Preparation of basolateral membranes that transport p-aminohippurate from primary cultures of rabbit kidney proximal tubule cells

The organic anion p-aminohippurate (PAH) is specifically secreted by the renal proximal tubule. The possibility was examined that the probenecid sensitive PAH transport system (which is involved in this secretory process in renal proximal tubule cells in vivo) is retained in primary cultures of rabbit kidney proximal tubule cells. Significant 3H-PAH uptake into primary cultures of proximal tubule cells was observed. After 10 min, 150 pmole PAH/mg protein had accumulated intracellularly. Given an intracellular fluid volume of 10 microliter/mg protein, the intracellular PAH concentration was estimated to be 15 microM. The initial rate of PAH uptake (when 50 microM PAH was in the uptake buffer) was inhibited 50% by 2 mM probenecid. Intact monolayers also exhibited Na+-dependent alpha methyl-D-glucoside uptake (an apical marker). Basolateral membranes were purified from primary rabbit kidney proximal tubule cell cultures. Probenecid sensitive PAH uptake into the membrane vesicles derived from the primary cultures was observed. The rate of PAH uptake was equivalent to that obtained with vesicles obtained from the rabbit renal cortex. No significant Na+-dependent D-glucose uptake into the vesicles was observed, indicating that primarily basolateral membrane vesicles had indeed been obtained.     

Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium

Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone- deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha- methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.     

Serum renotropic factor stimulates prostaglandin synthesis in primary cultures of rabbit kidney cells

Compensatory growth of the kidney occurs in response to a partial reduction in renal mass. This compensatory renal growth may be regulated by a circulating renotropic factor. Prostaglandin synthesis has been shown to be increased in kidneys undergoing compensatory renal growth in vivo. In the present study we observed that the addition of rabbit sera obtained after uninephrectomy enhanced DNA synthesis in primary cultures of rabbit kidney cells compared to sera obtained prenephrectomy. The stimulated kidney cells produced more prostaglandin E2 than control cells. Furthermore, the addition of prostaglandin E2 to rabbit kidney cells in the presence of control sera also stimulated DNA synthesis. These results provide further evidence that prostaglandins may participate in the biological events which regulate renal growth in response to a circulating renotropic factor.     

Growth and function of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium.

The properties of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium have been examined. Primary rabbit kidney proximal tubule cells were observed to grow at the same rate, 1.0 doublings/day, both in glucose-free and in glucose-supplemented medium. Growth in glucose-free medium was dependent upon the presence of an additional nutritional supplement, such as glutamine, pyruvate, palmitate, lactate, or beta hydroxybutyrate. Lactate, pyruvate, and glutamate are utilized for renal gluconeogenesis in vivo. The growth of the primary rabbit kidney proximal tubule cells in glucose-free medium was also dependent upon the presence of the three growth supplements insulin, transferrin, and hydrocortisone. Insulin was growth stimulatory to the primary proximal tubule cells in glucose-free medium, although insulin causes a reduction in the phosphoenolpyruvate carboxykinase (PEPCK) activity in these cells. PEPCK is a key regulatory enzyme in the gluconeogenic pathway. In order to evaluate whether or not the primary cells have gluconeogenic capacity, their glucose content was determined. The cells contained 5 pmoles D-glucose/mg protein. However, no significant glucose was detected in the medium. Presumably, the primary cells were either utilizing or storing the glucose made by the gluconeogenic pathway. Consistent with this latter possibility, cellular glycogen levels were observed to increase with time in culture. The effect of glucose on the expression of the alpha I(IV) collagen and laminin B1 chain genes was examined. Northern analysis indicated that the level of alpha I(IV) collagen mRNA was significantly elevated in glucose containing, as compared with glucose deficient, medium. In contrast, laminin B1 chain mRNA levels were not significantly affected by the glucose content of the medium.     

Stimulation by calcium and carbamoylcholine of the ouabain-sensitive uptake of 86Rb+ in isolated rat pancreatic acinar cells

The uptake of 86Rb+ was assayed in isolated rat pancreatic acinar cells to determine the effect of calcium and carbamoylcholine on the ouabain-sensitive and ouabain-insensitive components. The presence of calcium in the medium bathing the cells during the preincubation and the main incubation periods was needed to preserve in optimum conditions the uptake of 86Rb+, the stimulation by carbamoylcholine and the sensitivity to ouabain. In the presence of calcium, the ouabain-sensitive component of 86Rb+ uptake was higher than the ouabain-insensitive. The ouabain-sensitive component was 3-times lower in cells incubated in a medium lacking calcium and containing 1 mM EGTA, as compared to cells incubated in the presence of calcium. Carbamoylcholine, at 5 X 10(-4) M, stimulated the uptake of 86Rb+ and this effect depended on the presence of calcium in the bathing medium. Maximal stimulation by carbamoylcholine was reached at 0.2 mM calcium. The nett stimulation by carbamoylcholine was inhibited up to 85% by 1 mM ouabain. As judged by digitonin-disruption of plasma membrane, the above-indicated effects were limited to a cytoplasmic pool of 86Rb+ and a leaky plasma membrane could be ruled out. The results suggest that in rat pancreatic acinar cells, carbamoylcholine stimulated the ouabain-sensitive uptake of 86Rb+ and required the presence of calcium in the bathing medium.     

Interactions of intracellular pH and intracellular calcium in primary cultures of rabbit corneal epithelial cells

Summary Homeostasis of intracellular calcium ([Ca⁺⁺]_i) and pH (pH_i) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca⁺⁺]_i and pH_i on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca⁺⁺]_i with fura-2 and pH_i with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pH_i in these cells was 7.37±0.05 (n=20 cells) and resting [Ca⁺⁺]_i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH₄Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca⁺⁺]_i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH₄Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH₄Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pH_i decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca⁺⁺]_i, but produced a biphasic change in pH_i. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca⁺⁺]_i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pH_i decreased by 0.35±0.02 units. We conclude that an increase in pH_i leads to an increase in [Ca⁺⁺]_i in rabbit corneal epithelial cells; however, a decrease in pH_i leads to minor changes in [Ca⁺⁺]_i. The ability of CE cells to maintain proper calcium homeostasis when pH_i is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.     

Antigenic expression of aminopeptidase M, dipeptidyl-peptidase IV and endopeptidase by primary cultures from rabbit kidney proximal tubule

Techniques using microdissected tubules from rabbit kidney allow the isolation of well defined segments which can be cultured to obtain pure renal cell epithelia. From microdissected proximal tubules, we obtained epithelia the cells of which exhibit some of the antigenic expressions of the initial proximal cells. For this purpose, we used three monoclonal antibodies raised against apical brush border membranes of the proximal tubules. We determined with precision the identity and some of the molecular characteristics of the antigens bound by these three antibodies and found that they correspond to three hydrolases present in the brush borders of proximal renal cells (amino-peptidase, dipeptidyl-peptidase IV and endopeptidase). These apical markers are expressed by the growing cells of primary cultures from proximal tubules, suggesting strongly that they are effectively proximal cells and that no appreciable dedifferentiation occurred during the growth process. We have also shown that apical expression of these hydrolases on the plasma membrane of the epithelium occurred only after several days of culture and determined the complete polarization of the cells. Electron microscopy studies confirmed the degree of polarization of the cultured cells by the presence of numerous microvilli on their apical face.     

Antigenic expression of aminopeptidase M,dipeptidyl-peptidase IV and endopeptidase by primary cultures from rabbit kidney proximal tubule

Summary Techniques using microdissected tubules from rabbit kidney allow the isolation of well defined segments which can be cultured, to obtain pure renal cell epithelia. From microdissected proximal tubules, we obtained epithelia the cells of which exhibit some of the antigenic expressions of the initial proximal cells. For this purpose, we used three monoclonal antibodies raised against apical brush border membranes of the proximal tubules. We determined with precision the identity and some of the molecular characteristics of the antigens bound by these three antibodies and found that they correspond to three hydrolases present in the brush borders of proximal renal cells (amino-peptidase, dipeptidyl-peptidase IV and endopeptidase). These apical markers are expressed by the growing cells of primary cultures from proximal tubules, suggesting strongly that they are effectively proximal cells and that no appreciable dedifferentiation occured during the growth process. We have also shown that apical expression of these hydrolases on the plasma membrane of the epithelium occured only after several days of culture and determined the complete polarization of the cells. Electron microscopy studies confirmed the degree of polarization of the cultured cells by the presence of numerous microvilli on their apical face.     

Changes in expression and subcellular localization of annexin IV in rabbit kidney proximal tubule cells during primary culture

In the present study, we investigated the polarized expression of annexin IV at various stages in the growth of rabbit kidney proximal tubule cells (PTC) in primary cultures. The results of immunoblotting analysis and indirect immunofluorescence studies using a specific anti-annexin IV monoclonal antibody, indicated that annexin IV is expressed in proximal tubule cultured cells, although it was not detected in the proximal tubules present in frozen sections of kidney cortex and freshly isolated proximal tubule cells. In either non-confluent or confluent cells which remained attached to the collagen-coated support, annexin IV was mainly concentrated around the nucleus, whereas in PTC forming the monolayer of domes, it was restricted to the basolateral membrane domain. This basolateral localization was identical to that observed in other polarized epithelial cell types such as enterocytes. When the domes burst, the cells returned to the collagen-coated support and the annexin IV was again localized around the nuclei. The fact that the change of localization was very rapid suggested the existence of a considerable difference between the differentiation states of dome forming and adherent confluent cells. Moreover, a transient association of annexin IV with the basal body of apically located cilia also seemed to be correlated with a particular polarization state and/or differentiation states of adherent cultured cells, corresponding to the beginning of the polarized expression of aminopeptidase N, a hydrolase located in the apical brush border membrane, and to the falling of cells onto the support, subsequent to the bursting of the domes. In conclusion, these results provide evidence that annexin IV may constitute a new marker of the basolateral membrane domain of polarized epithelial renal cells in primary cultures. © 1995 Wiley-Liss, Inc.     

Effect of aldosterone on 86Rb fluxes in cultured kidney cells (A6)

This study was designed to evaluate the relative contributions of hormone induced changes in active and passive K+ transport in an epithelial cell line in continuous culture derived from toad kidney (A6) using 86Rb as a tracer for measuring unidirectional K+ fluxes. The effects of 24 h exposure to aldosterone (A) and aldosterone plus insulin (A+I) on unidirectional K+ fluxes were evaluated under short-circuited conditions and under open circuit conditions. In epithelia exposed to A, a small but significant amount of active K+ secretion was found, although it was not significantly greater than in control epithelia. The bidirectional fluxes in both A and A+I treated epithelia, under short-circuited conditions, increased by a similar amount over control values indicating an increase in apparent permeability of passive transepithelial K+ transport. Under open circuit conditions, A stimulated net K+ transport by about 5-fold over controls. The increase in K+ secretion produced by A under open circuit conditions could be explained by the combined effects of an increase in transepithelial K+ permeability and an increase in the transepithelial electrical potential difference (PD). The presence of I produced no additional effects to that of A on K+ transport under the conditions used in this study. It is concluded that the substantial increase in K+ secretion induced in A6 cells by 24 h exposure to A is primarily passive in nature. It is possible that the changes in both PD and transepithelial K+ permeability, which can account for the observed increase in K+ secretion, are secondary to the stimulation of active Na+ transport.     

Normal rat kidney proximal tubule cells in primary and multiple subcultures

Summary Anin vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys and separating the cortex, the cortical tissue is digested with the enzyme DNAse-collagenase (Type I) resulting in a high yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency in a serum-free, hormonally defined medium. The cell yield can be increased by transfering the conditioned medium on Day 1 to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces than on bovine collagen Type I coated surfaces. The culture medium was a 1∶1 mixture of Ham’s F-12 and Dulbecco’s modified Eagle’s medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal growth factor, and glutamine. The RPT cells became confluent in 7–10 d, at which point they could be subcultured by trypsinizing and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The cells were histochemically positive for alkaline phosphatase and γ-glutamyl transpeptidase activity and synthesized the intermediate filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence, the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture model will be a valuable tool for substrate uptake and nephrotoxicity studies.     

Streptomycin toxicity in primary cultures of flounder renal proximal tubule cells

Summary The aminoglycoside antibiotic streptomycin is a known nephrotoxin in vivo and a common component of cell culture media. The effects of streptomycin (100 μg/ml) on transepithelial electrical properties, glucose transport, glycolytic metabolism, and morphology were examined in primary proximal tubule cell cultures from winter flounder (Pseudopleuronectes americanus) kidney. Streptomycin treatment on either Days 2 to 12 or Days 8 to 13 abolished the transepithelial potential difference and short-circuit current across the monolayer but had no effect on transepithelial resistance in confluent 12 to 13-dcultures, suggesting the loss of active transepithelial transport. Consistent with these findings, mucosal-to-serosal glucose fluxes were greatly reduced in streptomycin-treated cultures and insensitive to the transport inhibitor phlorizin, indicating the absence of the apical Na-dependent glucose transport system associated with net glucose reabsorption. In addition to transport processes, antibiotic treatment also interfered with cellular energy metabolism as judged by the rapid reduction in glycolytic lactate production observed in the presence of streptomycin. Scanning and transmission electron microscopy revealed that streptomycin-treated culture were composed of cuboidal-to-columnar shaped cells which maintained intact tight junctions similar to control cultures. However, apical microvilli, the presumed sites of mucosal transport systems, were severely reduced in number in streptomycin-treated cultures. We concluded that streptomycin, at a dose commonly used in cell culture, inhibited the expression of differentiated function by flounder proximal tubule cell cultures. These cell cultures may provide a suitable model system for examination of the mechanisms of aminoglycoside nephrotoxicity. This investigation was supported by the University of Connecticut Research Foundation and by grant PCM-8003452 from the National Science Foundation, Washington, DC.     

Differential calcium dependence of contractile responses and 86Rb efflux from the rabbit aorta induced by vasoactive stimuli

⁸⁶Rb was used to monitor potassium movements in strips of rabbit aorta simultaneously with measurements of tension. Histamine, noradrenaline, the prostaglandin endoperoxide analogue U46619, angiotensin II, and 144 mM K⁺ each induced an increase in ⁸⁶Rb efflux concomitantly with contraction. For the first four agonists there was a rank-order correlation between the contractile response and ⁸⁶Rb efflux, but 144 mM K⁺ induced a massive increase in ⁸⁶Rb efflux although it was the weakest contractile stimulus. Contraction and increase in ⁸⁶Rb efflux-induced K⁺ were both reduced by verapamil, which blocks voltage-sensitive calcium channels, implying that both effects of K⁺ were mediated mainly by a depolarisation-induced influx of calcium. Noradrenaline increased both tension and ⁸⁶Rb efflux through an action on alpha-adrenoceptors, but its effect on efflux, unlike its effect on tension, was apparently totally dependent on the presence of extracellular calcium. Experiments performed in the presence of lanthanum, which blocks calcium influx, showed that the intracellular store of calcium released by noradrenaline apparently played no role in inducing ⁸⁶Rb efflux, although it could trigger contraction. Lanthanum also blocked contraction induced by K⁺ but less effect on the increase in ⁸⁶Rb efflux induced by K⁺. Thus, agonist-induced vascular contraction and ⁸⁶Rb efflux can be dissociated, but under normal conditions all the contractile stimuli tested induced ⁸⁶Rb efflux.     

Characterization of glucose transport by cultured rabbit kidney proximal convoluted and proximal straight tubule cells

Rabbit kidney proximal convoluted tubule (RPCT) and proximal straight tubule (RPST) cells were independently isolated and cultured. The kinetics of the sodium-dependent glucose transport was characterized by determining the uptake of the glucose analog alpha-methylglucopyranoside. Cell culture and assay conditions used in these experiments were based on previous experiments conducted on the renal cell line derived from the whole kidney of the Yorkshire pig (LLC-PK1). Results indicated the presence of two distinct sodium-dependent glucose transporters in rabbit renal cells: a relatively high-capacity, low-affinity transporter (V(max) = 2.28 +/- 0.099 nmoles/mg protein min, Km = 4.1 +/- 0.27 mM) in RPCT cells and a low-capacity, high-affinity transporter (V(max) = 0.45 +/- 0.076 nmoles/mg protein min, K(m) = 1.7 +/- 0.43 mM) in RPST cells. A relatively high-capacity, low-affinity transporter (V(max) = 1.68 +/- 0.215 nmoles/mg protein min, Km = 4.9 +/- 0.23 mM) was characterized in LLC-PK1 cells. Phlorizin inhibited the uptake of alpha-methylglucopyranoside in proximal convoluted, proximal straight, and LLC-PK1 cells by 90, 50, and 90%, respectively. Sodium-dependent glucose transport in all three cell types was specific for hexoses. These data are consistent with the kinetic heterogeneity of sodium-dependent glucose transport in the S1-S2 and S3 segments of the mammalian renal proximal tubule. The RPCT-RPST cultured cell model is novel, and this is the first report of sodium-dependent glucose transport characterization in primary cultures of proximal straight tubule cells. Our results support the use of cultured monolayers of RPCT and RPST cells as a model system to evaluate segment-specific differences in these renal cell types.     

Morphological and biochemical characterization of primary culture of rabbit proximal kidney tubule cells grown on collagen-IV coated millicell-cm

Summary The aim of this study was to better characterize rabbit proximal kidney tubule cells cultured on collagen IV-coated porous inserts, as compared to the same cells seeded in standard plastic wells. Total protein contents in confluent monolayers on permeable membranes were about twofold higher than those measured in confluent cultures in plastic wells. Microscopy examinations suggested that such a difference was probably due to a higher cell density and to an impressive development of the apical brush-border membrane. Moreover, measurement of unidirectional transport of p-aminohippuric acid and tetraethylammonium bromide confirmed the high polarization level of cultures on porous inserts. Results of methyl(α-d-[U-¹⁴C]glyco)pyranoside uptake suggested that cell phenotype was probably influenced by culture conditions. Analysis of different markers as a function of time in culture showed decreases of alkaline phosphatase (AP), γ-glutamyltranspeptidase (GGT), and Na⁺-K⁺-ATPase activities as well as increases in LDH, ATP, and glutathione levels, similar to those formerly reported for cells cultured in standard plastic plates. However, comparative data from 6-d-old monolayers have shown that AP, GGT, Na⁺-K⁺-ATPase, glutathione reductase (GRED), and selenium-dependent glutathione peroxidase (Se-GPX) activities were 2.8-, 2.6-, 1.6-, 1.2-, and 2.1-fold, respectively, better preserved on precoated permeable membranes. On the other hand, this paper reports for the first time in the literature that GRED and Se-GPX, two phase II detoxification enzymes, were well maintained in cultures of rabbit proximal kidney tubule cells. Our results show that culturing rabbit proximal kidney tubule cells on collagen IV-coated porous membranes was accompanied by an improvement of both morphological and biochemical properties of the cells.     

Primary cultures of rabbit renal proximal tubule cells: I. Growth and biochemical characteristics

Summay Before the usefulness of a new in vitro model can be ascertained, the model must be properly defined and characterized. This study presents the growth rate and biochemical characteristics of rabbit renal proximal tubule cells in primary culture over a 2-wk culture period. When grown in a hormonally defined, antibiotic-free medium these cells form confluent monolayer cultures within 7 d after plating. Multicellular done formation, an indicator of transepithelial solute transport, was expressed after confluent cultures were formed. The activity of the cytosolic enzyme, lactate dehydrogenase, and the lysosomal enzyme,N-acetyl-glucosaminidase, increased 14- and 2-fold during the first 8 d of culture. respectively. In contrast, the activity of a brush border enzyme, alkaline phosphatase, decreased 85% within the first 8 d of culture. Release of these enzyme markers into the culture medium, which are routinely used to measure cytoxicity, stabilized after 8 d in culture. The ratio of cellular protein to DNA changed according to the state of cellular growth. Values rose from 0.035 mg protein/μg DNA in preconfluent cultures to 0.059 mg protein/μg DNA in confluent cultures. These results document the characteristics of a primary proximal tubule cell culture system for future studies in in vitro toxicology. This paper was resented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society of Toxicology (SOT) and the Tissue Culture Association held at the 27th annual meeting of the SOT in Dallas, Texas in 1988. This work was supported by grants GM 07145, The Johns Hopkins Center for Alternatives to Animal Testing, and a Sigma Xi Grants-in-Aid of Research Award.     

Properties of Na-K pump in primary cultures of kidney cells.

Activities related to Na-K transport were measured in cell cultures of ground squirrel kidney cortex in order to compare these cells with those of intact kidney and of continuous cell lines. A microsomal preparation containing plasma membrane Na,K-ATPase from fresh kidney showed twice the activity of a similar preparation from 72-hour cultured cells. Na,K-ATPase of homogenates of 72-hour cells showed one-third to one-fourth the specific activity of that from 6-hour cultured cells. The associated K-dependent phosphatase activity also declined as a function of time in culture. The ouabain-sensitive influx of K into 6-hour cultured cells was twice as great as the K influx into 72-hour cells. The number of sites binding 3H-ouabain in intact cultured cells declined 81% on a cell protein basis between 6 and 72 hours in culture. This decline in ouabain binding sites was relatively greater than that of K influx, so that the K turnover number increased over this same time period. The decline in ouabain-sensitive K influx during culture was complementary to an increase in furosemide-sensitive K influx. Measurements of unidirectional and net K fluxes showed that there were three components of K influx into 3-day cultured cells: ouabain-sensitive Na:K exchange, furosemide-sensitive K:K exchange, and K diffusion. In the 6-hour cultures, however, there was no furosemide-sensitive K:K exchange. Thus, after three days in culture ground squirrel kidney cells lose a feature characteristic of the original parent cells (high Na,K-ATPase activity), and gain a feature common to many undifferentiated cultured cells (furosemide-sensitive K:K exchange).