Influence of protein kinase C activation by 4 beta-phorbol ester or 1-oleoyl-2-acetylglycerol on disaturated phosphatidylcholine synthesis and secretion, and protein phosphorylation in differentiating fetal rabbit type II alveolar cells

Abstract

Undifferentiated type II alveolar cells were isolated from the fetal rabbit lung on the 24th gestational day, grown in vitro for 2-3 days, and used to test the hypothesis that activation of protein kinase C by 4 beta-phorbol ester (TPA) or the diacylglycerol analogue, sn-1-oleoyl-2-acetylglycerol (OAG), stimulates disaturated phosphatidylcholine (DSPC) synthesis and secretion. To measure secretion, cells were prelabelled with [3H]choline in serum-free medium or medium with 10% carbon-stripped fetal bovine serum for 24 h. The radiolabel was removed and TPA (10(-6)-10(-9) M) or OAG (125, 250 or 500 microM) was incubated with the cells for 2 h. The medium was removed and filtered. Fresh medium with the same compound was added for an additional 16 h. To measure synthesis, cells were incubated with [3H]choline and concurrently TPA or OAG was added. Cells were removed at 2 or 18 h. After 2 h at concentrations of 10(-8) M, TPA augmented the release of 3H-labelled DSPC, the major component of the surfactant, by cells incubated in serum-free medium. In the presence of carbon-stripped fetal bovine serum, TPA (10(-7) and 10(-6) M) induced release of DSPC. The incorporation of [3H]choline into intracellular DSPC was increased after 2 or 18 h in fetal alveolar cells exposed to TPA at 10(-9) M or higher. OAG also significantly significantly stimulated the release of labelled DSPC after 2 h at all concentrations tested. In contrast, OAG-exposed cells displayed a reduction of [3H]choline incorporation into cellular DSPC. Characterization of radioactive material released by prelabelled fetal type II cells showed that phorbol ester stimulation increased the release of material which co-migrated with adult rabbit lung lamellar bodies on a sucrose gradient. Electrophoretic examination of [gamma-32P]ATP phosphorylation sites in fetal type II cells cells showed that TPA and OAG induced an increase in phosphorylation of a group of proteins with apparent molecular masses of 45, 50 and 55 kDa. Addition of phosphatidylserine to the incubations produced substantial increase in the phosphorylation of these proteins, particularly in the presence of TPA. Fetal type II cells also displayed a phosphorylation product with an apparent molecular mass of 97 kDa. This protein as well as two high-molecular-mass products appeared to be particular to cells incubated with TPA plus phosphatidylserine and may in part account for the different action of TPA compared to OAG with regard to synthesis and secretion of DSPC by the fetal type II cells.