Changing J774A.1 cells to new medium perturbs multiple signaling pathways, including the modulation of protein kinase...

Sphingosine, sphinganine, and other long-chain (sphingoid) bases are highly bioactive intermediates of sphingolipid metabolism that have diverse effects when added to cells, including the inhibition of protein kinase C (PKC) as evaluated by both enzymatic activity and [3H]phorbol dibutyrate ([3H]PDBu) binding. Nonetheless, changes in endogenous sphingoid bases have not been proven to affect PKC or other signal transduction pathways. We have discovered recently that changing J774A.1 cells to new medium results in up to 10-fold increases in sphingoid bases (Smith, E. R., and Merrill, A. H., Jr. (1995) J. Biol. Chem. 270, 18749-18758); therefore, this system was used to elevate sphingosine and sphinganine and determine if PKC was affected. Incubation of J774A.1 cells in new medium for 30 min increased the levels of these endogenous sphingoid bases to approximately 0.5 nmol/mg of protein and decreased [3H]PDBu binding by 40-60%. Addition of NH4Cl, which suppresses the change in sphingosine, restored [3H]PDBu binding. Elevation of endogenous sphinganine by a second method (addition of fumonisin B1, an inhibitor of ceramide synthase) also reduced [3H]PDBu binding; therefore, elevations in sphingosine and sphinganine can both affect PKC. The elevation in sphingoid bases was also associated with an increase in the amount of PKC-delta (the major PKC isozyme in J774A. 1 cells) in the cytosol, as determined by activity assays and immunoblot analyses. Changing the culture medium affected other PKC isozymes, increased cellular levels of diacylglycerol, dihydroceramide, and ceramide, and altered the expression of two genes (the expression of JE was increased, and the induction of MnSOD by TNF-alpha was potentiated). Thus, changing the culture medium has numerous effects on J774A.1 cells, including the modulation of PKC by endogenous sphingoid bases.