title: Mechanisms of AT1a receptor-mediated uptake of angiotensin II by proximal tubule cells: a novel role of the multiligand endocytic receptor megalin.
abstract: The present study tested the hypothesis that the multiligand endocytic receptor megalin is partially involved in the uptake of ANG II and downstream signaling responses in mouse proximal tubule cells (mPCT) by interacting with AT1a receptors. mPCT cells of wild-type (WT) and AT1a receptor-deficient (AT1a-KO) mice were treated with vehicle, the AT1 receptor blocker losartan (10 μM), or a selective megalin small interfering (si) RNA for 48 h. The uptake of fluorescein (FITC)-labeled ANG II (10 nM, 37°C) and downstream signaling responses were analyzed by fluorescence imaging and Western blotting. AT1a receptors and megalin were abundantly expressed in mPCT cells, whereas AT1a receptors were absent in AT1a-KO mPCT cells (P < 0.01). In WT mPCT cells, FITC-ANG II uptake was visualized at 30 min in the cytoplasm and in the nuclei 1 h after exposure. Losartan alone completely blocked the uptake of FITC-ANG II, whereas megalin siRNA inhibited only 30% of the response (P < 0.01). The remaining FITC-ANG II uptake in the presence of megalin siRNA was completely abolished by losartan. ANG II induced threefold increases in phosphorylated MAP kinases ERK1/2 and a onefold increase in phosphorylated sodium and hydrogen exchanger 3 (NHE3) proteins, which were also blocked by losartan and megalin-siRNA. By contrast, losartan and megalin siRNA had no effects on these signaling proteins in AT1a-KO mPCT cells. We conclude that the uptake of ANG II and downstream MAP kinases ERK1/2 and NHE3 signaling responses in mPCT cells are mediated primarily by AT1a receptors. However, megalin may also play a partial role in these responses to ANG II.
authors.affiliation: Laboratory of Receptor and Signal Transduction, Department of Pharmacology and Toxicology, Cardiovascular and Renal Research Center, Division of Nephrology, Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi.
authors.forename: Xiao C
authors.initials: XC
authors.lastname: Li
authors.affiliation: Laboratory of Receptor and Signal Transduction, Department of Pharmacology and Toxicology, Cardiovascular and Renal Research Center, Division of Nephrology, Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi jzhuo@umc.edu.
authors.forename: Jia L
authors.initials: JL
authors.lastname: Zhuo
mesh_terms: D000804:Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism*; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism*; D008024:Ligands; D019808:Losartan / Q000494:pharmacology; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010766:Phosphorylation; D034622:RNA Interference; D044140:Receptor, Angiotensin, Type 1 / Q000172:deficiency / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D014162:Transfection
chemical_list: C544066:Agtr1a protein, mouse; D047228:Angiotensin II Type 1 Receptor Blockers; D008024:Ligands; D026561:Low Density Lipoprotein Receptor-Related Protein-2; C055127:Lrp2 protein, mouse; D044140:Receptor, Angiotensin, Type 1; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D000804:Angiotensin II; D048049:Extracellular Signal-Regulated MAP Kinases; D016650:Fluorescein-5-isothiocyanate; D019808:Losartan
keywords: AT1 receptor-mediated endocytosis; angiotensin II; kidney; megalin; proximal tubule
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