ATP-dependent activation of phospholipase C by antigen, NECA, Na₃VO₄, and GTP-γ-S in permeabilized RBL cell ghosts: Differential augmentation by ATP, phosphoenolpyruvate and phosphocreatine

Abstract

Ghosts prepared from rat basophilic leukemia cells (RBL cell ghosts) and permeabilized with α-toxin fromS. aureus are a simplified system for the study of Fc_εRI-mediated activation of phospholipase C (PLC). This activity is dependent upon ATP and magnesium, and is enhanced by the addition of another compound containing an energetic phosphate group, either phosphoenolpyruvate (PEP) or phosphocreatine (PCr). This effect appears to be specific for PEP and PCr in that other compounds with energetic phosphate bonds including fructose 1,6-bisphosphate and additional ATP are not effective. On the contrary, GTP-γ-S, an activator of G proteins, activates PLC in the presence of ATP alone and this is not further enhanced by the addition of PEP. In addition to Fc_εRI and GTP-γ-S, two other stimuli lead to enhanced activity of PLC in permeabilized RBL cell ghosts: 1) an inhibitor of tyrosine phosphatases (Na₃VO₄) and 2) an analog of adenosine (NECA). Data presented here extend previous results to show that activation of PLC by GTP-γ-S is not enhanced either by the addition of PCr or by the addition of a more MgATP. Further new findings include the observations that activation of PLC by Na₃VO₄ is augmented by PEP and PCr in a fashion similar to that observed for Fc_εRI-mediated activation of PLC and that activation of PLC by NECA shows even more marked dependency on PEP than does activation by Fc_εRI or Na₃VO₄. Together, these experiments demonstrate that antigen, Na₃VO₄, GTP-γ-S, and NECA can all activate PLC in permeabilized RBL cell ghosts in the presence of ATP and that these activities are differentially affected by the addition of a second compound with an energetic phosphate bond.