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 Ca(2+)-stimulated exocytosis. 2. In
alphatoxin-permeabilized cells, p[NH]ppG up to 20 microM activated
Ca(2+)-stimulated exocytosis. Higher concentrations had little or
no effect. At a free Ca2+ concentration of 5 microM, 7
microM-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
Ca(2+)-stimulated exocytosis by p[NH]ppG. 3. In streptolysin
O-permeabilized cells, p[NH]ppG did not activate, but rather
inhibited Ca(2+)-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
Ca(2+)-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 Ca(2+)-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.