Nicotine Increases [Ca 2+ ] i in Rat Sublingual Mucous Acini by Stimulating Neurotransmitter Release from Presynaptic Terminals

Abstract

Abstract

The effects of nicotine on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) were examined using the Ca²⁺-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 Ca²⁺-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 Ca²⁺-ATPase inhibitor. Furthermore, inhibitors of the nicotine acetylcholine receptor (mecamylamine, decamethonium, hexamethonium, tubocurarine, and α-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)-octyl-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.