Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The minimal functional Na,K-ATPase unit is composed of a catalytic alpha-subunit and a glycosylated beta-subunit. So far three putative beta-isoforms have been described, but only beta 1-isoforms have been identified clearly as part of a purified active enzyme complex. In this study we provide evidence that a putative beta 3-isoform might be the functional component of Xenopus oocyte Na,K-ATPase. beta 3-isoforms are expressed in the oocyte plasma membrane together with alpha-subunits, but beta 3-isoforms are synthesized to a lesser extent than alpha-subunits. The unassembled oocyte alpha-subunits accumulate in an immature trypsin-sensitive form most likely in the endoplasmic reticulum (ER). Injection of both beta 1- and beta 3-cRNA into oocytes abolishes the transport constraint of the oocyte alpha-subunit, renders it trypsin-resistant, and finally leads to an increased number of functional pumps at the plasma membrane. In addition, beta 3-isoforms as beta 1-isoforms depend on the concomitant synthesis of alpha-subunits to be able to leave the ER and to become fully glycosylated. Finally, alpha-beta 1 and alpha-beta 3 complexes expressed at the plasma membrane appear to have similar transport properties as assessed by ouabain binding, rubidium uptake, and electrophysiological measurements in oocytes coexpressing exogenous alpha 1- and beta 1- or beta 3-isoforms. Thus our data indicate that beta 3-isoforms have functional qualities similar to beta 1-isoforms. They can assemble and impose a structural reorganization to newly synthesized alpha-subunits which permits the exit from the ER and the expression of functional Na,K-pumps at the plasma membrane.
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PMID:Processing, intracellular transport, and functional expression of endogenous and exogenous alpha-beta 3 Na,K-ATPase complexes in Xenopus oocytes. 130 55

Hydrogen peroxide (H2O2) is likely to play an important role in oxidant alveolar epithelium injury. We investigated the effect of H2O2 on uptake of phosphate, alanine in cultured rat alveolar type II cells. H2O2 induced inhibition of Na-dependent component of phosphate and alanine uptakes in time- and concentration-dependent manner. Twenty minutes exposure to 2.5 mM H2O2 decreased the maximum velocity (Vmax) of phosphate and alanine uptake by 50 and 62%, respectively, whereas Michaelis constant (Km) values were unchanged. H2O2 also decreased Na-K-ATPase activity, measured by ouabain-sensitive rubidium influx, and this effect was independent of H2O2-induced ATP depletion. A lipid-soluble antioxidant, d-alpha-tocopherol (20 microM, 24 h), prevented H2O2-induced decrease in Na-coupled uptake and Na-K-ATPase activity. These results indicate that H2O2 affects Na-dependent phosphate and alanine uptakes and suggest that this effect may be related at least, in part, to a decrease in Na transmembrane gradient, since H2O2 also affects Na-K-ATPase activity. The protective effect of d-alpha-tocopherol suggests that peroxidation of the membrane lipids is likely to be involved in the observed effects.
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PMID:Impairment of sodium-coupled uptakes by hydrogen peroxide in alveolar type II cells: protective effect of d-alpha-tocopherol. 131 14

In earlier studies Na(+)-K(+)-adenosinetriphosphatase (ATPase) and Na(+)-K(+)-2Cl- cotransport partially accounted for vascular smooth muscle cell (VSMC) K+ (Rb+) uptake. In other cells Rb+ is taken up by a K(+)-H(+)-ATPase that is sensitive to NC-1300-B, SCH28080, omeprazole, and N-ethylmaleimide (NEM). This study examines the effects of K(+)-H(+)-ATPase inhibitors on VSMC. Rubidium uptake by primary cultures of canine coronary artery (CCA) VSMC or cultured rat aortic (CRA) VSMC line A7r5 was reduced 19-37% by NC-1300-B, SCH28080, or omeprazole. N-ethylmaleimide reduced CCA VSMC K+ content from 1.55 +/- 0.02 to 1.24 +/- 0.06 mu eq/mg protein. The NC-1300-B-sensitive portion of CRA VSMC Rb+ uptake was not blocked by ouabain (0.1 mM) or bumetanide (0.1 mM), but was reduced by alkalinization with 7.5 mM NH4Cl, and increased by acidification with 7.5 mM Na-acetate. Intracellular pH (pHi) of CRA VSMC was reduced 0.14 +/- 0.03 U by NC-1300-B and 0.22 +/- 0.03 U by NEM. pHi of CCA VSMC was reduced 0.20 +/- 0.03 U by omeprazole (1 mM) and 0.20 +/- 0.03 U or 0.20 +/- 0.05 U by amiloride in the absence or presence of omeprazole, respectively. Fluorescence of 2',7'-bis(carboxyethyl)-5-(6')- carboxyethyl)-5-(6')-carboxyfluorescein due to excitation at 500:441 nm in rat aortic strips was reduced by 0.21 +/- 0.02 U by omeprazole and 0.22 +/- 0.03 U by K+ removal and increased by 0.21 +/- 0.06 U by K+ repletion. We conclude that VSMC possess a previously unknown Rb+ uptake mechanism. This newly discovered mechanism helps to maintain K+ gradient and pHi by extruding H+ in exchange for K+, and is presumably a K(+)-H(+)-ATPase similar to those described in other tissues.
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PMID:Evidence of a K(+)-H(+)-ATPase in vascular smooth muscle cells. 132 Mar 41

Previous studies suggest that enhancement of rubidium tracer (86Rb) lumen-to-bath efflux following removal of luminal Na is mediated in part by a Ba-insensitive pathway. To determine the role of a primary active K pump in this response, we examined the action of known inhibitors of H-K-ATPase (Sch 28080) and Na-K-ATPase (ouabain) on the 86Rb lumen-to-bath efflux coefficient (KRb). Luminal Sch 28080 (10 microM) significantly reduced KRb by 39 +/- 8.0% (P less than 0.05), whereas luminal ouabain (0.1 mM) reduced KRb by 10 +/- 14% (P = not significant), suggesting that a luminal H-K-ATPase mediates Rb efflux. To examine whether H-K-ATPase mediates Rb in KRb following removal of luminal Na, additional experiments were conducted to examine the effect of Sch 28080 on KRb in the presence and the absence of luminal Na. In the presence of luminal Na, 10 microM Sch 28080 reduced KRb by 15 +/- 5.0%. However, in the absence of luminal Na, 10 microM Sch 28080 decreased KRb by 48 +/- 8.2%. The percentage inhibition of KRb by Sch 28080 was significantly greater in the absence of luminal Na than in its presence (P less than 0.01), suggesting that the enhancement of KRb following removal of luminal Na is mediated in part by an H-K-ATPase pathway. In either case transepithelial voltage was not significantly altered. In contrast to the lack of effect of luminal ouabain, addition of 0.1 mM ouabain to the bath increased KRb (69.8 +/- 11.1 vs. 95.9 +/- 18.7 nm/s, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:H-K-ATPase enhancement of Rb efflux by cortical collecting duct. 132 55

Ultrafiltrate obtained by hemodialysis of patients with uremia who were not taking cardiac glycosides was used as a source of Na, K adenosine triphosphatase inhibitor for purification and further study. Inhibitory activity was measured by a linked-enzyme assay and by effect on rubidium 86 uptake in guinea pig aortic strips. Two approaches were used in purification: dialysis with a 500 dalton membrane followed by gel filtration with Sephadex G-25, and removal of protein by acidification and boiling followed by Sephadex G-10. The first procedure failed to separate the inhibitor from the salt fraction, whereas the second separated the inhibitor from the salt peak but resulted in partial coelution of the inhibitor with endogenous pyruvate, which interferes with the linked-enzyme assay. Pooled, concentrated G-10 elution fractions from the early part of the inhibitor peak, which were free of pyruvate, produced a dose-response relationship by enzymatic assay that was close to parallel with that for ouabain. Like ouabain, these fractions also inhibited 86Rb uptake in guinea pig aorta. Despite these properties, our previous work has demonstrated that the inhibitor, unlike some other ouabain-like or digitalis-like substances obtained from blood or urine, has no apparent role in body fluid homeostasis.
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PMID:An Na, K ATPase inhibitor from ultrafiltrate obtained by hemodialysis of patients with uremia. 132 35

A 6.5-kilobase murine genomic DNA fragment isolated by Levenson et al. (Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1489-1493) (called the ouabain resistance gene) has been shown to produce ouabain resistance in primate cells. Preliminary sequence information has revealed no homology with the coding sequence of the Na,K-ATPase. We have introduced this murine sequence into monkey and murine cells in an attempt to characterize its mechanism of action. In our experiments, transfection of this DNA fragment is associated with the low frequency (1 in 8 x 10(5) cells) appearance of ouabain-resistant clones of CV1, COS, and NIH 3T3 cells, an event not seen in control transfections. Characterization of a new clone of ouabain-resistant CV1 cells (called OR8 cells) revealed a 5-fold increase in the IC50 for ouabain inhibition of rubidium uptake and a 10-fold increase in cell survival on ouabain. Although the murine sequence was detectable in Southern blots of ouabain-resistant cells soon after transfection, this exogenous DNA was rapidly lost despite continued exposure to ouabain. Furthermore, we were unable to detect message expression by this genomic sequence in any of the three cell types tested. Instead, we found that all three ouabain-resistant cell lines exhibited point mutations in a domain of the alpha-subunit that has been implicated in ouabain sensitivity (H1-H2). One of these mutations (Asp121-Asn121 in OR8 cells) has been previously reported to cause ouabain resistance (Price, E.M., Rice, D.A., and Lingrel, J.B. (1989) J. Biol. Chem. 264, 21902-21906). Other novel mutations in the H2 transmembrane domain were also detected. We postulate that the "ouabain resistance gene" is important in the early selection process on ouabain but that the permanent ouabain-resistant phenotype is due to a stable mutation in one allele of the alpha-subunit of the Na,K-ATPase.
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PMID:Ouabain-resistant transfectants of the murine ouabain resistance gene contain mutations in the alpha-subunit of the Na,K-ATPase. 132 28

Cystinosis is an inherited metabolic disease characterized by accumulation of lysosomal cystine and renal impairment. In an attempt to better understand the link between cystine accumulation and renal functions, we studied the effects of cystine loading on the Na(+)-H+ antiporter and the sodium pump in renal epithelial cells (LLC-PK1) in culture. Incubation of LLC-PK1 with 1 mM cystine dimethyl ester (CDME) for 48 h caused lysosomal cystine loading and reduced by 22 +/- 2% the maximal velocity of sodium-hydrogen antiport with no significant change in the affinity of sodium for the transporter. Rubidium influx decreased to 46 +/- 5% of control. Ouabain binding experiments revealed a 10% reduction in the number of Na(+)-K(+)-ATPase units in the intact cells. Na(+)-K(+)-ATPase activity in the particulate fraction of the cells homogenate declined to 50 +/- 7.5% of controls. No significant change was observed in the activity of ouabain-insensitive phosphatases. The intracellular concentration of sodium increased from 20.6 +/- 3.7 to 64.8 +/- 10 mM, and potassium concentration decreased from 103 +/- 6 to 80 +/- 13 mM. In addition to the observed reduction in the sodium gradient and in agreement with the reduction in the intracellular potassium concentration, the membrane potential changed from -80.8 +/- 7.5 to -69.9 +/- 7.0 mV. The results suggest that intracellular accumulation of cystine is associated with reduction in the number and the activity of membrane transporters. The consequence of the changes in the activity of Na(+)-K(+)-ATPase is a reduction in the electrochemical forces that drive transport in the renal cells tested.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cystine dimethyl ester reduces the forces driving sodium-dependent transport in LLC-PK1 cells. 132 21

In this study we applied a method generally used for the study of Na+,K(+)-ATPase, as well as other systems of potassium transport, which makes use of a rubidium isotope (86Rb) as analogue of the potassium and is known as uptake of the 86Rb. This method proved to be particularly sensitive and versatile for kinetic studies of this pump system, allowing to assess possible alterations. Its application in the study of sodium and potassium transport in erythrocytes of uremic subjects in extracorporeal dialysis made it possible to reveal certain alterations due both to pump-dependent and pump-independent uptake. In fact, the results show the hypothesis of restoration of Na+,K(+)-pump activity for elimination during dialysis of one or more inhibitor present in the uremic plasma. Furthermore, a reduction in aspecific flows was noted which could be the result of more generalized damage of the membrane.
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PMID:[Uptake of 86Rb by human erythrocytes: modification of the method and applications]. 133 37

The expressions of mRNAs of hepatocyte growth factor (HGF) and its receptor (c-met) and its effects were examined in cultured renal epithelial cell lines (OK, LLCPK1, and MDCK cells) and rat mesangial cells in primary culture. Northern blot analysis revealed the presence of HGF mRNA in mesangial cells, but not in epithelial cells. c-met mRNA was detected in epithelial cells, but not in mesangial cells. HGF stimulated [3H]-thymidine incorporation (DNA synthesis) dose-dependently in OK and LLCPK1 cells, but not in MDCK and mesangial cells. Ouabaine sensitive rubidium uptake (Na,K-ATPase activity) was stimulated by 63% with HGF (10 ng/ml) treatment for 16hr in MDCK cells. The results suggest that HGF is produced in the kidney, at least in mesangial cells and works on epithelial cells to stimulate the proliferation and/or to modify cell functions in a paracrine manner.
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PMID:Hepatocyte growth factor is a paracrine factor for renal epithelial cells: stimulation of DNA synthesis and NA,K-ATPase activity. 137 Aug 95

Electron microprobe analysis on freeze-dried cryosections was used to determine the effect of the loop diuretics torasemide and furosemide on intracellular electrolyte concentrations in individual cells of the outer and inner stripe of the outer medulla and on cell rubidium uptake, the latter a measure of basolateral Na-K-ATPase activity. In addition, the organic osmolytes glycerophosphorylcholine (GPC), betaine, inositol and sorbitol in cortex, outer medulla and inner medulla were measured using HPLC. Both loop diuretics significantly reduced sodium and chloride concentrations and rubidium uptake in thick ascending limb cells, but did not affect sodium concentration or rubidium uptake in the proximal straight tubule (PST) cells or in the light or dark cells of the outer medullary collecting duct (OMCD). Chloride concentrations in these cells (that is, PST cells, OMCD light and dark cells) were lowered by loop diuretics, albeit less than in thick ascending limb cells. Administration of both loop diuretics for only 20 minutes was sufficient to significantly depress tissue concentrations of GPC, betaine, and myo-inositol in the outer medulla and of GPC, betaine and sorbitol at the papillary tip. These results indicate that loop diuretics, presumably by blocking apical sodium entry, decrease thick ascending limb cellular sodium concentration and, as a consequence, reduce Na-K-ATPase activity as assessed by cell rubidium uptake. Although this has been shown previously in in vitro preparations, the present study confirms this for the first time in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of loop diuretics on organic osmolytes and cell electrolytes in the renal outer medulla. 145 80


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