Inhibition of intracellular transport of newly synthesized prolactin by bafilomycin A1 in a pituitary tumor cell line, GH3 cells.

We have investigated the effects of bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, on the secretion and intracellular distribution of prolactin in cultured rat pituitary tumor cells (GH3 cells). Pulse-labeling the cells with L-[35S]methionine for 5 min and subsequently incubating in chase medium containing 1 microM bafilomycin A1 showed inhibition of basal secretion of labeled prolactin. The inhibition of the secretion by the drug was clearly observed when it was added within 7.5 min after the pulse-labeling, whereas inhibition was barely observed when added at 22 min. When the pulse-labeled cells were chased with 1 microM bafilomycin A1 for 1 h and then washed and incubated in the presence of 10 micrograms/ml brefeldin A (BFA) for 2 h, BFA barely affected the secretion during the latter 2 h period. This result suggested that the labeled prolactin had passed through a BFA-sensitive step(s) in the intracellular transport during the treatment with 1 microM bafilomycin A1. Immunofluorescence microscopy and immunogold electron microscopy revealed that, when the cells were incubated with 0.1 to 1.0 microM bafilomycin A1, small, dense secretory granules containing prolactin decreased markedly, and numerous large vacuoles appeared, which also contained prolactin, and were partially coated with clathrin-like materials. The Golgi apparatus itself was preserved except for some dilatation of the trans-Golgi cisternae and trans-Golgi network (TGN) where these vacuoles were likely to be formed. These results suggest that acidification in immature secretory granules generated by a vacuolar type H(+)-ATPase is necessary for the intracellular transport of prolactin, maturation and concentration processes of the secretory granules.