Alpha-subunit Of Na Research Articles

Ultrastructure and distribution dynamics of chloride cells in tilapia larvae in fresh water and sea water.

Integumental and branchial chloride cells of tilapia larvae (Oreochromis mossambicus) were studied at the light-microscopical and ultrastructural level. Total numbers and distribution of chloride cells were quantified after immunostaining of cross sections of the entire larvae with an antibody against the alpha-subunit of Na+/K+-ATPase. The majority (66%) of Na+/K+-ATPase-immunoreactive (ir) cells, i.e. chloride cells, of freshwater tilapia larvae were located extrabranchially up to 48 h after hatching. Five days after hatching, the majority (80%) of chloride cells were found in the buccal cavity. Transfer of 24-h-old larvae to 20% sea water speeded up this process; 24 h after transfer (i.e. 48 h after hatching), the majority (59%) of chloride cells were located in the buccal cavity. The branchial chloride cell population of 24-h- and 120-h-old larvae consisted of immature, mature, apoptotic and necrotic chloride cells. However, relatively more immature chloride cells were observed in freshwater larvae (42-63%) than in (previously studied) freshwater adults (21%), illustrating the developmental state of the gills. After transfer to sea water, the incidence of degenerative chloride cells did not change. Furthermore, the incidence of immature cells had decreased and a new subtype of chloride cells, the "mitochondria-poor" cells, appeared more frequently. These mitochondria-poor chloride cells were characterised by an abundant tubular system and relatively few mitochondria, which were aligned at the border or concentrated in one part of the cytoplasm. Most of these cells did not contact the water. The function of their enhanced appearance after seawater transfer is unknown.

Evidence for an allelic association between bipolar disorder and a Na +, K + adenosine triphosphatase alpha subunit gene (ATP1A3)

Background: Disturbances in central nervous system Na +, K + adenosine triphosphatase (ATPase) activity have previously been proposed as being involved in the pathophysiology of bipolar mood disorder. Methods: We have examined one particular alpha subunit of this enzyme for allelic association in a sample of 85 Irish bipolar patients and 85 matched controls. Results: There was evidence for an overall allelic association between the disease and a dinucleotide polymorphism within the ATP1A3 gene ( p = .022). Subjects were then analyzed on the basis of a number of criteria, and the significance of the association increased when cases were divided based on the nature of the first episode. Patients who presented with a depressive episode first showed a significant association ( p = .001) with this polymorphism. Conclusions: The results presented here provide preliminary evidence of an association between bipolar disorder and an alpha subunit of Na +, K + ATPase, the expression of which predominates in the brain.

Multiple Signal Transduction Pathways Link Na+/K+-ATPase to Growth-related Genes in Cardiac Myocytes: THE ROLES OF Ras AND MITOGEN-ACTIVATED PROTEIN KINASES

We showed before that in neonatal rat cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophic growth and transcriptional regulations of genes that are markers of cardiac hypertrophy. In view of the suggested roles of Ras and p42/44 mitogen-activated protein kinases (MAPKs) as key mediators of cardiac hypertrophy, the aim of this work was to explore their roles in ouabain-initiated signal pathways regulating four growth-related genes of these myocytes, i.e. those for c-Fos, skeletal alpha-actin, atrial natriuretic factor, and the alpha3-subunit of Na+/K+-ATPase. Ouabain caused rapid activations of Ras and p42/44 MAPKs; the latter was sustained longer than 90 min. Using high efficiency adenoviral-mediated expression of a dominant-negative Ras mutant, and a specific inhibitor of MAPK kinase (MEK), activation of Ras-Raf-MEK-p42/44 MAPK cascade by ouabain was shown. The effects of the mutant Ras, an inhibitor of Ras farnesylation, and the MEK inhibitor on ouabain-induced changes in mRNAs of the four genes indicated that (a) skeletal alpha-actin induction was dependent on Ras but not on p42/44 MAPKs, (b) alpha3 repression was dependent on the Ras-p42/44 MAPK cascade, and (c) induction of c-fos or atrial natriuretic factor gene occurred partly through the Ras-p42/44 MAPK cascade, and partly through pathways independent of Ras and p42/44 MAPKs. All ouabain effects required extracellular Ca2+, and were attenuated by a Ca2+/calmodulin antagonist or a protein kinase C inhibitor. The findings show that (a) signal pathways linked to sarcolemmal Na+/K+-ATPase share early segments involving Ca2+ and protein kinase C, but diverge into multiple branches only some of which involve Ras, or p42/44 MAPKs, or both; and (b) there are significant differences between this network and the related gene regulatory pathways activated by other hypertrophic stimuli, including those whose responses involve increases in intracellular free Ca2+ through different mechanisms.

Inhibition of Rat Na+/K+-ATPase Isoforms by Endogenous Digitalis Extracts from Neonatal Human Plasma

An unique endogeneous digitalis-like factor (EDLF) has been previously purified from human newborn cord plasma and its differential effects tested on the three well defined functional isoforms (α1, α2and α3) of the alpha subunits of Na+/K+-ATPase in ratEDLF specifically inhibits the enzymatic activity. It differs from ouabain by three criteria: a preincubation with the membranes is required for full activity, no effect on the rat cerebral α3isoform and a steep dose-response curve with the same apparent potency for rat α2and α1isoforms of high (10-7M) and low affinity (3 × 10-5M) for ouabain. These results indicate that the Na+/K+-ATPase inhibitor involved in the regulation of sodium and body fluid volume and present in neonate and adult human plasmas is distinct from ouabain

Keratinocyte growth factor stimulates CFTR-independent fluid secretion in the fetal lung in vitro.

Keratinocyte growth factor (KGF) caused cystic dilation of mouse fetal lung explants in vitro, markedly increasing the luminal volume of lung buds and disrupting branching morphogenesis. Effects of KGF were dose dependent, were detected within 4 h of treatment, and were blocked by cycloheximide but not by actinomycin D, indicating that de novo protein synthesis mediated the response. Effects of KGF were inhibited by bumetanide, an inhibitor of the Na(+)-K(+)-Cl- cotransporter, and ouabain, an inhibitor of the Na(+)-K(+)-ATPase. KGF stimulated fluid secretion equally in lung buds from cystic fibrosis transmembrane conductance regulators (CFTR) -/- and wild-type embryos, indicating that the effects were mediated by CFTR-independent Cl- transport. Microelectrode studies demonstrated that, whereas KGF did not acutely alter the transepithelial potential difference (PD) across the respiratory epithelium, the PD decreased while luminal volume increased during chronic exposure. KGF inhibited expression of alpha-subunit of epithelial Na+ channel (alpha-ENaC) mRNA, suggesting that KGF may inhibit Na+ absorption, which may contribute to KGF-induced fluid accumulation. KGF-induced fluid accumulation is driven by CFTR-independent Cl- transport and associated with decreased expression of alpha-ENaC.

Studies of renal injury. I. Gentamicin toxicity and expression of basolateral transporters.

Gentamicin nephrotoxicity may arise in part from alterations in the expression of genes critical for renal proximal tubule metabolism. We tested the hypothesis that gentamicin suppressed the gene expression of the Na+/Ca2+ exchanger (NaCaX), glucose transporter 1 (GLUT1) and alpha 1-subunit of Na(+)-K(+)-ATPase (alpha 1-NKA) in renal tubules. The products of these genes mediate Na(+)-dependent Ca2+ efflux, glucose efflux and influx, and ATP-dependent Na+ efflux across tubular basolateral membranes, respectively. After 10 days of gentamicin intoxication (40 mg/kg ip, twice daily), levels of mRNAs encoding NaCaX and the cognate protein declined. GLUT1 mRNA levels increased, although GLUT1 protein levels were also reduced. Moreover, whereas alpha 1-NKA mRNA levels remained unchanged, alpha 1-NKA protein levels were also reduced. We suggest that the higher GLUT1 mRNA level is part of the stress response to tubular injury. However, regardless of the mRNA level, the most consistent effect of gentamicin was reduction of specific protein levels. We propose that failure to translate high levels of mRNA into proportionally high levels of protein, as in the case of GLUT1, may attenuate the expression of stress response gene products, and thus diminish the possibility of recovery in gentamicin intoxication.

Photoaffinity labeling of the active site of the Na+/K(+)-ATPase with 4-azido-2-nitrophenyl phosphate.

Na+/K(+)-ATPase will hydrolyze small acylphosphates such as p-nitrophenyl phosphate (pNPP) in addition to ATP and can derive sufficient energy from the hydrolysis of these small molecules to catalyze active ion transport. In this report, 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive analog of pNPP, was used as a probe of the substrate binding site of dog renal Na+/K(+)-ATPase. ANPP was slowly hydrolyzed by Na+/K(+)-ATPase with a Vmax of 0.19 mumol mg-1 min-1 and with an apparent Km of 1.0 mM. The Km for hydrolysis of pNPP was 1.7 mM. ANPP competitively inhibited the hydrolysis of pNPP with a Ki of 0.37 mM. Both the ATPase and pNPPase activity of the Na+/K(+)-ATPase were irreversibly inhibited after photolysis of the enzyme and ANPP with UV light, although neither activity was completely inhibited by up to 200 microM ANPP. Inhibition of activity was prevented by including 0.2 mM ATP in the reaction or by excluding Mg2+ from the photolysis buffer. Photolysis with [32P]ANPP labeled only the alpha subunit of the Na+/K(+)-ATPase, and the amount of labeling was substantially reduced by 0.2 mM ATP or in the absence of Mg2+. The stoichiometry of labeling extrapolated to a maximum of about 1.2 nmol/mg of protein at 100% inhibition of Mg(2+)-dependent activity. Limited proteolytic digestion showed labeling sites on nonoverlapping tryptic peptides derived from the alpha subunit of Na+/K(+)-ATPase, and two radiolabeled peptides were purified from an exhaustive tryptic digest of [32P]ANPP-labeled Na+/K(+)-ATPase. One peptide contained amino acids Met-379 to Lys-406, and the second contained amino acids Ala-655 to Lys-676. Amino acids corresponding to Asn-398 and Pro-668 were missing from the sequences and may represent residues derivatized by ANPP from within the substrate binding site of Na+/K(+)-ATPase.

Modulation of brain Na+ channels by a G-protein-coupled pathway.

Na+ channels in acutely dissociated rat hippocampal neurons and in Chinese hamster ovary (CHO) cells transfected with a cDNA encoding the alpha subunit of rat brain type IIA Na+ channel (CNaIIA-1 cells) are modulated by guanine nucleotide binding protein (G protein)-coupled pathways under conditions of whole-cell voltage clamp. Activation of G proteins by 0.2-0.5 mM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), a nonhydrolyzable GTP analog, increased Na+ currents recorded in both cell types. The increase in current amplitude was caused by an 8- to 10-mV negative shift in the voltage dependence of both activation and inactivation. The effects of G-protein activators were blocked by treatment with pertussis toxin or guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), a nonhydrolyzable GDP analog, but not by cholera toxin. GDP[beta S] (2 mM) alone had effects opposite those of GTP[gamma S], shifting Na(+)-channel gating 8-10 mV toward more-positive membrane potentials and suggesting that basal activation of G proteins in the absence of stimulation is sufficient to modulate Na+ channels. In CNaIIA-1 cells, thrombin, which activates pertussis toxin-sensitive G proteins in CHO cells, caused a further negative shift in the voltage dependence of Na(+)-channel activation and inactivation beyond that observed with GTP alone. The results in CNaIIA-1 cells indicate that the alpha subunit of the Na+ channel alone is sufficient to mediate G protein effects on gating. The modulation of Na+ channels via a G-protein-coupled pathway acting on Na(+)-channel alpha subunits may regulate electrical excitability through integration of different G-protein-coupled synaptic inputs.

The influence of beta subunit structure on the stability of Na+/K(+)-ATPase complexes and interaction with K+.

Heterologous expression of the beta subunit of H+/K(+)-ATPase (HK beta) with alpha subunits of Na+/K(+)-ATPase (NK alpha) in yeast leads to the formation of ouabain binding complexes, indicating assembly of the two subunits into active ion pumps (Eakle, K. A., Kim, K. S., Kabalin, M. A., and Farley, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2834-2838). Complexes of NK alpha and HK beta are less sensitive to inhibition of ouabain binding by K+, suggesting that HK beta lowers the affinity of K+ binding sites. This effect is particularly pronounced when HK beta is combined with the alpha 3 isoform of NK alpha. In this case, titration with K+ yields a biphasic curve, suggesting that there are two nonequivalent sites for K+ binding. Attempts at purifying complexes formed with either alpha 1 + HK beta or alpha 3 + HK beta using SDS extraction of microsomal membranes resulted in the loss of ouabain binding. Controls show that alpha 1 + beta 1 and alpha 3 + beta 1 complexes still retain ouabain binding after SDS extraction under the same conditions. This suggests that the HK beta subunit forms a less stable complex with NK alpha subunits. We have created chimeric beta subunits comprised of the amino-terminal cytoplasmic and transmembrane regions of HK beta combined with the carboxyl-terminal extracellular region of Na+/K(+)-ATPase beta 1 (HN beta 1) and the complementary chimera with amino-terminal cytoplasmic and transmembrane regions of beta 1 combined with the carboxyl-terminal extracellular region of HK beta (NH beta 1). When NH beta 1 is combined with either alpha 1 or alpha 3, the complexes show profiles of K+ inhibition of ouabain binding that are very similar to HK beta combined with either alpha 1 or alpha 3. The data suggest that the extracellular region of HK beta is primarily responsible for the effect on apparent K+ affinity. When the HN beta 1 subunit is expressed with the alpha 3 subunit, less than 5% of the amount of ouabain binding complexes are formed compared with HN beta 1 + alpha 1. This observation suggests that the HN beta 1 subunit either assembles poorly or forms an unstable complex with alpha 3. After SDS extraction, complexes of alpha 1 + NH beta 1 and alpha 3 + NH beta 1 retain ouabain binding, while alpha 1 + HN beta 1 complexes are sensitive to SDS extraction.(ABSTRACT TRUNCATED AT 400 WORDS)

Pyridoxal 5'-phosphate probes at Lys-480 can sense the binding of ATP and the formation of phosphoenzymes in Na+,K(+)-ATPase.

The Lys-480 in the alpha-subunits of Na+,K(+)-ATPase from pig kidneys was specifically modified with pyridoxal 5'-phosphate (PLP) or pyridoxal 5'-diphospho-5'-adenosine (AP2PL) probes in the presence of NaCl. The site was shown to be the same as the ATP-protectable binding of these probes (Hinz, H.R., and Kirley, T.L. (1990) J. Biol. Chem. 265, 10260-10265). Modifications strongly reduced both Na+,K(+)-ATPase activity and the amount of Na(+)-dependent phosphoenzyme from [32P]ATP but not from [32P]acetyl-phosphate (AcP). Addition of AcP to the enzyme induced a slight decrease in the fluorescence of the PLP probe in the presence of 2 M NaCl and 4 mM MgCl2 but a single exponential increase in the presence of 16 mM NaCl and 4 mM MgCl2. The addition of ATP induced single exponential fluorescence increases at both Na+ concentrations. The data show that these probes can sense molecular events related to the formation of phosphoenzymes induced by AcP and presumably to the formation of Mg-Na-ATP-enzyme complex. The data also suggest that PLP or AP2PL probes at Lys-480 in the presence of Na+ and Mg2+ do not affect the transphosphorylation from AcP to Asp-369 to form phosphoenzymes but that they inhibit the transphosphorylation from the gamma-phosphoryl group of ATP and also ATP binding in the absence of Mg2+.

The significant conservative and variable regions of the homologous protein sequences.

A method of identification of significant conservative and variable regions in homologous protein sequences is presented. A set of aligned homologous sequences is divided into two groups consisting of m and n most related sequences. Each pair of sequences from different group is compared using unitary similarity matrix. The superposition of pairwise comparisons scanned by a window of 10 amino acid residues gives intergroup local variability profile (VP). Area S of the figure between the VP and its mean value line is compared with averaged area S(r) of 1000 VPs of artificial homologous protein families. The difference (S-S(r)) given in standard deviation units sigma r is believed to be the amino acid substitution overall irregularity along the homologous protein sequences OI = (S-S(r))/sigma r. If OI greater than 2, the real VP extrema containing the surplus of area S-(S(r) + 2 sigma r) are cut off. The cut off stretches are likely to be significant conservative and variable regions. The significant conservative and variable regions of six homologous sequence families (phospholipases A2, cytochromes b, alpha-subunits of Na, K-ATPase, L- and M-subunits of photosynthetic bacteria photoreaction centre and human rhodopsins) were identified. It was shown that for artificial homologous protein sequences derived by k-fold lengthening of natural proteins the OI value rises as square root of k. To compare the degree of substitution irregularity in homologous protein sequence families of different length L the value of standard substitution overall irregularity for L = 250 is proposed.

Chick heart cells with high intracellular calcium concentration have a higher affinity for cardiac glycosides than those with low intracellular calcium concentration, as revealed by affinity labelling with a digoxigenin derivative.

Digital-imaging fluorescence microscopy with fura-2 allows the determination of intracellular calcium concentration ([Ca2+]i) in single cells. At a cell density of 10(5) cells/petri dish 44% of the chick embryo heart cells had a high [Ca2+]i of 99.4 +/- 7.1 nM and 56% of the cells a low [Ca2+]i of 27.8 +/- 4.4 nM (mean +/- SE). This laboratory previously reported that high-[Ca2+]i and low-[Ca2+]i cells from chick embryo hearts differ in their sensitivity to cardiac glycosides, as shown by measuring the increase in [Ca2+]i to reach a new steady state [Ahlemeyer, B., Weintraut, H., Seibold, G. & Schoner, W. (1991) in The sodium pump: recent developments (Kaplan, J. H. & De Weer, P., eds) pp. 653-656, Rockefeller University Press, New York]. This time we used N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) which binds irreversibly to amino groups of the Na+/K(+)-ATPase, and sheep anti-digoxigenin Fab fragments coupled with fluorescein isothiocyanate to identify different cardiac glycoside-binding sites. Half-maximal labelling of high-[Ca2+]i cells was obtained at 0.36 nM HDMA, and at 12.0 nM with the low-[Ca2+]i cells. Specific labelling of the cells by HDMA was 91% and 80% in high-[Ca2+]i and low-[Ca2+]i cells, respectively, as revealed by competition experiments with a 1000-fold excess of ouabain. HDMA half-maximally elevated the [Ca2+]i of high-[Ca2+]i cells at a concentration of 50 pM and that of low-[Ca2+]i cells at 8.0 nM. Concentrations higher than 0.1 microM produced signs of intoxication. When the labelled cells were subjected to a SDS/PAGE, a 100-kDa band was found to contain HDMA. The electrophoretic mobility of a protein labelled at 10 nM HDMA was slightly higher than that of a protein labelled at 1.0 microM. The data suggest that different isoforms of the alpha-subunit of Na+/K(+)-ATPase may exist in low-[Ca2+]i and high-[Ca2+]i cells of chick embryo heart.