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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.

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 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 Cl- currents induced by cell swelling and forskolin were studied in primary cultures of rabbit distal convoluted tubule (DCTb) by the whole-cell patch clamp technique. We identified a Cl- conductance activated by cell swelling with an hyperosmotic pipette solution. The initial current exhibited an outwardly rectifying 1-V relationship, whereas steady state current showed strong decay at depolarized membrane potentials. The ion selectivity was I- > Br- > Cl- > > glutamate. The forskolin-activated Cl- conductance demonstrated a linear I-V relationship and its ion selectivity was Br- > Cl- > I- > glutamate. This last conductance could be related to the CFTR (cystic fibrosis transmembrane conductance regulator) previously identified in these cells. NPPB inhibited both Cl- currents, and DIDS inhibited only the swelling-activated Cl- current. Forskolin had no effect on the activation of the swelling-activated Cl- current. In DCTb cells which exhibited swelling-activated Cl- currents subsequently inhibited by DIDS, forskolin could activate CFTR related Cl- currents. In the continuous presence of I- which inhibited CFTR conductance, forskolin did not modify the swelling-activated current. The results suggest that both Cl- conductances could be co-expressed in the same DCTb cell and that CFTR did not modulate the swelling-activated conductance.

Chloride (Cl-) conductances were studied in primary cultures of rabbit distal convoluted tubule (very early distal "bright" convoluted tubule, DCTb) by the whole cell patch-clamp technique. We identified a Cl- current activated by 2 microM extracellular ionomycin. The kinetics of the macroscopic current were time dependent for depolarizing potentials with a slow developing component. The steady state current presented outward rectification, and the ion selectivity sequence was I- > Br- > > Cl > glutamate. The current was inhibited by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and 1 mM diphenylamine-2-carboxylate. To identify the location of the Cl- conductance, 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence experiments were carried out in confluent cultures developed on collagen-coated permeable filters. Cl- removal from the apical solution induced a Cl- efflux that was stimulated by 10 microM forskolin. Forskolin had no effect on the basolateral Cl- permeability Cl- substitution in the basolateral solution induced an efflux stimulated by 2 microM ionomycin or 50 microM extracellular ATP Ionomycin had no effect on the apical Cl- fluxes. Thus cultured DCTb cells exhibit Ca(2+)-activated Cl- channels located in the basolateral membrane. This Cl- permeability was active at a resting membrane potential and could participate in the Cl- reabsorption across the DCTb in control conditions.

Zinc, cadmium, and copper are known to interact in many transport processes, but the mechanism of inhibition is widely debated, being either competitive or noncompetitive according to the experimental model employed. We investigated the mechanisms of inhibition of zinc transport by cadmium and copper using renal proximal cells isolated from rabbit kidney. Initial rates of 65Zn uptake were assessed after 0.5 min of incubation. The kinetics parameters of zinc uptake obtained at 20 degrees C were a Jmax of 208.0 +/- 8.4 pmol.min-1.(mg protein)-1, a Km of 15.0 +/- 1.5 microM and an unsaturable constant of 0.259 +/- 0.104 (n = 8). Cadmium at 15 microM competitively inhibited zinc uptake. In the presence of 50 microM cadmium, or copper at both 15 and 50 microM, there was evidence of noncompetitive inhibition. These data suggest that zinc and cadmium enter renal proximal cells via a common, saturable, carrier-mediated process. The mechanisms of the noncompetitive inhibition observed at higher concentrations of cadmium or with copper require further investigation, but may involve a toxic effect on the cytoskeleton.

Isolated proximal cells from rabbit kidney were seeded on collagen-coated permeable supports. After 8 days, the cultured cells became organized as a confluent monolayer. The proximal origin of the monolayer was confirmed by enzymatic, immunological, electrical and electron microscopical studies. The epithelia exhibited a morphological polarity that allowed for measurements of effluxes across the apical or the basolateral membranes. 86Rb was used as an isotopic tracer to indicate potassium movements. The 86Rb+ efflux across the basolateral face was 1.93-times that across the apical face, and both effluxes were pH dependent. Apical and basolateral 86Rb+ effluxes increased when the Ca2+ ionophore ionomycin (3 microM) was applied and when monolayers were exposed to a hypotonic medium. A pharmacological study revealed that BaCl2 (5 mM), tetraethylammonium (TEA, 20 mM) and Leiurus quinquestriatus hebraeus scorpion venom (from which charybdotoxin is extracted) abolished both ionomycin and hypotonically-stimulated effluxes, whereas apamin had no significant effect on the hypotonically-stimulated 86Rb+ efflux. This stimulated efflux was also abolished when monolayers were preincubated with pertussis toxin, but did not decrease in a Ca2(+)-free medium.