Calcium-induced secretion from permeabilized rat mast cells: Requirements for guanine nucleotides

Abstract

Cells used in this work were permeabilized by streptolysin-O and then washed to remove freely soluble components. The secretory responsiveness of these cells to various combinations of calcium, MgATP and guanine nucleotide was characterized and in most respects was found to be similar to that of the metabolically inhibited (unwashed) cells. The content of adenosine and guanine nucleotides, which remain within the permeabilized cells after washing, was estimated as 0.83 and 0.12 mM (extrapolated to intact cells), which constitutes 18 and 25%, respectively, of the total nucleotide content of mast cells. High (> mM) concentrations of MgATP, required for the calcium-induced secretion, were reduced to μM levels by suboptimal concentrations of GTP, which also markedly increased both the rate and extent of the response. Similarily, μM concentrations of MgATP reduced the requirements of the calcium-dependent secretion for GTP. The synergy of the GTP and ATP effects suggests 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-γ-S were effective as substitutes for MgATP in the calcium-induced secretion, particularly that from metabolically inhibited cells. This indicates that MgATP does not act simply by providing GTP but is needed to maintain a phosphorylated state of the system. The synergistic effects of ATP and GTP were observed only in the presence of calcium. To test whether calcium/MgATP-induced secretion requires an activated G protein, the effects of G-protein inactivators were studied. GDP, deoxy GDP and GDP-β-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 inhibitors than that to GTP-γ-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-β-S. Thus, in the absence of any specific inhibitors for either G proteins or the transphosphorylation reaction, the degree of dependence of the calcium-induced secretion on a G protein remains unclear.