MODIFICATION OF ANNEXIN-II EXPRESSION IN PC12 CELL-LINES DOES NOT AFFECT CA2-DEPENDENT EXOCYTOSIS()

The Ca2+/phospholipd/cytoskeletal-binding protein annexin II has been proposed to play an important role in Ca2+-dependent exocytosis; howev er, the evidence for this role is inconclusive. More direct evidence o btained by manipulating annexin II levels in cells is still required. We have attempted to do this by generating stably transfected PC12 cel l lines expressing proteins which elevate or lower functional annexin II levels and using these cell lines to investigate Ca2+-dependent exo cytosis. Three cell lines were generated: one expressing an annexin II mutant which aggregates annexin II in at least a proportion of the ce lls, thereby removing functional protein from the cell; a mixed clonal cell line constitutively overexpressing human annexin II; and a clona l cell line capable of overexpressing annexin II in the presence of so dium butyrate. After digitonin permeabilization, Ca2+-dependent dopami ne release from these cell lines was compared with that from control n ontransfected cells, and, in addition, release was compared in induced to uninduced cells. There were no significant differences in Ca2+-dep endent exocytosis between any of the transfected cell lines before or after induction and the control cells. In addition, nontransfected PC1 2 cells treated with nerve growth factor, which elevates annexin II le vels severalfold, failed to increase Ca2+-dependent exocytosis after d igitonin permeabilization, compared with control cells. We conclude th at annexin II is not an important regulator of Ca2+-dependent exocytos is in PC12 cells.