Phospholipase A2-dependent and -independent pathways of arachidonate release from vascular smooth muscle cells☆

Abstract [Arg 8 ]vasopressin (AVP), through its V 1 receptor coupled to GTP-binding proteins, and aluminum fluoride (AlF 4 − ), which directly activates GTP-binding proteins, induced the release of [ 3 H]arachidonate from prelabeled A 7 r 5 vascular smooth muscle-like cells. Using fura-2-loaded cells, we observed that the release induced by AVP occurred concurrently with calcium (Ca 2+ ) mobilization from internal stores and entry of external Ca 2+ , whereas AlF 4 − -dependent arachidonate release was much slower and was not accompanied by intracellular Ca 2+ mobilization. Arachidonate transfer from phosphatidylcholine to phosphatidylethanolamine was an early event for both agonists, but phosphatidylinositol hydrolysis was an early event for AVP-stimulated cells and a late event for cells triggered with AlF 4 − . In addition, phospholipase inhibitors had no effect on arachidonate release induced by AlF 4 − . We investigated the enzymatic pathways involved in the releases of arachidonate, which occur in such different ways. Phospholipase A 2 activities were assayed in a cell-free system with various substrates, which made it possible to differentiate between cytosolic, secretory and Ca 2+ -independent phospholipases A 2 . THe specific activities were in the order alkenyl-AA-GPE > acyl-AA-GPE > acyl-AA-GPC in the presence of Ca 2+ . No significant activity was observed in the presence of Ca 2+ chelators and when dipalmitoyl-glycerophosphocholine was used as a substrate. Phospholipase 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. Imunodetected phosphorylated and unphosphorylated forms of cytosolic phospholipase A 2 (cPLA 2 ) also clearly increased in the membrane fractions of AVP-stimulated cells, and only the unphosphorylated form of cPLA 2 was present in AlF 4 − -triggered cells. We conclude that phospholipase C and translocation of cPLA 2 can account for arachidonate release with AVP stimulation, whereas neither phospholipase C nor any phospholipase A 2 activity appears to be implicated in AlF 4 − -dependent arachidonate release.