RAPID COMMUNICATION
Respiratory acidosis in carbonic anhydrase II-deficient mice

Yeong-Hau H. Lien and Li-Wen Lai

Department of Medicine, University of Arizona Health Sciences Center, Tucson, Arizona 85724

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 HCO<SUP>−</SUP><SUB>3</SUB> concentration ([HCO<SUP>−</SUP><SUB>3</SUB>]; 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 [HCO<SUP>−</SUP><SUB>3</SUB>]. The metabolic acidosis in CA II-deficient mice was corrected ([HCO<SUP>−</SUP><SUB>3</SUB>], 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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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).


View larger version (55K):
[in this window]
[in a new window]
 
Fig. 1.   Western blot of lung homogenates. Left lane, low-molecular-mass protein standard (GIBCO, Gaithersburg, MD) containing carbonic anhydrase (CA) II protein with a molecular mass of 29 kDa. Middle and right lanes, perfused lung homogenates (100 µg of total protein) from normal and CA II-deficient [CA II (-/-); homozygous mutant] mice, respectively.

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 HCO<SUP>−</SUP><SUB>3</SUB> concentration ([HCO<SUP>−</SUP><SUB>3</SUB>]; 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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Arterial blood gas analyses of normal, heterozygous, and homozygous carbonic anhydrase II-deficient mice

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 [HCO<SUP>−</SUP><SUB>3</SUB>] in normal mice. In CA II-deficient mice, the bicarbonate injection resulted in an increase in both [HCO<SUP>−</SUP><SUB>3</SUB>] 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.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Effects of NaHCO3 injection on arterial blood gas in normal and carbonic anhydrase II-deficient mice

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 [HCO<SUP>−</SUP><SUB>3</SUB>] 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 [HCO<SUP>−</SUP><SUB>3</SUB>] 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 [HCO<SUP>−</SUP><SUB>3</SUB>] 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-/HCO<SUP>−</SUP><SUB>3</SUB> 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 HCO<SUP>−</SUP><SUB>3</SUB> 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-HCO<SUP>−</SUP><SUB>3</SUB>-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.

    ACKNOWLEDGEMENTS

We thank Dr. John W. Bloom for helpful discussion.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Bidani, A. Analysis of abnormalities of capillary CO2 exchange in vivo. J. Appl. Physiol. 70: 1686-1699, 1991[Abstract/Free Full Text].

2.   Bradford, M. M. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976[Medline].

3.   Brion, L. P., W. Cammer, L. M. Stalin, C. Suarez, B. J. Zavilowitz, and V. L. Schuster. Expression of carbonic anhydrase IV in carbonic anhydrase II-deficient mice. Am. J. Physiol. 273 (Renal Physiol. 42): F234-F245, 1997[Abstract/Free Full Text].

4.   Cain, S. M., and A. B. Otis. Carbon dioxide transport in anesthetized dogs during inhibition of carbonic anhydrase. J. Appl. Physiol. 16: 1023-1028, 1961.

5.   Crandall, E. D., and J. E. O'Brasky. Direct evidence of participation of rat lung carbonic anhydrase in CO2 reactions. J. Clin. Invest. 62: 618-622, 1978[Medline].

6.   Enns, T., and E. P. Hill. CO2 diffusing capacity in isolated lung lobes and the role of carbonic anhydrase. J. Appl. Physiol. 54: 483-490, 1983[Abstract/Free Full Text].

7.   Fleming, R. E., M. A. Moxley, A. Waheed, E. C. Crouch, W. S. Sly, and W. J. Longmore. Carbonic anhydrase II expression in rat type II pneumocytes. Am. J. Respir. Cell Mol. Biol. 10: 499-505, 1994[Abstract].

8.   Fleming, R. E., C. A. Zuzicka, E. C. Crouch, and W. S. Sly. Pulmonary carbonic anhydrase IV: developmental regulation and cell-specific expression in the capillary endothelium. Am. J. Physiol. 265 (Lung Cell. Mol. Physiol. 9): L627-L635, 1993[Abstract/Free Full Text].

9.   Heming, T. A., and A. Bidani. Influence of proton availability on intracapillary CO2-HCO<SUP>−</SUP><SUB>3</SUB>-H+ reactions in isolated rat lungs. J. Appl. Physiol. 72: 2140-2148, 1992[Abstract/Free Full Text].

10.   Heming, T. A., C. Greers, G. Gros, A. Bidani, and E. D. Crandall. Effects of dextran-bound inhibitors on carbonic anhydrase in isolated rat lungs. J. Appl. Physiol. 61: 1849-1856, 1986[Abstract/Free Full Text].

11.   Heming, T. A., C. G. Vanoye, E. K. Stabenau, E. D. Roush, C. A. Fierke, and A. Bidani. Inhibitor sensitivity of pulmonary vascular carbonic anhydrase. J. Appl. Physiol. 75: 1642-1649, 1993[Abstract].

12.   Henry, R. P., S. J. Dodgson, R. E. Froster, and B. T. Storey. Rat lung carbonic anhydrase: activity, location, and isozymes. J. Appl. Physiol. 60: 638-645, 1986[Abstract/Free Full Text].

13.   Kemp, P. J., and C. A. R. Boyd. Anion exchange in type II pneumocytes freshly isolated from adult guinea-pig lung. Pflügers Arch. 425: 28-33, 1993[Medline].

14.   Lien, Y. H., S. J. Hsu, G. Moeckel, and L. Lai. Gene therapy partially corrects renal tubular acidosis in carbonic anhydrase II deficient mice (Abstract). J. Am. Soc. Nephrol. 6: 701, 1995.

15.   Lewis, S. E., R. P. Erickson, L. B. Barnett, P. J. Venta, and R. E. Tashian. N-ethyl-N-nitrosourea-induced null mutation at the mouse Car-2 locus: an animal model for human carbonic anhydrase II deficiency syndrome. Proc. Natl. Acad. Sci. USA 85: 1962-1966, 1988[Abstract].

16.   Nord, E. P., S. E. S. Brown, and E. D. Crandall. Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange modulates intracellular pH in rat type II alveolar epithelial cells. J. Biol. Chem. 263: 5599-5606, 1988[Abstract/Free Full Text].

17.   Ohlsson, A., W. A. Cumming, A. Paul, and W. S. Sly. Carbonic anhydrase II deficiency syndrome: recessive osteopetrosis with renal tubular acidosis and cerebral calcification. Pediatrics 77: 371-381, 1986[Abstract].

18.   Roush, E. D., and C. A. Fierke. Purification and characterization of a carbonic anhydrase II inhibitor from porcine plasma. Biochemistry 31: 12536-12542, 1992[Medline].

19.   Ryan, U. S., L. Whitney, and J. W. Ryan. Localization of carbonic anhydrase on pulmonary artery endothelial cells in cultures. J. Appl. Physiol. 53: 914-919, 1982[Abstract/Free Full Text].

20.   Siemann, D., R. Hill, and R. Bush. Analysis of blood gas values in mice following pulmonary irradiation. Radiat. Res. 81: 303-310, 1980[Medline].

21.   Sly, W. S., D. Hewett-Emmett, M. P. Whyte, Y.-S. L. Yu, and R. E. Tashian. Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proc. Natl. Acad. Sci. USA 80: 2752-2756, 1983[Abstract].

22.   Spicer, S. S., Z. H. Ge, R. E. Tashian, D. J. Hazen-Martin, and B. A. Schulte. Comparative distribution of carbonic anhydrase isozymes III and II in rodent tissues. Am. J. Anat. 187: 55-64, 1984.

23.   Swenson, E. R., J. Gronlund, J. Ohlsson, and M. P. Hlastala. In vivo quantitation of carbonic anhydrase and band 3 protein contributions to pulmonary gas exchange. J. Appl. Physiol. 74: 838-848, 1993[Abstract].

24.   Swenson, E. R., and T. H. Maren. A quantitative analysis of CO2 transport at rest and during maximal exercise. Respir. Physiol. 35: 129-159, 1978[Medline].

25.   Tashian, R. E., D. Hewett-Emmett, S. J. Dodgson, R. E. Forster II, and W. S. Sly. The value of inherited deficiencies of human carbonic anhydrase isozymes in understanding their cellular role. Ann. NY Acad. Sci. 429: 262-275, 1984[Abstract].

26.   Walden, T. L., Jr. Leukotriene C4-induced radioprotection: the role of hypoxia. Radiat. Res. 132: 359-367, 1992[Medline].

27.   Wistrand, P. J. The importance of carbonic anhydrase B and C for the unloading of CO2 by the human erythrocyte. Acta Physiol. Scand. 113: 417-426, 1981[Medline].


AJP Lung Cell Mol Physiol 274(2):L301-L304
1040-0605/98 $5.00 Copyright © 1998 the American Physiological Society