Why are there carbonic anhydrases in the liver?

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Why are there carbonic anhydrases in the liver? SUSANNA J. DODGSON

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Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6085, U.S.A. Received May 21, 1991

The transit of HCO; from the erythrocyte to the pulmonary capillary was believed to be too rapid to be accounted for by the uncatalysed hydration of CO,; the hunt for a catalytic factor resulted in the subsequent simultaneous discovery of carbonic anhydrase (CA; EC 4.2.1.1) at the Universities of Cambridge and Pennsylvania (Meldrum and Roughton, J. Physiol. (London), 80, 113-142, 1933; Stadie and O'Brien J. Biochem. (Tokyo), 103, 521-529, 1933). Within the following decade, a class of specific sulfonamide carbonic anhydrase inhibitors had been identified (Keilin and Mann, Biochem. J., 34, 1163-1 176, 1940) and interest in carbonic anhydrase activity for several decades became limited to a large extent to those who preferred it inactive. In the past two decades, there has been an upsurge of interest in the carbonic anhydrases as a result of the findings that there is a whole family of carbonic anhydrase isozymes. Erythrocyte carbonic anhydrase activity is now known to comprise two isozymes, CA I and CA 11, in humans and most other mammals; however, ruminants and cats have only CA 11. The five other characterized isozymes, CA 111, CA IV, CA V, CA VI, and CA VII, are distributed throughout the organs as are CA I and CA I1 (Dodgson et al. (Editors), The carbonic anhydrases: cellular physiology and molecular genetics, Plenum Press, 1991). In the past 15 years, CA I11 through CA VII have been discovered; all seven isozyrnes are known to be encoded by different genes. The CA isozymes differ kinetically in their values of K, (COJ, anion inhibition constants, and turnover numbers; e.g., human CA I1 has the highest turnover number of any known enzyme, which is two orders of magnitude higher than that of CA 111. The question as to why there are carbonic anhydrases in the mammalian liver is starting only now to be answered. CA VI, which is secreted into saliva (Fernley, In The carbonic anhydrases, 1991, op. cit.), and CA VII, which is localized to the parotid gland (reviewed in Dodgson et al., 1991, op. cit.) have not been found in the liver. All five other isozymes are known to be in the hepatocytes (Gros and Dodgson, Annu. Rev. Physiol., 50,669-694, 1988; Jeffery, In The carbonic anhydrases, 1991, op. cit.). In humans, CA I, CA 11, and CA 11, are encoded by separate, contiguous genes on chromosome 8 (Hewett-Emmett and Tashian, In The carbonic anhydrases, 1991, op. cit.); all are present in the hepatocytic cytosol; CA IV is on the plasma membrane (Carter et al., Biochirn. Biophys. Acta, 1026, 113-1 16, 1990) and CA V is inside the mitochondria (Dodgson et al., Proc. Nat. Acad. Sci. U.S.A. 77, 55625566, 1980). The only liver isozyme for which there is any evidence for a defined function is the mitochondrial CA V, which comprises less than 5% of the total hepatocytic CA activity (Dodgson and Watford, 277, 410-414, 1990). The Rinted in Canada / lrnprimt au Canada

possible functions of the cytosolic and membrane-bound isozymes in the liver are being currently addressed in my laboratory as well as other collaborating laboratories; the remainder of the review is involved with discussion of these possible functions. Mitochondria1 carbonic anhydrase Mitochondrial carbonic anhydrase, CA V, was first quantitated, characterized, and purified from guinea pig liver mitochondria (Dodgson, 1980, op. cit.; Hewett-Emmett et al., Isozyme Bull., 19, 13, 1986; Dodgson, J. Appl. Physiol., 63,2134-2141, 1987). The first suggestion that the guinea pig CA V was a unique isozyme came from the peculiarly high pH sensitivity of the CA activity of mitochondrial samples, but not erythrocyte lysates (Dodgson et al., 1980, op. cit.; Dodgson et al., J. Biol. Chem., 257, 1705-171 1, 1982). We have subsequently demonstrated that the rat CA V is no more pH sensitive than the total CA of rat erythrocyte hemolysates (Dodgson and Contino, Arch. Biophys. Biochem., 260, 334-341, 1988). The putative function of CA V was proposed within 3 years of its characterization when the rate of citrulline production by intact guinea pig liver mitochondria was reduced by the specific carbonic anhydrase inhibitor acetazolamide (Dodgson et al., J. Biol. Chem., 258,7696-7701,1983). Subsequent studies from this laboratory have provided further evidence for its function in providing HCO? for carbamy1 phosphate synthetase I (CPS 1) in guinea pig and rat liver (Dodgson et al., op. cit., 199 1) as well as in providing HCO< for pyntvate carboxylation (PC), and subsequently glucose synthesis in guinea pig liver and rat kidney (Dodgson and Forster, Arch. Biophys. Biochern., 251, 198-204, 1986; Dodgson and Cherian, Am. J. Physiol., 227, E791-E796, 1989; Arch. Biochem. Biophys., 282, 1-7, 1990). Recently we have obtained polyclonal antibodies against rat CA V (Carter et al., Biochim. Biophy. Acta, 1036, 237-241, 1990); Western blot analysis has indicated that CA V is specific to mitochondria, but that it is not unique to liver and kidney mitochondria. An immunohistochemical study using these antibodies (Vaananen et al., J. Histochem. Cytochem., 39, 451-459, 1991) has determined that CA V is present in the heart at several times the concentration found in the liver. Since there is no urea cycle in rat heart, this finding points to a different function for CA V in cardiac than in hepatic mitochondria. The putative function of CA V in controlling ureagenesis and pyruvate gluconeogenesis led to the prediction that its regulation should be tightly controlled, increasing when increased rates of these synthetic pathways are increased, such as in the acidotic and diabetic rat. Liver mitochondria were prepared from rats and guinea pigs that had either been normally fed and starved for 48 h, or else normally fed with


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only dilute acid as drinking water (Dodgson and Contino, 1988, op. cit.). Kidney CA V from the mildly acidotic rats doubled compared with the starved rats; liver CA V from rats and guinea pigs was unaffected. In a subsequent study, CA V activity in streptozotocin-administered male rats doubled in 6 days while CA I11 activity was halved, and the total liver CA activity remained constant (Dodgson and Watford Arch. Biochem. Biophys., 277, 410-414, 1990). An interesting finding was that the total CA activity of the rat liver homogenates remained constant; thus the other liver carbonic anhydrases considerably increased their activity. The question arises: is regulation of CA expression in diabetic rat liver a homeostatic response to minimize the effects of insulin deficiency, or do these changes in CA activity further aggravate these effects? It could be postulated that increased CA V activity results from the increased protein metabolism in diabetic rats, in line with the proposed function of CA V, in providing HCO; first for carbamyl phosphate synthetase I and ultimately, urea synthesis. Increased CA V activity may also result from the increased need for gluconeogenesis from lactate, since CA V is proposed to be also needed to provide HCO; for pyruvate carboxylation. If the cytosolic isozymes are required to provide HCO; for acetyl CoA carboxylase for de novo fatty acid synthesis as has been postulated (see below), then regulatory increases and decreases in the cytosolic isozymes would also be predicted in diabetic rats. Cytosolic carbonic anhydrases While the functions of the three soluble cytoplasmic isozyrnes in the liver are not at all clear, their regulation and localization are better understood. There is a peculiar sexual dimorphism of CA I1 and CA I11 occurring in rats, with little CA I11 in female or young male rats and a large concentration in adult male rats. More CA I1 activity is found in female rats, but the total activity in male and female rat livers remains much the same. CA I1 and CA I11 are believed to be controlled by the pulsatile release of growth hormone, which in turn is controlled by testosterone concentrations (Jeffery et al., Biosci. Rep., 5,735-738, 1985). The concentration of CA I11 in adult male rats is directly related to their age; the concentration in livers is the same in these rats and in age-related females at birth, but in males the concentration increases 200-fold at puberty and then declines less rapidly as the rat ages (Jeffery et al., J. Endocrinol., 110, 123-126, 1986). Whether this indicates a different function for the low-activity CA I11 in males than the function of the high-activity CA I1 in females, we do not yet know. The population of hepatocytes throughout the rat liver is not homogeneous; there is a small population of cells around the central vein, the perivenous hepatocytes, that have different characteristics from the cells distributed throughout the rest of the liver, the periportal hepatocytes. Immunohistochemical studies (Laurila et al., J. Histochem. Cytochem., 37, 1375-1382, 1989; Carter et al., Acta Physiol. Scand., 135, 163-167, 1989) are contradictory with respect to the heterogeneous distribution of cytosolic CA I, 11, and I11 in males and females; however, both reports indicate there is very little CA I in either sex, a little more CA I1 in females, and substantial amounts of CA I11 only in males, as data from radio-immuno-assay studies have shown, as discussed above.

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The ability to measure carbonic anhydrase activity is severely handicapped by the rapid rate at which C 0 2 is hydrated in the absence of carbonic anhydrase. Since the uncatalysed rate is considerably slowed at low temperatures (see Forster, Chap. 6 in Dodgson et al., 1991, op. cit.), carbonic anhydrase activity is generally assayed in ice-cold solutions. Only by the use of the 180.mass spectrometric technique is analysis of the carbonic anhydrase activity of multiple small samples possible at physiological temperatures (Itada and Forster, J. Biol. Chem., 252, 3881-3890, 1977; Dodgson et al., J. Appl. Physiol., 68, 2443-2450, 1990). In my laboratory, a study with cytosolic samples obtained by the dual-digitonin-pulse-perfusion technique (Quistorff, Biochem. J., 229,221-226, 1985) resulted in the finding that the total CA activity in samples containing glutamine synthetase (which has been reported to be present only in perivenous cytosols; Gebhardt and Mecke, EMBO J., 2, 567-570, 1983) was highest in fed rats, but was reduced 50 and 75% in 4-h-starved, and in 48-h-starved and subsequently refed rats, respectively (S.J. Dodgson and B. Quistorff, unpublished data). This latter study is the first to show that feeding, starving, and refeeding have dramatic effects on both the activity of cytosolic CA isozymes as well as their distribution throughout the liver. The finding that refeeding after starving reduced the CA activity in cytosolic samples still more than starving alone is perplexing, indicating that regulation of expression of the carbonic anhydrase isozymes is extremely complex. Cytosolic carbonic anhydrases have been suggested to have a function to provide H C 0 < for acetyl-CoA carboxylase, the first enzyme in the de novo synthesis of fatty acids (Dodgson et al., Ann. N.Y. Acad. Sci., 429,5 16-524,1984; Coulson and Herbert, Ann. N.Y. Acad. Sci., 429,525-527, 1984). These suggestions followed firstly from the observation that CA V was needed to provide HCOT for mitochondrial biosynthetic processes, and secondly, from a report that carbonic anhydrase inhibitors decrease the rate of fatty acid synthesis in mouse liver (Cao and Rous, Life Sci., 22, 2067-2072, 1978). Since concentrations of acetazolamide of 2 to 16 mM inhibited purified acetyl-CoA carboxylase, it was concluded that the observed decrease in fatty acid synthesis was not mediated through inhibition of carbonic anhydrase activity. In a whole animal study, injection of acetazolamide into lizards was observed to decrease the rates of lipid production (Coulson and Herbert, Ann. N.Y. Acad. Sci., 429,525-527, 1984). In my laboratory we have found that the rate of incorporation of [14c]acetate into total fatty acid is reduced 50% by 10 pM acetazolamide or less in male and female rats (Z.-G. Chu and S.J. Dodgson, unpublished data), indicating that the CA isozyme providing HCO; substrate is not CA 111. Proof that cytosolic carbonic anhydrases are providing HCOT for acetyl-CoA carboxylase awaits further experimental evidence. The possibility that rnitochondrial CA V functions in lipogenesis has not been addressed experimentally, but certainly should be considered. Membrane-bound carbonic anhydrase A membrane-bound carbonic anhydrase activity was functionally localized in plasma membranes of the hepatocyte (Lipsen and Effros, J. Appl. Physiol., 65,2736-2743, 1988) and was quantitated in plasma membranes by differential


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centrifugation (Garcia-Marin et al., Biochem. Biophys. Acta, 945,17-22, 1988). Recent immunohistochemicalstudies have provided evidence that this is CA IV, the membranebound carbonic anhydrase that has been isolated from renal plasma membranes (Wistrand and Knuuttila, Kidney Int., 35, 851-859, 1989; Zhu and Sly, J. Biol. Chem., 265, 8795-8801, 1990). In the kidney, CA IV is believed to be needed for bicarbonate resorption. Although bicarbonate resorption along the proximal tubule in the kidney does not have an equivalent in the liver, perhaps in a more limited way liver CA IV has a function in bicarbonate transport across hepatocytic plasma membranes.

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Conclusion It is probable that the carbonic anhydrase isozymes in the liver have important, but differing functions. Changes in CA isozyme distribution and concentration in rats fed different diets, or in rats made diabetic, point to extremely complex regulation of these isozymes. Acknowledgements The work in this laboratory is supported by US National Institutes of Health grant DK-38041 to S.J. Dodgson. The mass spectrometer facility is supported by HL 19737 to R.E. Forster 11.