PI3K signaling is required for prostaglandin-induced
mucosal recovery in ischemia-injured porcine
ileum
Dianne
Little1,
Rebecca A.
Dean1,
Karen M.
Young1,
Shaun A.
McKane2,
Linda D.
Martin2,
Samuel L.
Jones1, and
Anthony T.
Blikslager1
Departments of 1 Clinical Sciences and
2 Molecular Biomedical Sciences, North Carolina
State University, Raleigh, North Carolina 27606
 |
ABSTRACT |
We have previously shown
that PGE2 and PGI2 induce recovery of
transepithelial resistance (TER) in ischemia-injured porcine ileal mucosa, associated with initial increases in Cl
secretion. We believe that the latter generates an osmotic gradient that stimulates resealing of tight junctions. Because of evidence implicating phosphatidylinositol 3-kinase (PI3K) in regulating tight
junction assembly, we postulated that this signaling pathway is
involved in PG-induced mucosal recovery. Porcine ileum was subjected to
45 min of ischemia, after which TER was monitored for a 180-min
recovery period. Endogenous PG production was inhibited with
indomethacin (5 µM). PGE2 (1 µM) and PGI2
(1 µM) stimulated recovery of TER, which was inhibited by serosal
application of the osmotic agent urea (300 mosmol/kgH2O).
The PI3K inhibitor wortmannin (10 nM) blocked recovery of TER in
response to PGs or mucosal urea. Immunofluorescence imaging of
recovering epithelium revealed that PGs restored occludin and zonula
occludens-1 distribution to interepithelial junctions, and this pattern
was disrupted by pretreatment with wortmannin. These experiments
suggest that PGs stimulate recovery of paracellular resistance via a
mechanism involving transepithelial osmotic gradients and
PI3K-dependent restoration of tight junction protein distribution.
phosphatidylinositol 3-kinase; tight junction; occludin; zonula occludens-1
 |
INTRODUCTION |
RESTORATION OF
THE INTESTINAL mucosal barrier following a variety of injurious
or inflammatory events is a critical component of innate mucosal
defense (25). In previous studies, we have begun to
elucidate the pathways by which the prostanoids PGE2 and
PGI2 stimulate recovery of barrier function in porcine
ischemia-injured ileal mucosa. In particular, we have noted
that this recovery process appears to be related to events localized to
the paracellular space (4-6) rather than reparative
events such as epithelial restitution and villous contraction. However,
studies by other groups have shown that PGE2 is permissive
for growth factor-stimulated restitution (29) and
PGE2 stimulates contraction of uninjured villi and crypts
(9), suggesting that the reparative actions of PGs are
multiple and complex. In porcine ileal mucosa subjected to 45 min of
ischemia, villous contraction and epithelial restitution are
nearly complete within 60 min of injury, and yet PGs are able to
stimulate continued elevations in transepithelial resistance (TER)
after 60 min. These elevations in TER are correlated with decreased
transmucosal flux of the paracellular probes mannitol and inulin and
electron microscopic evidence of closure of paracellular spaces in
restituted epithelium (4, 6). Furthermore, PG-induced elevations in TER are inhibited by cytochalasin D (5), an
agent that initiates cytoskeletal contraction and opening of tight
junctions at the appropriate dosages (14).
The mechanisms by which PGs stimulate closure of paracellular spaces
are not fully characterized, although we know that sharp elevations in
Cl
secretion precede recovery and that inhibition of
Cl
secretion with the loop diuretic bumetanide attenuates
mucosal recovery (4). The role of Cl
secretion in recovery of paracellular resistance is unclear, although
it is conceivable that this event results in a transmucosal osmotic
gradient. Indeed, mucosal osmotic loads have been shown to stimulate
elevations in TER in normal guinea pig ileum (12) and
recovery of TER in ischemia-injured porcine ileal mucosa
(4). We have speculated that initial repair of tight
junctions would have to precede their subsequent closure and recovery
of TER (6).
PG signaling mechanisms that might result in tight junction repair
include their second messengers cAMP and Ca2+
(5), both of which have been shown to alter tight junction structure in Necturus gallbladder (7, 24).
Additional signaling intermediates that we have investigated are
tyrosine kinases and protein kinase C (6). Although
genistein augmented PG-induced mucosal recovery, this did not appear to
relate to its ability to inhibit tyrosine kinases, and inhibition of
protein kinase C had no effect on PG-stimulated mucosal recovery
(6). However, recent evidence suggests that
phosphatidylinositol 3-kinase (PI3K) is intimately involved in
regulation of tight junction assembly (27) and
preferentially binds to specific regions of the transmembrane protein
occludin via its p85 regulatory subunit (20). Therefore, in the present study, we sought to provide further evidence for a
selective action of PGs on recovery of paracellular resistance and to
determine if PI3K plays a role in this reparative process. Our data
show that inhibition of PI3K completely inhibits the action of PGs,
which is correlated with inhibition of the ability of PGs to restore
localization of the tight junction integral membrane protein occludin
and the cytoplasmic plaque protein zonula occludens-1 (ZO-1) to
interepithelial junctions.
 |
MATERIALS AND METHODS |
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 · kg
1 · h
1).
Pigs were placed on heating pads and ventilated with 100%
O2 via a tracheotomy by 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. Loops were randomly designated as control or
ischemic loops. The latter were 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 (2). 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), circulated in water-jacketed
reservoirs, and maintained at 37°C. The spontaneous potential
difference (PD) was measured by using Ringer-agar bridges connected to
calomel electrodes, and the PD was short-circuited through Ag-AgCl
electrodes by 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 180 min.
Experimental treatments.
Tissues were bathed in Ringer containing 5 µM indomethacin to prevent
PG production while stripping mucosa from the seromuscular tissues, and
indomethacin was added to the serosal and mucosal bathing solutions in
the same concentration before mounting tissues on Ussing chambers.
Other treatments that were added to the serosal and mucosal bathing
solutions before baseline electrical measurements were the PI3K
inhibitors wortmannin (10 nM) and LY-294002 (10 µM). Baseline
electrical readings were taken for 30 min, after which 1 µM
16,16-dimethyl-PGE2 (Sigma Chemical, St. Louis, MO) and 1 µM carbacyclin (the stable analog of PGI2) were added to the serosal bathing solution. In studies assessing the role of osmotic
gradients, 100-300 mosmol/kgH2O urea was added to
either the mucosal or serosal side of tissues. In studies in which
hydrostatic pressures were applied, the volume of fluid was
incrementally increased on the serosal side of tissues by 2-6 ml.
Isotopic mannitol and Na+ flux
studies.
All fluxes were conducted under short-circuit conditions (tissues
clamped to 0 mV). Dual transmucosal mannitol and Na+ fluxes
were performed on tissues paired according to their initial conductance
readings (within 25% of each other). [3H]mannitol (0.2 µCi/ml diluted in 10 mM mannitol) or [14C]inulin was
placed on the mucosal side of tissues and 0.3 µCi/ml 22Na
was placed on the serosal side of tissues following an initial 30-min
equilibration period. One 60-min flux was subsequently conducted from
60 to 120 min of the experimental recovery period by taking samples
from the side opposite to that of isotope addition and counted for
3H or 22Na in a scintillation counter.
Mucosal-to-serosal fluxes (Jms) of mannitol or
inulin and serosal-to-mucosal fluxes (Jsm) of
Na+ were calculated by using standard equations (1,
2).
Electron and light microscopy.
Tissues were taken at 0, 30, 60, 120, and 180 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 height of the villus and the width at the midpoint of the villus
were obtained by using a light microscope with an ocular micrometer.
For height measurements, the base of the villus was defined as the
intersection between adjacent villi at the opening of the crypt. For
villi in which the height of one side of the villus was disparate from
the other side, an average height was recorded. In addition, the height
of the epithelial-covered portion of each villus was measured. The
surface area of the villus was calculated by 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
(1). 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
percentage of denuded villous surface area was used as an index of
epithelial restitution.
In experiments designed to assess epithelial ultrastructure under the
influence of PGs, tissues were removed from Ussing chambers after 120 min (peak TER) during three separate experiments. Tissues were placed
in Trump's 4F:1G fixative and prepared for transmission electron
microscopy by using standard techniques (8). For each tissue evaluated, five well-oriented interepithelial junctions were
evaluated. A calibrated grid was placed over electron micrographs extending from the apical-most aspect of the interepithelial space to 3 µm deep to the apical membrane and 1.5 µm from either side of the
apical interepithelial space, so that the entire grid encompassed 9 µm2. The number of squares that were occupied by
paracellular space within this 9-µm2 grid was used to
calculate the area of the paracellular space.
Epithelial isolation.
Tissues were rinsed with 30 ml of cold CO2-saturated PBS
and subsequently dropped into a tube containing
CO2-saturated citrate phosphate buffer (in mM: 96 NaCl, 1.5 KCl, 27.0 Na citrate, 5.6 KH2PO4, and 8.0 Na2HPO4). The tube was capped immediately and incubated at 37°C in a water bath for 20 min. The tissue was then transferred to a tube containing CO2-EDTA buffer (in mM:
137 NaCl, 2.7 KCl, 1.5 KH2PO4, 8.0 Na2HPO4, 1.5 tetrasodium EDTA, and 2.5 glucose)
and incubated at 37°C in a water bath for 30 min. Tissues were
vortexed, after which a histological sample was submitted to check for
the degree of epithelial sloughing. Tissues were subsequently
centrifuged at 2,000 rpm for 10 min, and the pellet was solubilized in
EDTA buffer in preparation for Western blotting.
Gel electrophoresis and Western blotting.
Isolated epithelium from control and ischemia-injured mucosa
treated with indomethacin (5 µM), indomethacin (5 µM) and PGs (1 µM), or indomethacin (5 µM), PGs (1 µM), and wortmannin (10 nM)
and recovered for 120 min in oxygenated Ringer was snap frozen and
stored at
70°C before SDS-PAGE. Tissue aliquots were thawed at
4°C and added to 3 ml chilled lysis buffer, including protease inhibitors (0.5 mM Pefabloc, 0.1 mM 4-nitrophenyl phosphate, 0.04 mM
-glycerophosphate, 0.1 mM Na3VO4, 40 µg/ml
bestatin, 2 µg/ml aprotinin, 0.54 µg/ml leupeptin, and 0.7 µg/ml
pepstatin A) at 4°C. This mixture was homogenized on ice and then
centrifuged at 4°C, and the supernatant was saved. Protein analysis
of extract aliquots was performed (DC protein assay; Bio-Rad, Hercules,
CA). Tissue extracts (amounts equalized by protein concentration) were mixed with an equal volume of 2× SDS-PAGE sample buffer and boiled for
4 min. Lysates were loaded on a 10% SDS-polyacrylamide gel, and
electrophoresis was carried out according to standard protocols. Proteins were transferred to a nitrocellulose membrane (Hybond ECL;
Amersham Life Science, Birmingham, UK) by using an electroblotting minitransfer apparatus. Membranes were blocked at room temperature for
60 min in Tris-buffered saline plus 0.05% Tween 20 and 5% dry
powdered milk. Membranes were washed and then incubated for 60 min in
primary antibody. After being washed, the membranes were incubated for
45 min with horseradish peroxidase-conjugated secondary antibody. After
additional washes, the membranes were developed for visualization of
protein by the addition of enhanced chemiluminescence reagent
(Amersham, Piscataway, NJ). Densitometry was performed by using
appropriate software (IP gel; Scanalytics, Fairfax, VA).
Immunofluorescence microscopy.
Tissues were fixed in 10% neutral-buffered formalin for 24 h,
transferred to 70% ethanol, routinely processed for paraffin embedding, and cut into 5-µM sections. Slides were subsequently deparaffinized and rehydrated. Epitope retrieval was done by boiling the specimens in citrate buffer (pH 6.0) for 10 min, then allowing specimens to cool for 25 min at room temperature. Sections were blocked
with 2% BSA and washed with BLOTTO and PBS, after which they were
incubated in primary rabbit polyclonal anti-occludin, primary rabbit
polyclonal anti-ZO-1, or an isotype control for rabbit primary antibody
(negative control) for 1 h on ice. Sections were then incubated
with goat anti-rabbit IgG Cy3 conjugate for 30 min in the dark.
Sections were mounted, and well-oriented villi were examined with an
immunofluorescence microscope.
Data analysis.
Data were reported as means ± SE. All data were analyzed by using
an ANOVA for repeated measures, except where the peak response was
analyzed by using a standard one-way ANOVA or paired t-test (Sigmastat; Jandel Scientific, San Rafael, CA). A Tukey's test was
used to determine differences between treatments following ANOVA. Flux data was subjected to linear regression analysis, and the
correlation coefficient (R) was assessed for significance. P < 0.05 was considered significant for all analyses.
 |
RESULTS |
Application of 1 µM 16,16-dimethyl-PGE2 and 1 µM
carbacyclin (a stable analog of PGI2) to mucosal sheets of
porcine ileum injured by 45 min of ischemia and bathed in 5 µM indomethacin resulted in recovery of control levels of TER within
30 min, whereas ischemia-injured tissues exposed to
indomethacin alone showed minimal elevations of TER over a 180-min
recovery period (Fig. 1A). As
we have shown in previous reports, this PG-induced recovery was
preceded by a sharp elevation in Isc (Fig.
1B) attributable to secretion of Cl
(4). As in previous studies (6), there was no
difference in the histological appearance of repairing tissues treated
with indomethacin compared with those additionally treated with PGs (Fig. 2), which was confirmed by showing
no significant difference in the degree of epithelial restitution
(Table 1). In fact, restitution was
nearly complete within 60 min, suggesting that the peak effects of PGs
between 90 and 120 min were related to events localized to the
paracellular space.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Electrical responses of ischemia-injured tissues
treated with indomethacin (indo, 5 µM) and
16,16-dimethyl-PGE2 and carbacyclin (PGs, 1 µM).
A: serosal addition of PGs to ischemia-injured
tissues after an initial 30-min equilibration period resulted in rapid
recovery of control levels of transepithelial resistance (TER), whereas
tissues treated with indomethacin alone had little evidence of
recovery. B: elevations in TER were preceded by significant
elevations in short-circuit current (Isc), which
is associated with Cl secretion in this tissue. Plotted
values represent means ± SE; n = 8. The
significance of the elevations in TER and Isc in
the presence of PGs was determined by using 2-way ANOVA on repeated
measures (P < 0.05).
|
|

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 2.
Histological appearance of ischemia-injured
porcine ileal mucosa. A: ischemia for 45 min
resulted in lifting and sloughing of epithelium from the tips of villi.
B: after a 60-min recovery period in an Ussing chamber in
the presence of indomethacin (5 µM), villi have contracted and
epithelial restitution is nearly complete. C: tissues
treated with indomethacin and PGs (added after a 30-min equilibration
period and recovered for an additional 30 min) have a similar
histological appearance to tissues treated with indomethacin alone.
Bar = 100 µm.
|
|
To further explore the possibility that PG-induced changes in TER were
paracellular in nature, we measured Jms of the
paracellular probes [3H]mannitol and
[14C]inulin as well as Jsm of
22Na+ between 60 and 120 min of the recovery
period (when PG-treated tissues reached maximum TER values). Flux of
these probes was significantly greater in ischemia-injured
tissues treated with indomethacin alone compared with tissues treated
additionally with PGs (Fig. 3). We then
assessed the correlation between the flux of mannitol or inulin and
that of Na+ as a method of assessing the contribution of
changes in paracellular permeability (accounted for by mannitol or
inulin flux) to changes in TER (accounted for by
Jsm of Na+), as previously described
(15). We first confirmed that Jsm of Na+ closely correlated with changes in TER in tissues
treated with indomethacin or indomethacin/PGs (R = 0.76, P < 0.001, data not shown). We subsequently documented
a significant and linear correlation between fluxes of the paracellular
probes and Jsm of Na+ (Fig.
4), indicating that changes in resistance
were indeed reflective of changes in paracellular permeability.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 3.
Evaluation of the effects of indomethacin and PGs on
serosal-to-mucosal fluxes (Jsm) of
22Na and mucosal-to-serosal fluxes
(Jms) of [3H]mannitol and
[14C]inulin. Fluxes were commenced 30 min after the
addition of PGs and were conducted over a 1-h period. Tissues in the
presence of indomethacin (5 µM) and PGs (1 µM) had significantly
reduced Jsm 22Na over a 1-h time
period compared with tissues treated with indomethacin alone
(A). Similar results were obtained for
Jms of the paracellular probes
[3H]mannitol (B) and [14C]inulin
(C). Plotted values represent means ± SE;
n = 8. *P < 0.05 vs.
indomethacin-treated tissues.
|
|

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 4.
Correlation between Jsm
22Na and Jms
[3H]mannitol or Jms
[14C]inulin in ischemia-injured tissues treated
with indomethacin (5 µM) and PGs (1 µM) or indomethacin alone.
There was a significant correlation between Jsm
22Na and Jms mannitol (A)
or Jms inulin (B), suggesting that
changes in TER (which correlate closely with Jsm
22Na) were related to changes in paracellular permeability
(as indicated by changes in Jms of the
paracellular probes mannitol and inulin). The correlation coefficient
(R) and its significance (P values) are indicated adjacent
to linear regression plots.
|
|
Although the experiments thus far indicated an action of PGs on the
paracellular space, we wanted more direct evidence of the involvement
of the paracellular structures in the recovery response. Therefore, we
performed a series of experiments in which we added increasing levels
of serosal hydrostatic pressure by raising the fluid level of the
serosal reservoir. We postulated that this would dilate paracellular
spaces and apical tight junctions, thereby nullifying the effects of
PGs. Accordingly, there was a pressure-dependent decrease in the
PG-induced recovery of TER, with 6 cm serosal pressure nullifying the
effects of PGs on injured tissues (Fig.
5). This action was not attributable to
disruption of Cl
secretion, because there was no
significant reduction of Isc by 6 cm serosal
pressure. Tissues taken during peak TER levels in response to PGs
showed ultrastructural evidence of closely apposed tight junctions
compared with tissues treated with indomethacin alone. Furthermore,
tissues subjected to 6 cm of serosal pressure in the presence of PGs
also showed dilatation of paracellular structures (Fig.
6). These observations were confirmed
morphometrically by showing pressure-dependent increases in the area of
the paracellular space (Fig. 7). There
was no effect of hydrostatic pressure on normal tissues (data not
shown), suggesting that hydrostatic pressure selectively affected
tissues in the process of recovering paracellular resistance.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of serosal hydrostatic pressure on recovery of
TER. A: recovery response of ischemia-injured
tissues treated with indomethacin (5 µM) and PGs (1 µM) was
marginally reduced by 2-4 cmH2O of serosal pressure,
whereas 6 cmH2O of hydrostatic pressure fully and
significantly inhibited recovery of TER. B: changes in
Isc in response to serosal pressure did not
appear to be correlated with changes in TER. In particular, 6 cmH2O caused a small increase in Isc
but fully inhibited TER, suggesting that the effects of serosal
pressure are related to mechanical effects on the paracellular space
rather than alterations in Cl secretion. Plotted values
represent means ± SE; n = 8. The significant
reduction in TER in the presence of PGs/6 cmH2O compared
with tissues in the presence of PGs alone was determined by using 2-way
ANOVA on repeated measures (P < 0.05).
|
|

View larger version (179K):
[in this window]
[in a new window]
|
Fig. 6.
Electron micrographs of tissues exposed to indomethacin,
PGs, and serosal pressure. A: ischemia-injured
tissues after a 120-min in vitro recovery period in the presence of
indomethacin (5 µM) have dilated tight junctions (arrows) and
paracellular spaces. B: tissues additionally treated with
PGs (1 µM) have closely apposed tight junctions (arrows).
C: application of 6 cmH2O serosal hydrostatic
pressure to tissues treated with both indomethacin and PGs results in
dilatation of tight junctions (arrows) and paracellular spaces.
Bar = 2 µm. D: increased magnification of the
interepithelial junctional region of ischemia-injured tissues
treated with indomethacin for 120 min. Note dilatation of the tight
junction (arrow). E: same high power magnification of the
interepithelial junctional region of ischemia-injured tissues
treated with indomethacin and PGs revealed closely apposed tight
junctions (arrow), whereas tissues exposed to 6 cmH2O
serosal hydrostatic pressure had dilated tight junctions (arrow).
Bar = 1 µm.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 7.
Area of the paracellular space in the region of the
tight junction based on electron microscopic images. The area of the
paracellular space was dramatically reduced in tissues treated with
indomethacin (5 µM) and PGs (1 µM) compared with tissues treated
with indomethacin alone. Application of 2-4 cmH2O
serosal pressure had no significant effect on area of the paracellular
space compared with tissues treated with indomethacin and PGs, but 6 cmH2O hydrostatic pressure caused a significant elevation
in the area of the paracellular space. Plotted values represent
means ± SE; n = 8. *P < 0.05 vs.
ischemia/ indomethacin tissues. P < 0.05 vs. all other treatment groups, as determined by 1-way ANOVA and a
post-hoc Tukey's test.
|
|
In further experiments, we attempted to elucidate some of the
mechanisms involved in PG-induced recovery of paracellular resistance. In previous studies, we have suggested that increases in
Cl
secretion that precede recovery of TER may result in
development of an osmotic gradient across the mucosa (4,
6). To test this hypothesis, we applied increasing doses of urea
on the mucosal surface of ischemia-injured tissues treated with
indomethacin and compared the effects of these treatments with that of
the PGs. Accordingly, we noted dose-dependent increases in recovery of
TER with mucosal application of urea that peaked with application of
200 mosmol/kgH2O (Fig. 8).
Application of other osmotic agents to the mucosal surface of tissues,
including mannitol (300 mosmol/kgH2O) and lactulose (300 mosmol/kgH2O), resulted in similar increases in TER in
ischemia-injured mucosa (peak TER in response to mannitol, 66 ± 4
· cm2,
n = 6; peak TER in response to lactulose, 65 ± 2
· cm2, n = 3). To
demonstrate the importance of the direction of the osmotic gradient, we
applied 300 mosmol/kgH2O urea to the serosal surface of
ischemia-injured tissues and saw a reduction rather than an
increase in recovery of TER. We next reasoned that, if PGs were setting
up a mucosal-to-serosal osmotic gradient, the effect of the PGs should
be reversed with serosal application of urea. In support of this
premise, application of 300 mosmol/kgH2O urea to the
serosal surface of recovering tissues fully inhibited the action of PGs
on injured tissues (Fig. 8).

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 8.
Electrical responses of tissues subjected to
osmotic loads of urea. Dose-dependent increases in TER were noted in
response to 100-200 mosmol/kgH2O urea on the mucosal
surface of tissues, whereas 300 mosmol/kgH2O urea appeared
to have no further effect. Conversely, serosal application of 300 mosmol/kgH2O reduced TER below levels of tissues treated
with indomethacin alone and fully inhibited recovery of tissues in
response to PGs, suggesting that the orientation of osmotic gradients
in recovering tissue is critical. Plotted values represent means ± SE; n = 8. The significant increases in TER in the
presence of 200-300 mosmol/kgH2O mucosal urea and the
significant inhibitory effect of 300 mosmol/kgH2O serosal
urea were determined by using 2-way ANOVA on repeated measures
(P < 0.05).
|
|
In previous studies (6), we have postulated that tight
junction reassembly would be required to initiate recovery of TER. Because of studies (27) implicating PI3K in tight junction
assembly, we were particularly interested in this signaling pathway.
Application of the PI3K inhibitor wortmannin (10 nM) completely
inhibited PG-induced recovery but had no effect on
Isc in PG-treated tissues (Fig.
9). To rule out an effect of wortmannin
on restitution, we calculated the percentage of denuded mucosa during
in vitro recovery, as in our initial experiments. Following a 60-min
recovery period, there was no significant effect of wortmannin on
percentage of denuded mucosa (1.9 ± 1.4%) compared with other
treatment groups (Table 1), suggesting that wortmannin inhibited
paracellular effects of PG addition. However, wortmannin appeared to
reduce the small recovery response of ischemia-injured tissues
treated with indomethacin alone, suggesting the possibility of
nonspecific toxic effects of wortmannin. Therefore, we also assessed
the effects of the alternative PI3K inhibitor LY-294002 (10 µM). This
agent fully inhibited recovery of TER in PG-treated tissues. However, LY-294002 did not fully inhibit the PG-stimulated elevations in Isc, which remained significantly elevated
compared with tissues treated with indomethacin alone (Fig.
10). LY-294002 appeared to have no
effect on TER or Isc measurements when applied
to ischemia-injured tissues treated with indomethacin in the
absence of PGs.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 9.
Electrical responses of tissues to inhibition of
phosphatidylinositol 3-kinase (PI3K). A: pretreatment with
the PI3K inhibitor wortmannin (10 nM) fully inhibited recovery of TER
in tissues treated with indomethacin (5 µM) and PGs (1 µM).
B: Isc in tissues treated with
indomethacin and PGs was no different from Isc
levels in tissues additionally treated with wortmannin, suggesting that
inhibition of TER did not relate to blockade of
Isc. Significant inhibitory effect of wortmannin
on tissues treated with indomethacin and PGs was determined by using
2-way ANOVA on repeated measures (P < 0.05).
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 10.
Electrical responses of tissues to inhibition of PI3K
with LY-290042. A: tissues pretreated with the alternative
PI3K inhibitor LY-290042 (10 µM) had no evidence of recovery of TER
in response to treatment with PGs, whereas LY-290042 had no effect on
tissues treated solely with indomethacin. B:
Isc in tissues pretreated with indomethacin and
PGs was partially inhibited by LY-290042. However, in previous studies
(4), we have shown that complete inhibition of
Isc is required to inhibit recovery of TER,
suggesting that inhibition of recovery of TER by LY-290042 is related
to other mechanisms involving PI3K. Significant inhibitory effect of
LY-290042 on tissues treated with indomethacin and PGs was determined
by using 2-way ANOVA on repeated measures (P < 0.05).
|
|
Since we have postulated that PG-induced Cl
secretion
sets up an osmotic gradient that is in turn responsible for at least part of the recovery of paracellular resistance, we wanted to determine
whether the PI3K inhibitor LY-294002 would also inhibit urea-stimulated
recovery. Therefore, tissues were treated with 200 mosmol/kgH2O urea on their mucosal surface in the presence or absence of LY-294002 (10 µM). The PI3K inhibitor LY-294002 inhibited the effect of urea (Fig. 11),
suggesting that PI3K signaling is required for osmotic load-induced
recovery of TER, similar to that of PG-induced recovery of TER. Similar
results were obtained in tissues pretreated with wortmannin (data not
shown).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 11.
Effect of inhibition of PI3K on urea-induced recovery.
Treatment of ischemia-injured tissues with 200 mosmol/kgH2O urea on the mucosal side stimulated recovery
of TER, as in previous experiments. However, tissues pretreated with
the PI3K inhibitor LY-294002 (10 µM) failed to recover when subjected
to mucosal urea, suggesting an important role for PI3K in osmotic
load-stimulated mucosal recovery.
|
|
In additional experiments assessing PI3K-mediated events, we sought to
further define the role of PGs and PI3K inhibitors on select components
of the tight junction, since it is this structure that is largely
responsible for regulating paracellular permeability (13).
Therefore, we assessed the tissue expression of the tight junction
transmembrane proteins occludin and claudin-5 after 120 min of recovery
in the presence of PGs and wortmannin. We used a technique to isolate
epithelial cells from remaining mucosal elements to be sure that we
were not detecting tight junction proteins from other tissues such as
endothelium. Microscopic studies confirmed complete epithelial
separation from mucosal villi after the isolation procedure (data not
shown). Indomethacin appeared to reduce the expression of claudin-5 in
ischemia-injured mucosal epithelium, but there was no apparent
effect of any of our treatments on occludin expression (Fig.
12). However, in further studies using immunofluorescence microscopy, we noted differences in the distribution of occludin in the various treatment groups (Fig.
13). In particular, we noted
interepithelial localization of occludin labeling in control epithelium
that was disrupted in ischemia-injured tissue bathed in
indomethacin (5 µM) for 120 min. Treatment with PGs (1 µM) appeared
to restore the normal interepithelial junctional distribution of
occludin, whereas pretreatment of tissues with wortmannin (10 nM)
inhibited the ability of PGs to restore occludin distribution. To seek
further evidence of tight junction structural restoration in the
presence of PGs, we also performed immunofluorescence experiments to
assess the localization of the tight junction cytoplasmic plaque
protein ZO-1. These experiments revealed highly selective localization
of ZO-1 to the tight junction in control tissues and
ischemia-injured tissues exposed to PGs (1 µM). In contrast, ischemia-injured tissues recovered in the presence of
indomethacin (5 µM) alone or with wortmannin (10 nM) had evidence of
diffuse staining in the apical region of recovering epithelial cells
(Fig. 14).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 12.
Immunoblots for tight junction proteins. Tissues were
subjected to a 120-min in vitro recovery period, including control
tissue (cont) and ischemia-injured tissues treated with
indomethacin (indo, 5 µM), indomethacin and PGs (1 µM), or
indomethacin, PGs, and wortmannin (wort, 10 nM). Epithelium was
isolated from mucosa before Western blotting. A:
representative Western blot for claudin-5 (a 22-kDa protein located
between markers for 31.6 and 17.8 kDa) showed evidence of reduced
expression of claudin-5 in ischemia-injured epithelium treated
with indomethacin compared with other treatments. B:
occludin is a protein of variable molecular mass depending on the
degree of phosphorylation, typically in the range of 65-75 kDa
(26). There are no apparent differences in expression of
occludin (multiple bands located between markers for 79 and 41 kDa)
between the different treatment groups.
|
|

View larger version (133K):
[in this window]
[in a new window]
|
Fig. 13.
Immunofluorescence microscopic evaluation of
ischemia-injured tissues for occludin. A: normal
mucosa has evidence of accumulation of occludin at the lateral membrane
of cells, particularly toward the apical surface of the epithelium
where interepithelial junctions reside (arrows). B:
ischemia-injured mucosa following a 120-min in vitro recovery
period in the presence of indomethacin (5 µM). Note the disorganized
appearance of occludin fluorescence, with a lack of accumulation of
occludin at the region of the interepithelial junctions. C:
tissues treated with PGs (1 µM) have a pattern of occludin
fluorescence that resembles that of normal tissues (arrows).
D: tissues pretreated with indomethacin and wortmannin (10 nM) and subsequently treated with PGs have poorly organized occludin
fluorescence similar to that of tissues treated with indomethacin
alone. Bar = 5 µm.
|
|

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 14.
Immunofluorescence microscopic evaluation of
ischemia-injured tissues for zonula occludens-1 (ZO-1). Normal
mucosa has ZO-1 exclusively localized to the region of the tight
junction (A), whereas ischemia-injured mucosa
exposed to indomethacin (5 µM) for 120 min has evidence of diffuse
ZO-1 fluorescence at the apical region of recovering epithelial cells
(B). Treatment of ischemia-injured tissues with
indomethacin and PGs (1 µM) for 120 min restores localization of ZO-1
to the tight junctions (C), an effect that is inhibited by
pretreatment of tissues with wortmannin (D). Bar = 5 µm.
|
|
 |
DISCUSSION |
Mechanisms believed to be critical for recovery of injured
epithelium include restitution (18, 21) and, in the case
of small intestinal mucosa, villous contraction (19).
Restitution is a broad term that denotes recovery of an intact
monolayer of epithelium across a previously denuded region of the
mucosa (25). Thus restitution may be broken down into
epithelial migratory events and tight junction resealing events. PGs
have not been extensively linked to villous contraction or epithelial
migration, although there is evidence that PGE2 stimulates
contraction of villi in normal mucosa (9) and that
the cyclooxygenase inhibitor piroxicam suppresses epithelial migration
stimulated by growth factors in cultured intestinal epithelial cells
(29). However, we have not found any evidence for an
effect of PGs on either villous contraction or epithelial migration in
ischemia-injured porcine ileal mucosa. For example, tissues
exposed to 45 min of ischemia have histological evidence of a
complete epithelial monolayer after 60 min of in vitro recovery in
tissues regardless of whether they are treated with indomethacin alone
or indomethacin and exogenous PGs (Fig. 2). Nonetheless, PGs stimulate
significant elevations in TER and reductions in permeability to
mannitol and inulin, leading us to focus on potential effects of PGs on
paracellular structures. The present studies provide further evidence
for an effect of PGs on the paracellular space. For example,
Jsm of Na+ (which reflects changes
in TER) significantly correlated with Jms of the
paracellular probes inulin and mannitol. Furthermore, quantitation of
the dimensions of the junctional region of the paracellular space
revealed significant reductions in the area of this space in response
to PGs, whereas serosal hydrostatic pressure significantly increased
the area of the paracellular space and inhibited the actions of PGs on
recovery of TER.
Since tight junctions largely regulate paracellular permeability, it is
likely that at least a component of the action of PGs is directed at
these structures. Immunofluorescence of occludin and ZO-1 would tend to
support this conclusion, since localization of these tight junction
proteins to the region of the interepithelial junctions was associated
with peak TER in response to PGs. However, it is also possible that PGs
have an effect on the subjunctional paracellular space, the collapse of
which might be responsible for a component of the recovery of TER. The
importance of the proximity of epithelial lateral membranes has
previously been shown to influence measurements of TER (10,
11), and the experiments with serosal pressure support the idea
that dilating the paracellular space reduces the ability of PGs
to stimulate recovery of TER. However, electron micrographs also showed
evidence of dilation of the tight junction in response to serosal
pressure, making it difficult to separate the effects of this maneuver
on the paracellular space and the tight junction. Similarly,
ischemia-injured tissues treated with indomethacin alone had
dilated tight junctions and paracellular spaces, whereas those treated
with PGs had closely apposed tight junctions and paracellular spaces.
However, it is likely that tight junction resealing precedes collapse
of the subjacent paracellular space because the continued presence of a
dilated tight junction would allow extracellular fluid to enter the
paracellular space.
Mechanisms of tight junction resealing following ischemia have
not been fully characterized. First, it is likely that tight junctions
have to reassemble following ischemic injury, since ischemia or associated ATP depletion disrupts tight junction
integrity (16, 17). Reassembly of tight junctions
following events such as ATP depletion involves localization of
integral membrane proteins such as occludin to the apical-most aspect
of the lateral epithelial membrane, along with colocalization of
cytoplasmic proteins such as ZO-1 (28). Similarly, studies
utilizing a calcium switch (chelation and subsequent repletion of
calcium) to disrupt and allow recovery of tight junctions documented
the critical role of integral membrane proteins in orchestrating
reassembly of tight junctions (22). Although we do not
know if these same mechanisms are responsible for PG-stimulated
recovery of TER, we do have evidence on immunofluorescence that PGs
restore the distribution of the tight junction integral membrane
protein occludin and the cytoplasmic plaque protein ZO-1 to the region
of the interepithelial junction. However, there was no difference in
the expression of occludin in response to PG treatment, suggesting that
PGs stimulate movement of preexisting occludin and ZO-1 dispersed
throughout the cell during ischemic injury to the
interepithelial junction during recovery.
In the present study, we were able to use PI3K inhibitors to
functionally separate changes in Isc and TER,
both of which are stimulated by PGs. We know from previous studies that
inhibition of Isc and the associated secretion
of Cl
largely blocks the action of PGs on recovery of TER
(4). However, it now appears that PI3K-mediated events are
critical for recovery of TER despite the continued presence of
elevations in Isc. Inhibitors of PI3K also
blocked recovery of TER in response to mucosal osmotic loads of urea.
Together, this data suggests that PI3K-mediated events are downstream
of mechanisms resulting in a mucosal-to-serosal osmotic gradients,
including mucosal urea and PG-induced Cl
secretion
(proposed model shown in Fig. 15).
However, as an alternate possibility, it is also conceivable that PGs
require both elevations in Isc and intact PI3K
signaling to stimulate recovery of TER. As far as the specific
mechanisms involved in PI3K-sensitive recovery of TER, this will have
to await further study. However, previous studies demonstrating
preferential binding of the p38 regulatory domain of PI3K
to occludin (20) and a role for PI3K in junctional actin
rearrangement (23) suggest potential important functions of this enzyme in tight junction resealing.

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 15.
Proposed model of PG signaling pathways. We have
previously shown that PGE2 interacts with EP2
and EP3 receptors, which would be expected to activate
protein kinase A (PKA) or stimulate increases in intracellular
Ca2+, respectively (3). These second
messengers would then phosphorylate apical Cl and
Na+ channels, in the case of PKA, or result in opening of
basolateral K+ channels, in the case of increased
intracellular Ca2+ levels. The combined effect of these
second messenger-ion channel interactions is secretion of
Cl and blockade of Na+ absorption, which we
believe results in a mucosal-to-serosal osmotic gradient
(4). The effects of PGE2 can be enhanced with
the additional application of PGI2, which appears to
activate cholinergic nerves (5), and genistein, which
directly enhances Cl secretion via the CFTR
(6). As shown in the present studies, the addition of
PGE2 and PGI2 to the serosal surface of
ischemia-injured mucosa or the application of artificial
osmotic gradients to the mucosal surface of tissues stimulates recovery
of TER. These effects are prevented with the application of PI3K
inhibitors, suggesting that this enzyme is downstream of secretory and
osmotic processes. Furthermore, we have shown that PGs restore
interepithelial junctional localization of occludin and ZO-1, which is
inhibited by the PI3K blocker wortmannin, suggesting possible
mechanisms whereby PGs may stimulate tight junction recovery following
ischemic injury.
|
|
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-53284 (to A. T. Blikslager) and United States Department of Agriculture National Research Initiative Grant 0102490 (to A. T. Blikslager and S. L. Jones).
 |
FOOTNOTES |
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).
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.
September 25, 2002;10.1152/ajpgi.00121.2002
Received 27 March 2002; accepted in final form 10 September 2002.
 |
REFERENCES |
1.
Argenzio, RA,
Lecce J,
and
Powell DW.
Prostanoids inhibit intestinal NaCl absorption in experimental porcine cryptosporidiosis.
Gastroenterology
104:
440-447,
1993[ISI][Medline].
2.
Argenzio, RA,
and
Liacos JA.
Endogenous prostanoids control ion transport across neonatal porcine ileum in vitro.
Am J Vet Res
51:
747-751,
1990[ISI][Medline].
3.
Blikslager, AT,
Pell SM,
and
Young KM.
PGE2 triggers recovery of transmucosal resistance via EP receptor cross talk in porcine ischemia-injured ileum.
Am J Physiol Gastrointest Liver Physiol
281:
G375-G381,
2001[Abstract/Free Full Text].
4.
Blikslager, AT,
Roberts MC,
and
Argenzio RA.
Prostaglandin-induced recovery of barrier function in porcine ileum is triggered by chloride secretion.
Am J Physiol Gastrointest Liver Physiol
276:
G28-G36,
1999[Abstract/Free Full Text].
5.
Blikslager, AT,
Roberts MC,
Rhoads JM,
and
Argenzio RA.
Prostaglandins I2 and E2 have a synergistic role in rescuing epithelial barrier function in porcine ileum.
J Clin Invest
100:
1928-1933,
1997[Abstract/Free Full Text].
6.
Blikslager, AT,
Roberts MC,
Young KM,
Rhoads JM,
and
Argenzio RA.
Genistein augments prostaglandin-induced recovery of barrier function in ischemia-injured porcine ileum.
Am J Physiol Gastrointest Liver Physiol
278:
G207-G216,
2000[Abstract/Free Full Text].
7.
Duffey, ME,
Hainau B,
Ho S,
and
Bentzel CJ.
Regulation of epithelial tight junction permeability by cyclic AMP.
Nature
294:
451-453,
1981[ISI][Medline].
8.
Dykstra, MJ.
A manual of applied techniques for biological electron microscopy. New York: Plenum, 1994.
9.
Erickson, RA.
16,16-Dimethyl prostaglandin E2 induces villus contraction in rats without affecting intestinal restitution.
Gastroenterology
99:
708-716,
1990[ISI][Medline].
10.
Kottra, G,
and
Frömter E.
Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. II. Impedance measurements.
Pflügers Arch
425:
535-545,
1993[ISI][Medline].
11.
Kottra, G,
Haase W,
and
Frömter E.
Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. I. Microscopic and general electrophysiological observations.
Pflügers Arch
425:
528-534,
1993[ISI][Medline].
12.
Madara, JL.
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].
13.
Madara, JL.
Pathobiology of the intestinal epithelial barrier.
Am J Pathol
137:
1273-1281,
1990[Abstract].
14.
Madara, JL,
Moore R,
and
Carlson S.
Alteration of intestinal tight junction structure and permeability by cytoskeletal contraction.
Am J Physiol Cell Physiol
253:
C854-C861,
1987[Abstract/Free Full Text].
15.
Madara, JL,
Stafford J,
Barenberg D,
and
Carlson S.
Functional coupling of tight junctions and microfilaments in T84 monolayers.
Am J Physiol Gastrointest Liver Physiol
254:
G416-G423,
1988[Abstract/Free Full Text].
16.
Mandel, LJ,
Bacallao R,
and
Zampighi G.
Uncoupling of the molecular `fence' and paracellular `gate' functions in epithelial tight junctions.
Nature
361:
552-555,
1993[ISI][Medline].
17.
Molitoris, BA,
Falk SA,
and
Dahl RH.
Ischemia-induced loss of epithelial polarity. Role of the tight junction.
J Clin Invest
84:
1334-1339,
1989[ISI][Medline].
18.
Moore, R,
Carlson S,
and
Madara JL.
Rapid barrier restitution in an in vitro model of intestinal epithelial injury.
Lab Invest
60:
237-244,
1989[ISI][Medline].
19.
Moore, R,
Carlson S,
and
Madara JL.
Villus contraction aids repair of intestinal epithelium after injury.
Am J Physiol Gastrointest Liver Physiol
257:
G274-G283,
1989[Abstract/Free Full Text].
20.
Nusrat, A,
Chen JA,
Foley CS,
Liang TW,
Tom J,
Cromwell M,
Quan C,
and
Mrsny RJ.
The coiled-coil domain of occludin can act to organize structural and functional elements of the epithelial tight junction.
J Biol Chem
275:
29816-29822,
2000[Abstract/Free Full Text].
21.
Nusrat, A,
Delp C,
and
Madara JL.
Intestinal epithelial restitution. Characterization of a cell culture model and mapping of cytoskeletal elements in migrating cells.
J Clin Invest
89:
1501-1511,
1992[ISI][Medline].
22.
Nusrat, A,
Giry M,
Turner JR,
Colgan SP,
Parkos CA,
Carnes D,
Lemichez E,
Boquet P,
and
Madara JL.
Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia.
Proc Natl Acad Sci USA
92:
10629-10633,
1995[Abstract].
23.
Nybom, P,
and
Magnusson KE.
Studies with wortmannin and cytochalasins suggest a pivotal role of phosphatidylinositols in the regulation of tight junction integrity.
Biosci Rep
16:
265-272,
1996[ISI][Medline].
24.
Palant, CE,
Duffey ME,
Mookerjee BK,
Ho S,
and
Bentzel CJ.
Ca2+ regulation of tight-junction permeability and structure in Necturus gallbladder.
Am J Physiol Cell Physiol
245:
C203-C212,
1983[Abstract].
25.
Podolsky, DK.
Mucosal Immunity and Inflammation. V. Innate mechanisms of mucosal defense and repair: the best offense is a good defense.
Am J Physiol Gastrointest Liver Physiol
277:
G495-G499,
1999[Abstract/Free Full Text].
26.
Wong, V.
Phosphorylation of occludin correlates with occludin localization and function at the tight junction.
Am J Physiol Cell Physiol
273:
C1859-C1867,
1997[Abstract/Free Full Text].
27.
Woo, PL,
Ching D,
Guan Y,
and
Firestone GL.
Requirement for Ras and phosphatidylinositol 3-kinase signaling uncouples the glucocorticoid-induced junctional organization and transepithelial electrical resistance in mammary tumor cells.
J Biol Chem
274:
32818-32828,
1999[Abstract/Free Full Text].
28.
Ye, J,
Tsukamoto T,
Sun A,
and
Nigam SK.
A role for intracellular calcium in tight junction reassembly after ATP depletion-repletion.
Am J Physiol Renal Physiol
277:
F524-F532,
1999[Abstract/Free Full Text].
29.
Zushi, S.
Role of prostaglandins in intestinal epithelial restitution stimulated by growth factors.
Am J Physiol Gastrointest Liver Physiol
270:
G757-G762,
1996[Abstract/Free Full Text].
Am J Physiol Gastrointest Liver Physiol 284(1):G46-G56
0193-1857/03 $5.00
Copyright © 2003 the American Physiological Society