Synergistic activation of adenylyl cyclase is dependent upon phospholipase C-mediated processes in human neuroblastoma SK-N-BE(2)C cells.

1-[6-[17 beta-3-Methoxyestra-1,3.5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione (U-73122), an inhibitor of processes involved in the activation of phospholipase C, was used to assess the role of phospholipase C activation in the synergistic elevation of cAMP induced by carbachol and prostaglandin E2 in human neuroblastoma (SK-N-BE(2)C cells. Pre-treatment of the cells with U-73122 resulted in inhibition of carbachol-induced intracellular Ca2+ ([Ca2+]i) rise and inositol 1,4,5-trisphosphate (InsP3) generation, with maximal and half maximal inhibition (IC50) occurring at approximately 15 microM and 3.2 microM, respectively. U-731222 also inhibited the synergistic enhancement of cAMP accumulation induced by carbachol and prostaglandin E2 in a concentration-dependent manner with maximum and IC50 at 12 +/- 4 microM and 3.4 +/- 0.3 microM, respectively. However, U-73122 did significantly inhibit prostaglandin E2-induced production. While 1,2-bis(o-aminophenoxy)ethane-N,N,N,'N'-tetraacetic acid (BAPTA/AM) treatment decreased the synergistic cAMP accumulation by 28%m addition U-73122 further decreased it down to complete inhibition. Furthermore, GTP gamma S- and A1F4(-)-induced InsP3 generation in digitonin-mediated permeabilized cells was also inhibited by U-73122 treatment. Pre-treatment of the cells with neomycin, another blocker of the phospholipase C pathway, also resulted in inhibition of the carbachol-induced [Ca2+]i rise, InsP3 generation, and the enhancing effect on cAMP accumulation, to a comparable extent. But, Ca2+ chelation by BAPTA/AM in addition to neomycin treatment further decreased the cAMP accumulation. These results suggest that the increase in cytosolic Ca2+ and the coupling process between muscarinic receptor-like G-protein and phospholipase C are important for the synergistic activation of adenylyl cyclase in SK-N-BE(2)C cells.