CHANGING J774A.1 CELLS TO NEW MEDIUM PERTURBS MULTIPLE SIGNALING PATHWAYS, INCLUDING THE MODULATION OF PROTEIN-KINASE-C BY ENDOGENOUS SPHINGOID BASES

Sphingosine, sphinganine, and other long-chain (sphingoid) bases are h ighly bioactive intermediates of sphingolipid metabolism that have div erse effects when added to cells, including the inhibition of protein kinase C (PRC) as evaluated by both enzymatic activity and [H-3]phorbo l dibutyrate ([H-3]PDBu) binding. Nonetheless, changes in endogenous s phingoid bases have not been proven to affect PKC or other signal tran sduction pathways. We have discovered recently that changing J774A.1 c ells to new medium results in up to 10-fold increases in sphingoid bas es (Smith, E, R., and Merrill, A. H., Jr, (1995) J. Biol. Chem. 270, 1 8749-18758); therefore, this system was used to elevate sphingosine an d sphinganine and determine if PEC was affected. Incubation of J774A.1 cells in new medium for 30 min increased the levels of these endogeno us sphingoid bases to approximately 0.5 nmol/mg of protein and decreas ed [H-3]PDBu binding by 40-60%. Addition of NH4Cl, which suppresses th e change in sphingosine, restored [3H]PDBu binding. Elevation of endog enous sphinganine by a second method (addition of fumonisin B-1, an in hibitor of ceramide synthase) also reduced [H-3]PDBu binding; therefor e, elevations in sphingosine and sphinganine can both affect PKC. The elevation in sphingoid bases was also associated with an increase in t he amount of PRC-delta (the major PKC isozyme in J774A.1 cells) in the cytosol, as determined by activity assays and immunoblot analyses, Ch anging the culture medium affected other PKC isozymes, increased cellu lar levels of diacylglycerol, dihydroceramide, and ceramide, and alter ed the expression of two genes (the expression of JE was increased, an d the induction of MnSOD by TNF-alpha was potentiated). Thus, changing the culture medium has numerous effects on J774A.1 cells, including t he modulation of PKC by endogenous sphingoid bases.