Carbachol and nitric oxide inhibition of Na,K-ATPase activity in bovine ciliary processes.

PURPOSE: Nitric oxide (NO) donors and cholinergic agents decrease intraocular pressure, in part because they induce a decrease in aqueous humor production. Because Na,K-adenosine triphosphatase (ATPase) is involved in aqueous humor formation, this study was conducted to investigate the hypothesis that NO and cholinomimetics regulate its activity in bovine ciliary processes. METHODS: Bovine tissue slices were incubated with agonists and antagonists in a physiological buffer in vitro. Na,K-ATPase activity was determined by assaying hydrolysis of adenosine triphosphate (ATP) in suspended permeabilized tissue slices. RESULTS: Carbachol-induced inhibition of Na,K-ATPase activity correlated with increases in cGMP. This inhibition was abolished by the muscarinic blocker atropine, the NO inhibitor N(w)-nitro-L-arginine (L-NAME) and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Sodium nitroprusside (SNP) mimicked the actions of carbachol. The SNP-induced decrease in Na,K-ATPase activity correlated with an increase in cGMP and was also abolished by ODQ. Both 8-bromo (Br)-cGMP and okadaic acid also inhibited Na,K-ATPase activity. CONCLUSIONS: Carbachol-induced inhibition of Na,K-ATPase activity involves muscarinic receptor activation. That SNP mimics and L-NAME reverses carbachol's effect on Na,K-ATPase activity suggests that the actions of carbachol are mediated by NO. Carbachol's and SNP's effects on Na,K-ATPase activity involved soluble guanylate cyclase and cGMP. Inhibition of Na,K-ATPase activity by 8-Br-cGMP and okadaic acid indicates that protein phosphorylation events may mediate SNP-induced inhibition of Na,K-ATPase activity.

Immunocytochemical localization of Na-K-ATPase alpha- and gamma-subunits in rat kidney.

The gamma-subunit of the Na-K-ATPase is a single-span membrane protein that alters the kinetic properties of the enzyme. It is expressed in the kidney, but our initial observations indicated that it is not present in all nephron segments (Arystarkhova E, Wetzel RK, Asinovski NK, and Sweadner KJ. J Biol Chem 274: 33183-33185, 1999). Here we used triple-label confocal immunofluorescence microscopy in rat kidney with antibodies to Na-K-ATPase alpha1- and gamma-subunits and nephron segment-specific markers. Na-K-ATPase alpha1-subunit stain was low but unambiguous in proximal segments, moderate in macula densa, connecting tubules, and cortical collecting ducts, high in thick ascending limb and distal convoluted tubules, and nearly undetectable in glomeruli, descending and ascending thin limb, and medullary collecting ducts. The gamma-subunit colocalized at staining levels similar to alpha1-subunit in basolateral membranes in all segments except cortical thick ascending limb and cortical collecting ducts, which had alpha1-subunit but no detectable gamma-subunit stain. Selective gamma-subunit expression may contribute to the variations in Na-K-ATPase properties in different renal segments.

Structural similarities of Na,K-ATPase and SERCA, the Ca(2+)-ATPase of the sarcoplasmic reticulum.

The crystal structure of SERCA1a (skeletal-muscle sarcoplasmic-reticulum/endoplasmic-reticulum Ca(2+)-ATPase) has recently been determined at 2.6 A (note 1 A = 0.1 nm) resolution [Toyoshima, Nakasako, Nomura and Ogawa (2000) Nature (London) 405, 647-655]. Other P-type ATPases are thought to share key features of the ATP hydrolysis site and a central core of transmembrane helices. Outside of these most-conserved segments, structural similarities are less certain, and predicted transmembrane topology differs between subclasses. In the present review the homologous regions of several representative P-type ATPases are aligned with the SERCA sequence and mapped on to the SERCA structure for comparison. Homology between SERCA and the Na,K-ATPase is more extensive than with any other ATPase, even PMCA, the Ca(2+)-ATPase of plasma membrane. Structural features of the Na,K-ATPase are projected on to the Ca(2+)-ATPase crystal structure to assess the likelihood that they share the same fold. Homology extends through all ten transmembrane spans, and most insertions and deletions are predicted to be at the surface. The locations of specific residues are examined, such as proteolytic cleavage sites, intramolecular cross-linking sites, and the binding sites of certain other proteins. On the whole, the similarity supports a shared fold, with some particular exceptions.

Predicted location and limited accessibility of protein kinase A phosphorylation site on Na-K-ATPase.

Regulation of Na-K-ATPase by cAMP-dependent protein kinase occurs in a variety of tissues. Phosphorylation of the enzyme's catalytic subunit at a classical phosphorylation consensus motif has been observed with purified enzyme. Demonstration of phosphorylation at the same site in normal living cells or tissues has been more difficult, however, making it uncertain that the Na-K-ATPase is a direct physiological substrate of the kinase. Recently, the structure of the homologous sarco(endo)plasmic reticulum Ca-ATPase (SERCA1a) has been determined at 2.6 A resolution (Toyoshima C, Nakasako M, Nomura H, and Ogawa H. Nature 405: 647-655, 2000.), and the Na-K- ATPase should have the same fold. Here, the Na-K-ATPase sequence has been aligned with the Ca-ATPase structure to examine the predicted disposition of the phosphorylation site. The location is close to the membrane and partially buried by adjacent loops, and the site is unlikely to be accessible to the kinase in this conformation. Conditions that may expose the site or further bury it are discussed to highlight the issues facing future research on regulation of Na-K-ATPase by cAMP-dependent pathways.

Immunocytochemical localization of NaK-ATPase isoforms in the rat and mouse ocular ciliary epithelium.

PURPOSE: Ion gradients established by NaK-adenosine triphosphatase (ATPase) in the ocular ciliary epithelium (CE) contribute to the production of aqueous humor. Modulation of NaK-ATPase activity in the CE may alter aqueous inflow, aqueous turnover, and intraocular pressure. To understand the role of NaK-ATPase, it is necessary to examine the distribution of NaK-ATPase subunit isoforms within the epithelium. METHODS: Isoform-specific antibodies and scanning laser confocal microscopy were used to localize NaK-ATPase subunit isoforms in the CE of the mouse and rat. RESULTS: The nonpigmented epithelium (NPE) expressed alpha2 and beta3 at very high levels on its basolateral surface, and alpha1 and beta2 at much lower levels. The pigmented epithelium (PE) expressed alpha1 and beta1 subunits on its basolateral surface along its entire length, whereas alpha3 was expressed in the pars plana only. The distribution and apparent expression levels of isoforms were similar for mouse and rat, with only minor discrepancies, most likely caused by antibody sensitivity. CONCLUSIONS: The results indicate that sodium pumps in the NPE are primarily composed of alpha2 and beta3, whereas those in the PE are alpha1 and beta1. This specialization in isoform expression implies that NaK-ATPase has distinct physiological functions in the two epithelia and that its activity is likely to be regulated by different mechanisms.

Thermal denaturation of the Na,K-ATPase provides evidence for alpha-alpha oligomeric interaction and gamma subunit association with the C-terminal domain.

Thermal denaturation can help elucidate protein domain substructure. We previously showed that the Na,K-ATPase partially unfolded when heated to 55 degrees C (Arystarkhova, E., Gibbons, D. L., and Sweadner, K. J. (1995) J. Biol. Chem. 270, 8785-8796). The beta subunit unfolded without leaving the membrane, but three transmembrane spans (M8-M10) and the C terminus of the alpha subunit were extruded, while the rest of alpha retained its normal topology with respect to the lipid bilayer. Here we investigated thermal denaturation further, with several salient results. First, trypsin sensitivity at both surfaces of alpha was increased, but not sensitivity to V8 protease, suggesting that the cytoplasmic domains and extruded domain were less tightly packed but still retained secondary structure. Second, thermal denaturation was accompanied by SDS-resistant aggregation of alpha subunits as dimers, trimers, and tetramers without beta or gamma subunits. This implies specific alpha-alpha contact. Third, the gamma subunit, like the C-terminal spans of alpha, was selectively lost from the membrane. This suggests its association with M8-M10 rather than the more firmly anchored transmembrane spans. The picture that emerges is of a Na,K-ATPase complex of alpha, beta, and gamma subunits in which alpha can associate in assemblies as large as tetramers via its cytoplasmic domain, while beta and gamma subunits associate with alpha primarily in its C-terminal portion, which has a unique structure and thermal instability.

Genomic organization of the human FXYD2 gene encoding the gamma subunit of the Na,K-ATPase.

Although the gamma subunit of the Na,K-ATPase has only 66 or 68 amino acids, its human gene (FXYD2) was found to span 9.2 kb and have seven exons, including two alternatively spliced exons encoding different N-termini. Two candidate promoters with consensus sites for transcription factors Sp1, AP-1, and AP-2 are present, consistent with independent transcription of the splice variants. Multiple ESTs support the transcriptional competence of the identified gene elements. In the FXYD2 gene, there are two closely spaced polyadenylation signals, and both are used. A proposed third splice variant encoding a 31-residue N-terminal extension was not found in the gene, nor was the predicted larger protein found in human kidney Na,K-ATPase. Instead, evidence was found for the origin of the larger cDNA clone in homologous recombination with unrelated DNA from chromosome 2. FXYD2 is on chromosome 11q23 close to a site of tumorigenic chromosomal translocations, and has a number of repeat elements.

Carbachol inhibits Na(+)-K(+)-ATPase activity in choroid plexus via stimulation of the NO/cGMP pathway.

Secretion of cerebrospinal fluid by the choroid plexus can be inhibited by its cholinergic innervation. We demonstrated that carbachol inhibits the Na(+)-K(+)-ATPase in bovine choroid tissue slices and investigated the mechanism. Many of the actions of cholinergic agents are mediated by nitric oxide (NO), which plays important roles in fluid homeostasis. The inhibition of Na(+)-K(+)-ATPase was blocked by the NO synthase inhibitor [N(omega)-nitro-L-arginine methyl ester] and was quantitatively mimicked by the NO agonists sodium nitroprusside (SNP) and diethylenetriamine NO. Inhibition by SNP correlated with an increase in tissue cGMP and was abolished by 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. Inhibition was mimicked by the protein kinase G activator 8-bromo-cGMP and by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. cGMP-dependent protein kinase inhibitors Rp-8-pCPT-cGMP (0.5-5 microM) and KT-5823 (2.0 microM) did not block the effects of SNP, but higher concentrations of the more selective inhibitor (Rp-8-pCPT-cGMP) had a pharmacological inhibitory effect on Na(+)-K(+)-ATPase. The data suggest that cholinergic regulation of the Na(+)-K(+)-ATPase is mediated by NO and involves activation of guanylate cyclase and elevation of cGMP.

Interaction of protein kinase C and cAMP-dependent pathways in the phosphorylation of the Na,K-ATPase.

To test the hypothesis that there is cross-talk between the protein kinase C (PKC) and protein kinase A (PKA) pathways in the regulation of the Na,K-ATPase, we measured its phosphorylation in mammalian cell cultures. Phosphorylation of the PKC site, Ser-18, appeared to be due to the activation of the alpha isoform of the kinase. In NRK-52E and L6 cells, this phosphorylation was reduced by prior activation of a cAMP-dependent signaling pathway with forskolin. In principle this would be consistent with direct interaction between the two phosphorylation sites, but further investigation suggested a more indirect mechanism. First, phosphorylation of Ser-938, the PKA site, could not be detected despite the presence of active PKA. Second, there was a major reduction in the phosphorylation of unrelated phosphoproteins as a consequence of elevation of cAMP, suggesting generalized reduction of kinase activity or activation of phosphatase activity. In NRK-52E and L6, phosphorylation of the Na, K-ATPase at Ser-18 paralleled this global change. In C6 cells, in contrast, there was no cAMP effect on Na,K-ATPase phosphorylation at Ser-18 and no global cAMP effect on other phosphoproteins. The cross-talk is evidently mediated by events occurring at the cellular level.

Oligodendrocytes in brain and optic nerve express the beta3 subunit isoform of Na,K-ATPase.

The Na,K-ATPase, which catalyzes the active transport of Na(+) and K(+), has two principal subunits (alpha and beta) that have several genetically distinct isoforms. Most of these isoforms are expressed in the nervous system, but certain ones are preferentially expressed in glia and others in neurons. Of the beta isoforms, beta1 predominates in neurons and beta2 in astrocytes, although there are some exceptions. Here we demonstrate that beta3 is expressed in rat and mouse white matter oligodendrocytes. Immunofluorescence microscopy identified beta3 in oligodendrocytes of rat brain white matter in typical linear arrays of cell bodies between fascicles of axons. The intensity of stain peaked at 20 postnatal days. beta3 was identified in cortical oligodendrocytes grown in culture, where it was expressed in processes and colocalized with antibody to galactocerebroside. In the mouse and rat optic nerve, beta3 stain was seen in oligodendrocytes, where it colocalized with carbonic anhydrase II. For comparison, optic nerve was stained for the beta1 and beta2 subunits, showing distinct patterns of labelling of axons (beta1) and astrocytes (beta2). The C6 glioma cell line was also found to express the beta3 isoform preferentially. Since beta3 was not found at detectable levels in astrocytes, this suggests that C6 is closer to oligodendrocytes than astrocytes in the glial cell lineage.

The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression.

A gene family of small membrane proteins, represented by phospholemman and the gamma subunit of Na,K-ATPase, was defined and characterized by the analysis of more than 1000 related ESTs (expressed sequence tags). In addition to new and more complete cDNA sequence for known family members (including MAT-8, CHIF, and RIC), the findings included two new family members and new splicing variants. A large number of EST replicates made it possible to derive curated DNA sequence with higher confidence and accuracy than from the sequencing of individual clones. The family has a core motif of 35 invariant and conserved amino acids centered on a single transmembrane span. Features of each predicted protein product were compared, and tissue distributions were determined. The gene family was named FXYD (pronounced fix-id) in recognition of invariant amino acids in its signature motif. The abundant proteins are involved in the control of ion transport.

The gamma subunit modulates Na(+) and K(+) affinity of the renal Na,K-ATPase.

The Na(+),K(+)-ATPase catalyzes the active transport of ions. It has two necessary subunits, alpha and beta, but in kidney it is also associated with a 7.4-kDa protein, the gamma subunit. Stable transfection was used to determine the effect of gamma on Na, K-ATPase properties. When isolated from either kidney or transfected cells, alphabetagamma had lower affinities for both Na(+) and K(+) than alphabeta. A post-translational modification of gamma selectively eliminated the effect on Na(+) affinity, suggesting three configurations (alphabeta, alphabetagamma, and alphabetagamma*) conferring different stable properties to Na, K-ATPase. In the nephron, segment-specific differences in Na(+) affinity have been reported that cannot be explained by the known alpha and beta subunit isoforms of Na,K-ATPase. Immunofluorescence was used to detect gamma in rat renal cortex. Cortical ascending limb and some cortical collecting tubules lacked gamma, correlating with higher Na(+) affinities in those segments reported in the literature. Selective expression in different segments of the nephron is consistent with a modulatory role for the gamma subunit in renal physiology.

Cellular and subcellular specification of Na,K-ATPase alpha and beta isoforms in the postnatal development of mouse retina.

The Na,K-ATPase is a dominant factor in retinal energy metabolism, and unique combinations of isoforms of its alpha and beta subunits are expressed in different cell types and determine its functional properties. We used isoform-specific antibodies and fluorescence confocal microscopy to determine the expression of Na,K-ATPase alpha and beta subunits in the mouse and rat retina. In the adult retina, alpha1 was found in Müller and horizontal cells, alpha2 in some Müller glia, and alpha3 in photoreceptors and all retinal neurons. beta1 was largely restricted to horizontal, amacrine, and ganglion cells; beta2 was largely restricted to photoreceptors, bipolar cells, and Müller glia; and beta3 was largely restricted to photoreceptors. Photoreceptor inner segments have the highest concentration of Na,K-ATPase in adult retinas. Isoform distribution exhibited marked changes during postnatal development. alpha3 and beta2 were in undifferentiated photoreceptor somas at birth but only later were targeted to inner segments and synaptic terminals. beta3, in contrast, was expressed late in photoreceptor differentiation and was immediately targeted to inner segments. A high level of beta1 expression in horizontal cells preceded migration, whereas increases in beta2 expression in bipolar cells occurred very late, coinciding with synaptogenesis in the inner plexiform layer. Most of the spatial specification of Na,K-ATPase isoform expression was completed before eye opening and the onset of electroretinographic responses on postnatal day 13 (P13), but quantitative increase continued until P22 in parallel with synaptogenesis.

Rat skeletal muscle in culture expresses the alpha1 but not the alpha2 protein subunit isoform of the Na+/K+ pump.

Studies from this laboratory have shown that the physiological expression of the Na+/K+ pump in primary cultures of rat skeletal muscle increases with development. The molecular mechanisms underlying these changes are not known. Therefore, we have examined the expression of alpha and beta subunits of the Na+/K+ pump at both the protein and mRNA levels during myogenesis of primary skeletal muscle cell cultures obtained from newborn rats. Protein isoforms were identified by Western blotting techniques with specific monoclonal and polyclonal antibodies and subunit mRNA was studied with specific cDNA probes. Freshly isolated skeletal muscle from newborn rats expressed both alpha1 and alpha2 protein subunits. From day 1 after plating, primary cultures expressed only the alpha1 protein isoform. In contrast, both beta1 and beta2 isoforms were expressed in freshly isolated muscle and in primary cultures, with beta1 expression being stronger in both preparations. Studies on RNA expression showed that mRNA for alpha1, alpha2, beta1, and beta2 isoforms was identified both in freshly isolated muscle and after plating of cells in culture. These findings indicate that the lack of alpha2 protein expression in primary muscle cell cultures reflects a form of posttranscriptional regulation. There did not appear to be a quantitative difference in isoform expression as a function of age or of fusion in spite of developmental increases in Na+/K+ pump activity and its dependence on cell fusion. The lack of expression of the alpha2 subunit isoform suggests that the developmental changes in physiological expression of the Na+/K+ pump in primary cultures of skeletal muscle may be attributable either to the changes in activity of the alpha1 subunit or to differential activities of alphabeta complexes involving either of the beta subunits.

Plasticity of Na,K-ATPase isoform expression in cultures of flat astrocytes: species differences in gene expression.

The Na,K-ATPase plays an active role in glial physiology, contributing to K+ uptake as well as to the Na+ gradients used by other membrane carriers. There are multiple isoforms of Na,K-ATPase alpha and beta subunits, and different combinations result in different affinities for Na+ and K+. Isoform choice should thus influence K+ and Na+ homeostasis in astrocytes. Prior studies of astrocyte Na,K-ATPase subunit composition have produced apparently conflicting results, suggesting plasticity of gene expression. Purified flat astrocytes from the cerebral cortex and cerebellum of both mouse and rat were systematically investigated here. Using antibodies specific for the alpha1, alpha2, alpha3, beta1, beta2, and beta3 subunits, isoform level was assessed with Western blots, and cellular distribution was visualized with immunofluorescence. Although alpha1 was always expressed, differences were observed in the expression of alpha2 and beta2, subunits that can be expressed in astrocytes in vivo and in coculture with neurons. In addition, abundant alpha subunit was expressed in rat astrocytes and in mouse cerebellar astrocytes without an equivalent level of any of the known beta isoforms, suggesting that an additional beta subunit important for glia is yet to be discovered. Conditions that have been shown to increase Na,K-ATPase activity in astrocyte cultures, such as dibutyryl cAMP, high extracellular K+, and glutamate, did not specifically induce missing subunits, suggesting that cellular interactions are required to alter the ion transporter phenotype.

Tissue-specific expression of the Na,K-ATPase beta3 subunit. The presence of beta3 in lung and liver addresses the problem of the missing subunit.

The Na,K-ATPase belongs to a family of P-type ion-translocating ATPases sharing homologous catalytic subunits (alpha) that traverse the membrane several times and contain the binding sites for ATP and cations. In this family, only Na,K- and H,K-ATPases have been shown to have a second subunit, a single-span glycoprotein called beta. Recently a new isoform (beta3) has been identified in mammals. Here we describe structural features and tissue distribution of the beta3 protein, utilizing an antiserum specific for its N terminus. beta3 was the only beta detected in Na,K-ATPase purified from C6 glioma. Treatment with N-glycosidase F confirmed that beta3 is a glycoprotein containing N-linked carbohydrate chains. Molecular masses of the glycosylated protein and core protein were estimated to be 42 and 35 kDa, respectively, which are different from those of the beta1 and beta2 subunits. Detection of beta subunits has historically been difficult in certain tissues. Sensitivity was improved by deglycosylating, and expression was evaluated by obtaining estimates of beta3/alpha ratio. The proportion of beta3 protein in the rat was highest in lung and testis. It was also present in liver and skeletal muscle, whereas kidney, heart, and brain contained it only as a minor component of the Na,K-ATPase. In P7 rat, we found skeletal muscle and lung Na,K-ATPase to be the most enriched in beta3 subunit, whereas expression in liver was very low, illustrating developmentally regulated changes in expression. The substantial expression in lung and adult liver very likely explains long-standing puzzles about an apparent paucity of beta subunit in membranes or in discrete cellular or subcellular structures.

Hormonal and neurogenic control of Na-K-ATPase and myosin isoforms in neonatal rat cardiac myocytes.

In the rat heart there is a postnatal switch in the expression of isoforms of both Na-K-ATPase and myosin heavy chain (MHC). Here we investigated factors controlling isoform changes in cultures of neonatal cardiomyocytes. In serum-free medium, the compositions of either Na-K-ATPase or MHC isoforms resembled the neonatal phenotype. Thyroid hormone induced the MHC isoform switch but increased expression of all Na-K-ATPase isoforms to various extents. Dexamethasone failed to induce the MHC switch and inhibited Na-K-ATPase alpha 1 isoform expression without inducing the other isoforms. With both hormones, the adult phenotype for both MHC and Na-K-ATPase was seen but with low Na-K-ATPase alpha 2. The paucity of alpha 2 protein was not predicted by studies of mRNA levels. In serum, there was a gradual decline of Na-K-ATPase alpha 3 and the appearance of alpha 2, but again at a relatively low level. Expression of Na-K-ATPase alpha 2 was significantly upregulated when cardiomyocytes were cocultured with sympathetic neurons from superior cervical ganglia, without changes in the MHC isoforms. Thus innervation is postulated to play a specific role in modulating Na-K-ATPase gene expression.

Epitope and mimotope for an antibody to the Na, K-ATPase.

The epitope of a monoclonal antibody specific for the alpha 2 isoform of the Na,K-ATPase was determined and its accessibility in native enzyme was examined. Protein fragmentation with N-chlorosuccinimide, formic acid, trypsin, and leucine aminopeptidase indicated binding near the Na,K-ATPase N-terminus but did not unambiguously delineate the extent of the epitope. The ability of the antibody to bind to denatured enzyme made it a good candidate for screening a random peptide library displayed on M13 phage, but the consensus sequence that emerged was not found in the Na,K-ATPase, Full-length cDNA for the Na,K-ATPase was randomly fragmented and cloned into beta-galactosidase to create a lambda gt11 expression library; screening with the antibody yielded a set of overlaps spanning 23 amino acids at the N-terminus. Chimeras of Na,K-ATPase alpha 1 and alpha 2 narrowed down the epitope to 14-19 amino acids. The antibody did not recognize fusion proteins constructed with shorter segments of this epitope. It did recognize a fusion protein containing the M13 library consensus sequence, however, indicating that this sequence, which is rich in proline and hydrophobic amino acids (FPPNFLFPPPP), was a mimotope. The natural epitope, unique to the Na,K-ATPase alpha 2 isoform, was GREYSPAATTAENG. Reconstitution of antibody binding in a foreign context such as M13 PIII protein or beta-galactosidase thus required a relatively large number of amino acids, indicating that antibody mapping approaches must allow for epitopes of significant size. The epitope was accessible in native enzyme and exposed on the cytoplasmic side, documenting the surface exposure of a stretch of amino acids at the N-terminus, where the Na,K-ATPase isoforms differ most.