Essential role for IL-6 in postresuscitation inflammation in hemorrhagic shock

Zhi Hong Meng1, Kevin Dyer2, Timothy R. Billiar3, and David J. Tweardy1

1 Section of Infectious Diseases, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030; 2 University of Pittsburgh Cancer Institute, Pittsburgh 15213; and 3 Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Interleukin-6 (IL-6) is produced within multiple tissues and can be readily detected in the circulation in resuscitated hemorrhagic shock (HS). Instillation of IL-6 into lungs of normal rats induces polymorphonuclear neutrophilic granulocyte (PMN) infiltration and lung damage, while infusion of IL-6 into the systemic circulation of rats during resuscitation from HS reduces PMN recruitment and lung injury. The current study was designed to determine whether or not IL-6 makes an essential contribution to postresuscitation inflammation and which of the two effects of IL-6, its local proinflammatory effect or its systemic anti-inflammatory effect, is dominant in HS. Wild-type and IL-6-deficient mice were subjected to HS followed by resuscitation and death 4 h later. IL-6-deficient mice subjected to HS did not demonstrate any features of postresuscitation inflammation observed in wild-type mice, including increased PMN infiltration into the lungs, increased alveolar cross-sectional surface area, increased PMN infiltration into the liver, increased liver necrosis, increased signal transducer and activator of transcription 3 activation, and increased nuclear factor-kappa B activity. These findings indicate that IL-6 is an essential component of the postresuscitation inflammatory cascade in HS and that the local proinflammatory effects of IL-6 on PMN infiltration and organ damage in HS dominate over the anti-inflammatory effects of systemic IL-6.

signal transducers and activators of transcription proteins; nuclear factor-kappa B; neutrophils; myeloperoxidase; alveolar wall cross-sectional surface area; focal liver necrosis; interleukin-6


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INTERLEUKIN-6 (IL-6) is a 21-kDa cytokine that is produced by a variety of cells including fibroblasts, endothelial cells, mononuclear phagocytes, neutrophils, hepatocytes, and T and B lymphocytes. IL-6 receptor is composed of two chains, an alpha chain (gp80; CD126) specific for IL-6, and a beta chain (gp130) shared with all other members of the IL-6 family, including IL-11, oncostatin M, leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotropin 1. IL-6Ralpha occurs as a membrane-bound protein and in a soluble form. Soluble IL-6Ralpha results from alternative mRNA splicing or from C-reactive protein-controlled cleavage of the membrane-bound receptor. Membrane-bound IL-6Ralpha expression is cell type restricted, whereas gp130 expression is ubiquitous. Soluble IL-6Ralpha is detectable in the serum of normal individuals; circulating levels of soluble IL-6Ralpha increase in inflammatory states. Unlike other soluble receptors, soluble IL-6Ralpha is agonistic; consequently, in the presence of IL-6 and soluble IL-6Ralpha , virtually all cells become IL-6 responsive. IL-6 affects a variety of biological functions, including immunoglobulin production, the acute phase response, and inflammation. IL-6 has been shown to contribute to polymorphonuclear neutrophilic granulocyte (PMN) production in response to infection (9, 10) and to contribute to activation of PMN effector functions (12, 21).

We have previously demonstrated that IL-6 mRNA and protein were produced in the lungs, liver, and intestinal tracts of rats subjected to resuscitated hemorrhagic shock (HS) (18, 19) and that both the ischemic and reperfusion phases of resuscitated HS were required for their production (18). To investigate the consequence of this local production of IL-6 on inflammation in HS, we instilled IL-6 into the lungs of normal rats and observed increased PMN infiltration and lung damage (19). Others have demonstrated elevated circulating levels of IL-6 in humans and animals following trauma and HS (16, 30). In contrast to the effects of local instillation of IL-6, we observed that systemic infusion of IL-6 reduced inflammation and injury in both organs.

To determine which of the two effects of IL-6, its local proinflammatory effect or its systemic anti-inflammatory effect, is dominant in HS, we examined the extent of PMN infiltration, organ injury, and the activity levels of proinflammatory transcriptional factors in IL-6-deficient mice compared with wild-type mice. IL-6-deficient mice subjected to HS did not demonstrate any features of postresuscitation inflammation observed in wild-type mice, which included increased PMN infiltration into the lungs, increased alveolar cross-sectional surface area, increased PMN infiltration into the liver, increased liver necrosis, increased signal transducers and activators of transcription (STAT3) activation, and increased nuclear factor (NF)-kappa B activity. These findings indicate that IL-6 is an essential component of the postresuscitation inflammatory cascade in HS and that the local proinflammatory effects of IL-6 on PMN infiltration and organ damage in HS dominate over the anti-inflammatory effects of systemic IL-6.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Animal protocols were approved by the University of Pittsburgh Institutional Review Board for animal experimentation and were performed in accordance with the guidelines outlined by the National Institutes of Health for the care and use of laboratory animals. IL-6-deficient mice bred into the C57BL/6 background were generated as described (24) and generously provided by Dr. Valerie Poli (Univ. of Dundee). C57BL/6 mice were obtained from Charles River Laboratories (Southbridge, MA). Mice were bred in a specific pathogen-free animal barrier facility, maintained in standard conditions under a 12-h light-dark cycle, provided standard food and water ad libitum, and used at 8-10 wk of age.

HS protocol. Wild-type and IL-6-deficient mice were randomly subjected to either the HS or sham protocol as described (20). Briefly, mice were anesthetized initially using intraperitoneal pentobarbital sodium (50 mg/kg). Both femoral arteries were surgically prepared and cannulated. The left artery was used for continuous blood pressure monitoring. The right artery was used for blood withdrawal and blood and fluid administration. Animals were subjected to HS by withdrawal of blood (2.25 ml/100 g body wt) over 10 min to achieve a mean arterial pressure (MAP) of 30 mmHg. MAP was maintained at 30 mmHg for 3 h with continuous monitoring of blood pressure and withdrawal and return of blood as needed. Cannulas, syringes, and tubing were flushed with heparin sodium (1,000 U/ml) before all procedures. The animals were resuscitated to a MAP of >= 80 mmHg by administration of the remaining shed blood plus intra-arterial injection of 1 ml of saline over 30 min. Mice were killed by exsanguination 4 h after resuscitation. Time-matched sham control animals underwent cannulation, anesthesia, and monitoring procedures for an identical period of time as shock animals but were not bled. There were six animals in each shock and sham group.

Isolation and examination of lung and liver. After the mice were killed, the carcasses were flushed with cold (4°C) saline. The right lung was fixed with paraformaldehyde solution (4%) by inflation under a constant and fixed pressure (20 cm water). The left lung was frozen in liquid nitrogen. A portion of the liver was placed in paraformaldehyde (4%); another portion was frozen in liquid nitrogen. Paraformaldehyde-fixed specimens were sectioned and stained for myeloperoxidase (MPO) with hematoxylin and eosin (H+E) using standard procedures as described (17). The stained slides were examined at ×50-600 magnification. For quantitation of PMN infiltration, 10 randomly chosen (×400) fields of each lung and liver specimen stained with MPO were blindly scored for the number of intensely stained MPO-positive PMN as described (17).

Quantitation of alveolar wall cross-sectional surface area and focal liver necrosis. To quantitatively assess lung injury in sham and HS animals, we microscopically examined the H+E stained lung sections and measured the cross-sectional surface area of the alveoli. Five randomly chosen alveoli in each lung specimen were blindly measured by OPTIMAS identification techniques (Bioscan, Washington, DC) using a computer imaging device as described (25, 27). Images of the alveolar wall were optimized by adjusting contrast, gray scale, and exposure time and manually outlined for cross-sectional surface area quantitation. The same process was used to quantitate focal liver necrosis, except that the total area of focal liver necrosis on each slide section was measured and the area of focal liver necrosis was normalized for total area of liver examined.

Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) was performed using whole tissue extracts of frozen sections of liver as described (17). Briefly, binding reactions were performed using 20 µg of extracted protein and 32P-radiolabeled DNA binding elements. EMSA was performed using a 4% of polyacrylamide gel. STAT3 activity was assessed using the hSIE (high-affinity serum-inducible element) duplex oligonucleotide probe that is preferentially bound by homodimers or heterodimers of STAT3 and STAT1 (31). The mobility of the hSIE/STAT protein complex on EMSA gels depends on dimer composition with STAT3 homodimers [serum-inducible factor (SIF)-A complex] migrating slowest, STAT1 homodimers (SIF-C complex) migrating fastest, and STAT3/STAT1 heterodimers (SIF-B complex) having intermediate mobility. NF-kappa B activity was determined by EMSA using a 32P-radiolabeled duplex oligonucleotide based on the well-characterized NF-kappa B binding site within the murine inducible nitric oxide synthase promoter (11). The level of transcription factor activation of NF-kappa B and STAT3 were quantitated using PhosphorImager analysis of the appropriate gel shift band as described (17).

Statistical analysis. Data are presented as means ± SE. Differences within groups were determined using one-way ANOVA. Statistical significance of one-way ANOVA was assessed using Duncan's multiple range test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of HS on lung PMN infiltration and injury. We have previously shown that the lungs of rats harvested 4 h after resuscitated HS exhibited increased inflammation and injury compared with sham-treated animals (17). Wild-type mice subjected to HS also exhibited increased PMN infiltration and lung injury compared with sham controls (Fig. 1), as evidenced by increased pneumocyte swelling and infiltration of PMN into the interstitium and alveoli. In contrast, PMN infiltration and lung injury were less evident in IL-6-deficient mice subjected to HS (Fig. 1).


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Fig. 1.   Photomicrograph sections of inflated lungs. Wild-type (WT) and interleukin-6 (IL-6)-deficient mice (-/-) were subjected to the sham or hemorrhagic shock (HS) protocol (n = 6). Lungs were inflated and fixed in paraformaldehyde and then stained with hematoxylin and eosin (H+E; A) or myeloperoxidase (B). Slides were photographed at ×600 magnification.

PMN infiltration in the lungs of wild-type and IL-6-deficient mice was quantitated by counting MPO-positive PMN in 10 randomly selected lung fields (Fig. 2). HS increased lung PMN infiltration by 19-fold in wild-type mice (P < 0.05). In contrast, HS did not increase lung PMN infiltration in IL-6-deficient mice significantly above sham controls.


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Fig. 2.   Polymorphonuclear neutrophilic (PMN) infiltration into lungs of wild-type and IL-6-deficient mice following HS. Wild-type and IL-6-deficient mice were subjected to the sham (SH) or HS protocol (n = 6). Lungs of mice were inflated and fixed in paraformaldehyde, sectioned, and stained for myeloperoxidase (MPO). Slides were examined at ×400 magnification, and the number of MPO-positive PMN was counted in 10 randomly chosen fields. The data were pooled and are presented as group means ± SD. *Differs statistically from all other groups (P < 0.05).

To determine whether the effects of HS on PMN infiltration correlated with lung damage, we quantitated lung injury in each group (Fig. 3) by determining alveolar cross-sectional surface area as an aggregate measurement of pneumocyte swelling, interstitial edema, and cellular infiltration as previously described (23). HS increased alveolar cross-sectional surface area by 44% in wild-type animals compared with sham controls (P < 0.05). In contrast, alveolar cross-sectional surface area did not increase significantly in IL-6-deficient animals subjected to HS compared with sham controls.


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Fig. 3.   Alveolar wall cross-sectional surface area in wild-type and IL-6-deficient mice subjected to HS. Mice (n = 6) were subjected to sham or HS protocol. Lungs were inflated and fixed in paraformaldehyde and then sectioned and stained with H+E. Slides were examined at ×600 magnification. The alveolar wall cross-sectional surface area was quantitated by OPTIMAS technique using computer-assisted video imaging (A). The cross-sectional surface area of 5 random alveoli was measured in each lung specimen, the data were pooled from each group, and the means ± SD of each group are presented in B. *Differs statistically from each other (P < 0.05).

Effects of HS on liver PMN infiltration and necrosis. We previously demonstrated that HS caused liver damage measured by increased plasma transaminase levels in rats and mice (17) and by extent of focal liver necrosis in rats (23). In the current study, we demonstrate that the liver injury induced by HS in wild-type mice also consists of focal hepatocellular necrosis (Fig. 4), which is accompanied by PMN infiltration (Figs. 4 and 5). Wild-type mice subjected to HS demonstrated a 29-fold increase in liver PMN infiltration compared with sham controls (P < 0.01), whereas no significant difference in PMN infiltration was detected between IL-6-deficient animals subjected to the sham and HS protocol. Similarly, quantitation of focal liver necrosis in wild-type mice demonstrated a 58-fold increase in liver necrosis compared with sham controls (P < 0.01), whereas no significant difference was detected between IL-6-deficient sham and HS groups (Fig. 6).


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Fig. 4.   Photomicrograph sections of liver from wild-type and IL-6-deficient mice following sham or HS protocol. Liver were fixed in paraformaldehyde and stained with H+E (A) or MPO (B). Slides were photographed at ×200 magnification.



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Fig. 5.   PMN infiltration into liver of wild-type and IL-6-deficient mice following HS. The liver of sham and shock animals (n = 6) were fixed in paraformaldehyde, sectioned, and stained for MPO. Slides were examined at ×400 magnification, and the number of MPO-positive PMN were counted in 10 randomly chosen fields. The data were pooled and are presented as group means ± SD. **Differs statistically from all other groups (P < 0.01).



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Fig. 6.   Focal liver necrosis in wild-type and IL-6-deficient mice following HS. Paraformaldehyde-fixed portions of liver of each animal were sectioned and stained with H+E. Slides were examined, and each area of focal liver necrosis was measured by the OPTIMAS technique using computer-assisted video imaging (A). The amount of focal necrosis was totaled over the slide and normalized for total surface area of the section examined. The data from each group (n = 6) were pooled, and the group means ± SD plotted (B). **Differs statistically from all other groups (P < 0.01).

IL-6 is the predominant cytokine contributing to STAT3 activation in liver in HS. IL-6 is a central cytokine regulating the acute phase response of the liver. This effect is mediated, in part, through the activation of STAT3 by IL-6. We have previously demonstrated rapid and concurrent IL-6 production and STAT3 activation in liver of rats subjected to HS (18, 19). Previous studies demonstrated markedly diminished STAT3 activation in liver of IL-6-deficient mice following a localized inflammatory stimulus (subcutaneous turpentine injection) but near normal activation of STAT3 in liver of IL-6-deficient mice following a systemic inflammatory stimulus (intraperitoneal injection of bacterial lipopolysaccharide, LPS) (2). To directly assess if IL-6 is an important mediator of STAT3 activation in liver in HS, we measured STAT3 activity in whole liver extracts of wild-type and IL-6-deficient mice following HS (Fig. 7). Although STAT3 activity in wild-type mice subjected to HS was increased 35-fold over sham controls (P < 0.01), STAT3 activity in IL-6-deficient mice was not increased significantly in shock vs. sham animals, indicating that IL-6 alone is sufficient to account for STAT3 activation in liver following resuscitated HS.


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Fig. 7.   Signal transducers and activators of transcription (STAT3) activity within liver of wild-type and IL-6-deficient mice following HS. Whole liver extracts (20 µg) from 6 animals in each group were subjected to electrophoretic mobility shift assay (EMSA) using radiolabeled high-affinity serum-inducible element duplex oligonucleotide. Autoradiographs of the shifted bands (A). The positions of the STAT3 homodimer [serum-inducible factor (SIF)-A], STAT1 homodimer (SIF-C), and STAT3/STAT1 heterodimer (SIF-B) are shown (right). The amount of STAT3 homodimer was quantitated using PhosphorImager analysis of the SIF-A bands, and the means ± SD for each group are plotted (B). **Differs from the other groups (P < 0.01).

NF-kappa B activation following HS is reduced in IL-6-deficient mice. We have previously demonstrated that reduction of induced nitric oxide, either pharmacologically in rats or genetically in mice, results in decreased PMN recruitment and organ damage; this effect was mediated, in part, by reduced NF-kappa B activity (17). To assess whether or not the decrease in PMN infiltration and organ damage observed in IL-6-deficient mice may be mediated through impaired NF-kappa B activation, we measured NF-kappa B activity in liver extracts of wild-type and IL-6-deficient mice following HS (Fig. 8). As observed previously in the lungs of mice subjected to HS, HS induced a nearly threefold increase in NF-kappa B activity in the liver compared with sham controls (P < 0.05). In contrast, there was no increase in NF-kappa B activity in livers of IL-6-deficient mice following HS compared with sham controls (Fig. 8).


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Fig. 8.   Nuclear factor (NF)-kappa B activity within the liver of wild-type and IL-6-deficient mice following HS. Whole liver extracts (20 µg) from 6 animals in each group were subjected to EMSA using radiolabeled kappa B duplex oligonucleotide. Autoradiographs of the shifted bands (A). The amount of NF-kappa B band in each extract was quantitated using PhosphorImager analysis, and the means ± SD for each group are plotted (B). *Differs from the other groups (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our findings demonstrate that compared with HS in wild-type mice, HS in IL-6-deficient mice does not result in PMN infiltration into the lung or liver and does not increase lung alveolar cross-sectional surface area or focal liver necrosis. In addition, similar to its role in the acute phase response of liver in models of localized inflammation, IL-6 is the major mediator of STAT3 activation in liver following HS. Finally, activation of NF-kappa B was impaired in liver of IL-6-deficient mice following HS, which may contribute to the impaired inflammatory response seen in these animals.

Review of the literature on the role of IL-6 in inflammation suggests that the net effect of IL-6 on the host inflammatory response reflects a balance of two opposing effects, a paracrine effect of IL-6 that promotes inflammation, and an endocrine effect of IL-6 that diminishes inflammation. Previous studies with IL-6-deficient mice in infectious disease models demonstrated increased susceptibility to listeriosis (9, 22) and to infections with vaccinia virus (22), Escherichia coli (10), and Candida albicans. These studies indicated that IL-6 is an important component of the local inflammatory and immune response directed at limiting infection caused by a variety of infectious agents. In studies using noninfectious disease models and localized insults, IL-6-deficient mice demonstrated 1) a defective inflammatory response following subcutaneous injection of turpentine (13), 2) a defect in chemokine production resulting in diminished recruitment of neutrophils into tissue sites (26), 3) markedly reduced joint damage in a model of collagen-induced arthritis (3), and 4) resistance to splanchnic artery occlusion, a localized ischemic-reperfusion injury (8). In complementary types of studies, overexpression of IL-6 in the pancreas of transgenic mice promoted local inflammation (7) and instillation of IL-6 into the lungs of normal rats, resulting in lung inflammation and injury (19).

In addition to its local proinflammatory effects, several in vitro and in vivo studies have shown that IL-6 has anti-inflammatory effects. Exposure of cells to IL-6 or intraperitoneal injection of IL-6 was demonstrated to inhibit tumor necrosis factor-alpha (TNF-alpha ) and IL-1beta production in response to LPS and to protect against LPS toxicity, respectively (1, 4, 32). IL-6 also completely protected mice from mortality in a model of staphylococcal enterotoxin-induced toxic shock (5). We previously observed dramatically diminished lung and liver inflammation and injury in rats subjected to HS and resuscitated with fluids that contained IL-6 (23). IL-6 administration to cancer patients increased circulating levels of IL-1 receptor antagonist and soluble TNF-alpha receptor p55 (29). Overall, these findings are consistent with the role of circulating IL-6 in downmodulating the inflammatory response, especially in the setting of another local or systemic inflammatory insult.

Our findings reported here extend the findings of Cuzzocrea et al. (8) by demonstrating that IL-6 is essential not only for a localized ischemia-reperfusion injury but also a total body ischemia-reperfusion injury such as HS. In addition, our findings of reduced organ inflammation and injury in IL-6-deficient mice indicate that, in the case of HS, the local proinflammatory effects of endogenous IL-6 dominate over its systemic anti-inflammatory effects.

In a model of hepatic ischemia-reperfusion injury, Camargo et al. (6) demonstrated that systemic infusion of IL-6 resulted in reduced mortality, reduced hepatic injury, and reduced hepatic production of C-reactive protein and TNF-alpha , all consistent with an anti-inflammatory role of IL-6 in this model of liver insult. However, in contrast to our findings and those of Cuzzocrea et al. (8), in IL-6-deficient mice, Camargo et al. (6) demonstrated a small but significantly increased hepatic injury in IL-6-deficient mice subjected to hepatic ischemia-reperfusion injury. These results suggest that local endogenous production of IL-6 by liver may be protective of liver injury following a direct ischemia-reperfusion insult and raise the possibility that local IL-6 production in the setting of direct liver injury may serve predominantly an anti-inflammatory role.

IL-6 is the primary inducer of acute phase protein production by the liver in response to a variety of inflammatory insults (14). We have previously shown that STAT3 activation occurs in hepatocytes of Listeria-infected wild-type mice but not in IL-6-deficient mice and can be abrogated in wild-type mice by Kupffer cell depletion (15). Absence of acute phase protein production in IL-6-deficient mice following turpentine injection was accompanied by absence of STAT3 activation in the liver (2), supporting a critical role for STAT3 in acute phase protein production in response to a local inflammatory stimulus. Our finding of absent liver STAT3 activation in IL-6-deficient mice in HS indicates that IL-6 is the critical activator of STAT3 in HS. Studies are under way to determine whether or not upregulation of acute phase protein mRNA levels is also blunted in IL-6-deficient mice subjected to HS.

NF-kappa B is a member of the Rel transcription family that is activated in response to a variety of inflammatory stimuli including IL-1, TNF-alpha , oxidative stress, ultraviolet and ionizing radiation, and LPS. Activated NF-kappa B, in turn, transcriptionally activates a variety of proinflammatory genes including granulocyte colony-stimulating factor (G-CSF) and IL-6 (17, 28). Similar to IL-6-deficient mice, we have previously demonstrated reduced lung and liver inflammation and injury following HS in mice deficient in the gene for induced nitric oxide; this reduction in inflammation and injury was accompanied by reduced NF-kappa B activation and reduced G-CSF and IL-6 mRNA (17). Examinations are under way to determine whether impaired NF-kappa B activation in IL-6-deficient mice subjected to HS is accompanied by diminished induction of proinflammatory cytokines, such as G-CSF, and to determine whether or not physiological levels of IL-6 may be needed for normal levels of expression of components of the NF-kappa B activation and signaling cascade.


    ACKNOWLEDGEMENTS

We are grateful to Jeffrey Baust for excellent technical assistance in the hemorrhagic shock protocol.


    FOOTNOTES

This work was supported by National Institutes of Health Grants GM-53789 and CA-72261.

Address for reprint requests and other correspondence: D. J. Tweardy, Section of Infectious Diseases, Baylor College of Medicine, One Baylor Plaza, BCM 286, Rm. N1319, Houston TX 77030 (E-mail: dtweardy{at}bcm.tmc.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.

Received 8 June 2000; accepted in final form 6 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 280(2):C343-C351
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