Departments of 1 Food Animal and Equine Medicine and 2 Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606
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ABSTRACT |
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We have previously shown that
PGI2 and
PGE2 have a synergistic role in
restoring electrical transepithelial resistance
(R) in ischemia-injured
porcine ileum via the second messengers
Ca2+ and cAMP. Because
Ca2+ and cAMP stimulate
Cl secretion, we assessed
the role of PG-induced Cl
secretion in recovery of R. Mucosa
from porcine ileum subjected to ischemia for 45 min was mounted
in Ussing chambers and bathed in indomethacin and Ringer solution.
Addition of PGs stimulated a twofold increase in
R, which was preceded by elevations in
short-circuit current (increase of 25 µA/cm2). The PG-induced effect
on R was partially inhibited with
bumetanide, an inhibitor of
Cl
secretion. The remaining
elevations in R were similar in
magnitude to those induced in ischemic tissues by amiloride, an
inhibitor of Na+ absorption.
Treatment with 10
4 M
8-bromo-cGMP or 300 mosM mucosal urea resulted in elevations in
R similar to those attained with PG
treatment. PGs signal recovery of R
via induction of Cl
secretion and inhibition of Na+
absorption, possibly by establishing a transmucosal osmotic gradient.
tight junction; ischemia; mucosa
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INTRODUCTION |
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GUT BARRIER FUNCTION is of critical importance because it prevents bacteria and associated toxins from gaining access to subepithelial tissues and the circulation (7). This is a particular concern in patients undergoing breakdown of the intestinal barrier, such as that which occurs during intestinal ischemia-reperfusion injury (19, 24), because of the rapid and often irreversible onset of sepsis and multiple organ failure (30). PGs formed during breakdown of the intestinal barrier may amplify the inflammatory cascade (26). However, it is known that blockade of PG synthesis during early mucosal injury is detrimental, most likely because of the role of PGs in protecting and repairing the intestinal barrier (15).
We have recently developed (4) a model of mesenteric intestinal ischemia in the juvenile pig in which we have focused principally on mechanisms of epithelial repair. After brief periods of complete ileal ischemia (45-60 min), the epithelial barrier rapidly repairs when isolated mucosal tissues are mounted in Ussing chambers [as determined by recovery of transepithelial resistance (R)]. However, recovery of R is severely retarded when tissues are bathed in the nonspecific cyclooxygenase inhibitor indomethacin. Furthermore, we have shown that addition of PGI2 and PGE2 (PGs) to indomethacin-treated tissues synergistically stimulates recovery of R (4). We hypothesized that this action was mediated via second messenger-induced closure of tight junctions based on the following findings: there was no measurable effect of PGs on epithelial restitution despite a recent study showing that PGs augment cellular migration (34); macromolecules that traverse the paracellular space (mannitol and inulin) were largely excluded in injured tissues treated with PGs; PGE2 elevated intracellular cAMP levels, whereas PGI2 appeared to mediate its effects via cholinergic nerves, which utilize Ca2+ as a second messenger; addition of cAMP and a Ca2+ ionophore (A-23187) to ischemia-injured tissues mimicked the synergistic effect of PGI2 and PGE2; and the cytoskeletal contractile agent cytochalasin D completely inhibited the effect of PGs on R.
PG-induced changes in R were preceded
by dramatic elevations in short-circuit current
(Isc) (4),
which can be equated with Cl secretion in this tissue
(3). This finding is not surprising, since
Cl
secretion is triggered
by interactions between Ca2+ and
cAMP at the level of cellular ion transporters (5, 31). Therefore, we
postulated that PG-mediated increases in
R were triggered by
Cl
secretion.
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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 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 previously (3). For each Ussing chamber
experiment, 12 tissues were taken from one pig, but each of the 12 tissues was subjected to a different in vitro treatment. Tissues from
each of six pigs were used to assess the effect of each of the
treatments (n = 6). 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.
R (in
· cm2) was
calculated from the spontaneous PD and
Isc. If the
spontaneous PD was between
1.0 and 1.0 mV, tissues were voltage
clamped at ±100 µA for 5 s and the PD was recorded.
Isc and PD were
recorded every 15 min for 4 h.
Assessment of role of Cl
secretion.
Tissues were bathed in Ringer solution containing 5 × 10
6 M 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 tissue was mounted on Ussing
chambers. In further experiments, the secretory inhibitory agents
bumetanide (10
4 M), ouabain
(10
4 M), glibenclamide
(10
6-10
4
M), or diphenylamine-2-carboxylic acid
(10
4 M) were added to the
appropriate surface of tissues. Baseline electrical
readings were taken for 30 min, after which
10
6 M
16,16-dimethyl-PGE2 and
10
6 M of the
PGI2 analog carbacyclin were added
to the serosal surface of tissues to stimulate epithelial recovery. In
separate experiments, 10
4 M
8-bromo-cGMP (8-BrcGMP) was added to the serosal bathing solution at 30 min instead of PGs. In experiments utilizing
Cl
-free Ringer solution,
Cl
was replaced with
isethionate (136 mM), and in experiments performed with
bicarbonate-free Ringer solution, bicarbonate was replaced with HEPES
(5 mM) and isethionate (15 mM). In addition, bicarbonate-free solutions
were gassed with 100% O2 to
prevent production of bicarbonate from
CO2.
Isotopic flux studies.
These studies were performed at the same time as electrical
measurements were recorded. To assess transmucosal
Na+ and
Cl fluxes, we added
22Na or
36Cl to the mucosal or serosal
solutions of tissues paired according to their conductance (conductance
within 25% of each other). After a 15-min equilibration period and
before addition of treatments, standards were taken from the bathing
reservoirs. Thirty minutes after the addition of treatments, six
successive 30-min flux periods (from 60 to 240 min of the 4-h
experiments) were performed by taking samples from the bathing
reservoirs opposite to 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 was
determined and subtracted. Unidirectional
Na+ and
Cl
fluxes from mucosa to
serosa and serosa to mucosa and the net flux were determined using
standard equations. Similar methods were used to quantify
mucosal-to-serosal fluxes of
[3H]mannitol and
[14C]inulin. A single
30-min flux period was initiated after a 30-min equilibration period
after addition of treatments. The windows on the scintillation counter
were set to avoid counting overlap of
-emission from
3H and
14C.
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 and crypts were identified in each section. The length of the crypt and villus and the width at the midpoint of the villus were obtained using a micrometer in the eyepiece of a light microscope. 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 (2). The percentage of the villus 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 villus surface area was used as an index of epithelial restitution. For each crypt selected for morphometric analysis at 0 and 240 min, the number of crypt epithelial cells was counted and expressed as cells per micrometer as an index of crypt cell density.
In experiments designed to assess epithelial ultrastructure under the influence of PGs or mucosal osmotic loads, tissues were removed from Ussing chambers after 120 min (peak R) 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 intercellular junctions were evaluated, and the number of instances that the tight junctions and/or the intercellular spaces appeared to be dilated was recorded.Data analysis. Data are reported as means ± SE. All data recorded over a 4-h time course were analyzed using ANOVA for repeated measures (Sigmastat, Jandel Scientific, San Rafael, CA). A Tukey's test was used to determine differences between treatments after ANOVA. One-way ANOVA was used to compare the percentage of denuded villus surface area, number of crypt cells per micrometer, number of dilated tight junctions, and unidirectional NaCl, mannitol, and inulin fluxes between treatments. A post hoc Tukey's test was used when appropriate. For all analyses, P < 0.05 was considered significant.
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RESULTS |
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Application of 106 M
PGE2 and
10
6 M carbacyclin (a stable
analog of PGI2) to tissues
injured by 45 min of ischemia and bathed in indomethacin (5 × 10
6 M) resulted in
recovery of control levels of R within
30 min, whereas ischemic tissues exposed to indomethacin alone showed minimal elevations in R over a 4-h
period (Fig. 1,
top). In addition, PGs
stimulated a 30-fold increase in
Isc that preceded
recovery of R (Fig. 1,
bottom), which was suggestive of an
important role for Cl
secretion. To assess this possibility, the
Na+-K+-2Cl
transport inhibitor bumetanide
(10
4 M) was applied to the
basolateral surface of ischemia-injured tissues 30 min before
the addition of PGs. Bumetanide significantly reduced, but did not
abolish, the PG-induced increase in R
(Fig. 1, top), whereas the
PG-induced increases in
Isc were
completely inhibited by bumetanide (Fig. 1,
bottom).
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Because PGs have been shown both to stimulate
Cl secretion and inhibit
neutral NaCl absorption in porcine models of infectious diarrhea, we
reasoned that the failure of bumetanide treatment to fully inhibit the
PG-induced increases in R may be
associated with PG-induced inhibition of
Na+ absorption. Therefore, the
Na+/H+
apical exchange inhibitor amiloride
(10
3 M) was added to the
mucosal surface of indomethacin-treated tissues. Amiloride stimulated
elevations in R that were similar both
in magnitude and in time course to those induced by PGs in the presence of bumetanide (Fig. 1, top). In
addition, the effects of amiloride were not additive to those of the
PGs (Fig. 1, top), suggesting that
amiloride and PGs work via the same pathway. Collectively, these data
suggest that PGs trigger initial increases in
R by inducing
Cl
secretion and stimulate
sustained elevations in R by
inhibiting Na+ absorption.
The PG-induced recovery of R was
associated with significant reductions in mucosal-to-serosal fluxes of
[3H]mannitol and
[14C]inulin (Fig.
2), indicating a correlation between
recovery of R and recovery of barrier
function. Evaluation of tissues immediately after ischemia
revealed that denuded villus tips (Fig.
3A) were almost fully restituted within 60 min in the presence of indomethacin (Fig. 3B), indomethacin and PGs
(Fig. 3C), and indomethacin and amiloride (Fig. 3D). On
the basis of measurements of the percentage of the denuded villus
surface area, restitution was not enhanced by administration of PGs and
treatment with indomethacin alone did not retard restitution (Fig.
4). Therefore, we concluded that significant reductions in mucosal-to-serosal fluxes of mannitol and
inulin in the presence of PGs but not in the presence of indomethacin alone were attributable to changes in paracellular rather than transcellular resistance, as has been shown in our previous studies (4).
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Because recent studies indicate that PGs prolong crypt cell survival in acutely injured intestinal epithelium (6), we sought to determine if PGs augmented recovery of R by maintaining an increased crypt cell density compared with tissues treated with indomethacin alone. Crypts were significantly shorter in indomethacin-treated tissues at the end of the 4-h experiments compared with crypts from tissues immediately after ischemia (291.5 ± 12.7 vs. 233 ± 9.6 µm; P < 0.05), but there was no significant effect of PG treatment on crypt depth (239 ± 3.2 µm) compared with indomethacin alone. In addition, there were no differences in the number of crypt cells per micrometer between tissues immediately after ischemia (0.18 ± 0.003 cells/µm) vs. those treated with indomethacin alone for 4 h (0.19 ± 0.01 cells/µm) or tissues treated with indomethacin and PGs for 4 h (0.19 ± 0.01 cells/µm). These findings suggested that PGs did not augment recovery of R by changes in the secretory cell compartment.
Considering the apparent role of
Cl secretion and
Na+ absorption in recovery of
R, we reasoned that agents other than
PGs that stimulate Cl
secretion and inhibit Na+
absorption should also trigger increases in
R. For example, cGMP is not utilized
as a second messenger by either
PGE2 or
PGI2 (28) but produces the
identical transport response (8). The response of
ischemia-injured tissues to
10
4 M 8-BrcGMP was similar
to that of PGs (Fig. 5,
top). Furthermore, comparison of the
response of ischemia-injured ileum to 8-BrcGMP and 8-bromo-cAMP
(8-BrcAMP), a second messenger that is utilized by
PGE2 (4, 28), revealed similar
elevations in R (Fig. 5, top) associated with marked
elevations in Isc
(Fig. 5, bottom).
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Because Cl secretion is
driven by the electromotive force developed by the basolateral
Na+-K+-ATPase,
we performed experiments in which cellular
Na+-K+
ATPase was inhibited by ouabain
(10
4 M). Ischemic tissues
pretreated with ouabain and indomethacin demonstrated a significantly
reduced response to addition of PGs compared with tissues treated with
indomethacin and PGs alone (Fig. 6). The
reason for the initial elevation in R
in tissues treated with ouabain is unknown but may relate to cell
swelling in the absence of cellular electrolyte transport. In further
experiments, tissues treated with indomethacin and PGs under
Cl
-free conditions showed
marginal but nonsignificant elevations in
R (peak
R, 60 ± 3.9, 60 ± 6.2, and 69 ± 4.8
· cm2, in
control, indomethacin-treated ischemic, or indomethacin and PG-treated
ischemic tissue, respectively; n = 6, P > 0.05), supporting the hypothesis
that Cl
is necessary for
PG-stimulated recovery of R. It
should be noted that measurements of R
were artificially elevated by removing Cl
from the solutions as a
result of replacing Cl
with
a relatively impermeable ion (isethionate) that results in reduced
apical membrane conductance (17).
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Because bicarbonate has been shown to contribute to the anionic
secretion from porcine ileum (3), further experiments were performed
utilizing bicarbonate-free solutions. However, the lack of bicarbonate
had no effect on PG-stimulated elevations in
R (data not shown), suggesting that
Cl is the principal anion
associated with R recovery. To further assess the effect of Cl
secretion on recovery of barrier function, we pretreated
ischemia-injured tissues with the
Cl
channel inhibitor
diphenylamine-2-carboxylic acid
(10
4 M) and with various
doses
(10
4-10
6
M) of the highly specific cystic fibrosis transmembrane conductance regulator inhibitor glibenclamide (27). However, these agents had no
effect on either
Isc or
R in PG-treated tissues. A similar lack of effectiveness of a number of
Cl
channel inhibitors at
various dose ranges and by various routes has previously been
documented in weanling porcine ileum despite the effectiveness of these
agents in rodents and in cell culture (13).
Because of the apparent failure of the specific
Cl channel inhibitors, we
performed Na+ and
Cl
unidirectional flux
experiments to directly examine
Na+ and
Cl
transport. During the
first flux period (60-90 min), PGs triggered net secretion of
Cl
and
Na+, compared with net absorption
of Na+ and
Cl
in tissues exposed to
indomethacin alone (Table
1). During the intermediate
fluxes, PG-treated tissues showed lessening degrees of NaCl secretion
and increased NaCl absorption (data not shown). However, during the
last flux (210-240 min) indomethacin-treated tissues continued to
demonstrate net absorption of NaCl that significantly exceeded that of
PG-treated tissues (Table 1).
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The apparent role of both
Cl secretion and inhibition
of Na+ absorption in recovery of
R led us to postulate that the
development of a transepithelial osmotic gradient signals recovery of
R. Such a gradient might be expected
to develop across the apical junction, since secretion of
Cl
would draw
Na+ out of the paracellular space
across the tight junction. Previous studies have shown that increasing
mucosal osmotic loads stimulates significant increases in
R either by collapsing the
paracellular space (29) or by triggering closure of tight junctions
(20). To test this hypothesis, we added 300 mosM urea to the mucosal surface of ischemic tissues pretreated with indomethacin. This osmotic
gradient resulted in sustained elevations in
R that mimicked the effect of PGs
(Fig. 7).
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Because recovery of barrier function under the influence of PGs could
not be correlated with changes in epithelial restitution and osmotic
gradients across tissues have been associated with closure of
interepithelial tight junctions (20), we performed transmission
electron microscopy on tissues removed from the Ussing chamber during
peak R to evaluate the paracellular
spaces between restituted epithelial cells. In tissues treated with
indomethacin alone, 77.1 ± 2.9% of the intercellular spaces and
tight junctions appeared dilated (Fig.
8A), whereas in
tissues treated with PGs (Fig. 8B)
or urea (Fig. 8C), 6.7 ± 6.7 and
0 ± 0% of the intercellular spaces evaluated were dilated,
respectively. The number of dilated intercellular spaces was
significantly greater in tissues treated with indomethacin alone
compared with tissues treated with either PGs or urea
(P < 0.001, one-way ANOVA,
n = 3).
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DISCUSSION |
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The present studies confirm that PGs augment recovery of intestinal
barrier function, based on evidence of an early return to control
levels of R and a reduction in
mucosal-to-serosal fluxes of inulin and mannitol. Furthermore, the
present studies suggest that PGs signal recovery of barrier function
via mechanisms involving stimulation of
Cl secretion and inhibition
of Na+ absorption. Evidence in
favor of this supposition includes the findings that PG-induced
increases in R were partially
inhibited by bumetanide (an inhibitor of
Cl
secretion) and partially
simulated by amiloride (an inhibitor of
Na+ absorption that triggered
R increases similar to those of PGs in
the presence of bumetanide). Unidirectional
Na+ and
Cl
fluxes performed on
PG-treated tissues showed an early increase in
Cl
secretion and a
sustained inhibitory effect on Na+
absorption. Treatment of injured tissues with 8-BrcGMP, an agent that
stimulates Cl
secretion and
inhibits Na+ absorption, triggered
elevations in R similar in magnitude
to those of the PGs. Pretreatment with ouabain (which blocks both Cl
secretion and
Na+ absorption) inhibited
PG-induced recovery of R. Finally, the absence of Cl
in the tissue
bathing solutions resulted in marginal effects of PGs on
R, suggesting an important role for
Cl
in the response of
ischemia-injured porcine ileal mucosa to PGs. Studies using
specific Cl
channel
inhibitors were inconclusive, since neither
Isc nor
R were inhibited by these agents.
Previous studies suggest that the failure of these drugs to act on
porcine ileum relates to either species differences or the inability of
these agents to reach their target site (13).
The kinetic relationship between changes in
Cl secretion (as indicated
by Isc) and
changes in R in the present study was
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 that are
responsible for elevations in R are
relatively slow to respond or that a number of
Isc-stimulated intracellular signaling events precede changes in
R. As an example of the former
possibility, treatment of Necturus
gallbladder with 8-BrcAMP triggered maximal elevations in
Isc within 5 min,
whereas R was maximal by 30 min
associated with progressive increases in the number of junctional
fibrils (9). Alternatively, if Cl
secretion is involved in
physically collapsing the paracellular space, as has been shown in
previous studies (1, 17, 18), it is reasonable to suppose that some
time would be required for the secretory process to fully collapse a
dilated intercellular space. Likewise, inhibition of
Na+ absorption would serve to
collapse the paracellular space and prevent refilling during the latter
phases of recovery.
The relationship between PG-induced
Cl secretion and inhibition
of Na+ absorption with recovery of
R is best highlighted by experiments in which bumetanide and amiloride are utilized to inhibit
Cl
secretion and
Na+ absorption, respectively. In
experiments in which tissues are pretreated with bumetanide, the
recovery of R stimulated by the PGs is
partially but not fully inhibited. Because bumetanide fully inhibited
PG-induced elevations in
Isc (as an
indicator of Cl
secretion),
we interpret this to mean that PG-induced
Cl
secretion is responsible
for the initial dramatic recovery of R. However, careful inspection of Fig.
1 reveals that tissues treated with PGs and bumetanide have the same
R value at the end of the experiment
as tissues treated with PGs alone, despite the fact that the two groups
of tissues have significantly different peak
R values. We postulated that the
bumetanide-insensitive component of the PG effect on
R was attributable to inhibition of
Na+ absorption. We provided
indirect evidence for this hypothesis by treating tissues with
amiloride, an agent that, similar to the PGs, inhibits
Na+ absorption. Experiments that
show that amiloride simulated the effects of the PGs in the presence of
bumetanide and that amiloride and PGs do not have an additive effect on
recovery of R are consistent with this
hypothesis. In terms of the relative importance of PG-induced Cl
secretion and inhibition
of Na+ absorption, sustained
increases in R appear to be triggered
by inhibition of Na+ absorption,
whereas initial marked but transient elevations in R are associated with
Cl
secretion.
In contrast to the findings of the present study, experiments on
cultured epithelium have typically shown that increased
Cl secretion is associated
with decreases in R. For instance,
PGE1-stimulated Cl
secretion in T84 cells
is associated with increases in conductance (corresponding to decreases
in R) (32). In similar studies, synergistic elevations in both
Cl
secretion and
conductance were noted in T84 cells under the influence of vasoactive
intestinal polypeptide and the
Ca2+ ionophore A-23187 (5). The
critical difference between cultured epithelium such as T84 cells and
native epithelium from porcine ileum is the ~100-fold higher
R in T84 cells compared with porcine ileum (4, 5, 31). This high R results
largely from the proximity of cells to one another in confluent
monolayers so that the paracellular resistance is similar to that of
transcellular resistance (21). In contrast, paracellular resistance is
far lower in "leaky" epithelia than transcellular resistance,
reflected in a much reduced R (17,
18). Accordingly, changes in paracellular resistance may have more
dramatic effects on R than changes in transcellular resistance in leaky epithelia, such as mammalian small
intestinal mucosa (16) and Necturus
gallbladder (18). In other words, an expected drop in
transcellular resistance in the presence of a
Cl
secretogogue was likely
hidden by increases in paracellular resistance in
ischemia-injured porcine ileum.
We suspect that PGs enhance recovery of barrier function by stimulating closure of interepithelial spaces rather than by augmenting other critical reparative processes, including epithelial restitution (34) and crypt cell turnover (6). Our morphometric analyses indicate that tissues rapidly restitute within ~60 min. However, this early repair process was not altered by PG treatment. Similar findings (12) have been demonstrated in bile acid-injured rat jejunum, in which PGE2 had no effect on epithelial restitution. Because of the lack of morphological evidence of PGs on restitution, the reduction in mannitol and inulin fluxes in PG-treated tissues is likely attributable to recovery of paracellular permeability. Previous studies (22) have indicated a close correlation between fluxes of these macromolecules and changes in paracellular resistance rather than transcellular resistance. We also studied the effects of PGs on crypt morphology, because PGs have recently been shown to increase crypt cell survival in acutely injured epithelia. It is conceivable that increased crypt cell density could lead to increased R. However, there was no difference in the number of crypt cells per micrometer between the various treatments. Finally, we performed electronmicrography to evaluate the paracellular space of repairing epithelium. Our data indicate that this is a potential site of action of PGs, since ~7% of the paracellular spaces were dilated in tissues treated with PGs, whereas ~80% of the intercellular spaces were dilated in tissues treated with indomethacin alone.
We have previously reported that
PGE2 and
PGI2 signal recovery of barrier
function via cAMP and Ca2+,
respectively (4). Although previous studies suggest that cAMP (9) and
Ca2+ (23) signal changes in
R by direct effects on tight
junctions, the present studies suggest their effects may be mediated by
signaling Cl secretion and
inhibiting Na+ absorption. This
idea is highlighted by similar tissue responses to 8-BrcAMP and
8-BrcGMP, which both stimulate
Cl
secretion and inhibit
Na+ absorption. In addition, PGs
would be expected to elevate intracellular cAMP and
Ca2+ under
Cl
-free conditions
(although this was not measured in this study), but the absence of
Cl
prevented a full
response to PGs. There is previous evidence to support the possibility
that Ca2+ and cAMP signal
increases in R via
Cl
secretion in studies
performed in other species. For example, treatment of rabbit ileum with
the phosphodiesterase inhibitor theophylline caused an increase in
Isc and a
subsequent decrease in conductance that was dependent on the presence
of Cl
(25). Treatment of
Necturus gallbladder with cAMP
resulted in marked elevations in
Isc and
concomitant increases in R, but the
relationship between Cl
secretion and R was not further
explored (9).
We postulated that PGs may signal increases in R by creating a transmucosal osmotic gradient. To provide further evidence for this hypothesis, we treated ischemia-injured tissues with 300 mosM mucosal urea, which resulted in peak R similar to that of the PGs and 8-BrcGMP. However, the mechanism by which a PG-induced osmotic gradient might signal recovery of R is not clear. Ischemia-injured tissues treated with indomethacin alone had ultrastructural evidence of dilated intercellular spaces (including the region of the junctional complex), whereas tissues treated with PGs or urea had closely apposed intercellular spaces. It is possible that differences in the dimensions of the intercellular spaces occurred because of fixation artifacts (20) and potential experimental artifacts such as stretching of the mucosa during mounting of tissue in Ussing chambers (1). However, there are studies in Necturus gallbladder showing that cAMP-induced elevations in transepithelial R are inhibited by preventing collapse of the intercellular space with serosa-positive hydrostatic pressures, suggesting a potential role for the degree of intercellular space collapse in the regulation of R (18).
In contrast to evidence that transmucosal osmotic gradients induce
increases in R by collapsing the
intercellular space, Madara (20) has shown that the effects of osmotic
loads can be inhibited by pretreatment with the cytoskeletal
contractile agent cytochalasin D despite the continued presence of
collapsed intercellular spaces. Because cytochalasin D also results in
opening of tight junctions (22), these studies suggest that the tight
junction regulates R under conditions
of a transmucosal osmotic gradient. We have also shown that
cytochalasin D inhibits the effects of PGs on R, suggesting a possible signaling
mechanism via the cytoskeleton (4). However, cytochalasin D also
inhibits PG-induced Cl
secretion (data not shown). The cytoskeleton appears critical to the
Cl
secretory response by
stimulating apical secretory vacuoles to empty (14). Thus the precise
mechanism by which PGs augment recovery of paracellular
R in this tissue awaits further study.
Much of the data in the present study are strongly supported by
previous studies. For example, it is well known that
Ca2+ and cAMP stimulate increases
in R (9, 23) and
Isc (5, 31) and
that mucosal osmotic loads increase R
in intestinal epithelia (21, 29). The unique finding in this study is
that PGs, the physiological mediators that orchestrate recovery of R in ischemia-injured
intestine via Ca2+ and cAMP (4),
utilize second messenger-induced
Na+ and
Cl cellular transport
mechanisms to signal recovery of R,
possibly by generating transmucosal osmotic gradients. This may have
important clinical implications. For example, sepsis may result from a
breach of epithelial barrier function during a hypotensive episode,
allowing transepithelial passage of bacterial toxins (33), and PG
synthesis inhibitors, such as indomethacin, exacerbate intestinal
injury (15). However, PGs have often been considered to be
detrimental because they stimulate
Cl
secretion and diarrhea
(15, 32). The present studies suggest that PGs and the
Cl
secretion that they
stimulate provide important signals for the recovery of barrier
function in ischemia-injured mucosa.
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ACKNOWLEDGEMENTS |
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This study was supported by United States Department of Agriculture Grant 94-37204-0448. A. T. Blikslager was supported by a National Institutes of Health National Research Scholarship Award (F32 DK-09400-01).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: A. T. Blikslager, College of Veterinary Medicine, North Carolina State Univ., 4700 Hillsborough St., Raleigh, NC 27606.
Received 9 March 1998; accepted in final form 3 September 1998.
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