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
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ABSTRACT |
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
· 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
- and
-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
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INTRODUCTION |
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.
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METHODS |
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).
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 |
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 ),
tissue resistance values were multiplied by effective membrane area
(4.52 cm2) and are presented as
means ± SE (n = 42).
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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.
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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
-adrenergic receptor agonist isoproterenol, the
-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
-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.
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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
( Isc) were
determined in response to indicated concentrations of ATP ( ) and UTP
( ) applied apically or carbachol ( ) 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).
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The ionic basis of the
Isc and the role of
Ca2+.
As in many other cell types, P2Y2
nucleotide, muscarinic cholinergic, and
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).
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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).
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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.
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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).
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DISCUSSION |
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
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
-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
-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.
 |
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