Possible roles of neuropeptide Y Y₃-receptor subtype in rat aortic endothelial cell proliferation under hypoxia, and its specific signal transduction

The present study was undertaken to determine whether neuropeptide Y (NPY) induces proliferation of rat aortic endothelial cells (RAECs). Since NPY increased the permeability of RAEC monolayers to large molecules via the NPY Y₃ receptor, RAEC proliferation has been evaluated in terms of NPY-receptor subtypes and also intracellular mechanisms. RAECs were incubated with gases containing 20, 15, or 10% O₂ and a certain amount of N₂, depending on the O₂ content in 5% CO₂ incubators. NPY (10^-9-10^-6 M) increased the RAEC numbers under hypoxic conditions, such as 15 or 10% O₂. Peptide YY elicited no proliferative effect on RAEC, and NPY-(18-36) inhibited the NPY-induced increase in cell number, suggesting that NPY increases the RAEC count through the NPY Y₃ receptor. Pertussis toxin, U-73122, GF-109203X, myristorylated autocamtide-2-related inhibitory peptide, and wort-mannin inhibited the NPY-induced proliferation of RAEC concentration dependently. DY9760e little affected the proliferation caused by NPY. ML-9 and imatinib actually enhanced the NPY-induced proliferation of cells. These results indicated that the NPY Y₃ receptor is coupled with Gi protein, and that NPY-induced increases in RAEC proliferation are mediated by phospholipase C-protein kinase C and/or phosphatidylinositol 3-kinase pathways. In intracellular Ca²⁺-calmodulin-dependent pathways, calmodulin-dependent protein kinase II partly participates in the NPY-induced cell proliferation. Regarding the previously reported effect of NPY on the permeability of RAEC monolayers to large molecules, it is probable that protein kinase C and phosphatidylinositol 3-kinase pathways are activated for both permeability and cell proliferation induced by NPY under hypoxia, relevant to new insights into the roles of NPY in ischemia-hypoxia.