A rat parotid gland cell line, Par-C10, exhibits neurotransmitter-regulated transepithelial anion secretion

John T. Turner1, Robert S. Redman2, Jean M. Camden1, Linda A. Landon1, and David O. Quissell3

1 Department of Pharmacology, School of Medicine, University of Missouri, Columbia, Missouri 65212; 2 Oral Pathology Research Laboratory, Department of Veterans Affairs Medical Center, Washington, District of Columbia 20422; and 3 Department of Basic Sciences and Oral Research, School of Dentistry, University of Colorado Health Sciences Center, Denver, Colorado 80262

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Because of the lack of salivary gland cell lines suitable for Ussing chamber studies, a recently established rat parotid acinar cell line, Par-C10, was grown on permeable supports and evaluated for development of transcellular resistance, polarization, and changes in short-circuit current (Isc) in response to relevant receptor agonists. Par-C10 cultures reached confluence in 3-4 days and developed transcellular resistance values of >= 2,000 Omega · cm2. Morphological examination revealed that Par-C10 cells grew as polarized monolayers exhibiting tripartite junctional complexes and the acinar cell-specific characteristic of secretory canaliculi. Par-C10 Isc was increased in response to muscarinic cholinergic and alpha - and beta -adrenergic agonists on the basolateral aspect of the cultures and to ATP and UTP (through P2Y2 nucleotide receptors) applied apically. Ion replacement and inhibitor studies indicated that anion secretion was the primary factor in agonist-stimulated Isc. RT-PCR, which confirmed the presence of P2Y2 nucleotide receptor mRNA in Par-C10 cells, also revealed the presence of mRNA for the cystic fibrosis transmembrane conductance regulator and ClC-2 Cl- channel proteins. These findings establish Par-C10 cells as the first cell line of salivary gland origin useful in transcellular ion secretion studies in Ussing chambers.

parotid salivary gland; cell culture; Ussing chamber; ion secretion; P2Y2 receptors; polarized epithelia

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

USSING CHAMBER STUDIES WITH permanent cell lines of epithelial origin have contributed significantly to our understanding of ion secretory processes and their regulation. These cell lines include T84 (colon; Ref. 5) and Madin-Darby canine kidney (MDCK; Ref. 17) among others. However, no cell line of salivary gland origin, either acinar or ductal, has been reported to be useful in Ussing chamber studies of transcellular ion movements and their regulation by neurotransmitters.

Recently, we reported the establishment of simian virus 40-transformed cell lines of rat parotid (15) and submandibular (14) gland acinar origin. Although not without some inconsistencies, these cell lines exhibit a substantial degree of fidelity to the cell type of origin, including morphological, biochemical, and functional characteristics. Among these is the expression of receptors for salivary gland-relevant neurotransmitters, including norepinephrine, acetylcholine, vasoactive intestinal peptide, and extracellular nucleotides. In this report, we describe experiments indicating the suitability of one of the parotid gland cell lines, Par-C10, in Ussing chamber studies, which revealed the presence in these cells of an anion-dependent short-circuit current (Isc) that is regulated by neurotransmitters.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture. Par-C10 cells (5 × 105) were plated on Falcon cell culture inserts (diameter 2.4 cm, pore size 0.4 µm; Becton Dickinson, Franklin Lakes, NJ) coated with 0.10 mg/ml bovine type I collagen. The cultures were grown to confluence in DMEM-F12 (1:1) containing 2.5% fetal bovine serum (GIBCO BRL, Gaithersburg, MD) and the following supplements: 0.1 µM retinoic acid, 80 ng/ml epidermal growth factor, 2 nM triiodothyronine, 5 mM glutamine, 0.4 µg/ml hydrocortisone, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite, 50 µg/ml gentamicin, and 8.4 ng/ml cholera toxin. In some experiments, as indicated, the cholera toxin was omitted. Cells were cultured at 37°C in a humidified 95% air-5% CO2 atmosphere and used at confluence, typically 4-5 days after plating. Cells at passages 50-80 were utilized in these studies, with little change in most of the parameters examined, except as noted in the RESULTS and DISCUSSION sections.

Morphological evaluation. Par-C10 cells were prepared for microscopic evaluation as described previously (15) except that the cells were grown on permeable Falcon cell culture inserts. Briefly, confluent cultures were fixed for 1 h in 2.5% glutaraldehyde in 0.1 M NaPO4, washed three times, and placed in phosphate buffer containing 0.15 M sucrose. Fixation in OsO4 and other steps in the preparation of sections for transmission electron microscopy were as described previously (2).

Measurement of Isc in Par-C10 monolayers. Confluent cultures of Par-C10 cells on permeable supports were mounted in modified Ussing chambers for measurement of agonist-induced changes in Isc, as described previously for studies with T84 colonic epithelial cells (8). The standard medium for both the apical and basolateral reservoirs (volume 5 ml) was Krebs-Ringer-HCO-3 buffer, pH 7.5, containing (in mM) 118 NaCl, 3 KCl, 1.2 MgSO4, 25 NaHCO3, 1.0 CaCl2, and 10 glucose. HCO-3-free medium, pH 7.5, was buffered with 15 mM HEPES. Cl--free media utilized gluconate as the replacement anion with the Ca2+ concentration increased to 4 mM to counteract Ca2+ chelation by gluconate. Na+-free medium was prepared with N-methyl-D-glucamine or choline. Alterations to the apical or basolateral media for specific experiments are indicated in legends of Figs. 3-7. The Ussing apparatus included a water jacket to maintain the buffer temperature at 37°C, and the medium in both reservoirs was mixed and oxygenated by bubbling with 95% O2-5% CO2. Isc was measured continuously, and transepithelial potential difference was measured intermittently, using a VCC-600 automatic voltage-clamp apparatus (Physiologic Instruments, San Diego, CA) and calomel electrodes connected to the chamber baths with 4% agar-KCl bridges. Isc and automatic fluid resistance compensation current were applied through Ag-AgCl electrodes connected to chamber baths with 4% agar-KCl bridges. Resistance measurements were made by occasionally clamping the potential difference to a known voltage and measuring the current required to establish the potential. Resistance and conductance were calculated using Ohm's law. Isc values are expressed as the peak change obtained in response to agonist (µA/cm2) or alternatively as the area under the curve for the first 2 min following agonist addition (arbitrary units) to incorporate differences in the sustained phase of the response.

RT-PCR. Total RNA was prepared from confluent Par-C10 cultures in 100-mm-diameter dishes (passages 60-61) using an RNeasy kit (Qiagen, Chatsworth, CA). Cell lysates were treated with RNase-free DNase. cDNA was synthesized from ~1 µg of Par-C10 total RNA with random hexamer primers using an Advantage RT-for-PCR kit (Clonetech, Palo Alto, CA). The RT-PCR mixture included 2.5 units of Vent(exo-) DNA polymerase, 0.125 units of Vent DNA polymerase, 6 mM MgSO4, 400 µM dNTP, and 40 pmol of each primer in a 50-µl reaction volume. Template and primers were first denatured in a buffer with high salt concentration for 5 min at 95°C. The tubes were moved immediately to 4°C, the enzymes and dNTPs were added, and ultrapure water was used to give the final reaction volume. The PCR cycle consisted of denaturation at 95°C for 1 min, annealing for 1 min (the annealing temperature and number of reaction cycles varied with specific primer sets), and elongation at 72°C for 1 min followed by a 72°C final extension for 5 min. The primer sequences used and details of the RT-PCR reactions are given in Table 1. As a positive control for the P2Y6 primer set, cDNA was prepared from total RNA isolated from 1321N1 cells heterologously expressing the P2Y6 receptor and amplified as described above. Aliquots from the PCR reactions were electrophoresed on agarose gels. The remaining PCR products were purified directly from the reaction mixture using a Wizard PCR Preps DNA purification system (Promega, Madison, WI) and sequenced with specific internal primers and fluorescent chain terminator dNTPs on an Applied Biosystems sequencer (Perkin-Elmer, Foster City, CA).

                              
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Table 1.   Details of RT-PCR reactions

Data analysis. The statistical significance of differences between mean values was determined by unpaired Student's t-test or by one-way ANOVA and Student-Newman-Keuls post hoc test. Differences with P <=  0.05 were considered significant.

Materials. The following reagents were purchased from the indicated sources: fetal bovine serum, GIBCO BRL; epidermal growth factor, Collaborative Research (Bedford, MA); gentamicin, Fujisawa (Deerfield, IL); glutaraldehyde and OsO4, EMCorp (Chestnut Hill, MA); DNase, Boehringer-Mannheim (Indianapolis, IN); and Vent and Vent(exo-) DNA polymerases, New England Biolabs (Beverly, MA). All other reagents were obtained from Sigma Chemical (St. Louis, MO).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Development of transcellular resistance in Par-C10 cell cultures grown on permeable supports. As shown in Fig. 1, transcellular resistance across Par-C10 cell cultures grown on collagen-coated permeable supports increases as a function of time, approaching a maximum by 4 days. This time course was similar to that for cell proliferation, wherein confluence was typically attained after 3 days in culture. Par-C10 cultures grown on supports not coated with collagen or coated with other matrix components exhibited similar transcellular resistances at confluence, as did another parotid cell line, Par-C5, immortalized by the same technique used for Par-C10 (15). However, our initial investigations with these two parotid cell lines, as well as two immortalized submandibular gland cell lines, SMG-C6 and SMG-C10 (14), revealed that Par-C10 cells typically displayed the highest transcellular resistance values and exhibited the most polarized phenotype. As a result, we focused on the Par-C10 cell line for the studies described in this paper.


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Fig. 1.   Effect of time in culture on transcellular resistance of Par-C10 monolayers. Par-C10 cells were plated at a density of 5 × 105 cells on collagen-coated permeable supports, as described in METHODS. Transcellular resistance values at indicated times in culture were obtained with an EVOM (World Precision Instruments, New Haven, CT) volt-ohmmeter with miniature dual chopstick electrodes. After subtraction of medium resistance (115 Omega ), tissue resistance values were multiplied by effective membrane area (4.52 cm2) and are presented as means ± SE (n = 42).

Morphological evaluation. By transmission electron microscopy, confluent Par-C10 cell cultures consisted of a monolayer of plump cells attached to the collagen layer on the upper surface of the microporous membrane insert (Fig. 2A). The cells contained scattered secretory granules with a substructure that was mostly scanty but occasionally dense (Fig. 2, A and B). None of these was observed to be in the process of exocytosis. Cell processes extended into the pores of the membranes, some all the way to the bottom (Fig. 2, A and C), but did not spread out on the bottom surface. The cells were joined along their apical plasmalemmas by tripartite junctional complexes, consisting of tight and intermediate junctions and several desmosomes (Fig. 2, A and D). A terminal web was associated with the tight junctions (Fig. 2D). Numerous intercellular clefts were observed that had lumina segregated by junctional complexes (Fig. 2, A and D) and thus could be distinguished from ordinary intercellular spaces as secretory canaliculi (22). The plasmalemmas on the apical surfaces and lining the canaliculi were studded with small microvilli. The cytoplasm was rich in free ribosomes and also contained numerous mitochondria and dilated profiles of rough endoplasmic reticulum (Fig. 2, B and D). The only difference observed between the cells cultured with and without cholera toxin was that, in the latter, secretory granules occurred in clusters more frequently.


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Fig. 2.   Morphological aspects of Par-C10 cells cultured on permeable supports. Shown are transmission electron micrographs of Par-C10 cells cultured with (A, C, and D) or without (B) cholera toxin and processed for morphological evaluation as described in METHODS. Arrowhead, tight junction component of a tripartite junctional complex; arrows, membrane pores containing cell processes; c, secretory canaliculi; g, secretory granules; r, rough endoplasmic reticulum; w, terminal web. Magnifications: A, ×3,900; B, ×13,400; C, ×5,700; D, ×24,800.

Agonist-induced changes in Isc. We reported previously that Par-C10 cells are responsive, in terms of mobilization of second messengers, to salivary gland-relevant receptor agonists, including the muscarinic cholinergic agonist carbachol, the beta -adrenergic receptor agonist isoproterenol, the alpha -adrenergic receptor agonist phenylephrine, and the P2 nucleotide receptor agonists ATP and UTP (15). Representative tracings of Par-C10 monolayer Isc responses to maximally effective concentrations of three Ca2+-mobilizing agonists, UTP, carbachol, and phenylephrine, are presented in Fig. 3. As shown, UTP, when applied to the medium bathing the apical side of the monolayer, was the most efficacious of the three agents, whereas carbachol was more effective than phenylephrine when these latter two agents were applied basolaterally. The addition of UTP to the basolateral side or of carbachol or phenylephrine to the apical side was without effect on Isc. The patterns and magnitudes of response shown in Fig. 3 for these three agonists were consistent across a wide range of passage numbers of Par-C10 cells. Conversely, although initial studies with the cAMP-mobilizing agonist isoproterenol (applied basolaterally) revealed a strong enhancement of Isc, subsequent experiments revealed little or no Isc response to this beta -adrenergic receptor agonist, despite the fact that cAMP production in response to isoproterenol was similar among the various cell preparations used. Thus, although many of the characteristics of Par-C10 cells appear to be maintained, others appear to be more variable.


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Fig. 3.   Agonist-induced changes in Par-C10 short-circuit current (Isc). Confluent cultures of Par-C10 cells on permeable supports were placed in Ussing chambers and, following equilibration, were exposed to 100 µM UTP, carbachol, or phenylephrine. Isc as a function of time was recorded. In each case, addition of agonist to opposite side of cell monolayer had no effect on Isc.

The potency and efficacy of the naturally occurring nucleotide receptor agonists ATP and UTP for increasing Par-C10 cell monolayer Isc were similar following apical addition (Fig. 4). EC50 values were 0.46 ± 0.11 and 1.2 ± 0.4 µM for ATP and UTP, respectively. These results, combined with the lack of effect of UTP basolaterally, suggest that the P2Y2 subtype of nucleotide receptor is expressed in Par-C10 cells (see also Fig. 8), that this expression is limited to the apical membrane of these cells, and that this receptor can account for all of the effects of apically applied ATP and UTP on Isc. It is important to note that ATP, in contrast to UTP, is also effective in increasing Isc following basolateral addition (data not shown), an effect mediated by other P2 receptor subtypes that we are currently characterizing. Finally, Fig. 4 also shows the concentration-response curve for carbachol stimulation of Isc, which gave an EC50 value of 52 ± 29 µM, similar to values obtained for this agonist in a variety of tissues, including other salivary gland preparations (e.g., Refs. 3, 18).


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Fig. 4.   Concentration dependence of agonist-enhanced Isc. Changes in Par-C10 Isc (Delta Isc) were determined in response to indicated concentrations of ATP (open circle ) and UTP (bullet ) applied apically or carbachol (black-down-triangle ) applied basolaterally. Data represent peak increases in Isc and are means ± SE of a minimum of 3 determinations. Associated curves are best fits of cumulative data to sigmoidal dose response (variable slope) model (Prism, Graphpad, San Diego, CA).

The ionic basis of the Isc and the role of Ca2+. As in many other cell types, P2Y2 nucleotide, muscarinic cholinergic, and alpha 1-adrenergic receptors in Par-C10 cells are coupled through phospholipase C to increases in the intracellular Ca2+ concentration (15), a process that involves both the mobilization of intracellular stores and the influx of extracellular Ca2+. We therefore examined the effect of removal of Ca2+ from the basolateral and apical media on agonist-induced increases in Isc. As shown in Fig. 5, the magnitude of the response to apically applied UTP (A) and basolaterally applied carbachol (B), expressed as the area under the curve for the first 2 min following agonist addition, was unaffected when the Ca2+ concentration was lowered into the nanomolar range in the medium bathing the apical side of the monolayers. Conversely, lowering the basolateral medium Ca2+ concentration dramatically decreased, by 50% or more, the ability of both agonists to produce the sustained increases in Isc observed in the presence of Ca2+. The effect of lowering the Ca2+ concentration in both baths simultaneously was not greater than that observed with the decrease in only the apical medium. This finding indicates that the response to both agonists is dependent on the availability of extracellular Ca2+ and that this Ca2+ is available to the cell only from the basolateral side.


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Fig. 5.   Effect on agonist-stimulated Isc of Ca2+ removal from apical or basolateral incubation buffer. Par-C10 cell monolayers were placed in Ussing chambers and bathed on apical and basolateral sides with buffer containing either 1 mM Ca2+ (+) or 0.2 mM EGTA and no added Ca2+ (-), as indicated. Changes in Isc were determined in response to 10 µM UTP applied apically (A) or 100 µM carbachol applied basolaterally (B). Values are expressed as area under curve during first 2 min after agonist addition and are means ± SE of 4 (UTP) or 3 (carbachol) experiments. In each panel, differences in mean values were significant between bars labeled a and b (1-way ANOVA with Student-Newman-Keuls post hoc test; P <=  0.05).

The Isc-enhancing effects of Ca2+-mobilizing agonists in a variety of other epithelia are due to increases in apical anion secretion, i.e., Cl- and HCO-3. With a view toward defining the ionic nature of agonist-induced increases in Par-C10 cell Isc, responses to apically applied UTP in media lacking Cl- or HCO-3 or both were compared with the effects observed in the presence of both anions in the apical and basolateral media. As shown in Fig. 6A, the removal of Cl- decreased both the maximum level and the duration of the UTP-induced increase in Isc, compared with the response obtained in the presence of Cl- (Fig. 6A). Furthermore, the omission of HCO-3 from Cl--containing medium (Fig. 6B) also resulted in a marked decrease in the response to UTP, and the omission of both anions (Fig. 6B) resulted in only a slight, transient increase in Isc. As summarized in Fig. 6C, Isc responses to UTP were decreased by ~50% when either HCO-3 or Cl- was omitted and by ~90% in the absence of both anions. Conversely, the replacement of Na+ with N-methyl-D-glucamine or choline, or the inclusion of amiloride in the apical medium, had no effect on agonist-induced increases in Isc (data not shown).


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Fig. 6.   Effect on agonist-stimulated Isc of Cl- and HCO-3 removal from incubation buffer. Par-C10 cell monolayers were placed in Ussing chambers and incubated in HCO-3-buffered (A) or HEPES-buffered (B) medium containing 122 mM Cl- (solid tracings) or in which Cl- was replaced with gluconate (dashed tracings). Apical and basolateral buffer constituents were identical in each case. Changes in Isc in response to 10 µM UTP, applied apically, were then determined. Tracings shown in A and B are representative of a series of 3 experiments, summarized in C with data expressed as area under curve for first 2 min following UTP addition. Differences in mean values were significant between bars labeled a, b, and c (1-way ANOVA with Student-Newman-Keuls post hoc test; P <=  0.05).

To confirm the observations presented above, which suggest that Par-C10 Isc is dependent on HCO-3 and Cl-, the effects of three inhibitors of anion transport on UTP- and carbachol-stimulated Isc were examined. As shown in Fig. 7A, apically applied diphenylamine-2-carboxylic acid, DIDS, and 5-nitro-2-(3-phenylpropylamino)benzoic acid were all effective in decreasing significantly the Isc response to UTP, whereas basolateral addition of these inhibitors was without effect. The same inhibitory pattern was observed when carbachol (applied basolaterally) was used as the agonist. Taken together, the results presented in Figs. 6 and 7 and the lack of effect of amiloride or Na+ removal on agonist-stimulated Isc strongly suggest that apical anion secretion underlies the changes in Isc elicited by UTP and carbachol.


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Fig. 7.   Effect of Cl- transport inhibitors on agonist-induced increases in Isc. Par-C10 cell monolayers were placed in Ussing chambers and, after equilibration, were preincubated for 2 min with 500 µM diphenylamine-2-carboxylic acid (DPC), 100 µM DIDS, or 150 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in apical (a) or basolateral (b) medium or with no addition (agonist only). UTP (10 µM; A) or carbachol (100 µM; B) was then added in continued presence of preincubation agent, and changes in Isc were determined. Values represent maximal increase obtained and are expressed as means ± SE of 3 or more experiments. * Significant difference from control cells (agonist only) using Student's unpaired t-test at P <=  0.05.

RT-PCR analysis for P2Y receptor subtypes and anion transporters in Par-C10 cells. We have confirmed the presence of muscarinic cholinergic receptors in Par-C10 cells with radioligand binding assays and by demonstrating the effectiveness of atropine in blocking carbachol-stimulated Ca2+ mobilization and Isc (data not shown). Because there is no radioligand binding assay or high-affinity selective antagonist for the P2Y2 receptor, we used RT-PCR detection of the mRNA for the P2Y2 receptor to support the pharmacological identification (Fig. 4 and Ref. 15) of this subtype in Par-C10 cells. As shown in Fig. 8, RT-PCR with P2Y2 receptor mRNA-specific primers produced a product of the correct size (778 bp) that was >99% identical to the published rat P2Y2 receptor sequence. A recent study has indicated that another uridine-nucleotide-preferring P2Y receptor subtype, P2Y6, is expressed, along with P2Y2 receptors, in the apical membrane of another epithelial cell type (12). However, RT-PCR with primers specific for the P2Y6 receptor mRNA amplified no appropriately sized (871 bp) product from cDNA prepared from Par-C10 cell total RNA (Fig. 8), whereas a product of appropriate size and sequence was obtained with cDNA prepared from 1321N1 cells heterologously expressing the P2Y6 receptor. These results suggest that P2Y6 receptors are not expressed in Par-C10 cells. A faint smaller band, amplified from Par-C10 cell RNA with the P2Y6 primers in the presence of RT, is the same size as a more abundant band obtained with these primers in rat aortic smooth muscle cells. The sequence from the smooth muscle cells is unrelated to mRNAs encoding any known receptor proteins (data not shown). As is also shown in Fig. 8, RT-PCR with primers specific for two of the anion-transporting proteins identified previously in salivary gland acinar cells, the cystic fibrosis transmembrane conductance regulator (CFTR) (25) and the ClC-2 Cl- channel (1), gave products of the appropriate sizes (352 and 755 bp, respectively) and sequences, suggesting that these anion channels are also expressed in Par-C10 cells.


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Fig. 8.   Presence of P2Y2 receptor and Cl- channel mRNAs in Par-C10 cells. Par-C10 cell total RNA was examined by RT-PCR (see METHODS) for presence of mRNA for P2Y2 and P2Y6 subtypes of nucleotide receptors and for ClC-2 and cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. Effectiveness of P2Y6 primers, which generated no relevant product from Par-C10 mRNA, was confirmed by RT-PCR with total RNA from 1321N1 cells heterologously expressing P2Y6 receptor. Similar to results shown for P2Y2 and P2Y6, no amplification was obtained in absence of RT (-) when using ClC-2 and CFTR primers (data not shown).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

One of the goals of salivary gland research at the cellular level has been to understand the pathways involved in the ion transport processes essential to saliva formation and modification. The understanding of equivalent processes and their regulation in other epithelia has been facilitated by the availability of permanent cell lines that exhibit sufficient polarization, transcellular resistance, and other features that make them suitable for bioelectric measurements in Ussing chambers. Two particularly useful examples are the T84 (colon; Refs. 5, 11, 23) and MDCK (kidney; Refs. 6, 17, 21) cell lines. The Par-C10 cell line is, to our knowledge, the first cell line of salivary origin that can be reliably utilized in Ussing chamber studies. Although the phenotype of Par-C10 cells is not entirely consistent with that of fully differentiated parotid acinar cells, a number of acinar cell characteristics are exhibited by Par-C10 cells (Ref. 15 and as discussed below).

The morphological observations (Fig. 2) indicate that cultured Par-C10 cells are apically polarized and clearly are acinar, albeit modestly, in terms of cytodifferentiation. In addition, the presence of secretory canaliculi among the cells is evidence of acinar morphodifferentiation, as these structures occur only in the acini of most salivary glands, including the rat parotid gland (16, 22). Here it is important to note that the occasional cells with mitotic figures shared the cited features of acinar differentiation. Thus this cell line is apparently purely acinar in that no ductal or other "stem" cells are seen. The presence of secretory canaliculi may be a consequence of culturing the cells on permeable supports: morphological analysis of Par-C10 cells grown on plastic tissue culture dishes revealed no such structures (15).

The polarized morphology of Par-C10 cells is complemented by the consistency between these cells and other polarized epithelia with respect to apical vs. basolateral distribution of important ion secretory proteins and receptors. The data shown in Figs. 3 and 4 suggest that alpha 1-adrenergic and muscarinic cholinergic receptor expression is limited to the basolateral aspect of Par-C10 cells, whereas P2Y2 nucleotide receptor expression is apical, consistent with their distribution in other epithelia (7, 10, 13, 23). In addition, Par-C10 cells appear to express both CFTR and ClC-2 (Fig. 8) and possibly other anion transporting proteins relevant to normal salivary acinar cells (1, 25). The results with anion transport inhibitors (Fig. 7) suggest an exclusively apical distribution for these proteins, consistent with observations made in normal salivary acinar cells. Conversely, Par-C10 Isc is not diminished by inhibitors of Na+-K+-2Cl- cotransport such as bumetanide (Camden and Turner, unpublished observations), a finding considered more reflective of salivary duct cells than acinar cells (Ref. 24, but see Ref. 9). In addition, as reported previously (13), functional substance P receptors apparently are not expressed in Par-C10 cells, whereas evidence suggests that these receptors are found in normal salivary acinar but not ductal cells (4, 20). Finally, Par-C10 cell monolayers develop high transcellular resistances, similar to values obtained with MDCK (11, 23) and T84 cells (6, 21), and thus do not fit the classical definition of salivary acini as a "leaky" epithelium. Nonetheless, the suitability of Par-C10 cultures for studies of transcellular ion movement and its regulation promises to open new avenues for studying salivary gland secretion.

For the most part, agonist (UTP, carbachol, and phenylephrine) effects in terms of second messenger production (15) and increased Isc (Figs. 3-7) were found to be reasonably consistent across at least 30 cell passages. One marked exception to this observation was the effect of the beta -adrenergic receptor agonist isoproterenol on Isc, which varied from an increase similar to that obtained with maximally effective concentrations of UTP to no response at all, in various Par-C10 cultures (data not shown). This variability was paralleled by similar changes in the response to forskolin, whereas all of the cultures exhibited robust cAMP responses to isoproterenol and forskolin, suggesting that the expression of a component in the pathway downstream of the beta -adrenergic receptor, Gs, and adenylyl cyclase, possibly CFTR itself, may be altered. The decrease in the responsiveness to cAMP-mobilizing agents is not simply a function of passage number but apparently involves subtle differences in culture conditions or medium constituents, including cholera toxin, which we have used routinely in an attempt to promote a differentiated state of the Par-C10 cells. The basis for the differences in apparent CFTR activity requires investigation.

The above caveats notwithstanding, the Par-C10 parotid acinar cell line exhibits a number of acinar cell-like features as well as the characteristic, unique among salivary cell lines, of sufficient polarization and transcellular resistance to be useful in Ussing chambers. It is hoped that this cell line will prove as beneficial to salivary gland research as similar cell lines have been in studies of other organ systems with important epithelial components.

    ACKNOWLEDGEMENTS

We thank Rodney McNutt for assistance in preparing specimens for morphological evaluation and Dr. T. K. Harden for providing 1321N1 cells expressing the P2Y6 receptor.

    FOOTNOTES

This work was supported by National Institute of Dental Research Grant DE-07389 and the Department of Veteran Affairs.

Portions of this work have appeared in abstract form (19).

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. §1734 solely to indicate this fact.

Address for reprint requests: J. T. Turner, M561 Health Sciences Center, University of Missouri-Columbia, 1 Hospital Dr., Columbia, MO 65212.

Received 26 January 1998; accepted in final form 22 April 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Arreola, J., E. Park, J. E. Melvin, and T. Begenisich. Three distinct chloride channels control anion movements in rat parotid acinar cells. J. Physiol. (Lond.) 490: 351-362, 1996[Abstract].

2.   Ball, W. D., and R. S. Redman. Two independently regulated secretory systems within the acini of the submandibular gland of the perinatal rat. Eur. J. Cell Biol. 33: 112-122, 1984[Medline].

3.   Dai, Y., I. S. Ambudkar, V. J. Horn, C.-K. Yeh, E. E. Kousvelari, S. J. Wall, M. Li, R. P. Yasuda, B. B. Wolfe, and B. J. Baum. Evidence that M3 muscarinic receptors in rat parotid gland couple to two second messenger systems. Am. J. Physiol. 261 (Cell Physiol. 30): C1063-C1073, 1991[Abstract/Free Full Text].

4.   Dehaye, J. P., and R. J. Turner. Isolation and characterization of rat submandibular intralobular ducts. Am. J. Physiol. 261 (Cell Physiol. 30): C490-C496, 1991[Abstract/Free Full Text].

5.   Dharmsathaphorn, K., J. A. McRoberts, K. G. Mandel, L. D. Tisdale, and H. Masui. A human colonic tumor cell line that maintains vectorial electrolyte transport. Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9): G204-G208, 1984[Abstract/Free Full Text].

6.   Dho, S., K. Stewart, and J. K. Foskett. Purinergic activation of Cl- secretion in T84 cells. Am. J. Physiol. 262 (Cell Physiol. 31): C67-C74, 1992[Abstract/Free Full Text].

7.   Firestein, B. L., M. Z. Xing, R. J. Hughes, C. U. Corvera, and P. A. Insel. Heterogeneity of P2U- and P2Y-purinergic receptor regulation of phospholipases in MDCK cells. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F610-F618, 1996[Abstract/Free Full Text].

8.   Forte, L. R., S. L. Eber, J. T. Turner, R. H. Freeman, K. F. Fok, and M. G. Currie. Guanylin stimulation of Cl- secretion in human intestinal T84 cells via cyclic guanosine monophosphate. J. Clin. Invest. 91: 2423-2428, 1993[Medline].

9.   He, X., C.-M. Tse, M. Donowitz, S. J. Alper, S. E. Gabriel, and B. J. Baum. Polarized distribution of key membrane transport proteins in the rat submandibular gland. Pflügers Arch. 433: 260-268, 1997[Medline].

10.   Knowles, M. R., L. L. Clarke, and R. C. Boucher. Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis. N. Engl. J. Med. 325: 533-538, 1991[Abstract].

11.   Lavelle, J. P., H. O. Negrete, P. A. Poland, C. L. Kinlough, S. D. Meyers, R. P. Hughey, and M. L. Zeidel. Low permeabilities of MDCK cell monolayers: a model barrier epithelium. Am. J. Physiol. 273 (Renal Physiol. 42): F67-F75, 1997[Abstract/Free Full Text].

12.   Lazarowski, E. R., A. M. Paradiso, W. C. Watt, T. K. Harden, and R. C. Boucher. UDP activates a mucosal-restricted receptor on human nasal epithelial cells that is distinct from the P2Y2 receptor. Proc. Natl. Acad. Sci. USA 94: 2599-2603, 1997[Abstract/Free Full Text].

13.   Paradiso, A. M., S. J. Mason, E. R. Lazarowski, and R. C. Boucher. Membrane-restricted regulation of Ca2+ release and influx in polarized epithelia. Nature 377: 643-646, 1995[Medline].

14.   Quissell, D. O., K. B. Barzen, D. C. Gruenert, R. S. Redman, J. M. Camden, and J. T. Turner. Development and characterization of SV40 immortalized rat submandibular acinar cell lines. In Vitro Cell. Dev. Biol. 33: 164-173, 1997.

15.   Quissell, D. O., K. A. Barzen, R. S. Redman, J. M. Camden, and J. T. Turner. Development and characterization of SV40 immortalized rat parotid acinar cell lines. In Vitro Cell. Develop. Biol. 34: 58-67, 1998.

16.   Redman, R. S. Development of the salivary glands. In: The Salivary System, edited by L. M. Sreebny. Boca Raton, FL: CRC Press, 1987, p. 1-20.

17.   Simmons, N. L. Stimulation of Cl- secretion by exogenous ATP in cultured MDCK epithelial monolayers. Biochim. Biophys. Acta 646: 231-242, 1981[Medline].

18.   Sullivan, D. M., and J. T. Turner. Characterization of the muscarinic cholinergic receptor in the opossum (Didelphis virginiana, Kerr) submandibular gland: differences in receptor density and subtype compared with higher mammalian species. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 97C: 65-70, 1990.

19.   Turner, J. T., D. O. Quissell, and J. M. Camden. Regulation of transcellular Isc by nucleotides in parotid acinar cell lines (Abstract). J. Dent. Res. 75: 252, 1996.

20.   Valdez, I. H., M. Paulais, P. C. Fox, and R. J. Turner. Microfluorometric studies of intracellular Ca2+ and Na+ concentrations in normal human labial gland acini. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G601-G607, 1994[Abstract/Free Full Text].

21.   Weymer, A., P. Huott, W. Liu, J. A. McRoberts, and K. Dharmsathaphorn. Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line. J. Clin. Invest. 76: 1828-1836, 1985[Medline].

22.   Young, J. A., and E. W. van Lennep. The Morphology of the Salivary Glands. Boca Raton, FL: CRC Press, 1978.

23.   Zegarra-Moran, O., G. Romeo, and L. J. V. Galietta. Regulation of transepithelial ion transport by two different purinoceptors in the apical membrane of canine kidney (MDCK) cells. Br. J. Pharmacol. 114: 1052-1056, 1995[Abstract].

24.   Zeng, W., M. G. Lee, and S. Muallem. 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].

25.   Zeng, W. Z., M. G. Lee, M. Yan, J. Diaz, I. Benjamin, C. R. Marino, R. Kopito, S. Freedman, C. Cotton, S. Muallem, and P. Thomas. Immuno and functional characterization of CFTR in submandibular and pancreatic acinar and duct cells. Am. J. Physiol. 273 (Cell Physiol. 42): C442-C455, 1997[Abstract/Free Full Text].


Am J Physiol Cell Physiol 275(2):C367-C374
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