Increased prostacyclin and PGE2 stimulated cAMP production by macrophages from endotoxin-tolerant rats

Michel A. Makhlouf1, Lawrence P. Fernando1, Thomas W. Gettys2, Perry V. Halushka2,3, and James A. Cook1

Departments of 1 Physiology, 2 Medicine, and 3 Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Sublethal administration of lipopolysaccharide (LPS) renders rats tolerant to multiple lethal stimuli. Tolerant macrophages exhibit differential alterations in LPS-stimulated cytokine and inflammatory mediator release. Increased cAMP levels stimulated by PGE2 or prostacyclin (PGI2) result in differential effects on LPS-induced cytokine release and protect against the pathophysiological changes of endotoxemia. In the present studies, we sought to determine whether PGE2- and PGI2-stimulated cAMP levels are altered in tolerant macrophages. Incubation of macrophages with cicaprost or 11-deoxy-PGE1 in the presence of phosphodiesterase inhibitors resulted in significantly higher (2.5- to 6.5-fold) cAMP concentrations in tolerant macrophages compared with control. In contrast, isoproterenol-stimulated cAMP levels were not significantly different between control and tolerant cells. Also, incubation of tolerant macrophages with LPS did not result in significantly elevated cAMP levels. Prostacyclin (IP) receptor mRNA levels were significantly increased in tolerant cells compared with controls, whereas [3H]PGE2 binding and PGE2 EP4 receptor mRNA levels were not significantly changed. These studies suggest that LPS tolerance induces selective alterations in eicosanoid regulation of cAMP formation.

G proteins; pertussis toxin; forskolin; cicaprost; lipopolysaccharide; prostaglandin E2

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

REPEATED SUBLETHAL ADMINISTRATION of lipopolysaccharide (LPS) to humans and laboratory animals results in resistance to lethal doses of LPS and other noxious stimuli. This phenomenon is termed LPS tolerance (3, 8, 28). Peritoneal macrophages obtained from tolerant animals exhibit selective reductions in LPS-stimulated cytokine and inflammatory mediator production. Arachidonic acid (AA) metabolites are reduced (9), whereas the production of other mediators such as interleukin-6 and nitric oxide is maintained or even increased (6, 37). LPS tolerance does not appear to be because of changes in LPS receptors, but rather to altered intracellular signal transduction mechanisms (12, 20, 25).

cAMP is a central modulator of several macrophage functions. It affects cytokine release (14, 35), cytoskeletal function (17, 27), tumoricidal activity (33), ion channel function (30), immune complex phagocytosis (1), and procoagulant activity (7). PGE2- and prostacyclin (PGI2)-mediated increases in macrophage cAMP modulate the effect of LPS in this cell type. In addition, PGE2 and PGI2 analogs reduce LPS-stimulated tumor necrosis factor release in human mononuclear cells (10), increase nitric oxide production in liver macrophages (15), and ameliorate the pathophysiological changes in animal models of endotoxemia (4, 22).

Our previous studies demonstrated reduced tolerant macrophage membrane content of Gsalpha and Gialpha (23) compared with control cells. The changes in Gsalpha and Gialpha raised the possibility that agonist-stimulated cAMP production would be altered in macrophages obtained from LPS-tolerant rats. Additionally, stimulation of macrophages by LPS results in the release of inflammatory mediators known to increase cAMP levels. These include, among others, PGE2 and PGI2. In the present studies, we sought to determine whether PGE2- and PGI2-stimulated cAMP levels are changed in tolerant macrophages, and whether these changes affect macrophage cAMP levels after stimulation by LPS. Macrophages and macrophage membrane cAMP levels were determined after 15-min stimulations with cicaprost, a PGI2 analog, 11-deoxy-PGE1, a selective agonist for the PGE2 EP2 and EP4 receptor isoforms and forskolin. Macrophage cAMP levels were also measured after a 4-h stimulation by LPS. To determine whether LPS tolerance affects the expression of EP and prostacyclin (IP) receptors, membrane [3H]PGE2 binding and Northern blot analysis for the EP4 and IP receptors were also performed.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Unless stated otherwise, all materials were purchased from Sigma (St. Louis, MO) or Fisher (Pittsburgh, PA). Cicaprost was a gift from Schering (Germany), and 11-deoxy-PGE1 was purchased from Cayman Chemical (Ann Arbor, MI).

Tolerance induction. Male Long-Evans rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing 250-300 g were housed under controlled temperature and illumination and given food and water ad libitum. Tolerance was induced by intraperitoneal injection of Salmonella enteritidis endotoxin (Boivin preparation) for two consecutive days at doses of 100 and 500 µg/kg body wt, respectively. The experiments were performed 72 h after the final administration of endotoxin. All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina.

Cell culture. Macrophages were isolated by peritoneal lavage from ether-anesthetized rats with RPMI 1640 media. For measurements of cellular cAMP levels, macrophages were plated in 24-well plates at a density of 106 cells/well. Macrophages were allowed to adhere for 2 h at 37°C in the presence of 5% CO2. Adherent cells were washed three times with 5% dextrose and incubated with 100 µM IBMX, a phosphodiesterase inhibitor, and 10 µM indomethacin, a cyclooxygenase inhibitor for 15 min. Cells were then stimulated with various concentrations of cicaprost or isoproterenol (in the presence 0.2 mg/ml ascorbate) for 15 min. In separate experiments, macrophages were stimulated for 4 h with LPS (10 µg/ml) in the presence or absence or phosphodiesterase inhibitors. Cellular reactions were stopped by the addition of ice-cold ethanol. Samples were stored overnight at -20°C. The next day, cells and media were centrifuged at 2,000 g for 20 min, and the supernatant was dried in a Speed-Vac centrifuge. The dried samples were resuspended in 50 mM sodium acetate, pH 4.75, and assayed for cAMP as described below.

Preparation of membranes. Macrophage membranes were prepared by hypotonic lysis and centrifugation at 100,000 g as previously described (23).

Membrane adenylyl cyclase assay. The assay was performed as described by Gettys et al. (16) with modifications. The assay buffer consisted of 50 mM HEPES, pH 7.4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 0.5 µg/ml pepstatin A, 150 mM NaCl, 0.5 U/ml adenosine deaminase, 500 µM IBMX, 10 µM indomethacin, 1 mM EDTA, 25 U/ml creatine phosphokinase, 2 mM phosphocreatine, and 100 µM ATP. For forskolin-stimulated adenylyl cyclase activity, the buffer also contained 5 mM MnCl2. For cicaprost-, isoproterenol-, and 11-deoxy-PGE1-stimulated adenylyl cyclase activity, the buffer contained 1.5 mM MgCl2 and 5 µM GTP. The reaction was started by the addition of 50 µl membrane proteins (5-10 µg) to 250 µl assay buffer. Samples were incubated at 30°C for 15 min. The reaction was stopped by the addition of 50 µl of 25% trichloroacetic acid. The samples were centrifuged at 2,000 g for 20 min, and supernatant aliquots were taken for cAMP radioimmunoassay.

cAMP radioimmunoassay. The cAMP radioimmunoassay was performed according to the method described by Harper and Brooker and co-workers (5, 18) and as modified by Gettys et al. (16). cAMP standards (19 pM to 10 nM) and samples were prepared in 1 ml of 50 mM sodium acetate, pH 4.75. Samples and standards were acetylated by the addition of 20 µl triethylamine and 10 µl acetic anhydride. Aliquots (50 µl) were then transferred to new tubes, and 25 µl of 125I-cAMP (Linco, St. Louis, MO) and 25 µl of rabbit anti-cAMP antibody were added. After overnight incubation at 4°C, 50 µl of anti-rabbit IgG-coated magnetic beads were added. One hour later, 1 ml of a 12% polyethylene glycol (mol wt 8,000) solution was added to each tube. The samples were centrifuged at 1,800 g for 20 min. The pellet was washed with polyethylene glycol, and the tubes were centrifuged again. Radioactivity in the pellet was determined in a gamma counter. Sample cAMP levels were extrapolated from a log-logit standard curve.

Radioligand binding. Binding buffer consisted of 10 mM Tris · HCl, pH 6.5, 10 mM MgCl2, 1 mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin, and 0.5 µg/ml pepstatin A. Initial experiments demonstrated maximal binding at 1 h. The binding reaction was started by the addition of 35 µg of membrane proteins to binding buffer containing 0.5-5 nM [3H]PGE2 (sp act 171 Ci/mmol, New England Nuclear, Boston, MA) in the presence of 0-250 nM unlabeled PGE2 for 1 h. The binding reaction was stopped by the addition of 5 ml ice-cold binding buffer. The samples were rapidly vacuum filtered through GF/C Whatman glass filters, and the filters were washed twice to separate bound from unbound PGE2. Total [3H]PGE2 bound was determined after scintillation counting. Nonspecific binding was determined in the presence of 1 µM PGE2 and was 30% of total. Data were analyzed by the method of Scatchard (32) using the software program Ligand (26).

Northern blotting. Macrophages were scraped into 1 ml Ultraspec RNA (Biotecx, Houston, TX) solution. The suspension was passed through a 27-gauge needle to shear the DNA and then left on ice for 5 min. Chloroform (0.2 ml) was then added, and the samples were mixed well, left on ice for 5 min, and then centrifuged for 10 min at 10,000 g. The resulting supernatant was collected, and RNA was precipitated by the addition of an equal volume of isopropanol. The mixture was left on ice for 10 min and then centrifuged for 10 min at 10,000 g. The RNA pellet was washed twice with 70% ethanol, dried, and resuspended in diethylpyrocarbonate-treated water. The concentration of RNA in the sample was calculated from optical density (OD) values at 260 nm.

Electrophoresis and Northern blotting and hybridization were carried out as described by Sambrook et al. (31). Total RNA (10-15 µg) was separated on denaturing formaldehyde (2.2 M) agarose gels by electrophoresis in MOPS buffer (20 mM MOPS, 8 mM sodium acetate, and 1 mM EDTA pH 7.0). The RNA was transferred onto nylon membrane by capillary blotting. Hybridization of the membrane was carried out at 60°C in 3× SSC (0.45 M NaCl, 0.045 M sodium citrate, pH 7.0), 10× Denhardt's solution, salmon sperm DNA, 0.1% SDS, and 106 counts · min-1 (cpm) · ml-1 of the 32P-labeled probes for the rat IP or EP4 receptors. The membrane was washed with 1× SSC and 0.1% SDS, followed by another wash with 0.5× SSC and 0.1% SDS and exposed to film. Blots were densitometrically scanned, and the optical density of tolerant band was reported as percent of control. Equal loading of RNA was ascertained using ethidium bromide staining and visualization under ultraviolet light. We were unable to use glyceraldehyde-3-phosphate dehydrogenase or beta -actin to control for equal loading, since LPS has been shown to affect their mRNA levels (Cook, unpublished observations).

The cDNA for the rat IP and EP4 receptors was a kind gift from Dr. I Tanaka, Kyoto University, Japan. The 2.2-kb EcoR I cDNA fragment and 1.3-kb EcoR I fragment were purified from a 1% agarose gel. The probes were prepared by the random priming method using a kit from Bio-Rad (Hercules, CA) and [32P]dCTP from Amersham (Arlington Heights, IL). The specific activity of the probes was in the range of 108 to 109 cpm/µg DNA.

Statistical analysis. Statistical analysis of cAMP measurements was performed using the analysis of variance followed by Fisher's protected least-significant difference test. Statistical analysis for the binding data and Northern blot analysis were performed using an unpaired t-test. Data are expressed as means ± SE.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cicaprost and isoproterenol stimulated cAMP levels in macrophages. To determine whether cAMP production is altered in tolerant macrophages in response to extracellular agonists, control and tolerant macrophages were incubated in the presence of increasing concentrations of cicaprost or isoproterenol. Stimulation of macrophages with cicaprost resulted in a concentration-dependent increase in cAMP. Maximal cicaprost (1 µM)-stimulated cAMP levels were 2.5-fold higher (n = 7, P < 0.05) in tolerant macrophages compared with control cells (Fig. 1A). Isoproterenol increased cAMP in macrophages (n = 2 or 3), but the concentration did not differ between control and tolerant cells (Fig. 1B). In two separate experiments, 11-deoxy-PGE1 stimulated higher cAMP levels in tolerant macrophages compared with control (data not shown).


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Fig. 1.   Cicaprost (A) and isoproterenol (B) stimulated cAMP levels in control and tolerant macrophages. Macrophages were incubated with 100 µM IBMX and 10 µM indomethacin for 15 min, followed by 15-min stimulation with different concentrations of cicaprost or isoproterenol and in presence of IBMX and indomethacin. Data are expressed as means ± SE for 7-14 experiments (A) and 2 or 3 experiments (B). CON, control; TOL, tolerant. * P < 0.05 vs. respective control.

Macrophage membrane adenylyl cyclase activity. To determine receptor-dependent coupling in a broken cell preparation, agonist-dependent adenylyl cyclase activation was compared in macrophage membranes from control and tolerant rats. All three agonists increased cAMP levels in a concentration-dependent manner in control and tolerant membranes (Figs. 2, A and B, and 3A). Maximal cAMP levels were not significantly different between control and tolerant membranes; however, the increase over basal levels was significantly elevated in tolerant membranes treated with cicaprost (3.5- vs. 6.5-fold times basal for control and tolerant, respectively, n = 3, P < 0.05) and 11-deoxy-PGE1 (2.6- vs. 4.6-fold times basal for control and tolerant, respectively, n = 3, P < 0.05) but not with isoproterenol (4.7- vs. 4.8-fold times basal for control and tolerant, respectively, n = 2 or 3). Incubation of control and tolerant macrophage membranes with butaprost, a selective EP2 agonist, failed to increase cAMP levels (n = 4, data not shown).


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Fig. 2.   Cicaprost (A) and 11-deoxy-PGE1 (B) stimulated adenylyl cyclase activity in macrophage membranes. cAMP levels are expressed as fold times basal. Data are expressed as means ± SE of 3 experiments. * P < 0.05 vs. respective control.


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Fig. 3.   Isoproterenol (A) and forskolin (B) stimulated adenylyl cyclase activity in macrophage membranes. cAMP levels are expressed as pmol · mg-1 · min-1 for forskolin-stimulated adenylyl cyclase activity or as fold times basal for isoproterenol-stimulated activity. Data are expressed as means ± SE of 2 or 3 experiments (A) and 3-5 experiments (B). * P < 0.05 vs. respective control.

Because a change in adenylyl cyclase content could affect agonist-stimulated cAMP levels, macrophage membrane adenylyl cyclase activity was measured in the presence of forskolin, a direct activator of adenylyl cyclase. Forskolin-stimulated adenylyl cyclase activity was reduced by 40% (n = 3-5, P < 0.05) in tolerant membranes compared with control (Fig. 3B).

Effect of pertussis toxin. To determine whether the increased cicaprost-stimulated cAMP levels in tolerant macrophages are because of reduced inhibition of adenylyl cyclase by Gialpha , macrophages were preincubated with pertussis toxin for 4 h (9) and stimulated with 100 nM cicaprost. Once again, cicaprost-stimulated cAMP levels were increased in tolerant cells compared with control. Pertussis toxin pretreatment (0.1-10 ng/ml) did not significantly increase cAMP production stimulated by cicaprost (Fig. 4).


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Fig. 4.   Effect of pertussis toxin on cicaprost-stimulated cAMP levels in control and tolerant macrophages. Macrophages were incubated for 4 h with pertussis toxin, then incubated for 15 min with 100 µM IBMX and 10 µM indomethacin followed by stimulation with 100 nM cicaprost in presence of IBMX and indomethacin. Data are expressed as means ± SE of 3 experiments. * P < 0.05 vs. respective control.

[3H]PGE2 binding. The increased 11-deoxy-PGE1-stimulated cAMP levels in tolerant macrophages suggested the possibility of an upregulation of the expression of PGI2 and PGE2 receptors. Therefore, the density of EP receptors was determined using radioligand binding assays. [3H]PGE2 binding in macrophage membranes was saturable and displaceable. The equilibrium binding data were best fit by a single-site model (Fig. 5). The dissociation constant values for control and tolerant membranes were 1.2 ± 0.1 nM (n = 6) and 1.6 ± 0.2 nM (n = 6), respectively, and the maximum binding values were 42 ± 4 fmol/mg protein (n = 6) and 59 ± 9 fmol/mg protein (n = 6), respectively. These values, however, were not significantly different between control and tolerant cells.


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Fig. 5.   Representative Scatchard analysis of [3H]PGE2 binding to control and tolerant macrophage membranes. Membranes were incubated at 30°C in presence of 0.5-5 nM [3H]PGE2 and 0-250 µM unlabeled PGE2. Dissociation constant values for this experiment were 1.4 and 1.6 nM for control and tolerant membranes, respectively, and maximum binding values were 38 and 48 fmol/mg protein for control and tolerant membranes, respectively. Results are representative of 6 experiments.

Northern blot analysis. To determine whether EP4 and IP receptor mRNA levels are altered in tolerant macrophages, Northern blot analyses for EP4 and IP receptor mRNA were performed (Fig. 6A). Northern blots were densitometrically scanned, and the ODs of the control and tolerant bands were determined (Fig. 6B). There were no significant differences in the EP4 mRNA levels (tolerant OD/control OD = 99 ± 11%, n = 5); however, IP receptor mRNA levels were significantly elevated in tolerant cells (tolerant OD/control OD = 159 ± 26%, n = 5, P < 0.05).


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Fig. 6.   A: representative Northern blots of control and tolerant EP4 and IP receptors. Total RNA was isolated from macrophages, and Northern blotting was performed. Results are representative of 5 experiments. B: blots were densitometrically scanned, and optical densities of tolerant bands were expressed as percent of control. Data shown are means ± SE of 5 experiments.* P < 0.05 vs. control.

LPS-stimulated cAMP production. To determine whether tolerant macrophages produce more cAMP in response to LPS, macrophages were incubated with LPS for 4 h, and intracellular cAMP levels were determined. LPS caused a marginal, but significant, increase in cAMP levels in control cells treated with phosphodiesterase inhibitors (Fig. 7). There was no difference in basal or LPS-stimulated cAMP levels between control and tolerant macrophages not exposed to phosphodiesterase inhibitors.


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Fig. 7.   Effect of lipopolysaccharide (LPS) on macrophage cAMP levels. Macrophages were incubated for 4 h with media or 10 µg/ml LPS in presence or absence of 500 µM IBMX and 10 µM rolipram (ROL). Data are means ± SE of 3 experiments. * P < 0.05 vs. other IBMX + ROL groups.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Tolerant macrophages have been found to be associated with a 30-50% reduction in Gialpha , Gsalpha , and beta -subunit content (23). This observation raised the possibility that agonist-stimulated cAMP formation could be altered in LPS tolerance. The present study demonstrates that LPS tolerance leads to selective regulation of agonist-stimulated cAMP formation. Cicaprost and 11-deoxy-PGE1-stimulated cAMP levels were significantly greater in tolerant macrophages and macrophage membranes, whereas isoproterenol-stimulated cAMP levels were not significantly altered. These changes occurred in the presence of phosphodiesterase inhibitors, suggesting that they were not caused by increased degradation of cAMP. Additionally, the increased formation of cAMP in response to cicaprost and 11-deoxy-PGE1 was not because of a decreased inhibitory effect of Gialpha , since pertussis toxin did not alter the responses. Also, the response to isoproterenol, which is mediated by Gsalpha was not altered by tolerance. The response to forskolin, a direct stimulator of the catalytic subunit of adenylyl cyclase, was decreased in the tolerant macrophage membranes. Thus it would appear that some additional factor(s) may be responsible for producing the agonist-specific changes in cAMP responses. Alternatively, because there are multiple adenylyl cyclases in macrophages, it is possible that they may be coupled to different receptors, which in turn results in differential effects on cAMP formation.

Because cicaprost- and 11-deoxy-PGE1- but not isoproterenol-stimulated cAMP formation was increased in tolerant membranes, we determined the density of EP4 and IP receptors. Maximal specific PGE2 binding and EP4 mRNA levels were not significantly increased in tolerant cells. This suggests that the increased cAMP formation in response to 11-deoxy-PGE1 is because of covalent modification of the EP4 receptor, such as phosphorylation, resulting in enhanced signal transduction, or to cross-activation of the IP and/or other prostanoid receptors by 11-deoxy-PGE1. We were unable to determine IP receptor density in macrophage membranes because of the low specific activity of the PGI2 analog [3H]iloprost and apparent low density of receptors. However, IP receptor mRNA levels were increased in tolerant macrophages, raising the possibility that IP receptor density was increased. In contrast to our observations, incubation of RAW264.7 cells for 3 h with LPS resulted in increased EP4 and suppressed IP receptor mRNA levels (2). These differences in the effect of LPS may be because of the time of exposure and the mode of administration of LPS (in vivo vs. in vitro). However, the increased formation of cAMP in response to 11-deoxy-PGE1 is unlikely to be because of increases in receptor density.

Agents that increase cAMP levels in macrophages mimic the effect of tolerance. Increases in cAMP levels stimulated by PGE2 suppress endotoxin-stimulated tumor necrosis factor production in human monocytes (10, 11) and macrophages (13) and enhance the accumulation of nitrites in macrophages (34) and Kupffer cells (15). Furthermore, administration of PGE2 (4), phosphodiesterase inhibitors, or cAMP analogs (19) to animals confer protection against the lethal effects of endotoxin. This raised the possibility that the increased prostaglandin-stimulated cAMP levels in tolerant macrophages may contribute to the development of LPS tolerance. However, incubation of macrophages with LPS for 4 h, a time at which macrophage eicosanoid production is near maximal, increased cAMP levels in control macrophages only in the presence of phosphodiesterase inhibitors and had no effect on tolerant macrophage cAMP levels. Tolerant macrophages exhibit reduced basal and LPS-stimulated PGI2 and PGE2 (29). The increased response of these cells to PGE2 and PGI2 analogs may therefore be a compensatory mechanism for their reduced synthesis. Thus the altered response of tolerant macrophages to LPS is not because of an upregulation of cAMP production.

Several mechanisms have been proposed to explain LPS tolerance. These include downregulation of G proteins (23), induction of protein repressors (21), inactivation of transcription factors (36), or translational regulation of cytokine production (24). Our data demonstrate that LPS tolerance selectively alters eicosanoid regulation of the cAMP signal transduction pathway. Whether the changes in cAMP production in vitro contribute to LPS tolerance in vivo remains to be determined. This would necessitate selective inhibition of macrophage cAMP production in vivo and assessing its effect on LPS tolerance. These studies and others suggest that tolerance is not because of a change in a single intracellular pathway, but because of a collection of mechanisms, each contributing to the altered response to LPS. The functional significance of the changes in the cAMP signal transduction cascade in LPS tolerance merits further investigation.

    ACKNOWLEDGEMENTS

This work was supported in part by National Institute of General Medical Sciences Grant GM-27673.

    FOOTNOTES

Address for reprint requests: J. A. Cook, Dept. of Physiology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425.

Received 21 October 1997; accepted in final form 26 January 1998.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 274(5):C1238-C1244
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society




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