Carbonic Anhydrase in the Gills of Seawater-and Freshwater-acclimated Flounders Platichthys flesus: Purification,Characterization,and Immunohistochemical Localization

Abstract

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 (K_I) 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 inter-lamellar regions, and at the bases of the secondary lamellae.

Keywords

carbonic anhydrase flounder gills erythrocytes chloride cells mucous cells pavement cells primary lamellae secondary lamellae immunohistochemistry

The fish gill is a complex organ, known to be involved in respiratory gas exchange, ion transport, and acid-base regulation. Carbonic anhydrase (CA), abundantly present in gill epithelial cells, is assumed to play a role in these processes (see Perry and Laurent 1990 for review). However, according to the authors, gill CA has no role in CO₂ excretion under normal conditions. The erythrocyte CA is the primary and possibly the only site of HCO₃ ⁻ dehydration in fish, forming CO₂ which diffuses across the respiratory gill epithelium into the ventilatory water. A small portion of the CO₂ is hydrated by cytoplasmic branchial CA, supplying HCO₃ ⁻ and H⁺ for Na⁺/H⁺(NH₄ ⁺) and Cl⁻/HCO₃ ⁻ apical ionic exchangers. In view of these processes regulating acid-base and NaCl balance, the cell-specific localization of CA in fish gills has been investigated (Dimberg et al. 1981; Rahim et al. 1988; Flügel et al. 1991) and a large interspecies variability has been observed (Conley and Mallatt 1988). Considering the role of gill CA in osmoregulatory processes, the relation between branchial CA activity and/or distribution and ambient salinity has been also investigated in several species, yielding controversial results (Mashiter and Morgan 1975; Dimberg et al. 1981; Haswell et al. 1983; Lacy 1983; Zbanysek and Smith 1984; Perry and Laurent 1990; Flügel et al. 1991). In the flounder (Platichthys flesus), an euryhaline species, no significant differences in CA levels between seawater- and freshwater-adapted fish have been found in earlier studies in gills (Mashiter and Morgan 1975) and red cells (Carter et al. 1976). In this investigation we isolated and characterized the CA from flounder erythrocytes and from gills and determined their specific CA activities after seawater and freshwater acclimation. Polyclonal antibodies against purified erythrocyte and gill CA were raised. In gill sections from both freshwater and seawater flounders, CA was then localized immunohistochemically to visualize the cell-specific distribution of CA and possible adaptations to environmental salinity in the various cell types.

Materials and Methods

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 250-450 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 Whitney (1974) using p-aminomethylbenzenesulfonamide-agarose (pAMBS-gel; Sigma, Deisenhofen, Germany). CA was eluted from the column by 0.5 M NaClO₄, 0.1 M CH₃COONa (pH 5.6). By means of gel filtration (Sephadex G-75, particle size 40-120 μm; Pharmacia, Freiburg, Germany) NaClC₄ was removed and CA was equilibrated with 5 mM Tris-HCl, 0.15 M NaCl (pH 7.4). The purity of the enzyme was examined by SDS-PAGE (Laemmli 1970) in a 15% acrylamide gel. Proteins were stained with Coomassie Blue (PhastGel Blue R; Pharmacia).

Purification of Branchial CA

Before preparation, the gills were perfused with saline via the bulbus arteriosus until they appeared completely bloodfree. 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 Maren (1960) as modified by Bruns et al. (1986) in a barbital buffer system. Inhibition properties of erythrocyte and gill CA were studied by using increasing concentrations of acetazolamide (ACTZ; Sigma) and potassium iodide (KI; Merck, Darmstadt, Germany) in the assay. Inhibition constants (K_I) were then calculated according to Easson and Stedman (1937). Protein concentrations were analyzed according to Lowry et al. (1951).

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 (Kyhse-Anderson 1984; Wedge and Svenneby 1986). After transfer of the proteins from the gel to nitrocellulose membranes, they were immunostained using antisera and preimmune serum at 1:5000 dilution. Goat anti-rabbit IgG-peroxidase-conjugated secondary antibodies (Sigma) were diluted 1:1000, and peroxidase activity was visualized with 3,3′-diaminobenzidine tetrahydrochloride (Sigma).

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 5-6 hr in one of the following fixatives.

PLP (Periodate-Lysine-Paraformaldehyde). The fixative (0.01 M sodium periodate, 0.075 M lysine, 2% paraformaldehyde, 0.04 M phosphate buffer) was prepared according to McLean and Nakane (1974).

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 7-10-μm thick were mounted on gelatinor poly-L-lysine-coated slides (Sigma). After deparaffination, sections were immunostained by the avidin-biotin-peroxidase complex (ABC) technique (Hsu et al. 1981; Cetin et al. 1994) as described by Decker et al. (1996). Semithin sections were cut from epoxy-embedded tissue at 0.5 μm. After removal of the epoxy resin by sodium methoxide (Mayor et al. 1961), they were processed for immunostaining using the ABC method. The primary antibodies were applied at 1: 1000 dilution. Control sections were incubated with preimmune serum instead of immune serum at identical dilution.

Results

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).

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.

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.

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 K_I 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 Easson-Stedman 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.

Table 1

Subcellular distribution of gill CA activity (U…ml) in freshwater flounders^a

Hom	20,224
SI	14,820
PI	2718
SII	12,913
PII	338
SIII	177
PIII	39

Hom, homogenate; S, supernatant; P, pellet. The recovery values of CA activity after each centrifugation were 86.7% (SI + PI), 89.4% (SII + PII) and 63.9% (SIIII + PIII). Data are given for one preparation of pooled gills obtained from 20 flounders. SII and SIII represent cytosolic fractions with most of the CA activity; PIII is the washed membrane fraction with only minimal CA activity.

Table 2

Specific CA activity in gill fractions SII and SIII and erythrocytes (U…ml…mg⁻¹)^a

	Freshwater flounders	Saltwater flounders
SII	9.6	9.8
SIII	5.7	5.7
Erythrocytes	13.1	13.7

Data from pooled preparations of 20 freshwater and 20 saltwater flounders.

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 Laurent and Dunel 1980 for details).

Figure 1

Molecular and immunological characterization of flounder CA. Lane 1, purified gill CA; Lane 2, gill SII after centrifugation at 100,000 X g; Lane 3, purified erythrocyte CA; Lanes 4 and 5, erythrocyte lysate; Lane 6, purified erythrocyte CA; Lane 7, gill SII after centrifugation; Lane 8, purified gill CA. Primary antibody in A is anti-gill CA and in B is anti-erythrocyte CA. A molecular mass marker mix in the range from 10 kD to 200 kD was used. The molecular mass of bovine CA (30 kD) is marked. Each lane shows a single band of immunostained CA at 30 kD, indicating a specific reaction of the antibodies with both erythrocyte and gill CA.

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).

Figures 2-3

Easson-Stedman plots for inhibition of flounder erythrocyte CA (Figure 2) and branchial CA (Figure 3) by acetazolamide (Figures 2A and 3A) and potassium iodide (Figure 2B and 3B). i is the fractional inhibition of the enzyme activity at a total concentration of inhibitor I₀. The slope of the linear regression line gives the inhibition constant K_I. r ² was 0.77 (Figure 2A), 0.90 (Figure 2B), 0.49 (Figure 3A) and 0.92 (Figure 3B).

Figures 4-7 Paraffin sections from gill lamellae of flounders adapted to 30 ppt salinity. Tissue specimens were fixed in Bouin's solution (Figure 4) or in PLP (Figures 5–7). CC, chloride cells; PC, pillar cells; MC, mucous cells; PVC, pavement cells; SL, secondary lamellae; CVS, central venous sinus; C, cartilage. Bars = 50 μm.

Figure 4

Section incubated with anti-erythrocyte CA/avidin-biotin-peroxidase complex (ABC). CA-specific staining is apparent in chloride cells, pillar cells, cartilage, and in the membranes of some mucous cells.

Figure 5

Section immunostained with anti-erythrocyte CA/ABC. Chloride cells, pillar cells, and cells of unidentified type (arrowheads) show a strong reaction. Very weak staining is exhibited by pavement cells.

Figure 6

Section stained with anti-gill CA/ABC leading to a CA-specific reaction in chloride cells, pillar cells and pavement cells.

Figure 7

Control section incubated with preimmune serum/ABC, showing weak unspecific staining only.

Figures 8-9

Semithin sections of PLP fixed gills from a saltwater- and a freshwater-adapted flounder, respectively. The sections were incubated with anti-erythrocyte CA/ABC, showing staining of chloride cells, pillar cells, mucous cells, and pavement cells. Chloride cells appear to be rounder in outline in freshwater flounders. Bars = 50 mm.

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 (Figures 4–6). 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 (Figures 8 and 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 Figures 4–6), whereas the CCs of freshwater-acclimated fish appeared almost round.

Discussion

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) (Henry et al. 1988), rainbow trout, and lamprey (Petromyzon marinus) (Henry et al. 1993).

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 Carter et al. (1976) regarding CA levels in flounder erythrocytes. Mashiter and Morgan (1975) also found CA activity in flounder gills to be independent of environmental salinity. Contrary to our own results, however, they could not find any CA in flounder red blood cells. No relation to salinity was reported for the CA activity in erythrocytes and gills of the eel (Anguilla anguilla) (Haswell et al. 1983). However, the esterase activity of CA in the gills of young coho salmon (Oncorhynchus kisutch) was significantly higher in saltwater-adapted compared to freshwater fish (Zbanysek and Smith 1984). The specific CA activity in the gills of Oreochromis mossambicus increased in correlation to an enhanced environmental salinity as well (Kültz et al. 1992).

Inhibition Properties of Branchial and Erythrocyte CA

In this study the inhibition constants K_I 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. Maren et al. (1980) obtained a K_I value of 3…10⁻⁵ M for erythrocyte CA of the teleost Lophius americanus towards I⁻; the corresponding K_I value of the elasmobranch Squalus acanthias was 9…10⁻³ M. The K_I value for human CA II towards potassium iodide was given as 26…10⁻³ M (Maren et al. 1976). Towards ACTZ, the inhibition constants of flounder gill and erythrocyte CA were determined here to be 7.6…10⁻⁹ M and 8.4…10⁻⁹ M, respectively. A similar high affinity to sulfonamides was exhibited by the CA of gills (K_I = 2.01…10⁻⁹ M) and erythrocytes (K_I = 2.04…10⁻⁹ M) of the rainbow trout (Henry et al. 1993). A high affinity towards ACTZ was also found for the CA of the teleost Archosargus probatocephalus (K_I = 3…10^~8 M) (Sanyal 1984) and also for human CA II (K_I = 1…10⁻⁸ M; Sanyal et al. 1981). For benzolamide, Maren et al. (1980) determined a K_I value of 4…10⁻¹⁰ M with flounder and rainbow trout erythrocyte CA and proposed that the CA of teleost red blood cells might correspond to human CA II, which is in line with our present results.

Localization of Gill CA

The histochemical localization of branchial CA has been studied in several fish species using Hansson's technique (Hansson 1967). In gill lamellae from young salmon (Salmo salar), CA activity was found mainly in red blood cells and in the thin epithelial cells of the secondary lamellae (Dimberg et al. 1981). Chloride cells also showed a positive reaction. The staining of the epithelial cells was much stronger in fish exposed to saltwater compared to feshwater fish, predominantly in the apical parts. Conley and Mallat (1988) localized CA activity in the gills of 17 fish species. Pavement cells, chloride cells, or both were stained. The staining pattern showed little relation to taxonomy or to habitat salinity. Mucous cells remained always unstained; pillar cells showed positive staining in Salmo gairdneri. The branchial organs (gills and operculum) of the euryhaline killyfish Fundulus heteroclitus were investigated by Flügel et al. (1991). In seawater-adapted fish, CA was found predominantly in the CCs, which were considerably larger than in freshwater fish. Staining for CA in these cells appeared to be concentrated in the apical parts of the cytoplasm. Freshwater-acclimatized specimens showed no or only a faint reaction in CCs. In both groups, reaction products for CA were found in the respiratory cells of the secondary lamellae.

Rahim et al. (1988) purified one CA isozyme from red blood cells of the freshwater carp, Cyprinus carpio, and another isozyme from gills of the rainbow trout Salmo gairdneri (now Oncorhynchus mykiss). Both isozymes were shown to be immunologically distinct. This is in contrast to our results in the flounder, in which red cell CA and gill CA are identical. Immunohistochemically, Rahim et al. (1988) found heavy staining at the epithelial surface of the secondary lamellae, whereas pillar cells showed no reaction. In the primary epithelium, an intense immunoreaction was seen in the outer cell layer consisting mainly of CCs together with PVCs and MCs. The inner layers, which mostly consist of undifferentiated cells, showed weaker staining.

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 (Dimberg et al. 1981) and killyfish (Flügel et al. 1991), the staining intensity for CA varied with salinity in epithelial cells (salmon) and CCs (killyfish). These cells showed a stronger reaction in saltwater-adapted fish. This is in contrast to our results in the flounder, which show no difference in staining intensity and pattern between freshwater- and saltwater-adapted fish.

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 (Goss et al. 1992; Laurent et al. 1994; Morgan et al. 1994). Other studies (Laurent et al. 1985; Perry and Laurent 1989) suggest that both Na⁺ and Cl⁻ uptake occur in CCs. CCs in saltwater fish have been shown to be responsible for branchial ion excretion (Foskett and Scheffey 1982). Our present localization of cytoplasmic CA in pavement cells and in CCs both in freshwater and seawater flounders is in principle compatible with the idea that CA at this site is involved in osmoregulation. It has been known for several years that fish CCs may be affected by changes in environmental salinity (see Laurent and Dunel 1980 for review). In the flounder, the CCs of freshwater-adapted fish appear to be rounder in outline than in seawater fish. Whether or not this finding represents an osmoregulatory adaptation remains to be ascertained. Similar morphological adjustments to decreasing salinity have been described for the longjawed mudsucker, Gillichthys mirabilis (Yoshikawa et al. 1993).

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 Perry and Laurent (1990) for a role of this possible external CA.

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.

Footnotes

Acknowledgment

The expert technical assistance of Katrin Stoll and Martina Meyer is gratefully acknowledged.

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