title: The receptor-regulated calcium influx in mouse submandibular acinar cells is sodium dependent: a patch-clamp study.
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.
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008536:Meglumine / Q000494:pharmacology; D051379:Mice; D011188:Potassium / Q000378:metabolism*; D012964:Sodium / Q000502:physiology*; D013363:Submandibular Gland / Q000378:metabolism*
chemical_list: D007473:Ion Channels; D000001:Calcimycin; D008536:Meglumine; D001285:Atropine; D012964:Sodium; D000109:Acetylcholine; D011188:Potassium; D002118:Calcium