ARTICLE |
Correspondence to: Etienne Hollande, Laboratoire de Biologie Cellulaire et Moléculaire des Epithéliums, Université Paul Sabatier, 38 rue des 36 Ponts, 31400 Toulouse, France. E-mail: hollande@lmtg.ups-tlse.fr
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Summary |
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The subcellular distribution of carbonic anhydrase II, either throughout the cytosol or in the cytoplasm close to the apical plasma membrane or vesicular compartments, suggests that this enzyme may have different roles in the regulation of pH in intra- or extracellular compartments. To throw more light on the role of pancreatic carbonic anhydrase II, we examined its expression and subcellular distribution in Capan-1 cells. Immunocytochemical analysis by light, confocal, and electron microscopy, as well as immunoblotting of cell homogenates or purified plasma membranes, was performed. A carbonic anhydrase II of 29 kD associated by weak bonds to the inner leaflet of apical plasma membranes of polarized cells was detected. This enzyme was co-localized with markers of Golgi compartments. Moreover, the defect of its targeting to apical plasma membranes in cells treated with brefeldin A was indicative of its transport by the Golgi apparatus. We show here that a carbonic anhydrase II is associated with the inner leaflet of apical plasma membranes and with the cytosolic side of the endomembranes of human cancerous pancreatic duct cells (Capan-1). These observations point to a role for this enzyme in the regulation of intra- and extracellular pH. (J Histochem Cytochem 49:10451053, 2001)
Key Words: carbonic anhydrase II, HCO3- secretion, Golgi apparatus, intracellular trafficking, pancreatic duct cells, cell culture
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Introduction |
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THE FAMILY OF CARBONIC ANHYDRASES (CA, EC 4.2.1.1) comprises 14 isoforms characterized by tissue-specific expression and different subcellular localizations. These enzymes catalyze the reversible hydration reaction of carbon dioxide and regulate acidbase balance in various organs (
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Materials and Methods |
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Cell Culture
Cells of the Capan-1 cell line, isolated (
Detection of CA II
Cytoenzymological Detection of CA.
The enzymatic activity of CA was demonstrated according to the Hansson's method by both light and electron microscopic examination of cells fixed in situ with 2.5% glutaraldehyde (15 min, 4C) (
Immunocytochemistry on Fixed Cells. CA II was detected by immunoperoxidase and immunofluorescence. Reactions were performed on 1- to 6-day-old cells cultivated on glass slides in Leighton tubes, fixed in situ with paraformaldehyde (PFA 3%), then permeabilized or not with ethanol. Cells were incubated successively with polyclonal sheep immunserum directed against human CA II (Serotec, Oxford, UK; 1:100, 1 hr) containing 0.1% bovine serum albumin (BSA) followed by a serum of rabbit anti-sheep IgGs coupled to peroxidase (Pierce, Rockford, IL; 1:400, TrisBSA, pH 7.6, 45 min). Antigenantibody complexes were revealed using 3'-amino-9-ethyl carbazole (AEC) (Sigma Chemical; St Louis, MO) in the presence of hydrogen peroxide. Some immunoperoxidase reactions were carried out on cells fixed with paraformaldehyde and embedded in paraplast. For immunofluorescence, antigenantibody complexes were revealed using anti-sheep IgG coupled to fluorescein isothiocyanate (FITC) (Nordic Immunological Laboratories; Tilburg, The Netherlands). In this case, preparations were observed with a confocal laser scanning microscope (LSM410; Carl Zeiss, Oberkochen, Germany). Fluorescence excitation was produced by an argon laser at a wavelength of 488 nm.
Immunocytochemistry on Living Cells.
To detect CA II on the surface of Capan-1 cells, immunofluorescence reactions were carried out on living cells. Under these conditions, the cells were not permeabilized and only the extracellular epitopes of CA are revealed. Briefly, after blocking of nonspecific sites, cells were incubated with anti-CA II antibodies (1:50 in culture medium with fetal calf serum 10%, 2 hr, 4C), then rinsed and incubated with anti-sheep IgG antibodies coupled to FITC (1:400, 4C, 2 hr), and fixed with a mixture of methanol (95%)acetic acid (5%) (-20C, 7 min). Similar reactions were performed to compare the membrane distribution of CA II and CA IV. In this case, polyclonal antibodies directed against a peptide sequence (residues 118) of human GPI-anchored CA IV were used (
Controls were carried out on both living and fixed cells. Cells were incubated (a) with primary antibody previously fully bound to purified bovine CA II (10 µg/ml, 3 hr; Sigma), or (b) with only secondary antibody coupled to peroxidase or FITC.
Immunoblotting
Cell Homogenates.
To estimate the expression of CA II during growth of Capan-1 cells, immunoblots were made on cell homogenates of 16-day-old cultures. Cells were rinsed in PBS, then scraped off. After centrifugation (5 min, 2000 x g), the pellet was solubilized for 20 min at 4C in 1 ml of lysis buffer [HEPES 20 mM, NaCl 150 mM, EDTA 1 mM, Nonidet P-40 1%, aprotinin 25 µg/ml, leupeptin 10 µg/ml, pepstatin 15 µg/ml, phenylmethylsulfone (PMSF) 1 mM, DNase 2 µg/ml]. The lysate was centrifuged (5 min, 14,000 x g) and the supernatant collected. The supernatant was stored at -80C or used directly for immunoblotting. Proteins were assayed according to
Plasma Membranes.
Plasma membranes were isolated on a continuous Percoll gradient according to the method of
Intracellular Traffic of CA II
Two types of experiments were carried out to analyze the intracellular traffic of CA II. First, immunocytochemical reactions for co-labeling CA II and Golgi compartments using anti-CA II antibodies (1:100) and anti-Golgi zone antibodies (1:80) (Valbiotech; Paris, France) were performed on fixed and permeabilized 3-day-old cells. Antigenantibody complexes were revealed with anti-IgG coupled to FITC (1:400) for CA II and anti-IgG coupled with tetramethyl-rhodamine isothiocyanate (TRITC; Nordic Immunological) (1:200) for the Golgi marker. Preparations were observed by confocal microscopy. Second, CA II was examined on cells treated with brefeldin A (BFA). Six-day-old cells were treated for 2, 5, 9, 14, or 16 hr with BFA (5 µg/ml; Sigma) solubilized in ethanol. CA II was then detected by immunoperoxidase reactions. The percentage of CA II-immunoreactive cells was then determined. The following controls were carried out: (a) Immunocytochemical reactions were performed on the cultures to estimate the distribution and intensity of CA II immunolabeling before treatment with BFA or ethanol; (b) cells were cultured in the presence of ethanol (1 µl/ml), the solvent of BFA for each duration of treatment (2, 5, 9, 14, or 16 hr) followed by immunocytochemistry for detection of CA II; (c) check of reversibility of the action of BFA: cells treated with BFA for 2, 5, 9, 14, or 16 hr were then maintained in a complete medium without ethanol or BFA. At 48 hr later the cultures were fixed for immunoperoxidase staining.
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Results |
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Expression of CA II in Capan-1 Cells
The immunoblots of the cell homogenates of 16- day-old Capan-1 cells revealed the presence of a single CA II-immunoreactive protein of 29 kD irrespective of the age of the culture (Fig 1a1, Lanes 16). Densitometric analysis showed that the expression of this protein increased gradually during growth in culture; it was threefold higher in post-confluent cells (Day 6) than in the cells at the start of growth (Day 2) (Fig 1a2). The co-migration with the purified bovine CA II (Fig 1a1, Lane 0) demonstrated that this protein was a CA II. The absence of crossreaction of the anti-CA II antibody with the human CA IV, another isoform of CA expressed in Capan-1 cells, was also verified by immunoblotting. In Fig 1b1 it can be seen that the anti-CA II antibody that recognized the purified bovine CA II (Fig 1b1, Lane 2) and the CA II expressed by Capan-1 cells (Fig 1b1, Lane 1) did not recognize the purified human 35-kD CA IV (Fig 1b1, Lane 3). Conversely, the anti-CA IV antibody did not recognize the 29-kD CA II expressed by Capan-1 cells nor the purified CA II (Fig 1b2, Lanes 1 and 3). This antibody revealed the purified human 35-kD CA IV (Fig 1b2, Lane 2) and two 35-kD and 55-kD proteins in the Capan-1 cell homogenates (Fig 1b2, Lane 1).
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Distribution of CA II in Capan-1 Cells
The localizations of CA II were determined from immunocytochemical reactions on fixed and permeabilized cells. At confluence, some cells exhibited strong intracytoplasmic immunoreactivity. This was mostly throughout the cytoplasm (Fig 2a) and especially in the neighborhood of the nucleus (Fig 2a, arrows). Other cells presented small immunofluorescent spots in their periphery (Fig 2a, arrowheads). In young cultures (13 days), most of the cells were non-polarized and exhibited intracytoplasmic immunoreactivity. This immunoreactivity was seen in the nuclear envelope (Fig 2b and Fig 2c, arrows) and in the cytoplasm around the nuclei, either in a horseshoe or circular pattern (Fig 2c, small arrows) surrounded by many small CA II-immunoreactive granules. After Day 3, most of the cells were weakly labeled on the nuclear envelope but presented immunoreactivity in the form of granulations throughout the cytoplasm. On the other hand, in post-confluent cultures most cells had become polarized, in which the immunoreactivity was noted mainly in cytoplasmic digitations and microvilli (Fig 2d and Fig 2e, arrows). In cross-sections of the cell layer, the immunoreactivity was seen at the apical poles of polarized cells (Fig 2f, arrows). In some cases, labeling was also observed along basolateral membranes (Fig 2f, arrowheads). Electron microscopic examination of immunoperoxidase reactions demonstrated strong immunoreactivity on the plasma membranes of the microvilli on polarized cells (Fig 3a). CA activity was also revealed on apical plasma membranes after the Hansson's reaction. Precipitates from the enzyme reaction were noted on both the outer and the inner leaflets of the plasma membrane (Fig 3b and Fig 3c, arrows) as well as in the cytosol. Immunofluorescence studies were conducted on living cells in an attempt to determine whether the CA II lay on the inner or the outer side of the membrane. No CA II immunoreactivity was observed on the surface of Capan-1 cells (Fig 3d). On the other hand, strong CA IV immunoreactivity was detected at the cell surface (Fig 3e). Superimposition of the CA IV fluorescence image on the same field observed by interference microscopy (Fig 3f, small arrows) provided further support for this localization.
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The controls designed to check the specificity of the immunocytochemical reactions were all negative.
Demonstration of the Association of CA II with Plasma Membranes
As further support for the presence of CA II associated with the plasma membranes of polarized cells, immunoblots were made on fractions of plasma membranes prepared under conditions of different ionic strength. Fig 4 shows the presence of a 29-kD CA II-immunoreactive protein from plasma membranes purified under conditions of low ionic strength (Fig 4, Lane 3). This protein migrated in the same way as the CA II found in the cell homogenates (Fig 4, Lane 1) or the purified bovine CA II (Fig 4, Lane 2). On the other hand, in fractions of plasma membranes purified under conditions of high ionic strength, no CA II-immunoreactive protein was detected (Fig 4, Lane 4).
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CA II Traffic
Treatment of 6-day-old cultures with BFA led to a marked reduction in the number of cells showing membrane CA II immunoreactivity. In the untreated cultures (Fig 5a) or those treated with ethanol alone (114 hr) or in cells treated with BFA for short periods (14 hr), 48% of cells presented CA II immunoreactivity on their surface, whereas only 30% of cells presented such immunoreactivity after 9 hr of treatment with BFA (Fig 5b) and only 2.1% in cultures treated for 14 hr (Fig 5c). After 14 hr of treatment, immunoreactivity was observed in filamentous or elongated intracytoplasmic structures but not on plasma membranes (Fig 5c, arrows). Treatment at a concentration of 5 µg/ml BFA for more than 14 hr was found to be cytotoxic. To check the absence of toxicity of BFA, cells treated for 14 hr were maintained in a medium without ethanol and BFA. Immunocytochemistry showed the reappearance of CA II on apical plasma membranes.
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To find out whether CA II was associated with Golgi compartments, double-labeling experiments were conducted on 3-day-old cells using antibodies against CA II and Golgi compartments. Confocal microscopic study by merging the image of CA II immunofluorescence (Fig 6a, arrows) with that of the immunofluorescence from Golgi compartments (Fig 6b, arrows) showed that the two were largely co-localized (Fig 6c).
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Discussion |
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In this study, we demonstrated an association of CA II with apical plasma membranes of polarized cells and with membranes of Golgi compartments in human cancerous pancreatic duct cells of the Capan-1 cell line.
Capan-1 cells expressed a CA II-immunoreactive protein whose molecular weight of 29 kD corresponds to that of the human CA II described in various cell types (
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Acknowledgments |
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Supported by the Association Française de Lutte contre la Mucoviscidose (AFLM) and by the Ministère de l'Enseignement Supérieur et de la Recherche (MESR).
We would like to thank Dr Esclassan for his fruitful discussion and assistance, and Mr F. Stefani and Mr C. Baritaud for their technical assistance.
Received for publication October 13, 2000; accepted February 21, 2001.
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