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Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid, Spain.
1. Intracellular potentials, Cl- activity and membrane resistances were measured in Necturus small intestinal epithelium during Cl- replacement experiments using conventional or Cl- -selective double-barrelled microelectrodes. A Cl- conductance, located in the apical membrane and activated by cyclic nucleotides is demonstrated by ion-substitution experiments. 2. The mean mucosal membrane potential (Em) was -35.5 mV. Removal of Cl- from the mucosal medium by replacement with gluconate, evoked a sudden depolarization of Em and an immediate increase in the fractional resistance of the mucosal membrane (f(Rm)). The size of the change in Em varied between 3 and 65 mV, corresponding to Cl- to K+ permeability ratios between 0.2 and 20. It was inversely related to the initial f(Rm), which ranged from 0.04 to 0.50. 3. Prolonged incubation in low-Cl- solutions led to a reversal of the initial depolarization and to a sustained hyperpolarization accompanied by a marked increase in f(Rm). The new value of Em was close to the K+ equilibrium potential, consistent with a depletion of cellular Cl- and the preponderance of a K+ membrane permeability in the absence of Cl-. This emphasizes the role of Cl- in establishing Em. 4. Removal of mucosal Cl- produced a fast decrease in intracellular Cl-, as measured with Cl- -selective microelectrodes. The efflux was consistent with electrodiffusion across the mucosal membrane. Changes in Em paralleled changes in intracellular Cl- activity, indicating the presence of a large Cl- conductance. 5. Dibutyryl cyclic AMP or forskolin produced a slow depolarization, a decrease in f(Rm) and an increased change in intracellular potential in low mucosal Cl- which on average corresponds to an approximately 15-fold increase in the relative Cl- permeability. These results are consistent with an activation of apical Cl- conductance. 6. The selectivity of Cl- channels of Necturus enterocytes to different anions was obtained from potential measurements. The sequence of permeabilities was SCN- greater than I- greater than or equal to Br- greater than NO3- greater than Cl- much greater than HCO3- greater than gluconate. This is consistent with a model involving a weak interaction of the anions with the selectivity filter. 7. The selectivity of the anion conductance was maintained after activation with cyclic nucleotides, suggesting a single channel for the permeation of the different anions tested, rather than parallel channels. 8. Derivatives of 9-anthracene which are potent inhibitors of Cl- channels in other systems failed to block the apical Cl- conductance of Necturus enterocytes. Chloride conductance was also insensitive to furosemide.(ABSTRACT TRUNCATED AT 400 WORDS)
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