Chloride Currents Activated by Calcitonin and cAMP in Primary Cultures of Rabbit Distal Convoluted Tubule |
| |
Authors: | M Tauc M Bidet P Poujeol |
| |
Institution: | (1) URA CNRS 1938, Université de Nice Sophia Antipolis, Parc Valrose 06108 Nice Cedex 2, France, FR |
| |
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.
Received: 5 July 1995/Revised: 21 December 1995 |
| |
Keywords: | : Whole cell — Chloride conductance — SPQ fluorescence — cAMP — Calcitonin — Cultured distal tubule |
本文献已被 SpringerLink 等数据库收录! |
|