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• W2042679775 abstract "We have demonstrated previously the regulation of Cl−/HCO3− exchange activity by the cystic fibrosis transmembrane conductance regulator (CFTR) in model systems of cells stably or transiently transfected with CFTR (Lee, M. G., Wigley, W. C., Zeng, W., Noel, L. E., Marino, C. R., Thomas, P. J., and Muallem, S. (1999)J. Biol. Chem. 274, 3414–3421). In the present work we examine the significance of this regulation in cells naturally expressing CFTR. These include the human colonic T84 cell line and the mouse submandibular gland and pancreatic ducts, tissues that express high levels of CFTR in the luminal membrane. As in heterologous expression systems, stimulation of T84 cells with forskolin increased the Cl−/HCO3− exchange activity independently of CFTR Cl− channel activity. Freshly isolated submandibular gland ducts from wild type mice showed variable Cl−/HCO3−exchange activity. Measurement of [Cl−]i revealed that this was largely the result of variable steady-state [Cl−]i. Membrane depolarization with 5 mm Ba2+ or 100 mmK+ increased and stabilized [Cl−]i. Under depolarized conditions wild type and ΔF/ΔF mice had comparable basal Cl−/HCO3− exchange activity. Notably, stimulation with forskolin increased Cl−/HCO3− exchange activity in submandibular gland ducts from wild type but not ΔF/ΔF mice. Microperfusion of the main pancreatic duct showed Cl−/HCO3− exchange activity in both the basolateral and luminal membranes. Stimulation of ducts from wild type animals with forskolin had no effect on basolateral but markedly stimulated luminal Cl−/HCO3− exchange activity. By contrast, forskolin had no effect on either basolateral or luminal Cl−/HCO3−exchange activity of ducts from ΔF/ΔF animals. We conclude that CFTR regulates luminal Cl−/HCO3− exchange activity in CFTR-expressing cells, and we discuss the possible physiological significance of these findings regarding cystic fibrosis. We have demonstrated previously the regulation of Cl−/HCO3− exchange activity by the cystic fibrosis transmembrane conductance regulator (CFTR) in model systems of cells stably or transiently transfected with CFTR (Lee, M. G., Wigley, W. C., Zeng, W., Noel, L. E., Marino, C. R., Thomas, P. J., and Muallem, S. (1999)J. Biol. Chem. 274, 3414–3421). In the present work we examine the significance of this regulation in cells naturally expressing CFTR. These include the human colonic T84 cell line and the mouse submandibular gland and pancreatic ducts, tissues that express high levels of CFTR in the luminal membrane. As in heterologous expression systems, stimulation of T84 cells with forskolin increased the Cl−/HCO3− exchange activity independently of CFTR Cl− channel activity. Freshly isolated submandibular gland ducts from wild type mice showed variable Cl−/HCO3−exchange activity. Measurement of [Cl−]i revealed that this was largely the result of variable steady-state [Cl−]i. Membrane depolarization with 5 mm Ba2+ or 100 mmK+ increased and stabilized [Cl−]i. Under depolarized conditions wild type and ΔF/ΔF mice had comparable basal Cl−/HCO3− exchange activity. Notably, stimulation with forskolin increased Cl−/HCO3− exchange activity in submandibular gland ducts from wild type but not ΔF/ΔF mice. Microperfusion of the main pancreatic duct showed Cl−/HCO3− exchange activity in both the basolateral and luminal membranes. Stimulation of ducts from wild type animals with forskolin had no effect on basolateral but markedly stimulated luminal Cl−/HCO3− exchange activity. By contrast, forskolin had no effect on either basolateral or luminal Cl−/HCO3−exchange activity of ducts from ΔF/ΔF animals. We conclude that CFTR regulates luminal Cl−/HCO3− exchange activity in CFTR-expressing cells, and we discuss the possible physiological significance of these findings regarding cystic fibrosis. HCO3− secretion is a primary function of many CFTR 1The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; BLM, basolateral membrane; LM, luminal membrane; AE, Cl−/HCO3− (anion) exchanger; SMG, submandibular gland; WT, wild type; ΔF, deletion mutant of Phe-508 from WT CFTR; PSA, pancreatic solution A; BCECF-AM, 2′7′-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein acetoxymethyl ester; MQAE, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide; DIDS, 4,4′-diisothiocyanato- stilbene-2,2′-disulfonate. 1The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; BLM, basolateral membrane; LM, luminal membrane; AE, Cl−/HCO3− (anion) exchanger; SMG, submandibular gland; WT, wild type; ΔF, deletion mutant of Phe-508 from WT CFTR; PSA, pancreatic solution A; BCECF-AM, 2′7′-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein acetoxymethyl ester; MQAE, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide; DIDS, 4,4′-diisothiocyanato- stilbene-2,2′-disulfonate.-expressing cells (1Argent B.E. Case R.M. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1478-1498Google Scholar, 2Cook D.I. van Lennep E.W. Roberts M.L. Young J.A. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1061-1117Google Scholar, 3Greger R. Mall M. Bleich M. Ecke D. Warth R. Riedemann N. Kunzelmann K. J. Mol. Med. 1996; 74: 527-534Crossref PubMed Scopus (47) Google Scholar). Most of the HCO3− is secreted by duct or duct-like cells to the lumen and thus requires transductal HCO3− transport. Little is known about the pathways mediating HCO3− entry in the basolateral membrane (BLM). The best studies available to date are in the pancreatic duct, in which Case and co-workers (4Ishiguro H. Steward M.C. Lindsay A.R. Case R.M. J. Physiol. (Lond.). 1996; 495: 169-178Crossref Scopus (156) Google Scholar, 5Ishiguro H. Steward M.C. Wilson R.W. Case R.M. J. Physiol. (Lond.). 1996; 495: 179-191Crossref Scopus (117) Google Scholar, 6Ishiguro H. Naruse S. Steward M.C. Kitagawa M. Ko S.B.H. Hayakawa T. Case R.M. J. Physiol. (Lond.). 1998; 511: 407-422Crossref Scopus (75) Google Scholar) provided strong evidence that HCO3− influx is mediated largely by a BLM Na+-HCO3− cotransport. HCO3− efflux across the luminal membrane (LM) and its regulation are equally poorly understood. Most models assume that the electroneutral portion of HCO3− secretion is mediated by a luminal Cl−/HCO3−exchanger (AE, anion exchanger) (1Argent B.E. Case R.M. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1478-1498Google Scholar, 2Cook D.I. van Lennep E.W. Roberts M.L. Young J.A. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1061-1117Google Scholar, 3Greger R. Mall M. Bleich M. Ecke D. Warth R. Riedemann N. Kunzelmann K. J. Mol. Med. 1996; 74: 527-534Crossref PubMed Scopus (47) Google Scholar, 6Ishiguro H. Naruse S. Steward M.C. Kitagawa M. Ko S.B.H. Hayakawa T. Case R.M. J. Physiol. (Lond.). 1998; 511: 407-422Crossref Scopus (75) Google Scholar). This function is also believed to mediate part of Cl− absorption by the duct. Cl− is supplied to the duct lumen in the plasma-like primary fluid secreted by acinar cells (1Argent B.E. Case R.M. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1478-1498Google Scholar, 2Cook D.I. van Lennep E.W. Roberts M.L. Young J.A. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1061-1117Google Scholar, 3Greger R. Mall M. Bleich M. Ecke D. Warth R. Riedemann N. Kunzelmann K. J. Mol. Med. 1996; 74: 527-534Crossref PubMed Scopus (47) Google Scholar, 7Quinton P.M. Yankaskas J.R. Knowles J.R. Cystic Fibrosis in Adults. Lippincott, Philadelphia1998: 419-438Google Scholar). The pathophysiology of cystic fibrosis indicates that CFTR plays a critical, but poorly defined, role in HCO3− secretion and Cl− absorption. In tissues such as the salivary glands, in which acinar cells secrete the bulk of the fluid, CFTR is assumed to mediate the electrogenic part of Cl− absorption (2Cook D.I. van Lennep E.W. Roberts M.L. Young J.A. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1061-1117Google Scholar). The same role is attributed to CFTR in sweat glands (7Quinton P.M. Yankaskas J.R. Knowles J.R. Cystic Fibrosis in Adults. Lippincott, Philadelphia1998: 419-438Google Scholar) and intestinal epithelia (8Schultz S.G. Frizzell R.A. Gastroenterology. 1972; 63: 161-170Abstract Full Text PDF PubMed Google Scholar, 9Barrett K.E. Am. J. Physiol. 1997; 272: C1069-C1076Crossref PubMed Google Scholar). Recent work suggests that airway epithelia absorb Na+ and Cl− to produce a hypotonic airway surface fluid (Ref. 10Zabner J. Smith J.J. Karp P.H. Widdicombe J.H. Welsh M.J. Mol. Cell. 1998; 2: 397-403Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar and references within, but see Ref. 11Matsui H. Grubb B.R. Tarran R. Randell S.H. Gatzy J.T. Davis C.W. Boucher R.C. Cell. 1998; 95: 1005-1015Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar and references within). Because Na+ and Cl−concentrations in airway surface liquid produced by cystic fibrosis airway epithelium are isotonic (10Zabner J. Smith J.J. Karp P.H. Widdicombe J.H. Welsh M.J. Mol. Cell. 1998; 2: 397-403Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 11Matsui H. Grubb B.R. Tarran R. Randell S.H. Gatzy J.T. Davis C.W. Boucher R.C. Cell. 1998; 95: 1005-1015Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar), CFTR may mediate electrogenic Cl− absorption in airway epithelia (12Welsh M.J. Physiol. Rev. 1987; 67: 1143-1184Crossref PubMed Scopus (269) Google Scholar, 13Boucher R.C. Am. J. Respir. Crit. Care Med. 1994; 150: 581-593Crossref PubMed Scopus (198) Google Scholar). In glands like the pancreas, fluid secretion by acinar cells is limited, and the duct secretes the bulk of the fluid in pancreatic juice (1Argent B.E. Case R.M. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1478-1498Google Scholar). In this type of gland the limited supply of Cl− secreted by acinar cells led to the proposal that CFTR mediates Cl− secretion to the lumen of duct cells to fuel the Cl−/HCO3− exchanger (1Argent B.E. Case R.M. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1478-1498Google Scholar). However, a recent work showed that agonist- and cAMP-stimulated HCO3− secretion in guinea pig pancreatic duct is independent of luminal Cl− (6Ishiguro H. Naruse S. Steward M.C. Kitagawa M. Ko S.B.H. Hayakawa T. Case R.M. J. Physiol. (Lond.). 1998; 511: 407-422Crossref Scopus (75) Google Scholar). Hence, the role of CFTR in ion transport by these tissues remains obscure. To date direct evidence in support of the two models is meager indeed. Localization of CFTR in the luminal membrane of all CFTR-expressing epithelia is well documented (14Crawford I. Maloney P.C. Zeitlin P.L. Guggino W.B. Hyde S.C. Turley H. Gatter K.C. Harris A. Higgins C.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9262-9266Crossref PubMed Scopus (389) Google Scholar, 15Marino C.R. Matovcik L.M. Gorelik F.S. Cohn J.A. J. Clin. Invest. 1991; 88: 712-716Crossref PubMed Scopus (252) Google Scholar, 16Zeng W. Lee M.G. Yan M. Diaz J. Benjamin I. Marino C.R. Kopito R.R. Freedman S. Cotton C. Muallem S. Thomas P.J. Am. J. Physiol. 1997; 273: C442-C455Crossref PubMed Google Scholar). Cl−/HCO3− exchange activity was found in the BLM and LM of pancreatic (17Zhao H. Star R.A. Muallem S. J. Gen. Physiol. 1994; 104: 57-85Crossref PubMed Scopus (114) Google Scholar) and submandibular gland (SMG) ducts (18Zhao H. Xu X. Diaz J. Muallem S. J. Biol. Chem. 1995; 270: 19599-19605Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In SMG ducts the AE isoform 2 (AE2) was localized in the BLM (19He X. Tse C.-M. Donowitz M. Alper S.L. Gabriel S.E. Baum B.J. Pfluegers Arch. Eur. J. Physiol. 1997; 433: 260-268Crossref PubMed Scopus (141) Google Scholar). The isoform(s) expressed in the LM is not known. A relationship between HCO3− secretion and CFTR was documented in two cell lines and intestinal epithelia. In a human airway epithelial cell line CFTR-dependent HCO3− conductance (20Illek B. Yankaskas J.R. Machen T.E. Am. J. Physiol. 1997; 272: L752-L761PubMed Google Scholar, 21Lee M.C. Penland C.M. Widdicombe J.H. Wine J.J. Am. J. Physiol. 1998; 274: L450-L453Crossref PubMed Google Scholar) was proposed to be mediated by CFTR itself. By contrast, similar studies in a human pancreatic duct cell line concluded that electrogenic Cl−and HCO3− secretions are mediated by independent proteins (22Cheng H.S. Leung P.Y. Chew S.B.C. Leung P.S. Lam S.Y. Wong W.S. Wang Z.D. Chan H.C. J. Membr. Biol. 1998; 164: 155-167Crossref PubMed Scopus (35) Google Scholar). In duodenal epithelium basal and acid-stimulated HCO3− secretions were reduced or absent in CFTR −/− mice (23Hogan D.L. Crombie D.L. Isenberg J.I. Svendsen P. Schaffalitzky De Muckadell O.B. Ainsworth M.A. Gastroenterology. 1997; 113: 533-541Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Surprisingly, in a recent study Seidler et al. (24Seidler U. Blumenstein I. Kretz A. Viellard-Baron D. Rossmann H. Colledge W.H. Evans M. Ratcliff R. Gregor M. J. Physiol (Lond.). 1997; 505: 411-423Crossref Scopus (220) Google Scholar) showed that all forms of HCO3− secretion stimulated by agonists or agents that elevate cAMP, cGMP, and, in particular, [Ca2+]i were impaired in the intestinal epithelia of CFTR −/− mice. These studies suggest the likely involvement of CFTR in the electrogenic component of HCO3− secretion, which is particularly prominent in the intestine (25Allen A. Flemstrom G. Garner A. Kivilaakso E. Physiol. Rev. 1993; 73: 823-857Crossref PubMed Scopus (402) Google Scholar, 26Grubb B.R. Am. J. Physiol. 1995; 268: G505-G513PubMed Google Scholar). However, a large fraction of HCO3− secretion in tissues such as salivary glands (2Cook D.I. van Lennep E.W. Roberts M.L. Young J.A. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1061-1117Google Scholar) and the rat and mouse pancreas (1Argent B.E. Case R.M. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1478-1498Google Scholar) is mediated by an electroneutral HCO3− transport mechanism. The role of CFTR in this critical component of HCO3− secretion is unknown. The intimate relationship between CFTR expression and HCO3− secretion seen in intestinal (23Hogan D.L. Crombie D.L. Isenberg J.I. Svendsen P. Schaffalitzky De Muckadell O.B. Ainsworth M.A. Gastroenterology. 1997; 113: 533-541Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar,24Seidler U. Blumenstein I. Kretz A. Viellard-Baron D. Rossmann H. Colledge W.H. Evans M. Ratcliff R. Gregor M. J. Physiol (Lond.). 1997; 505: 411-423Crossref Scopus (220) Google Scholar) and airway epithelia (27Smith J.J. Welsh M.J. J. Clin. Invest. 1992; 89: 1148-1153Crossref PubMed Scopus (224) Google Scholar) raises the question of whether and how CFTR modulates HCO3− secretion in other CFTR-expressing tissues. In addition to its possible function as a regulator of a HCO3− conductive channel, CFTR may also regulate luminal Cl−/HCO3− exchange activity. In a recent study (28Lee M.G. Wigley W.C. Zeng W. Noel L.E. Marino C.R. Thomas P.J. Muallem S. J. Biol. Chem. 1999; 274: 3414-3421Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) we used cells stably transfected with CFTR and transient transfection of WT CFTR and several CFTR mutants to demonstrate regulation of AE activity by CFTR. To evaluate the physiological relevance of these findings, in the present work we report the regulation of the luminal AE activity by CFTR in the mouse SMG and pancreatic ducts. The SMG and pancreatic ducts were selected as model systems for several reasons. The fraction of electroneutral HCO3− secretion in these tissues is relatively high (1Argent B.E. Case R.M. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1478-1498Google Scholar, 2Cook D.I. van Lennep E.W. Roberts M.L. Young J.A. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1061-1117Google Scholar). Among all CFTR-expressing tissues, the mechanism of fluid and electrolyte secretion is understood best in the SMG (2Cook D.I. van Lennep E.W. Roberts M.L. Young J.A. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1061-1117Google Scholar). The SMG and pancreatic ducts express high levels of CFTR (15Marino C.R. Matovcik L.M. Gorelik F.S. Cohn J.A. J. Clin. Invest. 1991; 88: 712-716Crossref PubMed Scopus (252) Google Scholar), and the abundance of ducts in the SMG (2Cook D.I. van Lennep E.W. Roberts M.L. Young J.A. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1061-1117Google Scholar, 29Xu X. Zhao H. Diaz J. Muallem S. J. Biol. Chem. 1995; 270: 19606-19612Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) facilitates experimentation. Although the mouse is not the ideal species to study HCO3− secretion (1Argent B.E. Case R.M. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1478-1498Google Scholar, 6Ishiguro H. Naruse S. Steward M.C. Kitagawa M. Ko S.B.H. Hayakawa T. Case R.M. J. Physiol. (Lond.). 1998; 511: 407-422Crossref Scopus (75) Google Scholar, 30Chaturapanich G. Ishibashi H. Dinudom A. Young J.A. Cook D.I. J. Physiol. (Lond.). 1997; 503: 583-598Crossref Scopus (18) Google Scholar), it was selected for the present work because of the availability of ΔF/ΔF mice (31Zeiher B.G. Eichwald E. Zabner J. Smith J.J. Puga A.P. McCray Jr., P.B. Capecchi M.R. Welsh M.J. Thomas K.R. J. Clin. Invest. 1995; 96: 2051-2064Crossref PubMed Scopus (248) Google Scholar). To supplement the studies in the native ducts we also used the human colonic cell line T84 because these cells have been used extensively to study the properties of naturally occurring CFTR (32Barrett K.E. Am. J. Physiol. 1993; 265: C859-C868Crossref PubMed Google Scholar,33Illek B. Fischer H. Machen T.E. Am. J. Physiol. 1996; 270: C265-C275Crossref PubMed Google Scholar). We show here that stimulation of CFTR with cAMP increased Cl−/HCO3− exchange activity in T84 cells. More importantly, stimulation of CFTR by cAMP resulted in selective activation of luminal Cl−/HCO3− exchange activity in SMG and pancreatic ducts of WT mice, which was absent in ducts prepared from ΔF/ΔF mice. We conclude that CFTR regulates luminal Cl−/HCO3− exchange activity of SMG and pancreatic ducts and probably other CFTR-expressing cells. The standard perfusate was termed solution A and contained (in mm) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 Hepes (pH 7.4 with NaOH), and 10 glucose. The HCO3−-buffered NaCl solution B contained (in mm) 120 NaCl, 25 NaHCO3, 5 KCl, 1 MgCl2, 1 CaCl2, 5 Hepes (pH 7.4 with NaOH), and 10 glucose. The HCO3−-buffered Cl−-free solution C contained (in mm) 120 Na+-gluconate, 25 NaHCO3, 5 K+-gluconate, 1 MgSO4, 9.3 hemicalcium cyclamate, 5 Hepes (pH 7.4 with NaOH), and 10 glucose. To prepare HCO3−-buffered high KCl (100 mm K+) solution D, 95 mm NaCl in solution B was replaced with 95 mm KCl. To prepare HCO3−-buffered, high K+(100 mm K+), Cl−-free solution E, 95 mm Na+-gluconate was replaced with 95 mm K+-gluconate in solution C. For calibration of intracellular Cl−, solution E was supplemented with 5 μm nigericin and 10 μm tributyltin cyanide. The KSCN solution contained (in mm) 127 KSCN, 25 choline- HCO3−, and 5 Hepes (pH 7.4 with 2m Tris). The osmolarity of all solutions was adjusted to 310 mosm with the major salt prior to use. T84 cells were purchased from American Type Culture Collection (ATCC CCL 248, Rockville, MD) and maintained in a 1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum. The cells were plated on a sterile 22 × 40-mm coverslip at a density of 2.5 × 105 cells/cm2 for intracellular pH measurements. A cystic fibrosis mouse model in which the ΔF508 mutation was introduced in the mouse CFTR by gene targeting in ES cells (31Zeiher B.G. Eichwald E. Zabner J. Smith J.J. Puga A.P. McCray Jr., P.B. Capecchi M.R. Welsh M.J. Thomas K.R. J. Clin. Invest. 1995; 96: 2051-2064Crossref PubMed Scopus (248) Google Scholar) was obtained from Dr. Kirk R. Thomas (Eccles Institute of Human Genetics, HHMI, University of Utah School of Medicine, Salt Lake City). The mice were maintained on a standard diet, and genotyping was carried out on day 14 postpartum as described previously (16Zeng W. Lee M.G. Yan M. Diaz J. Benjamin I. Marino C.R. Kopito R.R. Freedman S. Cotton C. Muallem S. Thomas P.J. Am. J. Physiol. 1997; 273: C442-C455Crossref PubMed Google Scholar). Duct fragments from the mouse SMG were prepared by a slight modification of our published procedure (16Zeng W. Lee M.G. Yan M. Diaz J. Benjamin I. Marino C.R. Kopito R.R. Freedman S. Cotton C. Muallem S. Thomas P.J. Am. J. Physiol. 1997; 273: C442-C455Crossref PubMed Google Scholar). Mice were sacrificed by exposure to a methoxyflurane-saturated atmosphere and subsequent cervical dislocation, and the SMGs were removed to a cold pancreatic solution A (PSA). The composition of PSA was (in mm) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 Hepes (pH 7.4 with NaOH), 10 glucose, 10 pyruvate, 0.1% bovine serum albumin, and 0.02% soybean trypsin inhibitor. Each gland was cleaned by injection of 5 ml of PSA and minced. The minced tissue was transferred to 8 ml of PSA containing 2.5 mg of collagenase (CLS4, 254 units/mg; Worthington Biochemicals) and digested for 8–10 min at 37 °C. The dissociated cells were then washed twice with PSA, resuspended in 2 ml of PSA, and kept on ice until use. Microperfusion experiments were performed with microdissected pancreatic ducts from WT and ΔF/ΔF mice. The procedure for preparation and perfusion of the main pancreatic duct was identical to that used for perfusion of the rat pancreatic duct (17Zhao H. Star R.A. Muallem S. J. Gen. Physiol. 1994; 104: 57-85Crossref PubMed Scopus (114) Google Scholar). The ducts were dissected in PSA, cannulated, and perfused through the lumen and the bath with solution A. After completion of BCECF loading the ducts were perfused with HCO3−-buffered solution B for at least 10 min prior to manipulation of Cl−gradients. The procedure of pHi measurement in T84 cells was identical to that described in detail in our recent work (28Lee M.G. Wigley W.C. Zeng W. Noel L.E. Marino C.R. Thomas P.J. Muallem S. J. Biol. Chem. 1999; 274: 3414-3421Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In the case of SMG cells, the dissociated cells were loaded with BCECF by a 10-min incubation at room temperature in PSA containing 1 μm BCECF-AM. The cells were then washed with PSA and plated on a polylysine-coated coverslip that was assembled into a perfusion chamber. The chamber was placed on an inverted microscope, and intralobular ducts were identified by morphology. The BCECF fluorescence of 10–16 cells of a duct fragment was recorded at excitation wavelengths of 440 and 490 nm. Fluorescence ratios of 490/440 were calibrated using the procedures described previously (28Lee M.G. Wigley W.C. Zeng W. Noel L.E. Marino C.R. Thomas P.J. Muallem S. J. Biol. Chem. 1999; 274: 3414-3421Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In the case of the perfused pancreatic duct BCECF loading was accomplished by including 2.5 μm BCECF-AM in the luminal perfusate for 10 min. Changes in Cl−/HCO3−exchange activity were estimated from the initial rate of pHi changes (T84 cells and pancreatic ducts) or from the extent of pHi changes (SMG ducts). Initial rates of pHi changes were obtained from the first derivative of the traces using a single exponential fit. The extent of pHi changes was estimated by averaging the pHi changes measured as a result of Cl− removal and addition. All results are given as mean ± S.E. of the indicated number of experiments. [Cl−]i was measured with the aid of the Cl−-sensitive dye MQAE using the procedure described before for 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ; 18) with minor modifications. SMG cells were suspended in PSA containing 10 mm MQAE and incubated for 20 min at room temperature and 40 min at 0 °C before plating on coverslips. About 2 min after plating, unattached cells and external MQAE were washed by starting the perfusion with solution A. MQAE fluorescence was measured at an excitation wavelength of 360 nm with the dichroic mirror and emission cut-off filter set normally used to monitor Fura-2 fluorescence. At the end of each experiment a two-point calibration procedure was performed. To obtain the maximal fluorescence the cells were perfused with high K+, Cl−-free solution containing 5 μm nigericin and 10 μm tributyltin cyanide. Incubation in a Cl−-free solution without ionophores did not result in complete depletion of intracellular Cl−. To obtain the minimal fluorescence the cells were then exposed to a solution containing 127 mm KSCN. Significant dye leak, in particular after exposure to tributyltin cyanide, precluded a more extensive in vivo calibration. A Stern-Volmer constant of 12.4 m−1 reported before for rabbit SMG ducts (34Lau K.R. Evans R.L. Case R.M. Pfluegers Arch. Eur. J. Physiol. 1994; 427: 24-32Crossref PubMed Scopus (28) Google Scholar) was used to calculate [Cl−]i. The results of multiple experiments with each cell type and under the different conditions were analyzed using paired or nonpaired Student'st test, as appropriate. We have described previously the regulation of AE activity by CFTR in cells stably or transiently transfected with CFTR (28Lee M.G. Wigley W.C. Zeng W. Noel L.E. Marino C.R. Thomas P.J. Muallem S. J. Biol. Chem. 1999; 274: 3414-3421Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The purpose of the present work was to determine whether such regulation exists in a cell line and in native cells naturally expressing CFTR. The first set of experiments was performed with the human colonic cell line T84, which can serve as a suitable model system in future studies. This cell line has been used in the past in several studies as a model system to characterize natively expressed CFTR (32Barrett K.E. Am. J. Physiol. 1993; 265: C859-C868Crossref PubMed Google Scholar, 33Illek B. Fischer H. Machen T.E. Am. J. Physiol. 1996; 270: C265-C275Crossref PubMed Google Scholar). Fig. 1 shows representative experiments, and Fig. 2 summarizes the results under each experimental condition. DIDS-sensitive, Cl−- and HCO3−-dependent changes in pHi indicate the expression of relatively modest AE activity in T84 cells. Stimulation of the cells with 5 μm forskolin caused a reproducible reduction in pHi. This reduction in pHi was less pronounced than that observed in NIH 3T3 and HEK 293 cells expressing high levels of CFTR. Removal and addition of Cl− to the incubation medium showed that forskolin increased the rate of AE activity by about 2.2-fold or 0.051 ΔpH unit/min. As was found in NIH 3T3 and HEK 293 cells expressing CFTR (28Lee M.G. Wigley W.C. Zeng W. Noel L.E. Marino C.R. Thomas P.J. Muallem S. J. Biol. Chem. 1999; 274: 3414-3421Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), the AE activity stimulated by forskolin was not affected by inhibition of CFTR-mediated Cl− current with 0.1 mm N-phenylanthranilic acid (DPC in Fig. 1a) or 0.1 mm glibenclamide (Glib in Fig. 1b). On the other hand, the AE activity was nearly abolished by 0.5 mm DIDS (Fig. 1b), as was found in 293 cells expressing modest levels of CFTR (see Fig. 7b of Ref. 28Lee M.G. Wigley W.C. Zeng W. Noel L.E. Marino C.R. Thomas P.J. Muallem S. J. Biol. Chem. 1999; 274: 3414-3421Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar).Figure 2Properties of AE activity in T84 cells.The protocols of Fig. 1 were used to evaluate the effect of forskolin on AE activity in the presence of normal or high K+ media and the effect of DIDS on AE activity before and after stimulation with forskolin. The inset plots the forskolin-stimulated AE activity under normal and high K+ conditions. The figure shows the mean ± S.E. of the indicated number of experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The potential regulation or function of CFTR as a HCO3− channel (20Illek B. Yankaskas J.R. Machen T.E. Am. J. Physiol. 1997; 272: L752-L761PubMed Google Scholar, 35Gray M.A. Harris A. Coleman L. Greenwell J.R. Argent B.E. Am. J. Physiol. 1989; 257: C240-C251Crossref PubMed Google Scholar, 36Gray M.A. Winpenny J.P. McAlroy H. Argent B.E. Biosci. Rep. 1995; 15: 531-541Crossref PubMed Scopus (25) Google Scholar, 37Poulsen J.H. Fischer H. Illek B. Machen T.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5340-5344Crossref PubMed Scopus (354) Google Scholar) raised the possibility that the rate and extent of HCO3− influx during Cl−removal are underestimated because of the CFTR-dependent efflux of HCO3− which entered the cells through the anion exchanger. To test this possibility we measured the effect of membrane depolarization on HCO3− fluxes. In Figs. 1cand 2, T84 cells were depolarized by raising the external K+ concentration from 5 to 100 mm. This had a minor effect on pHi. Membrane depolarization nearly doubled the initial rate of HCO3− influx observed upon Cl− removal (Figs. 1c and Fig. 2,first and third bars fromleft). Stimulation with forskolin of cell" @default.
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• W2042679775 title "Cystic Fibrosis Transmembrane Conductance Regulator Regulates Luminal Cl−/HCO3−Exchange in Mouse Submandibular and Pancreatic Ducts" @default.
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