Role of Manganese Superoxide Dismutase and Protein Kinase C |
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Abstract |
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Abstract
Introduction
References
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The mechanism by which pertussis toxin (Ptx) causes lung edema is not clear. We investigated the role of
pulmonary manganese superoxide dismutase (MnSOD) and protein kinase C (PKC) in Ptx-induced lung
edema. We demonstrated that intraperitoneal injection of Ptx at a concentration of 5 µg/100 g body weight
caused a similar degree of lung edema in 2 d, as measured by lung wet weight/dry weight ratio, in heterozygous MnSOD gene (Sod2)-knockout mice (Sod2+/
) and in their wild-type littermates (Sod2+/+).
The level of lung MnSOD activity in Sod2+/ mice was approximately half that of Sod2+/ mice. Ptx had
no effect on levels of lung MnSOD messenger RNA, immunoreactive protein, or enzyme activity in either
Sod2+/+ or Sod2+/ mice. Ptx also had no effect on lung copper-zinc SOD, catalase, and glutathione peroxidase activities in these mice. On the other hand, Ptx caused the activation of lung PKC, for example, by
translocation of a 72-kD PKC isoform from the cytosolic fraction to the membrane fraction. Pretreatment
of mice with bisindolylmaleimide, a PKC inhibitor, prevented both the Ptx-induced activation of PKC and lung edema. These data suggest that Ptx-induced lung edema in mice is, at least in part, due to the activation of lung PKC.
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Introduction |
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Abstract
Introduction
References
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Pertussis toxin (Ptx) is an exotoxin produced by Bordetella pertussis, the bacterium that causes whooping cough in humans. In 1957, Andersen (1) reported that intranasal inoculation of the mouse-virulent strain Haemophilus pertussis or its extract caused lung edema in mice. Immunization either with a bacterial vaccine or with the extract of a mouse-virulent strain was unable to protect against lung edema induced by the extract, whereas it protected mice against infection with living organisms and hence prevented lung edema (1). The factor in the bacteria or the extract that causes lung edema is now known to be Ptx. However, the mechanism by which Ptx induces lung edema is not clear.
Recently, Clerch and colleagues (2) reported that intraperitoneal (i.p.) injection of Ptx (5 µg/100 g body weight [BW]) induced lung edema in young rats (21- to 35-d-old) within 12 h. Ptx-induced lung edema was associated with a selective reduction (50%) of pulmonary manganese superoxide dismutase (MnSOD) activity, was attenuated in a hypoxic (15% O2) environment, and was exaggerated by hyperoxic (95% O2) exposure. In addition, enhancing pulmonary MnSOD by endotoxin treatment attenuated Ptx-induced lung edema. It was therefore concluded that Ptx-induced lung edema was due to normoxic O2 toxicity; for example, a 50% reduction of lung MnSOD activity by Ptx rendered young rats sensitive to O2 toxicity even at the ambient O2 concentration (20% O2) (2).
More recently, Patterson and associates (3) reported that Ptx caused a marked increase in albumin permeability of bovine pulmonary artery endothelial cell monolayers, and that the Ptx-induced increase in endothelial permeability was mediated by the activation of protein kinase C (PKC). Activation of PKC by phorbol ester, diacylglycerol, or tumor necrosis factor has been shown to cause increased albumin permeability of endothelial monolayers (4, 5), as well as lung edema in the isolated, perfused lung (5) and in the whole animal (8, 9). Thus, activation of PKC by Ptx may be one mechanism by which Ptx causes lung edema.
In the current study we investigated the role of pulmonary MnSOD and PKC in Ptx-induced lung edema in
mice. We took advantage of the availability of MnSOD
gene (Sod2)-knockout mice (10, 11). Our results demonstrated that Ptx had no effect on pulmonary MnSOD in
both heterozygous (Sod2+/) mutant mice and their normal (Sod2+/+) littermates. On the other hand, Ptx activated
lung PKC, and inhibition of PKC activation prevented
the Ptx-induced lung edema, suggesting that Ptx-induced
lung edema in mice is, at least in part, due to PKC activation.
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Materials and Methods |
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Materials
Ptx was obtained from List Biological Laboratory, Inc. (Campbell, CA). The PKC inhibitor bisindolylmaleimide (BIM) (GF-109203X) and monoclonal anti-PKC antibody (mouse immunoglobulin G2a [IgG2a] isotype, clone MC5, mouse ascites fluid) were purchased from Sigma Chemical Co. (St. Louis, MO). Rabbit antihuman MnSOD antibody, which cross-reacts with mouse MnSOD, was a gift of Dr. Larry Oberley, University of Iowa (Iowa City, IA). Horseradish peroxidase (HRP)-labeled donkey antirabbit and sheep antimouse IgG antibodies and enhanced chemiluminescence (ECL) Western blotting detection reagents were obtained from Amersham Life Science (Arlington Heights, IL).
MnSOD Gene-Knockout Mice
MnSOD gene-knockout mice (Sod2mlucsf) were obtained
by deletion of exon 3 of the Sod2 gene using targeted homologous inactivation as described by Li and coworkers
(10). Male, heterozygous mutant mice (Sod2+/) back-crossed for six generations (N6) to C57BL/6 mice were obtained from Dr. Charles J. Epstein of the University of
California (San Francisco, CA) and were bred with normal
C57BL/6J female mice (Jackson Laboratories, Bar Harbor, ME) at the animal research facility of Stratton VA
Medical Center, Albany, NY (11). All cages, bedding,
feed, and water were autoclaved, and cages were placed in
a ventilated rack.
Genotypes of mutant mice were determined as described previously (11). Briefly, DNA was obtained by overnight digestion at room temperature of mouse toes with genomic DNA isolation reagent (DNAzol; Molecular Research Center, Inc., Cincinnati, OH) and amplified by polymerase chain reactions (PCRs) of mutant and normal fragments using one shared primer (5'-CGAGGGGCATCTAGTAGTGGAGAAG-3') and one primer for the normal (5'-TTAGGCTCAGGTTGTCCAGAA-3') or mutant (5'-CACACATCGGGAAAATGGTTG-3') allele. PCR was performed using the following reaction conditions: pre-PCR incubation at 95°C for 5 min followed by 40 cycles of: 95°C, 30 s; 60°C, 30 s; 72°C, 60 s in a 25-µl reaction mixture containing DNA (20 to 100 ng), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM deoxynucleotide triphosphates, 0.125 µM primer set, and 0.725 U AmpliTaq Gold DNA polymerase (Perkin-Elmer, Applied Biosystems Division, Foster City, CA), followed by agarose gel electrophoresis. The normal allele produced a ~ 500-base pair (bp) band, and the mutant allele produced a ~ 350-bp band (10, 11).
All homozygous mutant (Sod2/) mice died within 1 d
of birth (11), whereas the heterozygous mutant (Sod2+/)
mice were phenotypically normal (10, 11). Adult Sod2+/
mice and their normal littermates (Sod2+/+), 2 to 4 mo old,
were used in the current study.
Treatment of Mice
Mice were treated with i.p. injection of 0. 2 ml Hank's balanced salt solution (HBSS) containing various amounts of Ptx (3, 5, or 8 µg/100 g BW), or HBSS alone as control. In some experiments, animals were pretreated with tracheal insufflation of 0.05 ml HBSS containing 25 µM BIM, or HBSS alone as control, 1 h prior to i.p. injection of Ptx. Briefly, for tracheal insufflation, mice were anesthetized with methoxyflurane and intubated with a 21-gauge intravenous catheter (Angiocath; Becton Dickinson, Sandy, UT) as described previously (12). HBSS, 50 µl, containing 25 µM BIM, followed by 100 µl of air, was injected into the lungs through the intratracheal catheter. Control mice were injected with 50 µl HBSS alone, followed by 100 µl of air. Auscultation of the chest with a stethoscope was performed to ensure that the solution was injected into the lungs. Exposure of mice to 100% O2 was performed as described previously (11). Mice were given free access to water and food.
Measurement of Lung Edema
At 2 d after i.p. injection of Ptx or O2 exposure, mice were killed by pentobarbital sodium and exanguinated by transection of the inferior vena cava, and the chest was opened to obtain pleural effusion (PE) for measurement of PE volume. Lungs were removed, trimmed, blotted, and weighed to obtain lung wet weight (ww). They were then placed in a 65°C oven for 2 d and weighed to obtain lung dry weight (dw). The lung ww/dw ratio was then calculated.
Measurement of Pulmonary Antioxidant Enzyme Activities
Preparation of lung extracts for enzyme assays was performed as described previously (11). Briefly, mice were killed and exanguinated by transection of the inferior vena cava. Lungs were removed, trimmed, blotted, and weighed. The right lung was homogenized using a tissue homogenizer in 0.5 ml potassium phosphate buffer (0.05 M, pH 7.5) containing 1.0 mM ethylenediamenetetraacetic acid (EDTA), sonicated twice for 30 s, and centrifuged at 15,000 × g for 10 min at 4°C. The supernate was then assayed for protein content using bicinchoninic acid according to Smith and associates (13), and for enzyme activities and MnSOD immunoreactive protein. The left lung was homogenized and washed extensively in perchloric acid (0.5 N) at 4°C; the precipitate was extracted in 1 ml 1.5 N perchloric acid by boiling for 20 min, and assayed for DNA content using the diphenylamine reaction according to Richards (14).
The SOD activity was assayed using nondenaturing
polyacrylamide gel (PAG, 10%) electrophoresis according
to Beauchamp and Fridovich (15), as described previously
(11). The assay was based on the inhibitory effect of SOD
on the reduction of tetrazolium by O2 generated by photochemically reduced riboflavin. This method allowed simultaneous visualization and quantification of MnSOD and copper-zinc SOD (CuZnSOD) activities. The SOD
activity gel was then quantified using a computing densitometer (Molecular Dynamics, Sunnyvale, CA). In each assay, purified Escherichia coli MnSOD (Sigma; 4,000 U/mg
as determined according to McCord and Fridovich [16])
and bovine erythrocyte CuZnSOD (Sigma; 4,400 U/mg)
were used to obtain standard curves from which the lung extract MnSOD and CuZnSOD activities, respectively, were
derived. The catalase activity was determined according to
Bergmeyer (17) as modified previously (12), and the glutathione (GSH) peroxidase activity was determined according to Paglia and Valentine (18). The results were expressed
as the amount of enzyme activity (units) per milligram DNA
to minimize artifacts due to variations in lung size and lung
weight (12, 19), for example, Ptx-induced lung edema.
Immunoblot Analysis of Pulmonary MnSOD
The lung extracts were further analyzed for MnSOD immunoreactive protein using Western blot as described previously (19). Briefly, lung extracts (30 µg protein/lane) were electrophoresed in denaturing sodium dodecyl sulfate (SDS)-PAG (10%) according to Laemmli (20) and transferred electrophoretically to nitrocellulose membrane. The membrane was then blocked with 5% nonfat milk in phosphate-buffered saline plus 0.1% Tween 20, washed, and incubated overnight at 4°C with primary antibody, rabbit antihuman MnSOD (1:1,000). After washing, the membrane was incubated with HRP-labeled secondary antibody, donkey antirabbit IgG (1:1,000) for 1 h, followed by reaction with ECL Western blotting detection reagents according to the manufacturer's instructions (Amersham). The chemiluminescence signal was detected by exposure to X-ray film, quantified using a computing densitometer, and expressed as relative (densitometric) units per milligram DNA.
Northern Blot Analysis of Pulmonary MnSOD Messenger RNA (mRNA)
Northern blot analysis of pulmonary MnSOD mRNA was performed as described previously (19). Briefly, at 1 d after i.p. injection of Ptx, mice were killed and lungs were removed and extracted for total cellular RNA using an RNeasy Midi Kit (Qiagen, Inc., Santa Clarita, CA).
For Northern blots, denatured RNA samples (15 µg/ lane) were electrophoresed in 1.2% agarose-formaldehyde gels, transferred to nylon membrane (Genescreen Plus; New England Nuclear, Boston, MA) by capillary blotting, and stained with methylene blue to visualize the quality and size of 18S and 28S ribosomal RNA species. The membrane was then prehybridized as described previously (19). Hybridization was carried out with 100 µg/ml denatured salmon testis DNA and a murine MnSOD complementary DNA probe that had been labeled by random hexanucleotide priming (GIBCO BRL, Gaithersburg, MD) to a specific activity of > 109 cpm/µg DNA. After washing, autoradiographs were obtained and radioactive signals were quantified using a computing densitometer.
Immunoblot Analysis of Pulmonary PKC
At 2 h after i.p. injection of Ptx or HBSS, mice were killed
by i.p. injection of pentobarbital sodium and exanguinated
by transection of the inferior vena cava. Lungs and brain
were removed and homogenized in 1.0 ml modified radioimmunoprotection assay (RIPA) buffer (50 mM Tris-HCl,
pH 7.4; 150 mM NaCl; 1 mM EDTA; 1 mM 4-[2-aminoethyl] benzene sulfonyl fluoride; 1 mM Na3VO4; 1 mM NaF;
and 10 µg/ml each of aprotinin, leupeptin, and pepstatin) in ice. The homogenates were centrifuged at 2,900 × g for
20 min at 4°C in prechilled centrifuge tubes to remove cell
debris and nucleus, followed by centrifugation at 29,000 × g for 45 min at 4°C to obtain the cytosolic fraction. The
pellets were resuspended in 0.5 ml ice-cold RIPA buffer
containing detergents (1% NP-40 and 0.25% Na deoxycholate) by vortexing for 15 min, followed by centrifugation at 15,000 × g for 20 min at 4°C. The supernates, designated as solubilized membrane fraction, were then stored in aliquots at 
70°C.
Immunoblotting was performed as described previously. Briefly, cytosolic and membrane fractions, 10 µg protein/ lane, were electrophoresed in denaturing SDS-PAG (10%), transferred to nitrocellulose membrane, blocked, washed, and incubated with primary antibody, mouse monoclonal anti-PKC (1:1,000 dilution), for 1 h at room temperature. After washing, the membrane was incubated with HRP- labeled secondary antibody, sheep antimouse IgG (1:2,000 dilution) for 1 h, followed by reactions with ECL Western blotting detection reagents. The chemiluminescent signal was detected by exposure to X-ray film and quantified using a computing densitometer.
Statistical Analysis
Data from two groups were compared by a two-tailed t test, and those from more than two groups were compared by one-way analysis of variance with Bonferroni's correction for multiple comparisons (21), using a commercially available statistical analysis program (SPSS Inc., Arlington, VA). Differences were considered significant at P < 0.05.
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Results |
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Ptx-Induced Lung Edema
Injection of Ptx i.p. caused a dose (3 to 8 µg/100 g BW)- dependent increase in lung ww/dw ratio, an index of lung edema, in normal (Sod2+/+) mice (Figure 1). No pleural effusion was noted in any of the control or Ptx-treated mice. The degree of Ptx-induced lung edema in mice observed in this study was comparable to that in 21- to 35-d-old rats as reported by Clerch and associates (2). The lung ww/dw ratio at 2 d after i.p. injection of Ptx at 5 µg/100 g BW in rats reported by Clerch and colleagues (2) was 5.98 ± 0.08 versus control, 5.39 ± 0.06 (n = 5 in each group); similar figures for Sod2+/+ mice in our study were 5.03 ± 0.10 and 4.44 ± 0.10 (n = 5 in each group), respectively (Figure 1).
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To assess the role of pulmonary MnSOD in Ptx-induced
lung edema, we first examined the effect of Ptx in heterozygous Sod2-knockout mice (Sod2+/), which we have previously shown to have approximately 50% pulmonary MnSOD activity as compared with their normal littermates (Sod2+/+) (11). As shown in Figure 2, i.p. injection
of 5 µg/100 g BW Ptx also caused lung edema, (e.g., an increase in lung ww/dw ratio) in Sod2+/ mice (P value,
Sod2+/ Ptx versus Sod2+/ control, P < 0.005). However,
there was no difference between Sod2+/+ and Sod2+/
mice. Also shown in Figure 2 is the effect of hyperoxia on
lung ww/dw ratio in Sod2+/+ and Sod2+/ mice. Exposure
of mice to 100% O2 for 2 d caused only a slight increase in
lung ww/dw ratio, and there was no difference between Sod2+/+ and Sod2+/ mice, consistent with our previous
observation that Sod2+/ mice were not more susceptible
to pulmonary O2 toxicity than were Sod2+/+ mice (11). It is
evident that 5 µg/100 g BW of Ptx appeared to cause more
lung edema in mice than 2 d of 100% O2 exposure. All
subsequent experiments were performed using 5 µg Ptx/
100 g BW.
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Effect of Ptx on Pulmonary Antioxidant Enzyme Activities
Clerch and coworkers (2) reported that i.p. injection of Ptx in young (21- to 35-d-old) rats selectively reduced pulmonary MnSOD activity that was thought to be responsible for Ptx-induced lung edema. Therefore, we determined the effect of Ptx on pulmonary MnSOD and CuZnSOD activities in mice. This was performed using SOD activity gels that allowed simultaneous visualization and quantification of MnSOD and CuZnSOD activities.
As shown in Figure 3, mouse MnSOD and CuZnSOD
could be readily separated using PAG electrophoresis
(PAGE). Note that we used purified E. coli MnSOD and
bovine erythrocyte CuZnSOD, which migrated to different positions from those of mouse enzymes (11), to construct standard curves for the purpose of densitometric
quantification of mouse SOD activities. Because lung
CuZnSOD activity was much higher than that of MnSOD,
for the quantification of MnSOD activities, 200 µg protein
per lane was used in the presence of 5 mM KCN to inhibit
partially the activities of CuZnSOD (Figure 3A). For lung CuZnSOD activity, a separate gel using 12.5 µg protein
per lane was used (Figure 3B). It is obvious that Sod2+/
mice had reduced lung MnSOD activities (Figure 3A),
whereas CuZnSOD activities were comparable to those of
Sod2+/+ mice (Figure 3B). In addition, Ptx had no effect
on MnSOD or CuZnSOD activities in either Sod2+/+ or
Sod2+/ mice.
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Figure 4 summarizes the results of pulmonary antioxidant enzyme activities in mice treated with or without Ptx.
As expected, lung MnSOD activities in Sod2+/ mice were
approximately half those of Sod2+/+ mice, whereas CuZnSOD, catalase, and GSH peroxidase activities were similar
in both groups. At 2 d after injection, Ptx had no effect on
MnSOD, CuZnSOD, catalase and GSH peroxidase activities in either Sod2+/+ or Sod2+/ mice.
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Effect of Ptx on Pulmonary MnSOD Immunoreactive Protein and mRNA
The effect of Ptx on lung MnSOD was further studied using immunoblot and Northern blot analyses. As shown in
Figure 5, the level of MnSOD immunoreactive protein in
Sod2+/ mutants was reduced compared with that of
Sod2+/+ mice. Furthermore, Ptx had no effect on the level
of lung MnSOD immunoreactive protein in either Sod2+/+
or Sod2+/ mice. These data were consistent with the results for MnSOD enzyme activity as shown in Figures 3
and 4.
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Northern blot analysis revealed that the level of lung
MnSOD mRNA, expressed as MnSOD mRNA/18S RNA
ratio, in Sod2+/ mutants was similar to that of wild-type
Sod2+/+ mice. Furthermore, Ptx had no effect on the level
of pulmonary MnSOD mRNA in either Sod2+/+ or Sod2+/
mice (Figure 6).
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The observation that Sod2+/ mutants had levels of
lung MnSOD mRNA similar to that of Sod2+/+ mice,
whereas levels of MnSOD immunoreactive protein and
enzyme activity were significantly lower in Sod2+/ mice
than in Sod2+/+ mice, was surprising. The most likely explanation is that in this MnSOD gene-knockout mutant,
the mutant MnSOD gene is transcribed but the mutant
mRNA is nonfunctional, resulting in no MnSOD immunoreactive protein or enzyme activity. The murine MnSOD
gene consists of five exons, and in this knockout mutant
(Sod2mlucsf) exon 3, which encodes 39 amino acids involved
in homodimerization, tetramer formation, and manganese
binding, was deleted (10). As shown in Figure 6A, we were
unable to distinguish mutant MnSOD mRNA from normal mRNA by the size difference in Northern blotting, likely due to polyadenydation of the MnSOD mRNA (22).
To confirm this possibility, Northern blot of RNA extract
from fetal heart of one homozygous Sod2/ mutant revealed abundant MnSOD mRNA, whereas no MnSOD
immunoreactive protein or enzyme activity in the myocardial extract of Sod2/ mutant was detectable in the immunoblot and activity gel, respectively (data not shown).
Role of PKC in Ptx-Induced Lung Edema
Because Ptx had no effect on mouse lung MnSOD, we
next examined the role of PKC activation in Ptx-induced
lung edema. Because Sod2+/+ and Sod2+/ mice showed
similar responses to Ptx, we pooled the data from these two
groups together in this study. We used the PKC inhibitor BIM, which blocks the adenosine triphosphate-binding domain and thus inhibits all PKC isotypes (23). BIM was
given by tracheal insufflation 1 h prior to i.p. injection of
Ptx. As shown in Figure 7, BIM alone had no effect on
lung ww/dw ratio. However, it completely abolished the
Ptx-induced increase in lung ww/dw ratio, suggesting that
Ptx-induced lung edema might be mediated by PKC activation.
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Activation of lung PKC by Ptx was further confirmed by immunoblotting of lung cytosolic and membrane fractions. The PKC antibody used was a mouse monoclonal antibody raised against purified bovine brain PKC. This antibody recognizes an epitope located within the amino acid sequence 296-317, at the hinge region close to or at the trypsin cleavage site of PKC (24), and is known to cross-react with mouse PKC. As shown in Figures 8A and 8B, two bands of immunoreactive proteins were noted in control mouse lung (approximately 80 and 72 kD) (lanes 1 and 2) and brain (approximately 80 and 38 kD) (lane 9). In the lung cytosolic fraction (Figure 8A), only the 72-kD band was noted. In contrast, the 80-kD band was the dominant band in the membrane fraction (Figure 8B). On the other hand, in brain the 80-kD band was the dominant protein in both cytosolic and membrane fractions.
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At 2 h after i.p. injection of Ptx, there was a shift of 72-kD PKC from the cytosolic fraction to the membrane fraction (Figures 8A and 8B, lanes 3 and 4) as compared with controls (lanes 1 and 2), suggesting activation of PKC. The membrane-to-cytosol (M/C) ratio of the 72-kD band increased from 0.6 in the control group to 1.7 in the Ptx-treated group (Figure 8C). Pretreatment with BIM increased the 72-kD band in the cytosolic fraction (Figures 8A and 8B, lanes 5 and 6), resulting in a reduced M/C ratio. On the other hand, pretreatment of BIM abolished Ptx-induced translocation of 72-kD PKC from the cytosolic fraction to the membrane fraction (lanes 7 and 8).
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Discussion |
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The results presented in the current study demonstrated that
Ptx: (1) caused a similar degree of lung edema in Sod2+/+
and Sod2+/ mice; (2) had no effect on pulmonary antioxidant enzyme activities, including MnSOD; and (3) activated lung PKC. Furthermore, prevention of lung PKC activation by a PKC inhibitor, BIM, abolished Ptx-induced lung edema. These results suggest that Ptx-induced lung
edema in mice is, at least in part, due to PKC activation.
Clerch and associates (2) reported that Ptx, while stimulating the steady-state level of pulmonary MnSOD mRNA,
inhibited the enzyme activity. We were unable to demonstrate any effect of Ptx on the levels of pulmonary MnSOD
mRNA, immunoreactive protein, or enzyme activity. The
reason for the discrepancy between the current study and
that of Clerch and coworkers (2) is not clear. It could be
due to species differences. Clerch and associates used young
(21- to 35-d-old) rats, while we used 2 to 4-mo-old Sod2+/+
and Sod2+/ mice. In addition, the methods used for assaying SOD activity were different. In the current study, we
used an SOD activity gel, which allowed direct quantification of MnSOD and CuZnSOD activities individually after
separation of the enzymes by PAGE. On the other hand, in
the study of Clerch and colleagues (2), MnSOD activity was
assayed in the presence of diethyldithiocarbamate (DDC)
to inhibit the activity of CuZnSOD (25) in a whole-lung extract using the ferricytochrome c reduction method. Thus,
the accuracy of MnSOD activity measurement is dependent
on the complete and selective inhibition of CuZnSOD activity by the concentration of DDC used. In lung tissue,
CuZnSOD constitutes the majority of SOD, so any residual CuZnSOD activity will profoundly affect the measured
MnSOD activity. Unfortunately, the assay itself does not
allow investigators to ascertain whether all CuZnSOD activity has, in fact, been completely and selectively inhibited by DDC. Whether this difference in methodologies
for the SOD assay accounts for the observed difference in
the effect of Ptx on lung MnSOD is not clear.
The recent availability of SOD gene-knockout mice has
provided valuable insight into the role of various SOD
isozymes in the host defense against O2 toxicity. Despite
being the major cellular SOD, homozygous CuZnSOD
gene-knockout mice (Sod1/) with no CuZnSOD activity
develop and survive normally for months in room air with
no apparent abnormalities (26). Furthermore, they are no
more susceptible to 100% O2 toxicity than are their wild-type (Sod1+/+) littermates (27). Likewise, homozygous extracellular SOD gene-knockout mice (Sod3/) are phenotypically normal in room air; however, when exposed to
a lethal dose of O2 (> 95%), they develop severe lung injury earlier and have a shorter survival than their wild-type (Sod3+/+) littermates (28). In contrast, homozygous
MnSOD gene-knockout mice (Sod2/) die shortly after
birth in room air, with extensive mitochondrial injury and
dilated cardiomyopathy (10, 29). How long they survive
depends on the genetic background; for example, Sod2/
mutant mice on a CD-1 background die within 10 d (10);
on a C57BL/6J background (the one used in the current
study), they die within 1 d after birth (11); and on a mixed
background, they survive up to 18 d (29). Li and colleagues (10) reported that at 4 to 5 d after birth, the lung
water content of Sod2/ mice (on a CD-1 background)
was similar to that of normal Sod2+/+ littermates, suggesting that in mice with no MnSOD activity there was no evidence of lung edema even after 4 to 5 d of exposure to
room air (20% O2). We have demonstrated that there is no
increase in the susceptibility of Sod2+/ mice with 50% of
normal lung MnSOD activity to hyperoxia (e.g., 100% O2)
as compared with their Sod2+/+ littermates (11). These observations are in marked contrast to the conclusion by
Clerch and coworkers (2) that a 50% reduction of lung
MnSOD activity in 21- to 35-d-old rats rendered them susceptible to room air O2 toxicity with lung edema developing within 2 d. Whether this is due to species differences
needs further investigation.
Our observation that Ptx activated lung PKC and that
inhibition of PKC activation prevented Ptx-induced lung
edema suggests that Ptx-induced lung edema in mice is
mediated through PKC activation. This is consistent with
the report of Patterson and coworkers (3), who demonstrated that Ptx-induced increase in albumin permeability
of cultured bovine pulmonary endothelial cell monolayers was due to PKC activation. The mechanism by which Ptx
activates PKC is not clear. Ptx is a hexameric protein with
typical AB architecture, consisting of an A protomer (S1)
and a B oligomer (S2, S3, two S4's, and S5) (30). The A
protomer has adenosine diphosphate (ADP)-ribosyltransferase activity, which catalyzes the transfer of ADP-ribose
from nicotinamide adenine dinucleotide to a regulatory guanosine triphosphate-binding protein (G protein) in eukaryotic cells (31). The B oligomer also has some biologic
activities (independent of the enzymatic activity of the A
protomer), such as the activation of platelets and mitogenicity for lymphocytes (30, 32). Through ADP-ribosylation, Ptx inactivates a subclass (
i) of heterotrimeric G
proteins involved in transmembrane signal transduction.
Some of the functions of i subclass G proteins are the inhibition of adenylyl cyclase, the regulation of K+ and Ca2+
channels, and the activation of cyclic guanosine monophosphate phosphodiesterase (31, 33). Whether Ptx-induced activation of PKC, as demonstrated in the current study and
that of Patterson and colleagues (3), is mediated through
ADP-ribosylation and inactivation of G-proteins is not clear.
Ptx has been used extensively as a powerful tool to selectively inhibit Gi proteins in studying signal transduction
pathways (31, 33). The demonstration that Ptx can activate
PKC, leading to impaired endothelial barrier function and
lung edema, raises the important question of the specificity and interpretation of results obtained when Ptx is used
to inhibit G proteins.
In the current study we used a broad-spectrum PKC inhibitor, BIM, that inhibits all PKC isotypes (22). Likewise, the PKC monoclonal antibody used reacts with a number of PKC isotypes (24 and current study). Thus, we were unable to determine which PKC isotype(s) was activated by Ptx. However, we noticed that the molecular weight of the Ptx-sensitive PKC was approximately 72 kD. Further studies are necessary to identify the PKC isotype that is activated by Ptx, and the mechanism by which Ptx activates PKC.
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Footnotes |
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Address correspondence to: Min-Fu Tsan, M.D., Ph.D., Research Service (151), Stratton VA Medical Center, 113 Holland Ave., Albany, NY 12208. E-mail: tsan.min-fu{at}albany.va.gov
(Received in original form March 23, 1998 and in revised form July 13, 1998).
Abbreviations: adenosine diphosphate, ADP; bisindolylmaleimide, BIM; body weight, BW; copper-zinc SOD, CuZnSOD; diethyldithiocarbamate, DDC; enhanced chemiluminescence, ECL; glutathione, GSH; Hanks' balanced salt solution, HBSS; horseradish peroxidase, HRP; immunoglobulin, Ig; intraperitoneal, i.p.; membrane-to-cytosol ratio, M/C ratio; manganese SOD, MnSOD; messenger RNA, mRNA; polyacrylamide gel, PAG; PAG electrophoresis, PAGE; polymerase chain reaction, PCR; protein kinase C, PKC; pertussis toxin, Ptx; superoxide dismutase, SOD; lung wet weight/dry weight ratio, lung ww/dw ratio.Acknowledgments: The authors thank Arnold Johnson, Ph.D., for valuable advice on the PKC studies. This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.
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References |
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Abstract
Introduction
References
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