Protein Carbonylation of an Amino Acid Residue of the Na/K-ATPase ?1 Subunit Determines Na/K-ATPase Signaling and Sodium Transport in Renal Proximal Tubular Cells.
ABSTRACT: We have demonstrated that cardiotonic steroids, such as ouabain, signaling through the Na/K-ATPase, regulate sodium reabsorption in the renal proximal tubule. By direct carbonylation modification of the Pro222 residue in the actuator (A) domain of pig Na/K-ATPase ?1 subunit, reactive oxygen species are required for ouabain-stimulated Na/K-ATPase/c-Src signaling and subsequent regulation of active transepithelial (22)Na(+) transport. In the present study we sought to determine the functional role of Pro222 carbonylation in Na/K-ATPase signaling and sodium handling.Stable pig ?1 knockdown LLC-PK1-originated PY-17 cells were rescued by expressing wild-type rat ?1 and rat ?1 with a single mutation of Pro224 (corresponding to pig Pro222) to Ala. This mutation does not affect ouabain-induced inhibition of Na/K-ATPase activity, but abolishes the effects of ouabain on Na/K-ATPase/c-Src signaling, protein carbonylation, Na/K-ATPase endocytosis, and active transepithelial (22)Na(+) transport.Direct carbonylation modification of Pro224 in the rat ?1 subunit determines ouabain-mediated Na/K-ATPase signal transduction and subsequent regulation of renal proximal tubule sodium transport.
Project description:Cardiotonic steroids (such as ouabain) signaling through Na/K-ATPase regulate sodium reabsorption in the renal proximal tubule. We report here that reactive oxygen species are required to initiate ouabain-stimulated Na/K-ATPase·c-Src signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine prevented ouabain-stimulated Na/K-ATPase·c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase and sodium/proton exchanger isoform 3, and inhibition of active transepithelial (22)Na(+) transport. Disruption of the Na/K-ATPase·c-Src signaling complex attenuated ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of ouabain, and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, ouabain stimulated direct carbonylation of two amino acid residues in the actuator domain of the Na/K-ATPase α1 subunit. Taken together, the data indicate that carbonylation modification of the Na/K-ATPase α1 subunit is involved in a feed-forward mechanism of regulation of ouabain-mediated renal proximal tubule Na/K-ATPase signal transduction and subsequent sodium transport.
Project description:Na/K-ATPase signaling has been implicated in different physiological and pathophysiological conditions. Accumulating evidence indicates that oxidative stress not only regulates the Na/K-ATPase enzymatic activity, but also regulates its signaling and other functions. While cardiotonic steroids (CTS)-induced increase in reactive oxygen species (ROS) generation is an intermediate step in CTS-mediated Na/K-ATPase signaling, increase in ROS alone also stimulates Na/K-ATPase signaling. Based on literature and our observations, we hypothesize that ROS have biphasic effects on Na/K-ATPase signaling, transcellular sodium transport, and urinary sodium excretion. Oxidative modulation, in particular site specific carbonylation of the Na/K-ATPase ?1 subunit, is a critical step in proximal tubular Na/K-ATPase signaling and decreased transcellular sodium transport leading to increases in urinary sodium excretion. However, once this system is overstimulated, the signaling, and associated changes in sodium excretion are blunted. This review aims to evaluate ROS-mediated carbonylation of the Na/K-ATPase, and its potential role in the regulation of pump signaling and sodium reabsorption in the renal proximal tubule (RPT).
Project description:Transepithelial Na(+) transport is mediated by passive Na(+) entry across the luminal membrane and exit through the basolateral membrane by two active mechanisms: the Na(+)/K(+) pump and the second sodium pump. These processes are associated with the ouabain-sensitive Na(+)/K(+)-ATPase and the ouabain-insensitive, furosemide-inhibitable Na(+)-ATPase, respectively. Over the last 40 years, the second sodium pump has not been successfully associated with any particular membrane protein. Recently, however, purification and cloning of intestinal α-subunit of the Na(+)-ATPase from guinea pig allowed us to define it as a unique biochemical and molecular entity. The Na(+)- and Na(+)/K(+)-ATPase genes are at the same locus, atp1a1, but have independent promoters and some different exons. Herein, we spotlight the functional characteristics of the second sodium pump, and the associated Na(+)-ATPase, in the context of its role in transepithelial transport and its response to a variety of physiological and pathophysiological conditions. Identification of the Na(+)-ATPase gene (atna) allowed us, using a bioinformatics approach, to explore the tertiary structure of the protein in relation to other P-type ATPases and to predict regulatory sites in the promoter region. Potential regulatory sites linked to inflammation and cellular stress were identified in the atna gene. In addition, a human atna ortholog was recognized. Finally, experimental data obtained using spontaneously hypertensive rats suggest that the Na(+)-ATPase could play a role in the pathogenesis of essential hypertension. Thus, the participation of the second sodium pump in transepithelial Na(+) transport and cellular Na(+) homeostasis leads us to reconsider its role in health and disease.
Project description:BACKGROUND: Cellular quiescence is a state of reversible proliferation arrest that is induced by anti-mitogenic signals. The endogenous cardiac glycoside ouabain is a specific ligand of the ubiquitous sodium pump, Na,K-ATPase, also known to regulate cell growth through unknown signalling pathways. METHODS: To investigate the role of ouabain/Na,K-ATPase in uncontrolled neuroblastoma growth we used xenografts, flow cytometry, immunostaining, comet assay, real-time PCR, and electrophysiology after various treatment strategies. RESULTS: The ouabain/Na,K-ATPase complex induced quiescence in malignant neuroblastoma. Tumour growth was reduced by >50% when neuroblastoma cells were xenografted into immune-deficient mice that were fed with ouabain. Ouabain-induced S-G2 phase arrest, activated the DNA-damage response (DDR) pathway marker ?H2AX, increased the cell cycle regulator p21(Waf1/Cip1) and upregulated the quiescence-specific transcription factor hairy and enhancer of split1 (HES1), causing neuroblastoma cells to ultimately enter G0. Cells re-entered the cell cycle and resumed proliferation, without showing DNA damage, when ouabain was removed. CONCLUSION: These findings demonstrate a novel action of ouabain/Na,K-ATPase as a regulator of quiescence in neuroblastoma, suggesting that ouabain can be used in chemotherapies to suppress tumour growth and/or arrest cells to increase the therapeutic index in combination therapies.
Project description:Na,K-ATPase is a membrane protein that catalyzes ATP to maintain transmembrane sodium and potassium gradients. In addition, Na,K-ATPase also acts as a signal-transducing receptor for cardiotonic steroids such as ouabain and activates a number of signalling pathways. Several studies report that ouabain affects cell migration. Here we used ouabain at concentrations far below those required to block Na,K-ATPase pump activity and show that it significantly reduced RPE cell migration through two mechanisms. It causes dephosphorylation of a 130 kD protein, which we identify as p130cas. Src is involved, because Src inhibitors, but not inhibitors of other kinases tested, caused a similar reduction in p130cas phosphorylation and ouabain increased the association of Na,K-ATPase and Src. Knockdown of p130cas by siRNA reduced cell migration. Unexpectedly, ouabain induced separation of nucleus and centrosome, also leading to a block in cell migration. Inhibitor and siRNA experiments show that this effect is mediated by ERK1,2. This is the first report showing that ouabain can regulate cell migration by affecting nucleus-centrosome association.
Project description:Proatherogenic, hyperlipidemic states demonstrate increases in circulating ligands for scavenger receptor CD36 (eg, oxidized low-density lipoprotein [oxLDL]) and the Na/K-ATPase (eg, cardiotonic steroids). These factors increase inflammation, oxidative stress, and progression of chronic kidney disease. We hypothesized that diet-induced obesity and hyperlipidemia potentiate a CD36/Na/K-ATPase-dependent inflammatory paracrine loop between proximal tubule cells (PTCs) and their associated macrophages and thereby facilitate development of chronic inflammation and tubulointerstitial fibrosis. ApoE(-/-) and apoE(-/-)/cd36(-/-) mice were fed a high-fat diet for ?32 weeks and examined for physiologic and histologic changes in renal function. Compared with apoE(-/-), apoE(-/-)/cd36(-/-) mice had improved creatinine clearance and blood pressure which corresponded histologically with less glomerular and tubulointerstitial macrophage accumulation, foam cell formation, oxidant stress, and interstitial fibrosis. Coimmunopreciptation and a cell surface fluorescence-based crosslinking assay showed that CD36 and Na/K-ATPase ?-1 colocalized in PTCs and macrophages, and this association was increased by oxLDL or the cardiotonic steroid ouabain. OxLDL and ouabain also increased activation of Src and Lyn in PTCs. Cell-free conditioned medium from PTCs treated with oxLDL or ouabain increased macrophage migration. OxLDL, ouabain, or plasma isolated from high-fat diet-fed mice stimulated reactive oxygen species production in PTCs, which was inhibited by N-acetyl-cysteine, apocynin, or Na/K-ATPase ?-1 knockdown. These data suggest that ligands generated in hyperlipidemic states activate CD36 and the Na/K-ATPase and potentiate an inflammatory signaling loop involving PTCs and their associated macrophages, which facilitates the development of chronic inflammation, oxidant stress, and fibrosis underlying the renal dysfunction common to proatherogenic, hyperlipidemic states.
Project description:1. The role of protein kinase C (PKC) in agonist-induced contractions of guinea-pig ileum longitudinal smooth muscle has been investigated. 2. The phorbol esters, phorbol 12,13-dibutyrate (PDBu), phorbol 12,13-diacetate (PDA) and phorbol 12-myristate 13-acetate (PMA), relaxed tissues precontracted by submaximal concentrations of carbachol, histamine or substance P. 3. This inhibitory action of the phorbol esters was reversed following the application of ouabain, a specific inhibitor of Na(+)-K(+)-ATPase. Similarly, pretreatment with ouabain inhibited the ability of phorbol esters to relax tissues precontracted by the above agonists. 4. The slow relaxation of the tonic component of contraction induced by submaximal concentrations of carbachol and histamine, and all concentrations of substance P, was abolished in the presence of ouabain. 5. In Na(+)-loaded tissues, PDBu and carbachol caused a concentration-dependent increase of Na(+)-K(+)-ATPase activity, assessed by ouabain-sensitive 86Rb(+)-uptake. Extrusion of Na+, assessed by the cellular content of the ion, was also stimulated by PDBu (the effect of carbachol was not investigated). 6. We conclude that phorbol esters inhibit the tonic component of contractions induced by submaximal concentrations of these agonists through activation of Na(+)-K(+)-ATPase. We suggest that PKC may exert feedback control over the tonic component of agonist contractions through stimulation of the pump.
Project description:The sodium-potassium pump (Na(+),K(+)-ATPase) is responsible for establishing Na(+) and K(+) concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na(+),K(+)-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 A resolution in a state analogous to E2.2K(+).Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K(+), in marked contrast to previous models. Due to antagonism between ouabain and K(+), the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.
Project description:Purpose:Mutations in the RS1 gene, which encodes retinoschisin, cause X-linked juvenile retinoschisis, a retinal dystrophy in males. Retinoschisin specifically interacts with the retinal sodium-potassium adenosine triphosphatase (Na/K-ATPase), a transmembrane ion pump. Na/K-ATPases also bind cardiac glycosides, which control the activity of the pump and have been linked to disturbances in retinal homeostasis. In this study, we investigated the crosstalk between retinoschisin and cardiac glycosides at the retinal Na/K-ATPase and the consequences of this interplay on retinal integrity. Methods:The effect of cardiac glycosides (ouabain and digoxin) on the binding of retinoschisin to the retinal Na/K-ATPase was investigated via western blot and immunocytochemistry. Also, the influence of retinoschisin on the binding of cardiac glycosides was analyzed via enzymatic assays, which quantified cardiac glycoside-sensitive Na/K-ATPase pump activity. Moreover, retinoschisin-dependent binding of tritium-labeled ouabain to the Na/K-ATPase was determined. Finally, a reciprocal effect of retinoschisin and cardiac glycosides on Na/K-ATPase localization and photoreceptor degeneration was addressed using immunohistochemistry in retinoschisin-deficient murine retinal explants. Results:Cardiac glycosides displaced retinoschisin from the retinal Na/K-ATPase; however, retinoschisin did not affect cardiac glycoside binding. Notably, cardiac glycosides reduced the capacity of retinoschisin to regulate Na/K-ATPase localization and to protect against photoreceptor degeneration. Conclusions:Our findings reveal opposing effects of retinoschisin and cardiac glycosides on retinal Na/K-ATPase binding and on retinal integrity, suggesting that a fine-tuned interplay between both components is required to maintain retinal homeostasis. This observation provides new insight into the mechanisms underlying the pathological effects of cardiac glycoside treatment on retinal integrity.
Project description:The Na(+)/K(+) ATPase is an almost ubiquitous integral membrane protein within the animal kingdom. It is also the selective target for cardiotonic derivatives, widely prescribed inhibitors for patients with heart failure. Functional studies revealed that ouabain-sensitive residues distributed widely throughout the primary sequence of the protein. Recently, structural work has brought some consensus to the functional observations. Here, we use a spectroscopic approach to estimate distances between a fluorescent ouabain and a lanthanide binding tag (LBT), which was introduced at five different positions in the Na(+)/K(+) ATPase sequence. These five normally functional LBT-Na(+)/K(+) ATPase constructs were expressed in the cell membrane of Xenopus laevis oocytes, operating under physiological internal and external ion conditions. The spectroscopic data suggest two mutually exclusive distances between the LBT and the fluorescent ouabain. From the estimated distances and using homology models of the LBT-Na(+)/K(+) ATPase constructs, approximate ouabain positions could be determined. Our results suggest that ouabain binds at two sites along the ion permeation pathway of the Na(+)/K(+) ATPase. The external site (low apparent affinity) occupies the same region as previous structural findings. The high apparent affinity site is, however, slightly deeper toward the intracellular end of the protein. Interestingly, in both cases the lactone ring faces outward. We propose a sequential ouabain binding mechanism that is consistent with all functional and structural studies.