Phospholipase A2-dependent and -independent pathways of arachidonate release from vascular smooth muscle cells.
[Arg8]vasopressin (AVP), through its V1 receptor coupled to GTP-binding proteins, and aluminum fluoride (AlF4-), which directly activates GTP-binding proteins, induced the release of [3H]arachidonate from prelabeled A7r5 vascular smooth muscle-like cells. Using fura-2-loaded cells, we observed that the release induced by AVP occurred concurrently with calcium (Ca2+) mobilization from internal stores and entry of external Ca2+, whereas AlF4(-)-dependent arachidonate release was much slower and was not accompanied by intracellular Ca2+ 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 AlF4-. In addition, phospholipase inhibitors had no effect on arachidonate release induced by AlF4-. We investigated the enzymatic pathways involved in the releases of arachidonate, which occur in such different ways. Phospholipase A2 activities were assayed in a cell-free system with various substrates, which made it possible to differentiate between cytosolic, secretory and Ca2(+)-independent phospholipases A2. The specific activities were in the order alkenyl-AA-GPE > acyl-AA-GPE > acyl-AA-GPC in the presence of Ca2+. No significant activity was observed in the presence of Ca2+ chelators and when dipalmitoyl-glycerophosphocholine was used as a substrate. Phospholipase A2 activities did not change in homogenates from stimulated cells related to control cells. However, phospholipase A2 activity increased in membrane fractions from AVP-stimulated cells. Imunodetected phosphorylated and unphosphorylated forms of cytosolic phospholipase A2 (cPLA2) also clearly increased in the membrane fractions of AVP-stimulated cells, and only the unphosphorylated form of cPLA2 was present in AlF4(-)-triggered cells. We conclude that phospholipase C and translocation of cPLA2 can account for arachidonate release with AVP stimulation, whereas neither phospholipase C nor any phospholipase A2 activity appears to be implicated in AlF4(-)-dependent arachidonate release.
Cane
A
A
CNRS URA-1283, CHU Saint-Antione, Paris, France.
Breton
M
M
Béréziat
G
G
Colard
O
O
D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001127:Arginine Vasopressin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D005459:Fluorides / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010741:Phospholipases A / Q000502:physiology*; D054467:Phospholipases A2; D051381:Rats
D017607:Aluminum Compounds; D001127:Arginine Vasopressin; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D005459:Fluorides; D002118:Calcium; C032311:aluminum fluoride