Changes in actin network during calcium-induced exocytosis in permeabilized GH3 cells: calcium directly regulates F-actin disassembly.
Using digitonin-permeabilized GH3 cells, we investigated both the release of prolactin (PRL) and changes in the cytoskeleton. We determined that permeabilized GH3 cells released PRL in a dose-dependent manner upon addition of micromolar Ca(2+). Phalloidin, a filamentous actin (F-actin) stabilizing agent, inhibited both Ca(2+)-dependent and -independent PRL release, whereas cytochalasin B, a destabilizing agent, had almost no effect on the release. Observation with a confocal laser scanning microscope revealed that F-actin existed mainly in the cortical region in the quiescent state. Increased cytosolic Ca(2+) induced a change in F-actin distribution: F-actin in the cortical region decreased, whereas F-actin inside the cells increased. This change in F-actin distribution was not observed when phalloidin was added. Addition of cytochalasin B induced patchy F-actin spots, but the pattern of the changes of F-actin distribution did not change. The time course of change in F-actin distribution showed that the F-actin network in the cortical region was reduced within 1 min, and Ca(2+)-dependent release of PRL continued for up to 20 min. These results suggest that the F-actin network near the membrane acts as a barrier to exocytosis and that Ca(2+) directly controls the cytoskeletal changes.
Yoneda
M
M
Department of Obstetrics and Gynecology, Osaka University Medical School, 2-2, Yamadaoka Suita Osaka, 565-0871, Japan.
Nishizaki
T
T
Tasaka
K
K
Kurachi
H
H
Miyake
A
A
Murata
Y
Y
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D000199:Actins / Q000502:physiology*; D000236:Adenoma / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D003571:Cytochalasin B / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects* / Q000648:ultrastructure; D005089:Exocytosis; D018613:Microscopy, Confocal; D010590:Phalloidine / Q000494:pharmacology; D010911:Pituitary Neoplasms / Q000378:metabolism* / Q000648:ultrastructure; D011388:Prolactin / Q000378:metabolism*; D051381:Rats; D013268:Stimulation, Chemical; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure
D000199:Actins; D010590:Phalloidine; D003571:Cytochalasin B; D011388:Prolactin; D002118:Calcium