Calcium-G protein interactions in the regulation of macrophage secretion

The interplay between activated G proteins and intracellular calcium ([Ca(2+)](i)) in the regulation of secretion was studied in the macrophage, coupling membrane capacitance with calcium-sensitive microfluorimetry. Intracellular elevation of either the nonhydrolyzable analogue of GTP, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), or [Ca(2+)](i) enhanced the amplitude and shortened the time course of stimulus-induced secretion in a dose-dependent manner. Both the ionophore- and the stimulus-induced secretory response were abolished in the presence of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). The K(d) of Ca(2+)-driven secretion was independent of GTP gamma S concentration, whereas the K(d) of the GTP gamma S-driven response decreased from 63 to 31 microM in the presence of saturating concentrations of [Ca(2+)](i). The time course of stimulus-induced secretion was dependent upon the concentration of [Ca(2+)](i). The time course of GTP gamma S-driven secretion was concentration-independent at high levels of [Ca(2+)](i), suggesting that a calcium-dependent translocation/binding step was rate-limiting. Our data strongly support a model in which [Ca(2+)](i) and activated G proteins act independently of one another in the sequential regulation of macrophage secretion. [Ca(2+)](i) appears to play a role in the recruitment and priming of vesicles from reserve intracellular pools at a step that is upstream of G protein activation. While activated, G proteins appear to play a key role in fusion of docked vesicles. Thus, secretion can result either from activating more G proteins or from elevating [Ca(2+)](i) at basal levels of G protein activation.