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Abstract

Nonselective Ca²⁺-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca²⁺ on the cytosolic side, the mean open state probability P_o of one channel was about 0.5. In insideout oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 3′,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 μmol/l of NPPB or DPC decreased P_o from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 μmol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reduced P_o, but in contrast to the above mentioned substances, it induced fast flickering. Ba²⁺ (70 mmol/l) and tetraethylammonium (TEA⁺; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 4,4′-dinitro-2,2′-stilbenedisulfonate (DNDS). 10 μmol/l SITS applied to the cytosolic side increased P_o from 0.5 to 0.7 and with 100 μmol/l SITS the channels remained nearly permanently in its open state (P_o≅1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca²⁺ concentration to 0.1 μmol/l, addition of 100 μmol/l of SITS had no effect. Similarly, reducing bath Ca²⁺ concentration from 1.3 mmol/l in presence of 100 μmol/l SITS (channels are maximally activated) to 0.1 μmol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca²⁺. SITS had no effects, when applied to the extracellular side in outside out patches. In summary, the substances DPC, NPPB and DCDPC inhibit nonselective cation channels, where DCDPC has the most potent and NPPB the smallest effect; whereas SITS, DIDS and DNDS activate the channel when applied from the cytosolic side in the presence of Ca²⁺ ions.