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1 Department of Cellular Injury, Walter Reed Army Medical Institute of Research, Silver Spring, Maryland; 2 Department of Surgery, Walter Reed Army Medical Center, Washington, DC 20307; Departments of 3 Surgery, 4 Medicine, and 5 Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814; and 6 Beltsville Agricultural Research Center, Nutrient Requirements and Function Laboratory, Beltsville, Maryland 20705
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Prior induction of heat shock protein 70 (HSP70) protects against ischemia-reperfusion (I/R) mucosal injury, but the ability of HSP70 to affect I/R-induced alterations in epithelial cell function is unknown. Rats subjected to whole body hyperthermia (41.5-42°C for 6 min) increased HSP70 and heat shock factor 1 mRNA expression, reaching a maximum 2 h after heat stress and declining thereafter. HSP70 production was maximally elevated at 4 h after heat stress and remained elevated until after 12 h. Heat stress alone had no effect on mucosal function except to enhance secretion in response to ACh. Heat stress provided complete morphological protection against I/R-induced mucosal injury but did not confer a similar protection against I/R-induced decreases in mucosal resistance, sodium-linked glucose absorption, or tachykinin-mediated chloride secretion. Heat stress, however, attenuated the I/R-induced suppression of ACh response, and this effect was dependent on enteric nerves. Thus induction of heat shock protein 70 is associated with the preservation of mucosal architecture and attenuation of some specific functional alterations induced by I/R.
heat shock; intestinal function; ischemia-reperfusion
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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HEAT SHOCK PROTEINS (HSP) are a highly conserved family of constitutive (Hsc) and stress-inducible (Hsp) proteins that are classified by molecular weight (16). An important feature of HSP induction, particularly the Hsp70 family, is the acquisition of thermotolerance or cross tolerance, defined as the protection against subsequent heat stress or other noxious stimuli. Overexpression of Hsp70 confers protection and resistance to heat shock, oxidative stress, infections, and toxic molecules in many cell types (16, 18, 32, 33). This beneficial effect is attributed, in part, to the ability of Hsc70 to chaperone newly synthesized proteins until they are properly folded and in the proper cellular compartments (16, 18). Hsp70 can also provide protection by interference with apoptosis (3, 12) as well as by inhibiting the activation of stress proteins (2).
In unstressed cells, HSP are bound to heat shock factors (HSF) in the cytosol (8, 16). Exposure to heat stress results in the disassociation of HSF and HSP, leading to phosphorylation of HSF and formation of HSF trimers that enter the nucleus (8, 16). The HSF trimers bind to heat shock elements in the promoter region of the HSP gene and become further phosphorylated (16). This is followed by HSP mRNA transcription and protein translation. In mammalian cells, HSF1 responds to pathological stressors such as ischemia, inflammation, or toxins (16).
The splanchnic circulation is particularly susceptible to reduced blood flow, and the reperfusion subsequent to the ischemia causes a local inflammation characterized by mucosal injury, neutrophil infiltration, and production of inflammatory mediators including eicosanoids. Previously, we showed that thermal induction of Hsp70 conferred a morphological protection against ischemia-reperfusion (I/R) injury in rat small intestine in vivo (32, 33). Upregulation of HSP mRNA in the small intestine occurs as early as 30 min after injury, with maximum protein production observed at 4 h (34). The mechanism of this protective effect of HSP against ischemic injury has not been elucidated fully but may involve inhibition of neutrophil infiltration (32, 33), modulation of leukocyte-endothelial cell interactions (4), increases in cellular antioxidants (31), and reduced production of inflammatory molecules (18, 24). Hsc/Hsp70 is proposed to be involved in regulation of nitric oxide production (2). An unexplored aspect of Hsp70 induction in vivo is its effects on the physiological function of the intestine, and we hypothesized that Hsp70 induction is beneficial to both mucosal function and integrity. Many inflammatory and immune mediators, which are proposed to be upregulated or to be activated by Hsp70 production (26), have potent effects on mucosal function; however, it is not known whether the induction of HSP is associated with alterations in epithelial cell secretion and absorption. More importantly, it is unknown whether induction of Hsp70 protects the intestinal mucosal function against the deleterious effects of I/R in vivo. Therefore, the aim of this study was to assess the effects of hyperthermia-induced Hsp70 on mucosal function and to determine whether the Hsp70-induced protection against I/R-induced alterations in intestinal morphology is associated with similar preservation of the mucosal function.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Ischemia/Reperfusion
All experiments were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals (Institute of Animal Resources, National Resource Council). Adult male Sprague-Dawley rats (8-12 wk or 200-250 g), used in all studies, were fasted overnight with free access to water. Rats were anesthetized with ketamine (80 mg/kg im) and xylazine (16 mg/kg im). A midline laparotomy was performed, and, after a 60-min stabilization period, the superior mesenteric artery (SMA) was clamped. After 30 min, the clamp was removed and reperfusion of the intestine was confirmed by the return of pulsatile mesenteric blood flow. Occlusion of the SMA results in mucosal injury to the jejunum and ileum; therefore, these portions of the intestine were the focus of this study. After 1 h of reperfusion, sections of midjejunum or ileum were taken for histological evaluation, for in vitro Ussing chamber studies of epithelial cell function, and for determination of Hsp70 and HSF1 mRNA and Hsp70 protein. Animals in the sham group were treated identically, omitting the SMA occlusion. Animals remained anesthetized throughout the experiment.Heat Stress
Heat stress was performed as described previously (32, 33). Briefly, animals were anesthetized and placed in a prewarmed (47-50°C) humidified, aerated (room temperature at 92 ml/min) chamber until their rectal temperature reached 41.5-42.0°C. The average rate of heating was consistent among animals, ranging from 40 to 45 min to reach 41.5-42.0°C. After rectal temperature of 41.5°C was maintained for 6 min (model 4600, YSI, Yellow Springs, OH), the rats were removed from the heating chamber and placed on a 37°C heating pad and allowed to cool passively until all rats had a rectal temperature of 37°C (0.04°C/min). Next, a midline laparotomy was performed, and, after a 60-min stabilization period, heat-stressed animals were subjected randomly to I/R or sham operation (32, 33). In these experiments, tissue was harvested at 4 h after heat stress, the time of maximal Hsp70 protein production. For the time course experiments, animals were heat stressed as indicated above, and sections of intestine were obtained after the animals had cooled passively for 0 h (10 min after heating), 1, 2, 4, 8, 12, and 24 h.Ussing Chambers
Gently, the muscle was dissected from sections of midjejunum, and the stripped mucosae were mounted in Ussing chambers and incubated in oxygenated (95% O2-5% CO2) Krebs buffer (32, 33). The tissues were allowed to equilibrate for 20-30 min in Krebs buffer containing 12 mM glucose on the serosal side and 10 mM mannitol on the mucosal side before the addition of drugs. The basal short circuit current (ISC) and resistance were measured as well as concentration-dependent changes in ISC in response to glucose, ACh, neurokinin A (NKA), and substance P (SP). Every 50 s, the tissues were short-circuited at 1 V (World Precision Instruments DVC 1000 voltage clamp, Sarasota, FL), and resistance was calculated by using Ohm's law.The tissue was allowed to equilibrate for 20 min before cumulative addition of ACh, SP, or NKA to the serosal side. Responses were compared in the presence and absence of tetrodotoxin (TTX), a sodium-channel blocker that inhibits nerve conduction, to determine the contribution of enteric nerves. The tissue was exposed to TTX for 20 min before the addition of secretagogues. Glucose was added to the mucosal side to assess sodium-linked nutrient absorption. Responses of one or more intestinal segments exposed to glucose or secretagogues from an individual animal were averaged to yield a mean response per animal, and then the mean responses of four to seven animals were averaged to yield a mean ± SE for each group.
Solutions and Drugs
Krebs buffer contained (in mM) 4.74 KCl, 2.54 CaCl2, 18.5 NaCl, 1.19 NaH2PO4, 1.19 MgSO4, and 25.0 NaHCO3. Stock solutions of SP (0.1 mM) and NKA (0.1 mM) were dissolved and stored in 0.01 mM acetic acid and diluted on the day of the experiment in distilled water. Stock solutions of ACh chloride (100 mM) were prepared in water and subsequently diluted in distilled water on the day of the experiment. TTX was dissolved in citrate buffer to a stock solution of 1 mM. On the day of the experiment, appropriate dilutions of glucose and each secretagogue were made with distilled water. SP and NKA were purchased from Peninsula Laboratories (Belmont, CA). All other chemicals were purchased from Sigma Chemical (St. Louis, MO).Histology
At the end of each experiment, sections of midjejunum were formalin fixed, paraffin embedded, and sectioned (5 µm). Microscopic mucosal injury and polymorphonuclear neutrophil (PMN) infiltration (no./high power field) were evaluated in Giemsa-stained sections by two investigators who were unaware of the treatment. Injury was scored from 0 (normal) to 5 (severe) by use of a previously validated system (32, 33).Hsp70 and HSF-1
Western blot analysis.
One-centimeter sections of intestine were snap frozen in liquid
nitrogen and stored at 
70°C until analyzed. Briefly, the tissue
samples were solubilized by sonication in T-PER (Pierce Endogen,
Rockford, IL), and the proteins in the clarified supernatant were
resolved by SDS-PAGE before being blotted to nitrocellulose membrane.
The membrane was blocked and then probed with mouse monoclonal
antibodies against Hsp70 (StressGen, Victoria, BC, Canada) before
chemiluminescence development. The amount of heat stress protein
was determined by densitometry and was compared with expression of
actin protein.
RNA extraction and RT-PCR.
One milligram of rat intestine was minced, sonicated, and isolated with
Trizol reagent (GIBCO, Gaithersburg, MD). After isopropanol precipitation, the RNA pellets were washed and dissolved in water. The
RNA concentration was determined spectrophotometrically. The procedure
and primers used for RT-PCR to measure expression of Hsp70, HSF-1, and
-actin were published previously (7, 14). Briefly, the
total RNA was reversed transcribed with avian myeloblastosis virus
reverse transcriptase (Promega, Madison, WI) in a final volume of 20 µl. The mixture was incubated at 37°C for 10 min and then at 42°C
for 20 min. Heating the mixture at 95°C in a water bath for 10 min
before chilling on ice terminated the transcription reaction. The cDNA
was subjected to PCR with the use of AmpliTaq DNA polymerase
(Perkin-Elmer, Foster City, CA), and 30 PCR cycles were run (95°C for
1 min, 54°C for 1.5 min, and 72°C for 1.5 min). Identical
quantities (10 µl) of each PCR product were loaded onto 1% agarose
gels before being stained with 2 µl of ethidium bromide (stock
concentration: 1 µg/µl) in 100 ml of Tris borate and EDTA (TBE) buffer and photographed. The bands of interest
were quantitated densitometrically (15).
Data Analysis
Statistical analysis was performed by using t-tests to compare basal ISC and resistance. To compare the response to the secretogogues between treatment groups, concentration-response curves were constructed and subsequently analyzed with SYSTAT 5.2 using a multivariate analysis of variance test with repeated measures. Differences between groups were assessed by using a t-test designed for comparison of multiple means. A P < 0.05 was considered significant| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Induction of Hsp70 and Intestinal Morphology
The proximal (upper jejunum) and distal (midileum) intestinal tissue were compared with determine basal Hsc/Hsp70 mRNA production. There were no differences in Hsc/Hsp70 mRNA production between proximal and distal small intestine; therefore, in the remaining experiments, we used midjejunal intestinal tissue. Total body exposure to 41.5-42.0°C for 6 min increased HSF-1 and Hsc/Hsp70 mRNA expression within 10 min after exposure to heat stress (Fig. 1A). Maximal Hsc/Hsp70 and HSF-1 mRNAs were produced at 2 h after heat stress and returned to unheated levels by 12-24 h after heat stress. Similarly, heat stress induced increased Hsp70 protein expression (Fig. 1B) in the small intestine 4 h later, consistent with our previous results in this model (32, 33). This correlates with increased Hsc/Hsp70 mRNA expression at 2 h after heat stress. To allow for maximal HSP production, subsequent experiments were timed so that tissue was taken 4 h after heat stress.
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When compared with sham-treated animals, mesenteric I/R significantly
increased mucosal injury, which was characterized by submucosal edema,
loss of surface epithelial cells, exposed lamina propria and mucosal
hemorrhage, as well as PMN infiltration (Table 1). Similar to previous results
(32, 33), prior exposure of the animals to heat stress
completely prevented these effects (Table 1).
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Epithelial cell function. Indexes of epithelial cell function in healthy intestine include 1) a net flux of ions across the mucosa (ISC), 2) mucosal resistance, 3) secretion of chloride ion in response to agents added to the serosal (contraluminal) side, and 4) absorption of glucose from the luminal side via a sodium-linked transporter. These parameters were determined in sections of midjejunum after sham operation or mesenteric I/R, with or without prior exposure to heat stress.
Basal parameters. Neither I/R alone nor I/R after heat stress altered basal ISC, a measure of net active ion transport across the mucosa (Table 1). In contrast, I/R significantly decreased tissue resistance, an index of mucosal permeability, and this effect was not prevented by heat stress (Table 1).
Glucose absorption.
The addition of glucose to the mucosal side of the tissue stimulates
substrate-linked sodium absorption. Figure
2 shows that I/R significantly decreased
sodium-linked glucose absorption and that this effect was not prevented
by heat stress.
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Response to secretagogues.
Addition of ACh, SP, or NKA to the serosal side of the tissue
stimulates chloride secretion. Exposure to heat stress alone significantly increased responses to ACh (Figure
3) but had no effect on responses to NKA
(Fig. 4) or SP (Fig.
5). I/R alone significantly reduced
responses to all three secretagogues (Figs. 3-5). The I/R-induced
decrease in response to ACh was attenuated by heat stress; however, the
I/R-induced suppression of the responses to NKA and SP was unaltered by
heat stress.
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Neural dependence of responses to ACh.
Because heat stress alone significantly increased responses to ACh and
heat stress attenuated the inhibitory effect of I/R on this response,
we next determined whether these effects were due to a direct effect on
the cells or were mediated by enteric nerves. TTX, which blocks nerve
conduction, was added to the tissue before addition of ACh (Fig.
6). In sham-operated control and heat-stressed rats, responses to ACh were significantly reduced by TTX,
indicating that the ability of ACh to increase chloride secretion is
dependent on enteric nerves in both groups (Fig. 6A). The
ability of heat stress alone to elevate the response to ACh was not
observed in the presence of TTX, demonstrating that the effect of heat
stress is dependent on nerves (Fig. 6A). We also examined
the effect of TTX on responses in control and heat-stressed rats
subjected to I/R (Fig. 6B). TTX had no effect on the already
low responses in the I/R group; however, TTX significantly decreased
responses in the heat-stress + I/R group. In addition, the heat
stressed-induced increased in secretion in response to ACh was not
significant after treatment with TTX.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Previously, we demonstrated that whole body hyperthermia induced Hsp70 that was associated with protection against mesenteric I/R-induced microscopic injury and inflammation (33). These effects were associated with an inhibition of I/R-induced elevation in levels of inflammatory mediators, including leukotriene B4 and prostaglandin E2. In the present study, we demonstrated that despite a near-complete protection against I/R-induced histological injury, heat stress and the subsequent induction of Hsp70 provided limited protection against I/R-induced changes in mucosal function early in the reperfusion period. These data indicate that Hsp70-induced tolerance to oxidative stress is confined initially to a morphological cytoprotection with a subsequent extension to partial physiological function.
HSPs are intracellular proteins, and acquired cross tolerance, the ability of Hsp70 to protect against subsequent noxious conditions, is well documented in a number of cells types in vitro (2, 3, 12, 14, 38). In the present study, after in vivo heat stress, increased production of Hsp70 protein in the small intestine was observed as early as 2 h; after heat stress, it was maximal at 4 h and remained elevated until 12 h postexposure to heat. Furthermore, enhanced production HSF1-mRNA paralleled that of Hsc/Hsp70 mRNA. It should be noted that the return of HSF1 mRNA to baseline levels or below preceded that of Hsc/Hsp70 mRNA, suggesting that the downregulation of HSF-1 may play a role in the control of Hsp70 in response to heat stress in vivo.
There is also substantial evidence that Hsp70 functions to protect tissues and organs in vivo (4, 10, 11, 26, 28, 32, 33); however, traditionally the outcome of these studies is measured in terms of reduced animal mortality and macroscopic and/or microscopic cell damage. In contrast to this body of literature, the ability of Hsp70 to alter organ function or to protect against otherwise damaging or lethal stimuli is relatively unexplored with recent studies on the intestine, kidney, brain, and liver (1, 17, 26, 27, 35). In the present study, we subjected rats to 30 min of low-flow mesenteric ischemia, a nonlethal but damaging insult. A prominent feature of I/R damage is loss or reduction in surface epithelial cells with villus shortening resulting in a diminished absorptive surface (9, 20, 32, 33, 36). If left untreated, however, the resultant mucosal injury is resolved completely by 24 h into the reperfusion period (20). In the present study, we confirmed our previous data showing that prior induction of Hsp70 protected against microscopic mucosal injury and inflammation in response to mesenteric I/R (Fig. 2). The near complete microscopic protection against I/R-induced damage afforded by Hsp70 is impressive compared with other interventions that interfere with lipid peroxidation (27, 32) or complement activation (9) or that limit availability of reactive oxygen species (37).
Oxidative stress appears to play a role in I/R-induced cardiac injury, and Hsp70 may regulate the toxic reactive oxygen and nitrogen species by increasing the production of superoxide dismutase or inhibiting subsequent apoptosis (reviewed in Refs. 18 and 19). In contrast to HSP production in response to I/R in other organs such as the kidney, heart, and liver (1, 13, 17, 35), I/R did not increase Hsp70 in the small intestine in the present study, indicating that oxidative stress alone is an insufficient stimuli in this tissue (33). This difference may be a result of the time course of Hsp70 expression, which occurs within minutes to hours after heat stress in the small intestine and days to weeks after an ischemic event in other organs (13, 17, 35). This is supported by the fact that there is a rapid turnover of cells in the small intestine (every 7-10 days). The small intestine is disproportionately sensitive to changes in blood flow and may require more immediate activation of protective mechanisms.
A primary function of the small intestine is absorption of nutrients by the epithelial cells lining the gut lumen. These cells are particularly vulnerable to reduction in mesenteric blood flow because of the countercurrent arrangement of capillaries and venules in the intestinal villi. In the present study, we showed that I/R significantly decreased glucose absorption, reflecting the loss and/or damage to the epithelial cells (Fig. 2). Exposure to heat stress alone had no effect on glucose absorption and was unable to prevent the inhibition of glucose absorption after I/R, despite the presence of intact villi. These data demonstrate that heat-induced tolerance was not extended to protection of surface cells against I/R-induced decrements in epithelial cell absorption of nutrients early in the reperfusion period. The fact that 30 min of ischemia is nonlethal and that animals are completely recovered 24 h later (20) suggests that restoration of glucose absorption later in the reperfusion period is likely to be accelerated in heat-stressed rats compared with unstressed animals subjected to I/R.
An equally important function of the intestinal epithelia is the maintenance of the mucosal barrier that limits access of potentially harmful luminal contents such as bacterial toxins. Damage to surface epithelia is followed by reannealing of the mucosa and later replacement of surface cells by migration of immature cells from the base of the villus or crypt region. Thus protection of the cells in the crypt region is a critical factor for intestinal healing. Compared with unstressed animals, heat stress alone had no effect on basal ISC or resistance. I/R alone did not alter basal ISC, indicating that active ion transport, which is ultimately dependent on the energy-dependent removal of sodium in exchange for potassium, was unaffected. The decreased mucosal resistance after I/R alone is consistent with the severity of mucosal damage. This compromised tissue permeability may facilitate bacterial translocation, a phenomenon that is implicated in the extraintestinal complications of mesenteric ischemia (23). Surprisingly, heat stress did not alter the I/R-induced drop in resistance, even in morphologically intact tissue. Mucosal integrity, however, is dependent on the interactions between a number of cell types, as well as the competence of the vascular endothelium, and may be beyond the scope of the protection conferred by the elevated levels of intracellular Hsp70.
Villus crypt cells are also responsible for secretion of ions and fluid that facilitate digestion. In addition, intestinal secretion is important in the host response to enteric parasites (30) and cholera toxin (21, 22), helping to flush the intestinal lumen to remove harmful agents or minimize their contact with the epithelial lining. Chloride secretion is predominantly controlled by submucosal neurons that coordinate afferent and efferent activity. Intrinsic neurons in the submucosal plexus contain SP and ACh among other neurotransmitters. Thus addition of agonists such as ACh or the tachykinin SP to the serosal side reflects the ability of the intestinal mucosa to secrete chloride and fluid. SP binds to all known neurokinin receptors but has the highest affinity for NK1 receptors that are upregulated during inflammation (5, 6). NKA, another tachykinin, can also bind NK1 receptors. Heat stress alone did not alter responses to NKA or SP; however, responses to ACh were enhanced significantly. This is important because, in rats, a large component of the response to nerve stimulation is attributed to release of ACh which acts on muscarinic receptors on epithelial cells (25). These data are supported by previous reports showing increased chloride secretion, but no microscopic changes, in response to cold or restraint stress in rats (29). I/R decreased responses to all secretagogues in the present study, reflecting the significant loss of surface absorptive cells. Responses to ACh were restored in I/R, in part, by prior exposure to heat stress, demonstrating that heat stress upregulation of responses to ACh is retained during I/R.
To determine whether the protection afforded by heat stress on responses to ACh was due to a direct effect on the epithelial cells or to enteric nerves, the neurotoxin TTX was added to the tissue in vitro before addition of ACh. In both control and heat-stressed tissue obtained from sham-operated rats, TTX significantly reduced responses to 1 mM ACh by more than 90%, indicating a dependence on nerves (Fig. 6A). A comparison of responses in the presence of TTX revealed that the increased response to ACh in the sham-operated group exposed to heat stress alone was not observed in tissue treated with TTX, providing further support of a role for nerves (Fig. 6A). In rats subjected to I/R, responses to ACh were unaltered by TTX in the unstressed group but were significantly reduced in the heat-stressed group (Fig. 6B). Furthermore, the partial restoration of the changes in ISC to ACh in heat-stressed animals subjected to I/R was also absent in TTX-treated tissue. These data implicate a role for nerves in the protection afforded by heat stress against I/R-induced reductions in mucosal secretion. Thus the beneficial effect of heat stress in both the sham-operated and I/R groups is mediated by ACh acting within the enteric nervous system. We believe this is the first study to document that induction of Hsp70 is associated with protective effects on intestinal mucosal function that are neurally mediated and appear to selectively affect cholinergic nerves.
The mechanism underlying the effect of heat stress on nerves is unclear but may involve changes in ion movement across cells. Induction of Hsp70 is a calcium-dependent process (16), and increased cell injury is accompanied by elevated intracellular calcium that may lead to cell death. It is important to note, however, that prior heat stress attenuates the influx of calcium in response to subsequent noxious stimuli (16). We showed previously that I/R alone did not induce Hsp70 (33), but prior exposure to heat stress and limitation of calcium entry into cells likely contributed to the observed preservation of architectural and overall functional integrity of the mucosa. Neurotransmitter release depends on calcium entry into synaptic endings. The heat stress-induced elevation in response to ACh is not likely to be mediated by increased calcium because they were prevented by the sodium-channel blocker, TTX.
In conclusion, heat stress increased Hsp70 production, and this was preceded by upregulation of HSF-1 and Hsp70 mRNA expression. I/R induced significant mucosal injury and inflammation, increased mucosal permeability, and suppressed epithelial cell absorption and secretion. Prior exposure to heat stress provided complete protection against I/R-induced mucosal damage and PMN infiltration. In contrast, heat stress was unable to prevent most of the I/R-induced alterations in epithelial cell function but abrogated the I/R-induced decrease in the ACh response, an effect that was dependent on enteric nerves. These data indicate that heat stress-induced tolerance to a nonlethal oxidative stress is primarily a protection against cell damage that allows for a more rapid recovery of cell function, particularly those involving cholinergic nerves.
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ACKNOWLEDGEMENTS |
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Funding was provided by MRMC STOR, Complement Program and G183HO awarded to T. Shea-Donohue from WRAIR STOR.
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FOOTNOTES |
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The opinions contained herein are the private ones of the authors and are not to be construed as official policy or reflecting the views of the Department of Defense.
Original submission in response to a special call for papers on "Molecular Biology of Thermoregulation."
Address for reprint requests and other correspondence: T. Shea-Donohue, Dept. of Medicine, Uniformed Services Univ. of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814 (E-mail: tshea{at}usuhs.mil).
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.
10.1152/japplphysiol.01008.2001
Received 2 October 2001; accepted in final form 9 February 2002.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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