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

Abstract

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 E(2) 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 (InsP(3)) generation, with maximal and half maximal inhibition (IC50) occurring at approximately 15 mu M and 3.2 mu M; respectively. U-73122 also inhibited the synergistic enhancement of cAMP accumulation induced by carbachol and prostaglandin E(2) in a concentration-dependent manner with maximum and IC50 at 12 +/- 4 mu M and 3.4 +/- 0.3 mu M, respectively. However, U-73122 did significantly inhibit prostaglandin E(2)-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, GTP gamma S- and AlF4--induced InsP, 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, InsP(3) 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.