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 μM). In 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 dose-related increase in [Ca²⁺]_i which was blocked by 2 μM 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 μM) showed a dose-dependent inhibition of the Na⁺/K⁺-ATPase activity. The threshold decrease was at 1 μM Cch; it reached a maximum at 100 μM (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 established 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.