EVIDENCE THAT CERAMIDE SELECTIVELY INHIBITS PROTEIN-KINASE C-ALPHA TRANSLOCATION AND MODULATES BRADYKININ ACTIVATION OF PHOSPHOLIPASE-D

Sphingomyelinase (SMase) treatment (0.1 unit/ml for up to 30 min) of mouse epidermal (HEL 37) or human skin fibroblast (SF 3155) cells preincubated with [H-3]serine to label the sphingomyelin pool caused the accumulation of labeled ceramide but not sphingosine or ceramide 1-phosphate. Incubation of HEL-37 cells with dioctanoylglycerol (diC(8)) or SF 3155 cells with bradykinin caused translocation of calcium/phosphatidylserine-dependent protein kinase C (PKC) activity to particulate material. In both cell lines the translocation was blocked by SMase treatment of the cells or by incubation with the cell-permeable ceramide analogue N-acetylsphingosine (C-2-Cer). Western blot analysis indicated that treatment of HEL-37 cells with diC(8) or SF 3155 cells with bradykinin resulted in the translocation of both PKC-alpha and PKC-epsilon to particulate material. Treatment with SMase or C-2-Cer specifically blocked the translocation of PKC-alpha but not that of PKC-epsilon. Pretreatment of cells with SMase or C-2-Cer also inhibited the activation of phospholipase D activity induced by either diC(8) (HEL-37 cells) or bradykinin (SF 3155 cells). The data provide strong evidence that ceramide can negatively regulate the translocation of PKC-alpha but not PKC-epsilon and further suggest that PKC-alpha may be involved in regulating phospholipase D activity.