotonic pipette solution or by exposure to a hypotonic stress. Initial Cl- currents exhibited outwardly rectifying current-voltage relationship, whereas steady-state currents showed strong decay with depolarizing pulses. The ion selectivity sequence was I- = Br- > Cl- >> glutamate. Currents were inhibited by 0.1 mM 5-nitro-2-(3-phenylpropylamino) benzoic acid and 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and strongly blocked by 1 mM diphenylamine-2-carboxylate. Currents were insensitive to intracellular Ca2+ but required the presence of extracellular Ca2+. They were not activated in cells pretreated with 200 nM staurosporine, 50 microM LaCl3, 10 microM nifedipine, 100 microM verapamil, 5 microM tamoxifen, and 50 microM dideoxyforskolin. Staurosporine, tamoxifen, verapamil, or the absence of external Ca2+ was without effect on the fully developed Cl- currents. Osmotic shock also activated K+ currents in Cl- free conditions. These currents were time independent, activated at depolarized potentials, and inhibited by 5 mM BaCl2. The activation of Cl- and K+ currents by an osmotic shock may be implicated in regulatory volume decrease in DCTb cells.

Chloride (Cl-) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33 degrees C) contained (in mM): 140 NaCl, 1 CaCl2, 10 N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-D-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl- current activated by 10(-5) M forskolin, 10(-3) M 8-bromo adenosine 3',5'-cyclic monophophosphate (8 Br-cAMP), 10(-6) M phorbol 12-myristate 13-acetate (PMA), 10(-3) M intracellular adenosine 3',5'-cyclic monophophosphate (cAMP) and 10(-7) M calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br- > Cl- > > I- > glutamate. This current was inhibited by 10(-3) M diphenylamine-2-carboxylate (DPC) and 10(-4) M 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10(-3) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl- current blocked by DIDS. To determine the exact location of the Cl- conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mM SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl- removal from the apical solution induced a Cl- efflux which was stimulated by 10(-5) M forskolin, 10(-7) calcitonin and inhibited by 10(-5) M NPPB. In 140 mM NaBr, forskolin stimulated an apical Br- influx through the Cl- pathway. Forskolin and calcitonin had no effect on the basolateral Cl- permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl- conductance in the apical membrane through a cAMP-dependent mechanism.

Using the patch clamp technique on the apical membrane of primary cultures of rabbit distal bright convoluted tubule cells (DCTb), two types of Cl- channel were identified. A small channel of 9 pS was observed in 9% of the patches. Cells pretreated with 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or 5 microM forskolin increased the expression of Cl- channels by 26 and 37%, respectively. In cell-attached and excised inside-out patches, the current-voltage (I-V) relationships of the 9-pS channel were linear. In only 1 out of 47 active patches was the small-conductance Cl- channel still active 1 h after membrane excision. The addition of 0.1 microM of the catalytic subunit protein kinase A with 2 mM ATP to the cytoplasmic side restored channel activity in 8 out of 15 excised membrane patches. In 5 out of 467 patches of stimulated or nonstimulated cells, a larger Cl- conductance of 30 pS was also recorded. In excised inside-out patches this channel outwardly rectified and was activated by strong depolarization. In cultured DCTb cells, the small-conductance, cAMP-activated Cl- channel shares many properties with the cystic fibrosis transmembrane conductance regulator. Our results suggest that at least the small-conductance channel may participate in Cl- secretion across the apical membrane of DCTb in primary culture. This secretion may increase the rate of the apical Cl-/HCO3- exchange indirectly by enhancing the inwardly-directed Cl- gradient.