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Proceedings of the Society for Experimental Biology and Medicine 225:39-48 (2000)
© 2000 Society for Experimental Biology and Medicine

Original Article

Ion Transport in an Immortalized Rat Submandibular Cell Line SMG-C6

Robert Castro*,1, Lornell Barlow-Walden*, Trudi Woodson*, Jay D. Kerecman{dagger}, Guo H. Zhang* and J. Ricardo Martinez{ddagger}


* Department of Pediatrics, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284–7812;
{dagger} Department of Pediatrics, Wilford Hall Medical Center, San Antonio, Texas 78236–5000; and
{ddagger} National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892–2190


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The immortalized rat submandibular epithelial cell line, SMG-C6, cultured on porous tissue culture supports, forms polarized, tight-junction epithelia facilitating bioelectric characterization in Ussing chambers. The SMG-C6 epithelia generated transepithelial resistances of 956±84{Omega}.cm2 and potential differences (PD) of -16.9 ± 1.5mV (apical surface negative) with a basal short-circuit current (Isc) of 23.9 ± 1.7 µA/cm2 (n = 69). P2 nucleotide receptor agonists, ATP or UTP, applied apically or basolaterally induced a transient increase in Isc, followed by a sustained decreased below baseline value. The peak {Delta}Isc increase was partly sensitive to Cl- and K+ channel inhibitors, DPC, glibenclamide, and tetraethylammonium (TEA) and was completely abolished following Ca2+ chelation with BAPTA or bilateral substitution of gluconate for Cl-. The major component of basal Isc was sensitive to apical Na+ replacement or amiloride (half-maximal inhibitory concentration 392 nM). Following pretreatment with amiloride, ATP induced a significantly greater Isc; however, the poststimulatory decline was abolished, suggesting an ATP-induced inhibition of amiloride-sensitive Na+ transport. Consistent with the ion transport properties found in Ussing chambers, SMG-C6 cells express the rat epithelial Na+ channel {alpha}-subunit ({alpha}-rENaC). Thus, cultured SMG-C6 cells produce tight polarized epithelia on permeable support with stimulated Cl- secretory conductance and an inward Isc accounted for by amiloride-sensitive Na+ absorption.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Studies using intact salivary glands or freshly isolated cells have contributed to our understanding of salivary secretion and modification; however, elucidating the specific site(s) of ion transport regulation is difficult from heterogeneous cellular preparations. Isolated cultures of homogeneous salivary epithelial cells would allow for more precise measurements of regulatory ion transport under controlled conditions. Normal (nontumor-derived) epithelial cells from the salivary glands have been isolated and maintained in culture (1-3). Yet, even under optimal conditions, functional studies from primary salivary cultures are restricted due to limited life span or terminal differentiation (4). Continuous cell lines derived from rodent salivary glands have been established, demonstrating stable morphology and function (5), including tight junctions and polarized membrane proteins consistent with vectorial ion transport (6, 7). More recently Quissell et al. have reported the establishment of SV40-transformed rat parotid (8) and submandibular (9) gland cell lines of acinar origin. The parotid cells (Par-C10) grown on porous tissue culture supports formed tight, polarized epithelial monolayers, permitting characterization of neurotransmitter-regulated anion secretion (10).

The SMG-C6 cell line derived from rat submandibular acinar glands expresses specific acinar cell proteins and retains receptor-stimulated mobilization of intracellular signaling elements (Ca2+ and cAMP) with varying consistency compared with intact salivary glands or freshly isolated cells (9). Therefore, the current study was performed to characterize basal and stimulated ion transport processes from this immortalized homogeneous preparation and to compare them with properties found in acinar epithelium. SMG-C6 cells cultured on collagen-coated polycarbonate filters displayed clear evidence of polarization with stable transepithelial resistance, facilitating their bioelectric characterization in Ussing chambers. Under short-circuit conditions, the cell line exhibited agonist-stimulated Cl- secretory conductance. However, not commonly associated with acinar transport function, amiloride-sensitive apical Na+ absorption in conjunction with the expression of rat epithelial Na+ channels (rENaC) was demonstrated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials.
DMEM/F12, fetal bovine serum (FBS) and trace element mix were obtained from Gibco (Grand Island, NY). Transferrin and epidermal growth factor (EGF) were obtained from JRH Biosciences (Lenexa, KS). Insulin was obtained from Novo Nordisk Pharmaceuticals (Princeton, NJ). Polycarbonate Snapwell filters were obtained from Costar (Cambridge, MA). The drugs used were: amiloride, dimethylamiloride, benzamil, acetylcholine, ouabain, ATP, UTP, dibutyryl-cAMP (Bt2-cAMP), tetraethylammonium (TEA), bumetanide, isoproterenol, glibenclamide (Sigma Chemical Co, St. Louis, MO), diphenylamine-2-carboxylic acid (DPC; Fluka Chemical Corp, Ronkonkoma, NY), 1,2-bis-(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA-AM; Molecular Probes, Eugene, OR), and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; Research Biochemical, Natick, MA).

Cell Culture.
SMG-C6 cells (passages 22–34) were seeded at a density of 2.5 x 105 cells/cm2 on Snapwell tissue culture–treated filters (diameter 12mm, pore size 0.4 µm) coated with 1.0 µg/cm2 of human collagen type-I (Becton Dickinson, Franklin Lakes, NJ). The modified culture medium (8, 9) used was DMEM/F12 (1:1 mixture) with 2.5% FBS supplemented with trace element mix, 5 µg/ml transferrin, 1.1 µM hydrocortisone, 0.1 µM retinoic acid, 2.0 nM T3, 5 µg/ml insulin, 80 ng/ml EGF, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Transepithelial Transport Studies.
Transepithelial resistances (Rt) of the SMG-CG monolayers grown on permeable filters were serially measured by an EVOM-G (World Precision Instrument, WPI, Sarasota, FL). Filters exhibiting an Rt > 400 {Omega}.cm2 were placed in a modified Ussing chamber (Costar, Cambridge, MA) equipped with Ag/AgCl electrodes and bathed with Krebs-Ringer bicarbonate (KRB) medium containing (in mM): 120 NaCl, 2.5 K2HPO4, 0.6 KH2PO4, 1.2 CaCl2, 1.2 MgCl2, 20 NaHCO3, and 10 glucose at pH 7.4. For ion replacement studies, all Cl- and Na+ in KRB were replaced with an equivalent amount of gluconate or choline, respectively.

The media were air-lifted with 5% CO2/21% O2, and the temperature was maintained at 37°C. Using a DVC-1000 Voltage/Current Clamp (WPI) with automatic fluid resistance compensation, stable baseline potential difference (PD) was measured. After equilibration (10–15 min), the monolayer's PD was clamped to 0 mV, and short-circuit current (Isc) was continuously recorded. Every 30 sec, the monolayer was clamped to 1 mV for 0.5 sec so that measured changes in current enabled calculation of Rt using Ohm's law (Rt = {Delta}PD/{Delta}Isc). Reported Isc values refer to the movement of negative charge from the basolateral to apical side of the membrane, and the apical side PD is negative referenced to the basolateral side.

After reaching steady state Isc values, agents known to influence ion transport were instilled into either the apical or basolateral reservoir. Peak or treatment Isc values were measured at the point of maximal change immediately or following 20 min exposure, respectively. Amiloride and DPC were dissolved in DMSO and ethanol, respectively, with the final concentration of both diluents in the Ussing chamber fluid being 1:1000. Preliminary studies established the vehicle concentration at which no changes occurred in Rt. Therefore, all changes occurring in any of the transepithelial electrical parameters are due to an agent rather than its vehicle. Neither basal bioelectric properties nor the response to specific agents varied following each cell passage.

Western Blot Analysis.
The SMG-C6 monolayers grown on human collagen-coated culture dishes were treated with trypsin, washed, and pelleted at 4°C in balanced salt solution. Total protein from cell lysates were resolved on 12.5% SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to Optitran BA S-83 nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and incubated with primary monoclonal anticytokeratin mouse antibodies C11 (Santa Cruz Biotechnology, Santa Cruz, CA), a general epithelial (pan-cytokeratin) marker, or K4.62, specific for cytokeratin 19 (Sigma). In salivary glands, cytokeratin 19 expression is restricted to differentiated epithelial cells of ductal origin (6, 11). Horseradish peroxidase–labeled secondary antibody (Sigma) and the enhanced chemiluminescence detection system (ECL Plus, Amersham Pharmacia Biotech, Piscataway, NJ) were used to visualize proteins. Extracted proteins from rat salivary glands were blotted for comparison.

RNA Isolation and Northern Analyses.
Whole rat organs (submandibular glands, lung, kidney, and liver) or SMG-C6 epithelial (7 x 106 cells) RNA was extracted using Tri Reagent (Sigma Chemical Co., St. Louis, MO) and quantitated using an Ultrospec 2000 spectrophotometer (Amersham Pharmacia Biotech, Piscataway, NJ), RNA mode at 260/280nm (ratio > 1.8). The RNA was size-fractionated by electrophoresis on a 1.2% agarose gel containing 1 x MOPS and 1.1% formaldehyde. Total RNA of 20µg/lane was transferred to a Hybond-N(+) (Amersham Pharmacia Biotech, Piscataway, NJ) nylon membrane using the Turboblotter system (Schleichler & Schuell, Keene, NH). After prehybridization, blots were hybridized with 32P-labeled full length {alpha}-rat epithelial Na+ channel ({alpha}-rENaC) probe, washed with 0.2 x SSC + 0.01% SDS at 42°C for 30 min (12), and then subjected to autoradiography at -85°C for 3.5 hr (Lightening Plus intensifier screens and BioMax MS film; Eastman Kodak, Rochester, NY).

Statistical Analysis.
Data were expressed as mean ± standard error (SE). Changes in response to treatment with test agents were compared with initial values (control) by paired or unpaired t tests as appropriate or one-way analysis of variance followed by Student-Newman-Keul multiple comparison tests to determine statistical differences between groups. A P-value < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Baseline Bioelectric Properties.
Filters seeded with SMG-C6 cells (Days 3–5) exhibiting Rt values > 400 {Omega}.cm2 were mounted in Ussing chambers for electrophysiologic studies. Of the filters measured, >95% fit within these criteria. Under basal conditions, the Rtacross the SMG-C6 epithelial filters (n = 69) was 956 ± 84 {Omega}.cm2 and the PD was -16.9 ± 1.5 mV (referenced to basolateral reference electrode). Transepithelial current under short-circuit condition (Isc) measured 23.9 ± 1.7 µA/cm2.

Effect of Drugs Known to Modulate Cl- Transport.
The effects of ion transport inhibitors on basal Isc are summarized in Table IGo. The addition of the Na+-K+-ATPase inhibitor, ouabain (1 mM), to the basolateral fluid resulted in a rapid decrease in Isc (36.7 ± 1.8 to 4.3 ± 3.9 µA/cm2, P < 0.05). This inhibition of Isc was associated with a 10.7% increase in Rt. When ouabain was added to the apical fluid, Isc and Rt were not affected. Apical DPC (100 µM) or glibenclamide (100 µM), primary inhibitors of Ca2+- or cAMP-activated Cl- current (13, 14), respectively, or basolateral bumetanide (100 µM), an inhibitor of Na+-K+-2Cl- co-transport, did not significantly decrease basal Isc value. DIDS (100 µM), a known Cl-/HCO3- exchange blocker, added to the basolateral medium also did not alter basal Isc. Apical DIDS lowered the Isc by 23%; however, the decrease was variable and not significant. TEA, a K+ channel inhibitor added to the basolateral bath gradually decreased basal Isc by 27% (P < 0.05). TEA had minimal effects when added to the apical bath.


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  		Table I.  Effects of Ion-Transport Inhibitors on Basal and Drug-Altered Isc Values in SMG-C6 Monolayers
 
Effect of Drugs Known to Activate Ion Transport.
Addition of the ß-adrenergic agonist, isoproterenol (100 µM), into the basolateral bath did not significantly alter baseline Isc (peak {Delta}Isc, 2.0 ± 0.9 µA/cm2) (Table II)Go. Similarly, little change was noted when dibutyryl-cAMP (100 µM), a membrane permeant analog of cAMP, was added to the basolateral bath. The basolateral addition of the cholinergic agonist, acetylcholine (ACh, 100 µM), produced a transient Isc increase (peak {Delta}Isc, 6.9 ± 1.0 µA/cm2, P < 0.05) with a progressive decline in Isc to 79% of basal values. Addition of the P2 nucleotide receptor agonists, ATP or UTP, into the apical medium also induced a biphasic Isc response with an immediate transient increase (10.9 ± 2.4 µA/cm2 or 9.7 ± 2.4 µA/cm2, respectively) followed by a sustained suppression to {approx} 45%–55% below the basal values (Fig. 1)Go. Peak change in Isc values in response to apical ATP or UTP concentrations from 1.0 µM to 100 µM were similar and occurred within 36.7 ± 6.0 sec (Fig. 2)Go. The poststimulatory suppression by both P2 agonists equilibrated at 20.8 ± 1.6 min with the maximal suppression occurring at 100 µM for both agonists (Fig. 2)Go. The changes in Isc following basolateral ATP or UTP treatment did not differ from the apical biphasic response (Table II)Go. Simultaneous treatment with ATP (apical) and isoproterenol (basolateral) produced a comparable Isc peak following ATP treatment alone; however, the Isc following stimulation did not significantly decline below basal values (Table II)Go.


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  		Table II.  Effects of Ion Transport Stimulators on Basal and Drug-Altered Isc Values in SMG-C6 Monolayers
 


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  		Figure 1.  Representative Isc tracing of the apical (A) ATP (100 µM) and (B) UTP (100 µM) Isc (µA/cm2) response as a function of time. Filter seeded with SMG-C6 cells and mounted in Ussing chamber were treated with agents added to the apical medium (arrow).

 


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  		Figure 2.  Immediate peak (open bar) and poststimulatory (closed bar) changes in basal Isc ({Delta}Isc, µA/cm2) in SMG-C6 epithelium in response to apical administration of (A) ATP at 0.1 µM (n = 4), 1.0 µM (n = 5), 10 µM (n = 5), and 100 µM (n = 9); and (B) UTP at 0.1 µM (n = 4), 1.0 µM (n = 4), 10 µM (n = 5), and 100 µM (n = 7). The peak and poststimulatory changes in Isc values were measured within 60 sec and 20 min, respectively, following addition of P2 agonists. Values are mean ± SE.

 
The effects of various ion transport blockers on the ATP-induced changes in Isc are presented in Figure 3Go. Inhibiting the Cl-/HCO3- exchanger with apical DIDS (100 µM) did not alter the ATP response; however, blocking Cl- channels with apical DPC (100 µM) or glibenclamide (100 µM) significantly decreased the ATP-induced biphasic Isc responses. The Na+-K+-2Cl- co-transport inhibitors, bumetanide (100 µM), or K+ channel blocker, TEA (1 mM), added to the basolateral medium also decreased the ATP-induced peak; however, TEA did not alter the poststimulatory inhibition. Finally, incubating the SMG-C6 epithelia with the intracellular Ca2+ chelator, BAPTA-AM (10 µM, 20 min), abolished the transient and poststimulatory Isc responses to ATP (Fig. 3B)Go.



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  		Figure 3.  Immediate peak (open bar) and poststimulatory (closed bar) changes in basal Isc ({Delta}Isc, µA/cm2) across SMG-C6 epithelia in response to apical administration of ATP 100 µM alone (Control, n = 9) and (A) following pretreatment with DIDS (100 µM, n = 4), DPC (100 µM, n = 6), or glibenclamide (GLIB, 100 µM, n = 5) and (B) bumetanide (BUMET, 100 µM, n = 4), TEA (1 mM, n = 4), or BAPTA (10 µM, n = 5). All drugs were added to the apical media except for bumetanide and TEA. The peak and poststimulatory changes in Isc values were measured within 60 sec and 20 min, respectively, following addition of P2 agonists. Values are mean ± SE; *P < 0.05 versus control (ATP) Isc peak, {dagger}P < 0.05 versus control (ATP) poststimulation Isc.

 
Cl--free medium bathing both sides of SMG-C6 monolayers significantly increased basal Isc and Rt (Table III)Go and inhibited the ATP-induced Isc increase (Fig. 4)Go. In contrast, basolateral Cl--free medium reduced the basal Isc by 34% (28.3 ± 2.3 to 17.0 ± 3.3 µA/cm2) and reversed the apical ATP-induced Isc deflection ({Delta}Isc, -14.0 ± 1.8 µA/cm2), consistent with anion (Cl-) current movement from an apical to basolateral direction.


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  		Table III.  Effects of Ion Substitution on Basal Isc and Rt in SMG-C6 Monolayers
 


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  		Figure 4.  Change in ATP-stimulated (100 µM apical) peak Isc ({Delta}Isc, µA/cm2) across SMG-C6 epithelia bathed in Krebs-Bicarbonate (KB, Control, n = 8), bilateral Cl--free KB (n = 6), and basolateral Cl--free KB (n = 4) media. Cl- was replaced with gluconate. Apical amiloride (10 µM) was present in all experimental media to inhibit basal or stimulated Na+ transport. *P < 0.05 versus control.

 
Effect of Drugs Known to Modulate Na+ Transport.
Apical administration of amiloride at 100 mM completely abolished basal Isc from 39.8 ± 6.4 to 0.4 ± 0.4 µA/cm2 (Table I)Go. At the lower concentration of 10 µM, apical amiloride rapidly decreased Isc from 19.1 ± 4.9 to 0.3 ± 1.0 µA/cm2. The dose-dependent inhibitory effects of amiloride on basal Isc demonstrated a half-maximal inhibitory concentration (IC50) of 392 nM (Fig. 5A)Go. In contrast, basal Isc was unaffected by increasing concentration of EIPA, an inhibitor of amiloride-insensitive Na+ transport (15). At an apical EIPA concentration of 100 µM, Isc fell to 19.5% of basal values. Apical benzamil (100 µM), an amiloride analog with a high affinity for Na+ channels, similarly depressed basal Isc by 97% (Fig. 5B)Go. Dimethylamiloride (100 µM), an amiloride analog that nonspecifically blocks the Na+-H+ antiport (15), did not significantly lower basal Isc when applied apically. Basolateral addition of 100 mM amiloride or dimethylamiloride (100 µM) produced a smaller {approx} 30% decrease in basal Isc (Table I)Go.



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  		Figure 5.  Effect of Na+ transport inhibitor on basal Isc across SMG-C6 epithelia. (A) Percentage (%) of basal Isc inhibition across SMG-C6 epithelium in response to increasing concentrations of amiloride (closed circle, n = 5) and 5-(N-ethyl-N-isopropyl)-amiloride, EIPA (open circle, n = 4). (B) Effect of Amiloride 100 µM (AMIL, n = 8), Benzamil 100 µM (BENZ, n = 4) or dimethylamiloride 100 µM (DIMA, n = 4) on basal Isc (closed bar). Open bars are Isc values measured after apical treatment with the above drugs. *P < 0.05 versus basal Isc.

 
The peak change in basal Isc ({Delta}Isc) caused by apical ATP was significantly greater following preincubation with apical amiloride (19.6 ± 2.8 µA/cm2) or benzamil (34.6 ± 6.7 µA/cm2), compared with ATP alone (Fig. 6)Go. Furthermore, the poststimulatory Isc decrease was abolished in the presence of Na+ channel inhibition. Na+-free medium bathing both sides of the SMG-C6 monolayers abolished basal Isc (Table III)Go, whereas Na+-free apical medium reversed inward basal Isc to a negative value (-5.0 ± 1.1 µA/cm2), consistent with outward cation flow (i.e., Na+ movement from basolateral to apical bath, Table IIIGo). ATP (100 µM) added to the Na+-free apical bath induced a positive peak {Delta}Isc (18.7 ± 2.7 µA/cm2) with elimination of the poststimulatory Isc suppression (Fig. 7)Go. In contrast, Na+-free basolateral medium, while maintaining apical Na+ concentrations at {approx} 140 mM, did not significantly alter basal Isc or Rt values; however, ATP generated a negative peak {Delta}Isc (-7.4 ± 0.9 µA/cm2). Therefore, the SMG-C6 epithelia exhibit stimulus-induced apical Cl- secretion, dependent on basolateral Cl- and Na+; however, inward apical Na+ transport was the predominant source of the basal current.



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  		Figure 6.  (A) Representative tracing of sequential addition of apical amiloride (100 µM) and ATP (100 µM) induced changes in SMG-C6 epithelial-generated Isc as a function of time. (B) Peak (open bar) and poststimulatory (closed bar) changes in basal Isc ({Delta}Isc, µA/cm2) in response to apical administration of ATP 100 µM (Control, n = 9) in the presence of amiloride 100 µM (AMIL, n = 8) and benzamil 100 µM (BENZ, n = 5). The peak and poststimulatory changes in Isc values were measured within 60 sec and 20 min, respectively, following addition of P2 agonists. Values are mean ± SE; *P < 0.05 versus ATP peak response, {dagger}P < 0.05 versus ATP poststimulatory response.

 


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  		Figure 7.  Effect of Na+ replacement with choline on ATP stimulated peak and poststimulatory Isc response in SMG epithelium. Filters seeded with SMG-C6 cells, placed in Ussing chambers with the following media: Krebs-Bicarbonate bilaterally (Control, n = 9), Na+-free apical (n = 12), or Na+-free basolateral (n = 12). ATP (100 µM) was added apically, and the peak (open bar) and poststimulatory (closed bar) changes in Isc ({Delta}Isc, µA/cm2) are demonstrated. The peak and poststimulatory changes in Isc values were measured within 60sec and 20min, respectively, following addition of P2 agonist. Values are mean ± SE. *P < 0.05 versus control peak values, {dagger}P < 0.05 versus control poststimulatory values.

 
Expression of {alpha}-rENaC.
The SMG-C6 epithelium functionally demonstrates amiloride-sensitive Na+ transport; therefore, we determined if this cell line expresses the {alpha}-subunit from the rat epithelial Na+ channel ({alpha}-rENaC). rENaC is composed of three subunits ({alpha}, ß, and {gamma}) and is found in rat epithelial cells such as the kidney collecting duct, the duct of several exocrine glands, and the proximal and distal airways of the lung (16). The full-length {alpha}-rENaC clone was used as a probe to hybridize in a Northern blot consisting of RNA (20 µg) from various adult rat tissues and SMG-C6 cells. A 3.8-kb transcript was identified that is consistent with {alpha}-rENaC expression in the rat submandibular glands, lung, and kidneys and in the SMG-C6 epithelia (passage 26 and 29, Fig. 8Go). RNA obtained from rat livers (negative control) did not express {alpha}-rENaC.



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  		Figure 8.  Northern-blot containing 20 µg/lane total RNA isolated from SMG-C6 cells (passage 26 and 29) and rat submandibular gland (sub gland), lung, kidney, and liver, hybridized with the full-length 32P-labeled {alpha}-rENaC. A 3.5-kb transcript consistent with{alpha}-rENaC expression was detected in both passages from SMG-C6 cells and rat submandibular gland, lung, and kidney. No {alpha}-rENaC expression was detected in the liver.

 
Cytokeratin Expression.
Since the demonstration of amiloride-sensitive Isc and expression of {alpha}-ENaC was unexpected in a submandibular cell line of acinar origin, the expression of cytokeratin 19, specific for salivary ductal epithelium (6, 11) was determined using Western analysis. Protein extracted from the SMG-C6 cells and from isolated rat submandibular glands was positively labeled with the nonspecific antipancytokeratin antibody (Fig. 9)Go. However, cytokeratin 19 protein expression was found only in the whole submandibular gland and not identified in the SMG-C6 cells.



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  		Figure 9.  Western blot analysis of cytokeratin (CK) proteins from cell lysates obtained from whole rat salivary glands (S. gland) and SMG-C6 cells grown on collagen-coated culture dishes. Cell extracts were processed for Western blotting by SDS-PAGE and probed for (A) nonspecific cytokeratins (pancytokeratin-Pan CK) and (B) cytokeratin 19 (CK 19) expression. Position of molecular weight standard (in kDa) is indicated on left.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Salivary cells in culture serve as a useful model system to study in vitro the molecular and cellular biology of saliva production; however, the limited life span of primary cultured cells or the inability of tumor-derived cell lines to form polarized, tight-junction monolayers have prevented measurements of transepithelial ion movement (1, 3, 5, 17). Immortalized cell lines from rat salivary glands demonstrate structural and functional properties characteristic of in situ salivary epithelia, including tight junctions and localization of Na+-K+-ATPase proteins on the basolateral domain (6, 7). The recent characterization of neurotransmitter-regulated anion secretion in the parotid acinar cell line Par-C10 was the first report demonstrating vectorial ion transport across a salivary-derived epithelium in Ussing chambers (10).

The SMG-C6 cell line originates from the rat submandibular acinar epithelium and shares functional properties with those found in freshly excised salivary acinar cells including activation of the IP3-Ca2+ pathway by cholinergic, {alpha}-adrenergic, and P2 nucleotide receptor agonists and adenylate cyclase activation by ß-adrenergic agonists (9). Employing standard culture techniques on human collagen-coated permeable supports, we found that the SMG-C6 cell line formed polarized epithelia, having transepithelial resistances of {approx} 900 {Omega}.cm2 and a spontaneous PD of 16.9mV (apical negative). Under short-circuited conditions, ouabain added to the basolateral surface of the SMG-C6 epithelium inhibited the majority of the basal current, indicating ion transport systems dependent on Na+-K+-ATPase activity. Likewise, decreasing basolateral K+ permeability with TEA may also directly inhibit Na+-K+-ATPase activity, resulting in a decreased basal Isc (18). The lack of effect by Cl- transport inhibition with bumetanide or removal of extracellular Cl- suggests that basal Isc was not Cl--dependent.

The Isc response to P2 nucleotide receptor agonists ATP or UTP applied to either the apical or basolateral surface was biphasic, with an initial transient peak Isc, followed by a sustained Isc inhibition across the SMG-C6 epithelia. The P2Y2 nucleotide receptor subtype activated by UTP has been specifically localized to the apical surface of the rat salivary cell line Par-C10 (10). ATP also nonspecifically interacts with the P2Y2 and other P2 receptor subtypes (10, 19). The similar Isc response to UTP or ATP suggests the expression of P2Y2 receptors on the SMG-C6 epithelial apical and basolateral membranes. However, it is also possible that the agonists may have entered the contralateral medium and interacted with the receptor on the contralateral side from where they were added. In addition, without molecular biological probing for specific P2Y2 messages, the possibility of other P2 receptor subtypes cannot be excluded.

The peak {Delta}Isc responses across the SMG-C6 epithelium caused by P2 receptor agonists, representing an increase in either transepithelial cation absorption or anion secretion, were not amiloride-sensitive. However, the inhibitory effect of removing extracellular Cl- or using specific apical Cl- or basolateral K+ channel blockers, suggests activation of apical Cl- secretion (18). Cl- uptake is maintained by the action of the basolateral Na+-K+-2Cl- co-transporter or through Na+-H+ and Cl--HCO3- exchange (18). In the absence of Na+ or Cl- in the basolateral medium or presence of basolateral bumetanide, Cl- entry is decreased, and the ATP-stimulated Isc is diminished, further supporting activation of Cl- secretory pathways. The ATP response was further accentuated in the presence of Na+ channel inhibitors, amiloride and benzamil, consistent with apical membrane hyperpolarization resulting from Na+ entry blockade, thereby increasing the electrodiffusive driving force for apical Cl- efflux (20). Furthermore, establishing a Cl- gradient across the SMG-C6 epithelium in the basolateral direction, ATP stimulated an inward negative peak current, consistent with apical Cl- influx via ATP-activated channels (21).

P2 nucleotide receptor agonists stimulate intracellular Ca2+ mobilization in the SMG-C6 cells (9). Activation of the P2 receptors, coupled to phospholipase C, initiates increases in formation of IP3, resulting in a rapid increase in [Ca2+]i caused by release from internal stores and influx from the bathing solution (22). Since ATP induces a rapid and transient rise in both [Ca2+]i and Isc, activation of Ca2+-dependent Cl- channels are likely (18). Pre-incubating the SMG-C6 monolayers with BAPTA, an intracellular Ca2+ chelator, abolished the ATP-induced peak in Isc, providing further support for activation of a Ca2+-dependent Cl- conductance by P2 nucleotide receptor agonists.

The response to ACh, a potent stimulus of Ca2+ mobilization and Cl- and K+ efflux in isolated salivary cells and intact glands (23), produced an increase in Isc that was 45% smaller than that compared with either ATP or UTP. The smaller Isc response by cholinergic agonist stimulation is unclear; however, enhanced Ca2+ mobilization by upregulated P2 receptor subtypes has been reported in short-term culture of dispersed salivary gland cells (3). Likewise, SMG-C6 cells exposed to similar ATP or UTP concentrations caused higher elevations in [Ca2+]i levels compared with cholinergic stimulation (9). It is unclear if differences in [Ca2+]i elevation contribute to the differences in the Isc responses to these agonists.

Stimulation with a ß-adrenergic agonist generates intracellular increases in cAMP in SMG-C6 cells (9). Although ß-agonists regulate secretion of amylase in salivary acinar cells and are not thought to significantly increase Cl- conductance (18), cAMP and ATP have been shown to regulate a glibenclamide-sensitive current in salivary acinar and ductal cells (24, 25). In the present study, either isoproterenol or a cAMP-permeable analog did not alter basal Isc, and no synergism between ATP and isoproterenol was evident, although the Isc suppression following ATP administration alone was altered. Therefore, cAMP does not appear to alter Cl- secretion; however, the alteration in the ATP-induced Isc peak by glibenclamide found in this study suggests the presence of alternative Cl- channel expression on the SMG-C6 apical membrane.

In the absence of Na+ or with amiloride (10–100 µM) in the apical medium, basal Isc across the SMG-C6 epithelia was virtually abolished. The amiloride IC50 measured across the SMG-C6 epithelia is consistent with specific inhibition of Na+ apical channels (15, 26). Higher concentrations of amiloride also inhibit the Na+-H+ antiport (27) in rat submandibular cells. However, benzamil's significant reduction of basal Isc, in contrast with the response by amiloride analogs, dimethylamiloride or EIPA, further supports the presence of apical high-amiloride-affinity (H-type) channels (15). Amiloride or dimethylamiloride in the basolateral medium also lowered basal Isc, suggesting the presence of amiloride-sensitive transport systems on the basolateral membrane. However, in the absence of Na+ in the basolateral medium, basal Isc was not altered. Therefore, the basolateral effects of these agents on basal ion transport may reflect diffusion across the epithelium and inhibition of amiloride-sensitive Na+ channels on the apical side.

Consistent with vectorial Na+ transport found under Isc conditions, SMG-C6 cells express {alpha}-rENaC, the major subunit of the rat epithelial Na+ channel (28). Passive Na+ transport into the apical or luminal direction in response to Cl- secretion contributes to the secretory properties of the salivary acinar end pieces (18). However, amiloride-sensitive Na+ channels, localized to kidney, lung, and colonic epithelial apical membranes (16) are not naturally expressed in submandibular acinar epithelium. Apical Na+ channels are expressed in the salivary ducts epithelia, modifying the ion content of secondary saliva (29, 30). Despite the presence of amiloride-sensitive Na+ conductance and expression of {alpha}-rENaC, the SMG-C6 cells do not morphologically resemble duct cells (9). Furthermore, our inability to identify cytokeratin 19 protein expression, specific for salivary ductal cells (6, 11), also does not suggest a ductal origin for this cell line. Recently, the immortalized rat parotid acinar cell line Par-C5 (8) was also found to express {alpha}-rENaC (31).

The sustained decline in Isc following the transient ATP-stimulated peak Isc was eliminated with the Na+ channel blockers, amiloride or benzamil, or by removing extracellular apical Na+. Intracellular Ca2+-dependent inhibition of apical Na+ entry has been reported in epithelia from the airways (32-36), renal collecting duct (37), and MDCK cell line (38). Likewise, an increase in cytosolic Cl- also inhibits amiloride-sensitive Na+ transport in salivary duct cells (29, 39). The poststimulatory ATP response in the SMG-C6 epithelia following Ca2+ chelation or Cl- channel inhibition suggests a role in which changes in cytosolic Ca2+ or Cl- may activate intracellular signals, altering Na+ channel activity. Apical Na+ channel inhibition mediated by Ca2+- dependent protein kinase (40) or specific G proteins (39) has been demonstrated using patch-clamp techniques. In contrast, inhibition of Na+ transport across human bronchial epithelium by UTP was found to be independent of PKC or PLA2 activation (36). Increasing intracellular cAMP has also been shown to reverse partially the inhibitory effect of UTP-induced inhibition of Na+ transport in human bronchial epithelium (36). In this study, the co-administration of ATP and isoproterenol altered the poststimulatory decrease in Isc values, suggesting a similar inhibitory effect by cAMP on ATP-induced Na+ inhibition. Intracellular cAMP regulation of Na+ transport in this cell line is unclear; however, alteration in either ATP metabolism or desensitization of ATP-sensitive P2 receptors by cAMP occurs in tracheal submucosal epithelium (41). Finally, the entry of Na+ ion down its electrochemical gradient is dependent on Na+-K+-ATPase pump activity with the recycling of K+ across the basolateral membrane. The UTP inhibition of Na+ transport in human bronchial epithelium was partly due to downregulation of basolateral K+ conductance (36). In contrast, the TEA-sensitive K+ channel on the basolateral SMG-C6 membrane did not appear to be coupled to Na+ transport since TEA did not alter the ATP-induced sustained Isc inhibition. Further studies examining the specific roles of these specific intracellular signaling elements and Na+ transport inhibition across the SMG-C6 epithelia are needed to elucidate their underlying mechanism(s).

Salivary acini produce a Na+ and Cl- rich primary secretion, and the membrane components regulating fluid and electrolyte transport have been the subject of various reviews (18, 42). The SMG-C6 cell line epithelia share similar secretory properties, and the model depicted in Figure 10Go summarizes the coordinated active Cl- uptake and efflux via apical channels. Cholinergic (basolateral) and P2 nucleotide (apical and basolateral) receptor agonists exert their effects by stimulating the release of Ca2+ from intracellular stores, leading to the efflux of Cl- through apical channels and K+ via basolateral channels maintaining electroneutrality (23). This model also demonstrates the active reabsorption of Na+ via amiloride-sensitive Na+ channels that may be partly inhibited by intracellular Ca2+ or other downstream signaling elements. Thus, the secretory and absorptive properties exhibited by the SMG-C6 epithelia are indeed novel for in situ salivary epithelial ion transport processes and appear to share characteristics also found in salivary duct epithelia. Therefore, this cell line may prove to be a valuable in vitro model for understanding signaling mechanisms regulating fluid and electrolyte secretion and absorption in salivary and other epithelia.



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  		Figure 10.  Schematic drawing of putative SMG-C6 epithelial transport processes involved in active Na+ absorption and Cl- secretion. At baseline, Na+ enters the cells via specific apical channels along an inward-directed electrochemical gradient set up by basolaterally located Na+-K+-ATPase activity. Increased intracellular Ca+ appears to be a negative regulator of this channel whereas the regulatory role of increases in intracellular cAMP levels resulting from ß-adrenergic receptors (ß-AR) activation has not been determined. Uptake of Cl- is coupled to Na+ via the Na+-K+-2Cl- co-transporter in the basolateral membrane. Binding of P2 nucleotide or cholinergic (Ch) agonist to specific receptors coupled to G proteins on either basolateral or apical membrane, results in an increase in intracellular Ca2+, activating apical Cl- channels and secretion into the apical medium.

 


   Acknowledgments
 
We thank Dr. David O. Quissell (University of Colorado Health Science Center) for providing the SMG-C6 cell line and Dr. Hugh M. O'Brodovich and Bijan Rafii (The Hospital for Sick Children, Toronto, Ontario, Canada) for providing the {alpha}-ENaC mRNA probe and their technical assistance. We also acknowledge the excellent technical assistance of Dr. Shamin B. Mustafa and John H. Easton.


   Footnotes
 
This work was supported by the Department of Pediatrics and Medical Hispanic Center of Excellence, University of Texas Health Science Center, San Antonio, Texas.

1 To whom requests for reprints should be addressed at the Department of Pediatrics, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284–7812. E-mail: castror{at}uthscsa.edu Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Quissell DO, Redman RS, Mark MR. Short-term primary culture of acinar-intercalated duct complexes from rat submandibular glands. In Vitro Cell Dev Biol 22:469–480, 1986.[Medline]
  2. Sabatini LM, Allen-Hoffmann BL, Warner TF, Azen EA. Serial cultivation of epithelial cells from human and macaque salivary glands. In Vitro Cell Dev Biol 27A:939–948, 1991.
  3. Turner JT, Weisman GA, Camden JM. Upregulation of P2Y2 nucleotide receptors in rat salivary gland cells during short-term culture. Am J Physiol 273:C1100–C1107, 1997.[Abstract/Free Full Text]
  4. Yeh C-K, Mertz PM, Olivers C, Baum BJ, Kousvelari EE. Cellular characteristics of long-term cultured rat parotid acinar cells. In Vitro Cell Dev Biol 27A:707–712, 1991.
  5. Zhu Y, Aletta JM, Wen J, Zhang X, Higgins D, Rubin RP. Rat serum induces a differentiated phenotype in a rat parotid acinar cell line. Am J Physiol 275:G259–G268, 1998.[Abstract/Free Full Text]
  6. Laoide BM, Courty Y, Gastinne I, Thibaut C, Kellermann O, Rougeon F. Immortalized mouse submandibular epithelial cell lines retain polarized structural and functional properties. J Cell Sci 109:2789–2800, 1996.[Abstract]
  7. He X, Kuijpers GAJ, Goping G, Kulakusky JA, Zheng C, Delporte C, Tse CM, Redman RS, Donowitz M, Pollard HB, Baum BJ. A polarized salivary cell monolayer useful for studying transepithelial fluid movement in vitro. Pflugers Arch 435:375–381, 1998.[Medline]
  8. Quissell DO, Barzen KA, Redman RS, Camden JM, Turner JT. Development and characterization of SV40 immortalized rat parotid acinar cell lines. In Vitro Cell Dev Biol 34:58–67, 1998.
  9. Quissell DO, Barzen KA, Gruenert DC, Redman RS, Camden JM, Turner JT. Development and characterization of SV40 immortalized rat submandibular acinar cell lines. In Vitro Cell Dev Biol 33:164–173, 1997.
  10. Turner JT, Redman RS, Camden JM, Landon LA, Quissell DO. A rat parotid gland cell line, Par-C10, exhibits neurotransmitter-regulated transepithelial anion secretion. Am J Physiol 275:C367–C374, 1998.[Abstract/Free Full Text]
  11. Geiger S, Geiger B, Leitner O, Marshak G. Cytokeratin polypeptides expression in different epithelial elements of human salivary glands. Virchows Arch A Pathol Anat Histopathol 410:403–414, 1987.[Medline]
  12. O'Brodovich H, Canessa C, Ueda J, Rafii B, Rossier BC, Edelson J. Expression of the epithelial Na+ channel in the developing rat lung. Am J Physiol 265:C491–C496, 1993.[Abstract/Free Full Text]
  13. Dinudom A, Komwatana P, Young JA, Cook DI. A forskolin-activated Cl- current in mouse mandibular duct cells. Am J Physiol 268:G806–G812, 1995.[Abstract/Free Full Text]
  14. Ishikawa T, Cook DI. A Ca2+-activated Cl- current in sheep parotid secretory cells. J Membr Biol 135:261–271, 1993.[Medline]
  15. Kleyman TR, Cragoe EJ. Amiloride and its analogs, as tools in the study of ion transport. J Membr Biol 105:1–21, 1988.[Medline]
  16. Benos DJ, Awayda MS, Ismailov II, Johnson JP. Structure and function of amiloride-sensitive Na+ channels. J Membr Biol 143:1–18, 1995.[Medline]
  17. Horie K, Kagami H, Hiramatsu Y, Hata K, Shigetomi T, Ueda M. Selected salivary-gland cell culture and the effects of isoproterenol, vasoactive intestinal polypeptide, and substance P. Arch Oral Biol 41:243–252, 1996.[Medline]
  18. Cook DI, Van Lennep EW, Roberts ML, Young JA. Secretion by the major salivary glands. In: Johnson LR, Ed. Physiology of the Gastrointestinal Tract. New York: Raven Press, pp1061–1117, 1994.
  19. Turner JT, Landon LA, Gibbons SJ, Talamo BR. Salivary gland P2 nucleotide receptors. Crit Rev Oral Biol Med 10:210–224, 1999.[Abstract/Free Full Text]
  20. Cotton CU, Boucher RC, Gatzy JT. Paths of ion transport across canine fetal tracheal epithelium. J Appl Physiol 65:2376–2382, 1988.[Abstract/Free Full Text]
  21. Sheppard DN, Carson MR, Ostedgaard LS, Denning GM, Welsh MJ. Expression of cystic fibrosis transmembrane conductance regulator in a model epithelium. Am J Physiol 266:L405–L413, 1994.[Abstract/Free Full Text]
  22. Boarder MR, Weisman GA, Turner JT, Wilkinson GF. G-protein coupled P2 purinoceptors: From molecular biology to functional responses. Trends Pharmacol Sci 16:133–139, 1995.[Medline]
  23. Martinez JR, Camden J, Kingsbury MB. Effects of acetylcholine and transport inhibitors on K+ content in dispersed submandibular salivary cells of newborn and adult rats. Arch Oral Biol 33:203–207, 1988.[Medline]
  24. Zeng W, Lee MG, Yan M, Diaz J, Marino CR, Muallem S. Immuno and functional characterization of CFTR in submandibular and pancreatic acinar and duct cells. Am J Physiol 273:C442–C455, 1997.[Abstract/Free Full Text]
  25. Zeng W, Lee MG, Muallem S. Membrane-specific regulation of Cl- channels by purinergic receptors in rat submandibular gland acinar and duct cells. J Biol Chem 272:32956–32965, 1997.[Abstract/Free Full Text]
  26. Cheek JM, Kim K-J, Crandall ED. Tight monolayers of rat alveolar epithelial cells: Bioelectric properties and active sodium transport. Am J Physiol 256:C688–C693, 1989.[Abstract/Free Full Text]
  27. Lee MG, Schultheis PJ, Yan M, Shull GE, Bookstein C, Chang E, Tse M, Donowitz M, Park K, Muallem S. Membrane-limited expression and regulation of Na+-H+ exchanger isoforms by P2 receptors in the rat submandibular gland duct. J Physiol 513:341–357, 1998.[Abstract/Free Full Text]
  28. Champigny G, Voilley N, Lingueglia E, Friend V, Barbry P, Lazdunski M. Regulation of expression of the lung amiloride-sensitive Na+ channel by steroid hormones. EMBO J 13:2177–2181, 1994.[Medline]
  29. Bijman J, Cook DI, van Os CH. Effect of amiloride on electrolyte transport parameters of the main duct of the rabbit mandibular salivary gland. Pflugers Arch 398:96–102, 1983.[Medline]
  30. Dinudom A, Young JA, Cook DI. Amiloride-sensitive Na+ current in the granular duct cells of mouse mandibular glands. Pflugers Arch 423:164–166, 1993.[Medline]
  31. Zentner MD, Lin HH, Wen X, Kim KJ, Ann DK. The amiloride-sensitive epithelial sodium channel {alpha}-subunit is transcriptionally downregulated in rat parotid cells by the extracellular signal-regulated protein kinase pathway. J Biol Chem 273:30770–30776, 1998.[Abstract/Free Full Text]
  32. Al-Bazzaz FJ. Regulation of Na and Cl transport in sheep distal airways. Am J Physiol 267:L193–L198, 1994.[Abstract/Free Full Text]
  33. Iwase N, Sasaki T, Shimura S, Yanamoto M, Suzuki S, Shirato K. ATP-induced Cl- secretion with suppressed Na+ absorption in rabbit tracheal epithelium. Respir Physiol 107:173–180, 1997.[Medline]
  34. Acevedo M. Effect of acetyl choline on ion transport in sheep tracheal epithelium. Pflugers Arch 427:543–546, 1994.[Medline]
  35. Van Scott MR, Paradiso AM. Intracellular Ca2+ and regulation of ion transport across rabbit Clara cells. Am J Physiol 263:L122–L127, 1992.[Abstract/Free Full Text]
  36. Devor DC, Pilewski JM. UTP inhibits Na+ absorption in wild-type and {Delta}F508 CFTR-expressing human bronchial epithelia. Am J Physiol 276:C827–C837, 1999.
  37. Koster HP, Hartog A, van Os CH, Bindels RJ. Inhibition of Na+ and Ca2+ reabsorption by P2u purinoceptors requires PKC but not Ca2+ signaling. Am J Physiol 270:F53–F60, 1996.[Abstract/Free Full Text]
  38. Ishikawa T, Marunaka Y, Rotin D. Electrophysiological characterization of the rat epithelial Na+ channel (rENaC) expressed in MDCK cells. Effects of Na+ and Ca2+ J Gen Physiol 111:825–846, 1998.[Abstract/Free Full Text]
  39. Dinudom A, Komwatana P, Young JA, Cook DI. Control of the amiloride-sensitive Na+ current in mouse salivary ducts by intracellular anions is mediated by a G protein. J Physiol 487:549–555, 1995.[Medline]
  40. Frindt G, Palmer LG, Windhager EE. Feedback regulation of Na channels in rat CCT. IV. Mediation by activation of protein kinase C. Am J Physiol 270:F371–F376, 1996.[Abstract/Free Full Text]
  41. Shimura S, Sasaki T, Nagaki M, Takishima T, Shirato K. Extracellular ATP regulation of feline tracheal submucosal gland secretion. Am J Physiol 267:L159–L164, 1994.[Abstract/Free Full Text]
  42. Martinez JR. Cellular mechanisms underlying the production of primary secretory fluid in salivary glands. Crit Rev Oral Biol Med 1:67–78, 1990.[Free Full Text]
Received for publication December 2, 1999. Accepted for publication April 24, 2000.





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