Amiloride-resistant Madin-Darby canine kidney (MDCK) cells exhibit decreased cation transport

Cation transport systems were investigated in mutant Madin-Darby Canine Kidney (MDCK) cells resistant to the diuretic drug amiloride. The mutants were isolated previously as clones resistant to the cytotoxic effects of 3 X 10(-4) M amiloride. Decreased amiloride transport by the Na+ channel was implicated as the basis of the resistance (Taub, '78). Consistent with this hypothesis, Na+ accumulation was lower in amiloride-resistant cells than in normal sensitive MDCK cells. Kinetic studies indicated that Na+ uptake in MDCK cells occurs by a single ATP independent transport system--the Na+ channel. In several amiloride-resistant clones, including clone Amr2, the decreased Na+ uptake was associated with a decrease in both the Km and Vmax for Na+ uptake by the Na+ channel. In Amr2 cells no significant alteration in the inhibitory effect of amiloride on Na+ uptake was observed. As the Na+ channel may actually be a general uptake system for monovalent cations (a number of cations inhibit Na+ uptake), the uptake of these inhibitory cations was examined in Amr2 cells. Both Ca++ and ouabain-insensitive Rb+ uptake occurred at decreased rates in Amr2 cells as compared with normal MDCK cells. However, further uptake studies suggested that Na+, Ca++ and ouabain-insensitive Rb+ uptake all occur by different systems. Thus several transport systems may be defective in Amr2 cells. Amr2 cells were also resistant to the inhibitory effects of amiloride on CO2 evolution from pyruvate. These observations indicate that alterations at a number of molecular sites may be associated with defective Na+ transport via the Na+ channel in amiloride-resistant cells. Thus the amiloride-resistant cells are potentially valuable in examining the interrelationships between Na+ transport and other cellular functions.     

Modulation of the epithelial barrier by dexamethasone and prolactin in cultured Madin-Darby canine kidney (MDCK) cells

Glucocorticoids and prolactin (PRL) have a direct effect on the formation and maintenance of tight junctions (TJs) in cultured endothelial and mammary gland epithelial cells. In this work, we investigated the effect of a synthetic glucocorticoid dexamethasone (DEX) and PRL on the paracellular barrier function in MDCK renal epithelial cells. DEX (4 microM)+PRL (2 microg/ml) and DEX alone increased significantly the transepithelial electrical resistance after chronic treatment (4 days) of confluent MDCK monolayers or after 24 h treatment of subconfluent monolayers. Immunoblotting and immunocytochemistry revealed no changes in the expression and distribution of TJ-associated proteins occludin, ZO-1 and claudin-1 in confluent monolayers after hormone addition. However, a marked increase in junctional content for occludin and ZO-1 with no changes in their total expression was observed in subconfluent MDCK monolayers 24 h exposed to DEX or DEX+PRL. No change in cell proliferation/growth was detected at subconfluent conditions following hormone treatment. An increase in the total number of viable cells was observed only in confluent MDCK monolayers after exposure to DEX+PRL suggesting that the main effect of these hormones on already established barrier may be associated with the inhibition of cell death. In conclusion, our data suggest that these hormones (specially dexamethasone) have an effect on TJ structure and function only during the formation of MDCK epithelial barrier by probably modulating the localization, stability or assembly of TJ proteins to membrane sites of intercellular contact.     

Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells

To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filter-grown Madin-Darby canine kidney (MDCK) cells from liposomes at 0 degrees C. After penetration into the cell, the fluorescent probe accumulated in the Golgi area at temperatures between 0 and 20 degrees C. Chemical analysis showed that C6-NBD-ceramide was being converted into C6-NBD-sphingomyelin and C6-NBD-glucosyl-ceramide. An analysis of the fluorescence pattern after 1 h at 20 degrees C by means of a confocal scanning laser fluorescence microscope revealed that the fluorescent marker most likely concentrated in the Golgi complex itself. Little fluorescence was observed at the plasma membrane. Raising the temperature to 37 degrees C for 1 h resulted in intense plasma membrane staining and a loss of fluorescence from the Golgi complex. Addition of BSA to the apical medium cleared the fluorescence from the apical but not from the basolateral plasma membrane domain. The basolateral fluorescence could be depleted only by adding BSA to the basal side of a monolayer of MDCK cells grown on polycarbonate filters. We conclude that the fluorescent sphingomyelin and glucosylceramide were delivered from the Golgi complex to the plasma membrane where they accumulated in the external leaflet of the membrane bilayer. The results also demonstrated that the fatty acyl labeled lipids were unable to pass the tight junctions in either direction. Quantitation of the amount of NBD-lipids delivered to the apical and the basolateral plasma membranes during incubation for 1 h at 37 degrees C showed that the C6-NBD-glucosylceramide was two- to fourfold enriched on the apical as compared to the basolateral side, while C6-NBD-sphingomyelin was about equally distributed. Since the surface area of the apical plasma membrane is much smaller than that of the basolateral membrane, both lipids achieved a higher concentration on the apical surface. Altogether, our results suggest that the NBD-lipids are sorted in MDCK cells in a way similar to their natural counterparts.     

Characterization of insulin-like growth factor I receptors on Madin-Darby canine kidney (MDCK) cell line

Specific insulin-like growth factor I (IGF-I) receptors on the Madin-Darby canine kidney (MDCK) cell line were identified and characterized. [125I]IGF-I specifically bound to the cells, but [125I]insulin bindings to the cells was minimal. Unlabeled IGF-I displaced both the IGF-I and insulin bindings with potencies that were 100 and 10 times as great as insulin. By an affinity labeling technique, IGF type I receptors were present in the MDCK cells. IGF-I stimulated DNA synthesis and cell proliferation at physiological concentrations. On the other hand, insulin had a little effect on DNA synthesis. These data suggest that IGF type I receptors as demonstrated in MDCK cells are involved in DNA synthesis and cell proliferation.     

Development of cell surface polarity in the epithelial Madin-Darby canine kidney (MDCK) cell line.

The development of surface polarity has been studied in the epithelial Madin-Darby canine kidney (MDCK) cell line by examining two basolateral markers: a monoclonal antibody against a 58-kd protein and [35S]methionine uptake. The surface distribution of these markers was followed after plating the cells on coverslips or nitrocellulose filters. In subconfluent monolayers the apical surface of many cells was stained with the anti-58-kd antibody. Clearing of the apical surface occurred first after confluency had been reached in cells grown on coverslips. Similarly, in cells grown on filters the basolateral 58-kd protein disappeared from the apical surface concomitantly with the development of a measurable electrical resistance over the cell monolayer. The uptake of [35S]methionine was measured from both sides of filter-grown cells and began to polarize early after seeding, reaching a value of greater than 98% basolateral in the fully polarized monolayer. These results emphasize that the development of surface polarity in MDCK cells is a gradual process, and that extensive cell-cell contacts seem to be required for complete surface polarization.     

Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters.

Madin-Darby canine kidney (MDCK) cells (strain I) grown on 0.45 micron pore size nitrocellulose filters formed monolayers which were highly polarized and had high transepithelial electrical resistance (greater than 3000 ohm X cm2). Morphometric analysis showed that the area of the basolateral surface domain was 7.6 times larger than that of the apical. The uptake of fluid-phase markers [3H]inulin and horseradish peroxidase (HRP) was studied from the apical and the basal side of the monolayer. Uptake of [3H]inulin was biphasic and the rate during the first 40 min corresponded to a fluid phase uptake of 20.5 X 10(-8) nl/min per cell from the basolateral side, and 1.0 X 10(-8) nl/min per cell from the apical side. Electron micrographs of the monolayers after HRP uptake showed that the marker was rapidly delivered into endosome-like vesicles and into multivesicular bodies. No labelling of the Golgi complex could be observed during 2 h of uptake. Evidence was obtained for the transport of fluid phase markers across the cell. HRP and fluorescein isothiocyanate-dextran crossed the monolayers in either direction at a rate corresponding to approximately 3 X 10(-8) nl of fluid/min/cell. Adding the transcytosis rate to the rate of fluid accumulation into the cell yielded a total basolateral endocytic rate which was 6-fold greater than the apical rate. When the uptake rates were normalized for membrane area the apical and basolateral endocytic rates were about equal per unit cell surface area.     

Metabolic responses of sulfatide and related glycolipids in Madin-Darby canine kidney (MDCK) cells under osmotic stresses

Incorporation of (35)S-sulfate into the polar molecular species of sulfoglycolipids (SM4s) in Madin-Darby canine kidney cells increased in a hypertonic medium (500 mOsm/L) supplemented with sodium chloride. The unknown sulfoglycolipid (SX) was identified as GlcCer sulfate based on the results of TLC, GLC, and mass spectra. The synthesis of SX increased in the hypotonic medium unlike that of SM4s and SM3. TLC showed that hypertonic stress induced the accumulation of GalCer as a precursor of SM4s, whereas hypotonic stress increased GlcCer as a precursor of GlcCer sulfate. The level of ceramide as a precursor of both GalCer and GlcCer increased under hypertonic stress and decreased under hypotonic stress. Cerebroside sulfotransferase mRNA was shown to be elevated in the hyperosmotic condition but not in the hypotonic condition. The increase in SM4s under hypertonic stress was induced by the activation of both the ceramide galactosyltransferase and the cerebroside sulfotransferase genes, whereas the increase in GlcCer sulfate under hypotonic stress was caused by the accumulation of GlcCer as the result of activation of ceramide glucosyltransferase.     

Characterization of subclones of Madin-Darby canine kidney renal epithelial cell line

Heterogeneity in Madin-Darby canine kidney (MDCK) epithelial cells has been reported, however, its details have not been well described. In the present study, we show that subclones obtained from a MDCK cell line could be divided into two morphologically and biochemically distinct cell types with different hormonal responsiveness. Clones of the first type, motile clones, which had extended and flattened cytoplasm, were devoid of carbonic anhydrase activity. Clones of the second type, nonmotile clones, formed colonies of cuboidal cells and showed carbonic anhydrase activity. Motile clones synthesized cAMP in response to arginine vasopressin, prostaglandin E1, and isoproterenol but not glucagon. In contrast, nonmotile clones responded to all of these hormones. These findings suggest MDCK cells have multiple cellular origins. The motile clones have characteristics similar to the principal cells of the collecting system, whereas the nonmotile clones may be derived from the thick ascending limb or the intercalated cell. Our studies also demonstrate a significant influence of culture condition on MDCK cellular behavior (carbonic anhydrase activity, Na+/K+-ATPase activity and vasopressin responsiveness). Therefore, physiologic and biochemical experiments with MDCK cells must be interpreted with reservations about cellular heterogeneity as well as differences induced by culture conditions.     

Characterization of Zn(2+) transport in Madin-Darby canine kidney cells

The aim of this study was to characterize the mechanism implicated in Zn(2+) transport in MDCK cells. Trace elements such as Zn(2+), Cd(2+) or Cu(2+) induced MDCK cell depolarization at the micromolar level as demonstrated by bis-oxonol fluorescence and whole-cell patch experiments. This depolarization was inhibited by La(3+) and Gd(3+) and was not related to the activation of Na(+) or Cl(-) channels. Uptake of 65Zn was assessed under initial rate conditions. The kinetic parameters obtained at 37 degrees C were a K(m) of 18.9 microM and a V(max) of 0.48 nmol min(-1) (mg protein(-1)). Intracellular pH measurements using BCECF probe demonstrated that Zn(2+) transport induced a cytoplasmic acidification. The cytoplasmic acidification resulting from Zn(2+) uptake activated Na(+)/H(+) antiporter, which allowed for the recycling of protons. These data suggest that Zn(2+) enters MDCK cells through a proton-coupled metal-ion transporter, the characteristics of which are slightly different from those described for the metal transporter DCT1. This mechanism could be in part responsible of the metal transport evidenced in the distal parts of the renal tubule.     

Purification and characterization of the apically secreted 80 KDa glycoprotein from Madin-Darby canine kidney (MDCK) cells.

An 80 KDa glycoprotein (gp 80), known to be released predominantly from the apical surface by filter-grown Madin-Darby canine kidney cells, was purified to electrophoretic homogeneity. Purified gp 80 was found to have a disulfide-bonded dimeric structure, and appeared to exist in two molecular forms, a major (high-molecular weight) form consisting of a 46 KDa subunit and a 39 KDa subunit and a minor (low-molecular weight) form consisting of a 46 KDa subunit and a 33 KDa subunit. Upon de-glycosylation by N-glycanase treatment, the 46 KDa subunit was converted to a 25.6 KDa form, whereas both the 39 KDa and the 33 KDa subunit gave rise to a 21.1 KDa form. V8 protease mapping of deglycosylated polypeptides revealed the 39 KDa and the 33 KDa subunit to have nearly identical band patterns, which also exhibited a high degree of homology to that derived from the 46 KDa subunit. Radioimmunoassays revealed that the binding of the purified gp 80 to fibrinogen (or heparin) was dependent on both pH and divalent cations. Furthermore, binding of gp 80 to immobilized fibrinogen (or heparin) was inhibited in the presence of free fibrinogen (or heparin) added in the assay mixture.     

Sphingolipid metabolism is a crucial determinant of cellular fate in nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells

The present report was addressed to study the influence of sphingolipid metabolism in determining cellular fate. In nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells, sphingolipid de novo synthesis is branched mainly to a production of sphingomyelin and ceramide, with a minor production of sphingosylphosphocholine, ceramide 1-phosphate, and sphingosine 1-phosphate. Experiments with (32)P as a radioactive precursor showed that sphingosine 1-phosphate is produced mainly by a de novo independent pathway. Enzymatic inhibition of the de novo pathway and ceramide synthesis affected cell number and viability only slightly, without changing sphingosine 1-phosphate production. By contrast, inhibition of sphingosine kinase-1 activity provoked a significant reduction in both cell number and viability in a dose-dependent manner. When sphingolipid metabolism was studied, an increase in de novo formed ceramide was found, which correlated with the concentration of enzyme inhibitor and the reduction in cell number and viability. Knockdown of sphingosine kinase-1 expression also induced an accumulation of de novo synthesized ceramide, provoking a slight reduction in cell number and viability similar to that induced by a low concentration of the sphingosine kinase inhibitor. Taken together, our results indicate that the level of de novo formed ceramide is controlled by the synthesis of sphingosine 1-phosphate, which appears to occur through a de novo synthesis-independent pathway, most probably the salvage pathway, that is responsible for the MDCK cell fate, suggesting that under proliferating conditions, a dynamic interplay exists between the de novo synthesis and the salvage pathway.     

Involvement of VIP36 in intracellular transport and secretion of glycoproteins in polarized Madin-Darby canine kidney (MDCK) cells

VIP36, an intracellular lectin that recognizes high mannose-type glycans (Hara-Kuge, S., Ohkura, T., Seko, A., and Yamashita, K. (1999) Glycobiology 9, 833-839), was shown to localize not only to the early secretory pathway but also to the plasma membrane of Madin-Darby canine kidney (MDCK) cells. In the plasma membrane, VIP36 exhibited an apical-predominant distribution, the apical/basolateral ratio being approximately 2. Like VIP36, plasma membrane glycoproteins recognized by VIP36 were found in the apical and basolateral membranes in the ratio of approximately 2 to 1. In addition, secretory glycoproteins recognized by VIP36 were secreted approximately 2-fold more efficiently from the apical membrane than from the basolateral membrane. Thus, the apical/basolateral ratio of the transport of VIP36-recognized glycoproteins was correlated with that of VIP36 in MDCK cells. Upon overproduction of VIP36 in MDCK cells, the apical/basolateral ratios of both VIP36 and VIP36-recognized glycoproteins were changed from approximately 2 to approximately 4, and the secretion of VIP36-recognized glycoproteins was greatly stimulated. In contrast to the overproduction of VIP36, that of a mutant version of VIP36, which has no lectin activity, was of no effect on the distribution of glycoproteins to apical and basolateral membranes and inhibited the secretion of VIP36-recognized glycoproteins. Furthermore, the overproduction of VIP36 greatly stimulated the secretion of a major apical secretory glycoprotein of MDCK cells, clusterin, which was found to carry at least one high mannose-type glycan and to be recognized by VIP36. In contrast to the secretion of clusterin, that of a non-glycosylated apical-secretion protein, galectin-3, was not stimulated through the overproduction of VIP36. These results indicated that VIP36 was involved in the transport and sorting of glycoproteins carrying high mannose-type glycan(s).     

Increment of DNA topoisomerases in chemically and virally transformed cells

The activities of topoisomerases I and II were assayed in subcellular extracts obtained from nontumorigenic BALB/c 3T3 A31 and normal rat kidney (NRK) cell lines and from the same cells transformed by benzo[a]pyrene (BP-A31), Moloney (M-MSV-A31) and Kirsten (K-A31) sarcoma viruses, and simian virus 40 (SV-NRK). The enzymatic activity of topoisomerase I was monitored by the relaxation of negatively supercoiled pBR322 DNA and by the formation of covalent complexes between 32P-labeled DNA and topoisomerase I. Topoisomerase II activity was determined by decatenation of kinetoplast DNA (k-DNA). It was found that nuclear and cytoplasmic type I topoisomerase specific activities were higher in every transformed cell line than in the normal counterparts. These differences cannot be attributed to an inhibitory factor present in A31 cells. When chromatin was treated at increasing ionic strengths, the 0.4 M NaCl extract showed the highest topoisomerase I specific activity. Moreover, in this fraction the transformed cells exhibited the most significant increment in the enzymatic activity as compared with nontransformed cultures. Spontaneously transformed A31 cells showed topoisomerase I activity similar to that of extracts of cells transformed by benzo[a]pyrene. Topoisomerase II specific activity was also increased in SV-NRK cells, as judged by the assay for decatenation of k-DNA to yield minicircle DNA.     

Biosynthesis of the cell adhesion molecule uvomorulin (E-cadherin) in Madin-Darby canine kidney epithelial cells

The Ca(2+)-dependent cell adhesion molecule uvomorulin is a transmembrane glycoprotein that functions at the cell surface to regulate epithelial cell recognition and adhesion. We have investigated the temporal and spatial regulation of uvomorulin biosynthesis and cell surface expression in Madin-Darby canine kidney epithelial cells. We show that uvomorulin is synthesized as a precursor polypeptide (Mr 135,000) that is core glycosylated in the endoplasmic reticulum. The precursor is processed to the mature polypeptide (Mr 120,000) shortly after addition of complex carbohydrate groups in the late Golgi complex, but prior to delivery of the polypeptide to the cell surface. However, glycosylation is not required for either efficient processing of the precursor or transport of uvomorulin to the cell surface. At the cell surface, uvomorulin is turned over rapidly (t1/2 approximately 5 h). Induction of Ca(2+)-dependent cell-cell contact results in rapid localization of cell surface uvomorulin to regions of contact and an increase in the proportion of uvomorulin that is insoluble in buffers containing Triton X-100. These results indicate several regulatory steps in the biosynthesis and cell surface expression of uvomorulin in epithelial cells.     

Gangliosides of chemically and virally transformed rat embryo cells.

The gangliosides of control rat embryo cells, 3-methylcholanthrene, Rauscher leukemia virus, and combined 3-methylcholanthrene-Rauscher leukemia virus transformants were examined using [14 C]glucosamine as a tracer. All four cell lines exhibited a complex pattern of gangliosides. While N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosyl-glucosyl-ceramide was the major ganglioside in the control cell line, N-acetylneuraminyl-galactosyl-glucosyl-ceramide was the major ganglioside in the three transformants. The 3-methylcholanthrene transformant possessed a ganglioside pattern different from that of the Rauscher leukemia virus transformant. Hydrolysis of the gangliosides indicated that galactosamine, N-acetyl-and N-glycolylneuraminic acid were the labeled components in all cell lines.     

Increase of poly(L-lysine) uptake but not fluid phase endocytosis in neuraminidase pretreated Madin-Darby canine kidney (MDCK) cells

The uptake of Poly(L-lysine) conjugates in cultured cells has been used as a model for non-specific adsorptive endocytosis of cationic macromolecules. To study the effect of glycocalyx desialylation on the uptake of cationic macromolecules in epithelial cells, Madin-Darby canine kidney (MDCK) cell monolayers were treated with neuraminidase and incubated with Poly(L-lysine) conjugates. Neuraminidase pretreatment of MDCK cells resulted in a 40% increase in the uptake of Poly(L-lysine) whereas trypsin pretreatment did not. Neuraminidase pretreatment did not increase the endocytosis of fluid phase markers in MDCK cells, nor the uptake of Poly(L-lysine) in L929 fibroblasts. These results suggest that the negative charges, rather than the glycoprotein matrices of glycocalyx, play an important role in the control of the uptake of cationic macromolecules in epithelial cells.     

Dynamics of membrane-skeleton (fodrin) organization during development of polarity in Madin-Darby canine kidney epithelial cells

Madin-Darby canine kidney (MDCK) epithelial cells exhibit a polarized distribution of membrane proteins between the apical and basolateral domains of the plasma membrane. We have initiated studies to investigate whether the spectrin-based membrane skeleton plays a role in the establishment and maintenance of these membrane domains. MDCK cells express an isoform of spectrin composed of two subunits, Mr 240,000 (alpha-subunit) and Mr 235,000 (gamma-subunit). This isoform is immunologically and structurally related to fodrin in lens and brain cells, which is a functional and structural analog of alpha beta-spectrin, the major component of the erythrocyte membrane skeleton. Analysis of fodrin in MDCK cells by immunoblotting, immunofluorescence, and metabolic labeling revealed significant changes in the biophysical properties, subcellular distribution, steady-state levels, and turnover of the protein during development of a continuous monolayer of cells. The changes in the cellular organization of fodrin did not appear to coincide with the distributions of microfilaments, microtubules, or intermediate filaments. These changes result in the formation of a highly insoluble, relatively dense and stable layer of fodrin which appears to be localized to the cell periphery and predominantly in the region of the basolateral plasma membrane of MDCK cells in continuous monolayers. The formation of this structure coincides temporally and spatially with extensive cell-cell contact, and with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral plasma membrane.     

Modulation of fodrin (membrane skeleton) stability by cell-cell contact in Madin-Darby canine kidney epithelial cells

During growth of Madin-Darby canine kidney (MDCK) epithelial cells, there is a dramatic change in the stability, biophysical properties, and distribution of the membrane skeleton (fodrin) which coincides temporally and spatially with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral domain of the plasma membrane. These changes occur maximally upon the formation of a continuous monolayer of cells, indicating that extensive cell-cell contact may play an important role in the organization of polarized MDCK cells (Nelson, W. J., and P. J. Veshnock, 1986, J. Cell Biol., 103:1751-1766). To directly analyze the role of cell-cell contact in these events, we have used an assay in which the organization of fodrin and membrane proteins is analyzed in confluent monolayers of MDCK cells in the absence or presence of cell-cell contact by adjusting the concentration Ca++ in the growth medium. Our results on the stability and solubility properties of fodrin reported here show directly that there is a positive correlation between cell-cell contact and increased stability and insolubility of fodrin. Furthermore, we show that fodrin can be recruited from an unstable pool of protein to a stable pool during induction of cell-cell contact; significantly, the stabilization of fodrin is not affected by the addition of cyclohexamide, indicating that proteins normally synthesized during the induction of cell-cell contact are not required. Together these results indicate that cell-cell contact may play an important role in the development of polarity in MDCK cells by initiating the formation of a stable, insoluble matrix of fodrin with preexisting (membrane) proteins at the cell periphery. This matrix may function subsequently to trap proteins targeted to the membrane, resulting in the maintenance of membrane domains.     

Subtypes of Madin-Darby canine kidney (MDCK) cells defined by immunocytochemistry: Further evidence for properties of renal collecting duct cells

The Madin-Darby canine kidney (MDCK) cell line has been proposed as a model for studying intercalated (IC) cells of the renal cortical collecting duct. The IC cells are characterized by peanut lectin (PNA) binding capacity, carbonic anhydrase (CA) activity and Cl^-–HCO ₃ ^- exchange mediated by a band 3-related protein. It has been suggested that these properties are also expressed in MDCK cells. So far however, the nature of the specific protein involved in Cl^-–HCO ₃ ^- exchange, the type of CA isozyme and the relationship between these two characteristics and PNA binding, have not been investigated in MDCK cells by immunocytochemical methods. Using two antibodies raised against human erythrocyte band 3 protein and two against human erythrocyte CA I and II isozymes, our study provides evidence that a protein related to band 3 is expressed in about 5% of cultured MDCK cells; these band 3-positive cells do not bind PNA and are not reactive for CAI or CAII. About 30% of the MDCK cells bind PNA, two-thirds of which are also CAII-positive. A majority (about 65%) of MDCK cells is not reactive for the three markers used; their density is increased after incubation with aldosterone. These data indicate (i) that the Cl^-–HCO ₃ ^- exchanger of the MDCK cells could be related to human erythrocyte band 3, (ii) that the CA activity of the MDCK cell line bears antigenic identity with the erythrocyte CA II isozyme and (iii) that the latter is always co-localized with PNA binding. These results provide immunocytochemical evidence for the heterogeneity of the MDCK cell line, which might reflect the cellular heterogeneity encountered in the renal cortical collecting duct. Our data also indicate that clonal selection will be required for future functional studies of the MDCK cells.