ARTICLE |
Correspondence to: Susanne Sender, Vegetative Physiologie-4220, Medizinische Hochschule Hannover, 30623 Hannover, Germany..
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Summary |
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Flounders Platichthys flesus were investigated with respect to isolation, purification, and cellular localization of carbonic anhydrase (CA) in the respiratory system. CA was purified from gills and erythrocytes and was shown to exclusively represent a soluble enzyme with an apparent molecular weight of 30 kD. Inhibition constants (KI) towards acetazolamide (ACTZ) were 8.4·10-9 M for erythrocyte CA and 7.6·10-9 M for gill CA, indicating a high sensitivity to sulfonamides, as exhibited by human CA II. Specific CA activity did not differ significantly in seawater- and freshwater-acclimated fish. Antibodies were raised against purified gill and erythrocyte CA. Both antisera crossreacted and were used to localize CA in the gills of seawater and freshwater flounders at the light microscopic level. Independent of the salinity, a positive reaction of variable intensity was found in the following cell types: pavement cells (PVCs), forming the gill epithelial surface layer; mucous cells (MCs); pillar cells (PCs), bordering the vascular channels of the secondary lamellae; and chloride cells (CCs), mitochondria-rich cells located in the primary epithelium, the interlamellar regions, and at the bases of the secondary lamellae. (J Histochem Cytochem 47:4350, 1999)
Key Words: carbonic anhydrase, flounder, gills, erythrocytes, chloride cells, mucous cells, pavement cells, primary lamellae, secondary lamellae, immunohistochemistry
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Introduction |
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The fish gill is a complex organ, known to be involved in respiratory gas exchange, ion transport, and acidbase regulation. Carbonic anhydrase (CA), abundantly present in gill epithelial cells, is assumed to play a role in these processes (see
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Materials and Methods |
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Animals
Seawater flounders were obtained from the Biologische Anstalt Helgoland (German Bight) and kept at 30 ppt salinity. Flounders from brackish water at the mouth of the River Schlei (5 ppt salinity) were supplied by a local fisherman and kept in tapwater (freshwater flounders). Animal weight was 250450 g. Water temperature was kept at 12C and a 12-hr daylight period was applied. Before the experiments, flounders were acclimated for at least 2 weeks. The experiments were performed according to the national guidelines for care and use of laboratory animals.
Purification of Erythrocyte CA
Flounders were anesthetized in 0.02% 3-aminobenzoic acid ethylester-methanesulfonate (MS 222; Serva, Heidelberg, Germany). A total of 500 IU heparin was given IP. After 30 min the tail was cut off and blood was collected in heparinized Eppendorf tubes. Plasma was removed immediately by low-speed centrifugation. The erythrocytes of 20 flounders per preparation were pooled and washed twice in PBS (0.2 M, pH 7.3). They were lysed by freeze-thawing and centrifuged at 100,000 x g (1.5 hr, 4C) in a Kontron centrifuge TGA with a TFT 65.38 rotor (Kontron; Eching, Germany). The supernatant was mixed 1:2 with double-concentrated homogenizing buffer (20 mM Tris-HCl, 0.2 M Na2SO4, pH 8.7). Erythrocyte CA from freshwater flounders was purified by affinity chromatography according to
Purification of Branchial CA
Before preparation, the gills were perfused with saline via the bulbus arteriosus until they appeared completely blood-free. They were dissected and transferred into ice-cold saline, where the gills of 20 flounders per preparation were pooled. Filaments were separated from the gill arches and homogenized in ice-cold 10 mM Tris-HCl, 0.1 M Na2SO4 (pH 8.7), using the Ultra-Turrax (Janke & Kunkel; IKA-Werk, Staufen, Germany). The homogenate was centrifuged at 10,000 x g (20 min, 4C), leading to Pellet I (PI) and Supernatant I (SI). PI contains cell and bone fragments, nuclei and mitochondria; SI consists of cytosol and membranes. SI was then centrifuged at 100,000 x g (1.5 hr, 4C) to separate cytosol (SII) and membranes (PII). The resuspended Pellet II was centrifuged again at 100,000 x g to remove remaining cytosol (SIII) from the membrane fraction (PIII). SII exhibited the highest activities of branchial CA. SII from freshwater flounders was used to purify the enzyme. To remove remaining fragments of membranes or organelles, SII was then centrifuged again at 100,000 x g for 1.5 hr before affinity chromatography, which was carried out as described for erythrocyte CA above.
Determination of CA activity
During purification, CA activity was determined according to the micromethod of
Production of Antibodies and Test of Specificities
Purified erythrocyte CA and gill CA were used for antibody production in rabbits. Preimmune serum was taken from each animal before immunization. Six rabbits were immunized by SC injection of 100 µg CA, emulsified in complete Freund's adjuvant (Sigma). After 7 weeks the animals received an SC booster injection with 100 µg CA in incomplete Freund's adjuvant (Sigma). Twelve days later they were bled by heart puncture. Preimmune sera and antisera were stored at -20C.
The specificities of the antisera were tested by subjecting the purified antigens and the 100,000 x g supernatants of erythrocytes and gills to SDS-PAGE (15%), followed by Western blotting (
Tissue Preparation
After perfusion, the gills were dissected and the gill arches removed. For paraffin embedding, each gill was cut into three or four pieces. They were immersed for 56 hr in one of the following fixatives.
PLP (PeriodateLysineParaformaldehyde).
The fixative (0.01 M sodium periodate, 0.075 M lysine, 2% paraformaldehyde, 0.04 M phosphate buffer) was prepared according to
Bouin's Solution. This consisted of saturated picric acid: formalin (37%):glacial acetic acid, 15:5:1.
After fixation, the gills were washed in 35% ethanol several times, dehydrated in a graded series of ethanol, and embedded in paraffin.
For embedding in epoxy resin, gill lamellae were excised and immersed in PLP for 2 hr. They were then washed in PBS for 1 hr, dehydrated in a graded series of ethanol, and embedded in Araldite (Ciba-Geigy; Wehr, Germany).
Immunohistochemistry
Paraffin sections 710-µm thick were mounted on gelatin- or poly-L-lysine-coated slides (Sigma). After deparaffination, sections were immunostained by the avidinbiotinperoxidase complex (ABC) technique (
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Results |
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Subcellular Distribution of Branchial CA
Like erythrocyte CA, the branchial CA of flounders appears to be exclusively a soluble enzyme. PIII, the washed membrane fraction, showed only 0.2% of the CA activity found in the homogenate, excluding the presence of a significant fraction of a membrane-bound CA in flounder gills (Table 1). This conclusion still holds when the losses of CA activity during recentrifugations are considered (see legend to Table 1).
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CA Activity in Gills and Erythrocytes
Specific CA activity (U·ml·mg-1) was independent of the salinity to which the flounders were acclimated, as is shown in Table 2 for erythrocytes and the cytoplasmic gill fractions SII and SIII.
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Molecular Weight and Inhibition Kinetics
Purity of the isolated CA fractions was analyzed by Western blotting. After affinity chromatography and gel filtration, both gill and erythrocyte CA showed one band at 30 kD (Figure 1). Crossreactions were seen of anti-gill CA with erythrocyte CA and of anti-erythrocyte CA with gill CA.
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To distinguish CA I- and CA II-type isozymes, the sensitivities of gill and erythrocyte CAs towards ACTZ and potassium iodide (KI) were studied. The inhibition constant KI of erythrocyte CA was 8.4·10-9 M for ACTZ (Figure 2A) and 4.4·10-3 M for KI (Figure 2B). The inhibition constants of branchial CA were 7.6·10-9 M for ACTZ (Figure 3A) and 7.1·10-3 M for KI (Figure 3B). The EassonStedman plots were reasonably linear and monophasic. The high sensitivity to sulfonamides and the cytoplasmic localization suggest that the enzymes correspond to mammalian CA II. The sensitivity to iodide is intermediate between that of CA I and CA II. The identical inhibition properties and the immunological crossreactivities indicate that red cell and gill CA are identical.
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Immunohistochemical Localization of CA in Gills
CA localization was investigated in primary and secondary gill lamellae. The latter branch from the primary lamellae and carry the secondary (respiratory) epithelium, whereas primary lamellae and interlamellar region are covered by the primary epithelium. Both epithelia show differences concerning embryonic development, vascularization, and cell components (see
After incubation of gill sections with anti-CA/ABC, a positive reaction was seen in several cell types that could be identified by their shape and characteristic distribution pattern. CA was localized in pillar cells (PCs) separating the vascular channels as well as in chloride cells (CCs), which were of angular or ovoid shape and were scattered within the secondary lamellae (SL) and in the interlamellar region (Figure 4). Mucous cells (MCs) of rounded outline, which appeared to open to the surface at several sites, were sometimes stained at the cell membrane. In addition, cartilage (C) supporting the primary lamellae was found to be CA-positive. The central venous sinus (CVS) extending the length of the primary lamellae could be seen. In other areas of the flounder gills, PCs and CCs also displayed immunoreactivity for CA (Figure 5).
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In addition, a number of small round cells in the outer layer of the secondary epithelium were heavily stained. The type of these cells was difficult to identify. They may represent differentiating mucous cells. Pavement cells (PVCs), forming the external layer of secondary and primary epithelium, exhibited a variable staining pattern. In cells near the lateral end of the filaments, no immunoreactivity was found (Figure 4). In some areas, PVCs showed only a faint staining in the secondary lamellae (Figure 5). In other parts, however, they exhibited a prominent reaction up to the lamellar tip (Figure 6). Like PVCs, PCs also varied in their staining density for CA (Figure 4 Figure 5 Figure 6). However, PCs that extended into the base of the primary epithelium always exhibited a strong reaction. In controls, no immunostaining was observed (Figure 7).
To better visualize the fine structure of primary and secondary lamellae, semithin gill sections from seawater- and freshwater-adapted flounders were investigated (Figure 8 and Figure 9, respectively). PCs, PVCs, MCs and CCs were stained for CA, independent of the salinity to which the fish were adapted. The morphology of the CCs in the two fish groups appeared to be slightly different. The CCs of flounders adapted to seawater were mostly of angular outline (see also Figure 4 Figure 5 Figure 6), whereas the CCs of freshwater-acclimated fish appeared almost round.
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Discussion |
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Subcellular Distribution of Branchial CA
This study shows that CA in the gills of Platichthys flesus is exclusively a cytoplasmic enzyme. The majority of gill CA was also found to be soluble in other species, such as catfish (Ictalurus punctatus) (
CA Activity in Gills and Erythrocytes
Our measurements indicate that the CA activity in gills and red blood cells of Plathichthys flesus does not correlate with the salinity to which the fish are adapted. This confirms the findings of
Inhibition Properties of Branchial and Erythrocyte CA
In this study the inhibition constants KI of flounder branchial and erythrocyte CA towards potassium iodide (KI) were determined to be 7.1·10-3 M and 4.4·10-3 M, respectively.
Localization of Gill CA
The histochemical localization of branchial CA has been studied in several fish species using Hansson's technique (
Our own results show CA to be present in PVCs, MCs, CCs, and PCs. Together with the results cited above, they reflect the great interspecies variability in the cell type-specific distribution of branchial CA, a phenomenon that has been already pointed out by Conley and Mallat (1988). In the studies mentioned above which compared freshwater- and saltwater-adapted fish from the same species, i.e., from salmon (
Physiological Significance of CA in Fish Gills
Several investigations have provided evidence that in freshwater fish the CCs may be reponsible for Cl- uptake, the PVCs being the site of Na+ uptake (
The apical surface of the gill epithelium is covered by a film produced by mucous cells. Because it had probably been washed off during tissue processing, we were not able to demonstrate this mucous coat in the flounder. Because the mucous cells are CA-positive in this species, it is possible that the excreted mucous contains CA. CA at this site may enhance CO2 and NH3 excretion, as has been discussed by
We have no clue at the moment to the function of CA in pillar cells. To further elucidate the role of all four CA-positive cell types, detailed physiological experiments in flounder gills are required.
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Acknowledgments |
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The expert technical assistance of Katrin Stoll and Martina Meyer is gratefully acknowledged.
Received for publication May 8, 1998; accepted September 15, 1998.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bruns W, Dermietzel R, Gros G (1986) Carbonic anhydrase in the sarcoplasmic reticulum of rabbit skeletal muscle. J Physiol 371:351-364[Abstract]
Carter N, Auton J, Dando P (1976) Red cell carbonic anhydrase levels in flounders, Platichthys flesus L., from salt water and fresh water. Comp Biochem Physiol 55B:399-401
Cetin Y, Kuhn M, Kulaksiz H, Adermann K, Bargsten G, Grube D, Forssmann W-G (1994) Enterochromaffin cells of the digestive system: cellular source of guanylin, a guanylate cyclase-activating peptide. Proc Natl Acad Sci USA 91:2935-2939[Abstract]
Conley DM, Mallatt J (1988) Histochemical localization of Na+-K+ ATPase and carbonic anhydrase activity in gills of 17 fish species. Can J Zool 66:2398-2405
Decker B, Sender S, Gros G (1996) Membrane-associated carbonic anhydrase IV in skeletal musclesubcellular localization. Histochem Cell Biol 106:405-411[Medline]
Dimberg K, Höglund PG, Knutsson Ridderstråle Y (1981) Histochemical localization of carbonic anhydrase in gill lamellae from young salmon (Salmo salar L.) adapted to fresh and salt water. Acta Physiol Scand 112:218-220[Medline]
Easson LH, Stedman E (1937) The absolute activity of cholinesterase. Proc R Soc Lond [Ser B] 121:142-164
Flügel C, LütjenDrecoll E, Zadunaisky JA (1991) Histochemical demonstration of carbonic anhydrase in gills and opercular epithelium of seawater- and freshwater-adapted killyfish (Fundulus heteroclitus). Acta Histochem 91:67-75[Medline]
Foskett JK, Scheffey C (1982) The chloride cell: definitive identification as the salt-secretory cell in teleosts. Science 215:164-166[Medline]
Goss GG, Perry SF, Wood CM, Laurent P (1992) Mechanisms of ion and acid-base regulation at the gills of freshwater fish. J Exp Zool 263:143-159[Medline]
Hansson HPJ (1967) Histochemical demonstration of carbonic anhydrase activity. Histochemie 11:112-118[Medline]
Haswell MS, Raffin J-P, LeRay C (1983) An investigation of the carbonic anhydrase inhibitor in eel plasma. Comp Biochem Physiol 74A:175-177
Henry RP, Smatresk NJ, Cameron JN (1988) The distribution of branchial carbonic anhydrase and the effects of gill and erythrocyte carbonic anhydrase inhibition in the channel catfish Ictalurus punctatus. J Exp Biol 134:201-218[Abstract]
Henry RP, Tufts BL, Boutilier RG (1993) The distribution of carbonic anhydrase type I and II isozymes in lamprey and trout: possible co-evolution with erythrocyte chloride/bicarbonate exchange. J Comp Biochem [B] 163:380-388
Hsu S-M, Raine L, Fanger H (1981) Use of avidinbiotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580[Abstract]
Kültz D, Bastrop R, Jürss K, Siebers D (1992) Mitochonria-rich (MR) cells and the activities of the Na+/K+-ATPase and carbonic anhydrase in the gill and opercular epithelium of Oreochromis mossambicus adapted to various salinities. Comp Biochem Physiol 102B:293-301
KyhseAnderson J (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins to nitrocellulose. J Biochem Biophys Methods 10:203-209[Medline]
Lacy ER (1983) Histochemical and biochemical studies of carbonic anhydrase activity in the opercular epithelium of the euryhaline teleost, Fundulus heteroclitus. Am J Anat 166:19-39[Medline]
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-688[Medline]
Laurent P, Dunel S (1980) Morphology of gill epithelia in fish. Am J Physiol 238:R147-159[Medline]
Laurent PL, Goss GG, Perry SF (1994) Proton pumps in fish gill pavement cells? Arch Int Physiol Biochim Biophys 102:77-79[Medline]
Laurent PL, Höbe H, DunelErb S (1985) The role of environmental sodium chloride relative to calcium in gill morphology of freshwater salmonid fish. Cell Tissue Res 240:675-692
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275
Maren TH (1960) A simplified micromethod for the determination of carbonic anhydrase and its inhibitors. J Pharmacol Exp Ther 130:26-29
Maren TH, Friedland BR, Rittmaster RS (1980) Kinetic properties of primitive vertebrate carbonic anhydrases. Comp Biochem Physiol 67B:69-74
Maren TH, Rayburn CS, Liddell NE (1976) Inhibition by anions of human red cell carbonic anhydrase B: physiological and biochemical implications. Science 191:469-472[Medline]
Mashiter KE, Morgan MRJ (1975) Carbonic anhydrase levels in the tissues of flounders adapted to sea water and fresh water. Comp Biochem Physiol 52A:713-717
Mayor HD, Hampton JC, Rosario B (1961) A simple method for removing the resin from epoxy-embedded tissue. J Cell Biol 9:909-910
McLean IW, Nakane PK (1974) Periodatelysineparaformaldehyde fixative. A new fixative for immunoelectron microscopy. J Histochem Cytochem 22:1077-1083[Medline]
Morgan IJ, Potts WTW, Oates K (1994) Intracellular ion concentrations in branchial epithelial cells of brown trout (Salmo trutta L.) determined by X-ray microanalysis. J Exp Biol 194:139-151
Perry SF, Laurent PL (1989) Adaptational responses of rainbow trout to lowered external NaCl concentration: contribution of the branchial chloride cell. J Exp Biol 147:147-168
Perry SF, Laurent P (1990) The role of carbonic anhydrase in carbon dioxide excretion, acid-base balance and ionic regulation in aquatic gill breathers. In Truchot J-P, Lahlou B, eds. Animal Nutrition and Transport Processes. 2. Transport, Respiration and Excretion: Comparative and Environmental Aspects. Vol, 6-39. 57
Rahim SM, Delaunoy J-P, Laurent P (1988) Identification and immunocytochemical localization of two different carbonic anhydrase isoenzymes in teleostan fish erythrocytes and gill epithelia. Histochemistry 89:451-459[Medline]
Sanyal G (1984) Comparative carbon dioxide hydration kinetics and inhibition of carbonic anhydrase isozymes in vertebrates. Ann NY Acad Sci 429:165-178[Medline]
Sanyal G, Pessah NI, Maren TH (1981) Kinetics and inhibition of membrane-bound carbonic anhydrase from canine renal cortex. Biochim Biophys Acta 657:128-137[Medline]
Wedge E, Svenneby G (1986) Effects of the blocking agent bovine serum albumin and Tween 20 in different buffers on immunoblotting of brain proteins and marker proteins. J Immunol Methods 88:233-237[Medline]
Whitney PL (1974) Affinity chromatography of carbonic anhydrase. Anal Biochem 57:467-476[Medline]
Yoshikawa JSM, McCormick SD, Young G, Bern HA (1993) Effects of salinity on chloride cells and Na+, K+-ATPase activity in the teleost gillichthys mirabilis. Comp Biochem Physiol 105A:311-317
Zbanyszek R, Smith LS (1984) Changes in carbonic anhydrase activity in coho salmon smolts resulting from physical training and transfer into seawater. Comp Biochem Physiol A 79:229-233[Medline]