Stimulation of adenylyl cyclase mediated by phospholipase C-linked M₃ muscarinic receptor in human neuroblastoma SK-N-BE(2)C cells

Muscarinic receptor in human neuroblastoma SK-N-BE(2)C cells was identified and characterized. Treatment of the cells with carbachol evoked the generation of inositol 1,4,5-trisphosphate (IP₃) with a peak level reached at 1 min after stimulation. Carbachol increased intracellular Ca²⁺ ([Ca²⁺](i)) with an EC₅₀ value of 35 μM. In addition, carbachol produced a 1.3-3-fold increase in the cyclic AMP (cAMP) level compared with untreated control and elevated synergistically the cAMP level in the treatment with prostaglandin E₂ (PGE₂). The M₃ antagonist p-fluorohexahydrosiladifenidol (IC₅₀ = 0.5-0.8 μM) inhibited the increases in [Ca²⁺](i), IP₃, and cAMP more effectively than the M₁ antagonist pirenzepine (IC₅₀ = 59 μM) and the M₂ antagonist methoctramine (IC₅₀ = 20-30 μM). The involvements of [Ca²⁺](i) elevation and protein kinase C activation induced by phospholipase C activation were tested in the carbachol-induced cAMP production. The calcium chelator BAPTA/AM (75 μM) inhibited significantly the synergistic effects of carbachol and PGE₂ on the production of cAMP, whereas the Ca²⁺ ionophore ionomycin (1 μM) clearly enhanced PGE₂- induced cAMP production. However, phorbol 12-myristate 13-acetate did not enhance PGE₂-stimulated cAMP production. These data suggest that phospholipase C-linked M₃ receptors are present and that stimulation of the receptors activates adenylyl cyclase, at least in part, by the Ca²⁺- dependent system in the neuronal cells.