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• W2010468499 abstract "Measurement of [Cl−] i and the Cl− current in the rat salivary submandibular gland (SMG) acinar and duct cells was used to evaluate the role of Cl− channels in the regulation of [Cl−] i during purinergic stimulation. Under resting conditions [Cl−] i averaged 56 ± 8 and 26 ± 7 mm in acinar and duct cells, respectively. In both cells, stimulation with 1 mm ATP resulted in Cl− efflux and subsequent influx. Inhibition of NaKCl2 cotransport had no effect on [Cl−] i changes in duct cells and inhibited only about 50% of Cl− uptake in acinar cells. Accordingly, low levels of expression of NaKCl2 cotransporter protein were found in duct cells. Acinar cells expressed high levels of the cotransporter. Measurement of Cl− current under selective conditions revealed that acinar and duct cells express at least five distinct Cl− channels; a ClCO-like, volume-sensitive, inward rectifying, Ca2+-activated and CFTR-like Cl− currents. ATP acting on both cell types activated at least two channels, the Ca2+-activated Cl− channel and a Ca2+-independent glibenclamide-sensitive Cl−-current, possibly cystic fibrosis transmembrane regulator (CFTR). Of the many nucleotides tested only 2′-3′-benzoylbenzoyl (Bz)-ATP and UTP activated Cl− channels in SMG cells. Despite their relative potency in increasing [Ca2+] i, BzATP in both SMG cell types largely activated the Ca2+-independent, glibenclamide-sensitive Cl− current, whereas UTP activated only the Ca2+-dependent Cl− current. We interpret this to suggest that BzATP and UTP largely activate Cl− channels residing in the membrane expressing the receptor for the active nucleotide. The present studies reveal a potentially new mechanism for transcellular Cl− transport in a CFTR-expressing tissue, the SMG. Coordinated action of the P2z (luminal) and P2u (basolateral) receptors can mediate part of the transcellular Cl− transport by acinar and duct cells to determine the final electrolyte composition of salivary fluid. Measurement of [Cl−] i and the Cl− current in the rat salivary submandibular gland (SMG) acinar and duct cells was used to evaluate the role of Cl− channels in the regulation of [Cl−] i during purinergic stimulation. Under resting conditions [Cl−] i averaged 56 ± 8 and 26 ± 7 mm in acinar and duct cells, respectively. In both cells, stimulation with 1 mm ATP resulted in Cl− efflux and subsequent influx. Inhibition of NaKCl2 cotransport had no effect on [Cl−] i changes in duct cells and inhibited only about 50% of Cl− uptake in acinar cells. Accordingly, low levels of expression of NaKCl2 cotransporter protein were found in duct cells. Acinar cells expressed high levels of the cotransporter. Measurement of Cl− current under selective conditions revealed that acinar and duct cells express at least five distinct Cl− channels; a ClCO-like, volume-sensitive, inward rectifying, Ca2+-activated and CFTR-like Cl− currents. ATP acting on both cell types activated at least two channels, the Ca2+-activated Cl− channel and a Ca2+-independent glibenclamide-sensitive Cl−-current, possibly cystic fibrosis transmembrane regulator (CFTR). Of the many nucleotides tested only 2′-3′-benzoylbenzoyl (Bz)-ATP and UTP activated Cl− channels in SMG cells. Despite their relative potency in increasing [Ca2+] i, BzATP in both SMG cell types largely activated the Ca2+-independent, glibenclamide-sensitive Cl− current, whereas UTP activated only the Ca2+-dependent Cl− current. We interpret this to suggest that BzATP and UTP largely activate Cl− channels residing in the membrane expressing the receptor for the active nucleotide. The present studies reveal a potentially new mechanism for transcellular Cl− transport in a CFTR-expressing tissue, the SMG. Coordinated action of the P2z (luminal) and P2u (basolateral) receptors can mediate part of the transcellular Cl− transport by acinar and duct cells to determine the final electrolyte composition of salivary fluid. Salivary glands, in particular the submandibular gland (SMG), 1The abbreviations used are: SMG, submandibular gland; BLM, basolateral membrane; LM, luminal membrane; CFTR, cystic fibrosis transmembrane regulator; GLM, glibenclamide; BzATP, 2′-3′-benzoylbenzoyl-ATP; SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium; ORCC, outward rectifying Cl− channel; MAb, monoclonal antibody; ATPγS, adenosine 5′-O-(thiotriphosphate). 1The abbreviations used are: SMG, submandibular gland; BLM, basolateral membrane; LM, luminal membrane; CFTR, cystic fibrosis transmembrane regulator; GLM, glibenclamide; BzATP, 2′-3′-benzoylbenzoyl-ATP; SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium; ORCC, outward rectifying Cl− channel; MAb, monoclonal antibody; ATPγS, adenosine 5′-O-(thiotriphosphate). have been extensively used as a model system for fluid and electrolyte secretion by secretory epithelial cells (1Petersen O.H. J. Physiol. 1992; 448: 1-51Crossref PubMed Scopus (366) Google 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, 3Turner R.J. Dobrosielski-Vergona K. Biology of the Salivary Gland. CRC Press, Boca Raton, FL1993: 105-127Google Scholar). Salivary secretion occurs in two steps. Acinar cells secrete the primary isotonic, NaCl-rich fluid. The duct changes electrolyte composition and to some extent the osmolarity of the primary fluid by absorbing the NaCl and secreting KHCO3− (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, 3Turner R.J. Dobrosielski-Vergona K. Biology of the Salivary Gland. CRC Press, Boca Raton, FL1993: 105-127Google Scholar). The central feature of the accepted model of fluid and electrolyte secretion by salivary acinar cells is transepithelial Cl− movement as the driving force for fluid and electrolyte secretion (1Petersen O.H. J. Physiol. 1992; 448: 1-51Crossref PubMed Scopus (366) Google Scholar,3Turner R.J. Dobrosielski-Vergona K. Biology of the Salivary Gland. CRC Press, Boca Raton, FL1993: 105-127Google Scholar). Functional (4Martinez J.R. Cassity N. Am. J. Physiol. 1983; 245: G711-G716PubMed Google Scholar) and immunofluorescence localization (5Lytle C. Xu J.-C. Biemesderfer D. Forbush III, B. Am. J. Physiol. 1995; 269: C1496-C1505Crossref PubMed Google Scholar, 6He X. Tse C.-M. Donowitz M. Alper S.J. Gabriel S.E. Baum B.J. Pflugers Arch. 1997; 433: 260-268Crossref PubMed Scopus (141) Google Scholar) point to NaKCl2 cotransport as the Cl− entry mechanism in the basolateral membrane (BLM). A Ca2+-activated, outward rectifying Cl− channel, found in many salivary acinar cells (1Petersen O.H. J. Physiol. 1992; 448: 1-51Crossref PubMed Scopus (366) Google 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), is believed to be the Cl− exit pathway in the luminal membrane. This model, however, cannot account for a large fraction (∼40%) of Cl− transport by SMG acinar cells. A search for alternative Cl− pathways revealed the presence of at least three Cl− channels in parotid acinar cells; Ca2+-activated, volume-sensitive and hyperpolarization-activated Cl− channels (7Arreola J. Park K. Melvin J.E. Begenisich T. J. Physiol. 1996; 490: 351-362Crossref PubMed Scopus (66) Google Scholar, 8Arreola J. Melvin J.E. Begenisich T. J. Gen. Physiol. 1996; 108: 35-47Crossref PubMed Scopus (137) Google Scholar). Recently, we demonstrated the expression of CFTR in the luminal membrane (LM) of SMG acinar cells (9Zeng W.-Z. Lee M.G. Yan M. Diaz J. Benjamin I. Marino C.R. Kopito R. Freedman S. Cotton K. Muallem S. Thomas P. Am. J. Physiol. 1997; 273: C442-C455Crossref PubMed Google Scholar). Except for CFTR, the membrane localization, possible role in Cl− secretion, and regulation by agonists of the various Cl− channels are not known. Electrolyte transport by salivary ducts, including Cl− reabsorption, is not well understood on the molecular level. The bulk of Cl− entry in the LM and Cl− efflux across the BLM are assumed to be mediated by Cl−/HCO3− exchange and Cl− channels, respectively (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). Indeed, Cl−/HCO3− exchange activity was found in the LM of SMG duct cells (10Zhao H. Xu X. Diaz J. Muallem S. J. Biol. Chem. 1995; 270: 19599-19605Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Additional electrogenic Cl− transport, which is needed to balance the electrogenic Na+ reabsorption, may occur through luminal and basolateral Cl− channels (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). This prediction is based on the finding that changing LM and BLM Cl− concentration affects the transepithelial potential and resistance of the excretory SMG duct (11Augustus J. Bigman J. Van Os C.H. J. Membr. Biol. 1978; 43: 203-226Crossref PubMed Scopus (20) Google Scholar). Luminal Cl− permeability is likely to be in part mediated by CFTR. Duct cells of all salivary glands express CFTR (12Trezise A. Buchwald M. Nature. 1991; 353: 434-437Crossref PubMed Scopus (291) Google Scholar) in the LM (13Webster P. Vanacore L. Nairn A.C. Marino C.R. Am. J. Physiol. 1994; 267: C340-C348Crossref PubMed Google Scholar). Another Cl− channel found in SMG duct cells is a ClC2-like channel (14Komwatana P. Dinudom A. Young J.A. Cook D.I. Pflugers Arch. 1994; 428: 641-647Crossref PubMed Scopus (37) Google Scholar). The membrane localization and physiological function of this channel are not known, although it may participate in cell volume regulation, as in other cell types (15Valverde M.A. Hardy S.P. Sepulveda F.V. FASEB J. 1995; 9: 509-515Crossref PubMed Scopus (48) Google Scholar). How agonists regulate Cl− channels and Cl− transport in SMG and other salivary glands is only partially understood. In acinar cells Ca2+ mobilizing agonists activate the Ca2+-dependent Cl− channel (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, 3Turner R.J. Dobrosielski-Vergona K. Biology of the Salivary Gland. CRC Press, Boca Raton, FL1993: 105-127Google Scholar). Agonists acting through cAMP elevation can activate CFTR in acinar and duct cells (9Zeng W.-Z. Lee M.G. Yan M. Diaz J. Benjamin I. Marino C.R. Kopito R. Freedman S. Cotton K. Muallem S. Thomas P. Am. J. Physiol. 1997; 273: C442-C455Crossref PubMed Google Scholar, 16Dinudom A.P. Komwatana P. Young J.A. Cook D.A. Am. J. Physiol. 1995; 268: G806-G812PubMed Google Scholar). Salivary acinar and duct cells also respond to purinergic stimulation by a change in [Ca2+] i (17Gallacher D.V. Nature. 1982; 296: 83-86Crossref PubMed Scopus (89) Google Scholar, 18Soltoff S.P. McMillian M.K. Talamo B.R. Am. J. Physiol. 1992; 262: C934-C940Crossref PubMed Google Scholar, 19McMillian M.K. Soltoff S.P. Cantley L.C. Rudel R. Talamo B.R. Br. J. Pharmacol. 1993; 198: 453-461Crossref Scopus (58) Google Scholar, 20McMillian M.K. Soltoff S.P. Lechleiter J.D. Cantley L.C. Talamo B.R Biochem. J. 1994; 255: 291-300Google Scholar, 21Hurley T.W. Shoemaker D.D. Ryan M.P. Am. J. Physiol. 1993; 265: C1472-C1478Crossref PubMed Google Scholar, 22Hurley T.W. Ryan M.P. Shoemaker D.D. Arch. Oral Biol. 1994; 39: 205-212Crossref PubMed Scopus (21) Google Scholar). However, the identities of the receptors and whether they regulate Cl− channels in salivary gland cells are not known. In other epithelia, in particular airway and nasal epithelia, regulation of Cl− channels by purinergic receptors emerged as an important physiological activity with possible therapeutical implications (23Stutts M.J. Chinet T.C. Mason S.J. Fullton J.M. Clarke L.L. Boucher R.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1621-1625Crossref PubMed Scopus (144) Google Scholar, 24Stutts M.J. Fritz G.J. Paradiso A.M. Boucher R.C. Am. J. Physiol. 1994; 267: C1442-C1451Crossref PubMed Google Scholar, 25Boucher R.C. Am. J. Respir. Crit. Care Med. 1994; 150: 271-281Crossref PubMed Scopus (315) Google Scholar, 26Boucher R.C. Am. J. Respir. Crit. Care Med. 1994; 150: 581-593Crossref PubMed Scopus (198) Google Scholar, 27Hwang T.-H. Schwiebert E.M. Guggino W.B. Am. J. Physiol. 1996; 270: C1611-C1623Crossref PubMed Google Scholar). Different P2 receptors are expressed in the LM and BLM of these cells and appear to regulate multiple and different Cl− channels. Thus, apical ATP acting through P2Y2receptors activates the Ca2+-dependent Cl− channels, CFTR, and probably indirectly the outward rectifying Cl− channels (ORCC). Basolateral ATP acting through P2Y3 activates only CFTR in a Ca2+- and cAMP-independent manner (27Hwang T.-H. Schwiebert E.M. Guggino W.B. Am. J. Physiol. 1996; 270: C1611-C1623Crossref PubMed Google Scholar). More recently it was reported that the LM of nasal epithelial cells express UDP-sensitive receptors different from P2Y2, which are coupled to inositol 1,4,5-trisphosphate signaling and activate Cl− secretion in these cells (28Lazarowski E.R. Paradiso A.M. Watt W.C. Harden T.K. Boucher R.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2599-2603Crossref PubMed Scopus (100) Google Scholar). Due to our recent discovery of the expression of CFTR in SMG acinar cells, the localization of P2 receptors in the LM and BLM of SMG cells (29Lee M.G. Zeng W.Z. Muallem S. J. Biol. Chem. 1997; 272: 32951-32955Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and the importance of purinergic regulation of Cl− secretion in epithelia, in the present study we used [Cl−] i measurements, immunoanalysis, and recording of Cl− currents to characterize and evaluate the contribution of Cl− channels and the NaKCl2cotransporter to Cl− transport during purinergic stimulation of SMG acinar and duct cells. The combined results point to the central role of CFTR and the Ca2+-activated Cl− channel in regulating Cl− transport in SMG duct cells and their contribution to Cl− transport relative to that by the NaKCl2 cotransporter in acinar cells. The general methods are identical to those in our companion study (29Lee M.G. Zeng W.Z. Muallem S. J. Biol. Chem. 1997; 272: 32951-32955Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), except for the following. SPQ-loaded acini and duct fragments were plated on coverslips and perfused in a manner similar to that described for Fura 2-loaded cells (29Lee M.G. Zeng W.Z. Muallem S. J. Biol. Chem. 1997; 272: 32951-32955Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). SPQ fluorescence was measured at an excitation wavelength of 380 nm and was calibrated by incubating the cells with high K+ solutions containing different concentrations of Cl− (0–100 mm), 10 μmtributyltin, and 2.5 μm nigericin. A maximal fluorescence quench was obtained by exposing the cells to a solution containing 150 mm SCN− (for details, see Zhao and Muallem (30Zhao H. Muallem S. J. Gen. Physiol. 1995; 106: 1225-1242Crossref PubMed Scopus (21) Google Scholar)). The calibration curves were used to yield a Stern-Volmer constant of 15.7 ± 1.7 m−1(n = 4). The average is from four experiments with ducts and acini in the same recording field. The dye showed similar behavior in both cell types. As reported before for pancreatic acini (30Zhao H. Muallem S. J. Gen. Physiol. 1995; 106: 1225-1242Crossref PubMed Scopus (21) Google Scholar), SPQ fluorescence in SMG cells was minimally affected by changes in intracellular pH. 86Rb uptake was measured essentially as described previously (30Zhao H. Muallem S. J. Gen. Physiol. 1995; 106: 1225-1242Crossref PubMed Scopus (21) Google Scholar). SMG ducts and acini isolated by the Accudenz gradient were incubated in PSA buffer for 20 min at 37 °C. 86Rb uptake was initiated by diluting the cells (1:1) into warm PSA containing 86Rb (∼4.105 cpm/ml) and 0.2 mm bumetanide, 2 mm ouabain, or bumetanide and ouabain. At the indicated times triplicate samples of 0.5 ml were transferred to 8 ml of a cold stop solution containing 150 mm NaCl, 1 mmLaCl3, 10 mm Hepes (pH 7.4 with NaOH), and 1 mg/ml bovine serum albumin. The cells were washed three times with the same solution by centrifugation and resuspended in 0.5 ml of 0.2m NaOH, and 86Rb was determined by scintillation counting. 10–20-μl samples from at least six tubes were taken for measurement of protein, and the results were calculated in terms of nanomoles of K+/mg of protein. Western blot analysis of SMG proteins and immunolocalization were essentially as described in Lee et al. (31Lee M.G. Xu X. Zeng W. Diaz J. Wojcikiewicz R.J.H. Kuo T.H. Wuytack F. Racymaekers L. Muallem S. J. Biol. Chem. 1997; 272: 15765-15770Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). SMG ducts and acini were separated twice on an Accudenz gradient (32Xu X. Diaz J. Zhao H. Muallem S. J. Physiol. 1996; 491: 647-662Crossref PubMed Scopus (58) Google Scholar). Pancreatic and parotid acini were isolated by a standard collagenase digestion protocol (30Zhao H. Muallem S. J. Gen. Physiol. 1995; 106: 1225-1242Crossref PubMed Scopus (21) Google Scholar). The cells were pelleted and solubilized in an SDS-containing buffer, and the proteins were separated by SDS-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride membranes and the NaKCl2 cotransporter was detected with mAb T4(kindly provided by Dr. C. Lytle, Riverside, CA). The antibodies were detected by the ECL procedure. For immunolocalization, SMG embedded in OCT were used to cut 4-μm sections. The sections were plated on polylysine-coated coverslips, dried, and permeabilized with cold methanol. After blocking nonspecific sites by incubation with a buffer containing 5% goat serum, 1% bovine serum albumin, and 0.1% gelatin, the slices were incubated in the same buffer containing a 1:2000 dilution of the mAb for 1.5 h at room temperature. After washing, the antibodies were detected with 1:100 dilution of a secondary IgG tagged with fluorescein. Images were collected with a Bio-Rad MRC 1000 confocal microscope. To evaluate the role of different Cl− transporters in [Cl−] i regulation we first measured [Cl−] i with SPQ. Fig.1 shows that [Cl−] i was differently regulated in the SMG duct and acinar cells. In resting duct cells [Cl−] i averaged 26 ± 7 (n = 34) and in acinar cells 56 ± 8 mm (n = 29). Stimulation of duct cells with 1 mm ATP caused a rapid reduction in [Cl−] i to about 13.5 ± 4.5 mm. Subsequently [Cl−] i increased over 1.5–2 min and stabilized at 36 ± 6 mm (n = 11) (Fig. 1 a). Stimulation of acini in the same recording field with 1 mm ATP caused a reduction in [Cl−] i to 17 ± 5 mm, which was then increased to about 52 ± 5 mm (n= 11) (Fig. 1 d). The absence of HCO3− in the perfusion medium ensured that these [Cl−] i changes are not affected by Cl−/HCO3− exchange (10Zhao H. Xu X. Diaz J. Muallem S. J. Biol. Chem. 1995; 270: 19599-19605Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Another potential transporter mediating some of the agonist-dependent [Cl−] i changes is the NaKCl2 cotransporter (33Zhang B.-X. Cragoe Jr., G.H. Melvin J.E. Am. J. Physiol. 1993; 264: C54-C62Crossref PubMed Google Scholar, 34Evans R. Turner J.R. J. Physiol. 1997; 499: 351-359Crossref PubMed Scopus (57) Google Scholar). Fig. 1 e shows that in SMG acinar cells 0.1 mm bumetanide reduced the rate of Cl− uptake by about 46 ± 9% and [Cl−] i stabilized at 38 ± 8 mm(n = 9). In duct cells bumetanide failed to affect the [Cl−] i changes evoked by ATP stimulation (n = 9) (Fig. 1 b). On the other hand, Fig.1, c and f, shows that 100 μm of the general Cl− channel blocker diphenylamine-2-carboxylic acid largely inhibited Cl− efflux, and thus all [Cl−] i changes evoked by ATP (n= 4 for each cell type). In view of a recent report of the expression of the secretory NaKCl2 cotransporter in SMG duct cells (6He X. Tse C.-M. Donowitz M. Alper S.J. Gabriel S.E. Baum B.J. Pflugers Arch. 1997; 433: 260-268Crossref PubMed Scopus (141) Google Scholar) and the results in Fig. 1, we proceeded to examine more directly the expression and activity of NaKCl2 cotransport in the two cell types. Western blot analysis with the mAb T4, which recognizes multiple isoforms of the NaKCl2 cotransporter (5Lytle C. Xu J.-C. Biemesderfer D. Forbush III, B. Am. J. Physiol. 1995; 269: C1496-C1505Crossref PubMed Google Scholar), showed that acinar cells of various exocrine glands express different forms and levels of cotransporter protein (Fig.2 a). Parotid acinar cells express a 185–190-kDa protein. Interestingly, SMG acinar cells expressed at least 10- and 150-fold more NaKCl2cotransporter protein than did parotid and pancreatic acinar cells, respectively. The T4 mAb detected only low levels of cotransporter protein in the SMG ductal preparation. Densitometric analysis showed that intensity in the duct lane was about 6.8 ± 5% (n = 3) of that in SMG acinar lane. After correction for the amount of protein loaded in each lane, this value is within the contamination of the SMG duct preparation with acini (32Xu X. Diaz J. Zhao H. Muallem S. J. Physiol. 1996; 491: 647-662Crossref PubMed Scopus (58) Google Scholar). The results of the Western blot analysis were confirmed by immunolocalization studies. Fig. 2 b shows that the T4 mAb detected high levels of NaKCl2cotransport in the BLM of acinar cells. The LM of acinar cells and both membranes of duct cells did not stain with this antibody. Further evidence for low NaKCl2 cotransport activity in SMG duct cells was obtained by measuring 86Rb uptake. In SMG acinar cells bumetanide alone inhibited 86Rb uptake by about 60% and nearly all the 86Rb uptake in the presence of ouabain (Fig. 2 c). In duct cells bumetanide alone had no measurable effect on 86Rb uptake, ouabain alone inhibited the uptake by about 60%, and bumetanide further reduced this uptake by about 8% (Fig. 2 d). Although the latter fraction was consistently observed (n = 6), it never exceeded 10%. Excluding Cl−/HCO3− exchange and NaKCl2 cotransport in duct cells and finding a component of Cl− transport not mediated by these transporters in acinar cells stimulated with ATP suggested a role of Cl− channels in both cell types. To identify the Cl− channels participating in the [Cl−] i changes induced by ATP we attempted to characterize the various Cl− channels expressed in freshly isolated SMG duct and acinar cells. Following the voltage protocol of Ludewig et al. (35Ludewig U. Jentsch T. Pusch M. J. Physiol. 1997; 498: 691-702Crossref PubMed Scopus (57) Google Scholar) (Fig.3 a) revealed the presence of a voltage gated Cl− current with properties similar to those reported for ClCO (36Pusch M. Ludewig U. Rehfeldt A. Jentsch J.J. Nature. 1995; 373: 527-531Crossref PubMed Scopus (299) Google Scholar) (Fig. 3, traces 1 and 5). For the present studies the most characteristic feature of this current was the fast gating observed after maximal channel opening by hyperpolarizing prepulses. Typically, channel inactivation was faster at −140 than at −80 mV. Another channel found in both cell types is the volume-sensitive Cl− channel. Thus, swelling the cells revealed the presence of an outward rectifying Cl− current with time dependent inactivation in positive potentials (Fig. 3,traces 2 and 6), similar to that described in several other cell types (15Valverde M.A. Hardy S.P. Sepulveda F.V. FASEB J. 1995; 9: 509-515Crossref PubMed Scopus (48) Google Scholar). As reported before in SMG duct cells (14Komwatana P. Dinudom A. Young J.A. Cook D.I. Pflugers Arch. 1994; 428: 641-647Crossref PubMed Scopus (37) Google Scholar), SMG acinar cells also showed the presence of an inwardly rectifying Cl− current with voltage- and time-dependent activation (not shown). Elevating [Ca2+] i with the Ca2+ ionophore A23187 activated an outwardly rectifying Cl− current with a typical time-dependent activation and tail currents (Fig.3, traces 3 and 7). Finally, elevation of cellular cAMP activated a CFTR-like Cl− current in SMG duct and acinar cells (Fig. 3, traces 4 and 8) (see also Zeng et al. (9Zeng W.-Z. Lee M.G. Yan M. Diaz J. Benjamin I. Marino C.R. Kopito R. Freedman S. Cotton K. Muallem S. Thomas P. Am. J. Physiol. 1997; 273: C442-C455Crossref PubMed Google Scholar)). For the purpose of the present work the channels were not characterized further. However, as can be seen in Fig. 3 the Cl− channels expressed in both cell types are similar and have sufficiently distinct kinetic characteristics to aid in their identification during agonist stimulation. Stimulation with 1 mm ATP rapidly activated a Cl− current in single duct and acinar cells. Fig. 4shows the two patterns of Cl− current activation. In about 20% of experiments, after rapid activation by ATP the current returned to near resting level. Subsequent removal of ATP resulted in transient reactivation of the current (Fig. 4, a and e). In most experiments the current remained activated, and removal of ATP resulted in a small current rebounding before its complete inactivation. Determination of the current-voltage relationship at various times during and after ATP stimulation did not result in distinctive patterns (Fig. 4, c and g;lanes 2–4 in each panel). However, subtracting the current at period 3 from that measured at periods 2 and 4 suggests that in both cell types ATP activated at least two distinct Cl− channels (Fig. 4, d and h). Previous studies reported that ATP increases [Ca2+] i in SMG acinar (22Hurley T.W. Ryan M.P. Shoemaker D.D. Arch. Oral Biol. 1994; 39: 205-212Crossref PubMed Scopus (21) Google Scholar) and duct cells (32Xu X. Diaz J. Zhao H. Muallem S. J. Physiol. 1996; 491: 647-662Crossref PubMed Scopus (58) Google Scholar). To evaluate the contribution of the Ca2+-activated Cl− channels to the current activated by ATP we tested the effect of extracellular Ca2+ and intracellular EGTA on the current. An example of such experiments is shown in Fig.5 and the results of several experiments are summarized in Table I. In both cell types ATP activated Ca2+-dependent and Ca2+-independent Cl− currents. The Ca2+-dependent current was about 60 and 50% of the total current in acinar and duct cells, respectively. This was the case whether the current was calculated as the portion sensitive to external Ca2+ ((a) in Table I) or blocked by high intracellular [EGTA] ((b) in Table I). The kinetic properties of this current are similar to those induced by A23187 (Fig. 3). That is, the current showed outward rectification, time-dependent activation, and substantial tail currents (Fig. 5, b and f).Table ISummary of Cl− currents activated by purinergic stimulationConditionATP, 1 mmBzATP, 100 μmUTP, 100 μmAciniDuctAciniDuctAciniDuctcurrent in pA/pF*Resting/0.5 EGTA1.2 ± 0.34.3 ± 1.10.92 ± 0.342.5 ± 1.50.75 ± 0.271.7 ± 0.48Control26.9 ± 4.662.7 ± 7.718.8 ± 1.555.2 ± 6.711.4 ± 1.327.0 ± 4.9Ca2+-free11.7 ± 2.126.7 ± 5.313.6 ± 1.438.8 ± 2.60.84 ± 0.322.6 ± 1.2Ca2+-dependent (a)15.2 ± 5.036.0 ± 9.35.21 ± 2.116.4 ± 7.210.56 ± 1.224.4 ± 5.0n11105544Resting/5EGTA0.8 ± 0.33.2 ± 1.00.6 ± 0.31.6 ± 0.40.82 ± 0.251.31 ± 0.4Control10.6 ± 1.832.6 ± 4.412.8 ± 1.538.9 ± 4.10.93 ± 0.351.38 ± 0.5Ca2+-dependent (b)16.3 ± 4.930.1 ± 8.86.0 ± 1.715.3 ± 7.8Glibenclamide 100 μm1.1 ± 0.263.4 ± 1.40.8 ± 0.34.3 ± 2.6Glibenclamide-sensitive (a)9.48 ± 1.829.2 ± 4.612.0 ± 1.834.6 ± 4.8n 6 644Resting/0.5 EGTA1.1 ± 0.42.9 ± 1.30.7 ± 0.42.4 ± 0.51.2 ± 0.32.1 ± 0.6Control24.5 ± 3.672.2 ± 9.021.1 ± 2.766.7 ± 3.910.6 ± 1.428.51 ± 1.5Glibenclamide 100 μm11 ± 1.834.0 ± 4.45.4 ± 0.514.4 ± 1.910.4 ± 0.928.2 ± 1.65Glibenclamide-sensitive (b)13.5 ± 1.838.2 ± 10.015.7 ± 2.752.3 ± 4.10.2 ± 0.20.3 ± 0.2n10 95544* pF = picofarad. Open table in a new tab * pF = picofarad. The residual, Ca2+-independent current measured in the absence of external Ca2+ (Fig. 5, a and e) and in the presence of 5 mm internal EGTA (Fig. 5, c and g) or 2 mm1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (not shown) has kinetic properties resembling those of CFTR. Since we previously showed that glibenclamide (GLM) inhibits the CFTR-like current in SMG duct and acinar cells (9Zeng W.-Z. Lee M.G. Yan M. Diaz J. Benjamin I. Marino C.R. Kopito R. Freedman S. Cotton K. Muallem S. Thomas P. Am. J. Physiol. 1997; 273: C442-C455Crossref PubMed Google Scholar) we next tested the effect of GLM on the current stimulated by ATP. Fig.6 and Table I show that GLM completely inhibited the Ca2+-independent Cl− current. Furthermore, the Cl− current inhibited by GLM in the presence of low (Fig. 6, a and e) or high (Fig.6, c and g) EGTA concentration in the pipette solution has a CFTR-like kinetic characteristic (current/voltage curves 2-3 in b and f and curves 1-2 in d and h). The remaining current (current/voltage curves 3-1 in b and f) showed strong outward rectification, as expected from the current carried by the Ca2+-independent Cl− channel. Taking advantage of the membrane-specific action of BzATP and UTP (29Lee M.G. Zeng W.Z. Muallem S. J. Biol. Chem. 1997; 272: 32951-32955Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) we studied regulation of Cl− channels by P2 receptors localized in the LM and BLM, respectively. Fig. 7 shows the effect of 25 μm BzATP on Cl− currents in SMG duct and acinar cells and Table I summarizes between four and six such experiments. Despite the finding that BzATP in both cell types was the most active nucleotide in increasing [Ca2+] i, it activated largely the Ca2+-independent Cl− current. Thus, removal of ext" @default.
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• W2010468499 title "Membrane-specific Regulation of Cl− Channels by Purinergic Receptors in Rat Submandibular Gland Acinar and Duct Cells" @default.
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• W2010468499 cites W1961939200 @default.
• W2010468499 cites W1963761882 @default.
• W2010468499 cites W1969099915 @default.
• W2010468499 cites W1983792468 @default.
• W2010468499 cites W1986127988 @default.
• W2010468499 cites W1989949255 @default.
• W2010468499 cites W1990361339 @default.
• W2010468499 cites W1998773000 @default.
• W2010468499 cites W1998969662 @default.
• W2010468499 cites W1999274391 @default.
• W2010468499 cites W2010309531 @default.
• W2010468499 cites W2012246710 @default.
• W2010468499 cites W2023329018 @default.
• W2010468499 cites W2029381859 @default.
• W2010468499 cites W2033570433 @default.
• W2010468499 cites W2034861930 @default.
• W2010468499 cites W2036471807 @default.
• W2010468499 cites W2040783182 @default.
• W2010468499 cites W2056626469 @default.
• W2010468499 cites W2066784771 @default.
• W2010468499 cites W2087342108 @default.
• W2010468499 cites W2087479677 @default.
• W2010468499 cites W2092677752 @default.
• W2010468499 cites W2124052139 @default.
• W2010468499 cites W2134786188 @default.
• W2010468499 cites W2138710925 @default.
• W2010468499 cites W2142045692 @default.
• W2010468499 cites W2152969853 @default.
• W2010468499 cites W2161076328 @default.
• W2010468499 cites W2164404600 @default.
• W2010468499 cites W2171358667 @default.
• W2010468499 cites W2222376843 @default.
• W2010468499 cites W2287827025 @default.
• W2010468499 cites W2406672607 @default.
• W2010468499 cites W2415114169 @default.
• W2010468499 cites W2418617022 @default.
• W2010468499 cites W2472027441 @default.
• W2010468499 cites W4233719888 @default.
• W2010468499 cites W63235074 @default.
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