Crypt base cells show forskolin-induced Cl− secretion but no cation inward conductance

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Abstract
Whole-cell patch-clamp studies in base cells of isolated colonic crypts of rats pretreated with dexamethasone were performed to examine the effects of stimulation by forskolin (10 μmol/1). The experiments were designed in order to distinguish between two postulated effector mechanisms: the activation of a non-selective cation channel and the activation of Cl− channels. As shown in an accompanying report, forskolin depolarizes the membrane voltage (V m) by some 40–50 mV and enhances the whole-cell membrane conductance (G m) substantially in these cells. In this report all experiments were performed in the presence of forskolin. A reduction of the bath Na+ concentration from 145 to 2 mmol/1 led to a hyperpolarization ofV m by some 20–30 mV This hyperpolarization occurred very slowly suggesting that the hyperpolarization produced by the low-Na+ solution was caused indirectly and not by a change in the equilibrium potential for Na+,E Na +. A complete kinetic analysis of the effect on voltage of bath Na+ revealed a saturation-type relation with a high apparent affinity for Na+ of around 5–10 mmol/1. A reduction in bath Cl− concentration from 145 to 32 mmol/1 caused a depolarization ofV m from −34 ± 3 to −20 ± 4 mV (n = 13) in the presence of a high bath Na+ concentration, but had the opposite effect at low (5 mmol/1) Na+ concentrations:V m was hyperpolarized from −46 ± 4 to −62 ± 6 mV (n = 13). If the effect of Na+ onV m was caused by a non-selective cation channel the opposite would have been expected. To test directly whether the Na+2Cl−K+ cotransporter was responsible for the effects of changes in bath Na+ onV m, the effects of increasing concentrations of several loop diuretics were examined. Furosemide, piretanide, torasemide and burnetanide (up to 0.1–0.5 mmol/1) all hyperpolarizedV m, albeit only by less than 10 mV. Another subclass of loop diuretics containing a tetrazolate in position 1 [e.g. azosemide, no. 19A and no. 20A from Schlatter E, Greger R, Weidtke C (1983) Pflüger Arch 396: 210–217] were much more effective. Azosemide hyperpolarizedV m from −46 ± 3 to −74 ± 2 mV (n = 18) and reducedG m from 11 ± 1 to 4 ± 1 nS (n = 14). These data indicate that forskolin stimulates Cl− secretion in these cells by a mechanism fully compatible with the current scheme for exocrine secretion involving the Na+2Cl−K+ cotransporter.