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Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 -/- mice. In wild-type PCT cells in primary culture, a Cl- conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage (I-V) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I- > Br- > Cl- > > gluconate. This conductance was sensitive to 1 mM 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 mM diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K+ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 mM quinidine, 5 mM barium, and 10 microM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 microM 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl- and K+ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures.

The role of CFTR in the control of K(+) currents was studied in mouse kidney. Whole cell clamp was used to identify K(+) currents on the basis of pharmacological sensitivities in primary cultures of proximal (PCT) and distal convoluted tubule (DCT) and cortical collecting tubule (CCT) from wild-type (WT) and CFTR knockout (KO) mice. In DCT and CCT cells, forskolin activated a 293B-sensitive K(+) current in WT, but not in KO, mice. In these cells, a hypotonic shock induced K(+) currents blocked by charybdotoxin in WT, but not in KO, mice. In PCT cells from WT and KO mice, the hypotonicity-induced K(+) currents were insensitive to these toxins and were activated at extracellular pH 8.0 and inhibited at pH 6.0, suggesting that the corresponding channel was TASK2. In conclusion, CFTR is implicated in the control of KCNQ1 and Ca(2+)-sensitive swelling-activated K(+) conductances in DCT and CCT, but not in proximal convoluted tubule, cells. In KO mice, impairment of the regulatory volume decrease process in DCT and CCT could be due to the loss of an autocrine mechanism, implicating ATP and adenosine, which controls swelling-activated Cl(-) and K(+) channels.

We have previously investigated the effects of extracellular ATP on the concentration of free cytosolic calcium ([Ca2+]i) from rat cultured neurohypophysial astrocytes (pituicytes). We demonstrated that ATP acts via a P2Y receptor to increase [Ca2+]i. In the present study, we examine the effect of ATP on K+ efflux using 86Rb+ as an isotopic tracer, in order to characterize the possible presence of a Ca2+-activated K+ conductance and to establish the implications of pituicytes in the regulation of stimulus-secretion coupling. ATP evoked an increase in 86Rb+ efflux from cultured pituicytes. This effect was Ca2+ dependent, as indicated by the unresponsiveness of cells loaded with BAPTA/AM (20 microM). Furthermore, the effect of ATP was mimicked by 2-methylthio-adenosine-5'-triphosphate (2MeSATP), a P2 purinoceptor agonist, and abolished by Reactive Blue 2 (RB-2), a selective P2Y antagonist, implying a role for the P2Y purinoreceptor. A pharmacological study revealed that Ba2+ and tetraethylammonium (TEA), two inhibitors of K+ channels, both strongly reduced the ATP-stimulated 86Rb+ efflux. In addition, the effect of ATP was modulated by different peptidic toxins. Apamin (100 nM), an inhibitor of the small-conductance Ca2+-activated K+ channels, partly blocked ATP-induced 86Rb+ efflux. Leiurus quinquestriatus hebraeus (LQH) scorpion venom (20 microg/ml) and Buthus tamulus (BT) scorpion venom (20-200 microg/ml) inhibited ATP-induced 86Rb+ efflux. The specificity of the effects of the crude venoms was checked using charybdotoxin (100 nM) and iberiotoxin (1 pM), which are the active toxins extracted from the LQH and BT venoms, respectively. These data indicate the involvement of several types of Ca2+-activated K+ channels in the ATP-dependent K+ efflux, and lead to the proposal that, in the neurohypophysis, extracellular ATP released by nerve terminals may act directly on the pituicytes and induce a K+ efflux via a P2Y purinoreceptor.

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.

We have previously investigated the effects of extracellular ATP on the concentration of free cytosolic calcium ([Ca2+]i) from rat cultured neurohypophysial astrocytes (pituicytes). We demonstrated that ATP acts via a P2Y receptor to increase [Ca2+]i. In the present study, we examine the effect of ATP on K+ efflux using 86Rb+ as an isotopic tracer, in order to characterize the possible presence of a Ca2+-activated K+ conductance and to establish the implications of pituicytes in the regulation of stimulus–secretion coupling. ATP evoked an increase in 86Rb+ efflux from cultured pituicytes. This effect was Ca2+ dependent, as indicated by the unresponsiveness of cells loaded with BAPTA/AM (20 µM). Furthermore, the effect of ATP was mimicked by 2-methylthio-adenosine-5′-triphosphate (2MeSATP), a P2 purinoceptor agonist, and abolished by Reactive Blue 2 (RB-2), a selective P2Y antagonist, implying a role for the P2Y purinoreceptor. A pharmacological study revealed that Ba2+ and tetraethylammonium (TEA), two inhibitors of K+ channels, both strongly reduced the ATP-stimulated 86Rb+ efflux. In addition, the effect of ATP was modulated by different peptidic toxins. Apamin (100 nM), an inhibitor of the small-conductance Ca2+-activated K+ channels, partly blocked ATP-induced 86Rb+ efflux. Leiurus quinquestriatus hebraeus (LQH) scorpion venom (20 µg/ml) and Buthus tamulus (BT) scorpion venom (20–200 µg/ml) inhibited ATP-induced 86Rb+ efflux. The specificity of the effects of the crude venoms was checked using charybdotoxin (100 nM) and iberiotoxin (1 µM), which are the active toxins extracted from the LQH and BT venoms, respectively. These data indicate the involvement of several types of Ca2+-activated K+ channels in the ATP-dependent K+ efflux, and lead to the proposal that, in the neurohypophysis, extracellular ATP released by nerve terminals may act directly on the pituicytes and induce a K+ efflux via a P2Y purinoreceptor. [ABSTRACT FROM AUTHOR]