title: AT1 receptor-mediated uptake of angiotensin II and NHE-3 expression in proximal tubule cells through a microtubule-dependent endocytic pathway.
abstract: Angiotensin II (ANG II) is taken up by proximal tubule (PT) cells via AT1 (AT1a) receptor-mediated endocytosis, but the underlying cellular mechanisms remain poorly understood. The present study tested the hypothesis that the microtubule- rather than the clathrin-dependent endocytic pathway regulates AT1-mediated uptake of ANG II and ANG II-induced sodium and hydrogen exchanger-3 (NHE-3) expression in PT cells. The expression of AT1 receptors, clathrin light (LC) and heavy chain (HC) proteins, and type 1 microtubule-associated proteins (MAPs; MAP-1A and MAP-1B) in PT cells were knocked down by their respective small interfering (si) RNAs before AT1-mediated FITC-ANG II uptake and ANG II-induced NHE-3 expression were studied. AT1 siRNAs inhibited AT1 expression and blocked ANG II-induced NHE-3 expression in PT cells, as expected (P < 0.01). Clathrin LC or HC siRNAs knocked down their respective proteins by approximately 90% with a peak response at 24 h, and blocked the clathrin-dependent uptake of Alexa Fluor 594-transferrin (P < 0.01). However, neither LC nor HC siRNAs inhibited AT1-mediated uptake of FITC-ANG II or affected ANG II-induced NHE-3 expression. MAP-1A or MAP-1B siRNAs markedly knocked down MAP-1A or MAP-1B proteins in a time-dependent manner with peak inhibitions at 48 h (>76.8%, P < 0.01). MAP protein knockdown resulted in approximately 52% decreases in AT1-mediated FITC-ANG II uptake and approximately 66% decreases in ANG II-induced NHE-3 expression (P < 0.01). These effects were associated with threefold decreases in ANG II-induced MAP kinases ERK 1/2 activation (P < 0.01), but not with altered AT1 expression or clathrin-dependent transferrin uptake. Both losartan and AT1a receptor deletion in mouse PT cells completely abolished the effects of MAP-1A knockdown on ANG II-induced NHE-3 expression and activation of MAP kinases ERK1/2. Our findings suggest that the alternative microtubule-dependent endocytic pathway, rather than the canonical clathrin-dependent pathway, plays an important role in AT1 (AT1a)-mediated uptake of extracellular ANG II and ANG II-induced NHE-3 expression in PT cells.
authors.affiliation: Laboratory of Receptor and Signal Transduction, Division of Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202, USA.
authors.forename: Xiao C
authors.initials: XC
authors.lastname: Li
authors.forename: Ulrich
authors.initials: U
authors.lastname: Hopfer
authors.forename: Jia L
authors.initials: JL
authors.lastname: Zhuo
mesh_terms: D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D000804:Angiotensin II / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002966:Clathrin / Q000096:biosynthesis; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000235:genetics; D004705:Endocytosis / Q000502:physiology*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D007211:Indoles; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D011817:Rabbits; D044140:Receptor, Angiotensin, Type 1 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000199:Actins; D002966:Clathrin; D003598:Cytoskeletal Proteins; D005456:Fluorescent Dyes; D007211:Indoles; D034741:RNA, Small Interfering; D044140:Receptor, Angiotensin, Type 1; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D000804:Angiotensin II; C007293:DAPI; D016650:Fluorescein-5-isothiocyanate