title: Muscarinic acetylcholine receptor activation induces Ca2+ mobilization and Na+/K+-ATPase activity inhibition in eel enterocytes.
abstract: 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 microM). 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 microM 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 microM) showed a dose-dependent inhibition of the Na+/K+-ATPase activity. The threshold decrease was at 1 microM Cch; it reached a maximum at 100 microM (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.
authors: Physiology Laboratory, Department of Biology, University of Lecce, 73100 Lecce, Italy.
authors: A
authors: A
authors: Muscella
authors: S
authors: S
authors: Greco
authors: M G
authors: MG
authors: Elia
authors: E
authors: E
authors: Jiménez
authors: C
authors: C
authors: Storelli
authors: S
authors: S
authors: Marsigliante
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002217:Carbachol / Q000494:pharmacology*; D018679:Cholinergic Agonists / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004524:Eels; D020895:Enterocytes / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011950:Receptors, Cholinergic / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase; D016559:Tacrolimus / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
chemical_list: D065095:Calcineurin Inhibitors; D002121:Calcium Channel Blockers; D018679:Cholinergic Agonists; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D011950:Receptors, Cholinergic; C061465:calphostin complex; D019284:Thapsigargin; D002217:Carbachol; D012964:Sodium; D011493:Protein Kinase C; D010738:Type C Phospholipases; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D009543:Nifedipine; D002118:Calcium; D020123:Sirolimus; D016559:Tacrolimus