Proteinase Activated Receptor Signalling, Vascular Function, and Hyperglycaemia

Abstract

Proteinase-activated receptors (PARs) are a unique subfamily of G-protein coupled receptors (GPCRs), activated by proteolytic processing of their N-terminus to reveal a tethered ligand. The expression of PARs is promiscuous and they play important roles in physiology and pathophysiology. PAR signalling is critical in the regulation of thrombosis, inflammation, and immune function. Diabetes mellitus is a complex metabolic condition characterized by hyperglycaemia, a major pathological factor in the development of diabetic complications. Most of these complications are vascular and involve oxidative stress/inflammatory signalling. We hypothesize that PARs regulate vascular function and that hyperglycaemia-induced changes will change PAR-mediated vascular regulation. Methods: Freshly isolated vascular rings from porcine coronary artery (PCA) and male mouse aorta (MMA) have been used to assess PARs function, while rings cultured in high glucose (HG) were used to study the effect of hyperglycaemia on PAR-mediated signalling. Both contractile and relaxant PAR-mediated responses were assessed in the presence of signal pathway inhibitors using a force displacement bioassay method. Activation of MAPKinase was examined by Western blotting. Results: Activation of PARs 1 & 2 in PCA and MMA induced concentration- and endothelium-dependent relaxations that are not affected by inhibitors of MAPKinase, tyrosine kinases, Rhokinase, or COX, and were entirely NO-mediated in MMA, but NO- and EDHF–dependent in the PCA. On the other hand, activation of PARs 1 & 2 induced endothelium-independent COX-dependent (except for PAR1 in MMA) contractions in both preparations. These contractions were inhibited by inhibitors of EGFR and Src kinases (PAR2 >PAR1 in PCA), MAPKinases, and Rho kinase (only PAR2 in PCA), and by PKC inhibitors (only slightly, PAR2 in PCA). Transactivation of tyrosine kinase signalling was independent of MMPs in the PCA. In addition, high glucose (HG) cultured MMA showed compromised PAR2-mediated relaxations. Catalase, SOD, VAS270 (NADPH oxidase inhibitor), and kinase inhibitors preserved PAR2-relaxation in HG, indicating a role of oxidative stress. Conclusions: PARs regulate vascular endothelial and smooth muscle functions via activation of multiple signalling pathways. Endothelial dysfunction, as a result of HG-induced oxidative stress was manifested as reduced PAR2-mediated relaxations. These results show relevant roles of PAR1 and PAR2 in vascular biology and pathology.