Increased mitogenic and decreased contractile P2 receptors in smooth muscle cells by shear stress in human vessels with intact endothelium.

OBJECTIVE: We investigated the role of shear stress in regulating P2 receptors in human umbilical vein. METHODS AND RESULTS: Using a novel, computerized, biomechanical perfusion model, parallel vessel segments were randomized to simultaneous perfusion under high (25 dyn/cm2) or low (<4 dyn/cm2) shear stress at identical mean perfusion pressure (20 mm Hg) for 6 hours. In the endothelium, no significant P2 receptor mRNA differences were found. In smooth muscle cells (SMCs), high shear stress decreased P2X1 receptors, whereas P2Y2 and P2Y6 receptors were upregulated. These findings were consistent at the mRNA level (real-time reverse transcription-polymerase chain reaction), protein level (Western blot), and morphologically (immunohistochemistry). The changes were more pronounced in the subintimal layer of the media. CONCLUSIONS: Our findings suggest that shear stress might regulate gene expression in SMCs more than in the endothelium in intact vessels. Decreased expression of the contractile P2X1 receptor could lead to reduced vascular tonus and increased blood flow. Because P2Y2 and P2Y6 receptors stimulate growth and migration of SMCs, increased expression of these receptors could promote vascular remodeling induced by shear stress. The pattern of upregulation of mitogenic P2Y receptors and downregulation of contractile P2X1 receptor is similar to changes seen in the phenotypic shift from contractile to synthetic SMCs.

Important amino acids for the function of the human MT1 melatonin receptor.

Models of G protein-coupled melatonin receptor structure suggest that ligand recognition occurs in a binding pocket formed by transmembrane helices III, V and VII. Constitutively active mutations in G protein-coupled receptors have revealed that transmembrane helix III/intracellular loop 2 interface and transmembrane domain VI are critical regions in receptor activation. In this study, nine site-directed mutants of the human MT1 melatonin receptor were created to test the importance of specific amino acids in these regions in ligand recognition and receptor activation events. We analyzed ligand binding, G protein activation and subcellular localization of MT1 receptors transiently expressed in COS-7 cells. Receptor ELISA was employed to study expression levels of N-terminally HA epitope tagged wild-type and mutant MT1 receptors. Mutations in histidine H195 (His(5.46)) in transmembrane domain V reduced receptor affinity for 2-[125I]iodomelatonin. Several other mutants had diminished expression on the plasma membrane. Amino acids M107 (Met(3.32)) in transmembrane domain III and S280 (Ser(7.46)) in transmembrane domain VII were found not to participate in ligand recognition in human MT1 receptor. Constitutive activity was not obtained with mutations in N124 (Asn(3.49)) or P253 (Pro(6.50)). These mutants failed to bind 2-[125I]iodomelatonin and had reduced expression levels. The need to upgrade current melatonin receptor models has become evident. Several important amino acids for the human MT1 melatonin receptor function were revealed in the current study, with effects of mutations ranging from slightly reduced affinity or efficacy to complete loss of function.