PHOSPHOLIPASE A(2)-MEDIATED ACTIVATION OF PHOSPHOLIPASE-D IN RAT-HEART SARCOLEMMA

The effect of phospholipase A(2) (PLA(2))-dependent release of unsatur ated fatty acids (EA) on phospholipase D (PLD) function was examined i n purified sarcolemmal (SL) membranes isolated from rat heart. PLD hyd rolytic activity was determined by measuring either [C-14] phosphatidi c acid formation from exogenous [C-14] phosphatidylcholine (PtdCho) or [H-3] choline release from prelabelled SL Ptd[H-3]choline. SL membran es with endogenous [H-3] PtdCho that were prelabelled with [H-3] myris tic acid were used for testing PLD transphosphatidylation activity. Ex ogenous cis-unsaturated FA, arachidonate and oleate, significantly enh anced the [H-3] choline formation at 50 and 100 mu M, respectively; th eir effect was maximal at 250 mu M and declined at higher concentratio ns, Use of melittin (which stimulates membrane-bound PLA(2), thus rele asing FA) or exogenous PLA(2) reproduced the stimulatory effect of add ed arachidonate and oleate. Under melittin, PLA(2)-dependent FA releas e was strongly correlated (r = 0.99) to the PLD-dependent phosphatidic acid formation. Arachidonate- or melittin-enhanced PLD transphosphati dylation activity confirmed the augmented catalytic rate of PLD by the se agents. Melittin-evoked PLD activation was completely blocked by 1 mu M E-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyram-2-one , a selective inhibitor of Ca2+-independent nu Ca2+-dependent PLA(2), thus indicating that PLD stimulation under melittin occurred via PLA(2 ). Activity measurement and Western blotting studies revealed the pres ence of a Ca2+-independent, high molecular weight (110 kDa) PLA(2) in the SL membrane, and its immunoprecipitation by monoclonal antibodies significantly reduced the melittin-related PLD stimulation. These resu lts suggest that Ca2+-independent PLA(2) and subsequent endogenous mob ilization of sn-2 unsaturated FA modulate PLD activity in heart SL mem branes. This event may occur in physiological conditions via hormonal stimulation of membranal PLA(2) as well as in heart diseases character ized by PLA(2) pathological dysfunction. (C) 1998 Academic Press.