Departments of 1 Clinical Sciences, 2 Food Animal Health and Resource Management, and 3 Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, 27606; and 4 Department of Pediatrics, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina 27599.
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ABSTRACT |
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We have previously shown that PGE2 enhances
recovery of transmucosal resistance (R) in
ischemia-injured porcine ileum via a mechanism involving
chloride secretion. Because the tyrosine kinase inhibitor genistein
amplifies cAMP-induced Cl secretion, we postulated
that genistein would augment PGE2-induced recovery of
R. Porcine ileum subjected to 45 min of ischemia was mounted in Ussing chambers, and R and mucosal-to-serosal fluxes of [3H]N-formyl-methionyl-leucyl
phenylalanine (FMLP) and [3H]mannitol were
monitored as indicators of recovery of barrier function. Treatment with
genistein (10
4 M) and PGE2
(10
6 M) resulted in synergistic
elevations in R and additive reductions in mucosal-to-serosal
fluxes of [3H]FMLP and
[3H]mannitol, whereas treatment with genistein
alone had no effect. Treatment of injured tissues with genistein and
either 8-bromo-cAMP (10
4 M) or cGMP
(10
4 M) resulted in synergistic
increases in R. However, treatment of tissues with genistein
and the protein kinase C (PKC) agonist phorbol myristate acetate
(10
5-10
6
M) had no effect on R. Genistein augments recovery of R
in the presence of cAMP or cGMP but not in the presence of PKC agonists.
mucosa; chloride secretion; transmucosal resistance; tyrosine kinase
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INTRODUCTION |
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THE INTESTINAL MUCOSAL BARRIER is composed of a single layer of epithelial cells anchored to one another by a series of interepithelial junctions (25, 26). In terms of barrier function, the most critical of the interepithelial junctions is the apically situated tight junction because it regulates paracellular flux of solutes and bacterial toxins (25, 26). For example, it has recently been shown that the bacterial toxin N-formyl-methionyl-leucyl phenylalanine (FMLP), a potent neutrophil chemoattractant, may gain access to subepithelial tissues across submature tight junctions (35). Breakdown of the intestinal barrier, such as occurs during intestinal ischemia-reperfusion injury, increases the ability of bacteria and their toxins to traverse the gut and may lead to onset of sepsis and multiple organ failure (39). We have recently shown that PGE2 and PGI2 trigger recovery of barrier function in ischemia-injured intestinal mucosa via a pathway that principally involves closure of interepithelial spaces (8, 9).
PGE2 stimulates recovery of barrier function via cAMP (8).
This intracellular second messenger has diverse cellular effects, making it difficult to determine the exact role of this mediator in
recovery of barrier function. For example, cAMP may trigger elevations
in transepithelial resistance via direct effects on tight junctions
(16) but cAMP also signals Cl secretion (11, 41).
Our recent studies indicate that inhibition of
PGE2-triggered Cl
secretion in
ischemia-injured porcine ileum largely abolishes the reparative
action of PGE2 (8). Because of the apparent role of
Cl
secretion in recovery of barrier function, we
developed an interest in genistein, a novel Cl
secretagogue. There has been a great deal of recent interest in
isoflavinoids such as genistein because these compounds are antioxidants (10) and anticarcinogenic in the gut (21, 42) and may be
derived from dietary sources such as soybean meal (38). Genistein
augments Cl
secretion in a cAMP-dependent fashion,
possibly by interacting directly with phosphorylated cystic fibrosis
transmembrane conductance regulator (CFTR) (17). However, genistein is
a tyrosine kinase inhibitor that may have direct effects on recovery of
barrier function via its effects on tight junction-associated tyrosine kinases (13, 29, 36). In the present studies, we postulated that
genistein would augment PGE2-stimulated recovery of mucosal barrier function in acutely injured intestinal epithelium via a
signaling pathway that involved Cl
secretion rather
than an action involving inhibition of tyrosine kinases.
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MATERIALS AND METHODS |
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Experimental animal surgeries.
All studies were approved by the North Carolina State University
Institutional Animal Care and Use Committee. Six- to eight-week-old Yorkshire crossbred pigs of either sex were housed singularly and
maintained on a commercial pelleted feed. Pigs were held off feed for
24 h before experimental surgery. General anesthesia was induced with
xylazine (1.5 mg/kg im), ketamine (11 mg/kg im), and pentobarbital (15 mg/kg iv) and was maintained with intermittent infusion of
pentobarbital (6-8
mg · kg1 · h
1).
Pigs were placed on a heating pad and ventilated with 100% O2 via a tracheotomy using a time-cycled ventilator. The
jugular vein and carotid artery were cannulated, and blood gas analysis was performed to confirm normal pH and partial pressures of
CO2 and O2. Lactated Ringer solution was
administered intravenously at a maintenance rate of 15 ml · kg
1 · h
1.
Blood pressure was continuously monitored via a transducer connected to
the carotid artery. The ileum was approached via a ventral midline
incision. Ileal segments were delineated by ligating the intestinal
lumen at 10-cm intervals and subjected to ischemia by clamping
the local mesenteric blood supply for 45 min.
Ussing chamber studies.
After the ischemic period, the mucosa was stripped from the
seromuscular layer in oxygenated (95% O2/5%
CO2) Ringer solution and mounted in 3.14 cm2
aperture Ussing chambers, as described in a previous study
(5). Tissues were bathed on the serosal and mucosal sides
with 10 ml Ringer solution. The serosal bathing solution contained 10 mM glucose and was osmotically balanced on the mucosal side with 10 mM
mannitol. Bathing solutions were oxygenated (95% O2/5%
CO2) and circulated in water-jacketed reservoirs. The
spontaneous potential difference (PD) was measured using Ringer-agar
bridges connected to calomel electrodes, and the PD was short-circuited
through Ag-AgCl electrodes using a voltage clamp that corrected for
fluid resistance. Resistance ( · cm2)
was calculated from the spontaneous PD and short-circuit current (Isc). If the spontaneous PD was between
1.0
and 1.0 mV, tissues were current clamped at ±100 µA for 5 s and the
PD was recorded. Isc and PD were recorded every 15 min for 4 h.
Experimental treatments.
Tissues were bathed in Ringer containing 5 × 106 M indomethacin to prevent PG
production while the mucosa was stripped from the seromuscular tissues,
and indomethacin was added to the serosal and mucosal bathing solutions
in the same concentration before tissues were mounted on Ussing
chambers. In addition, bumetanide (10
4
M) was added to the serosal side of tissues at the beginning of the
experiment where appropriate. Baseline electrical readings were taken
for 30 min, after which further treatments were added to the tissues
depending on the study. Treatments added after the 30-min
equilibration included genistein (10
4 M,
serosal and mucosal), 16,16-dimethyl PGE2
(10
6 M, serosal), vasoactive intestinal
peptide (10
7 M, serosal),
8-bromo-cAMP (10
4 M, serosal and
mucosal), 8-bromo-cGMP (10
4 M, serosal
and mucosal), phorbol myristate acetate (PMA;
10
5
M-10
6 M, serosal and
mucosal), carbachol (10
5 M,
serosal), and theophylline (10
2 M,
serosal and mucosal).
Isotopic NaCl flux studies.
All fluxes were conducted under short-circuit conditions (tissues
clamped to 0 mV). To assess transmucosal Na+ and
Cl fluxes, 0.3 µCi/ml 22Na and
36Cl were added to the mucosal or serosal solutions of
tissues paired according to their conductance (conductance within 25%
of each other) (4, 5). After a 15-min equilibration period and before addition of treatments, standards were taken from the bathing reservoirs. A 30-min flux period was initiated at the same time as
addition of treatments by taking samples from the bathing reservoirs opposite from the side of isotope addition. Samples were counted for
22Na and 36Cl in a liquid scintillation
counter. The contribution of 22Na
-counts to
36Cl
-counts were determined and subtracted.
Unidirectional Na+ and Cl
fluxes from
mucosa to serosa (Jms) and serosa to mucosa
(Jsm) and the net flux (Jnet)
were determined using standard equations (4, 5).
[3H]FMLP and mannitol fluxes.
These studies were performed in much the same way as NaCl fluxes,
except that 0.2 µCi/ml [3H]FMLP or 0.2 µCi/ml [3H]mannitol (Sigma Chemical, St.
Louis, MO) was placed on the mucosal surface of affected tissues 15 min
after addition of treatments (PGE2 and/or genistein).
Radiolabeled probes were diluted in 104
M FMLP and 10
2 M mannitol, respectively.
Jms were calculated over a 1-h time period starting
30 min after the addition of treatments. Permeability was calculated
from flux data using the following formula
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Electron and light microscopy. Tissues were taken at 0, 15, 30, 60, 120, and 240 min for routine histological evaluation. Tissues were sectioned (5 µm) and stained with hematoxylin and eosin. For each tissue, three sections were evaluated. Four well-oriented villi were identified in each section. The length of the villus and the width at the midpoint of the villus were obtained using a light microscope with an ocular micrometer. In addition, the height of the epithelial-covered portion of each villus was measured. The surface area of the villus was calculated using the formula for the surface area of a cylinder. The formula was modified by subtracting the area of the base of the villus and multiplying by a factor accounting for the variable position at which each villus was cross-sectioned. In addition, the formula was modified by a factor that accounted for the hemispherical shape of the upper portion of the villus (4). The percentage of the villous surface area that remained denuded was calculated from the total surface area of the villus and the surface area of the villus covered by epithelium. The percent-denuded villous surface area was used as an index of epithelial restitution.
In experiments designed to assess epithelial ultrastructure under the influence of PGE2 and genistein, tissues were removed from Ussing chambers after 120 min (peak resistance) during three separate experiments (n = 3 for each treatment). Tissues were placed in Trump's 4F:1G fixative and prepared for transmission electron microscopy using standard techniques (10). For each tissue evaluated, five well-oriented interepithelial junctions were evaluated.cAMP RIA.
Tissues were removed from Ussing chambers once Isc
had peaked in response to genistein and PGE2 treatment, and
they were immediately frozen in liquid N2. Tissues were
stored at 70°C before extraction and RIA. One part tissue
(100 mg) was homogenized with nine parts 5% TCA. The homogenate was
centrifuged at 2,500 g at 4°C for 15 min and extracted
three times with 5 volumes of water-saturated ether. Excess ether was
discarded after each extraction, and the samples were evaporated to
dryness. RIA for cAMP was performed using a commercial kit according to
the manufacturer's instructions (Biomedical Technologies, Stoughton, MA).
Data analysis. Data were reported as means ± SE. All data were analyzed using ANOVA for repeated measures, except where peak or single time-point responses were analyzed using a standard one-way ANOVA (Sigmastat, Jandel Scientific, San Rafael, CA). A Tukey's test was used to determine differences between treatments after the ANOVA, and P < 0.05 was considered significant.
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RESULTS |
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Ischemia-injured tissues bathed in indomethacin (5 × 106 M) and treated with
16,16-dimethyl PGE2 (10
6 M)
after a 30-min equilibration period showed marked elevations in
transmucosal resistance (R) compared with tissues treated with indomethacin alone (Fig. 1A).
Additional treatment after the 30-min equilibration period with the
novel Cl
secretagogue genistein
(10
4 M) augmented the R
response to PGE2, whereas treatment with genistein and
indomethacin in the absence of PGE2 was without effect
(Fig. 1A). The response between PGE2 and genistein
was significantly greater than the additive effect of each
individually, indicating synergism between PGE2 and
genistein. Evaluation of Isc, which is closely
correlated with Cl
secretion in this tissue (5),
also showed a synergistic response (Fig. 1B).
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To determine whether the synergistic effects of PGE2 and
genistein on Isc were attributable to
Cl secretion, unidirectional Na+ and
Cl
fluxes were performed over a 30-min period
commencing 30 min after the addition of PGE2 and genistein.
These studies indicated that PGE2 and genistein had
synergistic effects on net Cl
secretion (Table
1). To directly assess the role of elevated Isc in the recovery of R,
ischemia-injured tissues were pretreated with the basolateral
Na+-K+-2Cl
transport
inhibitor bumetanide (10
4 M). Subsequent
stimulation of tissues with PGE2 resulted in significantly reduced recovery of R (Fig. 1C) and complete inhibition
of Isc (Fig. 1D) compared with tissues
treated with PGE2 in the absence of bumetanide (Fig. 1,
A and B). Furthermore,
genistein/PGE2-stimulated recovery of R was
completely abolished in the presence of bumetanide (Fig. 1C),
although limited elevations in Isc were detected
(Fig. 1D). These experiments suggested that
Isc (as a measure of Cl
secretion) is correlated with recovery of R.
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We have previously shown that PGE2-stimulated
Cl secretion is associated with closure of dilated
interepithelial spaces rather than an effect on epithelial restitution
(8). In the present studies, ~20% of the villous surface area was
denuded by the ischemic episode, but the denuded area was reduced to
~10% within 30 min (Table
2). Tissues continued to
restitute between 30 and 60 min in the presence indomethacin (resulting
in ~4% denuded villous surface). The addition of genistein,
PGE2, or both genistein and PGE2 to recovering
tissues at 30 min had no significant effect on denuded villous surface
area (Table 2). In addition, there were no apparent
morphological differences on light microscopy between recovering
tissues under the influence of the various treatments after 60 min of
recovery time (Fig. 2). Epithelium at the
tips of repairing villi appeared flattened regardless of treatment.
However, evaluation of restituted epithelium with electron microscopy
revealed dilatation of interepithelial spaces in tissues treated with
indomethacin alone, whereas tissues treated with PGE2 or
PGE2 and genistein had closely apposed interepithelial spaces (Fig. 3). As in previous studies
(8), ~75% of the interepithelial spaces were dilated in
indomethacin-treated tissues, whereas tissues additionally treated with
PGE2 had ~10% of the interepithelial spaces dilated, and
there was no apparent dilatation of interepithelial spaces in tissues
treated with both PGE2 and genistein.
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We have previously shown that PGE2 triggers increases in
Isc and R via the intracellular second
messenger cAMP (9). To explore the possibility that genistein's
synergism with PGE2 was mediated via an interaction with
cAMP, we assessed the effect of treating tissues with genistein and the
cAMP agonist VIP (107 M) after a 30-min
equilibration period. Similar to experiments using PGE2,
tissues treated with genistein and VIP showed synergistic elevations in
R and Isc, although these electrical
responses were transient (Fig. 4). To
expand on these results, we next determined the ability of genistein to
synergize with a range of putative Cl
secretagogues
that signal via distinct intracellular signaling pathways. Tissues were
treated with genistein and one of the following: 10
4 M 8-bromo-cAMP,
10
4 M 8-bromo-cGMP, the protein kinase C
stimulant PMA (10
6 M) (14), or the
Ca2+-mediated agonist carbachol
(10
5 M) (41). Tissues treated with
8-bromo-cGMP or 8-bromo-cAMP and genistein after a 30-min equilibration
period showed similar synergistic elevations in R and
Isc, whereas tissues treated with either carbachol
or PMA alone or in the presence of genistein had little or no
discernible effect on R or Isc (Fig. 4). To
rule out a dose-dependent lack of response of tissues to PMA and
carbachol, higher doses were used (10
5 M
PMA and 10
4 M carbachol). However, these
higher dosages produced no further effect on Isc
either alone or in the presence of genistein. PMA at higher doses
(10
5 M) tended to show reduced peaks in
Isc (peak Isc in the presence of 10
5 M PMA and
10
4 M genistein, 2 ± 1.7 µA/cm2, n = 4) compared with lower doses of PMA
(Fig. 5), possibly indicating near-toxic
levels.
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Because genistein had the most dramatic effects in tissues treated with
cAMP or cGMP, we questioned whether genistein was simply acting as a
phosphodiesterase inhibitor as has been demonstrated in pituitary (30)
and neural tissue (40). To assess the effect of phosphodiesterase
inhibition on recovery of ischemia-injured porcine mucosa,
tissues were treated after a 30-min equilibration period with
theophylline (102 M) or theophylline and
PGE2. In contrast to genistein, theophylline triggered
marked elevations in Isc and R when
administered alone. However, theophylline stimulated additive
elevations in Isc and synergistic elevations in
R when administered in combination with PGE2 (Fig.
6). These studies indicated that inhibition
of phosphodiesterase amplified the effects of PGE2,
presumably as a result of additive increases in intracellular cAMP
levels. To explore the potential of genistein to mediate its action on
mucosal recovery via this mechanism, we measured cAMP levels in
ischemia-injured tissues during the peak
Isc response (when the cAMP signal was presumed to
be maximal). Treatment with PGE2 resulted in approximately twofold increases in cAMP levels compared with tissues treated with
indomethacin alone (114.4 ± 7.7 vs. 47.8 ± 12.7 pmol/ml, respectively). Genistein tended to reduce rather than increase cAMP
levels in PGE2-treated tissues (83.1 ± 13.6 pmol/ml), but there was no statistically significant effect of genistein on cAMP
levels.
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Although it appeared that the synergistic effect of PGE2
and genistein on recovery of R was attributable to increased
Cl secretion, it has recently been shown that
genistein may stimulate recovery of barrier function via an action on
tight junction regulatory proteins (13, 29, 36). To assess the
potential role of genistein-mediated tyrosine kinase inhibition on
recovery of R in our model, we pretreated tissues with varying
doses (10
4
M-10
6 M) of the alternative
tyrosine kinase inhibitor tyrphostin 47 (37). However, tyrphostin 47 did not augment the effects of PGE2 on recovery of
R as genistein had done, and tyrphostin 47 had no effects of
its own on ischemia-injured mucosa (data not shown). In further
experiments, tissues were pretreated with the distinct tyrosine kinase
inhibitor herbimycin A (10
6 M), but,
similar to the results with tyrphostin 47, this agent had no effect on
recovery of R in the presence of PGE2 (data not shown).
To assess the ability of PGE2 and genistein to enhance
barrier function of ischemia-injured porcine ileum, we
performed Jms of the small bacterial toxin
[3H]-FMLP (5-6 Å, 180 Da). As
predicted from measurements of R, application of
PGE2 or genistein and PGE2 resulted in additive and significant reductions in Jms of FMLP compared
with tissues treated with indomethacin alone. However, genistein had no
effect in the absence of PGE2 (Fig.
7A). Although it was initially
thought that FMLP traversed the epithelium solely by the paracellular route (35), more recent studies indicate that FMLP may be transported across the epithelium by the hPepT1 transporter (27). Therefore, we
chose to further assess the effects of PGE2 and genistein
on paracellular permeability by performing Jms of
[3H]mannitol (Fig. 7B). Mannitol fluxes
showed similar trends to those of FMLP fluxes, but significant
reductions in mannitol flux were only noted in tissues treated with
both genistein and PGE2 compared with tissues treated with
indomethacin alone.
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To further assess the effect of PGE2 and genistein on
paracellular permeability, we calculated permeability from flux data for both Na+ and mannitol using standard equations (see
MATERIALS AND METHODS). We used Jsm of
Na+ to assess permeability to passive movement of
Na+ (rather than active transport).
Ischemia-injured tissues treated with indomethacin were
significantly more permeable to Na+
(PNa = 2.7 ± 0.07 × 105 cm/s) than mannitol
(Pmannitol = 1.4 ± 0.1 × 10
5 cm/s, P < 0.001 vs.
PNa), as might be expected on the basis of molecular size. Treatment with PGE2 and genistein resulted
in significant reductions in PNa (2.0 ± 0.2 × 10
5 cm/s, P < 0.01 vs.
PNa in tissues treated with indomethacin alone) and
Pmannitol (1.0 ± 0.1 × 10
5 cm/s, P < 0.05 vs.
Pmannitol in tissues treated with indomethacin alone). The degree of reduction in PNa (~26%)
was similar to that of mannitol (~29%).
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DISCUSSION |
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The present studies indicate that PGE2, but not genistein alone, enhances recovery of barrier function in ischemia-injured porcine mucosa. However, the reparative action of PGE2 is heightened in the presence of genistein, as determined by synergistic elevations in transmucosal resistance and additive reductions in Jms of FMLP and mannitol. In previous studies we have shown that PGE2 stimulates recovery of barrier function by triggering closure of interepithelial spaces (8), a process that would be expected to increase paracellular but not transcellular resistance. We believe a similar process is responsible for amplified recovery of R in the present studies because PGE2 and genistein had no effect on epithelial restitution but resulted in closure of dilated interepithelial spaces evident in tissues treated with indomethacin alone. In addition, PGE2 and PGE2 in combination with genistein incrementally reduced Jms of FMLP, a molecule that at least in part traverses the epithelium via the paracellular pathway (35), and Jms of mannitol, a molecule that exclusively traverses the epithelium via the paracellular route (24). Pmannitol was reduced by 29% by treatment with PGE2 and genistein, which correlated well with a 26% reduction in PNa after the same treatment. In previous studies, significant correlations between Jsm of Na+ and Jms of mannitol (which were used in the present studies to calculate permeability) were indicative of selective remodeling of interepithelial tight junctions (24). Reductions in mannitol fluxes (Fig. 7) were not as dramatic as the corresponding elevations in transepithelial resistance (Fig. 1) in tissues treated with PGE2 and genistein. However, mannitol fluxes were begun at 60 min and continued until 120 min recovery time, whereas R peaked at 60 min but was declining at 120 min. Because peak R was transient, it was not possible to directly correlate mannitol fluxes with maximal changes in R in the presence of PGE2 and genistein.
The predominant cellular activity assigned to genistein is that of a tyrosine kinase inhibitor (37), and it has recently been determined that tight junction regulatory proteins are acted on by tyrosine kinases (1). Accordingly, one plausible theory regarding the mechanism by which genistein augmented recovery of R in the present study is inhibition of tight junction-associated tyrosine kinases. For example, treatment of Madin-Darby canine kidney cells with vanadate and H2O2 resulted in rapid increases in paracellular permeability associated with increased phosphotyrosine immunofluorescence in tight junction-associated proteins. The effect of vanadate/H2O2 was inhibited by genistein or tyrphostin 25, both of which inhibited tyrosine phosphorylation (13). Conversely, the tyrosine kinase inhibitors tyrphostin 47 and herbimycin A had no effect on recovery of R in ischemia-injured porcine tissues, suggesting that genistein's action on recovery of barrier function is not attributable to inhibition of tyrosine kinases. However, we did not directly assess the action of genistein on tyrosine kinases in this study.
Another action that has been attributed to genistein is that of a phosphodiesterase inhibitor (30, 40). However, the action of genistein differed from that of theophylline, a known phosphodiesterase inhibitor in porcine intestinal epithelium (3, 6, 7), in that theophylline triggered recovery of R by itself, whereas genistein had no noticeable effect on R by itself. Although both genistein and theophylline caused synergistic elevations in R, measurements of cAMP levels in tissues treated with PGE2 and genistein revealed no additive action of genistein on cAMP levels, indicating an effect of genistein distinct from inhibition of phosphodiesterase.
The synergistic response between PGE2 and genistein on
recovery of barrier function was preceded by synergistic elevations in
Isc and net Cl secretion,
consistent with our previous work that has shown that changes in
epithelial Cl
transport trigger recovery of barrier
function (8). However, the kinetic relationship between changes in
Cl
secretion (as indicated by
Isc) and changes in R in the present studies were not direct. For instance, Fig. 1 demonstrates marked increases in Isc immediately after addition of the
treatment, whereas R is maximal ~1 h later. This might
suggest that the physical structures signaled by changes in
Isc and responsible for elevations in R are
relatively slow to respond. Such a hypothesis is supported by studies
in Necturus gallbladder, wherein addition of cAMP triggered maximal elevations in Isc within 5 min, whereas
R was maximal by 30 min associated with changes in
ultrastructural morphology of tight junctions (16). Additional
disparities in Isc and R elevations in the
present study included the fact that the magnitude of
Isc did not correlate with the magnitude of
elevations in R in tissues treated with PGE2 and
theophylline, whereas elevations in Isc and
R were closely correlated in tissues treated with
PGE2 and genistein. This likely relates to differences in
mechanisms of action between theophylline and genistein. For example,
we have previously shown that a significant proportion of
PGE2-stimulated elevations in R are associated with
inhibition of electroneutral Na+-Cl
absorption, a process that would not reflect in elevations in Isc (8). Theophylline would be expected to amplify
effects of PGE2 via the same signaling pathways because
theopylline increases intracellular cAMP, the second messenger
responsible for inhibition of Na+-Cl
absorption (4). Conversely, genistein had no demonstrable effects on
cAMP levels in the present study but appeared to have direct effects on
Isc and R in the presence of
PGE2. The mechanism of genistein's action on
Isc has not been conclusively determined (15, 18,
19, 22, 31, 37), although a recent study indicated that genistein
augmented Isc via a direct interaction with
activated CFTR (17).
The relative contribution of Cl secretion and
inhibition of Na+-Cl
absorption is
highlighted by experiments using bumetanide, which only partially
inhibited PGE2-stimulated elevations in R despite complete inhibition of Isc (Fig. 1C). The
bumetanide-insensitive component of PGE2-stimulated
R recovery has been shown in previous studies to be associated
with inhibition of neutral Na+-Cl
absorption by the PGE2 second messenger cAMP (8). However, the effect of bumetanide on tissues treated with both PGE2
and genistein was puzzling, since the
Na+-K+-2Cl
inhibitor fully
inhibited elevations in R in these tissues, suggesting the lack
of an important role for PGE2 inhibition of
Na+-Cl
absorption in the presence of
genistein. This may be explained by a recent study that indicates that
neutral Na+-Cl
absorption is intimately
associated with CFTR function (12), a function that may have been
altered by genistein. We speculate that treatment with both
PGE2 and genistein results in failure of PGE2
to inhibit Na+-Cl
absorption. Another
puzzling aspect of the effects of bumetanide on tissues treated with
PGE2 and genistein was the incomplete inhibition of
Isc. This could not be explained by
Cl
secretion, which is inhibited by bumetanide, or
changes in neutral Na+-Cl
absorption,
which contribute no net change in Isc. However, it is conceivable that tissues treated with PGE2 and genistein
secreted HCO
3, which would not be
inhibited by bumetanide. Electrogenic
HCO
3 secretion has previously been demonstrated in porcine ileum (2).
In addition to net Cl secretion in the presence of
PGE2 and genistein, there was also evidence for net
Na+ secretion (Table 1). Previous studies in
porcine jejunum (32, 33) and rabbit ileum (28) have shown a role for
the cAMP-signaling pathway in the stimulation of net Na+
secretion. Similarly in our studies, net Na+ secretion was
noted primarily in response to the cAMP agonist PGE2
(addition of genistein resulted in no significant additive effect on
net Na+ secretion). Na+ secretion in rabbit
ileum and porcine jejunum is dependent on the presence of
HCO
3 but not Cl
;
therefore, a Na+-HCO
3
electrogenic transport mechanism has been postulated (28). Whether or
not this transport mechanism exists in porcine ileum is not known (33,
34). However, net Na+ secretion is consistently present in
porcine jejunum under a variety of conditions, including rotavirus
infection (33) and porcine cryptosporidiosis (4).
In further studies, we showed that genistein increases
Isc and enhances recovery of R in the
presence of cGMP or cAMP-mediated agonists (including 8-bromo-cAMP and
VIP), but there was no evidence of enhanced recovery of R in
the presence of mediators that signal via protein kinase C (PMA) or
intracellular Ca2+ (carbachol). Recent studies
suggest that genistein has a direct action on CFTR by interacting with
select nucleotide-binding sites. However, phosphorylation of CFTR by
cyclic nucleotides is necessary for genistein to hyperactivate these
Cl channels (17). This may explain why genistein had
little or no effect in the presence of carbachol, an intracellular
Ca2+ agonist that mediates its action on
Cl
secretion via basolateral K+ channels
(41), or PMA, which may phosphorylate CFTR but at a distinct site from
that of the cyclic nucleotides (14).
The mechanisms by which genistein-augmented Cl
secretion in the presence of either cAMP or cGMP agonists results in
enhanced recovery of barrier function are unclear. Assuming that this
reparative process is a paracellular phenomenon (based on the
morphological appearance of the interepithelial spaces and a lack of an
effect on epithelial restitution), there are two general possibilities: physical collapse of interepithelial spaces as ion-rich fluid is drawn
out of the paracellular space or a transmucosal osmotic gradient
developed during apical secretion of Cl
that signals
closure of tight junctions (8). Madara (23) has previously concluded
that a transmucosal osmotic gradient results in rapid alterations in
tight junction structure, and we have also shown that placing an
osmotic load on the mucosal surface of injured tissues simulates the
action of Cl
secretagogues by inducing marked
increases in R (8). However, other investigators have induced
elevations in R in the presence of Cl
secretagogues and ascribed this action to collapse of lateral interepithelial spaces (20). Our previous finding that the cytoskeletal contractile agent cytochalasin D inhibits recovery of transmucosal resistance in the presence of PGE2 and PGI2
suggests that ischemia-injured tissues do not repair in the
presence of open tight junctions (9). However, we have documented
distinct morphological differences in the appearance of the
interepithelial spaces in tissues treated with indomethacin,
PGE2, and genistein compared with those treated with
indomethacin alone. Therefore, we have concluded that closure of both
tight junctions and interepithelial spaces likely contributes to
recovery of mucosal barrier function.
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ACKNOWLEDGEMENTS |
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We thank Julia Vorobiov and the Immunotechnologies Core of the University of North Carolina Center for Gastrointestinal Biology and Disease (National Institutes of Health Center Grant DK-34987) for assistance with cAMP assays.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grant DK-53284 and United States Department of Agriculture Grant 9802537 (both to A. T. Blikslager).
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 and other correspondence: A. T. Blikslager, College of Veterinary Medicine, North Carolina State Univ., 4700 Hillsborough St., Raleigh, NC 27606 (E-mail: anthony_blikslager{at}ncsu.edu).
Received 2 April 1999; accepted in final form 7 October 1999.
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REFERENCES |
---|
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---|
1.
Anderson, J. M.,
and
C. M. Van Itallie.
Tight junctions and the molecular basis for regulation of paracellular permeability.
Am. J. Physiol. Gastrointest. Liver Physiol.
269:
G467-G475,
1995
2.
Argenzio, R. A.
Peptide YY inhibits intestinal Cl secretion in experimental porcine cryptosporidiosis through a prostaglandin-activated neural pathway.
J. Pharmacol. Exp. Ther.
283:
692-697,
1997
3.
Argenzio, R. A.,
and
D. Lebo.
Ion transport by the pig colon: effects of theophylline and dietary sodium restriction.
Can. J. Physiol. Pharmacol.
60:
929-935,
1982[ISI][Medline].
4.
Argenzio, R. A.,
J. Lecce,
and
D. W. Powell.
Prostanoids inhibit intestinal NaCl absorption in experimental porcine cryptosporidiosis.
Gastroenterology
104:
440-447,
1993[ISI][Medline].
5.
Argenzio, R. A.,
and
J. A. Liacos.
Endogenous prostanoids control ion transport across neonatal porcine ileum in vitro.
Am. J. Vet. Res.
51:
747-751,
1990[ISI][Medline].
6.
Argenzio, R. A.,
J. Liacos,
H. M. Berschneider,
S. C. Whipp,
and
D. C. Robertson.
Effect of heat-stable enterotoxin of Escherichia coli and theophylline on ion transport in porcine small intestine.
Can. J. Comp. Med.
48:
14-22,
1984[ISI][Medline].
7.
Argenzio, R. A.,
and
S. C. Whipp.
Effect of theophylline and heat-stable enterotoxin of Escherichia coli on transcellular and paracellular ion movement across isolated porcine colon.
Can. J. Physiol. Pharmacol.
61:
1138-1148,
1983[ISI][Medline].
8.
Blikslager, A. T.,
M. C. Roberts,
and
R. A. Argenzio.
Prostaglandin-induced recovery of barrier function in porcine ileum is triggered by chloride secretion.
Am. J. Physiol. Gastrointest. Liver Physiol.
276:
G28-G36,
1999
9.
Blikslager, A. T.,
M. C. Roberts,
J. M. Rhoads,
and
R. A. Argenzio.
Prostaglandins I2 and E2 have a synergistic role in rescuing epithelial barrier function in porcine ileum.
J. Clin. Invest.
100:
1928-1933,
1997
10.
Cai, Q.,
and
H. Wei.
Effect of dietary genistein on antioxidant enzyme activities in SENCAR mice.
Nutr. Cancer
25:
1-7,
1996[ISI][Medline].
11.
Cartwright, C. A.,
J. A. McRoberts,
K. G. Mandel,
and
K. Dharmsathaphorn.
Synergistic action of cyclic adenosine monophosphate- and calcium-mediated chloride secretion in a colonic epithelial cell line.
J. Clin. Invest.
76:
1837-1842,
1985[ISI][Medline].
12.
Clarke, L. L.,
and
M. C. Harline.
CFTR is required for cAMP inhibition of intestinal Na+ absorption in a cystic fibrosis mouse model.
Am. J. Physiol. Gastrointest. Liver Physiol.
270:
G259-G267,
1996
13.
Collares-Buzato, C. B.,
M. A. Jepson,
N. L. Simmons,
and
B. H. Hirst.
Increased tyrosine phosphorylation causes redistribution of adherens junction and tight junction proteins and perturbs paracellular barrier function in MDCK epithelia.
Eur. J. Cell Biol.
76:
85-92,
1998[ISI][Medline].
14.
Dechecchi, M. C.,
A. Tamanini,
G. Berton,
and
G. Cabrini.
Protein kinase C activates chloride conductance in C127 cells stably expressing the cystic fibrosis gene.
J. Biol. Chem.
268:
11321-11325,
1993
15.
Diener, M.,
and
F. Hug.
Modulation of Cl secretion in rat distal colon by genistein, a protein tyrosine kinase inhibitor.
Eur. J. Pharmacol.
299:
161-170,
1996[ISI][Medline].
16.
Duffey, M. E.,
B. Hainau,
S. Ho,
and
C. J. Bentzel.
Regulation of epithelial tight junction permeability by cyclic AMP.
Nature
294:
451-453,
1981[ISI][Medline].
17.
French, P. J.,
J. Bijman,
A. G. Bot,
W. E. Boomaars,
B. J. Scholte,
and
H. R. de Jonge.
Genistein activates CFTR Cl channels via a tyrosine kinase- and protein phosphatase-independent mechanism.
Am. J. Physiol. Cell Physiol.
273:
C747-C753,
1997
18.
Illek, B.,
H. Fischer,
and
T. E. Machen.
Alternate stimulation of apical CFTR by genistein in epithelia.
Am. J. Physiol. Cell Physiol.
270:
C265-C275,
1996
19.
Illek, B.,
H. Fischer,
G. F. Santos,
J. H. Widdicombe,
T. E. Machen,
and
W. W. Reenstra.
cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein.
Am. J. Physiol. Cell Physiol.
268:
C886-C893,
1995
20.
Kottra, G.,
W. Haase,
and
E. Fromter.
Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. I. Microscopic and general electrophysiological observations.
Pflugers Arch.
425:
528-534,
1993[ISI][Medline].
21.
Kuo, S. M.
Antiproliferative potency of structurally distinct dietary flavonoids on human colon cancer cells.
Cancer Lett.
110:
41-48,
1996[ISI][Medline].
22.
Lehrich, R. W.,
and
J. N. J. Forrest.
Tyrosine phosphorylation is a novel pathway for regulation of chloride secretion in shark rectal gland.
Am. J. Physiol. Renal Fluid Electrolyte Physiol.
269:
F594-F600,
1995
23.
Madara, J. L.
Increases in guinea pig small intestinal transepithelial resistance induced by osmotic loads are accompanied by rapid alterations in absorptive-cell tight-junction structure.
J. Cell Biol.
97:
125-136,
1983[Abstract].
24.
Madara, J. L.
Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity.
J. Cell Biol.
102:
2125-2136,
1986[Abstract].
25.
Madara, J. L.
Loosening tight junctions. Lessons from the intestine.
J. Clin. Invest.
83:
1089-1094,
1989[ISI][Medline].
26.
Madara, J. L.
Warner-Lambert/Parke-Davis Award lecture. Pathobiology of the intestinal epithelial barrier.
Am. J. Pathol.
137:
1273-1281,
1990[Abstract].
27.
Merlin, D.,
A. Steel,
A. T. Gewirtz,
M. Si-Tahar,
M. A. Hediger,
and
J. L. Madara.
hPepT1-mediated epithelial transport of bacteria-derived chemotactic peptides enhances neutrophil-epithelial interactions.
J. Clin. Invest.
102:
2011-2018,
1998
28.
Minhas, B. S.,
S. K. Sullivan,
and
M. Field.
Bicarbonate secretion in rabbit ileum: electrogenicity, ion dependence, and effects of cyclic nucleotides.
Gastroenterology
105:
1617-1629,
1993[ISI][Medline].
29.
Mullin, J. M.,
K. V. Laughlin,
C. W. Marano,
L. M. Russo,
and
A. P. Soler.
Modulation of tumor necrosis factor-induced increase in renal (LLC-PK1) transepithelial permeability.
Am. J. Physiol. Renal Fluid Electrolyte Physiol.
263:
F915-F924,
1992
30.
Ogiwara, T.,
C. L. Chik,
and
A. K. Ho.
Tyrosine kinase inhibitors enhance GHRH-stimulated cAMP accumulation and GH release in rat anterior pituitary cells.
J. Endocrinol.
152:
193-199,
1997[Abstract].
31.
Reenstra, W. W.,
K. Yurko-Mauro,
A. Dam,
S. Raman,
and
S. Shorten.
CFTR chloride channel activation by genistein: the role of serine/threonine protein phosphatases.
Am. J. Physiol. Cell Physiol.
271:
C650-C657,
1996
32.
Rhoads, J. M.,
E. O. Keku,
L. E. Bennett,
J. Quinn,
and
J. G. Lecce.
Development of L-glutamine-stimulated electroneutral sodium absorption in piglet jejunum.
Am. J. Physiol. Gastrointest. Liver Physiol.
259:
G99-G107,
1990
33.
Rhoads, J. M.,
E. O. Keku,
J. Quinn,
J. Woosely,
and
J. G. Lecce.
L-Glutamine stimulates jejunal sodium and chloride absorption in pig rotavirus enteritis.
Gastroenterology
100:
683-691,
1991[ISI][Medline].
34.
Rhoads, J. M.,
E. O. Keku,
J. P. Woodard,
S. I. Bangdiwala,
J. G. Lecce,
and
J. T. Gatzy.
L-Glutamine with D-glucose stimulates oxidative metabolism and NaCl absorption in piglet jejunum.
Am. J. Physiol. Gastrointest. Liver Physiol.
263:
G960-G966,
1992
35.
Riehl, T. E.,
and
W. F. Stenson.
Mechanisms of transit of lipid mediators of inflammation and bacterial peptides across intestinal epithelia.
Am. J. Physiol. Gastrointest. Liver Physiol.
267:
G687-G695,
1994
36.
Schmitz, H.,
M. Fromm,
C. J. Bentzel,
P. Scholz,
K. Detjen,
J. Mankertz,
H. Bode,
H. J. Epple,
E. O. Riecken,
and
J. D. Schulzke.
Tumor necrosis factor-&agr regulates the epithelial barrier in the human intestinal cell line HT-29/B6
J. Cell Sci.
112:
137-146,
1998
37.
Sears, C. L.,
F. Firoozmand,
A. Mellander,
F. G. Chambers,
I. G. Eromar,
A. G. Bot,
B. Scholte,
H. R. de Jonge,
and
M. Donowitz.
Genistein and tyrphostin 47 stimulate CFTR-mediated Cl secretion in T84 cell monolayers.
Am. J. Physiol. Gastrointest. Liver Physiol.
269:
G874-G882,
1995
38.
Sfakianos, J.,
L. Coward,
M. Kirk,
and
S. Barnes.
Intestinal uptake and biliary excretion of the isoflavone genistein in rats.
J. Nutr.
127:
1260-1268,
1997
39.
Stoney, R. J.,
and
C. G. Cunningham.
Acute mesenteric ischemia.
Surgery
114:
489-490,
1993[ISI][Medline].
40.
Stringfield, T. M.,
and
B. H. Morimoto.
Modulation of cyclic AMP levels in a clonal neural cell line by inhibitors of tyrosine phosphorylation.
Biochem. Pharmacol.
53:
1271-1278,
1997[ISI][Medline].
41.
Vajanaphanich, M.,
C. Schultz,
R. Y. Tsien,
A. E. Traynor-Kaplan,
S. J. Pandol,
and
K. E. Barrett.
Cross-talk between calcium and cAMP-dependent intracellular signaling pathways. Implications for synergistic secretion in T84 colonic epithelial cells and rat pancreatic acinar cells.
J. Clin. Invest.
96:
386-393,
1995[ISI][Medline].
42.
Wei, H.,
Q. Cai,
and
R. O. Rahn.
Inhibition of UV light- and Fenton reaction-induced oxidative DNA damage by the soybean isoflavone genistein.
Carcinogenesis
17:
73-77,
1996[Abstract].