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

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 mu mol/l). 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/l led to a hyperpolarization of V-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/l. A reduction in bath Cl- concentration from 145 to 32 mmol/l caused a depolarization of V-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/l) Na+ concentrations: V-m was hyperpolarized from -46 +/- 4 to - 62 +/- 6 mV (n = 13). If the effect of Na+ on V-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+ on V-m, the effects of increasing concentrations of several loop diuretics were examined. Furosemide, piretanide, torasemide and bumetanide (up to 0.1-0.5 mmol/l) all hyperpolarized V-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) Pfluger Arch 396: 210-217] were much more effective. Azosemide hyperpolarized V-m from -46 + 3 to -74 +/- 2 mV (n = 18) and reduced G(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.