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• W2080440167 abstract "Water and solute transport across the plasma membrane of cells is a crucial biological function that is mediated mainly by aquaporins and aquaglyceroporins. The regulation of these membrane proteins is still incompletely understood. Using the male reproductive tract as a model system in which water and glycerol transport are critical for the establishment of fertility, we now report a novel pathway for the regulation of aquaporin 9 (AQP9) permeability. AQP9 is the major aquaglyceroporin of the epididymis, liver, and peripheral leukocytes, and its COOH-terminal portion contains a putative PDZ binding motif (SVIM). Here we show that NHERF1, cystic fibrosis transmembrane conductance regulator (CFTR), and AQP9 co-localize in the apical membrane of principal cells of the epididymis and the vas deferens, and that both NHERF1 and CFTR co-immunoprecipitate with AQP9. Overlay assays revealed that AQP9 binds to both the PDZ1 and PDZ2 domains of NHERF1, with an apparently higher affinity for PDZ1 versus PDZ2. Pull-down assays showed that the AQP9 COOH-terminal SVIM motif is essential for interaction with NHERF1. Functional assays on isolated tubules perfused in vitro showed a high permeability of the apical membrane to glycerol, which is inhibited by the AQP9 inhibitor, phloretin, and is markedly activated by cAMP. The CFTR inhibitors DPC, GlyH-101 and CFTRinh-172 all significantly reduced the cAMP-activated glycerol-induced cell swelling. We propose that CFTR is an important regulator of AQP9 and that the interaction between AQP9, NHERF1, and CFTR may facilitate the activation of AQP9 by cAMP. Water and solute transport across the plasma membrane of cells is a crucial biological function that is mediated mainly by aquaporins and aquaglyceroporins. The regulation of these membrane proteins is still incompletely understood. Using the male reproductive tract as a model system in which water and glycerol transport are critical for the establishment of fertility, we now report a novel pathway for the regulation of aquaporin 9 (AQP9) permeability. AQP9 is the major aquaglyceroporin of the epididymis, liver, and peripheral leukocytes, and its COOH-terminal portion contains a putative PDZ binding motif (SVIM). Here we show that NHERF1, cystic fibrosis transmembrane conductance regulator (CFTR), and AQP9 co-localize in the apical membrane of principal cells of the epididymis and the vas deferens, and that both NHERF1 and CFTR co-immunoprecipitate with AQP9. Overlay assays revealed that AQP9 binds to both the PDZ1 and PDZ2 domains of NHERF1, with an apparently higher affinity for PDZ1 versus PDZ2. Pull-down assays showed that the AQP9 COOH-terminal SVIM motif is essential for interaction with NHERF1. Functional assays on isolated tubules perfused in vitro showed a high permeability of the apical membrane to glycerol, which is inhibited by the AQP9 inhibitor, phloretin, and is markedly activated by cAMP. The CFTR inhibitors DPC, GlyH-101 and CFTRinh-172 all significantly reduced the cAMP-activated glycerol-induced cell swelling. We propose that CFTR is an important regulator of AQP9 and that the interaction between AQP9, NHERF1, and CFTR may facilitate the activation of AQP9 by cAMP. Epithelial cells lining the lumen of the excurrent duct of the male reproductive tract create a luminal environment that is optimal for sperm maturation and storage. The composition of the luminal fluid is progressively modified and is tightly regulated during transit from the testicular seminiferous tubules, into the efferent ducts, epididymis, and vas deferens (1Clulow J. Jones R.C. Hansen L.A. Man S.Y. J. Reprod. Fertil. Suppl. 1998; 53: 1-14PubMed Google Scholar, 2Crabo B. Acta Vet. Scand. 1965; 22: 1-94PubMed Google Scholar, 3Hansen L.A. Clulow J. Jones R.C. Exp. Physiol. 1999; 84: 521-527Crossref PubMed Scopus (42) Google Scholar, 4Hinton B.T. Palladino M.A. Microsc. Res. Tech. 1995; 30: 67-81Crossref PubMed Scopus (208) Google Scholar, 5Levine N. Marsh D.J. J. Physiol. 1971; 213: 557-570Crossref PubMed Scopus (286) Google Scholar, 6Robaire B. Hermo L. Knobil E. Neil J. The Physiology of Reproduction. Raven Press, New York1988: 999-1080Google Scholar, 7Robaire B. Viger R.S. Biol. Reprod. 1995; 52: 226-236Crossref PubMed Scopus (227) Google Scholar, 8Yeung C.H. Cooper T.G. Oberpenning F. Schulze H. Nieschlag E. Biol. Reprod. 1993; 49: 274-280Crossref PubMed Scopus (101) Google Scholar, 9Jones R.C. Murdoch R.N. Reprod. Fertil. Dev. 1996; 8: 553-568Crossref PubMed Scopus (100) Google Scholar, 10Turner T.T. J. Androl. 1995; 16: 292-298PubMed Google Scholar). Significant water reabsorption leading to a marked increase in sperm concentration and luminal hypertonicity occurs in the epididymis (5Levine N. Marsh D.J. J. Physiol. 1971; 213: 557-570Crossref PubMed Scopus (286) Google Scholar, 11Clulow J. Jones R.C. Hansen L.A. Exp. Physiol. 1994; 79: 915-928Crossref PubMed Scopus (114) Google Scholar, 12Turner T.T. Cesarini D.M. J. Androl. 1983; 4: 197-202Crossref PubMed Scopus (41) Google Scholar, 13Johnson A.L. Howards S.S. Science. 1977; 195: 492-493Crossref PubMed Scopus (34) Google Scholar). In addition, glycerol, a metabolic substrate for epididymal sperm, is accumulated in the lumen of the distal epididymis (14Cooper T.G. Brooks D.E. J. Reprod. Fertil. 1981; 61: 163-169Crossref PubMed Scopus (28) Google Scholar). In the more distal regions of the epididymis and in the vas deferens, water secretion driven by cystic fibrosis transmembrane conductance regulator (CFTR) 2The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorCFcystic fibrosisNHERF1Na/H exchanger regulatory factor 1cpt-cAMPchlorophenylthio cAMPIPimmunoprecipitationGSTglutathione S-transferasePBSphosphate-buffered salineBBMbrush border membranePVDFpolyvinylidene difluorideTBSTris-buffered salineaaamino acid(s)DPCdiphenylcarbamyl chloride. 2The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorCFcystic fibrosisNHERF1Na/H exchanger regulatory factor 1cpt-cAMPchlorophenylthio cAMPIPimmunoprecipitationGSTglutathione S-transferasePBSphosphate-buffered salineBBMbrush border membranePVDFpolyvinylidene difluorideTBSTris-buffered salineaaamino acid(s)DPCdiphenylcarbamyl chloride.-dependent chloride transport occurs and controls the fluidity of the luminal content (15Wong P.Y. Mol. Hum. Reprod. 1998; 4: 107-110Crossref PubMed Scopus (103) Google Scholar, 16Sedlacek R.L. Carlin R.W. Singh A.K. Schultz B.D. Am. J. Physiol. 2001; 281: F557-F570Crossref PubMed Google Scholar). Although the epididymis is among the most seriously affected organs in cystic fibrosis (CF), very little is known about the mechanisms that lead to the marked decrease in male fertility that occurs in this disease. Cystic fibrosis is one of the leading causes of male infertility (15Wong P.Y. Mol. Hum. Reprod. 1998; 4: 107-110Crossref PubMed Scopus (103) Google Scholar, 17van der Ven K. Messer L. van der Ven H. Jeyendran R.S. Ober C. Hum. Reprod. 1996; 11: 513-517Crossref PubMed Scopus (166) Google Scholar, 18Cuppens H. Cassiman J.J. Int. J. Androl. 2004; 27: 251-256Crossref PubMed Scopus (90) Google Scholar). CFTR plays a critical role in the anatomy and function of the epididymis and vas deferens. A large number of men with CF have no vas deferens, and/or absence or atrophy of some regions of the epididymis (19Anguiano A. Oates R.D. Amos J.A. Dean M. Gerrard B. Stewart C. Maher T.A. White M.B. Milunsky A. J. Am. Med. Assoc. 1992; 267: 1794-1797Crossref PubMed Scopus (477) Google Scholar, 20Oates R.D. Amos J.A. J. Androl. 1994; 15: 1-8PubMed Google Scholar). It was originally proposed that these abnormalities were the consequence of defective embryonic development. However, recent studies have indicated that dysfunction of the epididymis and vas deferens in patients with cystic fibrosis might be the results of a progressive atrophy of these tissues that may occur after birth and reach maximum intensity at adult age (21Gaillard D.A. Carre-Pigeon F. Lallemand A. J. Urol. 1997; 158: 1549-1552Crossref PubMed Scopus (60) Google Scholar, 22Blau H. Freud E. Mussaffi H. Werner M. Konen O. Rathaus V. Arch. Dis. Child. 2002; 87: 135-138Crossref PubMed Scopus (31) Google Scholar). These studies suggest that prevention strategies could be developed to help the CF-affected male population preserve their reproductive function.In a variety of epithelia, water channels (aquaporins) are involved in transepithelial bulk water flow driven by an osmotic gradient (reviewed in Ref. 23King L.S. Kozono D. Agre P. Nat. Rev. Mol. Cell. Biol. 2004; 5: 687-698Crossref PubMed Scopus (751) Google Scholar). In mammals, aquaporins are divided into two subgroups based on their permeability characteristics: the strict “aquaporins” (AQP0, 1, 2, 4, 5, 6, and 8) are selective for water and the “aquaglyceroporins” (AQP3, 7, 9, and 10) are permeable to neutral solutes in addition to water. AQP11 and AQP12 have recently been identified and are more distantly related to the other members of the aquaporin family (24Zardoya R. Biol. Cell. 2005; 97: 397-414Crossref PubMed Scopus (242) Google Scholar). Aquaporins and aquaglyceroporins show a wide range of distribution in organs that are actively involved in water movement (25Brown D. Katsura T. Kawashima M. Verkman A.S. Sabolic I. Histochem. Cell Biol. 1995; 104: 1-9Crossref PubMed Scopus (78) Google Scholar, 26Hamann S. Zeuthen T. La Cour M. Nagelhus E.A. Ottersen O.P. Agre P. Nielsen S. Am. J. Physiol. 1998; 274: C1332-C1345Crossref PubMed Google Scholar, 27Kreda S.M. Gynn M.C. Fenstermacher D.A. Boucher R.C. Gabriel S.E. Am. J. Respir. Cell Mol. Biol. 2001; 24: 224-234Crossref PubMed Scopus (217) Google Scholar, 28Nielsen S. Frokiaer J. Marples D. Kwon T.H. Agre P. Knepper M.A. Physiol. Rev. 2002; 82: 205-244Crossref PubMed Scopus (1020) Google Scholar, 29Nielsen S. King L.S. Christensen B.M. Agre P. Am. J. Physiol. 1997; 273: C1549-C1561Crossref PubMed Google Scholar). AQP9 has been identified as the major aquaglyceroporin in the excurrent duct of the male reproductive tract, the liver, and peripheral leukocytes (30Badran H.H. Hermo L.S. J. Androl. 2002; 23: 358-373PubMed Google Scholar, 31Da Silva N. Silberstein C. Beaulieu V. Pietrement C. Van Hoek A.N. Brown D. Breton S. Biol. Reprod. 2006; 74: 427-438Crossref PubMed Scopus (69) Google Scholar, 32Pastor-Soler N. Bagnis C. Sabolic I. Tyszkowski R. McKee M. Van Hoek A. Breton S. Brown D. Biol. Reprod. 2001; 65: 384-393Crossref PubMed Scopus (125) Google Scholar, 33Pastor-Soler N. Isnard-Bagnis C. Herak-Kramberger C. Sabolic I. Van Hoek A. Brown D. Breton S. Biol. Reprod. 2002; 66: 1716-1722Crossref PubMed Scopus (82) Google Scholar, 34Elkjaer M. Vajda Z. Nejsum L.N. Kwon T. Jensen U.B. Amiry-Moghaddam M. Frokiaer J. Nielsen S. Biochem. Biophys. Res. Commun. 2000; 276: 1118-1128Crossref PubMed Scopus (259) Google Scholar, 35Tsukaguchi H. Shayakul C. Berger U.V. Mackenzie B. Devidas S. Guggino W.B. van Hoek A.N. Hediger M.A. J. Biol. Chem. 1998; 273: 24737-24743Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 36Tsukaguchi H. Weremowicz S. Morton C.C. Hediger M.A. Am. J. Physiol. 1999; 277: F685-F696Crossref PubMed Google Scholar). In the male reproductive system, it is constitutively expressed in the apical stereocilia of principal cells along the entire length of the epididymis and vas deferens, as well as in the apical membrane of non-ciliated cells of the efferent ducts (32Pastor-Soler N. Bagnis C. Sabolic I. Tyszkowski R. McKee M. Van Hoek A. Breton S. Brown D. Biol. Reprod. 2001; 65: 384-393Crossref PubMed Scopus (125) Google Scholar). This aquaglyceroporin allows passage of a wide range of solutes, including glycerol, urea, mannitol, and sorbitol, in addition to water (35Tsukaguchi H. Shayakul C. Berger U.V. Mackenzie B. Devidas S. Guggino W.B. van Hoek A.N. Hediger M.A. J. Biol. Chem. 1998; 273: 24737-24743Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Thus, AQP9 provides a potential route for transepithelial fluid and solute transport in the epididymis. The promoter region of AQP9 contains a putative steroid hormone receptor-binding site (35Tsukaguchi H. Shayakul C. Berger U.V. Mackenzie B. Devidas S. Guggino W.B. van Hoek A.N. Hediger M.A. J. Biol. Chem. 1998; 273: 24737-24743Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar), and sex-linked differences in AQP9 expression were reported in the liver (37Nicchia G.P. Frigeri A. Nico B. Ribatti D. Svelto M. J. Histochem. Cytochem. 2001; 49: 1547-1556Crossref PubMed Scopus (99) Google Scholar). Androgens control AQP9 expression in the adult epididymis (30Badran H.H. Hermo L.S. J. Androl. 2002; 23: 358-373PubMed Google Scholar, 33Pastor-Soler N. Isnard-Bagnis C. Herak-Kramberger C. Sabolic I. Van Hoek A. Brown D. Breton S. Biol. Reprod. 2002; 66: 1716-1722Crossref PubMed Scopus (82) Google Scholar, 38Oliveira C.A. Carnes K. Franca L.R. Hermo L. Hess R.A. Biol. Cell. 2005; 97: 385-395Crossref PubMed Scopus (94) Google Scholar), and Aqp9 mRNA increases markedly during the first 4 weeks of postnatal development (31Da Silva N. Silberstein C. Beaulieu V. Pietrement C. Van Hoek A.N. Brown D. Breton S. Biol. Reprod. 2006; 74: 427-438Crossref PubMed Scopus (69) Google Scholar). However, the acute regulation of AQP9 function has not been well characterized. The presence of a putative PDZ (PSD-95, Drosophila discs large protein, ZO-1) binding motif, SVIM, in the COOH terminus of AQP9 indicates the potential intervention of PDZ proteins in its regulation. PDZ proteins are scaffolding proteins that facilitate the association of multiprotein complexes, a process that is essential for the phosphorylation of some transporters, channels, and receptors (39Brone B. Eggermont J. Am. J. Physiol. 2005; 288: C20-C29Crossref PubMed Scopus (86) Google Scholar, 40Shenolikar S. Voltz J.W. Cunningham R. Weinman E.J. Physiol. (Bethesda). 2004; 19: 362-369Crossref PubMed Scopus (135) Google Scholar). NHERF1 (Na/H exchanger regulatory factor; SLC9A3R1) is a major apical PDZ protein that contains three protein interaction domains: PDZ domain 1 (PDZ-1), PDZ domain 2 (PDZ-2), and a sequence located in the COOH terminus that binds to the family of Merlin/Ezrin/Radixin/Moesin (MERM) proteins (41Gonzalez-Agosti C. Wiederhold T. Herndon M.E. Gusella J. Ramesh V. J. Biol. Chem. 1999; 274: 34438-34442Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 42Bretscher A. Chambers D. Nguyen R. Reczek D. Annu. Rev. Cell Dev. Biol. 2000; 16: 113-143Crossref PubMed Scopus (323) Google Scholar, 43James M.F. Beauchamp R.L. Manchanda N. Kazlauskas A. Ramesh V. J. Cell Sci. 2004; 117: 2951-2961Crossref PubMed Scopus (64) Google Scholar). NHERF1 is involved in the cAMP regulation of a variety of transporters, including Na+/H+ exchanger type 3 (NHE3), CFTR, Na+-Pi cotransporter IIa (Npt2 or NaPi Iia) (reviewed in Refs. 39Brone B. Eggermont J. Am. J. Physiol. 2005; 288: C20-C29Crossref PubMed Scopus (86) Google Scholar, 40Shenolikar S. Voltz J.W. Cunningham R. Weinman E.J. Physiol. (Bethesda). 2004; 19: 362-369Crossref PubMed Scopus (135) Google Scholar, 44Weinman E.J. Cunningham R. Wade J.B. Shenolikar S. J. Physiol. 2005; 567: 27-32Crossref PubMed Scopus (66) Google Scholar, and 45Guggino W.B. Stanton B.A. Nat. Rev. Mol. Cell. Biol. 2006; 7: 426-436Crossref PubMed Scopus (344) Google Scholar)), and ROMK (46Yoo D. Flagg T.P. Olsen O. Raghuram V. Foskett J.K. Welling P.A. J. Biol. Chem. 2004; 279: 6863-6873Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar).Water transport and solute transport represent crucial events in the establishment and maintenance of male fertility, and we postulated that CFTR might be involved in their regulation. The present study is aimed at characterizing the functional contribution of AQP9 to apical glycerol permeability and at determining whether the apical PDZ protein NHERF1, and the PDZ-binding protein, CFTR, could participate in its regulation.EXPERIMENTAL PROCEDURESFunctional Studies on Epididymal Tubules Perfused in Vitro—Epididymal tubules were dissected from the initial segments of the epididymis in a cold preservation solution containing 56 mm Na2HPO4, 13 mm NaH2PO4, and 140 mm sucrose, as described previously (47Bagnis C. Marsolais M. Biemesderfer D. Laprade R. Breton S. Am. J. Physiol. 2001; 280: F426-F436PubMed Google Scholar). They were then transferred into a perfusion chamber mounted on the stage of an Olympus IMT-2 inverted microscope, and peritubular and luminal perfusions were performed (solutions in Table 1). The basolateral solution composition was based on normal plasma values, and the apical solution was based on previous epididymal micropuncture studies (5Levine N. Marsh D.J. J. Physiol. 1971; 213: 557-570Crossref PubMed Scopus (286) Google Scholar). After an initial control period, the apical membrane permeability to glycerol was estimated from the initial rate of increase in cellular volume induced upon isotonic replacement of either 60 or 120 mm raffinose (an impermeant solute in the epididymal tubule) with glycerol. Digital images of perfused tubules were captured at 15- or 30-s intervals, as described in the text, using a Nikon Coolpix 995 camera and were analyzed using IPLab software (Scanalytics, Fairfax, VA). For each time point, the height of epithelial cells was measured at 5-6 different locations along the tubule, and the values were averaged. Cell volume was assessed from these values and expressed as percentage of initial control volume, as we have previously published for kidney proximal tubules (48Breton S. Beck J.S. Cardinal J. Giebisch G. Laprade R. Am. J. Physiol. 1992; 263: F656-F664PubMed Google Scholar). Initial rates of cell swelling were determined from four cell volume values measured at 15-s intervals during the first minute of glycerol exposure. The effects of 500 μm phloretin, an AQP9 inhibitor, or 100 μm chlorophenylthio cAMP (cpt-cAMP) on glycerol-induced cell swelling were examined. We also examined the effects of three different CFTR inhibitors, DPC (500 μm), GlyH-101 (25 μm), and CFTRinh-172 (5 μm). GlyH-101 and CFTRinh-172 are previously characterized specific CFTR inhibitors kindly provided by Alan Verkman (University of California, San Francisco) (49Ma T. Thiagarajah J.R. Yang H. Sonawane N.D. Folli C. Galietta L.J. Verkman A.S. J. Clin. Investig. 2002; 110: 1651-1658Crossref PubMed Scopus (582) Google Scholar, 50Muanprasat C. Sonawane N.D. Salinas D. Taddei A. Galietta L.J. Verkman A.S. J. Gen. Physiol. 2004; 124: 125-137Crossref PubMed Scopus (228) Google Scholar). Statistical analysis was performed using the Student's t test for paired or unpaired experiments, as indicated in the text.TABLE 1Composition of luminal and basolateral solutionsControl luminal solutionGlycerol solutionBasolateral solutionmm NaCl5555100 KCl555 MgSO41.2 NaH2PO4111 Glucamine Cl10 Raffinose1200 (or 60) CaCl21.81.81.8 MgCl2 H2O1.21.2 Sodium acetate444 Na3 citrate111 Glucose5.55.55.5 Alanine666 Na2HPO4333 NaHCO35525 Glycerol120 (or 60) Sodium cyclamate2525 Osmolarity (mOsm/kg H2O)331334297 pH6.856.837.41 Open table in a new tab Antibodies and Peptides—An affinity purified rabbit polyclonal antibody was raised against a peptide corresponding to the last 15 amino acids (PSENNLEKHELSVIM) of the COOH-terminal tail of rat AQP9 (35Tsukaguchi H. Shayakul C. Berger U.V. Mackenzie B. Devidas S. Guggino W.B. van Hoek A.N. Hediger M.A. J. Biol. Chem. 1998; 273: 24737-24743Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). This antibody has been fully characterized previously (32Pastor-Soler N. Bagnis C. Sabolic I. Tyszkowski R. McKee M. Van Hoek A. Breton S. Brown D. Biol. Reprod. 2001; 65: 384-393Crossref PubMed Scopus (125) Google Scholar, 33Pastor-Soler N. Isnard-Bagnis C. Herak-Kramberger C. Sabolic I. Van Hoek A. Brown D. Breton S. Biol. Reprod. 2002; 66: 1716-1722Crossref PubMed Scopus (82) Google Scholar) and was used in this study for immunocytochemistry and for some immunoprecipitation (IP) assays. An affinity purified anti-rat AQP9 antibody raised in chicken (Chemicon International; Temecula, CA) was also used for Western blotting of material immunoprecipitated using our rabbit anti-AQP9 antibody. An affinity purified chicken polyclonal antibody was raised against a GST-NHERF1 fusion protein corresponding to amino acids 270-358 (IC270), which we used previously for the generation of a polyclonal rabbit antibody (41Gonzalez-Agosti C. Wiederhold T. Herndon M.E. Gusella J. Ramesh V. J. Biol. Chem. 1999; 274: 34438-34442Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). A peptide corresponding to the last 15 amino acids of AQP9 was generated in the Massachusetts General Hospital Peptide/Protein Core Facility, and some of the peptide was biotinylated. Three different anti-CFTR antibodies were used for immunofluorescence and Western blot detection. AME-4991 is the whole serum of a previously characterized antibody raised in rabbit against a synthetic 13-residue peptide of the carboxyl terminus of rat CFTR (51Golin-Bisello F. Bradbury N. Ameen N. Am. J. Physiol. 2005; 289: C708-C716Crossref PubMed Scopus (80) Google Scholar). A commercial rabbit affinity purified antibody against amino acid residues 1468-1480 of human CFTR (ACL-006 from Alomone) and a monoclonal antibody against amino acids 1377-1480 of human CFTR (Clone 24-1, MAB25031 from R&D Systems) were also used.Immunofluorescence Microscopy—Sexually mature male Sprague-Dawley rats were anesthetized with Nembutal (7.5 mg/100 g body weight intraperitoneal; Abbott Laboratories, North Chicago, IL) and perfused via the left ventricle with PBS (0.9% NaCl in 10 mm sodium phosphate buffer, pH 7.4) followed by a fixative containing 4% paraformaldehyde, 10 mm sodium periodate, 75 mm lysine, and 5% sucrose in 0.1 m sodium phosphate buffer, as described previously (32Pastor-Soler N. Bagnis C. Sabolic I. Tyszkowski R. McKee M. Van Hoek A. Breton S. Brown D. Biol. Reprod. 2001; 65: 384-393Crossref PubMed Scopus (125) Google Scholar, 33Pastor-Soler N. Isnard-Bagnis C. Herak-Kramberger C. Sabolic I. Van Hoek A. Brown D. Breton S. Biol. Reprod. 2002; 66: 1716-1722Crossref PubMed Scopus (82) Google Scholar), or with PBS containing 2% paraformaldehyde (for CFTR labeling). Epididymis and vas deferens were cryoprotected in 30% sucrose in PBS, mounted for cryosectioning in Tissue-Tek OCT compound 4583 (Sakura Fintek USA, Inc., Torrance, CA), and quick frozen. Sections were cut at a thickness of 5 μm using a Reichert-Jung 2800 Frigocut cryostat (Leica Microsystems, Inc., Bannockburn, IL) and picked up onto Superfrost/Plus microscope slides (Fisher Scientific). For indirect immunofluorescence microscopy, sections were hydrated for 15 min in PBS and treated for 4 min with 1% SDS in PBS, an antigen retrieval technique that we have previously described (52Brown D. Lydon J. McLaughlin M. Stuart-Tilley A. Tyszkowski R. Alper S. Histochem. Cell Biol. 1996; 105: 261-267Crossref PubMed Scopus (278) Google Scholar). Sections were washed in PBS 3 times for 5 min and then blocked in 1% bovine serum albumin/PBS for 15 min. Affinity purified rabbit anti-AQP9 antibody was applied at a dilution of 1:3200 in a moist chamber for 90 min at room temperature or overnight at 4 °C. Sections were washed in high salt PBS (PBS containing 2.7% NaCl) twice for 5 min and once in normal PBS. Goat anti-rabbit IgG coupled to CY3 was then applied for 1 h at room temperature followed by washes as above. Sections were double-stained by subsequent incubation with anti-NHERF1 antibody diluted 1:50 followed by donkey anti-chicken IgG conjugated to fluorescein isothiocyanate, or with anti-CFTR antibody MAB25031 diluted 1:10, followed by goat anti-mouse IgG conjugated to fluorescein isothiocyanate. Double labeling was also performed using anti-AQP9 chicken antibody diluted 1:50 followed by anti-CFTR AME-4991 antibody diluted 1:50.Slides were mounted in Vectashield medium (Vector Laboratories, Inc., Burlingame, CA). Digital images were acquired using a Nikon Eclipse 800 epifluorescence microscope (Nikon instruments, Inc., Melville, NY) using an Orca 100 CCD camera (Hamamatsu, Bridgewater, NJ), analyzed using IPLab scientific image processing software (Scanalytics, Inc., Fairfax, VA), and imported into Adobe Photoshop image editing software (Adobe Systems Inc., San Jose, CA).Apical Membrane Preparation—Epithelial cell apical membranes were isolated using the brush border membrane (BBM) Mg2+ precipitation technique, as previously described (32Pastor-Soler N. Bagnis C. Sabolic I. Tyszkowski R. McKee M. Van Hoek A. Breton S. Brown D. Biol. Reprod. 2001; 65: 384-393Crossref PubMed Scopus (125) Google Scholar, 33Pastor-Soler N. Isnard-Bagnis C. Herak-Kramberger C. Sabolic I. Van Hoek A. Brown D. Breton S. Biol. Reprod. 2002; 66: 1716-1722Crossref PubMed Scopus (82) Google Scholar). We have shown previously that AQP9 is significantly enriched in epididymal BBM (32Pastor-Soler N. Bagnis C. Sabolic I. Tyszkowski R. McKee M. Van Hoek A. Breton S. Brown D. Biol. Reprod. 2001; 65: 384-393Crossref PubMed Scopus (125) Google Scholar). Protein concentration was determined using the bicinchoninic acid assay (Pierce Biotechnology) using albumin as standard.IP and Co-IP Assays—Anti-AQP9 rabbit antibody was conjugated to magnetic beads (Dynabeads Protein A, Invitrogen) according to the manufacturer's protocol. The epididymal BBM preparation (250 μg) was pre-cleared by two consecutive 30-min incubations with non-conjugated magnetic beads. Immunoprecipitation assays were performed in 1 ml of IP buffer (1% Triton X-100, 150 mm NaCl, 10 mm Tris, pH 7.4, 1 mm EDTA, 1 mm EGTA, 0.2 mm sodium orthovanadate, 0.5 mm IGEPAL CA-630, 10% glycerol, 1% bovine serum albumin, complete protease inhibitors) for 2 h at 4 °C. After three washes in 1 ml of IP buffer, beads were resuspended in 50 μl of Laemmli reducing sample buffer, and incubated at room temperature for 45 min. Beads were captured using a magnetic particle concentrator (Invitrogen), and eluates were subjected to SDS-PAGE, as described below. For some experiments, the anti-AQP9 antibody was preincubated with the non-biotinylated AQP9 peptide prior to immobilization on the beads. In separate experiments, CFTR and AQP9 co-IP assays were performed using rabbit anti-CFTR antibody (Alomone) and our rabbit anti-AQP9 antibody, which were bound and cross-linked to immobilized protein A using the Seize X Protein A immunoprecipitation kit (Pierce). This procedure allowed for Western blot detection of proteins in the IP material using antibodies raised in the same species as that used for the IP. Total proteins from rat epididymis and lung were isolated using the ProFound lysis buffer (Pierce) complemented with protease inhibitors. CFTR co-IP was performed by incubating the immobilized anti-CFTR antibody sequentially with 1 mg of lung extract for 4 h, then with 1 mg of epididymis extract enriched with BBM overnight. This sequence was reversed to perform the AQP9 co-IP. After three washes, proteins were eluted in NuPAGE LDS sample buffer (Invitrogen) with reducing agent and protease inhibitors, incubated for 45 min at 23 °C, and analyzed by Western blotting.Immunoblotting (SDS-PAGE and Western Blotting)—Total epididymis homogenates, BBM samples, or IP eluates were diluted in sample buffer, and loaded onto Tris glycine polyacrylamide 4-20% gradient gels (Lonza, Rockland, ME). 4-12% NuPAGE gels (Invitrogen) were used for the analysis of CFTR/AQP9 co-IPs. After SDS-PAGE separation, proteins were transferred onto Immuno-Blot polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blocked in Tris-buffered saline (TBS) containing 5% nonfat dry milk and then incubated overnight at 4 °C with the primary antibody (anti-AQP9, -NHERF1, or -CFTR) diluted in TBS containing 2.5% milk. After three washes in TBS containing 0.1% Tween 20 (TBST), and a 15-min block in 5% milk/TBS, membranes were incubated with secondary antibodies (either goat anti-rabbit IgG or goat anti-chicken IgG) conjugated to horseradish peroxidase for 1 h at room temperature. After five further washes, antibody binding was detected with the Western Lightning Chemiluminescence reagent (PerkinElmer Life Sciences) and Kodak imaging films.Phosphatase Assay—330 μg (50 μl) of epididymis total homogenate was incubated with 100 μl of 1 mm Tris, 50 mm Tris-HCl, pH 7.5, for 10 min at 30 °C. 30 Units (30 μl) of calf intestine alkaline phosphatase (Calbiochem, Darmstadt, Germany) was then added (water was added in the control sample) and the solution was incubated for 15 min at 30 °C. Dephosphorylation was terminated by the addition of 180 μl of Laemmli sample buffer (2 times). 30 μl of each sample (containing 27 μg of protein) were then subjected to electrophoresis and Western blot, as described above.Preparation of Recombinant NHERF1—Human full-length NHERF1 (amino acids (aa) 1-358) and NHERF1 truncated fusion proteins containing PDZ1 (aa 11-97), PDZ2 (aa 149-236), PDZ1 and PDZ2 (aa 11-236), PDZ2 and the COOH-terminal portion (aa 149-358), and NHERF1 lacking both PDZ domains (aa 270-358) were amplified by PCR, subcloned into the BamHI-NotI sites of pGEX4T (Amersham Biosciences), and expressed in Escherichia coli, as described previously (43James M.F. Beauchamp R.L. Manchanda N. Kazlauskas A. Ramesh V. J. Cell Sci. 2004; 117: 2951-2961Crossref PubMed Scopus (64) Google Scholar). After a first step of purification using gluthathione-Sepharose 4B (Amersham Biosciences), GST fusion proteins were loaded onto Tris glycine polyacrylamide 4-20% gradient gels (PAGEr Duramide Precast Gels, 4-20% Tris glycine gels, Cambex, Rockland, ME). After SDS-PAGE separation, GST proteins were transferred onto a PVDF membrane. After stain" @default.
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• W2080440167 date "2008-02-01" @default.
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• W2080440167 title "Role of NHERF1, Cystic Fibrosis Transmembrane Conductance Regulator, and cAMP in the Regulation of Aquaporin 9" @default.
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