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

1-[6[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]1H-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 μM and 3.2 μM, respectively. U-73122 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 μM and 3.4 ± 0.3 μM, respectively. However, U-73122 did not 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%, addition of U-73122 further decreased it down to complete inhibition. Furthermore, GTPγS- and AlF₄^- 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-linked G-protein and phospholipase C are important for the synergistic activation of adenylyl cyclase in SK-N-BE(2)C cells.