Rat serum induces a differentiated phenotype in a rat parotid acinar cell line

Yingting Zhu, John M. Aletta, Jiayu Wen, Xuejun Zhang, Dennis Higgins, and Ronald P. Rubin

Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To establish a continuous cell line, freshly prepared rat parotid acinar cells were stably transfected with a plasmid vector containing the SV40 large T antigen. The acinar origin of these cells was confirmed by Western blotting, enzyme analysis, and morphological analysis. Transformed cells grown in 10% rat serum showed a modest reduction in cell number after 7 days and a concentration- and time-dependent increase in amylase levels ~16 times greater than those observed in fetal bovine serum-treated cells. Ultrastructural analysis revealed that cells grown in rat serum harbored protein-filled secretory granules localized adjacent to the endoplasmic reticulum, and punctate amylase-specific immunofluorescence distributed throughout the cytoplasm was consistent with the presence of amylase in secretory organelles. Clonal cells express tissue-specific proline-rich proteins and the four protein kinase C isozymes present in primary culture. Carbachol and isoproterenol stimulated [3H]protein secretion and isoproterenol enhanced amylase secretion from cells grown in rat serum. Moreover, norepinephrine, carbachol, and substance P produced a time- and concentration-dependent rise in cytoplasmic Ca2+. This continuous cell line of parotid acinar cells, which after treatment with rat serum retains the basic structural and functional properties of primary culture cells, will be utilized as a model system for studying long-term biological processes that regulate parotid cell function.

immortalization; parotid gland; alpha -amylase; plasmid vectors; cell culture; differentiation

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ALTHOUGH PRIMARY CULTURES are widely employed to characterize specific cell functions, their utility is limited by the number of viable cells that can be generated from a single preparation. Subculturing of primary cultures can be used to increase the quantity of cellular material available for biochemical analyses. This approach, however, generally leads to a loss or reduction of differentiated phenotypic properties of the isolated cells. An alternative means for increasing cell yields is the creation of an immortalized cell line. This strategy has allowed major advances in our knowledge of mechanisms involved in the regulation of cell type-specific differentiation and the functional responses to chemical agents (6, 11, 13).

The rat parotid gland has been extensively employed as an experimental model for studying agonist-activated ionic events and exocytotic protein and water secretion (22). The parotid gland is composed of acinar cells, which are responsible for the export of secretory proteins and fluid secretion and ductal cells that regulate the electrolyte composition of saliva. Although primary cultures of rat parotid acinar cells are useful for short-term experiments, these terminally differentiated epithelial cells do not maintain their phenotype in long-term culture (33). There is thus a critical need for a cell line appropriate for long-term studies on parotid acinar cell functions, such as growth and differentiation, as well as for isolating sufficient quantities of endogenous macromolecules.

Many immortalized cell lines have been created by the expression of a viral oncogene, such as the large T antigen from the SV40 genes of the human papilloma virus (3, 8). Although rat parotid acinar cell lines have been established using expression vectors containing large T antigen in the plasmid vector pSV3-neo, these cells at best exhibit barely detectable levels of alpha -amylase activity (20, 24). Furthermore, no quantitative data on the expression of alpha -amylase, the major parotid acinar protein, are available in these lines. We herein report the immortalization of rat parotid acinar cells derived from primary culture that shows stable differentiated characteristics in long-term culture, particularly in terms of calcium signaling, amylase content, and regulated secretion.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Collagenase was purchased from Worthington Biochemicals (Freehold, NJ). MCDB-153, F-12, fetal bovine serum (FBS), natural mouse laminin, and lipofectamine were purchased from GIBCO-BRL (Grand Island, NY). pSV3-neo vectors were obtained from American Tissue Culture Collection (Rockville, MD). The antibody for amylase and sheep anti-mouse and goat anti-rabbit antisera were purchased from Sigma Chemical (St. Louis, MO). The antibodies for protein kinase C (PKC)-alpha , PKC-epsilon , PKC-delta , and PKC-zeta were from Oxford Biomedical Research (Oxford, MI). Chemiluminescence reagents were from Amersham (Arlington Heights, IL). L-[methyl-3H]methionine (sp act 200 mCi/mmol) was purchased from DuPont NEN (Boston, MA). The amylase cDNA was a gift from Dr. G. H. Swift (University of Texas), and the anti-proline-rich protein (PRP) antibodies were generously provided by Dr. Lawrence Tabak (University of Rochester) and Dr. Don Carlson (University of California-Davis). Other biochemicals and reagents were obtained from Sigma Chemical. Rat serum was obtained from Sprague-Dawley rats by decapitation and exsanguination. After standing for ~1 h at room temperature, the blood samples were centrifuged twice at 3,500 rpm for 15 min. The clear supernatant was stored frozen at -20°C and thawed immediately before use. Rat serum obtained from commercial sources (GIBCO-BRL) showed comparable activity.

Cell isolation and culture. Acinar cells were prepared from male Sprague-Dawley rat parotid glands by a modification of the procedure of Terzian et al. (29). Rats weighing ~200 g were anesthetized with pentobarbital sodium. The parotid glands were excised and washed twice with calcium- and magnesium-free Hanks' balanced salt solution (HBSS) followed by trypsin (0.5 mg/ml) digestion in HBSS for 5 min at 37°C. After the clumps of tissue were incubated for 5 min with trypsin inhibitor (0.5 mg/ml) in HBSS at 37°C, they were washed twice with modified MCDB-153 medium comprised of excess (2-3×) amino acid concentrations (21) plus 10 ng/ml epidermal growth factor, 0.5 µM hydrocortisone, 10 nM triiodothyronine, 1× insulin-transferrin-selenium-X supplement, 100 U/ml penicillin, and 100 µg/ml streptomycin. After treatment with collagenase (0.75 mg/ml) for 30 min and 2 mM EGTA for 5 min, the cells were filtered through a sterile nylon filter (Nitex 150/51), washed twice, and suspended in modified MCDB-153 medium. Cells were then incubated for 2 h in 100-mm dishes to allow adhesion of fibroblasts. The supernatant containing the nonadherent acinar cells was then transferred to 35-mm dishes precoated with laminin (10 µg/ml) to provide a substratum for cell attachment.

Preparation of immortalized cells. Primary cultures of rat parotid cells were grown for 3 days at 35°C before transfection. pSV3-neo plasmid vectors containing SV40 promoter and large T antigen as well as two neomycin-resistance genes were used to immortalize the rat acinar cells by DNA transfection (20, 28). After the cells were rinsed with serum-free MCDB-153, they were exposed to the plasmid (30 µg) with 10 µl of lipofectamine in serum-free modified MCDB-153 (1 ml/35-mm dish). After 24 h the plasmid-lipofectamine mixture was removed and the cells were allowed to recover for 3 days. The recovery period was critical for the selection and survival of the transformed cells. Modified MCDB-153 containing 100 µg/ml of G418 was used during the first 7 days of selection, followed by 50 µg for maintenance of the cells. None of the cells in control, nontransfected cultures survived 4 wk of the treatment.

Transfected clones were subcultured to 12-well dishes by micropipetting under a microscope. Although three clones (designated 3-8, 3-9, and 2-10) were isolated, the 3-9 clone became the focus of our extended studies because of its more differentiated morphology and somewhat higher amylase content. The transfected acinar cells have been maintained for 50 passages without loss of viability. After the cell lines were established in the presence of 50 µg/ml G418 for 6 mo, the drug was removed entirely without detectable changes in amylase content, proliferation rate, or cell morphology. Three clones transfected with pSV5-neo vectors were also obtained; however, they grew very slowly, became very flat, and eventually died.

Initially, the primary acinar cells destined for transfection were kept in modified MCDB-153 plus 1 µM isoproterenol to purportedly facilitate cell survival (20). Subsequently, primary cells were found to survive in the absence of isoproterenol if the medium was changed every 1-2 days. Therefore transfections were generally carried out on cells not exposed to isoproterenol. Thus in our hands isoproterenol treatment is not a prerequisite for a successful transfection as previously proposed (20).

Clones 3-8, 3-9, and 2-10 were maintained in modified MCDB-153 plus 10% FBS and were passaged by gentle pipetting for the first three passages. They were subsequently passaged with 0.25% trypsin. Cell culture dishes precoated with laminin were initially required for attachment of the transformed cells. However, after three passages the cells attached and proliferated well in uncoated cell culture dishes, provided that the medium was changed every 3 days. The optimum temperature for cell culture proved to be 33°C. Media other than modified MCDB-153 (F-12, DMEM, MEM, and RPMI 1640) did not support the survival or differentiation of either primary cultures or transfected cells. All three cell lines are typically maintained in 10% FBS. A cryoprotectant medium composed of 10% glycerol, 50% FBS, and 40% MCDB-153 was adopted for storing acinar cell lines in liquid nitrogen, because DMSO (10%) was inadequate as a storage medium.

Determination of alpha -amylase activity. A cell aliquot (450 µl) from 10-day primary culture or clonal cells (containing ~105 cells) treated with 0.2% Triton X for total amylase activity was centrifuged through Nyosil oil in a microfuge at 12,000 g, and the supernatant fraction was analyzed for amylase activity at 25°C as previously described (14). Units of activity are defined as milligrams of maltose produced per hour per 105 cells.

Measurement of radiolabeled protein secretion. Tritiated protein release was determined by measuring the amount of [3H]methionine in a trichloroacetic acid-precipitable fraction of the medium as previously described (19). Briefly, clonal parotid acinar cells (106) were equilibrated for 20 min at 37°C in 2 ml of standard medium of the following composition (in mM): 120 NaCl, 5 KCl, 1.2 MgCl2, 1 CaCl2, 5 beta -hydroxybutyrate-Na, 20 Tris (pH 7.4), and 5 mg/ml BSA. After the cells were incubated for 20 min with 2 ml of standard medium plus 1.5 µCi [3H]methionine/ml, fresh medium devoid of radioactivity but supplemented with 1% essential and nonessential amino acids and 2 mM glutamine was added for 3 h. The cells were then washed three times with PBS and fresh standard medium containing either carbachol (10 µM) or isoproterenol (10 µM) was added. A 100-µl aliquot of each sample was then precipitated with 10% trichloroacetic acid, and the pellet was suspended in PBS and counted by liquid scintillation spectrometry using 5 ml of Ultima Gold (Packard, Meriden, CT).

Determination of alpha -amylase mRNA by Northern blotting. Total RNA was prepared using Trizol reagent (GIBCO, BRL) according to the manufacturer's instructions. RNA (30 µg) from each sample was fractionated on a 1.2% agarose gel and then transferred to a nitrocellulose membrane with 10× saline-sodium phosphate-EDTA buffer and fixed under ultraviolet light. The membrane was treated overnight with prehybridization buffer. The blot was then hybridized with [32P]dCTP labeled amylase cDNA, and autoradiography was conducted overnight.

Western blot analysis. Cell fractions of equivalent protein concentrations were analyzed by SDS-PAGE followed by electrophoretic protein transfer from gel to polyvinylidene difluoride membranes. The immunoblotting was carried out using specific polyclonal antibodies to amylase, PRP, and isoform-specific PKC and then developed with goat-anti-rabbit IgG linked to horseradish peroxidase. The relative amount of each protein was quantitated by densitometric scanning using an imaging densitometer (Bio-Rad).

Measurement of Ca2+ mobilization. Cellular Ca2+ levels were also determined in quiescent and stimulated 3-9 cells after loading with 5 µM fura 2-AM for 30 min as previously described (14).

Fluorescence confocal microscopy. Clonal cell cultures grown for 4-5 days were fixed in 4% formaldehyde for 20 min at room temperature. After the cells were washed three times with PBS, they were permeabilized with 0.5% Triton for 7 min. The cells were washed again and then blocked with 5% BSA for 30 min. The cells were then incubated for 2 h with rabbit anti-human alpha -amylase (1:1,000). After being washed three times, the cultures were incubated for 90 min with fluorescein conjugate goat anti-rabbit IgG (1:100; Molecular Probes, Eugene, OR). The cells were then washed four times and mounted. Cultures were examined using epifluorescence optics of a Nikon upright Optiphot microscope, followed by confocal imaging with a laser scanning microscopy system (model MRC-1024; Bio-Rad) (34).

Electron microscopy. Cultures were fixed with 3% glutaraldehyde, postfixed in 1% OsO4 for 1 h at 4°C, and dehydrated in a graded ethanol series. After the monolayer was detached from the plastic substratum using propylene oxide, the tissue was pelleted and embedded in Spurr's resin. Ultrathin sections were viewed in a Siemens 101 electron microscope.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell morphology. Clone 3-9 formed monolayers of flattened polygonal cells, which were often elongated (Fig. 1). Although the transfected cells appeared to resemble 10-day cultures of primary parotid cells, clear intra-cytoplasmic bodies were more prominent in the transfected cells and appeared to lack secretory organelles (Fig. 1, cf. A and B). Electron microscopy confirmed that 3-9 cells grown in the presence of FBS, which permits optimal cell proliferation, are devoid of secretory granules (Fig. 2A).


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Fig. 1.   Photomicrograph of primary cultures of parotid cells and transfected rat parotid acinar cells. Primary cultures of rat parotid acinar cells were transfected with pSV3-neo plasmid vector containing SV40 large T antigen using lipofectin. A: primary culture of parotid cells grown in 10% fetal bovine serum (FBS) for 10 days. B and C: cells isolated from clone 3-9 grown in 10% FBS (B) or 5% rat serum (RS, C).


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Fig. 2.   Electron micrographs of immortalized parotid cells. A: cells from clone 3-9 incubated in 10% FBS for 7 days. Note absence of secretory organelles. Bi: 3-9 cells incubated with 10% RS for 7 days. Arrows indicate presence of secretory organelles. Bii: higher magnification of cell depicted in Bi showing presence of organelles that appear identical to amylase-containing secretory granules. Calibration bars, 1 µm.

alpha -Amylase levels. Because of its relative abundance and functional role, alpha -amylase serves as a marker of acinar cell differentiation (12). The 3-9 cells express a 58-kDa band, which reacts with an antiserum to alpha -amylase (Fig. 3A). Clones 3-10 and 2-10 also expressed alpha -amylase (data not shown), but in all cases the alpha -amylase levels relative to total cellular protein were ~1,000-fold less than those present in freshly prepared extracts of parotid gland. Nevertheless, alpha -amylase expression was quite similar to that of primary parotid cells after 10 days of culture in 10% FBS (Fig. 3A). These findings confirm that amylase levels decrease dramatically within 10 days in primary culture and likewise in immortalized parotid cells (20, 33).


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Fig. 3.   Western and Northern blot identification of amylase immunoreative protein and mRNA in extracts of parotid acinar cells. A: cell extracts were processed for Western blotting by SDS-PAGE and using polyclonal antiserum raised against amylase. Representative immunoblots of parotid gland homogenate (100 ng, lane 1), 10-day-old primary culture of parotid cells incubated with 10% FBS (100 µg, lane 2), and 3-9 cells incubated for 10 days with 10% FBS (100 µg, lane 3). These results are representative of 3 independent experiments. B: Northern blot analysis was carried out using 30 µg of total cellular RNA from extracts of primary cells (lane 1) and transformed cells (lane 2) cultured in 10% FBS.

Despite the low levels of amylase activity, Northern blotting analysis indicated that the 3-9 cells cultured in 10% FBS express amylase mRNA levels that are similar to those found in primary acinar cells (Fig. 3B). Furthermore, 3-9 cells also exhibit an additional larger transcript not observed in primary cultures (see Ref. 27). These results indicate that the transcript for amylase is present in the clonal cells, but it is not efficiently translated into protein.

Induction of phenotypic markers by rat serum. We reasoned that the marked reduction in alpha -amylase was probably due to the lack of factors that regulate and/or maintain cell differentiation. The composition of the culture medium was therefore modified by the addition of various growth factors and substances that might influence differentiation. Transforming growth factor-beta (10 ng/ml), insulin-like growth factor 1 (10 ng/ml), nerve growth factor (10-100 ng/ml), interleukin-2 (20 ng/ml), interleukin-6 (100 ng/ml), and horse serum were ineffective in altering cell growth and amylase activity when tested after 3, 5, and 7 days of treatment. However, rat serum produced marked changes in growth rate, amylase content, and cellular morphology. Clonal 3-9 cells were initially plated in MCDB-153 medium containing 10% FBS. One day later the FBS was removed and replaced with MCDB-153 containing 10% rat serum. The cells in these cultures proliferated at the same rate as cells in FBS for the first 4 days in culture, but thereafter a modest reduction in cell number was consistently observed through 7 to 10 days in the cells treated with rat serum (data not shown). Furthermore, 3-9 cells incubated with two different concentrations of rat serum (5-10%) displayed a graded increase in cell amylase activity as assessed by enzymatic assay. The 3-9 cells cultured in medium containing 10% rat serum had ~16 times higher total amylase activity than cells grown in an equivalent concentration of FBS (Fig. 4A). The stimulatory effect of rat serum on amylase levels was time dependent, with detectable changes being observed within 1 day; maximum levels were reached after 7 days of exposure (Fig. 4, B and C).


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Fig. 4.   Concentration and time dependence of effect of RS on amylase levels in clonal 3-9 cells. A: amylase activity was measured by enzymatic analysis in 3-9 cells treated for 7 days with either 10% FBS, 5% RS, or 10% RS. Results shown are means ± SE of 4 independent experiments. B: time dependence of RS effects on amylase levels is demonstrated by representative immunoblots of cell extracts incubated with a 1:2,000 dilution of anti-amylase antibody. Extracts were derived from parotid gland (PG, lane 1), 3-9 cells grown in 10% FBS for 7 days (lane 2), or in 10% RS for 1 day (lane 3), 4 days (lane 4), and 7 days (lane 5). A 20-ng aliquot of protein was loaded in lane 1 and 20 µg protein was loaded in all other lanes. C: results (means ± SE) obtained after densitometric analysis of autoradiograms of 4 separate experiments are expressed in arbitrary densitometric units. * Difference between samples from FBS- and RS-treated cells was significant (P < 0.05 paired Student's t-test).

Additional markers of the parotid phenotype are the PRPs (4, 31). Intact salivary glands synthesize and secrete a multigene family of high- and lower molecular weight forms of these molecules. We thus attempted to detect the presence of these proteins in the cell lines as further confirmation of a parotid phenotype. A polyclonal antiserum raised against human 220-kDa PRP and an antibody raised against low-molecular weight PRPs (20-40 kDa) were used in Western blot analysis. In FBS, 3-9 cells exhibited immunoreactive bands of 220 kDa (Fig. 5A) and 42 kDa (Fig. 5B), reflecting the presence of both high- and lower molecular weight forms of PRP. Immunoreactivity of the 220-kDa form was markedly increased in the 3-9 cells grown in rat serum, thus indicating enhanced expression of another phenotypic factor (Fig. 5A). Lower molecular weight PRP was not affected by rat serum treatment (Fig. 5B).


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Fig. 5.   Western blot analysis of proline-rich protein (PRP) expression in 3-9 cells grown in presence and absence of RS. Cell extract was analyzed by SDS-PAGE, and immunoblotting was carried out using a 1:5,000 dilution of polyclonal antibodies against high molecular mass PRP (A) and lower molecular mass PRP (B). Equal amounts of protein (150 µg) were applied to 6% gel in A and 50 µg protein were applied to a 12% gel in B, except extracts of primary cell culture, in which only 5 µg of protein were applied to the gel. PC, primary culture of parotid cells in 10% RS; FBS, 10% FBS; RS, 10% RS. Molecular mass markers are provided at left. Results shown are representative of 2 other independent experiments.

In addition to the induction of alpha -amylase and 220-kDa PRP, rat serum was also found to promote the accumulation of secretory granules. Electron microscopic examination revealed that 3-9 cells incubated with 10% rat serum contain well-formed secretory granules localized adjacent to the endoplasmic reticulum (Fig. 2, Bi and Bii). By contrast, cells grown in FBS were devoid of these subcellular organelles (Fig. 2A). Immunofluorescence studies confirmed the presence of cellular amylase in cells grown in rat serum. Fluorescence was observed throughout the cytoplasm but appeared to be concentrated in a punctate pattern in many regions (Fig. 6C); by contrast immunoreactive granules were not apparent in cells treated with FBS (Fig. 6A).


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Fig. 6.   Confocal images of 3-9 cells. Cells were grown in either 10% FBS (A) or 5% RS (C) for 7 days before immunostaining with anti-amylase antibody followed by fluorescein-conjugated goat anti-rabbit IgG as described in MATERIALS AND METHODS. Control images were obtained from FBS (B)- and RS-treated (D) cells, which were stained with only secondary antibody.

PKC isoform expression. Members of the PKC family of serine-threonine kinases are ubiquitously distributed and are directly involved in cell proliferation, differentiation, and specific phenotype expression such as exocytotic secretion (17). Previous work has shown that freshly prepared primary cultures of rat parotid acinar cells contain four PKC isozymes: PKC-alpha , PKC-delta , PKC-epsilon , and PKC-zeta (29). It was therefore of interest to examine the profile of PKC isozymes present in the parotid cell lines, as well as the effect of serum on their expression. Western blot analysis demonstrated that these four isozymes were present in equivalent or even higher amounts in extracts of clonal cells incubated with FBS when compared with freshly prepared parotid gland extracts (Fig. 7). The 3-9 cells incubated with rat serum also expressed the same isozymes. Immunoreactivity of the bands representing PKC-alpha , PKC-delta , and PKC-zeta were increased in transformed cells grown in rat serum. On the other hand, the PKC-epsilon immunoreactive band was diminished by treatment with rat serum (Fig. 7), further reflecting the selectivity of protein induction by rat serum. The selective changes in PKC isoform expression that accompany the rat serum-induced increases in parotid phenotypic properties may serve to promote and/or maintain these properties.


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Fig. 7.   Western blot analysis of protein kinase C (PKC) isoforms. Primary parotid cells or 3-9 cells were incubated with either 10% FBS or 10% RS for 4 days. Immunoblot analysis was then carried out on cell extracts using 1:1,000 dilution of PKC isoform-specific antibodies. A: PKC-alpha . B: PKC-delta . C PKC-epsilon . D: PKC-zeta . A 50-µg aliquot of protein was loaded in each lane. Multiple bands observed in C are likely due to phosphorylated forms of PKC-epsilon (16). Abbreviations are defined in legends for Figs. 4 and 5.

Functional cellular responses. beta -Adrenergic agonists such as isoproterenol are potent activators of amylase secretion by parotid glands through the mediation of cAMP (22). Isoproterenol caused a time-dependent enhancement of amylase release from clonal 3-9 cells grown in rat serum (Fig. 8). The time course of secretion generally paralleled that observed in other parotid preparations in that an increase is observed within 5 min and maximum levels are approached within 30 min (1, 23). However, isoproterenol was ineffective in stimulating 3-9 cells not exposed to rat serum (data not shown). In primary cultures muscarinic cholinergic agonists such as carbachol elicit a modest secretory response that is mediated by a Ca2+-dependent mechanism (22). Not unexpectedly, because of the relatively low amounts of cell amylase, carbachol failed to augment amylase secretion from 3-9 cells as measured by enzymatic assay (data not shown). Secretion was also monitored in 3-9 cells by labeling exportable protein with [3H]methionine. Figure 9 shows that when the more sensitive radioactive measurement of protein secretion was utilized carbachol, like isoproterenol, produced a time-dependent increase in [3H]protein secretion. Thus clonal cells incubated with rat serum possess basic regulated secretory functions that parallel those observed in primary cultures (14).


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Fig. 8.   Isoproterenol-induced stimulation of amylase release from clonal 3-9 cells. Cells were grown in 10% RS for 7 days and then exposed to 10 µM isoproterenol for various intervals. Amylase release is expressed as percent of total amylase activity remaining in cells before stimulation. Each point represents mean ± SE for at least 4 separate experiments.


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Fig. 9.   Agonist-stimulated release of 3H-labeled protein from clonal cells. Cells grown in 10% RS for 7 days were exposed to [3H-CH3]methionine for 20 min, followed by 3-h period in nonradioactive medium. Cells were then washed and either 10 µM isoproterenol or 10 µM carbachol was added for various intervals. Data represent radioactivity obtained from aliquots of medium precipitated with trichloroacetic acid. Data are expressed as means ± SE for 4 separate experiments. * P < 0.05 (Student's paired t-test).

In addition to cholinergic muscarinic receptors, primary parotid acinar cells express alpha -adrenergic and substance P (tachykinin) receptors that when activated stimulate the inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ pathway (22). Furthermore, these clonal cells share properties of primary cultures in that they express receptors that mediate Ca2+ mobilization. Mean basal cytosolic Ca2+ in quiescent transformed cells was 66 ± 3 nM (n = 14), a value comparable to that determined for freshly isolated parotid acinar cells (86 ± 5 nM) (34). Figure 10 depicts the changes in cytosolic Ca2+ produced in 3-9 cells treated with rat serum after exposure to norepinephrine, carbachol, or substance P. The responses to all three agonists were rapid. However, the rise in cellular Ca2+ caused by substance P was transient even at maximally effective concentrations and within the first few minutes of stimulation, cytosolic Ca2+ returned to prestimulation levels (Fig. 10C). In contrast, 3-9 cells treated with either norepinephrine (Fig. 10A) or carbachol (Fig. 10B) exhibited a maintained secondary increase in intracellular Ca2+ that is due to Ca2+ entry. The transient nature of the response to substance P may be attributed to the rapid desensitization of receptors that this agonist produces in primary parotid cell cultures (15). Although the Ca2+ response to each agonist was concentration dependent (Fig. 10), the magnitude of each response was somewhat smaller (circa 25%) than that observed in primary cultures. It should also be noted that none of the agonists was effective in elevating cellular Ca2+ when incubated in FBS (data not shown). Nevertheless, these results demonstrate that the parotid cell line incubated in rat serum shares the properties of primary cultures in expressing functional muscarinic, alpha -adrenergic, and tachykinin receptors.


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Fig. 10.   Stimulatory effects of receptor-mediated agonists on cytoplasmic free Ca2+ in clonal 3-9 cells. Fura 2-loaded cells grown in 10% RS were incubated with various concentrations of norepinephrine (NE, A), carbachol (CCh, B), or substance P (SP, C) and cytoplasmic Ca2+ determined as described in MATERIALS AND METHODS. Traces are representative of at least 3 independent experiments.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study reports the establishment of a functional continuous cell line of rat parotid acinar cells. The 3-9 clonal acinar cells are not only more amenable to manipulation than primary cultures, but they can be maintained in culture indefinitely under the conditions that have been described. Future experiments to be conducted with this cell line will complement the extensive knowledge available regarding the biochemical and functional properties of freshly prepared parotid acinar cells. Long-term cultures of parotid cells developed previously (18, 33) have had limited utility because they lack the phenotypic and functional properties of primary parotid acinar cells and manifest limited viability. In a recent report, functional studies of two SV40 immortalized parotid cell lines demonstrated a beta -adrenergic receptor-mediated increase in cAMP and an alpha -adrenergic and muscarinic receptor-mediated increase in inositol phosphate production and Ca2+ release (24). On the other hand, in these cell lines the intracellular structures required for secretion were not well defined and alpha -amylase was not expressed (24).

Because epithelial cells immortalized by transfection with SV40 DNA constructs lose many of their characteristic phenotypic properties (8), it is not surprising that under certain conditions the 3-9 cells exhibited a rather undifferentiated phenotype, deficient in cellular amylase, secretory granules, and responsivity to cell surface agonists. However, the present work constitutes a significant advance in the development of clonal parotid acinar cells by demonstrating with the use of biochemical, immunological, and morphological markers that rat serum brings about a more differentiated phenotype by slowing mitogenic activity and promoting differentiation. After treatment with rat serum, 3-9 cells showed a modest reduction in their proliferative activity and a 16-fold increase in their amylase content. The latter effect of rat serum appears to be a consequence of an increase in amylase synthesis because it required 7 days to reach maximum levels. The more extensive, well-defined endoplasmic reticulum observed by electron microscopy in cells grown in rat serum is also a reflection of augmented protein synthesis.

Confocal microscopy confirmed the expression of amylase in cells grown in rat serum. The localization and punctate nature of the fluorescence were consistent with the presence of amylase within secretory organelles. Although we cannot rule out that the immunofluorescence was also a reflection of amylase localized to the endoplasmic reticulum, ultrastructural analysis showed that the rise in amylase content accompanying treatment with rat serum was associated with an increase in the number of secretory organelles. The additional findings that tissue-specific PRP was also detected in 3-9 cells and that the high-molecular mass PRP immunoreactivity increased when the cells were grown in rat serum are also a reflection of a more differentiated phenotype. Cells grown in rat serum, however, manifested increases in the immunoreactivity of only three of the four PKC isozymes found in primary cells, thus indicating that a generalized increase in protein synthesis is not a consequence of the action of rat serum.

A primary differentiated function of parotid acinar cells is the export of intracellular protein (mainly amylase); this process involves protein synthesis, intracellular transport and packaging, and regulated secretion (7). Experiments employing a radioactive amino acid precursor demonstrated that 3-9 cells grown in rat serum secrete tritiated protein in response to both isoproterenol and carbachol. The added findings that isoproterenol promoted amylase secretion, whereas carbachol was ineffective, indicate that the clonal cells behave like primary cultures in that they respond more effectively to isoproterenol, which acts via cAMP.

Various preparations of rat parotid gland (cells, slices, and acini) have been utilized as experimental models for studying Ca2+ regulation and its relation to phosphoinositide hydrolysis (22). The findings that norepinephrine, carbachol, and substance P were able to elevate Ca2+ levels in 3-9 cells treated with rat serum indicate that these cells behave like primary culture cells by expressing functional alpha -adrenergic, muscarinic, and tachykinin receptors (14, 15). Consistent with the notion that the Ca2+ response is mediated by IP3 is the additional finding that the 3-9 cells possess transcripts for all three of the known IP3 receptor subtypes (B. S. Lee and R. P. Rubin, unpublished observations; 32). The basis for the increased expression of cell surface receptors in rat serum-treated cells as opposed to those grown in FBS has not yet been determined. Whether the active component(s) of rat serum increases the number of receptors and/or the affinity of the receptors for agonist will be a subject for further study.

The effects of rat serum on the differentiated state of the clonal acinar cell line are reminiscent of those observed in rat pancreatoma (AR42J) cells, another exocrine cell line. These exocrine acinar cells exhibit a large increase in amylase content, the number of secretory organelles, and regulated amylase secretion when treated with dexamethasone (9, 10). However, a number of growth factors, hormones, and agents, including hydrocortisone, which is a component of MCDB-153, failed to promote the differentiated state of the parotid 3-9 cell line. On the other hand, the ability of rat serum to promote a more differentiated phenotype suggests that certain components of rat serum regulate the expression of specific gene products that are involved in parotid acinar cell differentiation. However, because the 3-9 cells grown in rat serum contain and secrete a reduced amount of amylase compared with the primary culture, these clonal cells exhibit a lower degree of differentiation than a freshly prepared parotid cell suspension. Whether differentiation as expressed by the synthesis and secretion of amylase can be further augmented through the enrichment of the culture medium remains to be determined.

Although our findings reveal that certain unidentified humoral factor(s) present in rat serum promote the development of a more differentiated phenotype in 3-9 cells, the role of neurotropic factors is still unclear. The importance of the sympathetic and parasympathetic nervous systems in parotid acinar cell growth and differentiation has been well documented (25, 26). Neuropeptides such as neuropeptide Y, neurotensin, substance P, calcitonin gene-related peptide, and vasoactive intestinal peptide have been identified as cotransmitters in autonomic nerves innervating the rat parotid gland (2, 5, 30). Thus additional experimentation is warranted to determine whether these neuropeptides regulate the development and function of parotid acinar cells.

In conclusion, we have established a continuous cell line of rat parotid acinar cells that exhibits differentiated functions and retains the intact beta - and alpha -adrenergic, muscarinic, cholinergic, and peptidergic receptor systems that exist in primary cultures. To our knowledge, this is the first report of immortalized rat parotid acinar cells that maintain structural and functional properties of primary culture cells in terms of regulated synthesis and secretion of the tissue-specific enzyme amylase. Although primary cultures of parotid cells have been widely employed for elucidating short-term signal transduction mechanisms, the cell line that we have developed will be utilized as a model system for studying long-term biological processes that regulate parotid cell function, including growth and differentiation.

    ACKNOWLEDGEMENTS

This work was supported by the National Institute of Dental Research Grant DE-05764. The electron microscopy was carried out in the Cell Analysis Facility at the Roswell Park Cancer Institute, which is supported by National Cancer Institute Grant CA-16056.

    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. §1734 solely to indicate this fact.

Address for reprint requests: R. P. Rubin, Dept. of Pharmacology and Toxicology, State Univ. of New York at Buffalo, School of Medicine and Biomedical Sciences, 102 Farber Hall, Buffalo, New York 14214.

Received 27 February 1998; accepted in final form 22 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(2):G259-G268
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