Stimulation of protease activated receptors on RT4 cells mediates arachidonic acid release via CA²⁺ independent phospholipase A₂

Purpose: 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. Materials and 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.