Alternation of cytosolic carbonic anhydrase isoenzymes during deciduomatal development in pregnant mice

FERTILITY AND STERILITY威 VOL. 82, SUPPL. 3, OCTOBER 2004 Copyright ©2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.

Alternation of cytosolic carbonic anhydrase isoenzymes during deciduomatal development in pregnant mice Whei-Ling Chiang, Ph.D.,a,,b,,d, Jer-Yuh Liu, Ph.D.b,,d ,Chu-Yu Liao, M.S.,b Shun-Fa Yang, M.S.b Yih-Shou Hsieh, Ph.D.,b and Shu-Chen Chu, Ph.D.c School of Medical Technology, Institute of Biochemistry, Chung Shan Medical University, and Department of Food Science, Chungtai Institute of Health Sciences and Technology, Taichung, Taiwan

Received October 15, 2003; revised and accepted March 2, 2004. Supported by National Science Council, Republic of China (NSC92-2320-B040-012 and NSC92-2745B-040-001). Reprint requests: Shu-Chen Chu, Ph.D., Department of Food Science, Chungtai Institute of Health Sciences and Technology, No. 11 Putzu Lane, Pu-tzu Road, Taichung 406, Taiwan (FAX: 886-4-2239-6771; ).E-mail: [email protected] a School of Medical Technology, Chung Shan Medical University. b Institute of Biochemistry, Chung Shan Medical University. c Department of Food Science, Chungtai Institute of Health Sciences and Technology. d These authors contributed equally. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2004. 03.033

Objective: To analyze the activity of cytosolic carbonic anhydrases (CAs) during the pre- and postimplantation stages of pregnancy in mice. Furthermore, to investigate the CA activity in mice in which abortion was induced by injection of substance P. Design: Controlled animal experiment. Setting: University research laboratory. Animal(s): A total of 75 ICR mice (weight between 20 –25 g) that showed two consecutive 4-day cycles. Intervention(s): Substance P (2.4 ␮g/g) or CA inhibitor (100 ␮g/g), or both, were administered. Decidualized uterine tissues were collected on days 0.5–10.5 after administration. Main Outcome Measure(s): The activity, protein expression pattern, and mRNA level of decidual cytosolic CAs. Results: The abortion induced by injection of substance P led to an aberrant expression of cytosolic CA and a decreased number of embryos. Furthermore, substance P-induced abortion could be effectively inhibited by CA inhibitors. Conclusions: Cytosolic CAs, especially CA II, may act as negative regulators in implantation, development, and maintenance of the pregnancy and therefore, this information could be further applied in developing therapies for human sterility. (Fertil Steril威 2004;82(Suppl 3):1095–1100. ©2004 by American Society for Reproductive Medicine.) Key Words: Carbonic anhydrases, deciduomata, substance P

Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc-containing enzymes, widely distributed, and present in at least 14 different isoforms. Some isoforms are cytosolic (including CA I, CA II, CA III, and CA VII), and others are membranebound (including CA IV, CA IX, CA XII, and CA XIV). Among them, CA V is mitochondrial and CA VI is secreted in the saliva. In addition, three acatalytic forms, CARP VIII, CARP X, and CARP XI, are identified. Carbonic anhydrase, which reversibly catalyses the hydration of carbon dioxide to bicarbonate and hydrogen ions, is widely distributed in mammalian tissues and has an important role in gas transport, acid/base regulation, calcification, and various secretory functions in tissues (1). A large number of novel CA inhibitors (such as sulfonamide) have been reported re-

cently, together with their potential applications as antiglaucoma, anticancer, and antiosteoporosis agents or for the management of a variety of neurological disorders (2). The presence of several CA isoenzymes has been reported in human placenta and reproductive tract (3– 6). The placenta functions as does the lung, liver, kidney, and other endocrine organs to guide the exchange of all factors necessary for a successful embryonic development and, later, fetal development and growth. In the placenta, the enzyme CA may provide ions for exchange with Na⫹, K⫹, and Cl⫺ in the transepithelial movement of ions and fluid, as well as facilitate carbon dioxide diffusion (7). It can also be active in intermediary metabolism, such as gluconeogenesis (8), urea (9), and fatty acid synthesis (10). In addition, CA II 1095

present in the endometrial and oviductal epithelia produces bicarbonate, which probably accelerates the migration of spermatozoa to the site of fertilization (11, 12). The concentrations of CA isoenzymes have been shown to be influenced by hormones, particularly steroids, in a number of tissues in various species (13). Previous immunohistochemical studies have also indicated that CA XII was highly expressed in the endometrial epithelium, suggesting that it may be functionally linked to the pH-dependent events in spermatozoa that precede fertilization (12).

in each group. Each group of mice was administered either substance P at a dose of 2.4 ␮g/g body weight (group SP), substance P (2.4 ␮g/g) plus CA inhibitor (sulfanilamide) (100 ␮g/g) (group SP/CAi), CA inhibitor (sulfanilamide) 100 ␮g/g only (group CAi), and phosphate-buffered saline (PBS) (control group). All these administrations were given on day 2.5 and the uteri were removed on day 8.5 after mating. The total number of resorbing sites was recorded, and decidual supernatants were prepared as described.

The decidual tissue is the transformed stromal part of the gestational endometrium fused with the chorion to form the maternal part of the materno-fetal interphase (placenta). Decidual tissues not only produce hormones, such as prolactin, but also have specific functions in implantation, development, and maintenance of the pregnancy (14). In addition, evidence has shown that endometrial CA may facilitate the implantation of blastocysts in rabbits (11). Therefore, in this study, decidual tissues were examined for the expression of cytosolic CAs in implantation of female mice.

All procedures were performed at 4°C. On days 0.5 to 10.5 of decidualization, the decidualized uterine tissue was slit open and harvested by mechanical scraping down to muscle with a glass slide. All decidualized uterine tissues were removed, weighed, thoroughly washed in PBS buffer (pH 7.4) and homogenized with a ratio of 0.5 g tissue/1 mL PBS buffer in a small glass homogenizer. After centrifugation (100,000 ⫻ g) at 4°C for 1 hour, supernatants were recovered and stored at ⫺70°C for the cytosolic fraction of CA and future assays.

The neurotransmitter substance P is one of the best known tachykinins (15) and is also considered to be an antiimplantation factor, as the peptides possibly exert a direct local alteration in uterine vascular permeability causing failure in implantation (16). At present, very little is known about the association of CA isoenzymes with substance P treatment during the implantation. It will be of interest to explore the physiologic role of cytosolic CA isoenzymes in the regulation of intracellular pH in the endometrium and to investigate the therapeutic potential of CA inhibitor in stressinduced murine abortion mediated by substance P.

Measurement of CA Activity

MATERIALS AND METHODS Chemicals Tris-HCl, ethylenediaminetetraacetic acid (EDTA), sodium dodecylsulfate (SDS), substance P, chloroform, and sulfanilamide, were purchased from Sigma Chemical Co. (St. Louis, MO). Other chemicals were of the highest purity available commercially.

Treatment of Animals Male and female ICR mice, weighing between 20 and 25 g, were purchased from National Science Council Animals Center (Taipei, Taiwan, ROC) and maintained in a temperature-controlled room (at 24°C) in an animal facility with a 12-hour light/dark cycle. All procedures were approved by Institutional Review Board, Chung Shan Medical University, and were performed in accordance with the Guiding Principles for Care and Use of Laboratory Animals. Vaginal smears were obtained daily and only those animals that showed two consecutive 4-day cycles were used. After overnight cohabitations (1 male ⫹ 2 females per cage), females with vaginal plugs (designated as day 0.5) were segregated and randomized into four groups with five mice 1096 Chiang et al.

Cytosolic CAs in pregnant mice

Preparation of Decidual Cytosolic Fraction

Carbonic anhydrase was stained using its presumptive native substrate, CO2 and water (17). The CA activity of uterine tissue homogenates was determined by activity stain on a 15% sodium dodecylsulfate polyacrylamide gel (SDSPAGE) with a modification as follows (18). After electrophoresis, the gel was immersed and shaken twice in 2.5% Trion X-100 for 30 minutes, followed by an immersion in 25% isopropanol at 4°C overnight, washed in 20 mM Tris buffer (pH 8.1) for 10 minutes, and finally stained by adding 6 to 8 drops of bromthymol blue (0.2% in 50% ethanol). After staining for about 30 minutes at 4°C, the gel was then placed in CO2 saturated water at 4°C. After 15–30 seconds, a distinct yellow band appeared at the position of CA in the gel. Relative photographic density was quantitated by scanning the photographic negatives on a gel documentation and analysis system (AlphaImager 2000, Alpha Innotech Corporation, San Leandro, CA).

Immunoblot Analysis Procedures for immunoblotting and antibody production have been described previously (19). Briefly, protein samples were separated in a 12.5% polyacrylamide gel, transferred onto a nitrocellulose membrane, and then incubated separately with CA I-, CA II-, and CA III-specific antibodies (19). The isoform-specific antibodies against CA III were generously provided by Dr. Chong-Kuei Lee in Department of Nutrition, Chung-Shan Medical University, Taichung, Taiwan. After incubation with horseradish peroxidase goat anti-rabbit IgG, the color signal was developed by 4-chloro1-napthol/ 3,3=-diaminobenzidine, 0.9% (wt/vol) NaCl in Tris-HCl. Relative photographic density was quantitated by scanning the photographic negatives on a gel documentation and analysis system (AlphaImager 2000, Alpha Innotech Corporation). Vol. 82, Suppl 3, October 2004

TABLE 1 Summary of cytosolic CA expression in decidualized uterine tissue analyzed by CA activity, immunoblotting analysis, and RT-PCR.

Day after mating Preimplantation 0.5 1.5 Implantation 2.5 3.5 4.5 5.5 6.5 Postimplantation 7.5 8.5 9.5 10.5

CA activity (% of day 0.5)

CA I

CA II

CA III

CA I

CA II

CA III

100 55 ⫾ 1.73

100 96 ⫾ 1.15

100 94 ⫾ 2.82

100 92 ⫾ 3.65

100 ND

100 99 ⫾ 3.46

100 50 ⫾ 5.77

16 ⫾ 3.46 15 ⫾ 1.72 8 ⫾ 0.57 12 ⫾ 2.66 15 ⫾ 1.12

20 ⫾ 0.88 23 ⫾ 1.2 15 ⫾ 1.73 13 ⫾ 1.16 9 ⫾ 2.3

45 ⫾ 6.35 50 ⫾ 2.88 45 ⫾ 5.76 47 ⫾ 2.44 40 ⫾ 4.04

19 ⫾ 2.76 14 ⫾ 2.3 13 ⫾ 3.46 10 ⫾ 1.15 7 ⫾ 1.02

ND ND ND ND ND

92 ⫾ 1.73 82 ⫾ 5.76 51 ⫾ 3.46 72 ⫾ 4.48 74 ⫾ 2.4

21 ⫾ 4.61 7.4 ⫾ 1.5 ND ND ND

49 ⫾ 5.19 63 ⫾ 8.66 75 ⫾ 2.88 97 ⫾ 1.15

125 ⫾ 11.54 130 ⫾ 2.88 133 ⫾ 3.6 220 ⫾ 11.52

79 ⫾ 6.35 83 ⫾ 4.86 95 ⫾ 4.33 115 ⫾ 8.96

46 ⫾ 6.92 64 ⫾ 5.77 120 ⫾ 11.5 115 ⫾ 8.69

ND ND ND 50 ⫾ 60.92

88 ⫾ 4.62 95 ⫾ 3.69 95 ⫾ 2.89 96 ⫾ 4.33

ND ND ND 15 ⫾ 4.04

Western blot (% of day 0.5)

RT-PCR (% of day 0.5)

ND ⫽ not detectable. Chiang. Cytosolic CAs in pregnant mice. Fertil Steril 2004.

Immunohistochemical Analyses The CA I, CA II, and CA III isoenzymes were localized on tissue sections of 5-␮m thickness using the avidin-biotinperoxidase complex technique (20, 21). Deparaffinized and rehydrated sections were treated with 1% hydrogen peroxide for 5 minutes at 25°C to inactivate endogenous peroxidase activity. The primary antiserum, a polyclonal antibody against CA I, CA II, or CA III was diluted with PBS at 1:600 for 1 hour at room temperature. After rinsing twice with Tris-buffered saline (TBS) for 20 minutes, the bound primary antibody was detected by sequential incubations with a biotinylated secondary antibody (LSAB kit from DAKO, Milan, Italy or ABC kit from Invitrogen, San Diego, CA) for 1 hour and DAB for 5–10 minutes, at room temperature with two rinses of TBS between incubations. After counterstaining with hematoxylin, sections were dehydrated through graded alcohols and cover glasses were applied using a histologic mounting medium.

Reverse Transcriptase–Polymerase Chain Reaction Total RNA was isolated from fresh tissues using a guanidinium chloride procedure (22). For reverse transcriptasepolymerase chain reaction (RT-PCR), 15 ␮g of RNA were used as templates for Mooney murine leukemia virus RT (300 units) in a 20-␮L reaction containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 20 units Rnasin (Promega, Madison, WI), and then amplification was conducted with two oligonucleotide primers based on the published cDNA sequence of cytosolic CA (23, 24). The RT-PCR products were analyzed through an electrophoresis FERTILITY & STERILITY威

in 1.8% agarose native gel and detected by ethidium bromide staining.

Statistical Analysis

Values were expressed as means ⫾ SE. The statistical analysis was performed by Mann–Whitney rank sum test between groups. SigmaStat software (Jandel Scientific Software, San Rafael, CA) was used for all statistical analyses. A P value of less than .05 was considered statistically significant.

RESULTS The proliferation of decidual tissues was evaluated by the expression of cyclin-dependent kinase (Cdks) families (cdc2). The Cdks are critical regulators in cell cycle progression and constitutively expressed throughout the cell cycle. Expression of cdc-2 was increased at day 2.5 after mating. However, it decreased from day 2.5– 6.5 and fluctuated from day 6.5–10.5 (data not shown). The CA staining with SDS-PAGE was conducted to determine the activities of CA I, CA II, and CA III, in decidualized uterine tissue of mice on days 0.5–10.5 after mating. There was a strong total CA activity on days 0.5–1.5 at the preimplantation stage, but CA activity decreased significantly (to 8%–16%) on days 2.5– 6.5 at the implantation stage as compared to day 0.5. Total CA activity at the postimplantation stage returned to the basal level of the preimplantation stage (Table 1, Fig. 1). Whether expression of CA protein was associated with total CA activity was determined by immunoblot analysis. 1097

FIGURE 1

FIGURE 2

The expression of carbonic anhydrases (CAs) in decidualized uterine tissue of mice on days 0.5–10.5 after mating. The uterine tissues homogenate was prepared and subjected to CA activity assay, immunoblotting analysis (with ␣-tubulin as an internal control), and reverse transcriptase-polymerase chain reaction (RT-PCR) (with glyceraldehyde-3-phosphatedehydrogenase [GAPDH] as an internal control).

Number of embryos in decidualized uterine tissue of mice from various groups on day 2.5 after mating. ***P⬍.001. SP ⫽ substance P; CAi ⫽ carbonic anhydrase inhibitor.

Chiang. Cytosolic CAs in pregnant mice. Fertil Steril 2004.

Chiang. Cytosolic CAs in pregnant mice. Fertil Steril 2004.

The expression of cytosolic CA was significantly less at the implantation stage compared to that on day 0.5 (9%–23% for CA I, 40%–50% for CA II, 7%–19% for CA III; Table 1, Fig. 1). The immunohistochemical results showed that the strongest signal for CA II was localized in the basolateral plasma membrane and gland of the decidualized cells in the pre- or postimplantation stage (data not shown). To confirm this observation we used RT-PCR to assay the mRNA level of cytosolic CA isoenzymes in decidualized uterine tissue. The results demonstrated that mRNA expression of CA II was reduced from 51%–92%, and that of CA III was reduced from 0%–7.4% of decidualized uterine tissue at implantation stage as compared to that on day 0.5 (Table 1, Fig. 1). These results suggested that CA isozymes, especially CA II, were down-regulated and then led to an enhanced cell proliferation at the implantation stage. In addition, the question of whether down-regulation of CAs plays a regulatory role in the process of implantation was investigated. Substance P (2.4 ␮g/g) was injected on day 2.5 and the data showed that abortion induced by substance P could lead to an aberrant expression of cytosolic CA and a decreased number of embryos (P⬍.001). 1098 Chiang et al.

Cytosolic CAs in pregnant mice

The CA activity in mice in groups SP/CAi and CAi were not significantly different from that of control (P ⫽ .8149, P ⫽ .391, respectively) (Fig. 2). Moreover, the mean total CA activity in the SP group was significantly greater than that in the SP/CAi and CAi groups (Fig. 3, P⬍.05). This increased activity of CA could be due to the higher expression of CA II in SP treatment group compared to that of CA I and CA III. The expression of CA II was significantly increased in the SP group (Fig. 4, P⬍.01).

DISCUSSION Decidualized uterine tissues are maternal in origin and functionally important not only for implantation, but also for

FIGURE 3 Analysis of the carbonic anhydrase (CA) protein level in decidualized uterine tissue of mice from four groups on day 2.5 after mating. The uterine tissues homogenate was prepared and subjected to immunoblotting analysis for CA I, CA II, and CA III. Here, ␣-tubulin was used as a control. SP ⫽ substance P; CAi ⫽ carbonic anhydrase inhibitor.

Chiang. Cytosolic CAs in pregnant mice. Fertil Steril 2004.

Vol. 82, Suppl 3, October 2004

FIGURE 4 Relative carbonic anhydrase (CA) protein level was quantified by densitometry in decidualized uterine tissue of mice from four groups (CA activity: dark red; CA I: cyan; CA II: dark blue; CA III: dark green) on day 2.5 after mating. *P⬍.05; **P⬍.01; ***P⬍.001. SP ⫽ substance P; CAi ⫽ carbonic anhydrase inhibitor.

Chiang. Cytosolic CAs in pregnant mice. Fertil Steril 2004.

the maintenance of pregnancy (14). The uterine endometrium in humans is a known source of CA and the activity of the endometrial CA doubles during the secretory (luteal) phase of the menstrual cycle (25). The presence of CA in the endometrium and maternal part of the placenta in other mammals (e.g., rabbit, sheep, and guinea-pig) is also well established (7). Chiang (26) and Kuo (27) and their colleagues suggested that the reduced expression of CA I and CA II may lead to a cellular acidic pH, thereby promoting tumor cell motility, and contribute to tumor growth and metastasis. In the present study, the reduction of CAs may have contributed to the cell growth in decidualized uterine of mice. In pregnant rabbits, at the preimplantation stage 6 to 7 days after mating, the blastocyst fluid was remarkably rich in bicarbonate and the endometrium, in CA (28). Our data showed that a stronger cytosolic CA activity was found in the decidualized uterine at the preimplantation stage on days 0.5–1.5 after mating and an essential event at this stage is fertilization. Fertilization consists in the activation of the ovum by a spermatozoon and the union of the male and female pronuclei, a process known as syngamy. Parkkila et al. (29) suggested that a physiological role of CA in the process of fertilization was to regulate the concentration of bicarbonate ions in the seminal plasma and spermatozoa. Bicarbonate ions not only maintain the motility of spermatozoa but also protect them in the acid environment of the vagina at midcycle. In fact, bicarbonate has been considered as an essential component for a successful IVF. Previous studies have also indicated that CA XII was highly expressed FERTILITY & STERILITY威

in the endometrial epithelium, suggesting that it may be functionally linked to the pH-dependent events in spermatozoa that precede fertilization. The CA IV, CA IX, or CA XII may produce bicarbonate ions outside of the cell that are transported inside by bicarbonate/chloride exchangers and used by the cytosolic CA II to titrate protons (12). The increased level of endometrial CA facilitates both the production and removal of carbon dioxide in endometrial cells. In addition, CA II binds to the carboxyl terminus of human band 3 protein, which catalyzes the electroneutral exchange of bicarbonate for chloride (30). Buffering protons facilitate proton secretion and protect cells from intracellular acidification. However, in the female genital tract, the endometrial and oviductal epithelium may produce an alkaline environment that maintains sperm motility. On the contrary, CA II have been shown to be downregulated at the implantation stage on days 2.5– 6.5. The protein and mRNA expression of CA I and CA II measured by Western blot and RT-PCR analyses have shown results in parallel with the expression of CA isoenzymes activity. This stage is for the early development of blastocysts. Blastocyst implantation involves a complex series of events occurring over time. In this study, we found that the expression of CA I and CA II at the implantation stage was significantly reduced compared to that of the preimplantation stage. There could be two ways to explain these findings: first, downregulation of cytosolic CAs will lead to an acidic environment in endometrium and our data suggested that CAs may be functionally involve in the pH-dependent blastocyst implantation in subsequent fertilization. Second, the reduced cytosolic CA expression may lead to an acidification to create a microenvironment conducive to cell growth and spread. However, we have also found the increased expression of Cdks families (cdc-2) in uterine cells (data not shown). Cdks are critical regulators of cell cycle progression and constitutively expressed throughout the cell cycle. The increased expression of cdc-2 has been demonstrated in decidual tissues, which are rapidly proliferating at the implantation stage of pregnancy. For a successful implantation, embryo– endometrial interactions must be initiated when the embryo and the endometrium have reached precise stages of development; the embryo must be at the blastocyst stage of development, and hormone-dependent changes resulting in the development of a short-lived receptive endometrium must have occurred (31). Hodgen and Falk (13) suggested that the abundance of cytosolic CA expression at postimplantation was in parallel with the estrogen (E) and P secretion. The regulation of CA activity and the appearance of this uterus-specific isoenzyme were influenced primarily by progestational stimulation in the rabbit and E in the guinea pig. The neurotransmitter substance P is one of the best known tachykinins and has been demonstrated to drive an immunological instability in the deciduas with increased levels of 1099

type 1 helper T-cell (Th 1) cytokines and decreased levels of type 2 helper T-cell (Th 2) cytokines. The Th 1 cytokines, such as interferon (INF)-␥ and tumor necrosis factor (TNF)-␣ have been suggested to induce abortion (32). Joachim et al. (33) suggested that substance P increases the production of decidual TNF-␣, which is involved in the stress-induced murine abortion. In the present study, substance P was used to induce abortion. The subsequent results showed that the number of embryos was significantly decreased after treatment with substance P to decidualized uterine tissues and also decreased cytosolic CAs mRNA and protein expression, as measured by RT-PCR and Western blot analyses. Furthermore, treatment with SP/CAi or CAi only showed a minor impact on implantation. We suggested that cytosolic CAs, as negative regulators of decidualized uterine tissues stability, function mainly on the regulation of acidic pH environment in the endometrium and may lead to an acidification to create a microenvironment conducive to cell growth and spread. Recently, a large number of structurally diverse CA inhibitors, including sulfanilamide, were found useful as diuretics, or in the treatment and prevention of a variety of diseases such as glaucoma, epilepsy, congestive heart failure, mountain sickness, gastric and duodenal ulcers, neurological disorders, and osteoporosis, as well as diagnositic tools (34, 35). In this report, we found that substance P-induced abortion could be effectively prevented by CA inhibitors, which might be useful in developing therapies for human sterility. These data also further supported our finding that cytosolic CAs are involved in implantation, development, and maintenance of the pregnancy. References 1. Supuran CT, Scozzafava A, Casini A. Carbonic anhydrase inhibitors. Med Res Rev 2003;23:146 – 89. 2. Supuran CT, Scozzafava A. Carbonic anhydrase inhibitors. Curr Med Chem Immunol Endocr Metas Ag 2001;1:61–97. 3. Aliakbar S, Brown PR, Jauniaux E, Bidwell DE, Nicolaides KH. Measurement of carbonic anhydrase isoenzymes in early human placental tissues. Biochem Soc Trans 1990;18:670. 4. Aliakbar S, Brown PR, Nicolaides KH. Localization of CAI and CAII isoenzymes in normal term human placenta by immunofluorescence techniques. Placenta 1990;11:35–9. 5. Mühlhauser J, Crescimanno C, Rajaniemi H, Parkkila S, Milovanov AP, Castellucci M, et al. Immunohistochemistry of carbonic anhydrase in human placenta and fetal membranes. Histochemistry 1994;101: 91– 8. 6. Lehtonen J, Shen B, Vihinen M, Casini A, Scozzafava A, Supuran CT, et al. Characterization of CA XIII, a novel member of the carbonic anhydrase isozyme family. J Biol Chem 2004;279:2719 –27. 7. Ridderstråle Y, Persson E, Dantzer V, Leiser R. Carbonic anhydrase activity in different placenta types: a comparative study of pig, horse, cow, mink, rat, and human. Microsc Res Tech 1997;38:115–24. 8. Dodgson SJ. Liver mitochondrial carbonic anhydrase (CA), gluconeogenesis, and ureagenesis in the hepatocyte. In: Dodgson SJ, Tashian RE, Gros G, Carter ND, eds. The carbonic anhydrases. Cellular physiology and molecular genetics. New York and London: Plenum Press, 1991:297–306. 9. Dodgson SJ, Forster RE 2d, Storey BT. The role of carbonic anhydrase in hepatocyte metabolism. Ann NY Acad Sci 1984;429:516 –24. 10. Herbert JD, Coulson RA. A role for carbonic anhydrase in de novo fatty acid synthesis in liver. Ann NY Acad Sci 1984;429:525–27.

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