The receptor‐regulated calcium influx in mouse submandibular acinar cells is sodium dependent: a patch‐clamp study.

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Abstract
1. Salivary acini were isolated enzymatically from submandibular glands of adult male mice. The patch‐clamp technique was employed to record K+ currents in cell‐attached patches of basolateral membrane. Application of acetylcholine (10(‐5) M) to the medium bathing the cells results in a pronounced and sustained activation of the K+ channels in the cell‐attached patches, an effect mediated by an intracellular second messenger. In the present study we investigate the mechanism by which acetylcholine achieves activation of K+ channels. 2. The effects of acetylcholine on single‐channel activity were shown to be dependent on extracellular Ca2+, i.e. due to Ca2+ influx. In cells bathed in Ca2+‐free medium acetylcholine activation resulted in no increase in single‐channel open probability. This blockade could be reversed by reintroduction of Ca2+ to the extracellular fluid in the continued presence of the agonist. The effects of acetylcholine in control medium (1.2 mM‐Ca2+) were mimicked by the Ca2+ ionophore, A23187 (10(‐8) M). 3. In K+‐depolarized cells (bathed in a Na+‐free, 145 mM‐KCl solution) there was no evidence of any voltage‐activated Ca2+ influx pathway. In the K+‐depolarized cells acetylcholine application was no longer associated with any increase in the open probability of the K+ channels. K+ channels could be activated by adding A23187 (10(‐8) M) to the high‐K+ solution. 4. Cells bathed in another Na+‐free (N‐methyl‐D‐glucamine substituted for Na+) but non‐depolarizing solution were also refractory to acetylcholine. K+ currents could, however, be activated in patches attached to these cells by application of A23187 (10(‐8) M) or by the introduction of 20 mM‐Na+ to the extracellular fluid in the presence of acetylcholine. The increased activity associated with the reintroduction of Na+ was totally reversed by atropine, i.e. it was receptor regulated. 5. The data presented above indicate that the cholinergic regulation of K+ channels is secondary to the receptor‐regulated activation of a Ca2+ influx pathway. There is no evidence of voltage‐activated Ca2+ influx in these cells. The cholinergic activation of Ca2+ influx is abolished in Na+‐depleted cells. We conclude that the Na+ dependency indicates either that Na+ is involved in the gating of some voltage‐independent Ca2+ channel or that Ca2+ entry is via a coupled Na+‐Ca2+ co‐ or countertransport pathway.