Mouse Beta-TC6 insulinoma cells possessing nicotinic receptor [Ohtani, M., Oka, T., Badyuk, M., Xiao, Y., Kellar, KJ., Daly, JW., 2006. Mouse beta-TC6 insulinoma cells: high expression of functional alpha3beta4 nicotinic receptors mediating membrane potential, intracellular calcium, and insulin release. Mol. Pharmacol. 69, 899-907.] also expressed M(3) and M(4) muscarinic receptors. Carbamylcholine, a mixed muscarinic/nicotinic receptor agonist, or oxotremorine M, a selective muscarinic agonist, elicited an elevation of cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and release of insulin. The maximal [Ca(2+)](i) response induced by carbamylcholine was larger than that of oxotremorine M or that of nicotine, suggesting that carbamylcholine enhanced the [Ca(2+)](i) response by stimulating two types of receptor. M(3) and M(4) muscarinic receptor antagonists inhibited the [Ca(2+)](i) responses to carbamylcholine and oxotremorine M, suggesting the involvement of these muscarinic receptors in the regulation of [Ca(2+)](i). In addition, pretreatment with carbamylcholine inhibited the [Ca(2+)](i) responses to oxotremorine M or nicotine, indicating that the effect of carbamylcholine on [Ca(2+)](i) was mediated by both muscarinic and nicotinic receptors. A phospholipase C (PLC) inhibitor U73122, a protein kinase C (PKC) inhibitor chelerythrine and a phospholipase A(2) (PLA(2)) inhibitor AACOCF(3) inhibited the [Ca(2+)](i) response to carbamylcholine or oxotremorine M, while these inhibitors did not block the effect of nicotine. Carbamylcholine induced a smaller extent of insulin secretion than oxotremorine M, suggesting that concomitant stimulation of muscarinic and nicotinic receptors by carbamylcholine resulted in the negative type of the receptor interaction.