Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin, and fodrin in Madin- Darby canine kidney epithelial cells

Cell-cell contact is an important determinant in the formation of functionally distinct plasma membrane domains during the development of epithelial cell polarity. In cultures of Madin-Darby canine kidney (MDCK) epithelial cells, cell-cell contact induces the assembly and accumulation of the Na+,K+-ATPase and elements of the membrane-cytoskeleton (ankyrin and fodrin) at the regions of cell-cell contact. Epithelial cell-cell contact appears to be regulated by the cell adhesion molecule uvomorulin (E-cadherin) which also becomes localized at the lateral plasma membrane of polarized cells. We have sought to determine whether the colocalization of these proteins reflects direct molecular interactions which may play roles in coordinating cell-cell contact and the assembly of the basal-lateral domain of the plasma membrane. Recently, we identified a complex of proteins containing the Na+,K+-ATPase, ankyrin, and fodrin in extracts of whole MDCK cells (Nelson, W.J., and R. W. Hammerton. 1989. J. Cell Biol. 108:893-902). We have now examined cell extracts for protein complexes containing the cell adhesion molecule uvomorulin. Proteins were solubilized from whole MDCK cells and fractionated in sucrose gradients. The sedimentation profile of solubilized uvomorulin is well separated from the majority of cell surface proteins, suggesting that uvomorulin occurs in a protein complex. A distinct portion of uvomorulin (30%) cosediments with ankyrin and fodrin (approximately 10.5S). Further fractionation of cosedimenting proteins in nondenaturing polyacrylamide gels reveals a discrete band of proteins that binds antibodies specific for uvomorulin, Na+,K+-ATPase, ankyrin, and fodrin. Significantly, ankyrin and fodrin, but not Na+K+-ATPase, coimmunoprecipitate in a complex with uvomorulin using uvomorulin antibodies. This result indicates that separate complexes exist containing ankyrin and fodrin with either uvomorulin or Na+,K+-ATPase. These results are discussed in the context of the possible roles of uvomorulin-induced cell-cell contact in the assembly of the membrane-cytoskeleton and associated membrane proteins (e.g., Na+,K+-ATPase) at the contact zone and in the development of cell polarity.     

Colocalization and coprecipitation of ankyrin and Na+,K+-ATPase in kidney epithelial cells

Interactions between integral proteins of the plasma membrane and the cytoskeleton may be important for localizing certain membrane proteins in a nonrandom fashion at specialized domains of the cell surface. Here, we show that ankyrin, the key protein for the linkage of the erythrocyte anion exchanger (band 3) to the spectrin-based membrane cytoskeleton, is also present in kidney distal tubular cells where ankyrin is precisely colocalized with Na+,K+-ATPase. Both proteins are confined to the basolateral plasma membrane and are absent from the apical membrane, the junctional complex and the membrane surface that contacts the basal lamina. Purified Na+,K+-ATPase of sheep and pig kidney contains a binding site for erythrocyte ankyrin as demonstrated by immunoprecipitation experiments. A band 3-like binding site for ankyrin is likely, since binding of ankyrin to Na+,K+-ATPase could be inhibited in a competitive fashion by the isolated cytoplasmic domain of erythrocyte band 3.     

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.     

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.     

(Na+,K+)-cotransport in the Madin-Darby canine kidney cell line. Kinetic characterization of the interaction between Na+ and K+

Confluent monolayer cultures of the differentiated kidney epithelial cell line, Madin-Darby canine kidney cells (MDCK), have been used to study ion transport mechanisms involved in transepithelial transport. We have investigated the previously reported K+-stimulation of 22Na+ uptake by confluent monolayers of Na+ depleted cells (Rindler, M. J., Taub, M., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 11431-11439). This component of Na+ uptake was insensitive to ouabain and amiloride, but was strongly inhibited by furosemide or bumetanide. Ouabain-insensitive 86Rb+ uptake was also inhibitable by furosemide or bumetanide and stimulated by extracellular Na+. The synergistic effect of Na+ and 86Rb+ uptake and K+ on 22Na+ uptake was reflected by an increase in the apparent Vmax and a decrease in the apparent Km as the concentration of the other cation was increased. The extrapolated Km for either 86Rb+ or 22Na+ uptake in the absence of the other cation was 30 mM while the Km in the presence of a saturating concentration of the other cation was 9 mM. The absolute Vmax values for 22Na+ and 86Rb+ uptake suggest a cotransport system with a stoichiometry of 2Na+:3K+. However, because of the experimental design, the actual ratio may be closer to 1:1. Competition with, and stimulation by, a variety of unlabeled cations indicated that Na+ could be partially replaced by Li+, while K+ could be fully replaced by Rb+ and partially replaced by NH4+ and CS+. Uptake by this system was dependent upon cellular ATP. Reduction of intracellular ATP to 3% of normal abolished both K+-stimulated 22Na+ uptake and Na+-stimulated 86Rb+ uptake.     

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.     

The lateral mobility of the (Na+,K+)-dependent ATPase in Madin-Darby canine kidney cells

Fluorescence microphotolysis (recovery after photobleaching) was used to determine the lateral mobility of the (Na+,K+)ATPase and a fluorescent lipid analogue in the plasma membrane of Madin-Darby canine kidney (MDCK) cells at different stages of development. Fluorescein-conjugated Fab' fragments prepared from rabbit anti-dog (Na+,K+)ATPase antibodies (IgG) and 5-(N-hexadecanoyl)aminofluorescein (HEDAF) were used to label the plasma membrane of confluent and subconfluent cultures of MDCK cells. Fractional fluorescence recovery was 50% and 80-90% for the protein and lipid probes, respectively, and was independent of developmental stage. The estimated diffusion constants of the mobile fraction were approximately 5 X 10(-10) cm2/s for the (Na+,K+)ATPase and approximately 2 X 10(-9) cm2/s for HEDAF. Only HEDAF diffusion showed dependency on developmental stage in that D for confluent cells was approximately twice that for subconfluent cells. These results indicate that (Na+,K+)ATPase is 50% immobilized in all developmental stages, whereas lipids in confluent MDCK cells are more mobile than in subconfluent cells. They suggest, furthermore, that the degree of immobilization of the (Na+,K+)ATPase is insufficient to explain its polar distribution, and they support restricted mobility of the ATPase through the tight junctions as the likely mechanism for preventing the diffusion of this protein into the apical domain of the plasma membrane in confluent cell cultures.     

Characterization of chemically and virally transformed variants of Madin-Darby canine kidney (MDCK) epithelial cells

Oncogenic derivatives of Madin-Darby canine kidney (MDCK) cells were isolated in the nude mouse, and nononcogenic anchorage-independent transformants were isolated in vitro following chemical mutagenesis in vitro. These transformed cell lines as well as a Moloney sarcoma virus (MSV) transformed line were characterized with respect to their serum and anchorage requirements, growth rates, final saturation densities, and sensitivities to contact inhibition. None of these in vitro growth characteristics were found to correlate with tumorigenicity in nude mice. One tumorigenic clone, MDCK-T1, was characterized with respect to serum-free growth requirements, cAMP production, and ornithine decarboxylase (ODC) activity. These cells exhibited a significant reduction in the PGE1 requirement for growth, they produced higher levels of cAMP, and they expressed a reduced level of ODC activity relative to the parental MDCK cells. These findings may reflect changes in growth control mechanisms which accompany kidney epithelial cell tumorigenesis and suggest that the study of transformed lines derived in this manner could lead to the identification of in vitro properties which are associated with malignancy.     

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.     

Coordinate expression of piretanide receptors and Na+,K+,Cl- cotransport activity in Madin-Darby canine kidney cell mutants

Two receptor sites for [3H]piretanide, a sulfamoylbenzoic acid loop diuretic, have been identified in intact Madin-Darby canine kidney cells, an epithelial cell line derived from dog kidney. The two receptor sites differed in their affinity for piretanide (KD1 = 2.1 +/- 1.4 nM and KD2 = 264 +/- 88 nM) and the maximal number of sites (Bmax1 = 11 +/- 4 and Bmax2 = 120 +/- 80 fmol/mg of protein). Madin-Darby canine kidney cells are known to possess a tightly coupled and highly cooperative Na+,K+,Cl- cotransporter which is sensitive to loop diuretics. Under ionic conditions identical to those used to study piretanide binding (30 mM Na+, 30 mM K+, 30 mM Cl-), the Ki for inhibition of the initial rate of 86Rb+ uptake by piretanide was 333 +/- 92 nM, a value not significantly different from the KD of the low affinity receptor site. [3H]Piretanide binding to three low K+-resistant mutants derived from this cell line was also studied. These mutants had been previously characterized as being partially or completely defective in Na+,K+,Cl- cotransport activity (McRoberts, J. A., Tran, C. T., and Saier, M. H., Jr. (1983) J. Biol. Chem. 258, 12320-12326). One of these mutants had undetectable levels of Na+,K+,Cl- cotransport activity and low to undetectable levels of specific piretanide binding. The second mutant had low but measurable levels of cotransport activity (11% of the wild-type levels) and displayed very low affinity (KD approximately 8000 nM) specific piretanide binding. In the third mutant, expression of Na+,K+,Cl- cotransport activity and both piretanide receptors was cell density-dependent. Subconfluent to just-confluent cultures of this mutant lacked detectable cotransport activity as well as specific piretanide binding, whereas very dense cultures displayed both piretanide receptors and had intermediate to nearly normal levels of cotransport activity. These results demonstrate that the Na+,K+,Cl- cotransporter is a receptor for loop diuretics, but they also raise questions about the functional significance of the two piretanide receptor sites.     

A blood group B-like pentaglycosylceramide is the major complex glycosphingolipid of the Madin-Darby canine kidney epithelial cell line I (MDCK I)

Two sublines of the epithelial cell line MDCK differ in glycosphingolipid composition (Hansson, G.C. et al. (1986) EMBO J. 5, 483-489). The Forssman pentaglycosylceramide was an abundant glycolipid in the MDCK II subline, but was absent in the MDCK I subline. The MDCK I line instead contained another five-sugar glycolipid in relatively large amounts. This component has now been isolated and characterized with mass spectrometry, methylation analysis, exoglycosidase digestion, and proton NMR spectroscopy. The structure was concluded to be Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 Cer. This is a blood group B-like glycolipid lacking fucose, earlier found in rabbit and bovine erythrocytes.     

Ouabain-sensitive 42K binding to Na+, K+-ATPase purified from canine kidney outer medulla.

Specific and ouabain-sensitive potassium binding to Na⁺, K⁺-ATPase was directly observed by centrifugation method with the purified enzyme and ⁴²K. The specific binding reached to saturation level at concentrations more than 0.2 mM KCl and the level was 6.2 nmol per mg ATPase with specific activity of 1470 μmol Pi/h·mg. The binding level, however, was proportional to the enzyme unit used. Simultaneous determination of ⁴²K binding and [³H]ouabain binding showed that two mol of potassium binding were blocked by one mol of ouabain binding per 3.2×10⁵ g enzyme. Although the apparent dissociation constant of the specific potassium binding was estimated at about 50 μM, Scatchard plot of the binding revealed non-linear relationship suggesting that the two potassium sites existed on one catalytic unit of enzyme would be not equivalent but cooperative.     

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.     

Quantitative immunocytochemical localization of Na+,K+-ATPase in rat ciliary epithelial cells

Ultrastructural localization of Na+,K+-ATPase in rat ciliary epithelium was investigated quantitatively by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of Na+,K+-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha-subunit of Na+,K+-ATPase in the ciliary body. Gold particles were found mainly on the basolateral surfaces of both the pigmented epithelial (PE) and nonpigmented epithelial (NPE) cells with an approximately twofold higher labeling density in the PE cells. A few gold particles were also found on the apical and ciliary channel surfaces of the PE cells, whereas no significant binding was found on the apical surfaces of the NPE cells. The basolateral surfaces of PE and NPE cells are markedly infolded and are much greater in area than the apical surfaces. This means that Na+,K+-ATPase is almost exclusively located on the basolateral surfaces of both the NPE and PE cells. We suggest that the Na+,K+-ATPase of both the NPE and PE cells play an important role in the formation of aqueous humor.     

Long-term regulation of Na+,K+-ATPase in opossum kidney cells by ouabain

Na(+),K(+)-ATPase, a basolateral transporter responsible for tubular reabsorption of Na(+) and for providing the driving force for vectorial transport of various solutes and ions, can also act as a signal transducer in response to the interaction with steroid hormones. At nanomolar concentrations ouabain binding to Na(+),K(+)-ATPase activates a signaling cascade that ultimately regulates several membrane transporters including Na(+),K(+)-ATPase. The present study evaluated the long-term effect of ouabain on Na(+),K(+)-ATPase activity (Na(+) transepithelial flux) and expression in opossum kidney (OK) cells with low (40) and high (80) number of passages in culture, which are known to overexpress Na(+),K(+)-ATPase (Silva et al., 2006, J Membr Biol 212, 163-175). Activation of a signal cascade was evaluated by quantification of ERK1/2 phosphorylation by Western blot. Na(+),K(+)-ATPase activity was determined by electrophysiological techniques and expression by Western blot. Incubation of cells with ouabain induced activation of ERK1/2. Long-term incubation with ouabain induced an increase in Na(+) transepithelial flux and Na(+),K(+)-ATPase expression only in OK cells with 80 passages in culture. This increase was prevented by incubation with inhibitors of MEK1/2 and PI-3K. In conclusion, ouabain-activated signaling cascade mediated by both MEK1/2 and PI-3K is responsible for long-term regulation of Na(+) transepithelial flux in epithelial renal cells. OK cell line with high number of passages is suggested to constitute a particular useful model for the understanding of ouabain-mediated regulation of Na(+) transport.     

Dog kidney (Na+, K+)-ATPase is more sensitive to inhibition by vanadate than human red cell (Na+, K+)-ATPase

(Na⁺, K⁺)-ATPase (EC 3.6.1.3) from kidney is more sensitive to inhibition by vanadate than red cell (Na⁺,K⁺)-ATPase. The difference appears to be in the apparent affinities of the two enzymes for K⁺ and Na⁺ at sites where K⁺ promotes and Na⁺ opposes vanadate binding. As a result of Na⁺-K⁺ competition at these sites, reversal of vanadate inhibition was accomplished at lower Na⁺ concentrations in red cell than in kidney (Na⁺,K⁺)-ATPase. It is possible that vanadate could selectively regulate Na⁺ transport in the kidney.     

Regulation of Na+, K+-ATPase activity in MDCK kidney epithelial cell cultures: Role of growth state,cyclic AMP,and chemical inducers of dome formation and differentiation

Na⁺,K⁺-ATPase activity was monitored in MDCK kidney epithelial cell monolayers and in cell extracts as a function of cell density, cAMP elevation, and exposure to hexamethylene bisacetamide (HMBA) and dimethylsulfoxide (Me₂SO). Ouabain-sensitive Na⁺,K⁺-ATPase and ⁸⁶Rb⁺ uptake activities, and the number of [³H]-ouabain binding sites were maximal in subconfluent cultures and decreased accompanying the development of a confluent monolayer. A sodium pump density of 8 × 10⁷ pumps/cell was estimated for subconfluent cultures, declining to 9 × 10⁵ pumps/cell at confluence. Previous studies have shown that dibutyryl cyclic AMP (Bt₂cAMP), 1-methyl-3-isobutylxanthine (IBMX), or the differentiation inducers HMBA and Me₂SO, which also caused cAMP elevation, all stimulated dome formation, a visible manifestation of active transepithelial Na⁺ and water transport (Lever, 1979). In the present study, all of these inducers were found to elevate intracellular Na⁺ content, implicating this variable in control of induction of dome formation. Operationally, inducers could be divided into two classes. HMBA and Me₂SO partially inhibited ouabain-sensitive ⁸⁶Rb⁺ influx. Ouabain, at a concentration that caused partial sodium pump inhibition and increased intracellular Na⁺ content, was also effective as an inducer. The second class, exemplified by IBMX and Bt₂cAMP caused a furosemide-sensitive increase in intracellular Na⁺ content. This class of inducers stimulated ouabain-sensitive ⁸⁶Rb⁺ uptake, presumably by substrate effects due to increased Na⁺ levels. The Na⁺ or ATP activation of Na⁺,K⁺-ATPase activity assayed in cell-free extracts, the affinity of the transport system for Rb⁺ in intact cells and intracellular ATP levels were unchanged by inducer treatment. Elevation of intracellular Na⁺ concentration, either by cAMP-stimulated, furosemide-sensitive mechanisms or by partial inhibition of the sodium pump may stimulate the induction of dome formation in MDCK cells.     

Dog kidney (Na+,K+)-ATPase is more sensitive to inhibition by vanadate than human red cell (Na+,K+)-ATPase

(Na+,K+)-ATPase (EC 3.6.1.3) from kidney is more sensitive to inhibition by vanadate than red cell (Na+,K+)-ATPase. The difference appears to be in the apparent affinities of the two enzymes for K+ and Na+ at sites where K+ promotes and Na+ opposes vanadate binding. As a result of Na+-K+ competition at these sites, reversal of vanadate inhibition was accomplished at lower Na+ concentrations in red cell than in kidney (NA+,K+)-ATPase. It is possible that vanadate could selectively regulate Na+ transport in the kidney.