PHOSPHOLIPASE A2-DEPENDENT AND A2-INDEPENDENT PATHWAYS OF ARACHIDONATE RELEASE FROM VASCULAR SMOOTH-MUSCLE CELLS

[Arg(8)]vasopressin (AVP), through its V-1 receptor coupled to GTP-bin ding proteins, and aluminum fluoride (AlF4-), which directly activates GTP-binding proteins, induced the release of [H-3]arachidonate from p relabeled A(7)r(5) vascular smooth muscle-like cells. Using fura-2-loa ded cells, we observed that the release induced by AVP occurred concur rently with calcium (Ca2+) mobilization from internal stores and entry of external Ca2+, whereas AlF4--dependent arachidonate release was mu ch slower and was not accompanied by intracellular Ca2+ mobilization. Arachidonate transfer from phosphatidylcholine to phosphatidyl ethanol amine was an early event for both agonists, but phosphatidylinositol h ydrolysis was an early event for AVP-stimulated cells and a late event for cells triggered with AlF4-. In addition, phospholipase inhibitors had no effect on arachidonate release induced by AlF4-. We investigat ed the enzymatic pathways involved in the releases of arachidonate, wh ich occur in such different ways. Phospholipase A(2) activities were a ssayed in a cell-free system with various substrates, which made it po ssible to differentiate between cytosolic, secretory and Ca2+-independ ent phospholipases A(2). THe specific activities were in the order alk enyl-AA-GPE > acyl-AA-GPE > acyl-PIA-GPC in the presence of Ca2+. No s ignificant activity was observed in the presence of Ca2+-chelators and when dipalmitoyl-glycerophosphocholine was used as a substrate. Phosp holipase A(2) activities did not change in homogenates from stimulated cells related to control cells. However, phospholipase A(2) activity increased in membrane fractions from AVP-stimulated cells. Imunodetect ed phosphorylated and unphosphorylated forms of cytosolic phospholipas e A(2) (cPLA(2)) also clearly increased in the membrane fractions of A VP-stimulated cells, and only the unphosphorylated form of cPLA(2) was present in AlF4--triggered cells. We conclude that phospholipase C an d translocation of cPLA(2) can account for arachidonate release with A VP stimulation, whereas neither phospholipase C nor any phospholipase A(2) activity appears to be implicated in AlF4--dependent arachidonate release. Copyright (C) 1997 Elsevier Science Inc.