Department of Medicine, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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ABSTRACT |
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To investigate the
role of carbonic anhydrase (CA) II on pulmonary
CO2 exchange, we analyzed arterial
blood gases from CA II-deficient and normal control mice. CA
II-deficient mice had a low arterial blood pH (7.18 ± 0.06) and
concentration
([
]; 17.5 ± 1.9 meq/l) and a high PCO2
(47.4 ± 5.3 mmHg), consistent with mixed respiratory and metabolic
acidosis. To eliminate the influence of metabolic acidosis on arterial
blood gases, NaHCO3 (4 mmol/kg body weight) was given intraperitoneally, and arterial blood gases were
analyzed 4 h later. Normal mice had a small increase in pH and were
able to maintain PCO2 and
[
]. The metabolic
acidosis in CA II-deficient mice was corrected
([
], 22.9 ± 2.4 meq/l), and respiratory acidosis became more profound (PCO2, 50.4 ± 2.4 mmHg). These results indicate that CA II-deficient mice have a partial
respiratory compensation for metabolic acidosis. We conclude that CA
II-deficient mice have a mixed respiratory and metabolic acidosis. It
is most likely that CO2 retention
in these animals is due to CA II deficiency in both red blood cells and
type II pneumocytes.
carbon dioxide exchange; arterial blood gas; metabolic acidosis; lung
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INTRODUCTION |
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CARBONIC ANHYDRASES (CAs; EC 3.2.1.1) are a family of metalloenzymes that catalyze the equilibration of CO2 and carbonic acid. It has been known that pulmonary CA plays an important role in pulmonary CO2 exchange. Several studies (4-6) using either animal models or isolated perfused lungs showed that inhibition of CA activity reduces the quantity of CO2 exchange significantly. There are a variety of CA isozymes in the lung. CA I is present in the lung cellular homogenate (12) and pulmonary arterial endothelial cells (19), CA III is in the apical pole of ciliated bronchiolar epithelia (22), and CA IV is in the membrane of pulmonary capillary endothelia (8). Recently, Fleming et al. (7) identified CA II in type II pneumocytes. Because of the unique location, it was speculated that CA II has important roles in pulmonary functions such as the regulation of fluid secretion and facilitation of CO2 elimination (7).
In humans, CA II deficiency results in renal tubular acidosis, osteopetrosis, and brain calcification (21). Ohlsson et al. (17) first reported mixed respiratory and metabolic acidosis in two CA II-deficient children who had low arterial blood pH and inappropriately high PCO2 for the metabolic acidosis. It was postulated that an abnormality of the rib cage secondary to osteopetrosis and/or CA II deficiency itself may cause respiratory acidosis. Lewis et al. (15) produced a strain of CA II-deficient mice by an induced mutagenesis. Initial studies established that the mice were not able to acidify urine after an acid load, consistent with renal tubular acidosis. Unlike human CA II deficiency, CA II-deficient mice do not show any evidence of osteopetrosis or brain calcification (15). Without any apparent abnormality of the rib cage, the CA II-deficient mouse can serve as an ideal animal model for investigating the role of CA II in pulmonary functions. In the present study, we undertook arterial blood gas analyses on CA II-deficient mice and demonstrated that CA II deficiency is associated with CO2 retention.
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MATERIALS AND METHODS |
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Animals. CA II-deficient mice were bred and maintained at the University of Arizona (Tucson) Animal Care Facility in accordance with National Institutes of Health guidelines. The original pair of heterozygous breeders, C57BL/6J CAR-2, were purchased from Jackson Laboratory (Bar Harbor, ME). The resulting F1 male homozygous mutants were crossed to normal female C3H strain mice, and the heterozygous F2 females were backcrossed to the F1 male mutants. The resulting homozygous deficient mice have a similar phenotype in terms of growth curves and urine pH values (both baseline and after acid load) compared with the original C57BL/6J CAR-2 mutant (14). Mice weighing 15-25 g were used for arterial blood gas analyses.
Western blot analysis. To confirm that CA II is present in the lung of the normal mouse and absent in CA II-deficient mice, a Western blot of lung homogenate was performed. Mice were anesthetized with pentobarbital sodium (60 mg/kg body weight intraperitoneally) and perfused through the heart with 30 ml of phosphate-buffered saline (pH 7.4) to remove red blood cells (RBCs) that contain CA II. The lung tissues from normal and CA II-deficient mice were homogenized separately in phosphate-buffered saline containing 0.2 mM phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO), freeze-thawed three times in liquid nitrogen and a water bath, and centrifuged at 14,000 g for 30 min at 4°C. The supernatant was collected, and the total protein concentration was determined with the method described by Bradford (2). An aliquot of 100 µg of total protein was resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane with an electroblotting apparatus (Hoefer Scientific Instruments, San Francisco, CA). Nonspecific protein binding was blocked by incubation of the blot for 1 h at room temperature in 5% nonfat milk in tris(hydroxymethyl)aminomethane (Tris)-buffered saline (TBS; 150 mM NaCl and 50 mM Tris · HCl, pH 7.4). The blot was incubated overnight at 4°C in sheep anti-human CA II (1:500 dilution; Binding Sites, San Diego, CA), which is known to cross-react with mouse CA II, in TBS-2% fetal calf serum-0.05% Tween 20, washed with TBS-0.05% Tween 20 three times, incubated for 1 h at room temperature with anti-sheep antibodies conjugated with alkaline phosphatase (1:10,000 dilution; Sigma), and washed with TBS-0.05% Tween 20 three times and TBS for the final wash. Color reaction was carried out in a 4-nitro blue tetrazolium chloride-5-bromo-4-chloro-3-indolyl phosphate solution (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's instructions.
Arterial blood gas analysis. Normal, CA II-deficient, and heterozygous mice were anesthetized with pentobarbital sodium. The mouse was placed on a warming pad to prevent loss of body heat. The carotid artery was exposed, and 0.15 ml of arterial blood was withdrawn into a heparinized syringe. Blood gas analysis was immediately performed on the sample with an IL1640 pH/blood gas/electrolytes analyzer (Instrumentation Laboratory, Lexington, MA). To eliminate the effects of metabolic acidosis on respiration, CA II-deficient and normal mice were given NaHCO3 (4 mmol/kg body weight) intraperitoneally. Four hours later, the mice were anesthetized, and arterial blood gas analysis was performed.
Statistics. The data are presented as means ± SD. Comparison of the group means with the control values was performed with Student's t-test for unpaired data employing Scheffé's method to correct for multiple comparisons. Effects of the bicarbonate injection were evaluated by comparison of the data obtained before and after treatment with a paired Student's t-test. Significance levels are reported at the P < 0.01 and P < 0.05 levels.
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RESULTS |
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Expression of CA II protein in the lung. Figure 1 shows a representative Western blot of perfused lung homogenates from normal and CA II-deficient mice using antibodies against human CA II. Normal mouse lung extract contained a single band with an estimated molecular mass of 29 kDa. This band was absent in the sample from CA II-deficient mice. The CA II protein was also absent in the RBCs of CA II-deficient mice (data not shown), as previously reported by Lewis et al. (15) and Brion et al. (3).
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Arterial blood gas analyses. Table
1 shows the results of arterial blood gas
analyses on normal, CA II-deficient, and heterozygous mice. CA
II-deficient mice had a lower pH (7.18 ± 0.06 vs. 7.38 ± 0.03;
P < 0.01), lower
concentration ([
]; 17.5 ± 1.9 vs. 22.6 ± 1.7 meq/l; P < 0.01), and higher PCO2 (47.4 ± 5.3 vs. 38.1 ± 3.4 mmHg; P < 0.01). There was no difference in
PO2 between the two groups. The
results from the heterozygous mice were not statistically different
from those of the normal mice. Therefore, CA II-deficient mice, but not
heterozygous mice, had a mixed respiratory and metabolic acidosis.
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Effects of alkaline load on arterial blood
gases. Metabolic acidosis stimulates respiration, which
reduces the PCO2 and thereby returns
the blood pH back toward the normal range. To eliminate the effects of
metabolic acidosis on respiration and to investigate the respiratory
compensation in CA II-deficient mice, we corrected metabolic acidosis
with an alkaline load. Table 2 shows that
an injection of NaHCO3 increased
arterial blood pH slightly but did not affect
PCO2 and
[] in normal mice. In
CA II-deficient mice, the bicarbonate injection resulted in an increase
in both [
] and
PCO2. There was a slight increase in
pH, but the difference was not statistically significant. Thus the
correction of metabolic acidosis resulted in a more profound
respiratory acidosis in CA II-deficient mice.
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DISCUSSION |
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In this report, we present evidence for the first time that demonstrates that CA II-deficient mice have a mixed respiratory and metabolic acidosis. CA II-deficient mice are unable to blow off CO2 to compensate for metabolic acidosis adequately. In fact, they retain CO2 in the presence of profound metabolic acidosis. When metabolic acidosis was corrected with an injection of NaHCO3, CA II-deficient mice developed a more severe respiratory acidosis. These results indicate that CA II plays an important role in pulmonary CO2 elimination.
Our results were obtained while mice were under anesthesia with
pentobarbital sodium. Although the anesthesia may alter blood gas
tensions, it is unlikely that this will preferentially affect CA
II-deficient mice. Our observation that the correction of metabolic acidosis worsens respiratory acidosis, as predicted, further confirms the validity of our methodology. The results of the arterial blood pH,
PCO2, and
[] analyses of normal mice in our study are within the normal limits of larger animals, whereas the PO2 values are
significantly lower. A literature review revealed that mouse arterial
PO2 has been reported to be in a wide
range from 60 to 90 mmHg (20, 26). It is possible that the variation is
due to the effects of anesthesia and/or difficulties associated
with obtaining samples. Our results of blood
[
] in CA II-deficient
mice are in agreement with those reported by Lewis et al. (15). More
recently, Brion et al. (3) reported that the blood pH in CA
II-deficient mice was 7.25 (normal, 7.37) and
[
] was 17.6 meq/l (normal, 21.1 meq/l). Again, our data are consistent with theirs.
We speculate that a deficiency of CA II in both RBCs and type II
pneumocytes contributes to the development of respiratory acidosis. As
mentioned earlier, inhibition of CA activity reduces pulmonary
CO2 exchange (4-6). The
extent of respiratory acidosis is in agreement with the degree of CA
inhibition (1, 24, 27). The role of different CA isozymes on
CO2 exchange has been studied
using different CA inhibitors. Swenson et al. (23) demonstrated that
ethoxyzolamide, a lipophilic CA inhibitor, caused a 67% reduction in
CO2 production by measuring
PCO2 in the course of single
prolonged breaths in dogs. Benzolamide, a hydrophilic CA inhibitor, had
minimal effects on CO2 production.
These results indicate that RBC CA and pulmonary intracellular CA such
as CA II, but not the membrane-associated CA such as CA IV, make
important contributions to pulmonary gas exchange. Heming et al. (10) have shown that in isolated perfused lung studies in which RBCs were
absent, lipophilic CA inhibitors reduced
CO2 production by 26%, whereas a
dextran-bound CA inhibitor, which inhibits only membrane-associated CA,
had no effects. Heming et al. (11) subsequently published further work
using the same system except that a non-bicarbonate buffer,
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, was added to the perfusate and a different extracellular CA
inhibitor, quarternary ammonium sulfanilamide, was used. Their newer
data pointed to CA IV, but not to intracellular CA, as being important in CO2 elimination (11). These
different results are due to the dependency of CA IV activity on proton
availability or non-bicarbonate buffer capacity in the perfusate (9).
Swenson et al. (23) have shown that CA IV has a minimal contribution to
CO2 elimination in vivo. It is
possible that pulmonary CA IV is inhibited by plasma CA inhibitors, one
of which was recently identified by Roush and Fierke (18). In addition,
RBC hemoglobin, the major non-bicarbonate buffer in the
blood, is not available to the membranous CA IV in the capillary
endothelium. Therefore, it is likely that, under physiological
conditions, pulmonary CA IV activity is suppressed, whereas CA II in
type II pneumocytes is contributory to pulmonary CO2 elimination. The mechanism by
which CA II mediates pulmonary CO2
exchange in type II pneumocytes is not well understood. We speculate
that the mechanism is similar to that in RBCs. Nord et al. (16)
reported that a
Cl/
exchanger exists in type II pneumocytes, which is confirmed by Kemp and
Boyd (13). If this anion exchanger is localized in the basolateral
membrane, it can move bicarbonate into the type II pneumocyte, and
CO2 will be produced from
bicarbonate by CA II and released into the alveolar space. At the same
time, Cl
enters the
interstitial space and is removed by the circulatory system to keep the
"dry" alveolar state. Further studies are needed to test this
hypothesis.
Another important point to be addressed here is whether CA II-deficient
RBCs are functionally normal. In an earlier report on CA II-deficient
patients (25), RBCs are not functionally impaired in
CO2 transport because the total CA
activity is ~55% of the normal level. This activity is attributed to
the CA I isozyme. However, in CA II-deficient mice, the total CA
activity in RBCs is only 2% of the normal level (3). Although, CAs I
and III are present in CA II-deficient mouse RBCs, there is no
upregulation of these cytosolic CA isozymes. Therefore, it is likely
that CA II-deficient mouse RBCs are functionally impaired, whereas
human ones are not. The residual CA activity in RBCs of CA II-deficient mice may prevent the respiratory acidosis from being considerably more
severe, particularly when the animal's ventilation is limited by the
effects of anesthesia (4). If RBC CA activity is totally absent,
alveolar PCO2 should still equal
end-capillary PCO2 but be much less
than the value theoretically in equilibrium with
H+ and
in the capillary blood (4). But once blood leaves the capillaries, the uncatalyzed reaction continues, CO2 will be formed, and
PCO2 will rise (9, 11). Thus it is
possible that the true PCO2 in
arterial blood may be lower than what we reported. We attempted to
minimize this uncatalyzed reaction by placing blood samples on ice and
by measuring the blood gases immediately. In addition, the residual RBC
CA activity in CA II-deficient mice may influence the postcapillary CO2-
-H+
reaction. Last, it is possible that the respiratory center of CA
II-deficient mice may be defective; thus it cannot stimulate alveolar
ventilation to lower alveolar PCO2.
However, the fact that after correction of metabolic acidosis, the
respiratory acidosis became worse indicates that the respiratory center
does sense the changes in blood pH and influences ventilation as
expected.
In summary, we conclude that CA II in both RBCs and type II pneumocytes is involved in pulmonary CO2 exchange. Using organ-specific gene therapy in the CA II-deficient mouse, we may be able to delineate the contribution of each CA II to pulmonary elimination of CO2.
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ACKNOWLEDGEMENTS |
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We thank Dr. John W. Bloom for helpful discussion.
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FOOTNOTES |
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This work was supported by Arizona Disease Control Research Commission Grants 9812 (to L.-W. Lai) and 9613 (to Y.-H. H. Lien) and National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-52358 (to Y.-H. H. Lien).
Address for reprint requests: Y.-H. H. Lien, Section of Nephrology, Dept. of Medicine, Univ. of Arizona Health Sciences Center, Tucson, AZ 85724.
Received 9 September 1997; accepted in final form 21 November 1997.
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