PAR2, not PAR1, regulates endothelium‐dependent vascular tone in resistance arteries

Protease activated receptors (PARs), a subfamily of G-protein-coupled receptors, are activated by cleavage of its extracellular domains. Four mammalian PAR members have been identified (PAR1-4). PAR1 is the thrombin receptor, while PAR2 is activated physiologically by peptides or N-terminal proteolytic cleavage. The resulting cleaved N-termini acts as a tethered ligand that initiates receptor signaling. PARs may regulate vascular tone, cell permeability and cell proliferation. However, the precise roles of PAR1 and PAR2 in regulating vascular tone via the endothelium, and how PAR-evoked signals transmit extracellularly and intracellularly, are incompletely understood. To explore the physiological functions, and the underlying signalling pathways in native endothelium, PAR1 (TFLLR-NH2 ) and PAR2 (2-Furoyl-LIGRLO-amide; 2fLI) activating peptides were used, and changes in intracellular Ca2+ and contractility measured in en face rat mesenteric arteries. Surprisingly, PAR1 activation (TFLLR-NH2 or trypsin) neither triggered Ca2+ signals in endothelial cells or alter vascular tone. On the other hand, PAR2 activation (2fLI) evoked large global propagating Ca2+ waves in endothelial cells and endothelium-dependent dilation of mesenteric arteries. The Ca2+ signals evoked by PAR2 activation were kinetically distinctive when compared to those of muscarinic receptor activation. PAR2 activation (2fLI) also evoked responses in different clusters of endothelial cells from those of muscarinic receptor (acetylcholine) activation. PAR2 activation with 2fLI or trypsin failed to evoke Ca2+ signals after depletion of the internal store with the Ca2+ -ATPase (SERCA) inhibitor, CPA. This result suggests that PAR2 evokes Ca2+ release from internal stores. Pharmacological block of inositol 1,4,5-trisphosphate (IP3 ) with either 2-APB or caffeine, also blocked PAR2-evoked Ca2+ signals. Phospholipase C inhibition (U73122) also blocked the Ca2+ signals evoked by PAR2 activation. Therefore, PAR2 is a PLC linked G protein coupled receptor which evokes Ca2+ release from the internal store via IP3 receptors in endothelial cells. The TRPC3 blocker, PYR3 and store-operated Ca2+ entry (SOCE) blocker, YM58483 each significantly supressed PAR2 mediated Ca2+ signals, suggesting internal store was replenished via TRPC3 channel and SOCE. In conclusion, PAR2 rather than PAR1, regulates vascular tone in mesenteric arteries by evoking Ca2+ release from the internal store via IP3 receptors to generate propagating Ca2+ waves in distinct sub-populations of endothelial cells.