Alison L. Woo

Identification of a Specific Role for the Na,K-ATPase α2 Isoform as a Regulator of Calcium in the Heart

It is well accepted that inhibition of the Na,K-ATPase in the heart, through effects on the Na/Ca exchanger, raises the intracellular Ca2+ concentration and strengthens cardiac contraction. However, the contribution that individual isoforms make to this calcium regulatory role is unknown. Assessing the phenotypes of mouse hearts with genetically reduced levels of Na,K-ATPase alpha 1 or alpha 2 isoforms clearly demonstrates different functional roles for these isoforms in vivo. Heterozygous alpha 2 hearts are hypercontractile as a result of increased calcium transients during the contractile cycle. In contrast, heterozygous alpha 1 hearts are hypocontractile. The different functional roles of these two isoforms are further demonstrated since inhibition of the alpha 2 isoform with ouabain increases the contractility of heterozygous alpha 1 hearts. These results definitively illustrate a specific role for the alpha 2 Na,K-ATPase isoform in Ca2+ signaling during cardiac contraction.

Sperm Motility Is Dependent on a Unique Isoform of the Na,K-ATPase

The Na,K-ATPase, a member of the P-type ATPases, is composed of two subunits, α and β, and is responsible for translocating Na+ out of the cell and K+into the cell using the energy of hydrolysis of one molecule of ATP. The electrochemical gradient it generates is necessary for many cellular functions, including establishment of the plasma membrane potential and transport of sugars and ions in and out of the cell. Families of isoforms for both the α and β subunits have been identified, and specific functional roles for individual isoforms are just beginning to emerge. The α4 isoform is the most recently identified Na,K-ATPase α isoform, and its expression has been found only in testis. Here we show that expression of the α4 isoform in testis is localized to spermatozoa and that inhibition of this isoform alone eliminates sperm motility. These data describe for the first time a biological function for the α4 isoform of the Na,K-ATPase, revealing a critical role for this isoform in sperm motility.

Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3- cotransporter

The NBC1 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Na}^{+}{/}\mathrm{HCO}_{3}^{-}\) \end{document} cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pHi) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pHi regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document}, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} revealed a sharp decrease in both cAMP-stimulated \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} uptake during cAMP-stimulated anion secretion in the proximal colon.

Mice with a Targeted Disruption of the AE2 Exchanger Are Achlorhydric

The AE2 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} exchanger is expressed in numerous cell types, including epithelial cells of the kidney, respiratory tract, and alimentary tract. In gastric epithelia, AE2 is particularly abundant in parietal cells, where it may be the predominant mechanism for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} efflux and Cl- influx across the basolateral membrane that is needed for acid secretion. To investigate the hypothesis that AE2 is critical for parietal cell function and to assess its importance in other tissues, homozygous null mutant (AE2-/-) mice were prepared by targeted disruption of the AE2 (Slc4a2) gene. AE2-/- mice were emaciated, edentulous (toothless), and exhibited severe growth retardation, and most of them died around the time of weaning. AE2-/- mice exhibited achlorhydria, and histological studies revealed abnormalities of the gastric epithelium, including moderate dilation of the gastric gland lumens and a reduction in the number of parietal cells. There was little evidence, however, that parietal cell viability was impaired. Ultrastructural analysis of AE2-/- gastric mucosa revealed abnormal parietal cell structure, with severely impaired development of secretory canaliculi and few tubulovesicles but normal apical microvilli. These results demonstrate that AE2 is essential for gastric acid secretion and for normal development of secretory canalicular and tubulovesicular membranes in mouse parietal cells.

Characterization of the Fourth α Isoform of the Na,K-ATPase

Abstract. The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical KD values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na+ and K+. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis.

Functional Roles of the α Isoforms of the Na,K-ATPase

Abstract: The Na,K‐ATPase is composed of two subunits, α and β, and each subunit consists of multiple isoforms. In the case of α, four isoforms, α1, α2, α3, and α4 are present in mammalian cells. The distribution of these isoforms is tissue‐ and developmental‐specific, suggesting that they may play specific roles, either during development or coupled to specific physiological processes. In order to understand the functional properties of each of these isoforms, we are using gene targeting, where animals are produced lacking either one copy or both copies of the corresponding gene or have a modified gene. To date, we have produced animals lacking the α1 and α2 isoform genes. Animals lacking both copies of the α1 isoform gene are not viable, while animals lacking both copies of the α2 isoform gene make it to birth, but are either born dead or die very soon after. In the case of animals lacking one copy of the α1 or α2 isoform gene, the animals survive and appear healthy. Heart and EDL muscle from animals lacking one copy of the α2 isoform exhibit an increase in force of contraction, while there is reduced force of contraction in both muscles from animals lacking one copy of the α1 isoform gene. These studies indicate that the α1 and α2 isoforms carry out different physiological roles. The α2 isoform appears to be involved in regulating Ca2+ transients involved in muscle contraction, while the α1 isoform probably plays a more generalized role. While we have not yet knocked out the α3 or α4 isoform genes, studies to date indicate that the α4 isoform is necessary to maintain sperm motility. It is thus possible that the α2, α3, and α4 isoforms are involved in specialized functions of various tissues, helping to explain their tissue‐ and developmental‐specific regulation.

In VivoEvidence for Interferon-γ-mediated Homeostatic Mechanisms in Small Intestine of the NHE3 Na+/H+Exchanger Knockout Model of Congenital Diarrhea

Mice lacking NHE3, the major absorptive Na+/H+ exchanger in the intestine, are the only animal model of congenital diarrhea. To identify molecular changes underlying compensatory mechanisms activated in chronic diarrheas, cDNA microarrays and Northern blot analyses were used to compare global mRNA expression patterns in small intestine of NHE3-deficient and wild-type mice. Among the genes identified were members of the RegIII family of growth factors, which may contribute to the increased absorptive area, and a large number of interferon-γ-responsive genes. The latter finding is of particular interest, since interferon-γ has been shown to regulate ion transporter activities in intestinal epithelial cells. Serum interferon-γ was elevated 5-fold in NHE3-deficient mice; however, there was no evidence of inflammation, and unlike conditions such as inflammatory bowel disease, levels of other cytokines were unchanged. In addition, quantitative PCR analysis showed that up-regulation of interferon-γ mRNA was localized to the small intestine and did not occur in the colon, spleen, or kidney. These in vivodata suggest that elevated interferon-γ, produced by gut-associated lymphoid tissue in the small intestine, is part of a homeostatic mechanism that is activated in response to the intestinal absorptive defect in order to regulate the fluidity of the intestinal tract.