Expression and Regulation of P2 Receptors in Human Circulation

Sammanfattning: P2 receptors mediate the actions of the extracellular nucleotides ATP, ADP, UTP and UDP regulating several physiological responses including cardiac function, vascular tone, smooth muscle cell (SMC) proliferation, platelet aggregation, and the release of endothelial factors. The aim of the present study was to investigate the mRNA and protein expression of P2 receptors in human SMCs, endothelial cells (ECs), erythrocytes and platelets, the regulation of P2 receptors under shear stress in human vessels, the changes of platelet P2 receptors in patients with systemic lupus erythematosus (SLE) and the regulation of plasma ATP levels by P2 receptor on erythrocytes. We established methods to quantify P2 receptors in mRNA and protein levels in human SMCs, ECs, erythrocytes and platelets. Using a novel computerized biomechanical perfusion model, we examined how shear stress regulates P2 receptor expression in human umbilical vein SMCs and ECs. Using microdialysis we established a method to measure ATP demonstrating an important negative feedback system in the control of plasma ATP levels. Our results indicate that P2X1, P2Y2, P2Y12 and P2Y6 are the most expressed P2 receptors in SMCs and are thus probably mediating the contractile and mitogenic actions of extracellular nucleotides. The P2X4, P2Y11, P2Y1 and P2Y2 are the most expressed P2 receptors in ECs and are most likely mediating release of nitric oxide, endothelium dependent hyperpolarizing factor (EDHF) and t-PA induced by extracellular nucleotides. An oligomerisation may occur for the P2Y1 receptor. Shear stress may regulate gene expression in SMCs more than in the endothelium in intact vessels. Decreased expression of the contractile P2X1 receptor in high shear stress could lead to reduced vascular tonus and increased blood flow. Because P2Y2 and P2Y6 receptors stimulate growth and migration of SMCs, the increased expression of these receptors in high shear stress could promote vascular remodeling induced by shear stress. The upregulation of mitogenic P2Y receptors and downregulation of contractile P2X1 receptors in high shear stress is similar to changes seen in the phenotypic shift from a contractile to synthetic SMCs. The first protocol for quantifying mRNA expression in human platelets was developed limiting the P2 receptor drug development targets to P2Y12, P2Y1 and P2X1. Furthermore, the method could be used to study platelet expression for any gene in human materials. mRNA levels in platelets from patients with SLE was compared with controls. A decreased P2Y12 expression at mRNA and protein levels could represent a protective response in SLE against thrombotic complications. Quantifying mRNA expression in human erythrocytes demonstrated selective high expression of P2Y13 receptors. The ATP degradation product ADP activated P2Y13 receptors resulting in inhibition of ATP release. This negative feedback system could be important in the control of plasma ATP levels and tissue circulation.