Differential expression of the Na,K-ATPase alpha 1 and alpha 2 subunit genes in a murine myogenic cell line. Induction of the alpha 2 isozyme during myocyte differentiation

Expression of Na,K-ATPase catalytic alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes in rodent muscle was investigated using the murine C2C12 myogenic cell line. RNA blot analyses of myoblasts revealed expression primarily of the alpha 1 mRNA and low levels of alpha 2 mRNA. Fusion of the proliferating myoblasts to form myotubes was accompanied by an approximate 12-fold induction of the alpha 2 mRNA. In contrast, expression of alpha 1 mRNA remained constant throughout myogenesis. The alpha 3 mRNA was not detected in either myoblasts or myotubes. The beta mRNA abundance also increased 2-3-fold during myotube formation. In rodent tissues, low and high affinity cardiac glycoside (e.g. ouabain) receptors have been shown to be associated with the Na,K-ATPase catalytic alpha 1 and alpha 2 isoform subunits, respectively. The existence of these two functional classes of Na,K-ATPase in myoblasts and myotubes correlated with the biphasic ouabain inhibition of Na,K-ATPase activity. Confluent myoblasts expressed primarily the alpha 1 isozyme (IC50 = 3.6 X 10(-5) M; 95% of total activity) and lesser amounts of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 5% of total activity). In contrast, the myotubes showed significant levels of the alpha 1 isozyme (IC50 = 4.0 X 10(-5) M; 68% of total activity) and, in addition, showed a 6-fold increase in the relative levels of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 32% of total activity). To quantitate further the expression of the high affinity, ouabain-sensitive alpha 2 isozyme, a whole cell [3H]ouabain-binding assay was used. Results revealed that myotubes have an approximately 6-fold greater concentration of [3H]ouabain-binding sites than myoblasts with an apparent dissociation constant (Kd) of 1.4 X 10(-7) M. The results indicate that muscle cells can express multiple isozymes of Na,K-ATPase and that expression of the alpha 2 isozyme is developmentally regulated during myogenesis.     

The alpha2 isoform of Na,K-ATPase mediates ouabain-induced cardiac inotropy in mice

Inhibition of Na,K-ATPase activity by cardiac glycosides is believed to be the major mechanism by which this class of drugs increases heart contractility. However, direct evidence demonstrating this is lacking. Furthermore it is unknown which specific alpha isoform of Na,K-ATPase is responsible for the effect of cardiac glycosides. Several studies also suggest that cardiac glycosides, such as ouabain, function by mechanisms other than inhibition of the Na,K-ATPase. To determine whether Na,K-ATPase, specifically the alpha2 Na,K-ATPase isozyme, mediates ouabain-induced cardiac inotropy, we developed animals expressing a ouabain-insensitive alpha2 isoform of the Na,K-ATPase using Cre-Lox technology and analyzed cardiac contractility after administration of ouabain. The homozygous knock-in animals were born in normal Mendelian ratio and developed normally to adulthood. Analysis of their cardiovascular function demonstrated normal heart function. Cardiac contractility analysis in isolated hearts and in intact animals demonstrated that ouabain-induced cardiac inotropy occurred in hearts from wild type but not from the targeted animals. These results clearly demonstrate that the Na,K-ATPase and specifically the alpha2 Na,K-ATPase isozyme mediates ouabain-induced cardiac contractility in mice.     

Thyroid hormone regulates alpha and alpha + isoforms of Na,K-ATPase during development in neonatal rat brain

The brain contains two molecular forms of Na,K-ATPase designated alpha found in non-neuronal cells and neuronal soma and alpha + found in axolemma. Previously we have shown that the abundance of both forms (determined by immunoblots) as well as Na,K-ATPase activity increases 10-fold between 4 days before and 20 days after birth (Schmitt, C. A., and McDonough, A. A. (1986) J. Biol. Chem. 261, 10439-10444). Hypothyroidism in neonates blunts these increases. Neonatal, but not adult brain Na,K-ATPase is thyroid hormone (triiodothyronine, T3) responsive. This study defines the period during which brain Na,K-ATPase responds to T3. The start of the critical period was defined by comparing Na,K-ATPase activity and alpha and alpha + abundance in hypothyroid and euthyroid neonates (birth to 30 days of age). For all parameters, euthyroid was significantly higher by 15 days of age. The end of the critical period was defined by dosing hypothyroid neonates with T3 daily (0.1 micrograms/g body weight) beginning at increasing days of age, and sacrificing all at 30 days then assaying enzyme activity and abundance. Those starting T3 treatment on or before day 19 were restored to euthyroid levels of Na,K-ATPase activity and abundance, while those starting T3 treatment on or after day 22 remained at hypothyroid levels of enzyme activity and abundance. We conclude that brain Na,K-ATPase alpha and alpha + isoforms are sensitive to T3 by as late as 15 days of age and that the period of thyroid hormone responsiveness is over by 22 days.     

Postnatal changes in Na,K-ATPase isoform expression in rat cardiac ventricle. Conservation of biphasic ouabain affinity.

The cardiac glycoside sensitivity of the rat heart changes during postnatal maturation and in response to certain pathological conditions. The Na,K-ATPase is thought to be the receptor for cardiac glycosides, and there are three isozymes of its catalytic (alpha) subunit with different cardiac glycoside affinities: alpha 1 (low affinity) and alpha 2 and alpha 3 (high affinity). We examined the developmental expression of the alpha subunit isozymes in rat ventricular membrane preparations by immunoblotting with isozyme-specific antibodies. The alpha 1 isozyme was present throughout all stages of maturation. A developmental switch from alpha 3 to alpha 2 occurred between 14 and 21 days after birth. Measurements of [3H]ouabain binding and inhibition of Na,K-ATPase activity indicated that alpha 2 and alpha 3 should make equivalent contributions to ion pump capacity; in both neonatal natal and adult preparations, ouabain interacted with a single class of high-affinity binding sites (KD = 15 or 40 nM, respectively; Bmax = 4-5 pmol/mg protein), and at low concentrations produced a similar degree of Na,K-ATPase inhibition (25%). The results indicate that the developmental difference in cardiac glycoside sensitivity cannot be explained by quantitative differences in the proportion of high-affinity isozymes of the Na,K-ATPase. The switch from alpha 3 to alpha 2 coincides with other major changes in cardiac electrophysiology and calcium metabolism.     

Structural peculiarities of Na+,K+-ATPase isozymes from the calf brain

Functionally active preparations of Na+,K(+)-ATPase isozymes from calf brain that contain catalytic subunits of three types (alpha 1, alpha 2, and alpha 3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of the membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na+ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na+,K(+)-ATPase of the alpha 1 beta 1 type and minor amounts of isozymes of the alpha 2 beta 2 (beta 1) and the alpha 3 beta 1 (beta 2) type. The axolemma contains alpha 2 beta 1- and alpha 3 beta 1 isozymes. A carbohydrate analysis indicated that alpha 1 beta 1 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the beta 1 isoform. An enhanced sensitivity of the alpha 3 catalytic subunit of Na+,K(+)-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR492 decreases Y493 was localized (residue numbering is that of the human alpha 3 subunit). This sequence corresponds to one of the regions of the greatest variability in alpha 1, alpha 2, alpha 3, and alpha 4-subunits, but at the same time, it is characteristic of the alpha 3 isoforms of various species. The presence of the beta 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na+,K(+)-ATPase alpha 3 beta 1 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the alpha 3 catalytic subunit was shown.     

Enzymatic properties of separated isozymes of the Na,K-ATPase. Substrate affinities, kinetic cooperativity, and ion transport stoichiometry

There are two isozymes of the Na,K-ATPase, which can be purified separately from rat renal medulla and brainstem axolemma. Here the basic kinetic properties of the two Na,K-ATPases have been compared in conditions permitting enzyme turnover. The two isozymes are half-maximally activated at different concentrations of ATP, the axolemma Na,K-ATPase having the higher affinity. They are half-maximally activated by Na+ and K+ at very similar concentrations but show differences in cooperativity toward Na+. The affinities of both isozymes for ATP and Na+ are affected in a qualitatively similar way by variations in the concentration of K+. Both isozymes transport 22Na+ and 42K+ in a ratio close to 3:2 in artificial lipid vesicles. The two isozymes differ most strikingly in the inhibition of ATPase activity by ouabain. The axolemma Na,K-ATPase has a high affinity for ouabain with positive cooperativity, while the renal medulla Na,K-ATPase has a lower affinity with negative cooperativity. It is likely that the cooperativity differences are due to kinetic effects, reflecting different rates of conformation transitions during enzyme turnover. The functional result of the contrasting cooperativities is that the difference in sensitivity to ouabain is amplified.     

Two isozymes of the Na,K-ATPase have distinct antigenic determinants

Two isozymes of the Na,K-ATPase were purified from rat renal medulla and rat brainstem axolemma, and antisera were raised in rabbits. When antibody titers were measured, two sera showed specificity for either the kidney or axolemma Na,K-ATPases and had limited cross-reactivity which could be removed by cross-adsorption. In blots of polyacrylamide gels, these sera reacted with only the alpha or alpha (+) Na,K-ATPase catalytic subunits, while they cross-reacted with both types of beta subunits. Two other sera each recognized both alpha and alpha (+), indicating that the catalytic subunit isozymes have additional shared antigenic determinants. A comparison of the Na,K-ATPases from the brains of different vertebrate species indicates that birds and fish differ from mammals and amphibians in the manifestation of Na,K-ATPases isozymes. Neither neuraminidase nor endoglycosidase F treatment eliminated specific antibody reaction or affected the electrophoretic mobilities of the alpha and alpha (+) subunits, although endoglycosidase F increased the mobilities of the two types of beta subunits to similar final apparent molecular weights. Blots of the peptide fragments produced by incomplete papain and trypsin digests of the alpha and alpha (+) subunits were stained with the specific sera, and the patterns of immunoreactive fragments were found to be markedly different. The results suggest that the antigenic differences reside in differences in the primary protein sequences of the two isozymes.     

Fine specificity mapping and topography of an isozyme-specific epitope of the Na,K-ATPase catalytic subunit

An isozyme-specific domain of the catalytic subunit of the Na,K-ATPase has been identified using a monoclonal antibody, McK1. The antibody's specificity was confirmed by its ability to stain proteolytic fingerprints of the Na,K-ATPase. The antibody recognized the alpha I isozyme of the rat Na,K-ATPase, but not the alpha II or alpha III isozymes. It recognized native and sodium dodecyl sulfate-denatured Na,K-ATPase and specifically stained basolateral membranes of the renal tubule. It bound to rat alpha I with highest affinity, but also cross-reacted with mouse, monkey, and human alpha I. It did not cross-react with sheep, pig, chicken, Torpedo, or dog alpha I. Fine specificity mapping was used to deduce the most likely antibody binding sites, based on comparison of eight amino acid sequences from cDNA clones. Two potential binding sites were found at widely separated locations. Limited tryptic digestion of the native enzyme was then used to demonstrate that the binding site was close to the N-terminal end of the Na,K-ATPase. The binding site is predicted to include the following essential amino acid sequence: Asp-Lys-Lys-Ser-Lys-Lys in rat alpha I or Asp-Lys-Lys-Gly-Lys-Lys in human alpha I. The antibody was found to bind to opened, but not to sealed right-side-out vesicles isolated from the rat renal medulla, demonstrating that the N-terminal end of the Na,K-ATPase is exposed at the interior of the cell.     

Deletion of the Na/K-ATPase alpha1-subunit gene (Atp1a1) does not prevent cavitation of the preimplantation mouse embryo

Increases in Na/K-ATPase activity occur concurrently with the onset of cavitation and are associated with increases in Na(+)-pump subunit mRNA and protein expression. We have hypothesized that the alpha1-isozyme of the Na/K-ATPase is required to mediate blastocyst formation. We have tested this hypothesis by characterizing preimplantation development in mice with a targeted disruption of the Na/K-ATPase alpha1-subunit (Atp1a1) using embryos acquired from matings between Atp1a1 heterozygous mice. Mouse embryos homozygous for a null mutation in the Na/K-ATPase alpha1-subunit gene are able to undergo compaction and cavitation. These findings demonstrate that trophectoderm transport mechanisms are maintained in the absence of the predominant isozyme of the Na(+)-pump that has previously been localized to the basolateral membranes of mammalian trophectoderm cells. The presence of multiple isoforms of Na/K-ATPase alpha- and beta-subunits at the time of cavitation suggests that there may be a degree of genetic redundancy amongst isoforms of the catalytic alpha-subunit that allows blastocyst formation to progress in the absence of the alpha1-subunit.     

Expression of the alpha3/beta1 isoform of human Na,K-ATPase in the methylotrophic yeast Pichia pastoris

Na,K-ATPase is a crucial enzyme for ion homeostasis in human tissues. Different isozymes are produced by assembly of four alpha- and three beta-subunits. The expression of the alpha3/beta1 isozyme is confined to brain and heart. Its heterologous production has so far never been attempted in a lower eukaryote. In this work we explored whether the methylotrophic yeast Pichia pastoris is capable of expressing the alpha3/beta1 isoform of human Na,K-ATPase. cDNAs encoding the alpha(3) and the beta(1)-subunits were cloned under the control of the inducible promoter of Pichia pastoris alcohol oxidase 1. Pichia pastoris could express the single alpha3- and beta1-subunits and even coexpress them after methanol induction. beta1-subunit was produced as a major 44-kDa glycosylated polypeptide and alpha3 as a 110-kDa unglycosylated polypeptide. Expression at the plasma membrane was limited in shaking flask cultures but by cultivating P. pastoris cells in a fermenter there was a 10-fold increase of the number of ouabain binding sites per cell. The exported enzyme was estimated to be about 0.230 mg L(-1) at the end of a bioreactor run. Na,K-ATPase proved active and the dissociation constant of the recombinant enzyme-ouabain interaction was determined.     

Functional characterization of a testes-specific alpha-subunit isoform of the sodium/potassium adenosinetriphosphatase.

Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) alpha and beta subunits have been identified in mammals. The association of the various alpha and beta polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase alpha4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha4 and the Na pump beta1 subunit are coexpressed in the cells, Na, K-ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na(+)-dependent, K(+)-sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K(+). Furthermore, the activity of alpha4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase alpha4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta1 and beta3 subunits. In insect cells, the alpha4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha4beta1 and alpha4beta3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na(+), K(+), ATP, and ouabain. The enzymatic properties of alpha4beta1 and alpha4beta3 are, however, distinct from the other Na pump isozymes. A Na, K-ATPase activity with similar properties as the alpha4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the alpha4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.     

Antisera specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the Na,K-ATPase: differential expression of alpha and beta subunits in rat tissue membranes

We have developed a panel of antibodies specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the rat Na,K-ATPase. TrpE-alpha subunit isoform fusion proteins were used to generate three antisera, each of which reacted specifically with a distinct alpha subunit isotype. Western blot analysis of rat tissue microsomes revealed that alpha 1 subunits were expressed in all tissues while alpha 2 subunits were expressed in brain, heart, and lung. The alpha 3 subunit, a protein whose existence had been inferred from cDNA cloning, was expressed primarily in brain and copurified with ouabain-inhibitable Na,K-ATPase activity. An antiserum specific for the rat Na,K-ATPase beta subunit was generated from a TrpE-beta subunit fusion protein. Western blot analysis showed that beta subunits were present in kidney, brain, and heart. However, no beta subunits were detected in liver, lung, spleen, thymus, or lactating mammary gland. The distinct tissue distributions of alpha and beta subunits suggest that different members of the Na,K-ATPase family may have specialized functions.     

RT-PCR detection of the Na,K-ATPase beta3-isoform in human heart.

The Na,K-ATPase is a heterodimer composed of an alpha-catalytic and a beta-glycoprotein subunit. At present, three different alpha-polypeptides (alpha1, alpha2, alpha3) and two distinct beta-isoforms (beta1 and beta2) have been detected in human heart. The aim of the present study was to determine whether or not the beta3-isoform of the Na,K-ATPase can be detected in human heart. Using the highly sensitive method of RT-PCR, we here show that human heart expresses the beta3-isoform of the Na,K-ATPase. Given the differences in pharmacological properties of the nine different Na,K-ATPase isoenzymes (containing all combinations of the subunit isoforms), the study of beta3-isoform regulation in human heart may be of interest in understanding the altered response of human myocardium to digitalis therapy during heart failure.     

Separate Na,K-ATPase genes are required for otolith formation and semicircular canal development in zebrafish

We have investigated the role of Na,K-ATPase genes in zebrafish ear development. Six Na,K-ATPase genes are differentially expressed in the developing zebrafish inner ear. Antisense morpholino knockdown of Na,K-ATPase alpha1a.1 expression blocked formation of otoliths. This effect was phenocopied by treatment of embryos with ouabain, an inhibitor of Na,K-ATPase activity. The otolith defect produced by morpholinos was rescued by microinjection of zebrafish alpha1a.1 or rat alpha1 mRNA, while the ouabain-induced defect was rescued by expression of ouabain-resistant zebrafish alpha1a.1 or rat alpha1 mRNA. Knockdown of a second zebrafish alpha subunit, alpha1a.2, disrupted development of the semicircular canals. Knockdown of Na,K-ATPase beta2b expression also caused an otolith defect, suggesting that the beta2b subunit partners with the alpha1a.1 subunit to form a Na,K-ATPase required for otolith formation. These results reveal novel roles for Na,K-ATPase genes in vestibular system development and indicate that different isoforms play distinct functional roles in formation of inner ear structures. Our results highlight zebrafish gene knockdown-mRNA rescue as an approach that can be used to dissect the functional properties of zebrafish and mammalian Na,K-ATPase genes.     

Cellular distribution and differential gene expression of the three alpha subunit isoforms of the Na,K-ATPase in the ocular ciliary epithelium.

We have investigated the localization and pattern of expression of the three alpha subunit isoforms of Na,K-ATPase in the transporting ciliary epithelium of the bovine eye. Using specific cDNA probes and antisera to the alpha 1, alpha 2, and alpha 3 isoforms of Na,K-ATPase, we demonstrated that mRNAs and polypeptides for the three distinct forms of the Na,K-ATPase alpha subunit (alpha 1, alpha 2, and alpha 3) were expressed in the ciliary epithelium in vivo. Immunochemical localization of the three alpha isoforms of Na,K-ATPase in two ultrastructurally different regions of the ciliary epithelium (namely, the pars plicata and pars plana) revealed that the three alpha isoforms of Na,K-ATPase were distributed in a distinct fashion in the basolateral plasma membrane domains of nonpigmented (NPE) and pigmented (PE) cells. The NPE cells in the pars plicata showed an immunoreactive signal to all the three alpha isoforms; in the pars plana, they showed immunoreactive signals only for the alpha 1 and alpha 2 isoforms but not for alpha 3. The PE cells, in both the pars plana and pars plicata regions, showed an immunoreactive signal only for the alpha 1 isoform; immunoreactive signals were not detected for alpha 2 and alpha 3. To verify the differential immunostaining patterns of NPE and PE cells, specific antibodies for each of the three alpha subunit isoforms of Na,K-ATPase were applied to immunoblots containing microsomal fractions from flow cytometric-sorted cells (NPE and PE). Our results indicate that alpha 1, alpha 2, and alpha 3 polypeptides were present in microsomal fractions of NPE cells of the pars plicata and pars plana and that the alpha 1 polypeptide was the only polypeptide present in the PE cells from both regions of the ciliary epithelium. These results also revealed that the alpha 3 isoform epitope recognized by the monoclonal antibody McB-X3.1 in the pars plicata is not readily accessible in the pars plana. A cell line was established from the ciliary epithelium of a bovine eye by viral transformation with simian virus 40. In culture, this cell line expressed all three alpha isoforms at the mRNA and polypeptide levels, suggesting that the line may have derived from the NPE layer.     

The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.     

Altered Pattern of Na,K-ATPase Activity and mRNA During Chronic Alcohol Consumption by Juvenile and Adolescent Rats

The effect of chronic ethanol consumption on cerebral cortical activity of Na,K-ATPase was determined in Long-Evans (LE) rats fed an ethanol-containing diet beginning at different stages of development. Na,K-ATPase activity was operationally resolved into α1 and α2/3 isozyme activities. There was no significant difference in Na,K-ATPase activities before and after alcohol consumption in the preparations from adult rats. However, for rats beginning alcohol consumption as adolescents, the α2/3 activity was significantly elevated following chronic alcohol consumption. Both LE and Sprague–Dawley rats showed this same selective increase in cortical α2/3 activity when rats began alcohol consumption as juveniles. The shift in cortical α2/3 activity was not observed in cerebellum or subcortical forebrain and was reversible when rats were fed ethanol throughout the normal adolescent period and then withdrawn and tested 2 weeks later (during the adult period). Levels of isoform-specific mRNA were determined in preparations of cerebral cortices of rats showing elevated α2/3 isozyme activities. In these preparations, isoform specific α2 and α3 mRNA was significantly elevated. There was no effect of ethanol feeding on cortical α1 mRNA. These findings indicate that the longer term effects of ethanol on the developing brain include elevated Na,K-ATPase activity and a mechanism that is pre-translational and isoform specific.     

RT-PCR detection of Na,K-ATPase subunit isoforms in human umbilical vein endothelial cells (HUVEC): evidence for the presence of alpha1 and beta3.

The endothelial Na,K-ATPase is an active component in maintaining a variety of normal vascular functions. The enzyme is characterized by a complex molecular heterogeneity that results from differential expression and association of multiple isoforms of both its alpha- and beta-subunits. The aim of the present study was to determine which isoforms of the Na,K-ATPase are expressed in human endothelial cells. HUVEC (human umbilical vein endothelial cells) were used as a model of well known human endothelial cells. The high sensitive method RT-PCR was used with primers specific for the various isoforms of the alpha- and beta-subunits of the Na,K-ATPase. The results show that HUVEC express alpha1-, but not alpha2-, alpha3- or alpha4-isoforms of the catalytic subunit and that beta3- but not beta2- or beta1-isoforms is present in these cells. These findings are in contradiction with our previous detection of Na,K-ATPase isoforms in HUVEC using antibodies (14). Such results raise the technical problem of the specificity of the available antibodies directed against the different isoforms as well as the question of the physiological relevance of the diversity of the Na,K-ATPase isoforms.     

Expression of Na,K-ATPase alpha subunit isoforms in the human ciliary body and cultured ciliary epithelial cells

We have analyzed the expression of Na,K-ATPase alpha subunit isoforms in the transporting ciliary processes of the human eye and in cultured cells derived from non-pigmented (NPE) and pigmented (PE) ciliary epithelium. Northern hybridization analysis shows that the mRNAs encoding all the three distinct forms of Na,K-ATPase alpha subunit [alpha 1, alpha 2, and alpha 3] are expressed in the human ciliary processes in vivo. Immunohistochemical analysis using antibodies specific for each of the three alpha subunit isoforms confirms that these polypeptides are present in the microsomal fraction from the human ciliary processes. The monoclonal antibody McB2, which is specific to the Na,K-ATPase alpha 2 subunit isoform, has been found to decorate specifically the basolateral membrane domains of NPE cells but not of the PE cells, suggesting its expression in vivo only in the ocular NPE ciliary epithelium. However, cultured cells derived from the NPE and PE layers exhibit a different pattern of expression of mRNA and protein for the Na,K-ATPase alpha subunit isoforms when compared to the tissue. Both the NPE and PE cells express alpha 1 and alpha 3 mRNA and polypeptide, whereas alpha 2 mRNA and polypeptide are undetectable in these cells. The established cell lines derived from the NPE layer express comparable levels of the alpha 1 and alpha 3 isoforms of Na,K-ATPase as detected in the primary culture. However, the established NPE cell lines are also distinguishable from the normal PE cells when analyzed by Western blot analysis with A x 2 antibodies. The results presented here clearly show that the NPE and PE cells in the ciliary body have a distinct expression of Na,K-ATPase alpha subunit isoforms as compared to cultured cells.