Nicotine increases [Ca2+]i in rat sublingual mucous acini by stimulating neurotransmitter release from presynaptic terminals.

The effects of nicotine on the intracellular free Ca2+ concentration ([Ca2+]i) were examined using the Ca(2+)-sensitive fluorescent dyes indo-1 and fura-2 in isolated rat sublingual mucous acini. Nicotine induced a dose-dependent increase in [Ca2+]i. In contrast to the muscarinic agonist carbachol-induced rise in [Ca2+]i, the nicotine-stimulated increase was abolished in a Ca(2+)-free medium, in the presence of L-type Ca2+ channel blockers (diltiazem and D888), by depolarization (high extracellular K+), and if the intracellular Ca2+ pool was first depleted with thapsigargin, an endoplasmic Ca(2+)-ATPase inhibitor. Furthermore, inhibitors of the nicotine acetylcholine receptor (mecamylamine, decamethonium, hexamethonium, tubocurarine, and alpha-bungarotoxin) blocked the nicotine-stimulated increase in [Ca2+]i without affecting the muscarinic-stimulated [Ca2+]i increase, whereas, muscarinic antagonists (atropine, pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide [4-DAMP]) inhibited both the nicotine- and carbachol-induced [Ca2+]i increases. Nicotine stimulation increased inositol 1,4,5-trisphosphate (IP3) content by 50%. Inhibition of the IP3-sensitive intracellular Ca2+ release pathway with 8-(diethylamino)-ocytl-3,4,5-trimethoxybenzoate (TMB-8) prevented the nicotine-induced increase in [Ca2+]i. Confocal imaging of [Ca2+]i indicated that the nicotine-induced and the carbachol-induced increases in [Ca2+]i occurred in the same cells within an acinus. However, in single sublingual acinar cells nicotine did not increase [Ca2+]i, whereas, carbachol did. Taken together, these results suggest that nicotine first triggers the release of acetylcholine from presynaptic nerve terminals associated with the dispersed sublingual acini which then activates muscarinic receptors.