Localization of Alkaline Phosphatase and Cathepsin D during Cell Restoration after Colchicine Treatment in Primary Cultures of Fetal Rat Hepatocytes

Localization of alkaline phosphatase (ALP) and cathepsin D (CAPD) in primary cultures of fetal rat hepatocytes was examined using double immunofluorescent staining in order to investigate the relationship between lysosome movement and the fate of ALP during cell restoration after microtubule disruption by colchicine. At 3 hr and 24 hr after colchicine treatment, numerous coarse dots containing ALP were observed throughout the cytoplasm, and some of these showed colocalization with CAPD. At 48 hr and 72 hr after colchicine treatment, although most of the dots containing ALP in the cytoplasm disappeared, dots containing CAPD remained. The present results suggest that the denatured ALP proteins remaining in the cytoplasm of hepatocytes during cell restoration after colchicine treatment are digested by lysosomes

Change in Localization of Alkaline Phosphatase and Mannosidase II by Colchicine Treatment of Primary Cultures of Fetal Rat Hepatocytes

We examined the changes in localization of alkaline phosphatase (ALP) and mannosidase II (man II), a Golgi marker, after colchicine treatment of primary cultures of fetal rat hepatocytes, using double immunofluorescence staining and confocal laser microscopy. In hepatocytes cultured in basal medium, ALP was localized in the perinuclear cytoplasm, and man II was observed in the Golgi region of the cytoplasm. When hepatocytes were cultured in dexamethasone-supplemented medium, ALP was also localized in the plasma membrane surrounding the bile canaliculus-like structure that was formed between adjacent cells. In hepatocytes cultured in the same medium containing colchicine, the structure of microtubules in the cytoplasm was lost, man II exhibited granular distribution scattering throughout the cytoplasm, and ALP was localized in coarse granular sites of the cytoplasm. However, ALP was not colocalized at the same sites as man II. The present study indicated that colchicine inhibits the dexamethasone-promoted translocation of ALP to the plasma membrane surrounding the bile canaliculus-like structure in primary cultures of fetal rat hepatocytes by disassembling microtubules and discomposing the Golgi complex