16-243 Sigma-AldrichAnti-Na+/K+ ATPase α-1 Antibody, clone C464.6, Alexa Fluor® 488

Antibody-based detection of protein distribution patterns both in wholemount and on sections revolutionized Xenopus research and ushered in the visual-based era of Xenopus data presentation. The ability to view the distribution of a gene product throughout an embryo makes it possible to rapidly map normal expression profiles and profiles that have been altered by an experimental intervention. The main limiting element in Xenopus immunostaining techniques has always been the availability of antibodies that work well on fixed whole embryos, a problem that persists. However, new antibodies are constantly being generated and improvements in detection systems allow antibodies that were once below the limits of detection to be utilized in multichannel immunofluorescence using tyramide amplification.

The primary transport system of sodium and potassium across the plasma cell membrane, the Na/K-ATPase, is a vital enzyme involved in numerous cellular events. This enzyme is the receptor for plant and amphibian steroids such as ouabain, digoxin and bufalin. In the past decade several endogenous steroids, identical or similar to the plant and amphibian steroids, termed here collectively digitalis-like compounds (DLC), have been identified in human tissues. This paper raises the hypothesis that alterations in the metabolism of endogenous DLC and in their interactions with the Na/K-ATPase may be associated with the development of malignancies. This hypothesis is based on the review of the literature pointing to: 1. An abnormal activity of the Na/K-ATPase and its sensitivity to DLC in malignant cells; 2. Abnormal plasma DLC concentrations in cancer patients; 3. Abnormal synthesis and release of DLC in immune compromised mice; and 4. Beneficial effects of DLC in the treatment of cancer.

The Na/K-ATPase is a complex of integral membrane proteins that carries out active transport of sodium and potassium across the cell plasma membrane, and maintains chemical gradients of these ions. The alpha subunit of the Na/K-ATPase has several isoforms that are expressed in a cell type- and tissue-dependent manner. In adult vertebrates, while kidney cells express mostly alpha1, muscle and glial cells -- alpha1 and alpha2, and sperm cells -- alpha1 and alpha4 isoforms of Na/K-ATPase, neurons may express alpha1, alpha2, alpha3 or any combination of these isoforms, and evidence suggests that neuronal type is the determining factor. The functional significance of multiple isoforms of the Na/K-ATPase and their non-uniform expression, and the link between neuron function and expression of a given isoform of the Na/K-ATPase in particular, remains unknown. Several hypotheses on this account were introduced, and in this work we will review the present status of these hypotheses, and their standing in application to recent data on the expression of isoforms of the Na/K-ATPase in the peripheral nervous system of vertebrate animals.

Na+,K(+)-ATPase is an ubiquitous membrane enzyme that allows the extrusion of three sodium ions from the cell and two potassium ions from the extracellular fluid. Its activity is decreased in many tissues of streptozotocin-induced diabetic animals. This impairment could be at least partly responsible for the development of diabetic complications. Na+,K(+)-ATPase activity is decreased in the red blood cell membranes of type 1 diabetic individuals, irrespective of the degree of diabetic control. It is less impaired or even normal in those of type 2 diabetic patients. The authors have shown that in the red blood cells of type 2 diabetic patients, Na+,K(+)-ATPase activity was strongly related to blood C-peptide levels in non-insulin-treated patients (in whom C-peptide concentration reflects that of insulin) as well as in insulin-treated patients. Furthermore, a gene-environment relationship has been observed. The alpha-1 isoform of the enzyme predominant in red blood cells and nerve tissue is encoded by the ATP1A1 gene. A polymorphism in the intron 1 of this gene is associated with lower enzyme activity in patients with C-peptide deficiency either with type 1 or type 2 diabetes, but not in normal individuals. There are several lines of evidence for a low C-peptide level being responsible for low Na+,K(+)-ATPase activity in the red blood cells. Short-term C-peptide infusion to type 1 diabetic patients restores normal Na+,K(+)-ATPase activity. Islet transplantation, which restores endogenous C-peptide secretion, enhances Na+,K(+)-ATPase activity proportionally to the rise in C-peptide. This C-peptide effect is not indirect. In fact, incubation of diabetic red blood cells with C-peptide at physiological concentration leads to an increase of Na+,K(+)-ATPase activity. In isolated proximal tubules of rats or in the medullary thick ascending limb of the kidney, C-peptide stimulates in a dose-dependent manner Na+,K(+)-ATPase activity. This impairment in Na+,K(+)-ATPase activity, mainly secondary to the lack of C-peptide, plays probably a role in the development of diabetic complications. Arguments have been developed showing that the diabetes-induced decrease in Na+,K(+)-ATPase activity compromises microvascular blood flow by two mechanisms: by affecting microvascular regulation and by decreasing red blood cell deformability, which leads to an increase in blood viscosity. C-peptide infusion restores red blood cell deformability and microvascular blood flow concomitantly with Na+,K(+)-ATPase activity. The defect in ATPase is strongly related to diabetic neuropathy. Patients with neuropathy have lower ATPase activity than those without. The diabetes-induced impairment in Na+,K(+)-ATPase activity is identical in red blood cells and neural tissue. Red blood cell ATPase activity is related to nerve conduction velocity in the peroneal and the tibial nerve of diabetic patients. C-peptide infusion to diabetic rats increases endoneural ATPase activity in rat. Because the defect in Na+,K(+)-ATPase activity is also probably involved in the development of diabetic nephropathy and cardiomyopathy, physiological C-peptide infusion could be beneficial for the prevention of diabetic complications.

The Na+-K+--ATPase, or Na+ pump, is a member of the P-type ATPase superfamily. In addition to pumping ions, Na+-K+--ATPase is engaged in assembly of multiple protein complexes that transmit signals to different intracellular compartments. The signaling function of the enzyme appears to have been acquired through the evolutionary incorporation of many specific binding motifs that interact with proteins and ligands. In some cell types the signaling Na+ --ATPase and its protein partners are compartmentalized in coated pits (i.e., caveolae) the plasma membrane. Binding of ouabain to the signaling Na+-K+--ATPase activates the cytoplasmic tyrosine kinase Src, resulting in the formation of an active "binary receptor" that phosphorylates and assembles other proteins into different signaling modules. This in turn activates multiple protein kinase cascades including mitogen-activated protein kinases and protein kinase C isozymes in a cell-specific manner. It also increases mitochondrial production of reactive oxygen species (ROS)and regulates intracellular calcium concentration. Crosstalk among the activated pathways eventually results in changes in the expression of a number of genes. Although ouabain stimulates hypertrophic growth in cardiac myocytes and proliferation in smooth muscle cells, it also induces apoptosis in many malignant cells. Finally, the signaling function of the enzyme is also pivotal to ouabain-induced nongenomic effects on cardiac myocytes.

The recently defined FXYD protein family contains seven members that are small, single-span membrane proteins characterized by a signature sequence containing an FXYD motif and three other conserved amino acid residues. Until recently, the functional role of FXYD proteins was largely unknown, with the exception of the gamma subunit of Na,K-ATPase, which was shown to be a specific regulator of renal alpha1-beta1 isozymes. We have investigated whether other members of the FXYD family may have a similar role as the gamma subunit and have found that CHIF (corticosteroid hormone-induced factor, FXYD4), FXYD7, as well as phospholemman (FXYD1) specifically associate with Na,K-ATPase and preferentially with alpha1-beta isozymes in native tissues, and produce distinct effects on the transport properties of Na,K-ATPase that are adapted to the physiological demands of the tissues in which they are expressed. These results provide evidence for a unique and novel mode of regulation of Na,K-ATPase by FXYD proteins that involves a tissue-specific expression of an auxiliary subunit of distinct Na,K-ATPase isozymes.