Ca 2+ mobilization and Cl efflux in submandibular salivary cells of adult and newborn rats

Abstract

These cells were isolated by enzymatic digestion from glands of 1-day-old and fully mature rats. The effects of exposure to acetylcholine on cytosolic Ca 2+ and Cl efflux were studied by, respectively, spectrofluorimetry of fura-2 and the net efflux of the isotopic tracer 36Cl from preloaded cells. In both types of cells, when incubated in Ca 2+-containing solutions, acetylcholine initially caused a rapid, significant increase in cytosolic Ca 2+ (from approx. 90 to 480–570 nmol), followed by a slower decrease to a plateau value of 280–290 nmol. The initial peak persisted (315–339 nmol) in Ca 2+-free solutions but the cytosolic Ca 2+ concentrations decreased rapidly to levels below prestimulation values (30 nmol). 36Cl efflux in tracer preloaded cells incubated in Ca 2+-containing medium in the presence of acetylcholine was 18% in cells of new born animals and 35% in adult cells. In Ca 2+-free medium, mature cells showed a transient but significant (26%) efflux of 36Cl. Cells of 1-day-old rats did not show a net efflux of 36Cl under these conditions, but subsequent addition of Ca 2+ caused a 15% reduction (i.e. efflux) in tracer content. The antagonist 3,4,5-trimethoxybenzoate-8( N, N-dithylamino)octyl ester (TMB-8), which blocks internal Ca 2+ release, inhibited both the initial increase in cell Ca 2+ in both types of cells and the transient efflux of 36Cl seen in mature cells when incubated in Ca 2+-free solutions. At high concentrations (5 mM), LaCl 3 inhibited efflux of 36Cl in mature cells but not in those of newborn rats. However, at lower concentrations (0.1 mM), which do not interfere with fluorescence spectra, LaCl 3 did not inhibit the effect of acetylcholine on cell Ca 2+. These results suggest that Cl efflux in adult submandibular cells is regulated by an increase in cell Ca 2+ arising from release of internal Ca 2+ and from influx of external Ca 2+. Both of these responses are evident in cells of newborn animals but Cl efflux is either decreased or absent. This is likely to be associated with a deficiency in the Cl channels or in the coupling between Ca 2+ and the channel substrate through regulatory molecules associated with phosphorylation of the channel protein.