EXOCYTOSIS FROM PERMEABILIZED BOVINE ADRENAL CHROMAFFIN CELLS IS DIFFERENTLY MODULATED BY GUANOSINE 5'-[GAMMA-THIO]TRIPHOSPHATE AND GUANOSINE 5'-[BETA-GAMMA-IMIDO]TRIPHOSPHATE

1. In bovine adrenal chromaffin cells made permeable either to molecules less-than-or-equal-to 3 kDa with alphatoxin or to proteins less-than-or-equal-to 150 kDa with streptolysin O, the GTP analogues guanosine 5'[beta-gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[gamma-thio triphosphate (GTP[S]) differently modulated Ca2+-stimulated exocytosis. 2. In alphatoxin-permeabilized cells, p[NH]ppG up to 20-mu-M activated Ca2+-stimulated exocytosis. Higher concentrations had little or no effect. At a free Ca2+ concentration of 5-mu-M, 7-mu-M-p[NH]ppG stimulated exocytosis 6-fold. Increasing the free Ca2+ concentration reduced the effect of p[NH]ppG. Pretreatment of the cells with pertussis toxin prevented the activation of the Ca2+-stimulated exocytosis by p[NH]ppG. 3. In streptolysin O-permeabilized cells, p[NH]ppG did not activate, but rather inhibited Ca2+-dependent catecholamine release under all conditions studied. In the soluble cytoplasmic material that escaped during permeabilization with streptolysin O, different G-protein alpha-subunits were detected using an appropriate antibody. Around 15% of the cellular alpha-subunits were detected in the supernatant of permeabilized control cells. p[NH]ppG or GTP[S] stimulated the release of alpha-subunits 2-fold, causing a loss of about 30% of the cellular G-protein alpha-subunits under these conditions. Two of the alpha-subunits in the supernatant belonged to the G(o) type, as revealed by an antibody specific for G(o)-alpha. 4. GTP[S], when present alone during stimulation with Ca2+, activated exocytosis in a similar manner to p[NH]ppG. Upon prolonged incubation, GTP[S], in contrast to p[NH]ppG, inhibited Ca2+-induced exocytosis from cells permeabilized by either of the pore-forming toxins. This effect was resistant to pertussin toxin. 5. The p[NH]ppG-induced activation of Ca2+-stimulated release from alphatoxin-permeabilized chromaffin cells may be attributed to one of the heterotrimeric G-proteins lost during permeabilization with streptolysin O. The inhibitory effect of GTP[S] on exocytosis is apparently not mediated by G-protein alpha-subunits, but by another GTP-dependent process still occurring after permeabilization with streptolysin O.