Phospholipase A2-mediated activation of phospholipase D in rat heart sarcolemma.
The effect of phospholipase A2 (PLA2)-dependent release of unsaturated fatty acids (FA) on phospholipase D (PLD) function was examined in purified sarcolemmal (SL) membranes isolated from rat heart. PLD hydrolytic activity was determined by measuring either [14C] phosphatidic acid formation from exogenous [14C] phosphatidylcholine (PtdCho) or [3H] choline release from prelabelled SL Ptd[3H]choline. SL membranes with endogenous [3H] PtdCho that were prelabelled with [3H] myristic acid were used for testing PLD transphosphatidylation activity. Exogenous cis-unsaturated FA, arachidonate and oleate, significantly enhanced the [3H] choline formation at 50 and 100 microM, respectively; their effect was maximal at 250 microM and declined at higher concentrations. Use of melittin (which stimulates membrane-bound PLA2, thus releasing FA) or exogenous PLA2 reproduced the stimulatory effect of added arachidonate and oleate. Under melittin, PLA2-dependent FA release was strongly correlated (r = 0.99) to the PLD-dependent phosphatidic acid formation. Arachidonate- or melittin-enhanced PLD transphosphatidylation activity confirmed the augmented catalytic rate of PLD by these agents. Melittin-evoked PLD activation was completely blocked by 1 microM E-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, a selective inhibitor of Ca(2+)-independent v Ca(2-)-dependent PLA2, thus indicating that PLD stimulation under melittin occurred via PLA2. Activity measurement and Western blotting studies revealed the presence of a Ca(2+)-independent, high molecular weight (110 kDa) PLA2 in the SL membrane, and its immunoprecipitation by monoclonal antibodies significantly reduced the melittin-related PLD stimulation. These results suggest that Ca(2+)-independent PLA2 and subsequent endogenous mobilization of sn-2 unsaturated FA modulate PLD activity in heart SL membranes. This event may occur in physiological conditions via hormonal stimulation of membranal PLA2 as well as in heart diseases characterized by PLA2 pathological dysfunction.
Laboratory of Membrane Biology, St Boniface General Hospital Research Centre, Winnipeg, Canada.
S Y
SY
Liu
P S
PS
Tappia
J
J
Dai
S A
SA
Williams
V
V
Panagia
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D016718:Arachidonic Acid / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D008555:Melitten / Q000494:pharmacology; D009206:Myocardium / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D019301:Oleic Acid / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism / Q000493:pharmacokinetics; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010739:Phospholipase D / Q000187:drug effects / Q000378:metabolism*; D010741:Phospholipases A / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D054467:Phospholipases A2; D011753:Pyrones / Q000494:pharmacology; D051381:Rats; D012508:Sarcolemma / Q000187:drug effects / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D009281:Naphthalenes; D010713:Phosphatidylcholines; D010726:Phosphodiesterase Inhibitors; D011753:Pyrones; D008555:Melitten; D016718:Arachidonic Acid; D019301:Oleic Acid; D004533:Egtazic Acid; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; D010741:Phospholipases A; D054467:Phospholipases A2; D010739:Phospholipase D; D002118:Calcium