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Journal of Endocrinology (2000) 166, 677-687       DOI: 10.1677/joe.0.1660677
© 2000 Society for Endocrinology
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Journal of Endocrinology, Vol 166, Issue 3, 677-687
Copyright © 2000 by Society for Endocrinology

Articles

Changes in actin network during calcium-induced exocytosis in permeabilized GH3 cells: calcium directly regulates F-actin disassembly

M Yoneda, T Nishizaki, K Tasaka, H Kurachi, A Miyake, and Y Murata


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.


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