Amphibian Cell Line Research Articles

The Number of Ethylene Oxide Groups of Sulphate-Based Surfactants Influences the Cytotoxicity of Mixed Micelles to an Amphibian Cell Line

Sodium lauryl ether sulphate (SLEnS) is an anionic surfactant that is widely used in many fields, such as cosmetics and detergents, among others. This study evaluated the influence of the number of ethylene oxide (EO) units, present in the head group of SLEnS, on its cytotoxicity to the A6 cell line of Xenopus laevis using MTT and resazurin assays. The A6 cell line was exposed for 48 h to six SLEnS variants: SLE0S, SLE1S, SLE4S, SLE11S, SLE30S, and SLE50S (subscript values correspond to the number of EO units). Overall, the six variants impaired the A6 cells’ viability at low concentrations for the MTT assay, with the median lethal concentrations (LC50,48h) ranging between 0.398 and 0.554 mg/L and for the resazurin assay between 0.557 and 0.969 mg/L. Further, the obtained results indicate SLEnS variants with fewer EO units to be the most cytotoxic in the resazurin assay; although a similar cytotoxicity pattern was observed with the MTT assay, a significant association between the number of EO units and the values of LC50 was not found. This result highlights the usefulness of in vitro assays with A6 cell lines as a first screening tool for assessing the structure–toxicity relationship of this type of surfactant, also providing a baseline for the development of environmentally friendlier chemical compounds while still maintaining their efficiency.

The amphibian invitrome: Past, present, and future contributions to our understanding of amphibian immunity

Many amphibian populations are declining worldwide, and infectious diseases are a leading cause. Given the eminent threat infectious diseases pose to amphibian populations, there is a need to understand the host-pathogen-environment interactions that govern amphibian susceptibility to disease and mortality events. However, using animals in research raises an ethical dilemma, which is magnified by the alarming rates at which many amphibian populations are declining. Thus, in vitro study systems such as cell lines represent valuable tools for furthering our understanding of amphibian immune systems. In this review, we curate a list of the amphibian cell lines established to date (the amphibian invitrome), highlight how research using amphibian cell lines has advanced our understanding of the amphibian immune system, anti-ranaviral defence mechanisms, and Batrachochytrium dendrobatidis replication in host cells, and offer our perspective on how future use of amphibian cell lines can advance the field of amphibian immunology.

Following Ussing's legacy: from amphibian models to mammalian kidney and brain.

Professor Hans H. Ussing (1911-2000) was one of the founding members of the field of epithelial cell biology. He is most famous for the electrophysiological technique that he developed to measure electrogenic ion flux across epithelial tissues. Ussing-style electrophysiology has been applied to multiple tissues and has informed fields as diverse as amphibian biology and medicine. In the latter, this technique has contributed to a basic understanding of maladies such as hypertension, polycystic kidney disease, cystic fibrosis, and diarrheal diseases to mention but a few. In addition to this valuable contribution to biological methods, Prof. Ussing also provided strong evidence for the concept of active transport several years before the elucidation of Na+K+ATPase. In addition, he provided cell biologists with the important concept of polarized epithelia with specific and different transporters found in the apical and basolateral membranes, thus providing these cells with the ability to conduct directional, active and passive transepithelial transport. My studies have used Ussing chamber electrophysiology to study the toad urinary bladder, an amphibian cell line, renal cell lines, and, most recently, choroid plexus cell lines. This technique has formed the basis of our in vitro mechanistic studies that are used in an iterative manner with animal models to better understand disease progress and treatment. I was honored to be invited to deliver the 2022 Hans Ussing Lecture sponsored by the Epithelial Transport Group of the American Physiological Society. This manuscript is a version of the material presented in that lecture.

So, you want to create a frog cell line? A guide to establishing frog skin cell lines from tissue explants

Skin is an important interface with the external environment and investigating amphibian skin cell biology will improve our understanding of how environmental factors such as pathogens and pollutants are contributing to global amphibian declines. There is a critical need for in vitro systems to facilitate conservation research in model and non-model amphibians and the creation of new amphibian cell lines will play a significant role in reducing or even replacing the use of live animals for in vivo studies by providing an in vitro alternative. Here, we detail an adapted protocol for the generation of spontaneously arising cell lines from frog skin tissues, without the need for immortalization steps. Expanding the amphibian invitrome will foster and expedite new research in amphibian gene function, cellular responses, host-pathogen interactions, and toxicology. The following customizations to traditional tissue explant generation procedures have facilitated the successful generation of adherent skin epithelial-like cell lines from Xenopus laevis and can be further adapted for use with different frog species, such as Rana sylvatica, and different tissues:•Osmotic adjustment of culture medium and solutions for different amphibian species.•Use of small tissue explants, instead of enzymatic digestion of tissues, and gentle spotting of these tissue explants onto the growth surface of tissue culture flasks to promote better tissue adherence.•Partial replacement of medium to allow accumulation of potential endogenous growth factors in cultures.

Amphibian thrombocyte-derived extracellular vesicles, including microRNAs, induce angiogenesis-related genes in endothelial cells.

Thrombocytes circulate in the blood of nonmammalian vertebrates and are involved in hemostasis; however, many detailed characteristics of thrombocytes remain unclear. Recently, we established an amphibian thrombocyte cell line. Here, we report the finding that thrombocytes produce integrin alpha IIb (CD41)-positive extracellular vesicles (EVs), which include microRNAs (miRs). Flow cytometric analysis showed the expression of CD41+ and phosphatidylserine on the surface of EVs. Nanotracking analysis showed that these CD41+ EVs were approximately 100nm in diameter. As CD41+ EVs were also observed from African clawed frogs, the production of CD41+ EVs might be common to amphibians. Microarray analysis showed that the CD41+ EVs contain many kinds of miRs. These CD41+ EVs were phagocytosed by endothelial cells and macrophages. qPCR analysis showed that many angiogenesis-related genes were up-regulated in CD41+ EV-treated endothelial cells. Over-expression of some miRs in the CD41+ EVs increased the proliferation of endothelial cells. These results indicated that thrombocytes produced CD41+ EVs, including miRs, that were received by endothelial cells to induce the expression of angiogenesis-related genes. These results indicated that the CD41+ EVs produced from thrombocytes act as signaling molecules to repair damaged blood vessels.

Replication of Rift Valley Fever Virus in Amphibian and Reptile-Derived Cell Lines.

Rift Valley fever phlebovirus (RVFV) is a zoonotic arthropod-borne virus, which has led to devastating epidemics in African countries and on the Arabian Peninsula. Results of in-vivo, in-vitro and field studies suggested that amphibians and reptiles may play a role as reservoir hosts of RVFV, promoting its maintenance during inter-epidemic periods. To elucidate this hypothesis, we examined two newly established reptile-derived cell lines (Egyptian cobra and Chinese pond turtle) and five previously generated reptile- and amphibian-derived cell lines for their replicative capacity for three low- and high-pathogenic RVFV strains. At different time points after infection, viral loads (TCID50), genome loads and the presence of intracellular viral antigen (immunofluorescence) were assessed. Additionally, the influence of temperatures on the replication was examined. Except for one cell line (read-eared slider), all seven cell lines were infected by all three RVFV strains. Two different terrapin-derived cell lines (Common box turtle, Chinese pond turtle) were highly susceptible. A temperature-dependent replication of RVFV was detected for both amphibian and reptile cells. In conclusion, the results of this study indicate the general permissiveness of amphibian and reptile cell lines to RVFV and propose a potential involvement of terrapins in the virus ecology.

Bid is involved in apoptosis induced by Chinese giant salamander iridovirus and contributes to the viral replication in an amphibian cell line

Bid is a pro-apoptotic BH3-only member of the Bcl-2 superfamily that functions to link the extrinsic apoptotic pathway and the mitochondrial amplification loop of the intrinsic pathway. In this study, the expression and functions of Chinese giant salamander (Andrias davidianus) Bid (AdBid) were investigated. The AdBid cDNA sequence contains an open reading frame (ORF) of 576 nucleotides, encoding a putative protein of 191 aa. AdBid possesses the conserved BH3 interacting domain and shared 34–52% sequence identities with other amphibian Bid. mRNA expression of AdBid was most abundant in muscle. The expression level of AdBid in Chinese giant salamander muscle, kidney and spleen significantly increased after Chinese giant salamander iridovirus (GSIV) infection. Additionally, a plasmid expressing AdBid was constructed and transfected into the Chinese giant salamander muscle cell line (GSM cells). The morphology and cytopathic effect (CPE) and apoptotic process in AdBid over-expressed GSM cells was significantly enhanced during GSIV infection compared with that in control cells. Moreover, a higher level of the virus major capsid protein (MCP) gene copies and protein synthesis was confirmed in the AdBid over-expressed cells. These results indicated that AdBid played a positive role in GSIV induced apoptosis and the viral replication. This study may contribute to the better understanding on the infection mechanism of iridovirus-induced apoptosis.

In vitro and in silico modeling of perfluoroalkyl substances mixture toxicity in an amphibian fibroblast cell line

Poly and perfluoroalkyl substances (PFAS) are a large group of emerging organic pollutants that can persist in the environment and bioaccumulate in biota. They are found in complex mixtures, and although the exact number of PFAS is unknown, it has been estimated to be in the thousands. The objective of this study was two-fold. First, we examined the cytotoxicity of PFAS singly and in binary mixtures using an amphibian fibroblast cell line. Second, we used this experimental data to develop quantitative structure-activity relationship (QSAR) models for single and binary mixtures. We tested the cytotoxicity of four common PFAS: perfluorooctane sulfonate (PFOS); perfluorooctanoic acid (PFOA); perfluorohexane sulfonate (PFHxS); and perfluorohexanoic acid (PFHxA). PFOS was the most toxic and PFHxA the least cytotoxic. Binary mixtures allowed for the construction of isobolograms to test for additivity, synergism, or antagonism. Using this data, QSAR modeling was used for predicting the toxicity of 24 single and 1380 binary mixtures (theoretically generated). Overall, our experimental and modeling results showed that mixtures were approximately additive, with the exception of PFOS and PFOA, which were found to be weakly synergistic. This data shows that certain mixtures of PFAS may have increased toxicity potential above what the simple sum of PFAS concentrations would suggest. More studies are needed that test the toxicity of PFAS mixtures.

Infrared spectroscopy detects changes in an amphibian cell line induced by fungicides: Comparison of single and mixture effects

Amphibians are regarded as sensitive sentinels of environmental pollution due to their permeable skin and complex life cycle, which usually involves reproduction and development in the aquatic environment. Fungicides are widely applied agrochemicals and have been associated with developmental defects in amphibians; thus, it is important to determine chronic effects of environmentally-relevant concentrations of such contaminants in target cells. Infrared (IR) spectroscopy has been employed to signature the biological effects of environmental contaminants through extracting key features in IR spectra with chemometric methods. Herein, the Xenopus laevis (A6) cell line was exposed to low concentrations of carbendazim (a benzimidazole fungicide) or flusilazole (a triazole fungicide) either singly or as a binary mixture. Cells were then examined using attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy coupled with multivariate analysis. Results indicate significant changes in the IR spectra of cells induced by both agents at all concentrations following single exposures, primarily in regions associated with protein and phospholipids. Distinct differences were apparent in the IR spectra of cells exposed to carbendazim and those exposed to flusilazole, suggesting different mechanisms of action. Exposure to binary mixtures of carbendazim and flusilazole also induced significant spectral alterations, again in regions associated with phospholipids and proteins, but also in regions associated with DNA and carbohydrates. Overall these findings demonstrate that IR spectroscopy is a sensitive technique for examining the effects of environmentally-relevant levels of fungicides at the cellular level. The combination of IR spectroscopy with the A6 cell line could serve as a useful model to identify agents that might threaten amphibian health in a rapid and high throughput manner.

Morphological Changes in Amphibian and Fish Cell Lines Infected with Andrias davidianus Ranavirus

Andrias davidianus ranavirus (ADRV) is an emerging viral pathogen that causes severe disease in Chinese giant salamanders, the largest extant amphibian in the world. A fish cell line, Epithelioma papulosum cyprinid (EPC), and a new amphibian cell line, Chinese giant salamander spleen cell (GSSC), were infected with ADRV and observed by light and electron microscopy. The morphological changes in these two cell lines infected with ADRV were compared. Cytopathic effect (CPE) began with rounding of the cells, progressing to cell detachment in the cell monolayer, followed by cell lysis. Significant CPE was visualized as early as 24 h post infection (hpi) in EPC cells and at 36 hpi in GSSC cells. Microscopical examination showed clear and significant CPE in EPC cells, while less extensive and irregular CPE with some adherent cells remaining was observed in GSSC cells. Following ADRV infection, CPE became more extensive. Transmission electron micrographs showed many virus particles around cytoplasmic vacuoles, formed as crystalline arrays or scattered in the cytoplasm of infected cells. Infected cells showed alteration in nuclear morphology, with condensed and marginalized nuclear chromatin on the inner aspect of the nuclear membrane and formation of a cytoplasmic viromatrix adjacent to the nucleus in both cell lines. Some virus particles were also detected in the nucleus of infected GSSC cells. Both cell lines are able to support replication of ADRV and can therefore be used to investigate amphibian ranaviruses.

Growth ofRickettsia felisinDrosophila melanogasterS2 Cells

Rickettsia felis is an obligate, intracellular, Gram-negative bacterium and a member of the transitional group rickettsiae. This bacterium has been shown to grow in vitro in amphibian, tick, and mosquito cell lines. Here, we present data to show the growth of R. felis strain LSU in Drosophila melanogaster S2 cells, an embryonic, hemocytic cell line with phagocytic properties. R. felis LSU was isolated from Ixodes scapularis E6 (ISE6) cells and used to infect S2 cells, grown at 25°C. By 19 days postinfection, the S2 cells were 100% infected with R. felis as determined by Acridine Orange and Diff-Quik staining. A species-specific R. felis qPCR assay was used to demonstrate that the kinetics associated with the S2 cell culture infection involved a lag/adaptation phase, followed by continued growth to 20 days postinfection. Moreover, R. felis organisms were observed in the S2 cells using transmission electron microscopy and a polyclonal antibody against spotted fever rickettsiae. The ability to use D. melanogaster S2 cells for growing rickettsial agents is a useful tool owing to the ease of manipulation of the S2 cultures and the wide-ranging possibility of Drosophila resources available for future studies.

Development and characterization of Xl1, a Xenopus laevis chondrocyte-like cell culture

We describe the development and characterization of a new cell line, designated Xl1, derived from vertebra and long bones of Xenopus laevis. These cells can mineralize their extracellular matrix upon addition of an inorganic phosphate donor and vitamin C, as characterized by von Kossa staining. In addition they express genes such as matrix gla protein (mgp), alkaline phosphatase, type II collagen, and retinoic acid receptors, representing a valuable tool to analyze expression and regulation of Xenopus cartilage-associated genes. Continuous treatment with retinoic acid (RA) inhibited mineralization, alkaline phosphatase expression and its activity, suggesting that RA is a potential negative regulator of Xl1 cell differentiation. These cells are receptive to efficient transfer of DNA using conventional methods including calcium phosphate, liposome-mediated transfer or electroporation and were found to express basal levels of mgp at least 50-fold higher than the routinely used Xenopus A6 cell line, as seen by transcription assays with the distal X. laevis mgp promoter. Being the first amphibian cell line derived from bone tissue, the Xl1 culture provides an excellent in vitro tool for functional promoter studies, being suitable, among other uses, for identifying promoter elements mediating cartilage-expressed genes as shown here for mgp.

Inhibition of αENaC expression and ENaC activity following blockade of the circadian clock‐regulatory kinases CKIδ/ε

Increasing evidence suggests that the circadian clock plays an important role in the control of renal function. We previously showed that the circadian clock protein Per1 positively regulates the expression of the alpha subunit of the renal epithelial sodium channel (αENaC). Casein kinase I is a critical regulator of clock proteins. In order to enter the nucleus, Per1 must be phosphorylated by casein kinase Iε (CKIε). Renal cortical collecting duct (mpkCCDc14) cells were treated with the CKIδ/ε inhibitor PF670462. DNA affinity purification assays showed that CKIδ/ε inhibition prevented Per1 from interacting with a target E‐box from the αENaC promoter. Steady state αENaC mRNA levels were reduced more than 60% following CKIδ/ε inhibition. CKIδ/ε inhibition significantly reduced αENaC protein levels. ENaC activity is tightly controlled by aldosterone and αENaC is a well‐known transcriptional target of aldosterone. Inhibition of CKIδ/ε effectively prevented the aldosterone response of αENaC, suggesting an interaction between Per1 and aldosterone‐mediated regulation of this gene. Importantly, single channel analysis in the amphibian renal cell line 2F3 demonstrated that CKIδ/ε inhibition resulted in decreased ENaC NPo. The present study shows for the first time that CKIδ/ε inhibition represses αENaC expression and ENaC activity, providing further evidence for direct control of ENaC by the circadian clock.

A Novel Obligate Intracellular Gamma-Proteobacterium Associated with Ixodid Ticks, Diplorickettsia massiliensis, Gen. Nov., Sp. Nov

BackgroundObligate intracellular bacteria of arthropods often exhibit a significant role in either human health or arthropod ecology.Methodology/Principal FindingsAn obligate intracellular gamma-proteobacterium was isolated from the actively questing hard tick Ixodes ricinus using mammalian and amphibian cell lines. Transmission electron microscopy revealed a unique morphology of the bacterium, including intravacuolar localization of bacteria grouped predominantly in pairs and internal structures composed of electron-dense crystal-like structures and regular multilayer sheath-like structures. The isolate 20B was characterized to determine its taxonomic position using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that this strain belongs to the family Coxiellaceae, order Legionellales of Gamma-proteobacteria, and the closest relatives are different Rickettsiella spp. The level of 16S rRNA gene sequence similarity between strain 20B and other recognized species of the family was below 94.5%. Partial sequences of the rpoB, parC and ftsY genes confirmed the phylogenetic position of the new isolate. The G+C content estimated on the basis of whole genome analysis of strain 20B was 37.88%. On the basis of its phenotypic and genotypic properties, together with phylogenetic distinctiveness, we propose that strain 20B to be classified in the new genus Diplorickettsia as the type strain of a novel species named Diplorickettsia massiliensis sp. nov.Conclusions/SignificanceConsidering the source of its isolation (hard tick, often biting humans) the role of this bacterium in the pathology of humans, animals and ticks should be further investigated.

The Frog Prince: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells.

Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrates are transpositionally inactive due to the accumulation of mutations in their transposase genes. A novel open reading frame-trapping method was used to isolate uninterrupted transposase coding regions from the genome of the frog species Rana pipiens. The isolated clones were approximately 90% identical to a predicted transposase gene sequence from Xenopus laevis, but contained an unpredicted, approximately 180 bp region encoding the N-terminus of the putative transposase. None of these native genes was found to be active. Therefore, a consensus sequence of the transposase gene was derived. This engineered transposase and the transposon inverted repeats together constitute the components of a novel transposon system that we named Frog Prince (FP). FP has only approximately 50% sequence similarity to Sleeping Beauty (SB), and catalyzes efficient cut-and-paste transposition in fish, amphibian and mammalian cell lines. We demonstrate high-efficiency gene trapping in human cells using FP transposition. FP is the most efficient DNA-based transposon from vertebrates described to date, and shows approximately 70% higher activity in zebrafish cells than SB. Frog Prince can greatly extend our possibilities for genetic analyses in vertebrates.

Comparative Functional Analysis of Rat TGF-?1 and Xenopus laevis TGF-?5 Promoters Suggest Differential Regulations

We have carried out a comparative functional analysis of the rat TGF-beta1 and Xenopus laevis TGF-beta5 promoters across several mammalian and amphibian cell lines. Progressive deletion constructs of both the promoters have been made using a PCR based approach and the basal promoter activities studied in Xenopus tadpole cell line (XTC), Xenopus adult kidney fibroblast cell line (A6), human hepatoma cell line (HepG2), normal rat kidney cell line (NRK), and Chinese hamster ovary cell line (CHO). Data suggests that the basal promoter activity of TGF-beta1 is low as compared to TGF-beta5 promoter in XTC cells but comparable in A6 cells, while TGF-beta5 promoter shows nearly negligible activity as compared to TGF-beta5 promoter in all the tested mammalian cell lines. Moreover, TGF-beta5 promoter is found to be repressed in XTC cells on treatment with TGF-beta5 protein. Thus, the regulation of TGF-beta1 and TGF-beta5 promoters is distinct in amphibian and mammalian species. We therefore suggest that contrary to the suggested functional equivalence of TGF-beta1 and TGF-beta5 proteins, TGF-beta1 and TGF-beta5 genes have distinct functions in their respective species.

The environmental pathogen Mycobacterium ulcerans grows in amphibian cells at low temperatures.

Mycobacterium ulcerans, the etiological agent of Buruli ulcers, is an environmental pathogen. We cultivated it in an amphibian (XTC-2) cell line that grows at 28 degrees C. By counting of Ziehl-Neelsen-stained mycobacteria and by quantitative PCR analysis, we found that M. ulcerans multiplies rapidly in association with XTC-2 cells. Transmission electron microscopy demonstrated the presence of intracellular M. ulcerans microorganisms. These data suggest an intracellular environmental niche, and we propose use of XTC-2 cells for isolation of M. ulcerans from environmental sources.

Regulation of the cell cycle by p53 after DNA damage in an amphibian cell line.

In mammalian cells, the p53 protein is a key regulator of the cell cycle following DNA damage. In the present study, we investigated the function of p53 in the A6 amphibian cell line. Using various specific Xenopus p53 monoclonal antibodies, we showed that Xenopus p53 accumulates after DNA damage, including gamma and UV irradiation or treatment with adriamycin. Such accumulation is accompanied by an increase in the apparent molecular weight of the protein. This change was shown to be the result of a phosphorylation event that occurs after DNA damage. Accumulation of Xenopus p53 is parallel to a drastic change in the cell cycle distribution. Brief exposure to adriamycin or gamma irradiation induces reversible growth arrest, whereas long-term exposure to adriamycin leads to apoptosis. Taken together, these results indicate that p53 has a similar behaviour in frog cells and mammalian cells, and that it conserves two activities, cell cycle arrest and apoptosis.

Rapid upregulation of serum and glucocorticoid-regulated kinase ( sgk) gene expression by corticosteroids in vivo

The molecular mechanisms by which corticosteroids regulate epithelial sodium transport remain to be fully elucidated. Expression of the serum and glucocorticoid-regulated kinase ( sgk) has recently been reported to be regulated acutely by corticosteroids in the amphibian A6 cell line and in cortical collecting tubule cells in vitro. In order to extend this observation to a mammalian system in vivo, the acute response of the sgk gene to a single parenteral dose of aldosterone or dexamethasone was examined in the rat kidney and distal colon. The sgk mRNA levels were significantly elevated by both steroids by 30 min in the distal colon, reaching a peak at 2 h. A more modest increase in sgk mRNA levels was also seen in the kidney in response to both steroids. In both tissues, sgk mRNA has a very short half-life. As for other corticosteroid-regulated genes, the response appears to be mediated by both the mineralocorticoid and glucocorticoid receptors. The response to aldosterone in the distal colon in the presence of cycloheximide was superinduced, strongly suggesting that this is a primary response. The responses to both adrenalectomy and carbenoxolone sodium treatment suggest that the observed responses to corticosteroids can occur in the physiological range of endogenous circulating corticosteroids. These studies provide strong evidence that sgk is an aldosterone-induced gene in vivo in a mammalian system.

Delivery of newly synthesized Na(+)-K(+)-ATPase to the plasma membrane of A6 epithelia.

Na(+)-K(+)-ATPase is localized to the basolateral cell surface of most epithelial cells. Conflicting results regarding the intracellular trafficking of Na(+)-K(+)-ATPase in Madin-Darby canine kidney cells have been reported, with delivery to both apical and basolateral membranes or exclusively to the basolateral cell surface. We examined the delivery and steady-state distribution of Na(+)-K(+)-ATPase in the amphibian epithelial cell line A6 using an antibody raised against Na(+)-K(+)-ATPase alpha-subunit and sulfo-N-hydroxysuccinimidobiotin to tag cell surface proteins. The steady-state distribution of the Na(+)-K(+)-ATPase was basolateral, as confirmed by immunocytochemistry. Delivery of newly synthesized Na(+)-K(+)-ATPase to the cell surface was examined using [35S]methionine and [35S]cysteine in a pulse-chase protocol. After a 20-min pulse, the alpha-subunit and core glycosylated beta-subunit were present at both apical and basolateral cell surfaces. The alpha-subunit and core glycosylated beta-subunit delivered to the apical cell surface were degraded within 2 h. Mature alpha/beta-heterodimer was found almost exclusively at the basolateral surface after a 1- to 24-h chase. These data suggest that immature Na(+)-K(+)-ATPase alpha-subunit and core glycosylated beta-subunits are not retained in the endoplasmic reticulum of A6 cells and apparently lack sorting signals. Mature Na(+)-K(+)-ATPase is targeted to the basolateral surface, suggesting that basolateral targeting of the protein is conformation dependent.