CALCIUM-INDUCED SECRETION FROM PERMEABILIZED RAT MAST-CELLS - REQUIREMENTS FOR GUANINE-NUCLEOTIDES

Cells used in this work were permeabilized by streptolysin-O and then washed to remove freely soluble components. The secretory responsivene ss of these cells to various combinations of calcium, MgATP and guanin e nucleotide was characterized and in most respects was found to be si milar to that of the metabolically inhibited (unwashed) cells. The con tent of adenosine and guanine nucleotides, which remain within the per meabilized cells after washing, was estimated as 0.83 and 0.12 mM (ext rapolated to intact cells), which constitutes 18 and 25%, respectively , of the total nucleotide content of mast cells. High (> mM) concentra tions of MgATP, required for the calcium-induced secretion, were reduc ed to muM levels by suboptimal concentrations of GTP, which also marke dly increased both the rate and extent of the response. Similarily, mu M concentrations of MgATP reduced the requirements of the calcium-depe ndent secretion for GTP. The synergy of the GTP and ATP effects sugges ts that, together, the two nucleotides can maintain a pool of free GTP , presumably as a result of transphosphorylation from ATP to GDP. Thus , MgATP may work by transphosphorylating the endogenous GDP. However, neither GTP nor GTP-gamma-S were effective as substitutes for MgATP in the calcium-induced secretion, particularly that from metabolically i nhibited cells. This indicates that MgATP does not act simply by provi ding GTP but is needed to maintain a phosphorylated state of the syste m. The synergistic effects of ATP and GTP were observed only in the pr esence of calcium. To test whether calcium/MgATP-induced secretion req uires an activated G protein, the effects of G-protein inactivators we re studied. GDP, deoxy GDP and GDP-beta-S exerted differing degrees of inhibition on secretory responses induced by various combinations of effectors. The response to calcium/MgATP was less sensitive to these i nhibitors than that to GTP-gamma-S (with or without calcium). However, all three 'inhibitors' were also capable of stimulating calcium/MgATP -dependent secretion, indicating a transphosphorylation, producing GTP , dGTP and GTP-beta-S. Thus, in the absence of any specific inhibitors for either G proteins or the transphosphorylation reaction, the degre e of dependence of the calcium-induced secretion on a G protein remain s unclear.