Department of Biomedical Sciences, Cornell University, Ithaca, New York
Submitted 12 December 2003 ; accepted in final form 12 November 2004
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ABSTRACT |
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canine jejunal cell culture; epithelial sodium channel; sodium-glucose cotransporter 1; sodium absorption; chloride secretion
In the present study, we introduce a new primary culture of the normal jejunum with a focus on the method of producing that culture from crypt cells isolated from the dog jejunum. In addition, we provide a preliminary structural and functional characterization of the epithelial monolayer. Primary cultures and up to seven subcultures consistently formed confluent epithelial monolayers on Snapwell filters. The monolayers exhibit the morphological and functional polarization expected of the normal jejunum, including a prominent apical brush border. The tight junction presents claudin-3 and -4 as in the normal jejunum. The expression of epithelial Na+ channels (ENaC) and an amiloride-sensitive short-circuit current (Isc) can be attributed to the presence of culture-stimulating agents (epidermal growth factor, hydrocortisone, insulin), because this current disappears in plain Ringer solution lacking culture-stimulating agents. Fortuitously, the expression of an amiloride-sensitive Isc allows an estimate of the electrical resistance of transcellular and paracellular pathways. The estimates reveal a leaky epithelium with a paracellular pathway 13 times as conductive as the transcellular pathway. Measures of the unidirectional isotopic Na+ fluxes confirmed the leaky nature of the cultured monolayers and pointed to Na+ transport systems in addition to that mediated by ENaC. One such transporter is the Na+-D-glucose cotransporter SGLT1 identified by Western blot. When electrogenic Na+ absorption via ENaC is minimized by the use of plain Ringer solution containing mucosal amiloride, the addition of dibutyryl-cAMP (DBcAMP) to the serosal side activated a Cl-dependent Isc consistent with the stimulation of transepithelial Cl secretion. The culture can be studied for hours in Ussing chambers, thus affording detailed investigations of jejunal transport across a single layer of epithelial jejunal cells under well-defined experimental conditions.
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MATERIALS AND METHODS |
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For the culture of monolayers and for their initial physiological characterization, we used fortified OptiMEM, which we define as serum-free OptiMEM supplemented with 10 mM HEPES, pH 6.5, 2.5 mM glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM GlutaMAX-I (Invitrogen-GIBCO-BRL), 20 ng/ml epidermal growth factor (human recombinant; Upstate Biotechnology), 150 nM hydrocortisone 21-hemisuccinate sodium salt (Sigma), 10 µg/ml insulin (human recombinant; Sigma), and 4% FBS. After 3 h, fortified OptiMEM was aspirated, and the attached cells (consisting almost exclusively of intact or large portions of crypts) were rinsed four times, refed with fortified OptiMEM, and incubated at 37°C. After 24 h, the incubator temperature was changed to 3234°C for the rest of the culture period.
After the primary cultures had grown in 100-mm-diameter dishes, between 300,000 and 400,000 cells in 0.5 ml fortified OptiMEM were subcultured onto Snapwell permeable filters (insert growth area 1.13 cm2, 0.4 µm pore size; Costar, Cambridge, MA) and incubated at 32°C and 6% CO2 to form dog intestinal epithelial cell (DIEC) monolayers.
We evaluated the growth of DIEC monolayers by visual inspection under the microscope (Nikon Diaphot) and by daily measurements of the resistance across the filter area with a hand-held Millicell-ERS volt- and ohmmeter (Millipore, Billerica, MA). Because this resistance measurement (between two points with heterogeneous current distribution) is meant to monitor culture growth to confluence, we call this resistance "growth area resistance" (see Fig. 4e). For measurements of the transepithelial resistance (Rt) under more homogeneous conditions of transepithelial current distribution, we used current-voltage (I-V) plots measured across confluent monolayers in the Ussing chamber.
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Cells were processed for transmission and scanning electron microscopy as previously described (41).
Western blot. Total cell lysates solubilized in SDS-PAGE sample buffer were subjected to SDS-PAGE and Western blot as described previously (49). The DNA concentration was determined using the Hoechst-33258 DNA assay and a mini-fluorometer (Hoefer; Pharmacia Biotech, Piscataway, NJ). The amount of cell lysate applied to each well was normalized to DNA (39). Lysates obtained from 0.2 x 106 cells/sample were subjected to SDS-PAGE on a 7.510% acrylamide gel. The proteins were transferred to a nitrocellulose membrane (High-bond nitrocellulose; Amersham Life Science, Arlington Heights, IL) using a transblot system (Bio-Rad, Hercules, CA) at 100 V and 5°C for 90 min. The membranes were then blocked in 80 mM Na2HPO4, 20 mM NaH2PO4·2H2O, 100 mM NaCl, and 0.1% Tween 20 containing 3% BSA at 4°C overnight. Incubation with antibodies, washing, protein detection, antibody stripping, and reprobing were performed according to the ECL-Plus Western blotting protocol from Amersham Life Science. Blots were scanned with a Molecular Dynamics Storm 840 scanner (Amersham Pharmacia Biotech, Sunnyvale, CA) in the fluorescence mode. In other experiments, we employed fluorescent secondary antibodies and the Li-COR Odyssey Infrared Imaging System (Lincoln, NE). After transfer of the proteins to a Hybond-P/polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech) and blocking for 4 h in Odyssey Blocker, incubation with primary antibodies was done in Odyssey Blocker containing 0.1% Tween 20 overnight at 4°C. After four washes with Tris-buffered saline (TBS: 20 mM Tris base, 137 mM NaCl, pH 6) containing 0.1% Tween 20, the blots were incubated with Alexa Fluor 680 goat anti-mouse IgG (1:2,500 dilution in Odyssey Blocker, 0.1% Tween 20) for 1 h at room temperature and shielded from light. After being washed, the blots were scanned in the Odyssey Infrared scanner. The primary antibodies used are the same as listed above.
In a separate experiment, the presence of SGLT1 in DIEC cell lysates was examined by Western blot with an antibody specific to rabbit SGLT1. In these studies, SDS-PAGE and the transfer of protein from gel to PVDF membrane was done as described previously (55). After the membrane was incubated for 2 h in blocking solution consisting of TBST (20 mM Tris·HCl, 137 mM NaCl, and 0.1% Tween, pH 7.6) fortified with 3% BSA, the membrane was then treated for 2 h with antibody diluted in TBST plus 1% BSA (1:1,000). To verify immunoreactivity of SGLT1, the antibody 8821 was first preincubated with 0.5 µg/ml of the immunizing peptide for 2 h, which yielded a negative Western blot. Both antibody 8821 and immunizing peptide were gifts of Dr. E. M. Wright (University of California Los Angeles). The antibody 8821 has been used successfully in the past in investigations of SGLT1 in the dog jejunum in vivo (22).
Electrophysiological studies in Ussing chambers. Electrophysiological measurements were performed on DIEC monolayers mounted in Ussing chambers (CHM5; World Precision Instruments, Sarasota, FL). Monolayers of passage 1 through 7 were studied. Finding consistent electrophysiological properties among them, we did most experiments on passages 1 to 3.
In most transport experiments, fortified OptiMEM (10 ml) was present on both sides of the epithelium. Using the methods of atomic absorption spectrophotometry, flame photometry, and coulometry, we measured the following partial composition of the OptiMEM culture medium: Na+ concentration, 157.0 ± 3.7 mM (n = 3); K+ concentration, 4.6 ± 0.3 mM (n = 3); Cl concentration, 123.7 ± 4.7 mM (n = 3); and protein, 0.56 ± 0.01 mg/ml. According to the manufacturer of OptiMEM culture medium, the glucose concentration is 13.9 mM (personal communication); other ingredients of OptiMEM culture medium are proprietary information.
In experiments examining the ion dependence of the measured Isc, confluent monolayers were taken from their cultures in fortified OptiMEM and mounted in plain Ringer solution lacking culture-stimulating agents. Plain Ringer solution contained (in mM): 120 NaCl, 5 KCl, 25 NaHCO3, 1.2 CaCl2, 1.2 MgCl2, and 5 glucose, pH 7.3. Low-Cl Ringer was prepared by replacing 116.8 mM NaCl with sodium isothionate. Glucose-free Ringer solution contained no glucose.
Mucosal and serosal solutions were circulated at a rate of 0.2 ml/s by air-lifting with 95% O2-5% CO2. The pH was maintained between 7.35 and 7.38. The temperature was maintained at 32°C with a water bath (Neslab Instruments, Portsmouth, NH). For voltage and current recording, we used the multichannel voltage/current clamp of World Precision Instruments (EVC-4000) and Ag/AgCl voltage/current electrodes embedded in 4% agar and 150 mM NaCl. Voltage and current were measured with respect to ground in the serosal compartment. To measure the Rt, we clamped monolayers at 5-mV voltage steps from 20 to +20 mV for 5 s each and recorded the transepithelial current. The Rt was calculated as the inverse of the slope of the I-V plot.
After mounting of the DIEC monolayers in the Ussing chamber, the transepithelial voltage (Vt) was monitored for 2030 min until Vt had become stable. Thereafter, the bath temperature was gradually increased to 38°C for the next 5 min. The Isc was measured at a Vt clamp of 0 mV. After Isc had stabilized after
10 min, experiments were begun. Amiloride (Merck, Whitehouse Station, NJ) was applied to the mucosal or serosal side. The effect of mucosal amiloride concentration on electrical variables was examined in detail, since serosal amiloride had no effect. Ouabain and DBcAMP were purchased from Sigma (St. Louis, MO) and presented from the serosal side only. Amiloride and ouabain were dissolved in DMSO, and stored as stock solutions at 1,000 times their final concentration in the experiment. The experimental DMSO concentration of 0.1% had no effect on measured electrical variables.
Transepithelial Na+ flux measurements. We used 22Na+ (Amersham Biosciences) to measure unidirectional Na+ fluxes across DIEC monolayers. Radioactivity in known and unknown samples was measured with a Beckman gamma counter (Gamma 300). In the typical isotopic flux experiment, the DIEC monolayer was mounted in the Ussing chamber and bathed with fortified OptiMEM on both sides. After Vt had reached steady state at 38°C, the monolayer was voltage clamped at 0 mV and maintained in the short-circuit condition for the rest of the experiment. To begin, 10 µCi 22Na+ were added to the mucosal or serosal side. At intervals of 10 min, 1 ml sample solution was taken from the other side for the measurement of 22Na+ that had crossed the DIEC monolayer. The counted sample was then returned to the side from where it had been taken. After the 40-min flux period, amiloride (10 µM) was added to the mucosal solution, and the flux measurements were repeated, again under short-circuit conditions.
When 1 ml was removed from 10 ml of apical or basolateral solution, the water column dropped by 0.4 cm of water, i.e., 1.5%. The transepithelial hydrostatic pressure difference thus introduced did not cause measurable transepithelial water flows, since water levels remained constant. Moreover, there were no transepithelial streaming potentials, and no effects on Isc and Rt. The unidirectional isotopic Na+ flux from mucosa to serosa was measured in one set of DIEC monolayers and the reverse flux in another set. Accordingly, the net transepithelial Na+ flux is the difference between values of two experimental groups (Table 1).
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For RT-PCR, 1 µl RNA (1.55 µg/µl) sample was used. The mRNA was reverse-transcribed to cDNA and further amplified by PCR reactions (SuperScript III One-Step RT-PCR System; Invitrogen). The 25-µl reaction mixture for RT-PCR included 12.5 µl of 2x reaction buffer, 1 µl total RNA sample, 1 µl forward primer (10 µM), 1 µl reverse primer (10 µM), 1 µl RT/Taq enzyme mixture, and 8.5 µl H2O. The first cDNA strands were reverse-transcribed at 52°C for 30 min. After denaturation at 94°C for 2 min, the cDNA strands were amplified by PCR reactions for 40 cycles. Each cycle entailed incubation at 94°C for 15 s, then 52°C for 30 s, and 72°C for 45 s. Finally, the DNA strands were extended at 72°C for 5 min. During the reactions, the following primers were used: ENaC forward: 5'-AACCTGCCTTTATGGACGAT-3' and
ENaC reverse: 5'-AGGTTGGACAGGAGGGTGAC-3'.
To identify the PCR product, 10 µl of the PCR product were mixed with 2 µl 6x DNA loading buffer (0.25% bromphenol blue, 0.25% xylene cyanol FF, and 15% Ficoll in H2O), applied to 1.5% agarose gel dissolved in 40 mM Tris-acetate and 1 mM EDTA and separated under 100 V with a horizontal elctrophoresis apparatus (MINI SUB DNA CELL; Bio-Rad). The PCR product was purified and submitted for sequencing with the ENaC forward primer to the Bioresource Center of Cornell University.
Statistics. All data are presented as means ± SE. The paired Student's t-test was used for the significant difference (P < 0.05) between control and experimental groups, whereas the z-test was used for the significant difference between specific data and a group mean value (P < 0.05).
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RESULTS |
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Although the culture steps producing DIEC are straightforward and reliable, a few steps proved particularly important. First, it was crucial to start cultures with intact or nearly intact crypts because isolated epithelial cells or fully differentiated villus cells did not attach to the substrate, nor did they survive for more than 23 days. As shown in Fig. 1a, essentially intact crypt cells attach to the substrate after only 4 h of incubation, and cells at the border of the cluster start to spread out and migrate. The use of ECL-coated dishes significantly improved (2- to 3-fold) the number of crypt cells attaching to substrate, but ECL-coated dishes were not essential, since qualitatively similar results were obtained with uncoated dishes. Nonepithelial cells, large tissue fragments, and clumps of villus cells did not adhere strongly and were washed out. Proliferative (mitotic) cells could be detected by staining mitotic apparatuses with anti-tubulin antibodies after only one night of incubation. However, mitotic apparatuses were relatively rare and flat and therefore difficult to detect by direct examination of unstained cultures.
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A second crucial step proved to be temperature control. When cultures were maintained at 37°C, they remained viable for at least 34 wk, but cell proliferation ceased entirely after 1 wk. In contrast, when cultures were grown at 3234°C starting on day 2, regions containing rapidly proliferative cells became evident after 34 days (Fig. 1e). Proliferative cells incorporated bromodeoxyuridine, demonstrating rapid cell division and expansion in the cell population (Fig. 1f). Older, nonproliferative cells became enlarged and apoptotic after 2 wk. By that time, the smaller proliferative cells represented the main cell type that could also be serially passaged. We have estimated a population doubling time to be 1827 h in nine cultures. Proliferative cells could be readily subcultured at least six times, yielding epithelial cells that were characterized and used in all subsequent studies. We have called these cells DIEC.
When confluent, DIEC maintained a typical epithelial morphology and formed tightly confluent DIEC monolayers with frequent, obvious domes when grown on solid (plastic) support, indicating transepithelial ion and water transport in the direction of absorption. The presence of tight junctions could be readily demonstrated by microscopic studies and by Western blotting with antibodies to junctional proteins. As shown in Fig. 2a, of the five claudins examined, only claudin-3 and -4 were detected in primary, first-, and second-passage cultures. In some experiments, small amounts of claudin-1 were also seen. Another tight junction protein, occludin-1, could also be identified by Western blot (Fig. 2a). Claudin-2 and claudin-5 were not detected in any primary culture or passage. In these experiments, -tubulin was used as a loading control (Fig. 2a).
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Electron microscopy. When DIEC were cultured in plastic dishes in the presence of fortified OptiMEM, both primary cultures and subcultures formed monolayers, with the basolateral membrane adhering to the substrate and microvilli on the apical membrane facing the culture medium (Fig. 3a). Next to this polarization, transmission electron micrographs revealed extensive infoldings of the basolateral membrane (Fig. 3, be), tight junctions near the apical border (Fig. 3d), and desmosomes near the serosal border (Fig. 3e). Some cells of the DIEC monolayers contain granules in the apical cytoplasm, possibly identifying primitive goblet cells (Fig. 3, ac). Our attempt to identify goblet cells with antibodies to mucin did not succeed, since the rat and human antibodies apparently did not recognize mucin in the dog. Cells without granules are probably epithelial cells with the properties of transepithelial transport.
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Daily measurements of the electrical resistance across the insert growth area revealed an exponential rise in resistance as DIEC monolayers became confluent (Fig. 4e). After 34 days, the electrical resistance increased sharply, reaching peak resistances 67 days after seeding the culture. Thereafter, resistance decreased together with increasing variability. In view of the transient nature of the Rt, we confined our study on transepithelial transport in Ussing chambers to monolayers between 4 and 6 days old.
Electrophysiology of DIEC monolayers in fortified OptiMEM culture medium.
Primary cultures of DIEC were not studied. Instead, our electrophysiological observations focused largely on confluent DIEC monolayers of the first, second, and third passage after noting no major differences with subsequent passages. Monolayers showing signs of apoptosis or degeneration were not used in electrophysiological studies. Under control conditions in fortified OptiMEM containing culture-stimulating agents (epidermal growth factor, hydrocortisone, and insulin), the DIEC monolayers had an open-circuit voltage (Vt) of 6.8 ± 0.6 mV (n = 36) with a range from 2.0 to 14.4 mV, apical side negative. Apical side positive voltages were never observed. Rt was 1,050 ± 105 ·cm2 on average with a range from 429 to 2,173
·cm2 (n = 22). When DIEC monolayers were voltage clamped at 0 mV, Isc was 8.1 ± 0.4 µA/cm2 (n = 36), with positive current flowing from the apical to the basolateral side. The Isc ranged from 5.3 to 13.3 µA/cm2.
Amiloride-sensitive Isc and expression of ENaC in DIEC monolayers grown and studied in fortified OptiMEM culture medium.
The effects of amiloride were studied in the presence of fortified OptiMEM (Fig. 5). The control Vt was 3.6 ± 0.4 mV (n = 10), the Rt was 694 ± 72 ·cm2 (n = 7), and the Isc was 8.4 ± 0.6 µA/cm2 (n = 10; Fig. 5A). After amiloride (10 µM) was added to the apical side, Vt dropped immediately and significantly to 0.7 ± 0.2 mV (P < 0.001), and Isc significantly decreased to 1.3 ± 0.4 µA/cm2 (P < 0.001, Fig. 5A). The inhibitory effects of amiloride were immediate, requiring 12 s to reach full effect. Even though amiloride significantly inhibited the Vt and Isc, it had no significant effect on Rt, which was 694 ± 72
·cm2 under control conditions and 687 ± 62
·cm2 (n = 7) in the presence of amiloride (Fig. 5A).
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Amiloride (10 µM) added to the basolateral solution had no significant effect on Vt, Rt, and Isc (Fig. 5B). In these nine DIEC monolayers, the control Vt was 6.6 ± 0.8 mV, the Rt was 887 ± 82 ·cm2, and the Isc was 7.2 ± 0.3 µA/cm2. After amiloride was added to the serosal side, Vt decreased to 5.1 ± 0.6 mV, Rt decreased to 731 ± 80
·cm2, and Isc decreased to 7.0 ± 0.3 µA/cm2. None of these changes was significant.
We also examined the effect of amiloride on Vt, Isc, and Rt at 32°C, the temperature at which monolayers were cultured. At this temperature, 10 µM amiloride added to the apical side of the monolayer inhibited Vt and Isc, again with no effects on Rt (data not shown).
Figure 6A shows the linear I-V plot measured across DIEC monolayers in the absence and presence of amiloride. The slope of the I-V plots is the transepithelial conductance (1/Rt), and the y-intercept is the Isc. Apical amiloride (10 µM) significantly reduced the Isc from 7.5 ± 0.3 to 0.9 ± 0.2 µA/cm2 (P < 0.001), without affecting transepithelial conductance (1.49 and 1.42 mS/cm2 before and after amiloride, respectively). Significant effects on Vt and Isc but not on Rt suggest that the paracellular conductance is much greater than the transcellular conductance such that changes in transepithelial conductance are undetectable when a transcellular transport pathway is blocked.
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Figure 6C shows the separation of the RT-PCR product of ENaC mRNA in agarose gel, revealing a DNA band of 500 bp. After being sequenced, the band turned out to be a DNA strand with 547 bp, 100% identical to the reported canine
ENaC cDNA (GenBank accession no. AF209748).
Effects of amiloride on transepithelial Na+ fluxes in monolayers grown in fortified OptiMEM culture medium. To examine whether the amiloride-sensitive Isc is carried by Na+, we measured unidirectional, transepithelial isotopic 22Na+ fluxes in the absence and presence of apical amiloride (Table 1).
Monolayers were bathed on both sides with fortified OptiMEM containing culture-stimulating agents. Under control conditions, the unidirectional Na+ flux from the mucosal to the serosal side (Jms) was 18.6 nmol·min1·cm2 and the reverse flux, from serosa to mucosa (Js
m) was 9.7 nmol·min1·cm2. The net transepithelial Na+ flux was 8.9 nmol·min1·cm2 from mucosa to serosa, which is equivalent to a current of 14.3 µA/cm2. Because the Isc measured in parallel was only 8.7 ± 1.1 µA/cm2, the transepithelial Na+ flux significantly exceeds the Isc by a factor of 1.6 (z = 5.09, P < 0.001).
In the presence of amiloride (10 µM), the Jms Na+ flux remained near control values, 18.9 ± 2.6 nmol·min1·cm2 (n = 6). Likewise, Js
m Na+ flux, 11.6 ± 1.7 nmol·min1·cm2, remained near control values (n = 6). Accordingly, the net transepithelial Na+ flux was 7.3 nmol·min1·cm2, or 11.7 µA/cm2, which was not significantly different from control (Table 1). In contrast, amiloride caused Isc to drop significantly to 1.1 ± 0.2 µA/cm2. Thus amiloride substantially reduced the measured Isc to 13% of control values without affecting the net transepithelial Na+ absorptive flux (Table 1).
Effects of ouabain on the electrophysiology of monolayers grown in fortified OptiMEM culture medium.
In monolayers studied in the presence of fortified OptiMEM containing culture-stimulating agents, the addition of ouabain (1 mM) to the basolateral side inhibited Isc and Vt and decreased Rt (Fig. 7). Unlike the immediate effects of amiloride, the effects of ouabain developed slowly. Under control conditions, Vt was 9.7 ± 1.9 mV, Rt was 1,708 ± 151 ·cm2, and Isc was 7.2 ± 0.8 µA/cm2 in six DIEC monolayers. After addition of ouabain to the serosal solution (12 s), Vt significantly decreased to 8.2 ± 1.4 mV (P < 0.001), and Isc significantly decreased to 5.9 ± 0.8 µA/cm2 (P < 0.001). Later (20 min), Vt had decreased further to 0.5 ± 0.3 mV (P < 0.001, compared with control), Isc had fallen to values not significantly different from zero, and Rt had decreased significantly to 1,106 ± 111
·cm2 (P < 0.01).
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The same experiment repeated in the presence of low-Cl Ringer solution (13 mM Cl) revealed a marked decrease in the response to DBcAMP (Fig. 8C). In the presence of low-Cl Ringer on both sides of the monolayer and apical amiloride (10 µM), the addition of DBcAMP (1 mM) to the serosal side significantly increased Isc from zero to 2.5 ± 0.1 µA/cm2 (P < 0.001); it increased Vt from 1.2 ± 0.1 to 3.6 ± 0.3 mV (P < 0.001), and it significantly decreased Rt from 3,883 ± 388 to 1,827 ± 255 ·cm2 (P < 0.001) in six monolayers. Although the effects of DBcAMP were statistically significant, they were less than those observed in the presence of plain Ringer solution with a Cl concentration of 129.8 mM (Fig. 8, B and C). These results show that the DBcAMP stimulation of Isc and Vt was dependent on the presence of Cl.
The same conclusion was reached in studies of DIEC monolayers in glucose-free Ringer solution and in the presence of apical amiloride (10 µM). Under these conditions, the addition of 1 mM DBcAMP to the serosal Ringer solution significantly increased Vt from 1.3 ± 0.1 to 7.1 ± 0.1 mV (P < 0.001), it significantly increased Isc from 0.1 ± 0.1 to 4.6 ± 0.4 µA/cm2 (P < 0. 0.001), and it significantly reduced Rt from 2,718 ± 229 to 1,532 ± 134 ·cm2 (P < 0.001), again in six monolayers. Because these effects are quantitatively similar to those observed in the presence of glucose, the significant effects of DBcAMP are not the result of stimulation of electrogenic SGLT.
Expression of SGLT1 in DIEC monolayers. Western blot analysis confirmed the presence of SGLT1 in DIEC cultures (Fig. 9A). In a protein extract of DIEC, the antibody 8821 specific to mammalian SGLT1 recognized a protein band at 75 kDa, where SGLT1 is expected to locate (Fig. 9A, left). Antibody specificity was confirmed by tying up the antibody with immunizing peptide, which prevented it from binding to SGLT1 in the DIEC extract (Fig. 9A, right).
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DISCUSSION |
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In the present study, we introduced an intestinal cell culture derived from the normal, healthy intestinal mucosa of the dog jejunum. The DIEC culture was directly produced from jejunal crypts, apparently from committed crypt cells. The culture could be carried through at least six passages without apparent morphological changes or loss of ability to form polarized monolayers on filters. When monolayers are grown on filters, epithelial cells display long and dense microvilli at the apical membrane, extensive basolateral membrane infoldings, and well-defined tight junctions and desmosomes (Figs. 3 and 4). The expression of keratin no. 18 (Fig. 1c) and the presence of several tight junction proteins confirm the epithelial nature of the culture (Fig. 2). The expression of the epithelial Na+ channel ENaC (Figs. 5 and 6) was unexpected and most likely the result of the presence of epidermal growth factor, corticosterone, and insulin in the culture medium (vide infra).
Proteins of the tight junction and the paracellular pathway. To date, 24 members of the claudin protein family have been identified in mice and men (50). The claudins have four transmembrane domains with NH2 and COOH terminals located in the cytoplasm. Associated with other junctional proteins (occludin, ZO proteins, and junctional adhesion proteins), the claudins define the barrier and permselectivity functions of the paracellular pathway (29, 34). For example, the presence of claudin-16 in tight junctions of the thick ascending limb of the Loop of Henle defines the paracellular Ca2+ and Mg2+ permselectivity in this part of the nephron (46). More than two claudins may coexist in a tight junction, forming heteropolymers that appear to increase the structural and functional diversity of the paracellular pathway (17).
So far, claudin-3 and claudin-4 have been found in the jejunum (43). Significantly, the same claudins are expressed in the DIEC culture of the dog jejunum, indicating a cultured tight junction that resembles the junction of the normal jejunum. A receptor function has been attributed to these two claudins (5, 26). Binding to the enterotoxin of Clostridium perfringens, claudin-3 and claudin-4 are thought to participate in triggering the diarrhea of the Clostridium infection. Apparently, claudins can have functions in addition to permissive paracellular transport.
Occludin is another tight junction protein with four transmembrane domains. The discrete distribution of occludin in a narrow band surrounding the cells of DIEC monolayers is consistent with its location in tight junctions (Fig. 2b). Likewise, the distribution of ZO-1 is limited to the region of the tight junction (Fig. 2c).
On first examination, the distribution of claudin-4 suggests its presence in the cytoplasm of cell (Fig. 2d). However, upon closer inspection, it appears that the microtome cut takes a tangent to tight junctions. Leaving one epithelial cell, the cut encounters a sharp edge of claudins, suggesting its entry into the tight junction. Passing obliquely through the tight junction, the cut gives rise to the diffuse distribution of claudin-4. In other regions of Fig. 2b, claudin is as discretely confined to the cell border, like occludin and ZO-1, consistent with the expression of claudins at plasma membranes (35). Nevertheless, claudins have been observed inside cells. When exogenous claudin-2 and -4 were experimentally expressed in Madin-Darby canine kidney II cells, they were detected in some intracellular, vacuole-like structures in addition to their expression at the cell border, whereas native claudins were restricted to the cell border (9). Studies by Kobayashi et al. (30) suggest that foreign claudins can induce the formation of vacuole-like structures in the cytoplasm.
Desmosomes are "welding spots" that hold cells together and resist tears in epithelial tissues. Desmosomes are abundantly expressed in DIEC monolayers where they outline the lateral interstitial space between epithelial cells and beyond the tight junction (Fig. 2e).
DIEC, a leaky epithelium in spite of appreciable Rt values.
In 1972, Frömter and Diamond introduced the concepts of "leaky" and "tight" epithelia (16). In general, leaky epithelia have 1) low values of Vt (from 0 to 11 mV) and Rt (from 6 to 200 ·cm2), 2) a brush-border apical membrane, and 3) the functional properties of isosmotic fluid transport at high rates. In contrast, tight epithelia have high values of Vt (30100 mV) and Rt (3002,000
·cm2). Tight epithelia lack a brush border at apical surfaces. The main function of tight epithelia is to generate and maintain high transepithelial concentration differences of solute and water for storage.
In the present study, DIEC monolayers had on average a Rt of 1,050 ·cm2 consistent with a tight epithelium when compared with 50
·cm2, the resistance of the small intestine in vivo (15, 36). On the other hand, a low Vt and the presence of a dense brush border indicate a leaky epithelium. Instead of measures of the Vt and Rt, Boulpaep (6) has proposed a more appropriate criterion of epithelial leakiness or tightness, namely the ratio of transcellular and paracellular transport in general and the ratio of transcellular and paracellular resistance in particular. To estimate this resistance ratio in DIEC monolayers, we have assumed that the electromotive force of the active transport pathway passing through epithelial cells is 120 mV. Because Isc is 8.1 µA/cm2, it follows that the tanscellular resistance is 14,815
·cm2. In view of a Rt (transcellular and paracellular resistance in parallel) of 1,050
·cm2, the paracellular resistance is 1,131
·cm2. Thus the ratio of transcellular and paracellular resistance indicates a paracellular conductance 13 times greater than the transcellular conductance consistent with a leaky epithelium. A high paracellular conductance may obscure the effects of amiloride on the Rt (Figs. 5 and 6).
Functional characterization of DIEC.
Absorption of salt, water, and dietary nutrients is the hallmark function of the mammalian small intestine. The human intestine absorbs 600 mmol Na+ and 6.5 liters H2O/day. The absorption of water is coupled to the absorption of solute. In turn, the absorption of solute relies largely on Na+-dependent transport mechanisms such as Na+-glucose cotransport, Na+-amino acid cotransport, Na+/H+ exchange transport, and NaCl absorption via parallel Na+/H+ and Cl/HCO3 transport. The transport of Na+ across the small intestine varies 1) along the length of the intestine (radial heterogeneity), 2) along the length from crypt to the tip of a villus (axial heterogeneity), and 3) between individual epithelial cells in regions of the villus tip and crypt that express transport systems to varying degree (cellular heterogeneity). DIEC monolayers derived from the normal jejunum appear to illustrate some of these heterogeneities. For one reason, the net absorptive isotopic Na+ flux was nearly two times the Isc measured in parallel (Table 1), suggesting the presence of electroneutral Na+ transport mechanisms such as NaCl absorption via parallel Na+/H+ and Cl/HCO3 transport. For another reason, the inequality of current and flux suggests that flux and current do not derive from the same monolayer area, i.e., the monolayer is heterogeneous like the normal jejunum.
Our preliminary functional characterization of DIEC monolayers illustrated the influence of environmental factors on the expression of transport systems. When DIEC monolayers are grown and studied in OptiMEM solution containing the culture-stimulating agents epidermal growth factor, hydrocortisone, and insulin, the monolayers display an amiloride-sensitive Isc mediated by ENaC (Fig. 6). This current disappears in plain Ringer solution (Fig. 8A). The subsequent addition of cAMP to the serosal side activates a Cl-dependent current consistent with transepithelial secretion of Cl (Fig. 8, B and C). Thus DIEC monolayers can be manipulated to express electrogenic Na+ absorption under one set of conditions and electrogenic Cl secretion under another.
When grown and studied in fortified OptiMEM solution, the inhibition of the Isc with amiloride presented from the mucosal side (and not the serosal side, Fig. 5) and the inhibitory effects of serosal ouabain on voltage and Isc (Fig. 7) outline the rudiments of the Ussing model of epithelial Na+ transport (51). The amiloride concentration-response curve revealed an IC50 of 0.76 µM (Fig. 6B), similar to that measured in other epithelia expressing ENaC (4). The immediate effect of ouabain on Vt and Isc probably results from the blockade of the electrogenic Na+-K+-ATPase operating with a stoichiometric exchange of three Na+ for two K+. The ouabain-sensitive pump current was measured as 1.3 ± 0.2 µA/cm2, which is significantly different from zero (P < 0.001). The gradual decline of Vt and Isc in the presence of ouabain probably reflects the dissipation of Na+, K+, and voltage gradients across cell membranes.
Isotopic Na+ flux measurements in DIEC monolayers grown and studied in fortified OptiMEM show that ENaC-mediated transepithelial Na+ transport accounts for only 18% of the net absorptive transepithelial Na+ transport (Table 1). Because these measurements were made in the absence of Vt and concentration differences, DIEC monolayers must possess additional transepithelial active transport mechanisms for Na+. Indeed, an antibody specific to SGLT1 recognizes a protein in DIEC lysates that localizes at the expected position (Fig. 9A). That the same antibody recognizes SGLT1 in the dog jejunum in vivo indicates that our culture expresses Na+/D-glucose cotransport like the normal jejunum (22). Next to Na+/D-glucose cotransport, DIEC monolayers may express other Na+-dependent solute transport systems that were not investigated in the present study.
Significantly, amiloride-sensitive Vt and Isc disappeared in monolayers when fortified OptiMEM, containing growth factor, hydrocortisone, and insulin, was replaced with plain Ringer solution (Fig. 8A). In the presence of plain Ringer solution on both sides of the monolayer, the Vt and Isc decayed with a time course expected from the endocytic deactivation of ENaC (7, 8). The decay of voltage and current suggests that the presence of growth factor, glucocorticoid, and insulin in the culture medium had activated the expression of ENaC (38, 45). Still, the presence of ENaC in a cell culture of the small intestine may not be so unusual as it first appears. In rats, the surgical removal of the colon (ileoanal anastomosis) induces ENaC expression in the ileum (31). Furthermore, the induction of ENaC activity has been attributed to serum- and glucocorticoid-induced kinase that is present in both jejunum and ileum (10).
Next to transepithelial Na+ absorption, the transepithelial secretion of fluid driven by secretory Cl transport is a functional hallmark of the jejunum. In particular, Cl-driven fluid secretion is thought to take place in the crypt region of villi and is mediated by epithelial cells that harbor Cl channels in the apical membrane and the Na+-K+-2Cl cotransporter in the basolateral membrane (13, 54). Up to now, at least the following three different Cl channels have been found in the small intestine: the stretch-activated Cl channel, the Ca2+-activated Cl channel, and cystic fibrosis transmembrane conductance regulator (CFTR) that is activated by phosphorylation via protein kinase A (PKA) and cAMP (3). DIEC monolayers apparently exhibit Cl secretion via CFTR in view of the effects of DBcAMP (Fig. 8, B and C). The nucleotide induced a significant increase in Isc (from mucosa to serosa) when ENaC channels were blocked with amiloride. The cAMP-stimulated Isc was dependent on the presence of Cl in the bath medium consistent with the stimulation of transepithelial Cl secretion. Furthermore, the sustained stimulation of Isc distinguishes CFTR channels from Ca2+-activated and stretch-activated Cl channels, which respond only transiently to activation (3, 13).
Of note is the significant decrease of the Rt upon stimulation with cAMP (Fig. 8, B and C). The activation of CFTR in the apical membrane of DIEC monolayers was not expected to decrease the Rt too much in view of the low paracellular resistance relative to the transcellular resistance that prevented a significant increase in Rt in the presence of amiloride (Figs. 5A and 6A). Thus cAMP may have affected the paracellular pathway in addition to CFTR in the apical membrane of DIEC. Indeed, the nucleotide is thought to increase tight junction permeability (25). The molecular mechanism is not completely understood, but the PKA phosphorylation of Thr207 of claudin-5, one of the tight junction proteins, is known to trigger the rapid decrease in transendothelial resistance in rat lung endothelium (47).
Na+-dependent glucose cotransport via SGLT1 is another hallmark of the jejunum. Glucose enters the cell from the intestinal lumen against its chemical gradient at the expense of the Na+ electrochemical gradient across the apical membrane. Glucose leaves the cells across the basolateral membrane down its chemical gradient through another sugar transporter of the family of GLUT glucose transporters (48).
Western blot analysis revealed the presence of SGLT1 in DIEC monolayers. The electrophysiological evidence for SGLT1 was less clear. The addition of 10 mM glucose to glucose-free medium tended to increase the Isc, but the change was not statistically significant (Fig. 9B). SGLT1 is also present in other cultures of the mammalian intestine, such as Caco-2, HT29 cl.19A, and HT29-D4. However, it is active only in fully differentiated HT29-D4 cell monolayers (12, 18, 20), suggesting that SGLT1 activity correlates with cell differentiation (11). Indeed, SGLT genes are transcribed and translated only in mature enterocytes at villus tips (23). Thus it is likely that the number of tip cells present in DIEC monolayers may be sufficient to yield a positive Western blot but insufficient to yield significant transepithelial electrical changes when stimulated with glucose.
Na+-D-glucose cotransport can be activated by cAMP, as in HRT-18 cells (37). However, in DIEC monolayers, the cAMP stimulation of the Isc was primarily the result of stimulation of a Cl-dependent current (Figs. 8 and 9).
As a good model for the study of intestinal transport, a monolayer must resemble the in vivo condition physically, morphologically, and biochemically. In the present study, we have examined the morphological and electrophysiological characteristics of a new intestinal culture derived from the normal dog jejunum. DIEC monolayers resemble normal epithelium to a remarkable degree. They display the morphological polarization of the jejunum with a prominent brush border, basolateral membrane infoldings, and the functional polarization of transporters in expected basolateral and apical membrane domains of the jejunum. Likewise, DIEC tight junctions possess the claudin proteins of the normal jejunum. Yet we leave many transport questions open to further studies, and the expression of digestive enzymes has yet to be examined. As a culture of the normal small intestine, DIEC monolayers may find wide application serving basic and applied motivations, from studies of the mechanism and regulation of transepithelial solute and water transport to drug absorption, intestinal clearance of xenobiotics, and high throughput evaluations of pharmaceutical agents.
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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.
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