AY-NH2

PURPOSE: ;There is growing evidence for a role of proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, in cancer. We have previously shown that PAR1 and PAR4 are able to promote the migration of hepatocellular carcinoma (HCC) cells suggesting a function in HCC progression. In this study, we assessed the underlying signalling mechanisms.;METHODS: ;Using Hep3B liver carcinoma cells, RTK activation was assessed by Western blot employing phospho-RTK specific antibodies, ROS level were estimated by H2DCF-DA using confocal laser scanning microscopy, and measurement of PTP activity was performed in cell lysates using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as a substrate.;RESULTS: ;Thrombin, the PAR1 selective agonist peptide TFLLRN-NH2 (PAR1-AP), and the PAR4 selective agonist peptide, AYPGKF-NH2 (PAR4-AP), induced a significant increase in Hep3B cell migration that could be blocked by inhibitors targeting formation of reactive oxygen species (ROS), or activation of hepatocyte-growth factor receptor (Met), or platelet-derived growth factor receptor (PDGFR), respectively. The involvement of these intracellular effectors in PAR1/4-initiated migratory signalling was further supported by the findings that individual stimulation of Hep3B cells with the PAR1-AP and the PAR4-AP induced an increase in ROS production and the transactivation of Met and PDGFR.

Protease-activated receptor-1 (PAR1), PAR2 and PAR4 activation can alter the gastrointestinal motility. To investigate effects mediated by PARs in the lower esophageal sphincter, we measured contraction or relaxation of transverse strips from the guinea-pig lower esophageal sphincter caused by PAR1 (TFLLR-NH2 and SFLLRN-NH2), PAR2 (SLIGKV-NH2 and SLIGRL-NH2) and PAR4 peptide agonists (GYPGKF-NH2, GYPGQV-NH2 and AYPGKF-NH2) as well as PAR protease activators (thrombin and trypsin). In resting lower esophageal sphincter strips, TFLLR-NH2 and SFLLRN-NH2 caused moderate concentration-dependent relaxation whereas thrombin did not cause any relaxation or contraction. Furthermore, in carbachol-contracted strips, TFLLR-NH2 and SFLLRN-NH2 caused marked whereas thrombin caused mild concentration-dependent relaxation. These indicate the existence of PAR1 mediating relaxation. Similarly, in resting lower esophageal sphincter strips, trypsin caused moderate concentration-dependent relaxation whereas SLIGRL-NH2 and SLIGKV-NH2 did not cause any relaxation or contraction. In addition, in carbachol-contracted strips, trypsin caused marked whereas SLIGRL-NH2 and SLIGKV-NH2 caused mild concentration-dependent relaxation.

Proteinase-activated receptors (PARs) are activated by either serine proteinases or synthetic peptides corresponding to the NH2-terminal tethered ligand sequences that are unmasked by proteolytic cleavage. Although PARs are highly expressed in the kidney, their roles in regulating renal function are not known. In the present study, we evaluated the impact of PAR activation on renal hemodynamics using PAR1- and PAR2-activating peptides (TFLLR-NH2 and SLIGRL-NH2) and proteinases (thrombin and trypsin) as PAR agonists in the isolated perfused rat kidney preparation. PAR1 activation resulted in renal vasoconstriction and a marked reduction in the glomerular filtration rate (GFR). In contrast, PAR2 activation caused vasodilation, partially reversing the vasoconstriction induced by TFLLR-NH2 and ANG II and increasing GFR that had been prereduced by ANG II. The vasoconstrictor actions of PAR1 activation were abolished by protein kinase C inhibition. The PAR2-induced vasodilation was only partially blocked by NG-nitro-l-arginine methyl ester, suggesting both nitric oxide-dependent and -independent mechanisms. Although PAR4 mRNA was detected in renal parenchyma, the PAR4-activating peptide AYPGKF-NH2 had no effect on renal perfusion flow rate.