ISSN:
1432-1424
Keywords:
Key words: Whole cell — Chloride conductance — SPQ fluorescence — cAMP — Calcitonin — Cultured distal tubule
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Abstract. 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°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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s002329900049
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