Endothelin-1 activates endothelial cell nitric-oxide synthase via heterotrimeric G-protein betagamma subunit signaling to protein jinase B/Akt.
Endothelin-1 has dual vasoactive effects, mediating vasoconstriction via ETA receptor activation of vascular smooth muscle cells and vasorelaxation via ETB receptor activation of endothelial cells. Although it is commonly accepted that endothelin-1 binding to endothelial cell ETB receptors stimulates nitric oxide (NO) synthesis and subsequent smooth muscle relaxation, the signaling pathways downstream of ETB receptor activation are unknown. Here, using a model in which we have utilized isolated primary endothelial cells, we demonstrate that ET-1 binding to sinusoidal endothelial cell ETB receptors led to increased protein kinase B/Akt phosphorylation, endothelial cell nitric-oxide synthase (eNOS) phosphorylation, and NO synthesis. Furthermore, eNOS activation was not dependent on tyrosine phosphorylation, and pretreatment of endothelial cells with pertussis toxin as well as overexpression of a dominant negative G-protein-coupled receptor kinase construct that sequesters betagamma subunits inhibited Akt phosphorylation and NO synthesis. Taken together, the data elucidate a G-protein-coupled receptor signaling pathway for ETB receptor-mediated NO production and call attention to the absolute requirement for heterotrimeric G-protein betagamma subunits in this cascade.
Liu
Songling
S
Duke University Liver Center, Duke University Medical Center, Durham, North Carolina 27710, USA.
Premont
Richard T
RT
Kontos
Christopher D
CD
Huang
Jianhua
J
Rockey
Don C
DC
D000818:Animals; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D042783:Endothelial Cells / Q000201:enzymology*; D019332:Endothelin-1 / Q000378:metabolism*; D004789:Enzyme Activation; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D017466:Receptors, Endothelin / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
D016895:Culture Media, Serum-Free; D019332:Endothelin-1; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D017466:Receptors, Endothelin; D014443:Tyrosine; C118981:G-protein Beta gamma; C494920:Akt1 protein, rat