1 Department of Biology, Spelman College, and 2 Department of Physiology, Morehouse School of Medicine, Atlanta, Georgia 30314
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ABSTRACT |
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We have developed a cell culture
of guinea pig gallbladder epithelial cells with which to study ion
transport. When grown on permeable supports, the cultured epithelia
developed a transepithelial resistance (Rt) of
~500 · cm2. The epithelial cell origin of the
cell culture was further confirmed by immunocytochemical localization
of cytokeratin. Ionomycin and forskolin increased transepithelial
voltage and short-circuit current (Isc) and
decreased Rt. The response to ionomycin was transient, whereas that to forskolin was sustained. Both were attenuated by replacement of Cl
and/or
HCO3
. Mucosal addition of the anion transport
inhibitors DIDS or diphenylamine-2-carboxylic acid (DPC) blocked the
response to ionomycin. The response to forskolin was blocked by DPC but
not by DIDS. Ionomycin, but not forskolin, increased intracellular
Ca2+ concentration in fura 2-loaded cells.
PGE2, histamine, vasoactive intestinal polypeptide, and
secretin elicited a sustained increase in Isc.
Responses to ATP and CCK were transient. Thus cultured guinea pig
gallbladder epithelia display the range of responses observed in the
native tissue and are an appropriate model for studies of ion transport
in gallbladder and intestinal epithelia.
cultured cells; histamine; ATP; vasoactive intestinal polypeptide; prostaglandin E2; secretin; cholecystokinin; anion secretion; diphenylamine-2-carboxylic acid; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; ionomycin; intracellular Ca2+; forskolin; cAMP
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INTRODUCTION |
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CELL CULTURE SYSTEMS PROVIDE a homogeneous population of cells that can be grown and studied under defined conditions and manipulated to express foreign proteins. As such, the availability of tissue culture cells has enhanced the ability of investigators to study a wide range of biological and biochemical processes. In gastrointestinal epithelia, cell cultures have been used to study ion transport processes and their regulation by second-messenger pathways. Many of the cell culture systems widely used to study intestinal ion transport, such as T84 (15), Caco-2 (20, 21), and HT-29 (63), are derived from colonic carcinomas. Although these cells display many of the transport characteristics of the normal colonic tissue type from which they were derived, some investigators have suggested caution in their use because of their tumor origin (17).
Obtaining established cell lines or primary cultures of cells derived from the small intestine that display the transport characteristics of more proximal regions of the intestine has been more problematic. For example, the established non-tumor-derived cell line IEC-6 forms a confluent monolayer but maintains many characteristics of poorly differentiated immature cells (45). The development of transepithelial electric parameters typical of epithelial tissues has not been demonstrated for IEC-6 or the closely related cell line IEC-18 (32, 45).
The gallbladder is an absorptive epithelium that elaborates and stores
concentrated bile subsequent to isosmotic absorption of Na+
and Cl. The gallbladder shares many transport
characteristics with those observed in the small intestine (5,
49). Both the small intestine and gallbladder are "leaky"
epithelia, having a low transepithelial resistance
(Rt) and voltage (Vt).
Na+ and Cl
absorption involves either a
neutral Na-Cl carrier and/or dual exchange proteins for
Na+/H+ and
Cl
/HCO3
operating in parallel
(11, 13, 19, 27, 36). Agents that elevate intracellular
cAMP [e.g., theophylline, prostaglandin, and possibly vasoactive
intestinal polypeptide (VIP) and secretin] and in some cases
intracellular Ca2+ (e.g., ionomycin and ATP) stimulate
anion secretion (8, 41-43, 50).
The goal of the present study was to develop a primary culture of guinea pig gallbladder epithelial cells to facilitate studies of the regulation of ion transport by this tissue. Other groups have developed primary cultures of gallbladder epithelia from several species (6, 8, 28, 29, 31, 44), including guinea pig (39). However, to our knowledge there are no previous reports of a systematic study in which these cell cultures have been shown to both develop transepithelial electric parameters and respond to hormones characteristic of the native tissue. The results of this study demonstrate that we have successfully developed a primary culture of guinea pig gallbladder cells that can be used for the systematic investigation of the effects of secretagogues on ion transport in gallbladder epithelia. Given the similarities between the transport processes of leaky epithelia such as gallbladder and small intestine, this cell culture may serve as a useful model with which to study transport processes common to these epithelial cell types.
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MATERIALS AND METHODS |
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Preparation of guinea pig gallbladder epithelial cell cultures. Excised gallbladders from male Hartley guinea pigs (200-300 g, Harlan Sprague-Dawley, Indianapolis, IN) were stripped of underlying muscle and minced. The tissue was incubated in an isolation solution containing 0.005% pronase and 0.1% BSA at 37°C, and the solution was triturated at intervals to facilitate cell isolation. After 1 h, the isolated cells were decanted and washed in three changes of sterile DMEM-Ham's F-12 media containing 20% FBS (Life Technologies, Rockville, MD), ITS media supplement (5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenium), 0.05 mg/ml hydrocortisone, 2 mM glutamine (Fisher Scientific, Atlanta, GA), and 0.1% penicillin-streptomycin (Life Technologies) (47). Cell viability following isolation as assessed by trypan blue exclusion was >95%. Before seeding on glass coverslips or permeable Transwell supports (Costar, Cambridge, MA), the isolated cells were briefly placed in plastic culture flasks to decrease fibroblast contamination. Transwells (0.78 cm2; Costar 3407) were coated with the basement membrane extract Matrigel (Becton Dickinson Labware, Bedford, MA) before seeding. Cells isolated from the gallbladders of two guinea pigs were pooled and yielded a sufficient number of cells to seed 18 Transwells at a density of 3.0 × 105 cells/Transwell in 200 µl of supplemented DMEM-Ham's F-12 medium.
Immunocytochemistry. Cells grown on glass coverslips were fixed and permeabilized with ice-cold methanol. Cells were first incubated with a primary antibody that recognizes cytokeratin (mouse anti-keratin monoclonal AE1/AE3; Boehringer Mannheim, Indianapolis, IN). The secondary antibody (anti-mouse IgG, Fab-specific TRITC conjugate) was then applied. Cells to which only the secondary antibody was applied without prior addition of primary antibody served as controls. Fluorescence localization of cytokeratin was observed using a Molecular Dynamics Multiprobe 2001 inverted confocal laser microscope with the appropriate setting for Cy3 fluorescence. Laser intensity was adjusted to the lowest attenuation that permitted a strong specific signal and minimal background without significant photobleaching of the dye. The images were digitized, stored, and analyzed using a Silicon Graphics Iris Indigo workstation and printed using a Mitsubishi color video copy processor.
Transepithelial electric parameters of cultured monolayers.
Gallbladder epithelial cell cultures grown on Transwells were mounted
into Lucite Ussing chambers (Costar) and bathed by a physiological
Ringer solution. The Vt and short-circuit
current (Isc) were monitored by means of an
automatic voltage clamp device (Model VCC-600; Physiologic Instruments,
San Diego, CA) in contact with the bathing solutions via Ag-AgCl
electrodes inserted into KCl- or Ringer-agar bridges. Experiments were
conducted under open-circuit [transepithelial current
(It) = 0] or short-circuit conditions
(Vt = 0) except for brief periods during
which Vt or It was
clamped to ±5 mV or 5 µA, respectively. Under both conditions, reported values of Vt and
Isc are measured rather than calculated values.
Rt was calculated from deflections in
Vt produced by bipolar Vt/It pulses using the
relation Rt = Vt/
It.
Secretagogues were added directly to both half-chambers from stock
solutions to give appropriate final concentrations. Anion transport
inhibitors were added to the apical/mucosal bathing solution. When
secretagogues or inhibitors were dissolved in DMSO or ethanol, an equal
volume of solvent was added to control cultures. Cultures were
equilibrated in the chamber for 20-30 min before the addition of
secretagogues or inhibitors. Normal Ringer solution contained (in mM)
140 Na+, 5 K+, 25 HCO3
, 1 Ca2+, 1 Mg2+, and 124 Cl
.
HCO3
-free solutions contained (in mM) 140 Na+, 5 K+, 1 Ca 2+, 1 Mg
2+, 124 Cl
, and 10 HEPES. For
low-Cl
solutions, gluconate replaced Cl
to
a final Cl
concentration of 4 mM.
HCO3
-containing solutions were gassed with 95%
O2-5% CO2. HCO3
-free
solutions were gassed with 100% O2. Experiments were
conducted at 37°C (pH = 7.4).
Measurement of intracellular Ca2+. Cultured gallbladder cells plated on coverslips were incubated with fura 2-AM (Molecular Probes, Eugene, OR) for 30 min at 37°C. In some cases, the loading solution contained 5 mM Probenecid to enhance dye uptake. The cells were washed once with HEPES-based Ringer and placed on the stage of a digital-imaging microscope. The dual-wavelength ratiometric technique (23, 55) was used to measure the intracellular Ca2+ concentration ([Ca2+]i). Cellular images were acquired on a Zeiss IM-35 microscope (Carl Zeiss Instruments, Norcross, GA) using a Nikon 40× objective (Southern Micro Instruments, Atlanta, GA) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. The images were collected with a Dage ISIT 66 video camera (Dage, Michigan City, IN) and analyzed using Inovison IC300 software (Inovision, Research Triangle, NC) running on a SPARCstation2 workstation (Sun Microsystems, Mountain View, CA). Digitized images were corrected for background fluorescence. The dye within the guinea pig gallbladder cells appeared to be fully Ca2+ sensitive. Thus [Ca2+]i was calculated by extrapolation from a standard curve that had been generated from the measurement of the fluorescent ratio of aliquots of fura 2-free acid over a range of Ca2+ concentrations. Fluorescent ratios of the aliquots were acquired at the same gain setting on the camera that was used for the acquisition of cellular fluorescent ratios.
Reagents. All were purchased from Sigma-Aldrich Chemical (St. Louis, MO) unless otherwise specified.
Statistics. Results are given as means ± SE. The statistical significance of differences was determined from paired or unpaired Student's t-test or ANOVA as appropriate. Values of P < 0.05 were considered statistically significant. In all cases, n represents the number of experiments.
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RESULTS |
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When cultured under appropriate conditions, isolated guinea pig gallbladder epithelial cells developed a confluent monolayer. Contamination by nonepithelial cells was minimized by mechanically stripping the mucosal layer of underlying musculature and preplating cells on plastic to remove fibroblasts. Cell proliferation required the presence of serum in the culture media because cells cultured in a similar defined serum-free medium supplemented with epidermal growth factor remained viable for several days but failed to proliferate. With the inclusion of 20% FBS in the culture medium cell, nidi (5-10 cells) appeared in 1 day that subsequently became confluent in 4-5 days.
The epithelial origin of the resulting cell culture was confirmed from
the localization of the epithelial cell marker cytokeratin in the
cultured cells by immunocytochemistry using a fluorescently labeled
antibody and confocal microscopy. Figure
1 shows the filamentous pattern of
intracellular cytokeratin typical of epithelial cells. No fluorescence
was observed in control cells, to which only the secondary antibody was
applied (data not shown).
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To evaluate functional similarities between cultured epithelia and the
native tissue, cells were grown on Transwells for assessment of
transepithelial electric characteristics. Rt
increased with time in culture, reaching a maximal resistance of
650 ± 99 in 4 days, corresponding to the time of confluence
(Fig. 2). Given a Transwell surface area
of 0.78 cm2, this corresponds to a
Rt of ~500
· cm2, a
value higher than that reported for the native tissue (24, 27). Rt remained stable up to day
14 and then declined. The time course of changes in
Rt was also used to evaluate the effect of serum
concentration on proliferation and confluence. Although there was no
effect of serum concentration in the range of 5-20% FBS on the
time at which the cultures reached the maximal
Rt, Rt was significantly
higher at day 14 for cells grown in 20 vs. 5% FBS
(P < 0.05).
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Cultures grown on Transwells having a Rt > 300 were placed in modified Ussing chambers, and the responses to
either 1 × 10
6 M ionomycin or 5 × 10
5 M forskolin (which elevate intracellular
Ca2+ and cAMP, respectively) were evaluated. Figure
3 and Table
1 show that ionomycin produced transient
changes in Vt and in Isc, respectively, whereas forskolin produced a biphasic, sustained increase. In both cases, Rt tended to decrease
at the peak of the changes in Isc and then
returned toward normal (Fig. 4). These changes are similar to the changes observed in native tissue that result from a stimulation of anion secretion (26, 42, 51).
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Ion substitution experiments were conducted to determine the ionic
dependency of the ionomycin- and forskolin-stimulated
Isc. Figure 5
shows that replacing both Cl and HCO3
significantly inhibited ionomycin and forskolin responses. The forskolin-stimulated Isc was significantly
attenuated when either Cl
or HCO3
was
replaced. These results are expected if anion secretion underlies the
changes in Isc.
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To further evaluate the contribution of anion secretion to the
ionomycin- and forskolin-stimulated Isc, we
determined the effect of anion transport inhibitors (Fig.
6). Anion transport inhibitors were added
to the mucosal bathing solution. The response to ionomycin was blocked
by DIDS (1 × 104 M) and diphenylamine-2-carboxylic
acid (DPC; 5 × 10
5 M), whereas the response to
forskolin was blocked by DPC but not DIDS. This sensitivity to anion
transport inhibitors provides additional evidence that the ionomycin-
and forskolin-stimulated Isc represents anion
secretion.
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Changes in Isc following the addition of
ionomycin and forskolin should reflect independent activation of
Ca2+- and cAMP-dependent pathways, respectively. To confirm
this, we evaluated the effect of ionomycin and forskolin on
[Ca2+]i in cells loaded with the fluorescent
Ca2+ indicator fura 2 with the use of fluorescent video
microscopy (Fig. 7). Resting
[Ca2+]i was 60 nM. Ionomycin caused a
sustained increased [Ca2+]i to 1.3 µM,
whereas forskolin had no effect.
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Ionomycin and forskolin modulate ion transport by affecting
intracellular Ca2+ and cAMP levels, respectively, at
intracellular sites downstream of the binding of a secretagogue or
transport modulator to its plasma membrane receptor. To evaluate the
plasma membrane receptor profile of cultured gallbladder epithelia, we
assessed the effect of several modulators of gastrointestinal ion
transport on Vt and Isc.
Figure 8 shows typical changes in
Vt in response to the addition (indicated by the
arrows) of PGE2 (106 M), histamine
(10
4 M), VIP (10
8 M), secretin
(10
7 M), and ATP (10
4 M). The effect of
these secretagogues on mean Vt and
Isc is shown in Table 1. Responses to
PGE2, histamine, VIP, and secretin were sustained as was
the case for forskolin. ATP and CCK elicited transient responses
similar to that observed for ionomycin. Carbachol (10
3 M)
and glucagon (10
6 M) had no effect on the transepithelial
electric parameters (data not shown).
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DISCUSSION |
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The goal of this study was to develop a primary culture of guinea pig gallbladder epithelial cells with which to study the regulation of ion transport in mammalian gallbladder. Our criteria for achieving this goal included demonstrating that the cell culture 1) develops a confluent monolayer of epithelial cells that is stable over time, 2) develops a Rt, Vt, and Isc, 3) lends itself to the application of several different experimental techniques, and, importantly, 4) displays the range of responses associated with the native tissue. The data support our success in developing a primary cell culture that meets these criteria.
First, immunocytochemistry demonstrated an intracellular distribution of cytokeratin in cultured gallbladder cells typical of epithelial cells. The localization of cytokeratin is widely used as a marker for epithelial cell origin (18, 39, 40). The cell culture reaches confluency in 4 days and is stable for 5-7 days. Second, cell cultures grown on permeable supports develop transepithelial electric parameters Vt, Rt, and Isc. Thus the effects of various transport modulators on ion transport can be assessed from changes in these parameters. Third, we have demonstrated that the primary culture can be used in a number of techniques required for studies of the regulation of ion transport. This includes the Ussing Isc technique and the use of fluorescence indicator dyes that offer a convenient measure of intracellular ion concentrations such as Ca2+. Although not shown in this study, we have also successfully initiated patch clamp studies in both whole cell and cell-attached configurations to further characterize cells derived from cultured monolayers.
Fourth, and importantly, the primary cultures of guinea pig gallbladder
epithelial cells display responses to transport modulators observed for
the native tissue. For example, agents that elevate intracellular
cAMP (e.g., theophylline, forskolin, prostaglandin, and possibly VIP
and secretin) and in some cases Ca2+ (e.g., ionomycin and
ATP) stimulate anion secretion by Necturus, guinea pig,
porcine, and human gallbladder (8, 28, 41-43). Mechanisms underlying secretion are typical of secretory epithelia in
that Ca2+ and cAMP open anion-conductive pathways in the
apical membrane (28, 42, 43, 50, 51). Our results clearly
demonstrate appropriate responses of cultured monolayers to agents that
elevate Ca2+ and cAMP, ionomycin, and forskolin,
respectively. These agents increased Vt and
Isc and reduced Rt. The
ionomycin- and forskolin-stimulated Isc were
attenuated by replacement of bath Cl and/or
HCO3
and blocked by anion transport inhibitors,
consistent with stimulation of anion secretion. The ionomycin- and
forskolin-stimulated responses differed in anion transport inhibitor
sensitivity, ability to elevate [Ca2+]i, and
time course, suggesting activation of distinct pathways. In the absence
of measurements of ion fluxes, we cannot exclude alternative
interpretations for the results of anion substitution and blocker
experiments (e.g., effects on cation fluxes subsequent to changes in
intracellular pH). However, activation of anion secretion is the most
plausible explanation consistent with our data and the literature
(51).
The different sensitivity of ionomycin- and forskolin-stimulated
responses to the anion transport inhibitors DPC and DIDS observed in
this study suggests similarities between Cl efflux
pathways in human and guinea pig gallbladder epithelia. In our studies,
DPC and DIDS blocked the ionomycin-stimulated increase in
Isc. Although the forskolin-stimulated response
was blocked by DPC, DIDS was ineffective. Inhibition by DPC of
cAMP-stimulated HCO3
secretion in the native
gallbladder epithelia has been attributed to an effect on apical
Cl
/HCO3
exchange (42). In
human gallbladder epithelia (8), differential sensitivity
of Ca2+- and cAMP-dependent Cl
secretion to
anion transport inhibitors was suggested to reflect activation of
separate Cl
efflux pathways. This appears to be the case
in a number of secretory epithelia, in which DIDS has been observed to
block Ca 2+-dependent Cl
currents and
channels (1, 2) but has no effect on cAMP-dependent Cl
currents and channels (1, 2, 4, 7, 9, 14,
16). On the other hand, DPC blocks both Ca2+- and
cAMP-dependent Cl
currents and channels (1, 4,
7). The molecular species underlying DIDS-sensitive currents are
unclear; however, DPC-sensitive, cAMP-dependent currents appear to be
associated with the cystic fibrosis transmembrane conductance regulator
CFTR (4, 12, 48). In Necturus gallbladder,
anion transport inhibitors (28, 30, 53) did not block
cAMP-dependent Cl
currents. Thus cultured guinea pig
gallbladder epithelial cells closely resemble cultured human
gallbladder epithelial cells (8).
Given that anion secretion appears to underlie both ionomycin- and forskolin-stimulated Isc, the observed differences in the time course of changes in Isc would not be expected. Forskolin elicited a biphasic, sustained increase in Vt and Isc and a fall in Rt. Ionomycin elicited similar but transient changes in transepithelial electric parameters. In bovine pancreatic duct cells and T84 cells, agents that increase [Ca2+]i elicit a transient and rapid increase in Isc, whereas the response to agents that elevate cAMP is delayed but sustained (1, 14, 16). The mechanisms underlying these differences are unclear. One possible explanation is that K+ efflux occurring concomitantly with anion secretion blunts the change in Isc related to anion secretion. In support of this, ionomycin-stimulated K+ efflux is five times that elicited by PGE2 in native guinea pig gallbladder (26). An alternative explanation for the transient nature of the Isc response to ionomycin is that some inhibitory pathway activated by Ca2+ turns off secretion, as suggested by Barrett (3) and Wang et al. (60) for T84 cells.
To evaluate the plasma membrane receptor profile of cultured
gallbladder epithelia, we assessed the effect of several modulators of
gallbladder ion transport on Vt and
Isc. PGE2, histamine, VIP, secretin,
and ATP stimulated Isc at concentrations
observed to be effective on native guinea pig gallbladder epithelia and
other anion secreting epithelia (33-35, 41, 43, 46, 51, 52, 58). CCK also stimulated the Isc, but
carbachol and glucagon had no effect on the transepithelial electric
parameters. To our knowledge, carbachol and glucagon have not been
reported to stimulate Isc in native guinea pig
gallbladder. ATP and CCK elicited a transient response similar to that
observed for ionomycin. Responses to PGE2, histamine, VIP,
and secretin were sustained, as was that for forskolin. These
similarities suggest that PGE2, histamine, VIP, and
secretin activate cAMP-dependent pathways, whereas ATP and CCK activate
Ca2+-dependent pathways in cultured guinea pig gallbladder
epithelial cells. Elevation of cAMP by PGE2, VIP, and
secretin has been observed for gallbladder epithelial cells from
several species (41, 43). Likewise, elevation of
[Ca2+]i by ATP was observed for several anion
secretory epithelia in which ATP acts at plasma membrane purinergic
receptors (8, 59, 64). In Necturus gallbladder,
ATP increased apical and basolateral K+ conductance and
increased apical Cl permeability (10).
Chinet and colleagues (8) recently demonstrated the
presence of P2Y2 purinoceptors on cultured human gallbladder cells, the
activation of which elicits anion secretion through activation of
Ca2+-dependent pathways. In preliminary studies, we have
observed increased [Ca2+]i following ATP
addition to fura 2-loaded cells (25).
The response of cultured gallbladder epithelial cells to histamine and CCK also resembles that of forskolin and ionomycin, respectively. However, the second-messenger pathways activated by histamine and CCK in gallbladder epithelial cells are less clear. In some epithelial cell types, histamine elicits anion secretion by binding to H1 receptors and activating Ca2+-dependent signaling pathways (46, 52), whereas in others both Ca2+- and cAMP-dependent pathways are activated (33, 54, 61). The increase in cAMP was shown to result from eicosanoid production, which then stimulated ion transport. Although the effect of CCK on anion secretion in gallbladder epithelial cells has not been reported, in pancreatic acinar cells CCK stimulates secretion via activation a Ca2+-dependent pathway (62). Together, these results demonstrate a physiological role for these agents in regulating cultured gallbladder epithelial ion transport and, importantly, that the cell culture is an appropriate model system for the native tissue.
Although the responses of cultured cells to secretagogues were similar
to those of the native tissue, the cultured guinea pig gallbladder
epithelia differed from the native tissue with respect to
Rt. Rt was considerably
higher in cultured epithelia, at 500 vs. 50 · cm2 (24, 51). Presently, we do
not have an explanation for this observation. However, a comparison of
the resistance of cultures grown on filters to resistance of native
tissue is complicated by the elaboration of the surface area of native
tissues by the presence of folds and ridges. This would lead to an
underestimate of true surface area and, therefore, resistance in native
tissue. We also cannot discount the possibility that our culture
conditions (e.g., matrix, growth factors, etc.) select for a specific
cell type or change some of the monolayer characteristics.
Alternatively, the difference in Rt may reflect
the absence of a factor that normally regulates junctional
permeability. It is important to note, however, that the response of
the Rt to secretagogues is identical to that of
native gallbladder epithelia.
To our knowledge, this is the first systematic study in which a gallbladder epithelial cell culture has been shown to develop transepithelial electric parameters and to respond to hormones characteristic of the native tissue. Other groups have developed primary cultures of gallbladder epithelia from several species for use in a variety of different studies (6, 8, 28, 29, 31, 39, 44). Most pertinent to the present study, O'Brien et al. (39) demonstrated that epithelioid outgrowths from guinea pig gallbladder explants expressing cytokeratin could be maintained for 10-14 days in culture. These cells failed to form confluent monolayers and were not characterized beyond preliminary single-channel measurements. Details concerning the culture conditions were not given; thus we cannot speculate as to differences in our culture conditions.
Many cell culture systems presently used to study regulation of
intestinal epithelial ion transport are of colonic tumor origin (17). In addition, small intestinal cell lines derived
from fetal tissue such as IEC-6 and IEC-18 cells display immature
phenotypes (32, 45). Gallbladder and small intestinal
epithelial cells have a number of similarities with respect to ion
transport mechanisms and regulation. Like guinea pig gallbladder, the
mammalian small intestine absorbs Na+ by an electroneutral
process, secretes HCO3, and responds similarly to
intestinal hormones and transport modulators such as VIP, secretin,
histamine, and PGE2 (reviewed in Refs. 5 and 49). Given
this, the primary gallbladder epithelial cell culture described in this
study may provide a new model with which to examine mechanisms and
regulation of ion transport common to these epithelia.
In summary, our laboratory has developed a primary culture of guinea pig gallbladder cell that displays transport processes and hormonal responses typical of the native tissue. The cultured guinea pig gallbladder cells can be studied using the wide range of techniques currently available to study epithelial ion transport and its regulation. As such, the preparation provides a useful model system with which to study transport in gallbladder and intestinal epithelial cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. Catherine S. Chew for helpful discussions, Andrew Shaw for assistance with confocal microscopy, and Erin Booker for technical assistance.
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FOOTNOTES |
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This work was supported by grant GM-08241-13 and grant RR-11598 from the National Institutes of Health. L. Hammonds-Odie was supported by NASA cooperative agreement NCC8-179.
Address for reprint requests and other correspondence: P. J. Gunter-Smith, Dept. of Biology, Spelman College, Atlanta, GA 30314 (E-mail: pgunter{at}spelman.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 October 1999; accepted in final form 8 May 2000.
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