COMPLETE DISCRIMINATION OF DOCOSAHEXAENOATE FROM ARACHIDONATE BY 85 KDA CYTOSOLIC PHOSPHOLIPASE A(2) DURING THE HYDROLYSIS OF DIACYL AND ALKENYLACYLGLYCEROPHOSPHOETHANOLAMINE

In our previous report (Shikano, M., Masuzawa, Y. and Yazawa, K. (1993 ) J. Immunol. 150, 3525-3533), we described that the enrichment of doc osahexaenoic acid (DHA, 22:6(n - 3)) reduces both arachidonic acid (AA , 20:4(n - 6)) release and platelet-activating factor (PAF) synthesis in human eosinophilic leukemia cells, Eol-1. Since no DHA release was observed in response to Ca-ionophore stimulation, we presumed that the phospholipase A(2) (PLA(2)) responsible for AA release and PAF synthe sis can not hydrolyze the DHA moiety of phospholipids. In the present paper, we examined whether DHA-containing diacyl- and alkenylacylglyce rophosphoethanolamine (DHA-diacylGPE and DHA-alkenylacyGPE) are suscep tible to the action of AA-preferential 85 kDa cytosolic phospholipase A(2) (cPLA(2)) from rabbit platelets in comparison with AA and eicosap entaenoic acid (EPA, 20:5(n - 3)) derivatives. When diacylGPE was used as a substrate, DHA release was almost negligible under the assay con dition that allowed AA and EPA to be liberated at the rates of 4.3 mu mol/min per mg protein and 2.5 mu mol/min per mg protein, respectively . On the other hand, 14 kDa type II PLA, hydrolyzed DHA-diacylGPE as w ell as AA-diacylGPE and EPA-diacylGPE. When DHA-diacylGPE and AA-diacy lGPE were mixed at equimolar concentrations, DHA release by cPLA(2) wa s not observed and AA release was reduced to 32% in the case without D HA-diacylGPE. This indicated that DHA-diacylGPE is a poor substrate bu t possesses the inhibitory activity for cPLA(2). cPLA(2) does not clea rly discriminate between AA-alkenylacylGPE and AA-diacylGPE. As in the case using diacylGPE as a substrate, DHA-alkenylacylGPE was completel y discriminated from AA-alkenylacylGPE by cPLA(2). The roles of DHA an d cPLA(2) in the synthesis of lipid mediators will be discussed in rel ation to the new aspects of the substrate specificity of cPLA(2) provi ded here.