FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2044612296> ?p ?o ?g. }

• W2044612296 endingPage "26915" @default.
• W2044612296 startingPage "26906" @default.
• W2044612296 abstract "Dopamine (DA) is a key hormone in mammalian sodium homeostasis. DA induces natriuresis via acute inhibition of the renal proximal tubule apical membrane Na+/H+ exchanger NHE3. We examined the mechanism by which DA inhibits NHE3 in a renal cell line. DA acutely decreases surface NHE3 antigen in dose- and time-dependent fashion without altering total cellular NHE3. Although DA1 receptor agonist alone decreases surface NHE3, simultaneous DA2 agonist synergistically enhances the effect of DA1. Decreased surface NHE3 antigen, caused by stimulation of NHE3 endocytosis, is dependent on intact functioning of the GTPase dynamin and involves increased binding of NHE3 to the adaptor protein AP2. DA-stimulated NHE3 endocytosis can be blocked by pharmacologic or genetic protein kinase A inhibition or by mutation of two protein kinase A target serines (Ser-560 and Ser-613) on NHE3. We conclude that one mechanism by which DA induces natriuresis is via protein kinase A-mediated phosphorylation of proximal tubule NHE3 leading to endocytosis of NHE3 via clathrin-coated vesicles. Dopamine (DA) is a key hormone in mammalian sodium homeostasis. DA induces natriuresis via acute inhibition of the renal proximal tubule apical membrane Na+/H+ exchanger NHE3. We examined the mechanism by which DA inhibits NHE3 in a renal cell line. DA acutely decreases surface NHE3 antigen in dose- and time-dependent fashion without altering total cellular NHE3. Although DA1 receptor agonist alone decreases surface NHE3, simultaneous DA2 agonist synergistically enhances the effect of DA1. Decreased surface NHE3 antigen, caused by stimulation of NHE3 endocytosis, is dependent on intact functioning of the GTPase dynamin and involves increased binding of NHE3 to the adaptor protein AP2. DA-stimulated NHE3 endocytosis can be blocked by pharmacologic or genetic protein kinase A inhibition or by mutation of two protein kinase A target serines (Ser-560 and Ser-613) on NHE3. We conclude that one mechanism by which DA induces natriuresis is via protein kinase A-mediated phosphorylation of proximal tubule NHE3 leading to endocytosis of NHE3 via clathrin-coated vesicles. dopamine Na+/H+ exchanger protein kinase A opossum kidney hexahistidine enhanced green fluorescent protein wild type cAMP binding-defective regulatory subunit of protein kinase A phosphate-buffered saline 4-morpholineethanesulfonic acid Tris(2-carboxyethyl)phosphine hydrochloride adaptor protein 2 analysis of variance Extracellular fluid volume and to a certain extent blood pressure in mammals are determined by the balance between sodium intake and renal sodium excretion (1Hollenberg N.K. Kidney Int. 1980; 17: 423-429Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 2Simpson F.O. Lancet. 1988; 2: 25-29Abstract PubMed Scopus (59) Google Scholar). As regulator of sodium excretion, the intrarenal autocrine-paracrine dopamine (DA)1 system assumes far greater importance than circulating endocrine or neurogenic dopamine (3Ball S.G. Lee M.R. Br. J. Clin. Pharmacol. 1977; 4: 115-118Crossref PubMed Scopus (103) Google Scholar, 4Jose P.A. Felder R.A. Holloway R.R. Eisner G.M. Am. J. Physiol. 1986; 250: F1033-F1038PubMed Google Scholar, 5Jose P.A. Jolloway R.R. Campbell T.W. Eisner G.M. Nephron. 1988; 48: 54-57Crossref PubMed Scopus (3) Google Scholar, 6Siragy H.M. Felder R.A. Howell N.L. Chevalier R.L. Peach M.J. Carey R.M. Am. J. Physiol. 1989; 257: F469-F477PubMed Google Scholar). DA is produced in the proximal tubule via decarboxylation of its precursor l-dihydroxyphenylalanine derived from the plasma and glomerular filtrate (7Chan Y.L. J. Pharmacol. Exp. Ther. 1976; 199: 17-24PubMed Google Scholar, 8Baines A.D. Chan W. Life Sci. 1980; 26: 253-259Crossref PubMed Scopus (141) Google Scholar, 9Hayashi M. Yamaji Y. Kitajima W. Saruta T. Am. J. Physiol. 1990; 258: F28-F33PubMed Google Scholar) and is then secreted into the tubular lumen where it exerts its effects on multiple nephron segments, which cumulates in inhibition of tubular sodium absorption and natriuresis. Renal DA synthesis and excretion are increased by increased dietary salt and an intravenous saline load (10Alexander R.W. Gill J.R. Tamabe H. Lovernberg W. Keiser H.R. J. Clin. Invest. 1974; 54: 194-200Crossref PubMed Scopus (245) Google Scholar, 11Sowers J.R. Crane P.D. Beck F.W. McClanahan M. King M.E. Mohanty P.K. Endocrinology. 1984; 115: 2085-2090Crossref PubMed Scopus (64) Google Scholar, 12Goldstein D.S. Stull R. Eisenhofer G. Gill Jr., J.R. J. Clin. Sci. 1989; 76: 517-522Crossref PubMed Scopus (68) Google Scholar, 13Hayashi M. Yamaji Y. Kitajima W. Saruta T. Am. J. Physiol. 1991; 260: E675-E679PubMed Google Scholar), and blockade of DA synthesis or DA receptor significantly blunts the natriuretic response (14Pelayo J.C. Fildes R.D. Eisner G.M. Jose P.A. Am. J. Physiol. 1983; 245: F247-F253PubMed Google Scholar, 15Krishna G.G. Danovitch G.M. Beck F.W. Sowers J.R. J. Lab. Clin. Med. 1985; 105: 214-218PubMed Google Scholar, 16Corruzi P. Biggi A. Musiari L. Ravanetti C. Vescovi P.P. Novarini A. Clin. Sci. (Lond.). 1986; 70: 523-526Crossref PubMed Scopus (19) Google Scholar, 17Hedge S.S. Jadhav A.L. Lokhandwala M.F. Hypertension. 1989; 13: 828-834Crossref PubMed Scopus (98) Google Scholar, 18Hansell P. Fasching A. Kidney Int. 1991; 39: 253-258Abstract Full Text PDF PubMed Scopus (87) Google Scholar). Quantitatively, the most significant inhibition of sodium transport occurs in the proximal tubule. DA inhibits proximal tubule sodium absorption partially by hemodynamic alterations (19McDonald R.H. Goldberg L.I. McNay J.L. Tuttle E.P. J. Clin. Invest. 1964; 43: 1116-1124Crossref PubMed Scopus (392) Google Scholar, 20Davis B.B. Walter M.J. Murdaugh H.V. Proc. Soc. Exp. Biol. Med. 1968; 129: 210-213Crossref PubMed Scopus (55) Google Scholar, 21Hollenberg N.K. Adams D.F. Mendell P. Abrams H.L. Merrill J. Clin. Sci. Mol. Med. 1973; 45: 733-742PubMed Google Scholar, 22Chapman B.J. Horn N.M. Munday K.A. Robertson M.J. J. Physiol. (Lond.). 1980; 298: 437-452Crossref Scopus (54) Google Scholar), but the major effect is directly on the tubule epithelium (23Bello-Reuss E. Pastoraiza-Munoz E. Colindres R.E. Am. J. Physiol. 1977; 232: F26-F32PubMed Google Scholar, 24Laradi A. Sakhrani L.M. Massry S.G. Miner. Electrolyte Metab. 1986; 12: 303-307PubMed Google Scholar, 25Hagiwara N. Kubota T. Kubokawa M. Fujimoto M. Jpn. J. Physiol. 1990; 40: 351-368Crossref PubMed Scopus (11) Google Scholar, 26Baum M. Quigley R. Kidney Int. 1998; 54: 1593-1600Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) via inhibition of two principal sodium transporters: the apical membrane Na+/K+exchanger (NHE3) (27Felder C.C. Campbell T. Albrecht F. Jose P. Am. J. Physiol. 1990; 259: F297-F303PubMed Google Scholar, 28Gesek F. Schoolworth A. Am. J. Physiol. 1990; 258: F514-F521PubMed Google Scholar, 29Winaver J. Burnett J.C. Tyce G.M. Dousa T.P. Kidney Int. 1990; 38: 1133-1140Abstract Full Text PDF PubMed Scopus (65) Google Scholar, 30Jadhav A.L. Liu Q. Clin. Exp. Hypertens. [A]. 1992; 14: 653-666PubMed Google Scholar, 31Felder C.C. Albrecht F.E. Campbell T. Eisner G.M. Jose P.A. Am. J. Physiol. 1993; 263: F1031-F1037Google Scholar, 32Sheikh-Hamad D. Wang Y.P. Jo O.D. Yanagawa N. Am. J. Physiol. 1993; 264: F737-F743PubMed Google Scholar, 33Wiederkehr M.R. Di Sole F. Fan L. Hu M.C. Collazo R. Murer H. Helmle-Kolb C. Moe O.W. Kidney Int. 2001; 59: 197-209Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and the basolateral Na+,K+-ATPase (34Aperia A. Bertorello A. Seri I. Am. J. Physiol. 1987; 252: F39-F45PubMed Google Scholar, 35Bertorello A.M. Hopfield J.F. Aperia A. Greengard P. Am. J. Physiol. 1988; 254: F795-F801PubMed Google Scholar, 36Bertorello A. Aperia A. Acta Physiol. Scand. 1988; 132: 441-443Crossref PubMed Scopus (33) Google Scholar, 37Bertorello A. Aperia A. Am. J. Physiol. 1990; 259: F924-F928PubMed Google Scholar, 38Bertorello A. Aperia A. Am. J. Hypertens. 1990; 3: 51S-54SCrossref PubMed Google Scholar). These effects are mediated by the DA receptor where five molecular isoforms (DR1-like receptors: DR1 and DR5, and DR2-like receptors DR2, DR3, and DR4) have been identified to date; all five isoforms are known to be present in the renal tubular epithelium (39Felder C.C. McKelvey A.M. Gitler M.S. Eisner G.M. Jose P.A. Kidney Int. 1989; 36: 183-193Abstract Full Text PDF PubMed Scopus (90) Google Scholar, 40Takemoto F. Satoh T. Cohen H.T. Katz A.I. Pflügers Arch. Eur. J. Physiol. 1991; 419: 243-248Crossref PubMed Scopus (32) Google Scholar, 41Lokhandwala M.F. Amenta F. FASEB J. 1991; 5: 3023-3030Crossref PubMed Scopus (144) Google Scholar, 42Jose P.A. Raymond J.R. Bates M.D. Aperia A. Felder R.A. Carey R.M. J. Am. Soc. Nephrol. 1992; 12: 1265-1278Google Scholar, 43Amenta F. Clin. Exp. Hypertens. 1997; 19: 27-41Crossref PubMed Scopus (43) Google Scholar). Previous studies in isolated apical membrane vesicles have shown that DA inhibits proximal tubule apical membrane Na+/H+ exchange activity mainly via DR1-like receptors (25Hagiwara N. Kubota T. Kubokawa M. Fujimoto M. Jpn. J. Physiol. 1990; 40: 351-368Crossref PubMed Scopus (11) Google Scholar, 26Baum M. Quigley R. Kidney Int. 1998; 54: 1593-1600Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 27Felder C.C. Campbell T. Albrecht F. Jose P. Am. J. Physiol. 1990; 259: F297-F303PubMed Google Scholar, 28Gesek F. Schoolworth A. Am. J. Physiol. 1990; 258: F514-F521PubMed Google Scholar, 30Jadhav A.L. Liu Q. Clin. Exp. Hypertens. [A]. 1992; 14: 653-666PubMed Google Scholar, 31Felder C.C. Albrecht F.E. Campbell T. Eisner G.M. Jose P.A. Am. J. Physiol. 1993; 263: F1031-F1037Google Scholar, 33Wiederkehr M.R. Di Sole F. Fan L. Hu M.C. Collazo R. Murer H. Helmle-Kolb C. Moe O.W. Kidney Int. 2001; 59: 197-209Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) through both PKA-dependent and PKA-independent mechanisms (27Felder C.C. Campbell T. Albrecht F. Jose P. Am. J. Physiol. 1990; 259: F297-F303PubMed Google Scholar, 31Felder C.C. Albrecht F.E. Campbell T. Eisner G.M. Jose P.A. Am. J. Physiol. 1993; 263: F1031-F1037Google Scholar). The Na+/H+ exchanger on the apical membrane of the renal proximal tubule is encoded by NHE3 (44Biemesderfer D. Pizzonia J. Abu-Alfa A. Exner M. Reilly R. Igarashi P. Aronson P.S. Am. J. Physiol. 1993; 265: F736-F742PubMed Google Scholar, 45Amemiya M. Loffing J. Lötscher M. Kaissling B. Alpern R.J. Moe O.W. Kidney Int. 1995; 48: 1206-1215Abstract Full Text PDF PubMed Scopus (355) Google Scholar, 46Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar), one of the seven members of the NHE gene family (47Wakabayashi S. Shigekawa M. Pouyssegur J. Physiol. Rev. 1997; 77: 51-74Crossref PubMed Scopus (562) Google Scholar). We have shown in opossum kidney (OK) cells that the DA1-like and DA2-like receptors have synergistic actions on NHE3 activity and that inhibition of NHE3 activity by DA is accompanied by complex changes in NHE3 phosphorylation and dephosphorylation (33Wiederkehr M.R. Di Sole F. Fan L. Hu M.C. Collazo R. Murer H. Helmle-Kolb C. Moe O.W. Kidney Int. 2001; 59: 197-209Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). However, the mechanisms by which DA acutely reduces NHE3 activity have not been examined. Redistribution of NHE3 transporters has been shown to mediate regulation of NHE3 activity in intact kidney (48Zhang Y. Mircheff A.K. Hensley C.B. Makyar C.E. Warnock D.G. Chambrey R. Yip K.P. Marsh D.J. Holstein-Rathlou N.H. McDonough A.A. Am. J. Physiol. 1996; 270: F1004-F1014Crossref PubMed Google Scholar, 49Zhang Y. Magyar C.E. Norian J.M. Holstein-Rathlou N.H. Mircheff A.K. McDonough A.A. Am. J. Physiol. 1998; 274: C1090-C1100Crossref PubMed Google Scholar, 50Zhang Y.B. Magyar C.E. Holstein-Rathlou N.H. McDonough A.A. J. Am. Soc. Nephrol. 1998; 9: 531-537PubMed Google Scholar, 51Yip K.P. Tse C.M. McDonough A.A. Marsh D.J. Am. J. Physiol. 1998; 275: F565-F575PubMed Google Scholar, 52Fan L. Wiederkehr M.R. Collazo R. Huang H. Crowder L.A. Moe O.W. J. Biol. Chem. 1999; 274: 11289-11295Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 53Zhang Y. Norian J.M. Magyar C.E. Holstein-Rathlou N.H. Mircheff A.K. McDonough A.A. Am. J. Physiol. 1999; 276: F711-719Crossref PubMed Google Scholar, 54Magyar C.E. Zhang Y. Holstein-Rathlou N.H. McDonough A.A. Am. J. Physiol. 2000; 279: F358-F369PubMed Google Scholar), in cultured renal epithelial cells (55Yang X. Amemiya M. Peng Y. Moe O.W. Preisig P.A. Alpern R.J. Am. J. Physiol. 2000; 279: C410-C419Crossref PubMed Google Scholar, 56Collazo R. Fan L. Hu M.C. Zhao H. Wiederkehr M.R. Moe O.W. J. Biol. Chem. 2000; 275: 31601-31608Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), and in transfected fibroblasts (57D'Souza S. Garcia-Cabado A., Yu, F. Teter K. Lukacs G. Skorecki K. Moore H.P. Orlowski J. Grinstein S. J. Biol. Chem. 1998; 273: 2035-2043Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 58Kurashima K. Szabó E.Z. Lukacs G. Orlowski J. Grinstein S. J. Biol. Chem. 1998; 273: 20828-20836Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 59Janecki A.J. Montrose M.H. Zimniak P. Zweibaum A. Tse C.M. Khurana S. Donowitz M. J. Biol. Chem. 1998; 273: 8790-8798Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 60Kurashima K. D'Souza S. Szaszi K. Ramjeesingh R. Orlowski J. Grinstein S. J. Biol. Chem. 1999; 274: 29843-29849Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 61Chow C.W. Khurana S. Woodside M. Grinstein S. Orlowski J. J. Biol. Chem. 1999; 274: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 62Janecki A.J. Janecki M. Akhter S. Donowitz M. J. Biol. Chem. 2000; 275: 8133-8142Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 63Akhter S. Cavet M.E. Tse C.M. Donowitz M. Biochemistry. 2000; 39: 1990-2000Crossref PubMed Scopus (50) Google Scholar, 64Szaszi K. Kurashima K. Kapus A. Paulsen A. Kaibuchi K. Grinstein S. Orlowski J. J. Biol. Chem. 2000; 275: 28599-28606Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In addition, although NHE phosphorylation has been associated with changes in NHE3 activity (52Fan L. Wiederkehr M.R. Collazo R. Huang H. Crowder L.A. Moe O.W. J. Biol. Chem. 1999; 274: 11289-11295Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 65Kurashima K., Yu, F.H. Cabado A.G. Szabó E.Z. Grinstein S. Orlowski J. J. Biol. Chem. 1997; 272: 28672-28679Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 66Zhao H. Wiederkehr M.R. Collazo R. Fan L. Crowder L.A. Moe O.W. J. Biol. Chem. 1999; 274: 3978-3987Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 67Wiederkehr M.R. Zhao H. Moe O.W. Am. J. Physiol. 1999; 276: C1205-C1217Crossref PubMed Google Scholar, 68Peng, Y., Moe, O. W., Chu, T. S., Preisig, P. A., Yanagisawa, M., and Alpern, R. J. (1999) Am. J. Physiol. C938–C945.Google Scholar), and phosphorylation appears to be functionally important for regulation of NHE3 activity by pharmacologic activators of protein kinases in transfected fibroblasts (65Kurashima K., Yu, F.H. Cabado A.G. Szabó E.Z. Grinstein S. Orlowski J. J. Biol. Chem. 1997; 272: 28672-28679Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 66Zhao H. Wiederkehr M.R. Collazo R. Fan L. Crowder L.A. Moe O.W. J. Biol. Chem. 1999; 274: 3978-3987Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 67Wiederkehr M.R. Zhao H. Moe O.W. Am. J. Physiol. 1999; 276: C1205-C1217Crossref PubMed Google Scholar), the physiologic significance of NHE3 phosphorylation is still undetermined. In this paper we characterize one mechanism by which DA acutely inhibits NHE3: the internalization of NHE3 secondary to PKA-mediated NHE3 phosphorylation and NHE3 endocytosis via clathrin-coated vesicles. OK cells were maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 1 mm sodium pyruvate, 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin and were rendered quiescent postconfluence by serum removal for 48 h prior to experimentation. Transient transfections were performed with LipofectAMINE (Life Technologies, Inc.). Transfection efficiency was monitored by cotransfection with β-galactosidase and staining cells with 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside as well as staining with anti-epitope (c-Myc) antibody (typically 80%). Mammalian expression plasmids used in this study include: 1) C-terminal c-Myc- and hexahistidine (6His)-tagged wild type opossum (NHE3/c-Myc/6H); 2) C-terminal enhanced green fluorescent protein (eGFP)-tagged NHE3 (NHE3/eGFP); 3) C-terminal c-Myc- and 6His-tagged NHE3 with two mutated serines, S560A and S613A (NHE3S560A/S613A/c-Myc/6H); 4) wild type dynamin (dynWT); 5) dominant-negative GTP binding-defective dynamin (dynK44A); 6) cyclic AMP binding-defective regulatory subunit of protein kinase A (RIImut). Agonists used include dopamine (stabilized with 1.1 mm sodium ascorbate) (Sigma), the DR1-specific agonist SKF38393 (Tocris, St. Louis, MO), the DR2-specific agonist quinpirole (Tocris), DR1-specific antagonist SCH23390 (Research Biochemicals, Natick, MA), the DR2-specific antagonist sulpiride (Tocris), 8-bromo-cAMP, and the PKA inhibitor H89. Commercial antisera include anti-c-Myc (Invitrogen, Carlsbad CA) and anti-adaptin a (Santa Cruz Biotechnology, Santa Cruz, CA). To disrupt activation of PKA by cAMP by RIImut, a 5-min pulse of 5 mm8-bromo-cAMP was given to the cells 16 h post-transfection to allow the dominant-negative RIImut to engage the native PKA-catalytic subunit. Experiments with dopamine were performed at 48 h post-transfection. These assays were performed as described previously (56Collazo R. Fan L. Hu M.C. Zhao H. Wiederkehr M.R. Moe O.W. J. Biol. Chem. 2000; 275: 31601-31608Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). To measure surface NHE3, OK cells were surface-labeled with biotin using a modification of the method of Gottardi after the addition of agonists (56Collazo R. Fan L. Hu M.C. Zhao H. Wiederkehr M.R. Moe O.W. J. Biol. Chem. 2000; 275: 31601-31608Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 69Gottardi C.J. Dunbar L.A. Caplan M.J. Am. J. Physiol. 1995; 268: F285-F295Crossref PubMed Google Scholar). After rinsing in PBS/calcium/magnesium (150 mm NaCl, 10 mmNa2HPO4, pH 7.40, 0.1 mmCaCl2, 1 mm MgCl2), cells were incubated with the arginine and lysine reactive of NHS-SS-biotin (2 mg/ml; Pierce) in buffer (150 mm NaCl, 10 mmtriethanolamine, pH 7.4, 2 mm CaCl2), quenched (PBS/calcium/magnesium with 100 mm glycine), and lysed in biotin-RIPA (150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 5 mm EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS). Lysates were centrifuged (109,000 × g for 25 min at 2 °C, Beckman TLX/TLA 100.3 rotor, Fullerton, CA), and protein content in the supernatant was quantified by the method of Bradford. Equal amounts of cell lysate were equilibrated with streptavidin-agarose (Pierce) at 4 °C. Beads were rinsed sequentially with solutions A (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 5 mm EDTA), B (50 mm Tris-HCl, pH 7.4, 500 mm NaCl), and C (50 mm Tris-HCl, pH 7.4), and biotinylated proteins were liberated by reduction incubation in 100 mm dithiothreitol, reconstituted in Laemmli's buffer, resolved by SDS-polyacrylamide gel electrophoresis, and electrotransferred to Imobilin. NHE3 antigen was quantified by labeling with either anti-NHE3 antiserum 5683 or for experiments with exogenous c-Myc-tagged NHE3, a monoclonal anti-c-Myc was used (Invitrogen, Carlsbad, CA). Endocytosis was measured by a protocol adapted and modified from the stage-specific MesNa-resistant and avidin-protection endocytosis assays originally described by Carter and co-workers (56Collazo R. Fan L. Hu M.C. Zhao H. Wiederkehr M.R. Moe O.W. J. Biol. Chem. 2000; 275: 31601-31608Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 70Carter L.L. Redelmeier T.E. Woollenweber L.A. Schmid S.L. J. Cell Biol. 1993; 120: 37-40Crossref PubMed Scopus (139) Google Scholar). OK cells were surface-labeled with NHS-SS-biotin and quenched as described above. Cells were then warmed to 37 °C in the presence of DA or vehicle to allow endocytosis to occur over 30 min. Surface biotin was then saturated with avidin (50 mg/ml PBS) and washed with biocytin (50 mg/ml PBS), or alternatively, surface biotin was cleaved with the small cell-impermeant reducing agent MesNa (50 mm in 50 mm Tris, pH 7.4). The freshly endocytosed proteins bearing biotin were protected from either avidin saturation or MesNa cleavage. Cells were then solubilized in RIPA, and biotinylated proteins were retrieved and assayed for NHE3 as described above. Avidin-protected fraction measures early and late endocytosis because avidin cannot enter the constricted necks of clathrin-coated pits. TCEP-protected fraction measures late endocytosis because complete excommunication from the exterior is required to prevent TCEP access. OK cells were plated on glass coverslips and transfected with NHE3/eGFP. Cultures of transfected OK cells were maintained at 37 °C for 48 h, and fresh medium was replenished 2 h prior to the experiment. Using a fluorescence microscope, living green fluorescent cells were selected before the treatment as described previously (71Karim-Jimenez Z. Hernando N. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12896-12901Crossref PubMed Scopus (50) Google Scholar). DA or vehicle was then added to the cell medium. After the stated period of incubation at 37 °C, the same selected cells were identified again, and the pattern of expression of the different cotransporters was compared with that before treatment. At least six experiments were performed for each condition to evaluate the results. 80% confluent OK cells were transfected with either wild type NHE3/c-Myc/6H or NHE3S560A/S613A/c-Myc/6H. 48 h post-transfection, OK cells were treated with either vehicle or DA. After washing with ice-cold PBS, cells were lysed with ice-cold RIPA buffer (150 mm NaCl, 50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 1 mm EGTA, 1% (v/v) Triton X-100, 100 mg/ml phenylmethylsulfonyl fluoride, 4 mg/ml leupeptin, 4 mg/ml aprotinin, 10 mg/ml pepstatin). The slurry was cleared by centrifugation (109,000 × g for 25 min at 4 °C in a Beckman TLX/TLA 100.3 rotor), and the adaptin AP2 was immunoprecipitated with anti-adaptin α (1:500 dilution) and protein G-Sepharose. After washing with RIPA buffer, the antibody-antigen complex was eluted in SDS buffer (5 mm Tris-HCl, pH 6.8, 10% (v/v) glycerol, 1% (w/v) β-mercaptoethanol, 0.1% (w/v) SDS, 0.01% (w/v) bromphenol blue), resolved on SDS-polyacrylamide gel, and transferred to nitrocellulose membrane. NHE3 was quantified by immunoblot with anti-c-Myc. In the OK cell, DA decreases NHE3 protein in a dose-dependent (Fig.1) and time-dependent (Fig.2) fashion. Typical experiments are shown in Figs. 1 A and 2 A, and the summarized data are shown in Figs. 1 B and 2 B. The dose dependence (Fig. 1) of a decrease in surface NHE3 (half-maximal decrease at 10−5m and maximal decrease of 70% at 10−4m measured after 30 min DA) is similar to that of DA-induced inhibition of NHE3 activity described previously (33Wiederkehr M.R. Di Sole F. Fan L. Hu M.C. Collazo R. Murer H. Helmle-Kolb C. Moe O.W. Kidney Int. 2001; 59: 197-209Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The time dependence (Fig. 2) of decrease in surface NHE3 however is discrepant with changes in NHE3 activity. Whereas a decrease in NHE3 activity is evident after 5 min of DA (33Wiederkehr M.R. Di Sole F. Fan L. Hu M.C. Collazo R. Murer H. Helmle-Kolb C. Moe O.W. Kidney Int. 2001; 59: 197-209Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), a decrease in surface NHE3 is not detectable until after 20 to 30 min. There was no change in total cellular NHE3 within the experimental period (Fig. 1 A). Identical results were obtained from studying native OK NHE3 protein or with OK cells transiently expressing NHE3/c-Myc/6H (not shown).Figure 2Effect of DA on surface NHE3 in OK cells: time dependence. OK cells were rendered quiescent, and 10−5m dopamine was added for the stated period of time. Monolayers were biotinylated, and surface proteins were retrieved from the cell lysate by streptavidin precipitation. NHE3 protein abundance was quantified by immunoblot with anti-OK NHE3. n = sets of time responses.Bars and error bars indicate mean and S.E.Panel A, typical experiment. Panel B, summary of all experiments. n = 4. Asterisks indicatep < 0.05 compared with control (ANOVA).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition to the biochemical biotin assay, we also examined the effect of DA on NHE3 by imaging live cells. OK cells were transiently transfected with NHE3/eGFP, and fluorescent microscopy on live transfected cells showed NHE3 to be a typical brush-border protein with the characteristic punctate staining (Fig.3). The addition of DA caused a time-dependent decrease in surface NHE3 with the appearance of a characteristic intracellular staining pattern (Fig. 3). Within the 2 h of fluorescent microscopic examination, no significant decrease in total cellular NHE3 was appreciated. This is compatible with the biochemical data presented above. The addition of vehicle served as a time control where no change in NHE3 distribution was seen (Fig. 3). Previous studies have shown synergistic roles for DA1 and DA2 receptor agonism on NHE3 activity (33Wiederkehr M.R. Di Sole F. Fan L. Hu M.C. Collazo R. Murer H. Helmle-Kolb C. Moe O.W. Kidney Int. 2001; 59: 197-209Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). We next examined the relative roles of DA1 and DA2 receptors on surface NHE3 antigen. DA1agonist alone was effective in reducing surface NHE3, whereas DA2 alone was ineffective. The combination of DA1 and DA2 resulted in greater reduction of surface NHE3 than DA1 alone. (Fig. 4,A and B). To confirm these results further, we used subtype-specific inhibitors to try to block the effect of DA on surface NHE3 (Fig. 4, C andD). DA1 blockade abolished most of the DA-induced decrease in surface NHE3, whereas DA2 blockade had minimal effect. These findings are similar to the previously reported synergistic effect observed with the inhibition of NHE3 activity by DA1 and DA2 agonists (33Wiederkehr M.R. Di Sole F. Fan L. Hu M.C. Collazo R. Murer H. Helmle-Kolb C. Moe O.W. Kidney Int. 2001; 59: 197-209Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). NHE3 has been visualized in both the plasma membrane and in intracellular compartments in both native kidney tissue and culture cells (48–64). The decrease in surface NHE3 in response to DA can be caused by decreased exocytotic insertion or increased endocytotic retrieval. We quantified endocytotic rate biochemically with the MesNa protection assay. Fig. 5,A and B, shows that DA stimulated NHE3 endocytosis by 58%. When cells were kept at 4 °C to arrest trafficking, a small amount of MesNa-protected NHE3 was visible. This likely reflects incomplete cleavage of the NHS-biotin rather than endocytosis at 4 °C (Fig. 5 A). This usually represents <10% of the signal. The GTPase dynamin is required for both clathrin- and caveolin-mediated endocytosis (72Schmid S.L. Annu. Rev. Biochem. 1997; 66: 511-548Crossref PubMed Scopus (674) Google Scholar, 73Vallee R.B. Herskovits J.S. Aghajanian J.G. Burgess C.C. Shpetner H.S. CIBA Found. Symp. 1993; 176: 185-193PubMed Google Scholar). We next cotransfected the GTP binding-defective dominant-negative dynamin I (dynK44A) along with c-Myc-tagged NHE3 into OK cells and studied the effect of DA on surface NHE3 protein (Fig.6 A). Because the read-out in this assay is with the anti-c-Myc antiserum, which does not react with the native NHE3, only transfected cells were selectively studied. Whereas cells transfected with wild type dynamin (dynWT) showed normal down-regulation of surface NHE3 by DA, cells transfected with dynK44A failed to respond to DA (Fig. 6, Aand B). A key component of the clathrin-coated vesicle endocytotic pathway is the family of adaptor proteins (APs) (73Vallee R.B. Herskovits J.S. Aghajanian J.G. Burgess C.C. Shpetner H.S. CIBA Found. Symp. 1993; 176: 185-193PubMed Google Scholar, 74Robinson M.S. Hirst J. Biochim. Biophys. Acta. 1998; 1404: 173-193Crossref PubMed Scopus (329) Google Scholar). We next examined the association of NHE3 with the adaptor protein AP2 by coimmunoprecipitation. Fig.7 A shows that DA increased the amount of NHE3 bound to total cellular AP2. In fact, the association of NHE3 to AP2 is barely detectable without DA. In sum, our data indicate that DA shifts NHE3 from the plasma membrane to endocytic vesicles via a dynamin- and AP2-dependent process.Figure 6Effect of DA on surface NHE3 : dependence on dynamin. OK cells were cotransfected with NHE3/c-Myc along with either wild type dynamin (dynWT) or K44A dominant-negative dynamin (dynK44A). DA (10−5m 30 min) was added. Monolayers were biotinylated, and surface proteins were retrieved from the cell lysate by streptavidin precipitation. NHE3 protein abundance was quantified by immunoblot with anti-c-Myc. n = sets of experiments. Barsand error bars indicate mean and S.E. Asterisksindicate p < 0.05 compared with control (ANOVA).Panel A, typical experiment. Panel B, summary of all experiments. n = 4.View Large Image" @default.
• W2044612296 created "2016-06-24" @default.
• W2044612296 creator A5005535610 @default.
• W2044612296 creator A5023261848 @default.
• W2044612296 creator A5023488815 @default.
• W2044612296 creator A5034776433 @default.
• W2044612296 creator A5086640425 @default.
• W2044612296 creator A5089345665 @default.
• W2044612296 date "2001-07-01" @default.
• W2044612296 modified "2023-10-15" @default.
• W2044612296 title "Dopamine Acutely Stimulates Na+/H+Exchanger (NHE3) Endocytosis via Clathrin-coated Vesicles" @default.
• W2044612296 cites W13069992 @default.
• W2044612296 cites W1550813715 @default.
• W2044612296 cites W1597035911 @default.
• W2044612296 cites W1924833612 @default.
• W2044612296 cites W1967965496 @default.
• W2044612296 cites W1971052608 @default.
• W2044612296 cites W1971407543 @default.
• W2044612296 cites W1973480659 @default.
• W2044612296 cites W1973745925 @default.
• W2044612296 cites W1979135046 @default.
• W2044612296 cites W1981295957 @default.
• W2044612296 cites W1986542931 @default.
• W2044612296 cites W1990259051 @default.
• W2044612296 cites W1991460269 @default.
• W2044612296 cites W1994154830 @default.
• W2044612296 cites W1996355148 @default.
• W2044612296 cites W1998626317 @default.
• W2044612296 cites W2003014671 @default.
• W2044612296 cites W2004104084 @default.
• W2044612296 cites W2004347894 @default.
• W2044612296 cites W2014616031 @default.
• W2044612296 cites W2014837122 @default.
• W2044612296 cites W2018054062 @default.
• W2044612296 cites W2019490673 @default.
• W2044612296 cites W2025929812 @default.
• W2044612296 cites W2029399045 @default.
• W2044612296 cites W2029707570 @default.
• W2044612296 cites W2030965408 @default.
• W2044612296 cites W2031974543 @default.
• W2044612296 cites W2033139011 @default.
• W2044612296 cites W2040966940 @default.
• W2044612296 cites W2042776102 @default.
• W2044612296 cites W2046828489 @default.
• W2044612296 cites W2049647741 @default.
• W2044612296 cites W2050280508 @default.
• W2044612296 cites W2053302368 @default.
• W2044612296 cites W2053382782 @default.
• W2044612296 cites W2053972094 @default.
• W2044612296 cites W2057734442 @default.
• W2044612296 cites W2058241031 @default.
• W2044612296 cites W2061476447 @default.
• W2044612296 cites W2069966111 @default.
• W2044612296 cites W2071523941 @default.
• W2044612296 cites W2071688598 @default.
• W2044612296 cites W2079804960 @default.
• W2044612296 cites W2079993971 @default.
• W2044612296 cites W2085340755 @default.
• W2044612296 cites W2091005407 @default.
• W2044612296 cites W2095942814 @default.
• W2044612296 cites W2112874964 @default.
• W2044612296 cites W2113526842 @default.
• W2044612296 cites W2115210138 @default.
• W2044612296 cites W2120031794 @default.
• W2044612296 cites W2122412269 @default.
• W2044612296 cites W2134341522 @default.
• W2044612296 cites W2142498139 @default.
• W2044612296 cites W2143205348 @default.
• W2044612296 cites W2148851012 @default.
• W2044612296 cites W2150846836 @default.
• W2044612296 cites W2151783154 @default.
• W2044612296 cites W2153689647 @default.
• W2044612296 cites W2154292480 @default.
• W2044612296 cites W2156630856 @default.
• W2044612296 cites W2167312071 @default.
• W2044612296 cites W2167897285 @default.
• W2044612296 cites W2224689137 @default.
• W2044612296 cites W2273095057 @default.
• W2044612296 cites W2303879656 @default.
• W2044612296 cites W2315253906 @default.
• W2044612296 cites W2320431948 @default.
• W2044612296 cites W2336633857 @default.
• W2044612296 cites W2396562217 @default.
• W2044612296 cites W2397137157 @default.
• W2044612296 cites W2404142064 @default.
• W2044612296 cites W2410028840 @default.
• W2044612296 cites W2419285849 @default.
• W2044612296 cites W2431348053 @default.
• W2044612296 cites W2437246389 @default.
• W2044612296 cites W2467782684 @default.
• W2044612296 doi "https://doi.org/10.1074/jbc.m011338200" @default.
• W2044612296 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11328806" @default.
• W2044612296 hasPublicationYear "2001" @default.
• W2044612296 type Work @default.
• W2044612296 sameAs 2044612296 @default.
• W2044612296 citedByCount "143" @default.
• W2044612296 countsByYear W20446122962012 @default.
• W2044612296 countsByYear W20446122962013 @default.

• next