Muscarinic acetylcholine receptor activation induces Ca2+ mobilization and Na+/K+ATPase activity inhibition in eel enterocytes.

The effect of carbachol (Cch) on intracellular calcium concentration ([Ca2+](i)) in eel enterocytes was examined using the fluorescent Ca2+ indicator fura-2, Cch caused a biphasic increase in [Ca2+](i), with an initial spike followed by a progressively decreasing level (over 6 min) to the initial. pre-stimulated, level. The effect of Cch was dose-dependent with a 7(.)5-fold increase in [Ca2+](i) over basal level induced by the maximal dose of Cch (100 muM). Ca2+-free/EGTA buffer the effect of Cch was less pronounced and the [Ca2+](i) returned rapidly to basal levels. The increment of [Ca2+](i) was dose-dependently attenuated in cells pre-treated with U73122. a Specific inhibitor of phospholipase C, suggesting that the Cch-stimulated increment of [Ca2+](i) required inositol triphosphate formation. In the presence of extracellular Ca2+, thapsigargin (TG), a specific microsomal Ca2+-ATPase inhibitor, caused a sustained rise in [Ca2+](i) whereas in Ca2+-free medium the increase in [Ca2+](i) was transient; in both cases, subsequent addition of Cch was without effect. When 2 mM CaCl2 were added to the cells stimulated with TG or with Cch in Ca2+-free medium, a rapid increase in [Ca2+](i) was detected. corresponding to the capacitative Ca2+ entry. Thus, both TG and Cch depleted intracellular Ca2+ stores and stimulated influx of extracellular Ca2+ consistent with capacitative Ca2+ entry. K+ depolarization obtained with increasing concentrations of KCl in the extracellular medium induced a close-related increase in [Ca2+](i) which was blocked by 2 muM nifedipine, a non-specific L-type Ca2+ channel blocker, Nifedipine also changed significantly the height of the Ca2+ transient, and the rate of decrement to the pre-stimulated [Ca2+](i) level, indicating that Ca2+ entry into enterocytes also occurs through an L-type voltage-dependent calcium channel pathway. We also show that isolated enterocytes stimulated with increasing Cch concentrations (0(.)1-1000 muM) showed a close-dependent inhibition of the Na+/K+-ATPase activity. The threshold decrease was at 1 muM Cch: it reached a maximum at 100 muM (50(.)5% inhibition) and did not decrease further with the use of higher dose. The effect of Cch on Na+/K(+)ATPase activity was dependent on both protein kinase C (PKC) and protein phosphatase calcineurin activation since the PKC inhibitor calphostin C abolished Cch effects, while the calcineurin inhibitor FK506 augmented Cch effect. Collectively, these data establish a functional pathway by which Cch can modulate the activity of the Na+/K+-ATPase through a PKC-dependent (calphostin C-sensitive) pathway and a calcineurin-dependent (FK506-sensitive) pathway.