PHOSPHOLIPASE-D HYDROLYSIS OF CHOLINE PHOSPHOGLYCERIDES IS SELECTIVE FOR THE ALKYL-LINKED SUBCLASS OF MADIN-DARBY CANINE KIDNEY-CELLS

Madin-Darby canine kidney (MDCK) cells were used to study the synthesi s of diglycerides from choline phospholipids (PC) in response to 12-O- tetradecanoylphorbol-13-acetate (TPA). In this system, diglyceride for mation was blocked in the presence of ethanol (0.5%), and a correspond ing amount of phosphatidylethanol (PEt) was formed, indicating that ph ospholipase D is responsible for the diglyceride production. Analysis of the subclasses of phosphatidylethanol revealed 1-O-alkyl-(alkyl), 1 -O-alk-1'-enyl-(alkenyl), and 1-acyl species of PEt (38.0,8.3, and 53. 7%, respectively). The molecular species of the alkyl-PEt most closely matched the alkyl-PC. No change in the relative amounts of alkyl- ver sus acyl-PEt was observed with time after stimulation. Comparison of t he alkyl content of PEt (38.0%) and the parent PC (15.2%) indicated a marked selectivity for the alkyl subclass of PC. A cell-free assay (Hu ang, C., Wykle, R. L., Daniel, L. W., and Cabot, M. C. (1992) J. Biol. Chem. 267, 16859-16865) for phospholipase D was also used to confirm the selectivity of the enzyme for alkyl-PC versus acyl-PC. The predomi nant molecular species of PEt contained saturated acyl or alkyl chains in position-1 and monounsaturated residues in position-2 accounting f or approximately 50% of the total PEt. 1-O-Octadecyl-2-oleoyl-sn-glyce rol, a representative alkyl molecular species, was synthesized and tes ted for its effect upon protein kinase C derived from MDCK cells. This alkyl-diglyceride (DG) neither stimulated protein kinase C nor inhibi ted its activation by diacylglycerol. In summary, TPA-stimulated phosp holipase D is selective for the alkyl-PC subclass in MDCK cells. The a lkyl-DG subsequently formed does not appear to function as a second-me ssenger in activating protein kinase C.