STIMULATION OF ADENYLYL-CYCLASE MEDIATED BY PHOSPHOLIPASE C-LINKED M(3) MUSCARINIC RECEPTOR IN HUMAN NEUROBLASTOMA SK-N-BE(2)C CELLS

Abstract

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 EC(50) value of 35 mu M. 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 E(2) (PGE(2)). The M(3) antagonist p-fluorohexahydrosiladifenidol (IC50 = 0.5-0.8 mu M) inhibited the increases in [Ca2+](i), IP3, and cAMP more effectively than the M(1) antagonist pirenzepine (IC50 = 5-9 mu M) and the M(2) antagonist methoctramine (IC50 = 20-30 mu M) 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 mu M) inhibited significantly the synergistic effects of carbachol and PGE(2) on the production of cAMP, whereas the Ca2+ ionophore ionomycin (1 mu M) clearly enhanced PGE(2)-induced cAMP production. However, phorbol 12-myristate 13-acetate did not enhance PGE(2)-stimulated cAMP production. These data suggest that phospholipase C-linked M(3) 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.