Stimulation of protease activated receptors on RT4 cells mediates arachidonic acid release via Ca2+ independent phospholipase A2.
OBJECTIVE
Protease activated receptors (PAR) represent a family of G protein coupled receptors with 7 membrane spanning domains that are activated by proteolysis of the N-terminus of the receptor by serine proteases. The presence of multiple PARs on the same cell is thought to extend the range of proteases a cell responds to rather than expand the range of intracellular responses. We investigated arachidonic acid and prostaglandin E2 release in the human urothelial carcinoma cell line RT4 in response to stimulation with thrombin, which activates PAR-1, and tryptase, which activates PAR-2.


METHODS
RT4 cells were incubated with thrombin, tryptase or PAR agonist peptides and intracellular phospholipase A2 (PLA2) activity, arachidonic acid and prostaglandin E2 release were measured. Pretreatment with bromoenol lactone, a selective inhibitor for Ca2+ independent PLA2 (iPLA2), was also investigated.


RESULTS
Thrombin and tryptase stimulation resulted in a 2 to 3-fold increase in membrane associated iPLA2 that was accompanied by comparative increases in arachidonic acid and prostaglandin E2 release. These responses were also observed when synthetic peptides representing the tethered ligand for each receptor were incubated with RT4 cells. Arachidonic acid and prostaglandin E2 release, and iPLA2 activation were completely inhibited by pretreatment with bromoenol lactone.


CONCLUSIONS
Stimulating RT4 cells with PAR-1 or PAR-2 leads to the selective activation of iPLA2 as well as the release of arachidonic acid and prostaglandin E2, which may provide cytoprotection during an acute inflammatory reaction.
McHowat
J
J
Department of Pathology, Saint Louis University School of Medicine, St. Louis, Missouri, USA.
Creer
M H
MH
Rickard
A
A
D016718:Arachidonic Acid / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins; D002295:Carcinoma, Transitional Cell / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology; D015232:Dinoprostone / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015801:Helminth Proteins / Q000378:metabolism; D006801:Humans; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012697:Serine Endopeptidases / Q000494:pharmacology / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology; D053802:Tryptases; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000378:metabolism*; D019459:Urothelium / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D015801:Helminth Proteins; C087977:par-2 protein, C elegans; D016718:Arachidonic Acid; D017346:Protein Serine-Threonine Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D012697:Serine Endopeptidases; D013917:Thrombin; D053802:Tryptases; D019204:GTP-Binding Proteins; D015232:Dinoprostone