Two G-Proteins Act in Series to Control Stimulus-Secretion Coupling in Mast Cells: Use of Neomycin to Distinguish between G-Proteins Controlling Polyphosphoinositide Phosphodiesterase and Exocytosis

Provision of GTP (or other nucleotides capable of acting as ligands for activation of G-proteins) together with Ca2+ (at micromolar concentrations) is both necessary and sufficient to stimulate exocytotic secretion from mast cells permeabilized with streptolysin-O. GTP and its analogues, through their interactions with G P, also activate polyphosphoinositide-phosphodiesterase (PPI-pde generating inositol 1,4,5-trisphosphate and diglyceride [DG]). We have used mast cells labeled with [3 H]inositol to test whether the requirement for GTP in exocytosis is an expression of G P activity through the generation of DG and consequent activation of protein kinase C, or whether GTP is required at a later stage in the stimulus secretion sequence. Neomycin (0.3 mM) inhibits activation of PPI-pde, but maximal secretion due to optimal concentrations of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S) can still be evoked in its presence. When ATP is also provided the concentration requirement for GTP-γ-S in support of exocytosis is reduced. This sparing effect of ATP is nullified when the PPI-pde reaction is inhibited by neomycin. We argue that the sparing effect of ATP occurs as a result of enhancement of DG production and through its action as a phosphoryl donor in the reactions catalyzed by protein kinase C.