| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
METHODS IN CELL PHYSIOLOGY
1Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles; 2Ocular Surface Center, Doheny Eye Institute, Los Angeles; 3La Jolla Laboratories, Pfizer Inc., San Diego; Departments of 4Medicine, 5Ophthalmology, 6Physiology and Biophysics, and 7Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, California
Submitted 16 May 2007 ; accepted in final form 12 August 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
-hexosaminidase catalytic activity in the AP culture medium in response to 100 µM basal CCh. In summary, rabbit lacrimal acinar cell monolayers generate a Cl–-dependent, ouabain-sensitive AP 
BL Isc in response to CCh, consistent with current models for Na+-dependent Cl– secretion. lacrimal gland; short-circuit current; epithelial ion channels; Na+/H+ exchangers; Na+-K+-2Cl– symporters
, and tumor necrosis factor-
(13, 26, 32, 49, 52). It generally is thought that these immune mediators are responsible for parenchymal atrophy and dysfunction of the surviving tissue (17, 53, 60). However, it also has been proposed that chronic stimulation of M3 muscarinic acetylcholine receptors by agonistic autoantibodies causes functional quiescence by downregulating downstream signaling mediators such as Gq and G11 (38). KCS is one of the most commonly treated eye conditions in the United States, affecting as many as 10 million people, approximately two-thirds of whom are women. Treatment strategies aimed at rehydrating the ocular surface with electrolyte-balanced lubricant eye drops and ointments provide temporary relief but usually do not arrest or reverse eye damage (15, 20, 29).
The secretory parenchyma of the lacrimal gland consists of acini and a converging system of ducts. The acinar epithelium accounts for 
80% of the volume of the gland and is believed to produce most of the fluid that flows through the ducts to the ocular surface. Acinar cells employ classic exocytotic mechanisms to secrete tear-specific proteins, secretory component, and secretory IgA, and they employ an array of transmembrane ion transporters and aquaporins to secrete electrolytes and water. Ex vivo models have been devised to study the release of proteins and secretory component into culture media bathing isolated and primary cultured acini. The availability of these ex vivo models has made it possible to formulate a detailed understanding of the signal transduction pathways that regulate protein secretion on a moment-to-moment basis and to pose specific hypotheses to explain why these mechanisms become quiescent during states of autoimmune activation associated with lacrimal insufficiency (38, 60). In contrast, it has not been possible to study transepithelial electrolyte and water transport processes in ex vivo models. Consequently, many features of the mechanisms acinar cells use to produce fluid remain uncertain, and little is known about how these mechanisms are acutely regulated or why they become quiescent in chronic, pathophysiological states.
The reason it has heretofore been impossible to study lacrimal epithelial electrolyte and water transport ex vivo is that isolated acinar cells have a powerful tendency to reorganize themselves into acinus-like structures, recapitulating their histiotypic structure. We have tried to devise methods for establishing primary cultured lacrimal acinar cells as confluent monolayers. Such a model would be extremely useful for efforts to address important unanswered questions about lacrimal epithelial transport physiology. Over the longer term, it could represent a critical step forward in the bioengineering of a functional lacrimal gland prosthesis (45).
A previous study by our group (44) demonstrated that rabbit lacrimal gland acinar cells established subconfluent monolayers but otherwise retained histiotypic morphology and cell function when cultured on various polymeric substrata in the presence of an extracellular matrix protein, Matrigel. In the present study, we established epithelial cell monolayers on polyester membrane Transwell inserts and used the classic Ussing short-circuit methods to evaluate their transepithelial electrophysiological behavior. We based our experimental design on the currently accepted working hypothesis of how primary active transport of Na+ and K+ and secondary active transport of Cl– produce an osmotic gradient that drives a flow of water across the acinar epithelium (Fig. 1) (11, 33, 48, 50, 54). According to this hypothesis, the Na+ pump enzyme Na+,K+-ATPase uses energy derived from the hydrolysis of ATP to drive the electrochemically unfavorable efflux of Na+ and influx of K+ through the basal-lateral membrane. Na+/H+ (proton) exchangers and Cl–/HCO3– (anion) exchangers use energy of the Na+ electrochemical gradient to drive Cl– influx and establish an outwardly directed Cl– electrochemical gradient. Cl–-selective channels in the apical membrane then facilitate efflux of Cl–, which generates a lumen-negative transepithelial voltage difference, driving a secretory flux of Na+ through the paracellular pathway. K+-selective channels allow K+ ions to recycle across the basal-lateral membrane so that the acini produce a Na+-Cl–-rich fluid. The corresponding working hypothesis describing transepithelial transport in the ducts differs from this model primarily in that it places K+-selective channels in parallel with Cl–-selective channels in the apical membrane, allowing formation of a K+-Cl–-rich fluid (33).
|
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
4 kg, were purchased from Irish Farms (Norco, CA). All animals used in this study were treated in accordance with the ARVO Resolution on Use of Animals in Ophthalmology and Vision Research. Animals were maintained in a facility fully accredited by the American Association for Laboratory Animal Science. Animals were narcotized with a mixture of ketamine (20–40 mg/ml) and xylazine (5–9 mg/ml), 1–1.5 ml, and euthanized with an overdose of Eutha-6CII (120 mg/ml). Materials. Standard 6-well Snapwell and 12-well Transwell polyester membrane cell culture inserts were obtained from Costar (Corning, Corning, NJ), and Hepato-STIM culture medium (HSM) was purchased from BD Biosciences (Medford, MA) (43). Fetal bovine serum (FBS) was purchased from Omega Scientific (Tarzana, CA).
Purified lacrimal gland acinar cell monolayers. The procedures for isolation of purified lacrimal gland acinar cells (pLGACs) were as previously described (21). Briefly, after anesthesia, inferior lacrimal glands were removed aseptically and finely minced with a pair of scalpel blades (no. 18) in a petri dish with Ham's complete medium. Ham's complete medium consists of Ham's F-12 supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine, 2 mM butyrate, 0.084 mg/l linoleic acid, 0.05 mg/ml trypsin inhibitor, 10 mM HEPES, and 5 mg/ml bovine serum albumin (BSA). The minced tissue was then washed and digested with collagenase, DNase, and hyaluronidase for 25 min at 37°C in 95% O2-5% CO2. Gland digests were then centrifuged (700 rpm, 5 min), filtered through a 70-µm pore size cell strainer, and washed twice with Hanks' and Ham's complete media. The cell suspension was then further purified by centrifuging in a Ficoll gradient (190 rpm, 15 min) to reduce contamination by fibroblasts. The resultant cellular fraction was resuspended in HSM at a density of 1 x 106 cells/ml. HSM is a serum-free, defined medium containing dexamethasone, ITS (insulin, transferrin, and selenium), and a proprietary formulation of hormones and metabolites. For cell culture procedures, the medium was supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, 0.25 µg/ml fungizone, and 1 ng/ml epidermal growth factor (EGF).
The upper chambers of six-well culture inserts, which were used for transport studies in Ussing chambers, received 0.4 ml of the cell suspension, and the lower chambers received 2.5 ml of the culture medium. The upper chambers of 12-well culture inserts, which were used for all other studies, received 0.5 ml of the cell suspension, and the lower chambers received 1.5 ml of the culture medium. The plates were then left undisturbed in a 37°C incubator in 95% O2-5% CO2 for 2 days to facilitate cell attachment. After 2 days, cells were observed under an inverted light microscope (Nikon, Garden City, NY). Subconfluent cell monolayers received fresh culture medium with EGF every 2 days. Inserts with confluent monolayers received fresh medium without EGF. Confluent pLGAC monolayers (pLGACMs) were typically obtained in 3–5 days. The monolayers were prescreened for transepithelial resistance (TER) with a Millicell ERS epithelial Voltohmeter (Millipore, Allen TX). TER readings from culture inserts without cells were subtracted from readings obtained from inserts with pLGACMs. Cell monolayers with TER in the range of 200–1,500 
/cm2 were used for transport studies.
Transmission electron microscopy.
On day 5, pLGACMs on culture inserts were fixed with half-strength Karnovsky's fixative solution and left at 4°C overnight for transmission electron microscopy (TEM). After fixing, the samples were washed three times with PBS solution, rinsed three times in 0.1 M cacodylate buffer, and then postfixed in 2% OsO4 for 1 h. After three rinses at 10-min intervals in 100 mM sodium acetate buffer, the samples were stained en bloc with 1% uranyl acetate in 50 mM sodium acetate buffer overnight. The samples were rinsed with buffer and dehydrated through a graded series of ethanol rinses and then infiltrated and embedded in Eponate 12 resin (Ted Pella, Redding, CA). Thin sections (70 nm) were cut with a diamond knife and placed on copper grids. The sections were later stained with uranyl acetate and lead citrate for viewing in a JEOL 1200 EX transmission electron microscope (Tokyo, Japan).
Immunofluorescence staining.
pLGACMs on culture inserts were sectioned and fixed with 100% methyl alcohol for 15 min. They were then washed three times with PBS at 5-min intervals and blocked with 5% BSA in PBS for 15 min to decrease nonspecific antibody binding. The samples were then incubated with primary antibodies against Na+,K+-ATPase (Upstate USA, Chicago, IL) at a dilution of 1:10 in 1% BSA solution for 1 h at 37°C. Sections of monolayers were also incubated with either anti-occludin antibody (Invitrogen, Carlsbad, CA) at a dilution of 1:50 or mouse IgG1 
-isotype control antibody (Sigma-Aldrich, St. Louis, MO) at a dilution of 1:100 in 1% BSA solution overnight at 4°C. The samples were later washed with PBS and incubated with rhodamine red-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:100 in PBS for 45 min at room temperature. After they were washed with PBS, the samples were mounted onto slides with antifade mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) and analyzed under a confocal imaging system (Carl Zeiss Meditec, Oberkochen, Germany).
Ussing chamber studies.
Transport studies were conducted in bicarbonated Ringer buffer solutions maintained at 37°C and equilibrated with 95% O2-5% CO2. The composition of the buffer solutions was as follows: normal bicarbonated Ringer buffer solution (BRS) contained (in mM) 116.4 NaCl, 5.4 KCl, 25 NaHCO3, 0.78 NaH2PO4, 1.8 CaCl2·2H2O, 0.81 MgCl2·6H2O, 5.55 D-glucose, and 15 HEPES. The Cl–-free bicarbonated Ringer buffer solution was prepared by replacing NaCl, KCl, and CaCl2·2H2O in BRS with (in mM) 116.4 Na-isethionate, 1.5 K2SO4, and 1 Ca-gluconate, respectively. The pH of the Ringer solutions was adjusted to 7.5 using 2 N hydrochloric acid. Chemicals and inhibitors used in this study were purchased from Sigma-Aldrich.
The setup of the Ussing chamber has been previously described (3). Briefly, six-well culture inserts with pLGACMs were equilibrated in BRS buffer for 1 h and gently mounted in Ussing chambers (Physiologic Instruments, San Diego, CA). The samples were allowed to stabilize for 15–30 min. The reservoir on each side of the cell monolayer was filled with 4 ml of BRS fluid. The chambers were warmed to 37°C by a water jacket fed from a circulating water bath and continuously bubbled and stirred with 95% O2-5% CO2 gas lifts.
Voltage-sensing electrodes consisting of Ag-AgCl pellets and current-passing electrodes of Ag wire were connected by agar bridges containing 3 M KCl and interfaced via head-stage amplifiers (DM-MC6; Physiologic Instruments) to a microcomputer-controlled voltage-current clamp (VCC-MC6; Physiologic Instruments). Voltage-sensing electrodes were matched to within 1 mV asymmetry and corrected by an offset-removal circuit. The fluid resistance was determined in the absence of a filter; resistances of blank filter inserts were negligible and were electrically compensated using the series compensation circuit on the clamp. Following convention, transepithelial voltages reported are referenced to the basal side. Short-circuit current (Isc) flowing in the apical-to-basal direction was considered negative. All experiments were performed under short-circuit conditions with voltage clamped to zero.
Once the bioelectric properties of the pLGACMs attained steady-state values, a cholinergic agonist, carbachol, was added (100 µM) to the basal chambers. Active efflux of Na+ across the cell monolayer, driven by Na+-K+-ATPase, was evaluated by the addition of 100 µM ouabain to the bathing fluid on the basal chamber with four replicate samples.
The effects of Cl–-free conditions on bioelectric properties were evaluated using the superfusion technique described previously (8). This method of buffer exchange reduces the adverse effects induced by sudden changes in hydrostatic pressure encountered when bathing fluids were completely removed and then replenished. The bathing fluids in both sides of the Ussing chambers were replaced simultaneously by gravity feeding at a rate of 20 ml/min to the bottoms of the chambers. The apical bathing fluid received Cl–-free BRS buffer, while the basal side fluid received Cl–-free BRS buffer containing 100 µM carbachol. The excess volume was suctioned off simultaneously at the surface, thus keeping the bathing fluid volume constant. Isc and TER were continually monitored during the whole superfusion process.
The Na+/H+ exchanger inhibitor amiloride was dissolved in dimethyl sulfoxide (1 mM), and the Na+-K+-2Cl– cotransporter inhibitor bumetanide (0.1 mM) was dissolved in dimethyl sulfoxide. Equal volumes of the vehicles were used as controls. Four replicate samples were used for the control group, five for the amiloride group, and six for bumetanide and amiloride + bumetanide groups.
Measurement of -hexosaminidase secretion.
Secretion assays were carried out in 12-well culture inserts on day 4 with four replicate samples. The culture medium in each well was aspirated, and 0.5 and 1.5 ml of fresh, serum-free Dulbecco's modified Eagle's medium was added on the upper (apical) and lower (basal) chambers, respectively. The plates were then incubated at 37°C for 2–3 h; 200 µl of the apical and basal medium (unstimulated) were then removed. The basal medium was completely aspirated, and 0.6 ml of freshly prepared 100 µM carbachol solution was added to the basal chamber. The plate was incubated at 37°C under 5% CO2 for 30 min. After incubation, 150 µl of the medium from the apical and basal chambers were collected and centrifuged at 700 rpm for 5 min at room temperature. The resulting supernatants were removed and stored frozen until they could be analyzed for -hexosaminidase catalytic activity. The assay used a GENios Plus microplate reader (Phenix, Hayward, CA) to detect hydrolysis of the artificial substrate 4-methylumbelliferyl--D-glucosaminide. Data are expressed as the relative increase above basal value. Data were analyzed using a Student's t-test for unpaired samples assuming equal variance when comparing two group means. Data are means ± SE.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
-isotype control (Fig. 3B). Immunofluorescence staining showed that pLGACs expressed Na+-K+-ATPase on both the apical and basal-lateral membranes (Fig. 4). Significant differences in Na+-K+-ATPase staining patterns were observed between carbachol-stimulated and unstimulated cells (Fig. 5). Both the carbachol-stimulated and unstimulated cells expressed a plasma membrane stain for Na+-K+-ATPase. However, a weak, diffuse intracellular staining for Na+-K+-ATPase was observed in the cytoplasm of carbachol-stimulated cells, whereas no intracellular staining for Na+-K+-ATPase was seen in the cytoplasm of unstimulated cells.
|
|
|
A positive carbachol-dependent Isc would be consistent with either the Ussing model for Na+ absorption or the model for Cl– secretion first proposed by Silva et al. (46). The shared feature of both models is the active efflux of Na+ across the basal-lateral membrane, driven by Na+-K+-ATPase. Addition of 100 µM ouabain to the basal-lateral chamber resulted in the complete return of carbachol-dependent Isc to baseline values (Fig. 6). The rapid decrease in Isc was accompanied by a rapid increase in TER (data not shown).
|
|
|
-Hexosaminidase assay.
Of the proteins rabbit lacrimal gland acinar cells secrete in response to cholinergic stimulation, -hexosaminidase is the most easily measured (2). In the unstimulated state, secretion of -hexosaminidase to the apical medium was nearly sixfold greater than secretion to the basal medium. Stimulation of 100 µM carbachol doubled secretion of -hexosaminidase to the apical bathing medium (P 
0.05) but had no significant effect on secretion to the basal medium (Fig. 9).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Na+-K+-ATPase generates chemiosmotic energy that drives a variety of transport processes in the plasma membranes of all vertebrate cells (42, 47). In most epithelia, it is distributed asymmetrically, and immunocytochemical or immunofluorescence microscopy usually detects it exclusively in the basal-lateral plasma membrane. The lacrimal acinar cell appears to be an exception to this generalization. Both subcellular fractionation analyses and immunolocalization methods indicate that, although it is expressed at highest specific activity in the basal-lateral membrane, it also is expressed in the apical plasma membrane (55), but it is preponderantly localized in intracellular pools (7, 34) now known to be associated with the early endosome, recycling endosome, and trans-Golgi network (58). Immunofluorescence staining of the cultured lacrimal acinar cell monolayers demonstrated the localization of Na+-K+-ATPase at both the basal-lateral and apical plasma membranes, with greater intensity of labeling at the basal-lateral plasma membranes.
Cholinergic stimulation triggers a number of events in isolated and primary cultured lacrimal acinar cells related to their diverse secretory functions. These include activation of apical Cl– channels (14, 16, 39), activation of basal-lateral Na+/H+ antiporters (27, 40), release of macromolecular secretory products and internalization and recycling of secretory vesicle membrane constituents (23, 56), release of the polymeric IgA receptor secretory component (41), acceleration of the vesicle traffic recycling between the plasma membranes and endosomes (19, 28), and a net translocation of Na+-K+-ATPase pumps from the intracellular pools to the basal-lateral plasma membranes (59). The detection of Na+-K+-ATPase in the cytoplasm of cells in the monolayers after carbachol stimulation but not in nonstimulated cells may indicate that Na+-K+-ATPase subcellular localization and traffic differ from the models that have been studied previously, or it might result from technical issues related to the density of Na+-K+-ATPase being near the threshold of detection in the intracellular compartments.
It is the Na+-K+-ATPase pump units expressed in the basal-lateral plasma membrane that are thought to provide the energy necessary for net fluxes of electrolytes in either the absorptive or the secretory direction (18, 46). The cardiac glycoside ouabain, an inhibitor of Na+-K+-ATPase, has been shown to reduce fluid secretion by rabbit lacrimal gland in vivo (5, 10), and our observation that addition of ouabain to the basal medium completely abolishes the carbachol-induced Isc is in accord with this concept.
A positive Isc such as we observed after stimulation with carbachol is formally consistent with either active electrogenic absorption of a cation, i.e., Na+, or active electrogenic secretion of an anion, i.e., Cl–. Replacement of Cl– in the apical and basal bathing media abolished the carbachol-induced Isc, as predicted by the model of anion secretion. However, this observation could not exclude a model of Na+ absorption in which Na+ influx across the apical plasma membrane is a Cl–-dependent, rather than Na+ channel-mediated, process. Models for absorption and secretion differ only in the subcellular localization of the transporters that couple Na+ and Cl– influxes. Two types of coupled Na+ and Cl–-transport mechanism have been characterized, Na+-K+-2Cl– symporters and a parallel array of Na+/H+ exchangers and Cl–/HCO3– exchangers (31). Neither amiloride, an inhibitor of Na+/H+ exchangers, nor bumetanide, an inhibitor of Na+-K+-2Cl– symporters, perceptibly altered Isc when added to the basal medium singly. However, the two inhibitors in combination reduced Isc by 65%. This observation indicates that Na+-coupled Cl– influx across the basal-lateral membrane is mediated both by Na+/H+ exchangers and Cl–/HCO3– exchangers and by Na+-K+-2Cl– symporters. It also suggests that acinar cells contain large intracellular reserves of the transporters and can recruit one Na+ transporter to the plasma membrane when the other is inhibited. Analytical subcellular fractionation studies demonstrated the existence of large intracellular pools of Na+/H+ exchangers (59) and Cl–/HCO3– exchangers (27). Similar experiments were unable to detect evidence for Na+-K+-2Cl– symporters in isolated membrane vesicles but could not exclude their presence in intact cells. The failure of the combination of inhibitors to completely inhibit Isc might have been due to relatively low sensitivity of various Na+/H+ exchanger isoforms to amiloride (36, 37). Alternatively, it might have been due to the ongoing recycling traffic, which internalizes transporters to acidic compartments, where they dissociate inhibitors, and then returns them to the plasma membrane in an active state.
Secretagogues are known to stimulate isolated and primary cultured lacrimal acinar cells to release secretory proteins to their ambient media (59), but with those models it was necessary to employ sophisticated live-cell imaging methods to verify that the exocytotic process occurred preferentially at the apical plasma membrane (25). Our studies with the acinar cell monolayer model confirm that carbachol-induced -hexosaminidase secretion is directed to the apical plasma membrane. However, our observations also reveal that acinar cells maintain a constitutive secretory process directed to the basal-lateral membrane, such as has been predicted on the basis of data obtained through subcellular fractionation analyses (58).
In summary, we report the first successful ex vivo reconstitution of an electrophysiologically functional lacrimal gland epithelial tissue. When grown on polyester membrane scaffolds, acinar cells from rabbit lacrimal glands established continuous monolayers with transepithelial resistances typical of the "leaky" epithelia. The reconstituted epithelia generate a carbacholinduced, ouabain-sensitive, Cl–-dependent, apical basal-lateral Isc consistent with current cellular models in which Na+-coupled cotransporters use the energy of Na+ electrochemical potential gradient, established by Na+-K+-ATPase, to mediate active secretion of Cl– (12, 50). We anticipate that this model will be useful for studying basic mechanisms in lacrimal cell physiology in addition to electrolyte and water transport. It has been proposed that lacrimal acinar cells exocytotically secrete partially processed autoantigen peptides to the underlying tissue space by way of the same membrane recycling traffic that mediates the constitutive secretion of -hexosaminidase. According to this scenario, alterations or reorientations of traffic during chronic stimulation with carbachol, prolactin, or other cytokines or with inflammatory mediators lead to the exposure of autoantigen epitopes that potentially provoke autoimmune responses that will manifest clinically as Sjögren's syndrome. The reconstituted lacrimal epithelial monolayer should be extremely useful as an experimental model for studying these mechanisms.
At the same time, harnessing the principles of tissue engineering to develop bioengineered tissue constructs is a rapidly emerging alternative in a clinical setting when tissue or organ transplantation is not possible or feasible. The lacrimal gland presents a classic example, since no description of a surgical transplantation procedure for the gland in humans has been reported in the literature. Hence, a tissue-engineered tear secretory system would be an attractive alterative for patients suffering from severe dry eyes due to nonfunctioning lacrimal glands or blocked ducts (such as those with Stevens-Johnson syndrome), chemical or thermal injuries, or ocular cicatricial pemphigoid (an inflammatory syndrome involving the ocular mucous membranes). For a tissue-engineered system to produce fluid, the critical requirements that have to be met (45) include that the cellular component in the construct establishes continuous epithelial monolayers with functional tight junctions, an asymmetric distribution of transport proteins mediating active secretion of Cl–, and an apically oriented pathway for exocytotic secretion of proteins. The present study indicates that this goal may be attainable.
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
2. Andersson SV, Edman MC, Bekmezian A, Holmberg J, Mircheff AK, Peter Gierow J. Characterization of -hexosaminidase secretion in rabbit lacrimal gland. Exp Eye Res 83: 1081–1088, 2006.[CrossRef][ISI][Medline]
3. Angelow S, Kim KJ, Yu AS. Claudin-8 modulates paracellular permeability to acidic and basic ions in MDCK II cells. J Physiol 571: 15–26, 2006.
4. Bisbee CA. Prolactin effects on ion transport across cultured mouse mammary epithelium. Am J Physiol Cell Physiol 240: C110–C115, 1981.
5. Botelho SY, Fuenmayor N. Lacrimal gland flow and potentials during dinitrophenol, ouabain, and ethacrynic acid perfusion. Invest Ophthalmol Vis Sci 20: 515–521, 1981.
6. Bovell DL, Clunes MT, Elder HY, Wong CH, Ko WH. Nucleotide-evoked ion transport and [Ca2+]i changes in normal and hyperhidrotic human sweat gland cells. Eur J Pharmacol 403: 45–48, 2000.[CrossRef][ISI][Medline]
7. Bradley ME, Azuma KK, McDonough AA, Mircheff AK, Wood RL. Surface and intracellular pools of Na,K-ATPase catalytic and immuno-activities in rat exorbital lacrimal gland. Exp Eye Res 57: 403–413, 1993.[CrossRef][ISI][Medline]
8. Chang-Lin JE, Kim KJ, Lee VH. Characterization of active ion transport across primary rabbit corneal epithelial cell layers (RCrECL) cultured at an air-interface. Exp Eye Res 80: 827–836, 2005.[CrossRef][ISI][Medline]
9. Culp DJ, Lee DK, Penney DP, Marin MG. Cat tracheal gland cells in primary culture. Am J Physiol Lung Cell Mol Physiol 263: L264–L275, 1992.
10. Dartt DA, Moller M, Poulsen JH. Lacrimal gland electrolyte and water secretion in the rabbit: localization and role of (Na+ + K+)-activated ATPase. J Physiol 321: 557–569, 1981.
11. Do CW, To CH. Chloride secretion by bovine ciliary epithelium: a model of aqueous humor formation. Invest Ophthalmol Vis Sci 41: 1853–1860, 2000.
12. Dubyak GR. Ion homeostasis, channels, and transporters: an update on cellular mechanisms. Adv Physiol Educ 28: 143–154, 2004.
13. Esch TR. Pathogenetic factors in Sjögren's syndrome: recent developments. Crit Rev Oral Biol Med 12: 244–251, 2001.[Abstract]
14. Evans MG, Marty A, Tan YP, Trautmann A. Blockage of Ca-activated Cl conductance by furosemide in rat lacrimal glands. Pflügers Arch 406: 65–68, 1986.[CrossRef][ISI][Medline]
15. Fante RG, Snyder RW, Metrikin DC. Treating dry eye. CLAO J 18: 14, 1992.[Medline]
16. Findlay I, Petersen OH. Acetylcholine stimulates a Ca2+-dependent C1– conductance in mouse lacrimal acinar cells. Pflügers Arch 403: 328–330, 1985.[CrossRef][ISI][Medline]
17. Fox RI, Kang HI. Pathogenesis of Sjögren's syndrome. Rheum Dis Clin North Am 18: 517–538, 1992.[ISI][Medline]
18. Frizzell RA, Field M, Schultz SG. Sodium-coupled chloride transport by epithelial tissues. Am J Physiol Renal Fluid Electrolyte Physiol 236: F1–F8, 1979.
19. Gierow JP, Lambert RW, Mircheff AK. Fluid phase endocytosis by isolated rabbit lacrimal gland acinar cells. Exp Eye Res 60: 511–525, 1995.[CrossRef][ISI][Medline]
20. Goto E, Dogru M, Fukagawa K, Uchino M, Matsumoto Y, Saiki M, Tsubota K. Successful tear lipid layer treatment for refractory dry eye in office workers by low-dose lipid application on the full-length eyelid margin. Am J Ophthalmol 142: 264–270, 2006.[CrossRef][ISI][Medline]
21. Guo Z, Song D, Azzarolo AM, Schechter JE, Warren DW, Wood RL, Mircheff AK, Kaslow HR. Autologous lacrimal-lymphoid mixed-cell reactions induce dacryoadenitis in rabbits. Exp Eye Res 71: 23–31, 2000.[CrossRef][ISI][Medline]
22. Handler JS, Perkins FM, Johnson JP. Studies of renal cell function using cell culture techniques. Am J Physiol Renal Fluid Electrolyte Physiol 238: F1–F9, 1980.
23. Herzog V, Farquhar MG. Luminal membrane retrieved after exocytosis reaches most Golgi cisternae in secretory cells. Proc Natl Acad Sci USA 74: 5073–5077, 1977.
24. Holly FJ, Lemp MA. Tear physiology and dry eyes. Surv Ophthalmol 22: 69–87, 1977.[CrossRef][ISI][Medline]
25. Jerdeva GV, Wu K, Yarber FA, Rhodes CJ, Kalman D, Schechter JE, Hamm-Alvarez SF. Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells. J Cell Sci 118: 4797–4812, 2005.
26. Kolkowski EC, Reth P, Pelusa F, Bosch J, Pujol-Borrell R, Coll J, Jaraquemada D. Th1 predominance and perforin expression in minor salivary glands from patients with primary Sjögren's syndrome. J Autoimmun 13: 155–162, 1999.[CrossRef][ISI][Medline]
27. Lambert RW, Bradley ME, Mircheff AK. pH-sensitive anion exchanger in rat lacrimal acinar cells. Am J Physiol Gastrointest Liver Physiol 260: G517–G523, 1991.
28. Lambert RW, Maves CA, Gierow JP, Wood RL, Mircheff AK. Plasma membrane internalization and recycling in rabbit lacrimal acinar cells. Invest Ophthalmol Vis Sci 34: 305–316, 1993.
29. Lamberts DW. Topical hyperosmotic agents and secretory stimulants. Int Ophthalmol Clin 20: 163–169, 1980.[CrossRef][Medline]
30. Larsen EH, Novak I, Pedersen PS. Cation transport by sweat ducts in primary culture. Ionic mechanism of cholinergically evoked current oscillations. J Physiol 424: 109–131, 1990.
31. Lau KR, Howorth AJ, Case RM. The effects of bumetanide, amiloride and Ba2+ on fluid and electrolyte secretion in rabbit salivary gland. J Physiol 425: 407–427, 1990.
32. Magnusson V, Nakken B, Bolstad AI, Alarcon-Riquelme ME. Cytokine polymorphisms in systemic lupus erythematosus and Sjögren's syndrome. Scand J Immunol 54: 55–61, 2001.[CrossRef][ISI][Medline]
33. Mircheff AK. Lacrimal fluid and electrolyte secretion: a review. Curr Eye Res 8: 607–617, 1989.[ISI][Medline]
34. Mircheff AK, Lu CC, Conteas CN. Resolution of apical and basal-lateral membrane populations from rat exorbital gland. Am J Physiol Gastrointest Liver Physiol 245: G661–G667, 1983.
35. Mircheff AK, Wang Y, Jean MD, Ding C, Trousdale MD, Hamm-Alvarez SF, Schechter JE. Mucosal immunity and self-tolerance in the ocular surface system. Ocul Surf 3: 182–192, 2005.[ISI][Medline]
36. Noel J, Roux D, Pouyssegur J. Differential localization of Na+/H+ exchanger isoforms (NHE1 and NHE3) in polarized epithelial cell lines. J Cell Sci 109: 929–939, 1996.[Abstract]
37. Park K, Olschowka JA, Richardson LA, Bookstein C, Chang EB, Melvin JE. Expression of multiple Na+/H+ exchanger isoforms in rat parotid acinar and ductal cells. Am J Physiol Gastrointest Liver Physiol 276: G470–G478, 1999.
38. Qian L, Wang Y, Xie J, Rose CM, Yang T, Nakamura T, Sandberg M, Zeng H, Schechter JE, Chow RH, Hamm-Alvarez SF, Mircheff AK. Biochemical changes contributing to functional quiescence in lacrimal gland acinar cells after chronic ex vivo exposure to a muscarinic agonist. Scand J Immunol 58: 550–565, 2003.[CrossRef][ISI][Medline]
39. Saito Y, Ozawa T, Hayashi H, Nishiyama A. Acetylcholine-induced change in intracellular Cl– activity of the mouse lacrimal acinar cells. Pflügers Arch 405: 108–111, 1985.[CrossRef][ISI][Medline]
40. Saito Y, Ozawa T, Nishiyama A. Acetylcholine-induced Na+ influx in the mouse lacrimal gland acinar cells: demonstration of multiple Na+ transport mechanisms by intracellular Na+ activity measurements. J Membr Biol 98: 135–144, 1987.[CrossRef][ISI][Medline]
41. Schechter J, Stevenson D, Chang D, Chang N, Pidgeon M, Nakamura T, Okamoto CT, Trousdale MD, Mircheff AK. Growth of purified lacrimal acinar cells in Matrigel raft cultures. Exp Eye Res 74: 349–360, 2002.[CrossRef][ISI][Medline]
42. Scheiner-Bobis G. The sodium pump. Its molecular properties and mechanics of ion transport. Eur J Biochem 269: 2424–2433, 2002.[ISI][Medline]
43. Schonthal AH, Warren DW, Stevenson D, Schecter JE, Azzarolo AM, Mircheff AK, Trousdale MD. Proliferation of lacrimal gland acinar cells in primary culture. Stimulation by extracellular matrix, EGF, and DHT. Exp Eye Res 70: 639–649, 2000.[CrossRef][ISI][Medline]
44. Selvam S, Thomas PB, Trousdale MD, Stevenson D, Schechter JE, Mircheff AK, Jacob JT, Smith RE, Yiu SC. Tissue-engineered tear secretory system: functional lacrimal gland acinar cells cultured on matrix protein-coated substrata. J Biomed Mater Res B Appl Biomater 80: 192–200, 2007.[Medline]
45. Selvam S, Thomas PB, Yiu SC. Tissue engineering: current and future approaches to ocular surface reconstruction. Ocul Surf 4: 120–136, 2006.[ISI][Medline]
46. Silva P, Stoff J, Field M, Fine L, Forrest JN, Epstein FH. Mechanism of active chloride secretion by shark rectal gland: role of Na-K-ATPase in chloride transport. Am J Physiol Renal Fluid Electrolyte Physiol 233: F298–F306, 1977.
47. Skou JC. The sodium, potassium-pump. Scand J Clin Lab Invest Suppl 180: 11–23, 1986.[Medline]
48. Smith JJ, Welsh MJ. Fluid and electrolyte transport by cultured human airway epithelia. J Clin Invest 91: 1590–1597, 1993.[ISI][Medline]
49. Solomon A, Dursun D, Liu Z, Xie Y, Macri A, Pflugfelder SC. Pro- and anti-inflammatory forms of interleukin-1 in the tear fluid and conjunctiva of patients with dry-eye disease. Invest Ophthalmol Vis Sci 42: 2283–2292, 2001.
50. Speake T, Whitwell C, Kajita H, Majid A, Brown PD. Mechanisms of CSF secretion by the choroid plexus. Microsc Res Tech 52: 49–59, 2001.[CrossRef][ISI][Medline]
51. Stott DI, Hiepe F, Hummel M, Steinhauser G, Berek C. Antigen-driven clonal proliferation of B cells within the target tissue of an autoimmune disease. The salivary glands of patients with Sjögren's syndrome. J Clin Invest 102: 938–946, 1998.[ISI][Medline]
52. Sun D, Emmert-Buck MR, Fox PC. Differential cytokine mRNA expression in human labial minor salivary glands in primary Sjögren's syndrome. Autoimmunity 28: 125–137, 1998.[ISI][Medline]
53. Tsubota K, Toda I, Yagi Y, Ogawa Y, Ono M, Yoshino K. Three different types of dry eye syndrome. Cornea 13: 202–209, 1994.[CrossRef][ISI][Medline]
54. Ussing HH, Lind F, Larsen EH. Ion secretion and isotonic transport in frog skin glands. J Membr Biol 152: 101–110, 1996.[CrossRef][ISI][Medline]
55. Wood RL, Mircheff AK. Apical and basal-lateral Na/K-ATPase in rat lacrimal gland acinar cells. Invest Ophthalmol Vis Sci 27: 1293–1296, 1986.
56. Wu K, Jerdeva GV, da Costa SR, Sou E, Schechter JE, Hamm-Alvarez SF. Molecular mechanisms of lacrimal acinar secretory vesicle exocytosis. Exp Eye Res 83: 84–96, 2006.[CrossRef][ISI][Medline]
57. Xanthou G, Tapinos NI, Polihronis M, Nezis IP, Margaritis LH, Moutsopoulos HM. CD4 cytotoxic and dendritic cells in the immunopathologic lesion of Sjögren's syndrome. Clin Exp Immunol 118: 154–163, 1999.[CrossRef][ISI][Medline]
58. Xie J, Qian L, Wang Y, Rose CM, Yang T, Nakamura T, Hamm-Alvarez SF, Mircheff AK. Novel biphasic traffic of endocytosed EGF to recycling and degradative compartments in lacrimal gland acinar cells. J Cell Physiol 199: 108–125, 2004.[CrossRef][ISI][Medline]
59. Yiu SC, Lambert RW, Tortoriello PJ, Mircheff AK. Secretagogue-induced redistributions of Na,K-ATPase in rat lacrimal acini. Invest Ophthalmol Vis Sci 32: 2976–2984, 1991.
60. Zoukhri D. Effect of inflammation on lacrimal gland function. Exp Eye Res 82: 885–898, 2006.[CrossRef][ISI][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME |
