Journal articles on the topic '(Na+,K+)-dependent ATPase'
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1
Aizman, R. I., G. Celsi, L. Grahnquist, Z. M. Wang, Y. Finkel, and A. Aperia. "Ontogeny of K+ transport in rat distal colon." American Journal of Physiology-Gastrointestinal and Liver Physiology 271, no. 2 (August 1, 1996): G268—G274. http://dx.doi.org/10.1152/ajpgi.1996.271.2.g268.
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Infants need to retain more K+ than adults to avoid growth retardation. Since the K+ requirements are different in infants (I) and in adults (A), the mechanisms regulating K+ homeostasis should also be different. The colon plays an important role for the regulation of K+ homeostasis. Colonic K+ transport is bidirectional. In this study we have examined the development of colonic K+ transport with special reference to the contribution of different K(+)-transporting pathways. The net colonic K+ uptake, as determined by in vivo perfusion studies and by 86Rb uptake, was significantly higher in I than in A rats. In both I and A colon, approximately 80% of total 86Rb uptake was dependent on vanadate-sensitive P-type adenosinetriphosphatases (ATPases), but the contribution of these different ATPases changes during development. The activity of colonic Na(+)-K(+)-ATPase, measured as ouabain-sensitive Na(+)-dependent ATP hydrolysis and as 86Rb uptake, was lower in I than in A rats. In contrast, the activity of K(+)-ATPases located in apical membrane and measured as ouabain insensitive and SCH-28080 sensitive, as ouabain-sensitive Na(+)-independent ATP hydrolysis, and as 86Rb uptake was significantly higher in I than in A rats. The ratio between apically located K(+)-ATPases and basolateral Na(+)-K(+)-ATPase activities was almost 3.2-fold higher in I than in A colon. We identified with Northern blot the expression of the colonic H(+)-K(+)-ATPase and the Na(+)-K(+)-ATPase alpha-subunits. The alpha-mRNA expression of both ATPases was significantly higher in I than in A rats. The pH and K+ sensitivity of the ouabain-insensitive, SCH-28080-sensitive K(+)-ATPase was the same in I and A colons. In conclusion, the relative activity of apical K+ absorbing ATPases is higher in the I than in the A colon, which should aid infants in retaining K+.
2
Benders, A. A., J. A. Timmermans, A. Oosterhof, H. J. Ter Laak, T. H. M. S. M. van Kuppevelt, R. A. Wevers, and J. H. Veerkamp. "Deficiency of Na+/K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle and cultured muscle cells of myotonic dystrophy patients." Biochemical Journal 293, no. 1 (July 1, 1993): 269–74. http://dx.doi.org/10.1042/bj2930269.
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Since defective regulation of ion transport could initiate or contribute to the abnormal cellular function in myotonic dystrophy (MyD), Na+/K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase were examined in skeletal muscle and cultured skeletal muscle cells of controls and MyD patients. Na+/K(+)-ATPase was investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphate (3-O-MFPase). SR Ca(2+)-ATPase was analysed by e.l.i.s.a., Ca(2+)-dependent phosphorylation and its activities with ATP and 3-O-methylfluorescein phosphatase (3-O-MFP). In MyD muscle the K(+)-dependent 3-O-MFPase activity and the activity and concentration of SR Ca(2+)-ATPase were decreased by 40%. In cultured muscle cells from MyD patients the activities as well as the concentration of both Na+/K(+)-ATPase and SR Ca(2+)-ATPase were reduced by about 30-40%. The ouabain-binding constant and the molecular activities, i.e. catalytic-centre activities with ATP or 3-O-MFP, of Na+/K(+)-ATPase and SR Ca(2+)-ATPase were similar in muscle as well as in cultured cells from both controls and MyD patients. Thus the decreased activity of both ATPases in MyD muscle is caused by a reduction in the number of their molecules. To check whether the deficiency of ATP-dependent ion pumps is a general feature of the pathology of MyD, we examined erythrocytes from the same patients. In these cells the Ca2+ uptake rate and the Ca(2+)-ATPase activity were lower than in controls, but the Ca(2+)-ATPase concentration was normal. Thus the reduced Ca(2+)-ATPase activity is caused by a decrease in the molecular activity of the ion pump. The Na+/K(+)-ATPase activity is also lower in erythrocytes of MyD patients. It is concluded that the observed alterations in ion pumps may contribute to the pathological phenomena in the muscle and other tissues in patients with MyD.
3
Mathews, P. M., D. Claeys, F. Jaisser, K. Geering, J. D. Horisberger, J. P. Kraehenbuhl, and B. C. Rossier. "Primary structure and functional expression of the mouse and frog alpha-subunit of the gastric H(+)-K(+)-ATPase." American Journal of Physiology-Cell Physiology 268, no. 5 (May 1, 1995): C1207—C1214. http://dx.doi.org/10.1152/ajpcell.1995.268.5.c1207.
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The H(+)-K(+)-ATPase of the gastric parietal cells is responsible for the acidification of the stomach lumen. This heterodimeric protein belongs to the family of cation-translocating P-type ATPases, which includes the closely related Na(+)-ATPase. We have cloned the alpha-subunit cDNA of the Xenopus and murine gastric H(+)-K(+)-ATPase (alpha H-K). We have expressed Xenopus and murine alpha H-K along with the previously cloned gastric H(+)-K(+)-ATPase beta-subunit of rabbit (beta H-K) in Xenopus oocytes by cRNA injection. An antibody directed against the beta H-K coimmunoprecipitates under nondenaturing conditions the alpha H-K of both species, demonstrating assembly of the alpha/beta complex. Additionally, we demonstrate the presence of K(+)-transporting H(+)-K(+)-ATPase in the plasma membrane of oocytes by 86Rb- uptake. The H(+)-K(+)-ATPase-mediated K+ uptake was inhibited by the gastric H(+)-K(+)-ATPase inhibitor Sch-28080, but not by ouabain, and shows K(+)-dependent activation (K1/2 approximately 2 mM). Furthermore, H(+)-K(+)-ATPase-expressing oocytes show a Sch-28080 inhibitable proton extrusion. Our data indicate that the expressed H(+)-K(+)-ATPase behaves functionally in oocytes as in the gastric gland.
4
Missiaen, L., F. Wuytack, H. De Smedt, M. Vrolix, and R. Casteels. "AlF4- reversibly inhibits ‘P’-type cation-transport ATPases, possibly by interacting with the phosphate-binding site of the ATPase." Biochemical Journal 253, no. 3 (August 1, 1988): 827–33. http://dx.doi.org/10.1042/bj2530827.
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The only known cellular action of AlF4- is to stimulate the G-proteins. The aim of the present work is to demonstrate that AlF4- also inhibits ‘P’-type cation-transport ATPases. NaF plus AlCl3 completely and reversibly inhibits the activity of the purified (Na+ + K+)-ATPase (Na+- and K+-activated ATPase) and of the purified plasmalemmal (Ca2+ + Mg2+)-ATPase (Ca2+-stimulated and Mg2+-dependent ATPase). It partially inhibits the activity of the sarcoplasmic-reticulum (Ca2+ + Mg2+)-ATPase, whereas it does not affect the mitochondrial H+-transporting ATPase. The inhibitory substances are neither F- nor Al3+ but rather fluoroaluminate complexes. Because AlF4- still inhibits the ATPase in the presence of guanosine 5′-[beta-thio]diphosphate, and because guanosine 5′-[beta gamma-imido]triphosphate does not inhibit the ATPase, it is unlikely that the inhibition could be due to the activation of an unknown G-protein. The time course of inhibition and the concentrations of NaF and AlCl3 required for this inhibition differ for the different ATPases. AlF4- inhibits the (Na+ + K+)-ATPase and the plasmalemmal (Ca2+ + Mg2+)-ATPase noncompetitively with respect to ATP and to their respective cationic substrates, Na+ and Ca2+. AlF4- probably binds to the phosphate-binding site of the ATPase, as the Ki for inhibition of the (Na+ + K+)-ATPase and of the plasmalemmal (Ca2+ + Mg2+)-ATPase is shifted in the presence of respectively 5 and 50 mM-Pi to higher concentrations of NaF. Moreover, AlF4- inhibits the K+-activated p-nitrophenylphosphatase of the (Na+ + K+)-ATPase competitively with respect to p-nitrophenyl phosphate. This AlF4- –induced inhibition of ‘P’-type cation-transport ATPases warns us against explaining all the effects of AlF4- on intact cells by an activation of G-proteins.
5
Asano, Shinji, Satomi Hoshina, Yumi Nakaie, Toshiyuki Watanabe, Michihiko Sato, Yuichi Suzuki, and Noriaki Takeguchi. "Functional expression of putative H+-K+-ATPase from guinea pig distal colon." American Journal of Physiology-Cell Physiology 275, no. 3 (September 1, 1998): C669—C674. http://dx.doi.org/10.1152/ajpcell.1998.275.3.c669.
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A guinea pig cDNA encoding the putative colonic H+-K+-ATPase α-subunit (T. Watanabe, M. Sato, K. Kaneko, T. Suzuki, T. Yoshida, and Y. Suzuki; GenBank accession no. D21854 ) was functionally expressed in HEK-293, a human kidney cell line. The cDNA for the putative colonic H+-K+-ATPase was cotransfected with cDNA for either rabbit gastric H+-K+-ATPase or TorpedoNa+-K+-ATPase β-subunit. In both expressions, Na+-independent, K+-dependent ATPase (K+-ATPase) activity was detected in the membrane fraction of the cells, with a Michaelis-Menten constant for K+ of 0.68 mM. The expressed K+-ATPase activity was inhibited by ouabain, with its IC50 value being 52 μM. However, the activity was resistant to Sch-28080, an inhibitor specific for gastric H+-K+-ATPase. The ATPase was not functionally expressed in the absence of the β-subunits. Therefore, it is concluded that the cDNA encodes the catalytic subunit (α-subunit) of the colonic H+-K+-ATPase. Although the β-subunit of the colonic H+-K+-ATPase has not been identified yet, both gastric H+-K+-ATPase and Na+-K+-ATPase β-subunits were found to act as a surrogate for the colonic β-subunit for the functional expression of the ATPase. The present colonic H+-K+-ATPase first expressed in mammalian cells showed the highest ouabain sensitivity in expressed colonic H+-K+-ATPases so far reported (rat colonic in Xenopus oocytes had an IC 50 = 0.4–1 mM; rat colonic in Sf9 cells had no ouabain sensitivity).
6
Kraut, J. A., F. Starr, G. Sachs, and M. Reuben. "Expression of gastric and colonic H(+)-K(+)-ATPase in the rat kidney." American Journal of Physiology-Renal Physiology 268, no. 4 (April 1, 1995): F581—F587. http://dx.doi.org/10.1152/ajprenal.1995.268.4.f581.
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Enzymatic and microperfusion studies have indicated that an ATP-dependent H+/K+ exchange process is present in the collecting duct of the mammalian kidney. Immunochemical staining has also provided evidence for expression of a gastric-type H(+)-K+ adenosine triphosphatase (H(+)-K(+)-ATPase). Rat kidney mRNA was probed with use of the polymerase chain reaction (PCR) to determine the presence of an H(+)-K(+)-ATPase. cDNA made with mRNA isolated from the kidneys of rats maintained on a low-K diet was used as template in PCR reactions with primers encompassing the cDNA sequence of the alpha-subunit of the gastric H(+)-K(+)-ATPase and the 5' and 3' ends of the colonic H(+)-K(+)-ATPase. The resulting products, 300–700 bp in size, hybridized with probes directed against either the gastric or colonic sequences of the H(+)-K(+)-ATPase. Sequencing of the individual PCR products showed identity with the appropriate regions of the alpha-subunits of the gastric H(+)-K(+)-ATPase and colonic H(+)-K(+)-ATPase. These data indicate that the rat kidney expresses mRNAs encoding both gastric and colonic H(+)-K(+)-ATPases.
7
Crambert, Gilles, Ciming Li, Dirk Claeys, and Käthi Geering. "FXYD3 (Mat-8), a New Regulator of Na,K-ATPase." Molecular Biology of the Cell 16, no. 5 (May 2005): 2363–71. http://dx.doi.org/10.1091/mbc.e04-10-0878.
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Four of the seven members of the FXYD protein family have been identified as specific regulators of Na,K-ATPase. In this study, we show that FXYD3, also known as Mat-8, is able to associate with and to modify the transport properties of Na,K-ATPase. In addition to this shared function, FXYD3 displays some uncommon characteristics. First, in contrast to other FXYD proteins, which were shown to be type I membrane proteins, FXYD3 may have a second transmembrane-like domain because of the presence of a noncleavable signal peptide. Second, FXYD3 can associate with Na,K- as well as H,K-ATPases when expressed in Xenopus oocytes. However, in situ (stomach), FXYD3 is associated only with Na,K-ATPase because its expression is restricted to mucous cells in which H,K-ATPase is absent. Coexpressed in Xenopus oocytes, FXYD3 modulates the glycosylation processing of the β subunit of X,K-ATPase dependent on the presence of the signal peptide. Finally, FXYD3 decreases both the apparent affinity for Na+ and K+ of Na,K-ATPase.
8
Ono, S., J. Guntupalli, and T. D. DuBose. "Role of H(+)-K(+)-ATPase in pHi regulation in inner medullary collecting duct cells in culture." American Journal of Physiology-Renal Physiology 270, no. 5 (May 1, 1996): F852—F861. http://dx.doi.org/10.1152/ajprenal.1996.270.5.f852.
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Studies in inner medullary collecting duct (IMCD) cells in primary culture have proposed two mechanisms for Na(+)-independent hydrogen ion transport: an H(+)-adenosinetriphosphatase (H(+)-ATPase) and an H(+)-K(+)-ATPase. In the present study, we have employed two sources of IMCD cells, cells in primary culture derived from the terminal papilla of the Munich-Wistar rat (IMCDp) and an established murine cell line (mIMCD-3), to define the predominant mechanism(s) of Na(+)-independent intracellular pH (pHi) recovery in the IMCD. In confluent monolayers of IMCDp and mIMCD-3 cells, pHi was measured using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) following addition and withdrawal of NH4Cl. Removal of K+ completely abolished Na(+)-independent pHi recovery in both IMCDp (delta pHi/min = 0.039 +/- 0.006 to 0.005 +/- 0.003; P < 0.001) and in mIMCD-3 (delta pHi/min = 0.055 +/- 0.009 to -0.003 +/- 0.002; P < 0.001) cells, respectively. In mIMCD-3 cells, K(+)-dependent pHi recovery was abolished by either of two specific inhibitors of the H(+)-K(+)-ATPase, Sch-28080 (5 or 10 microM) or A-80915A (10 microM). In contrast, bafilomycin A1 (2.5 and 10 nM), an inhibitor of the H(+)-ATPase, failed to attenuate K(+)-dependent pHi recovery. Moreover, sequence verified mouse gastric and colonic alpha-H(+)-K(+)-ATPase probes hybridized to total RNA from mIMCD-3 cells. Based on these findings, we conclude that Na(+)-independent pHi recovery from an acid load in both IMCDp and mIMCD-3 cells in critically dependent on extracellular K(+)-That K(+)-dependent pHi recovery was inhibited by both Sch-28080 and A-80915A but not by bafilomycin A1 suggests that the predominant mechanism by which Na(+)-independent pHi recovery is accomplished in IMCD is through the H(+)-K(+)-ATPase. Expression of both gastric and colonic alpha-H(+)-K(+)-ATPase mRNA in mIMCD-3 cells suggests that one or both of these H(+)-K(+)-ATPases may be responsible for proton secretion in the IMCD.
9
Vujisic, Ljubica, Danijela Krstic, and Jovan Vucetic. "Chemical aspect of the influence of cobalt ions on atpase activity." Journal of the Serbian Chemical Society 65, no. 7 (2000): 507–15. http://dx.doi.org/10.2298/jsc0007507v.
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The influence of Co 2+ ions on the activities of Na+/K+-ATPase and Mg2+ -ATPase, enzymes from rat brain synaptic plasma membrane, was studied. The aim of this study was to investigate the inhibition of both ATPases activities byexposure tocobalt ions as a function of experimentally added CoSO4. The "free" Co2+ concentrations in the reaction mixturewere also calculated and discussed. CoSO4 induced a dose-dependent inhibition of both enzymes. The IC50 values of Co 2+, as calculated from the experimental curves, were 168 mM for Na+/K+-ATPase and 262 mMfor Mg 2+-ATPase, and for the recalculated free Co 2+ concentration 75.4 mM for Na+/K+-ATPase and 136 mM for Mg 2+-ATPase. The obtained linear Dixon's plot for Na+/K+-ATPase implies equilibium binding of cobalt with inhibitory sites on the enzyme. The kinetic parameters for both enzymes in presence and absence of CoSO4 were calculated from the experimental data. The results of the kinetic analysis show that inhibition of Na+/K+-ATPase induced by CoSO4 is non-competitive, and for Mg 2+-ATPase that there are two sites of different sensitivities or two different enzymes.
10
Reinhardt, J., M. Kosch, M. Lerner, H. Bertram, D. Lemke, and H. Oberleithner. "Stimulation of protein kinase C pathway mediates endocytosis of human nongastric H+-K+-ATPase, ATP1AL1." American Journal of Physiology-Renal Physiology 283, no. 2 (August 1, 2002): F335—F343. http://dx.doi.org/10.1152/ajprenal.00226.2001.
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The human nongastric H+-K+-ATPase, ATP1AL1, shown to reabsorb K+ in exchange for H+ or Na+, is localized in the luminal plasma membrane of renal epithelial cells. It is presumed that renal H+-K+-ATPases can be regulated by endocytosis. However, little is known about the molecular mechanisms that control plasma membrane expression of renal H+-K+-ATPases. In our study, activation of protein kinase C (PKC) using phorbol esters (phorbol 12-myristate 13-acetate) leads to clathrin-dependent internalization and intracellular accumulation of the ion pump in stably transfected Madin-Darby canine kidney cells. Functional inactivation of the H+-K+-ATPase by PKC activation is shown by intracellular pH measurements. Proton extrusion capacity of ATP1AL1-transfected cells is drastically reduced after phorbol 12-myristate 13-acetate incubation and can be prevented with the PKC blocker bisindolylmaleimide. Ion pump internalization and inactivation are specifically mediated by the PKC pathway, whereas activation of the protein kinase A pathway has no influence. Our results show that the nongastric H+-K+-ATPase is a specific target for the PKC pathway. Therefore, PKC-mediated phosphorylation is a potential regulatory mechanism for apical nongastric H+-K+-ATPase plasma membrane expression.
11
Crambert, Gilles, Jean-Daniel Horisberger, Nikolai N. Modyanov, and Käthi Geering. "Human nongastric H+-K+-ATPase: transport properties of ATP1al1 assembled with different β-subunits." American Journal of Physiology-Cell Physiology 283, no. 1 (July 1, 2002): C305—C314. http://dx.doi.org/10.1152/ajpcell.00590.2001.
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To investigate whether nongastric H+-K+-ATPases transport Na+ in exchange for K+ and whether different β-isoforms influence their transport properties, we compared the functional properties of the catalytic subunit of human nongastric H+-K+-ATPase, ATP1al1 (AL1), and of the Na+-K+-ATPase α1-subunit (α1) expressed in Xenopus oocytes, with different β-subunits. Our results show that βHK and β1-NK can produce functional AL1/β complexes at the oocyte cell surface that, in contrast to α1/β1 NK and α1/βHK complexes, exhibit a similar apparent K+ affinity. Similar to Na+-K+-ATPase, AL1/β complexes are able to decrease intracellular Na+ concentrations in Na+-loaded oocytes, and their K+ transport depends on intra- and extracellular Na+ concentrations. Finally, controlled trypsinolysis reveals that β-isoforms influence the protease sensitivity of AL1 and α1 and that AL1/β complexes, similar to the Na+-K+-ATPase, can undergo distinct K+-Na+- and ouabain-dependent conformational changes. These results provide new evidence that the human nongastric H+-K+-ATPase interacts with and transports Na+ in exchange for K+ and that β-isoforms have a distinct effect on the overall structural integrity of AL1 but influence its transport properties less than those of the Na+-K+-ATPase α-subunit.
12
McBride, B. W., and L. P. Milligan. "Influence of feed intake and starvation on the magnitude of Na+,K+-ATPase(EC 3.6.1.3)-dependent respiration in duodenal mucosa of sheep." British Journal of Nutrition 53, no. 3 (May 1985): 605–14. http://dx.doi.org/10.1079/bjn19850070.
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1. Oxygen consumption and Na+,K+-ATPase(EC 3.6.1.3)-dependent (ouabain-sensitive) and -independent respiration were measured for duodenal mucosa biopsies from 10-month-old sheep given two levels of digestible energy (DE) intake (7.6–7.7 and 14.8 MJ lucerne (Medicago sativa) pellets/d) and following 48 h of starvation.2. The mucosal biopsies were determined to be structurally intact and free of adherent bacteria on histological and scanning-electron-microscope examinations.3. The use of D-glucose as a substrate during incubations did not elevate (P > 0.05) the respiration indices of the biopsies over those measured during acetate incubations.4. Glucose uptake did not (P > 0.05) influence the Na+,K+-ATPase-dependent respiration of the mucosal biopsies.5. Na+,K+-ATPase-dependent respiration accounted for 50% of the total O2, consumption of the mucosal biopsies of sheep given the lower level of DE.6. Total O2, consumption of the duodenalmucosa was not (P > 0.05) increased when sheep were given the higher level of DE but Na+,K+-ATPase-dependent respiration of the mucosa was elevated (P < 0.01) by 37% during this period.7. When sheep were starved for 48 h, total O2, consumption of the mucosal biopsies was not (P > 0.05) affected, however, Na+,K+-ATPase-dependent respiration of the biopsies dropped (P < 0.01) by 45%.8. Na+,K+-ATPase-dependknt respiration accounted for 61.3% of the O2, uptakes of mucosa from the sheep given the higher level of DE and 28.3% of the 02, uptake of mucosa from fasted sheep.
13
Sabolic, I., D. Brown, J. M. Verbavatz, and J. Kleinman. "H(+)-ATPases of renal cortical and medullary endosomes are differentially sensitive to Sch-28080 and omeprazole." American Journal of Physiology-Renal Physiology 266, no. 6 (June 1, 1994): F868—F877. http://dx.doi.org/10.1152/ajprenal.1994.266.6.f868.
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Adenosinetriphosphatase (ATPase) activity stimulated by K+ and inhibited by Sch-28080 (SCH), omeprazole (OME), and vanadate has been measured in microsomes from mammalian renal medulla and attributed to a kidney isoform of the H(+)-K(+)-ATPase. To determine whether the H(+)-K(+)-ATPase inhibitors could also inhibit the vacuolar (V)-type H(+)-adenosinetriphosphatase (H(+)-ATPase, i.e., H+ pump) in mammalian intracellular vesicles, we examined their effects on bafilomycin-sensitive acidification in renal cortical vesicles (CEV) and medullary endocytic vesicles (MEV). Rats were injected with fluorescein isothiocyanate-labeled dextran, and labeled endosomes were enriched from kidney tissue homogenates by differential and Percoll density gradient centrifugation. In the CEV, the V-type H+ pump was inhibited 25% by SCH and 30% by OME (100 microM each). Whereas the inhibition by OME was concentration and time dependent, the inhibition by SCH was only concentration dependent. Inhibition by these compounds was similar in the presence of 50 mM K+ (in = out) and in the complete absence of K+, thus ruling out a significant involvement of H(+)-K(+)-ATPase-mediated acidification. Inhibition, however, was not observed with 10 microM SCH and OME. The sensitivity of the V-type H+ pump to 100 microM SCH and OME in CEV was confirmed by the comparable inhibitions of intravesicular acidification observed in acridine orange fluorescence quench studies and by inhibition of Pi liberation in an ATPase assay. We also found that the V-type H+ pump in isolated rat liver endosomes is sensitive to 100 microM SCH and OME to a similar degree. In the MEV, acidification was only weakly affected by 100 microM SCH and OME, thus suggesting that H(+)-ATPases in endosomes from cortical and medullary tubules are different, possibly due to a previously described selective expression of subunit isoforms. Our finding indicates the importance of using low concentrations (< 10 microM) of OME and SCH in studies of H(+)-K(+)-ATPase in nongastric tissues to avoid misinterpretation of the data due to nonspecific inhibition of V-type H(+)-ATPases.
14
Modyanov, N. N., P. M. Mathews, A. V. Grishin, P. Beguin, A. T. Beggah, B. C. Rossier, J. D. Horisberger, and K. Geering. "Human ATP1AL1 gene encodes a ouabain-sensitive H-K-ATPase." American Journal of Physiology-Cell Physiology 269, no. 4 (October 1, 1995): C992—C997. http://dx.doi.org/10.1152/ajpcell.1995.269.4.c992.
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The cDNA for ATP1AL1, the fifth member of the human Na-K-adenosinetriphosphatase (ATPase)/H-K-ATPase gene family, was recently cloned (A. V. Grishin, V. E. Sverdlov, M. B. Kostina, and N. N. Modyanov. FEBS Lett. 349: 144-150, 1994). The encoded protein (ATP1AL1) has all the primary structural features common to the catalytic alpha-subunit of ion-transporting P-type ATPases and is similar (63-64% identity) to the Na-K-ATPase alpha-subunit isoforms and the gastric H-K-ATPase alpha-subunit. In this study, ATP1AL1 was expressed in Xenopus laevis oocytes in combination with the beta-subunit of rabbit gastric H-K-ATPase. The functional properties of the stable alpha/beta-complex were studied by 86Rb+ uptake and demonstrated that ATP1AL1 is a novel human K(+)-dependent ATPase [apparent half-constant activation/(K1/2) for K+ approximately 375 microM)]. ATP1AL1-mediated inward K+ transport was inhibited by ouabain (inhibition constant approximately 13 microM) and was found to be inhibited by high concentrations of SCH-28080 (approximately 70% at 500 microM). ATP1AL1 expression resulted in the alkalinization of the oocytes' cytoplasm and ouabain-sensitive proton extrusion, as measured with pH-sensitive microelectrodes. These data argue that ATP1AL1 is the catalytic alpha-subunit of a human nongastric P-type ATPase capable of exchanging extracellular potassium for intracellular protons.
15
Feraille, E., M. L. Carranza, B. Buffin-Meyer, M. Rousselot, A. Doucet, and H. Favre. "Protein kinase C-dependent stimulation of Na(+)-K(+)-ATP epsilon in rat proximal convoluted tubules." American Journal of Physiology-Cell Physiology 268, no. 5 (May 1, 1995): C1277—C1283. http://dx.doi.org/10.1152/ajpcell.1995.268.5.c1277.
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In rat proximal convoluted tubule (PCT), activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) was previously reported to inhibit Na(+)-K(+)-ATPase, a paradoxical finding in view of the known stimulatory effect of PKC on Na+ reabsorption. Because this inhibition occurs via phospholipase A2 activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on PKC action on Na(+)-K(+)-ATPase. Results confirmed that PDBu inhibited PCT Na(+)-K(+)-ATPase activity under usual conditions. In contrast, when oxygen supply was increased, PDBu had no effect on Na(+)-K(+)-ATPase hydrolytic activity, but it dose-dependently stimulated ouabain-sensitive 86Rb+ uptake. This latter effect, which was abolished by PKC inhibitors, resulted from an increment of the Na+ sensitivity of Na(+)-K(+)-ATPase. Thus, in oxygenated rat PCTs, activation of PKC primarily stimulated Na(+)-K(+)-ATPase. This likely contributes to increase solute reabsorption. Inhibition of Na(+)-K(+)-ATPase was observed only under hypoxic conditions. It may represent an adaptation to protect PCTs against deleterious effects of hypoxia.
16
Haley, C., and M. Donnell. "K+ reabsorption by the lower Malpighian tubule of Rhodnius prolixus: inhibition by Ba2+ and blockers of H+/K+-ATPases." Journal of Experimental Biology 200, no. 1 (January 1, 1997): 139–47. http://dx.doi.org/10.1242/jeb.200.1.139.
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Active K+ reabsorption by the lower Malpighian tubule of the blood-feeding hemipteran Rhodnius prolixus does not involve the amiloride-sensitive K+/H+ exchangers or V-type H+-ATPases implicated in secretion of ions from haemolymph to lumen in the upper tubule. Amiloride, N-ethylmaleimide, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol and bafilomycin A1 inhibit haemolymph-to-lumen secretion of Na+ and K+ by the upper Malpighian tubule, but have little or no effect on lumen-to-haemolymph reabsorption of K+ by the lower tubule. The effects of inhibitors of H+/K+-ATPases, including omeprazole and SCH 28080, suggest that a pump similar to the H+/K+-ATPase of the gastric mucosa is involved in KCl reabsorption. The presence of K+ channels in the basolateral membrane in the lower Malpighian tubule is suggested by inhibition of KCl reabsorption by basolateral but not apical application of the K+ channel blocker Ba2+, and by blockade of K+-dependent changes in membrane potential by Ba2+. It is proposed, therefore, that K+ is pumped from lumen to cell by an ATP-dependent pump resembling the H+/K+-ATPase of the gastric mucosa, and that K+ leaks from cell to bathing saline (haemolymph) via an electrodiffusive pathway (i.e. K+ channels).
17
Rajendran, Vazhaikkurichi M., Satish K. Singh, John Geibel, and Henry J. Binder. "Differential localization of colonic H+-K+-ATPase isoforms in surface and crypt cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 274, no. 2 (February 1, 1998): G424—G429. http://dx.doi.org/10.1152/ajpgi.1998.274.2.g424.
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Two distinct colonic H+-K+-adenosinetriphosphatase (H+-K+-ATPase) isoforms can be identified in part on the basis of their sensitivity to ouabain. The colonic H+-K+-ATPase α-subunit (HKcα) was recently cloned, and its message and protein are present in surface (and the upper 20% of crypt) cells in the rat distal colon. These studies were performed to establish the spatial distribution of the ouabain-sensitive and ouabain-insensitive components of both H+-K+-ATPase activity in apical membranes prepared from surface and crypt cells and K+-dependent intracellular pH (pHi) recovery from an acid load both in isolated perfused colonic crypts and in surface epithelial cells. Whereas H+-K+-ATPase activity in apical membranes from surface cells was 46% ouabain sensitive, its activity in crypt apical membranes was 96% ouabain sensitive. Similarly, K+-dependent pHi recovery in isolated crypts was completely ouabain sensitive, whereas in surface cells K+-dependent pHi recovery was insensitive to ouabain. These studies provide compelling evidence that HKcα encodes the colonic ouabain-insensitive H+-K+-ATPase and that a colonic ouabain-sensitive H+-K+-ATPase isoform is present in colonic crypts and remains to be cloned and identified.
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Van der Hijden, H. T. W. M., S. Kramer-Schmitt, E. Grell, and J. J. H. H. M. de Pont. "The basal Mg2+-dependent ATPase activity is not part of the (H++K+)-transporting ATPase reaction cycle." Biochemical Journal 267, no. 3 (May 1, 1990): 565–72. http://dx.doi.org/10.1042/bj2670565.
Full textAbstract:
Purified gastric (H(+)+K+)-transporting ATPase [(H(+)+K+)-ATPase] from the parietal cells always contains a certain amount of basal Mg2(+)-dependent ATPase (Mg2(+)-ATPase) activity. lin-Benzo-ATP (the prefix lin refers to the linear disposition of the pyrimidine, benzene and imidazole rings in the ‘stretched-out’ version of the adenine nucleus), an ATP analogue with a benzene ring formally inserted between the two rings composing the adenosine moiety, is an interesting substrate not only because of its fluorescent behaviour, but also because of its geometric properties. lin-Benzo-ATP was used in the present study to elucidate the possible role of the basal Mg2(+)-ATPase activity in the gastric (H(+)+K+)-ATPase preparation. With lin-benzo-ATP the enzyme can be phosphorylated such that a conventional phosphoenzyme intermediate is formed. The rate of the phosphorylation reaction, however, is so low that this reaction with subsequent dephosphorylation cannot account for the much higher rate of hydrolysis of lin-benzo-ATP by the enzyme. This apparent kinetic discrepancy indicates that lin-benzo-ATP is not a substrate for the (H(+)+K+)-ATPase reaction cycle. This idea was further supported by the finding that lin-benzo-ATP was unable to catalyse H+ uptake by gastric-mucosa vesicles. The breakdown of lin-benzo-ATP by the (H(+)+K+)-ATPase preparation must be due to a hydrolytic activity which is not involved in the ion-transporting reaction cycle of the (H(+)+K+)-ATPase itself. Comparison of the basal Mg2(+)-ATPase activity (with ATP as substrate) with the hydrolytic activity of (H(+)+K+)-ATPase using lin-benzo-ATP as substrate and the effect of the inhibitors omeprazole and SCH 28080 support the notion that lin-benzo-ATP is not hydrolysed by the (H(+)+K+)-ATPase, but by the basal Mg2(+)-ATPase, and that the activity of the latter enzyme is not involved in the (H(+)+K+)-transporting reaction cycle (according to the Albers-Post formalism) of (H(+)+K+)-ATPase.
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Marciniak, Andrzej, Anna Jamroz-Wiśniewska, Ewelina Borkowska, and Jerzy Bełtowski. "Time-dependent effect of leptin on renal Na+,K+-ATPase activity." Acta Biochimica Polonica 52, no. 4 (August 4, 2005): 803–10. http://dx.doi.org/10.18388/abp.2005_3392.
Full textAbstract:
Leptin, secreted by adipose tissue, is involved in the pathogenesis of arterial hypertension, however, the mechanisms through which leptin increases blood pressure are incompletely elucidated. We investigated the effect of leptin, administered for different time periods, on renal Na(+),K(+)-ATPase activity in the rat. Leptin was infused under anesthesia into the abdominal aorta proximally to the renal arteries for 0.5-3 h. Leptin administered at doses of 1 and 10 microg/min per kg for 30 min decreased the Na(+),K(+)-ATPase activity in the renal medulla. This effect disappeared when the hormone was infused for > or =1 h. Leptin infused for 3 h increased the Na(+),K(+)-ATPase activity in the renal cortex and medulla. The stimulatory effect was abolished by a specific inhibitor of Janus kinases (JAKs), tyrphostin AG490, as well as by an NAD(P)H oxidase inhibitor, apocynin. Leptin increased urinary excretion of hydrogen peroxide (H(2)O(2)) between 2 and 3 h of infusion. The effect of leptin on renal Na(+),K(+)-ATPase and urinary H(2)O(2) was augmented by a superoxide dismutase mimetic, tempol, and was abolished by catalase. In addition, infusion of H(2)O(2) for 30 min increased the Na(+),K(+)-ATPase activity. Inhibitors of extracellular signal regulated kinases (ERKs), PD98059 or U0126, prevented Na(+),K(+)-ATPase stimulation by leptin and H(2)O(2). These data indicate that leptin, by acting directly within the kidney, has a delayed stimulatory effect on Na(+),K(+)-ATPase, mediated by JAKs, H(2)O(2) and ERKs. This mechanism may contribute to the abnormal renal Na(+) handling in diseases associated with chronic hyperleptinemia such as diabetes and obesity.
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Lei, Jianxun, Cary N. Mariash, Maneesh Bhargava, Elizabeth V. Wattenberg, and David H. Ingbar. "T3 increases Na-K-ATPase activity via a MAPK/ERK1/2-dependent pathway in rat adult alveolar epithelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 294, no. 4 (April 2008): L749—L754. http://dx.doi.org/10.1152/ajplung.00335.2007.
Full textAbstract:
Thyroid hormone (T3) increases Na-K-ATPase activity in rat adult alveolar type II cells via a PI3K-dependent pathway. In these cells, dopamine and β-adrenergic agonists can stimulate Na-K-ATPase activity through either PI3K or MAPK pathways. We assessed the role of the MAPK pathway in the stimulation of Na-K-ATPase by T3. In the adult rat alveolar type II-like cell line MP48, T3 enhanced MAPK/ERK1/2 activity in a dose-dependent manner. Increased ERK1/2 phosphorylation was observed within 5 min, peaked at 20 min, and then decreased. Two MEK1/2 inhibitors, U0126 and PD-98059, each abolished the T3-induced increase in the quantity of Na-K-ATPase α1-subunit plasma membrane protein and Na-K-ATPase activity. T3 also increased the phosphorylation of MAPK/p38; however, SB-203580, a specific inhibitor of MAPK/p38 activity, did not prevent the T3-induced Na-K-ATPase activity. SP-600125, a specific inhibitor of the MAPK/JNK pathway, also did not block the T3-induced Na-K-ATPase activity. Phorbol 12-myristate 13-acetate (PMA) significantly increased ERK1/2 phosphorylation and Na-K-ATPase activity. The PMA-induced Na-K-ATPase activity was inhibited by U0126. These data indicate that activation of MAPK-ERK1/2 was required for the T3-induced increase in Na-K-ATPase activity in addition to the requirement for the PI3K pathway.
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Carranza, M. L., E. Feraille, and H. Favre. "Protein kinase C-dependent phosphorylation of Na(+)-K(+)-ATPase alpha-subunit in rat kidney cortical tubules." American Journal of Physiology-Cell Physiology 271, no. 1 (July 1, 1996): C136—C143. http://dx.doi.org/10.1152/ajpcell.1996.271.1.c136.
Full textAbstract:
We have previously shown that, in oxygenated rat kidney proximal convoluted tubules (PCT), activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) directly stimulates Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity. PKC modulation of Na(+)-K(+)-ATPase activity by phosphorylation of its alpha-subunit was the postulated mechanism. The present study was therefore designed to investigate the relationship between PKC-mediated phosphorylation of the catalytic alpha-subunit and the cation transport activity of the Na(+)-K(+)-ATPase. In a suspension of rat kidney cortical tubules, activation of PKC by 10(-7) M PDBu increased the level of phosphorylation of the Na(+)-K(+)-ATPase alpha-subunit and stimulated the ouabain-sensitive 86Rb uptake by 47 and 42%, respectively. Time and dose dependence of the PDBu-induced increase in Na(+)-K(+)-ATPase activity and phosphorylation was strongly linearly correlated. The effects of PDBu on phosphorylation and activity of Na(+)-K(+)-ATPase were prevented by GF-109203X, a specific PKC inhibitor, whereas H-89, a specific PKA inhibitor, was ineffective. These results demonstrate that PKC activation induces phosphorylation of the catalytic alpha-subunit of Na(+)-K(+)-ATPase, which may participate in the stimulation of its cation transport activity in the rat PCT.
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Casare, Fernando, Daiane Milan, and Ricardo Fernandez. "Stimulation of calcium-sensing receptor increases biochemical H+-ATPase activity in mouse cortex and outer medullary regions." Canadian Journal of Physiology and Pharmacology 92, no. 3 (March 2014): 181–88. http://dx.doi.org/10.1139/cjpp-2013-0256.
Full textAbstract:
The aim of this project was to investigate the interaction between the calcium-sensing receptor (CaSR) and proton extrusion by the V-ATPase and gastric-like isoform of the H+/K+-ATPase in the mouse nephron. Biochemical activity of H+- ATPases was analysed using a partially purified membrane fraction of mouse cortex and outer medullary region. The V-ATPase activity (sensitive to 10−7 mol·L−1 bafilomycin) from the cortical and outer medullary region was significantly stimulated by increasing the [Formula: see text] (outside Ca2+), in a dose-dependent pattern. Gastric H+/K+-ATPase activity (sensitive to 10−5 mol·L−1 Schering 28080) was also sensitive to changes in [Formula: see text] levels. A significant increase in V-ATPase activity was also observed when CaSR was stimulated with agonists such as 300 μmol·L−1 Gd3+ and 200 μmol·L−1 neomycin, both in the cortex and outer medulla. The cortical and outer medullary gastric H+/K+-ATPase activity was also stimulated by Gd3+ and neomycin. Finally, cortical V-ATPase activity was significantly stimulated by 10−9 mol·L−1 angiotensin II, and the stimulation of CaSR in the presence of angiotensin significantly enhanced this effect, suggesting that an interaction in the intracellular signaling pathways is involved. In summary, CaSR stimulation enhances the biochemical activity of V-ATPase and gastric H+/K+-ATPase in both the cortical and outer medullary region of mouse kidney.
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Urushidani, T., and J. G. Forte. "Stimulation-associated redistribution of H+-K+-ATPase activity in isolated gastric glands." American Journal of Physiology-Gastrointestinal and Liver Physiology 252, no. 4 (April 1, 1987): G458—G465. http://dx.doi.org/10.1152/ajpgi.1987.252.4.g458.
Full textAbstract:
The objective of this work is to establish a procedure to study the stimulation-dependent membrane redistribution and properties of H+-K+-ATPase in an in vitro model system, rabbit isolated gastric glands. Stimulated (10(-4) M histamine plus 10(-5) M forskolin) and resting (10(-4) M metiamide) glands were homogenized and fractionated into PO (40 g, 5 min), P1 (400 g, 10 min), P2 (14,500 g, 10 min), P3 (48,200 g, 90 min), and supernatant, S3. Significant changes occurred in the distribution of our marker for H+-K+-ATPase (K+-p-nitrophenyl phosphatase) activity: a reduction in activity of P3 and a compensatory increment in P1. P3 showed valinomycin (Val)-dependent vesicular H+ uptake, while H+ uptake in P1 was Val independent. Direct measurements of ATPase revealed that H+-K+-ATPase activity of P3 was Val dependent and decreased by stimulation; H+-K+-ATPase activity of P1 was Val independent and increased by stimulation. Further density gradient purification of P1 showed that membranes lighter than 17% Ficoll contained higher specific H+-K+-ATPase activity, and the observed increase in H+-K+-ATPase associated with stimulation was more pronounced. Also, the lighter fractions from stimulated P1 had much latent H+-K+-ATPase activity that was unmasked by n-octylglucoside. The properties of membrane fractions from isolated glands were consistent with results obtained in vivo: high H+-K+-ATPase activity of P3 from resting glands corresponds to cytoplasmic tubulovesicles lacking KCl transport pathways; high activity of P1 from stimulated glands corresponds to apical plasma membrane vesicles containing KCl transport in addition to the H+-K+-ATPase, and full competency for the generation of HCl.
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Alves, Daiane S., Glen A. Farr, Patricia Seo-Mayer, and Michael J. Caplan. "AS160 Associates with the Na+,K+-ATPase and Mediates the Adenosine Monophosphate-stimulated Protein Kinase-dependent Regulation of Sodium Pump Surface Expression." Molecular Biology of the Cell 21, no. 24 (December 15, 2010): 4400–4408. http://dx.doi.org/10.1091/mbc.e10-06-0507.
Full textAbstract:
The Na+,K+-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na+,K+-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na+,K+-ATPase binding partners revealed a direct association between the Na+,K+-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na+,K+-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na+,K+-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na+,K+-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na+,K+-ATPase.
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Anner, B. M., M. Moosmayer, and E. Imesch. "Mercury blocks Na-K-ATPase by a ligand-dependent and reversible mechanism." American Journal of Physiology-Renal Physiology 262, no. 5 (May 1, 1992): F830—F836. http://dx.doi.org/10.1152/ajprenal.1992.262.5.f830.
Full textAbstract:
An inhibitory receptor for cardioactive steroids such as digoxin and ouabain is located at the extracellular surface of the Na-K-adenosinetriphosphatase (ATPase) molecule. Besides cardioactive steroids, mercury is a potent inhibitor of the Na-K-ATPase activity. The half-maximal inhibitory concentration (IC50), determined within 30 min at 37 degrees C at 1 microgram protein/ml, was 200 nM, despite the presence of 1 mM EDTA; the IC50 decreased with increasing protein/inhibitor ratio, and it reached 2.7 microM at 0.1 mg protein/ml and 20 microM at 1 mg protein/ml. The IC50 for Na-K-ATPase inhibition by the diuretic compound mersalyl was 4 and 5 microM for the nondiuretic p-chloromercuribenzenesulfonic acid at 0.1 mg protein/ml. The IC50 for HgCl2 inhibition was modulated by the presence of EDTA as well as by the pump ligands Mg, Na, K, and ATP. The E2 conformation of the Na-K-ATPase molecule was more sensitive to HgCl2 than the E1 conformation. The mercury antidote 2,3-dimercapto-1-propanesulfonic acid was able to reactivate approximately 70% of the blocked enzyme. In conclusion, a metal-binding domain of the Na-K-ATPase molecule with particular high affinity for Hg(II) was described functionally in the present work. Therefore Na-K-ATPase belongs to the metal-binding proteins. Metals may modulate the cellular expression and activity of the system by interacting with its metal-binding interface.
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Khundmiri, Syed J., Mohammed Ameen, Nicholas A. Delamere, and Eleanor D. Lederer. "PTH-mediated regulation of Na+-K+-ATPase requires Src kinase-dependent ERK phosphorylation." American Journal of Physiology-Renal Physiology 295, no. 2 (August 2008): F426—F437. http://dx.doi.org/10.1152/ajprenal.00516.2007.
Full textAbstract:
Parathyroid hormone (PTH) inhibits Na+-K+-ATPase activity by serine phosphorylation of the α1-subunit through ERK-dependent phosphorylation and translocation of protein kinase Cα (PKCα). On the basis of previous studies, we postulated that PTH regulates sodium pump activity through Src kinase, PLC, and calcium-dependent ERK phosphorylation. In the present work utilizing opossum kidney cells, a model of renal proximal tubule, PTH-stimulated ERK phosphorylation and membrane translocation of PKCα were prevented by inhibition of Src kinase, PLC, and calcium entry. Pharmacological inhibition of PLA2 did not prevent PTH-stimulated ERK phosphorylation but completely prevented PKCα translocation. Silencing the expression of cytosolic or calcium-independent PLA2 also prevented PTH-mediated phosphorylation of Na+-K+-ATPase α1-subunit and PKCα without blocking ERK phosphorylation. Inhibition of Na+-K+-ATPase activity by the PLA2 metabolites arachidonic acid and 20-hydroxyeicosatetraenoic acid was prevented by specific inhibition of PKCα but not by U0126, a MEK-1 inhibitor. Transient transfection of constitutively active MEK-1 cDNA induced phosphorylation of Na+-K+-ATPase α1-subunit and PKCα, which was prevented by PLA2 inhibition. We conclude that PTH stimulates Na+-K+-ATPase phosphorylation and decreases the activity of Na+-K+-ATPase by a sequential activation of a signaling pathway involving Src kinase, PLC, ERK, PLA2, and PKCα.
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Holthouser, Kristine A., Amritlal Mandal, Michael L. Merchant, Jeffrey R. Schelling, Nicholas A. Delamere, Ronald R. Valdes, Suresh C. Tyagi, Eleanor D. Lederer, and Syed J. Khundmiri. "Ouabain stimulates Na-K-ATPase through a sodium/hydrogen exchanger-1 (NHE-1)-dependent mechanism in human kidney proximal tubule cells." American Journal of Physiology-Renal Physiology 299, no. 1 (July 2010): F77—F90. http://dx.doi.org/10.1152/ajprenal.00581.2009.
Full textAbstract:
Recent investigations demonstrate increased Na/H exchanger-1 (NHE-1) activity and plasma levels of ouabain-like factor in spontaneously hypertensive rats. At nanomolar concentrations, ouabain increases Na-K-ATPase activity, induces cell proliferation, and activates complex signaling cascades. We hypothesize that the activity of NHE-1 and Na-K-ATPase are interdependent. To test whether treatment with picomolar ouabain regulates Na-K-ATPase through an NHE-1-dependent mechanism, we examined the role of NHE-1 in ouabain-mediated stimulation of Na-K-ATPase in kidney proximal tubule cell lines [opossum kidney (OK), HK-2, HKC-5, and HKC-11] and rat kidney basolateral membranes. Ouabain stimulated Na-K-ATPase activity and tyrosine phosphorylation in cells that express NHE-1 (OK, HKC-5, and HKC-11) but not in HK-2 cells that express very low levels of NHE-1. Inhibition of NHE-1 with 5 μM EIPA, a NHE-1-specific inhibitor, prevented ouabain-mediated stimulation of86Rb uptake and Na-K-ATPase phosphorylation in OK, HKC-5, and HKC-11 cells. Expression of wild-type NHE-1 in HK2 cells restored regulation of Na-K-ATPase by picomolar ouabain. Treatment with picomolar ouabain increased membrane expression of Na-K-ATPase and enhanced NHE-1-Na-K-ATPase α1-subunit association. Treatment with ouabain (1 μg·kg body wt−1·day−1) increased Na-K-ATPase activity, expression, phosphorylation, and association with NHE-1 increased in rat kidney cortical basolateral membranes. Eight days' treatment with ouabain (1 μg·kg body wt−1·day−1) resulted in increased blood pressure in these rats. These results suggest that the association of NHE-1 with Na-K-ATPase is critical for ouabain-mediated regulation of Na-K-ATPase and that these effects may play a role in cardioglycoside-stimulated hypertension.
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Akopova, O. V., O. N. Kharlamova, A. V. Kotsiuruba, Yu P. Korkach, and V. F. Sagach. "THE STUDY OF NITRIC OXIDE ACTION IN VIVO ON NA+ , K+ -ÀÒPASE IN RAT AORTA." Fiziolohichnyĭ zhurnal 55, no. 1 (February 4, 2009): 27–35. http://dx.doi.org/10.15407/fz55.01.027.
Full textAbstract:
The influence of nitric oxide on Na+,K+-ATPase activity in rat aorta was studied by means of stimulation of endogenous NO synthesis after injections of bacterial lipopolysaccharide (LPS) and pharmacological NO donor nitroglycerine (NG). It was shown that NO action on Na+,K+-ATPase in vivo is dose-dependent. Stimulation of the endogenous NO synthesis by LPS as well as the administration of low doses of NG lead to the activation of Na+,K+-ATPase and favor the conclusion that NO-dependent Na+,K+-ATPase stimulation mediates vasodilatory and hypotensive action of nitric oxide. The Na+,K+-ATPase activity in rat aorta depends on the balance between the level of reactive oxygen and nitrogen species (ROS and RNS), formation of NO depots in the tissue of aorta as high- and low molecular weight nitrosothiols, and also on the intensity of free-radical reactions resulting in the generation of hydroperoxide radicals. The results obtained suggest that NOS- and cGMP-dependent pathway takes part in Na+,K+-ATPase activation by LPS and NG, but the enzyme inhibition by nitric oxide in vivo is not cGMP-dependent and is determined by the activation of free-radical reactions and dramatic enhancement of nitrosylation level in rat aorta tissue.
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Klein, U. "THE INSECT V-ATPase, A PLASMA MEMBRANE PROTON PUMP ENERGIZING SECONDARY ACTIVE TRANSPORT: IMMUNOLOGICAL EVIDENCE FOR THE OCCURRENCE OF A V-ATPase IN INSECT ION-TRANSPORTING EPITHELIA." Journal of Experimental Biology 172, no. 1 (November 1, 1992): 345–54. http://dx.doi.org/10.1242/jeb.172.1.345.
Full textAbstract:
Active electrogenic K+ transport in insects serves as the energy source for secretion or absorption in gastrointestinal epithelia or for the receptor current in sensory epithelia. In the larval midgut of the tobacco hornworm Manduca sexta, a vacuolar-type proton pump (V-ATPase) and a K+/nH+ antiport represent the functional elements of the potassium pump. Several immunological findings support the hypothesis that active K+ transport in other insect epithelia may also be energized by a V-ATPase. In immunoblots, crude homogenates of sensilla-rich antennae and Malpighian tubules of M. sexta cross reacted with an immune serum directed to the purified plasma membrane V-ATPase from the midgut; the M. sexta midgut V-ATPase cross reacted with polyclonal antibodies to endomembrane V-ATPases from xenic origin. In immunocytochemical investigations of larvae of M. sexta and adults of Antheraea pernyi, monoclonal antibodies to defined subunits of the purified midgut V-ATPase or polyclonal antibodies to xenic endomembrane V-ATPase labelled the sites of active K+ transport: the goblet cell apical membrane in the midgut, the brush border of Malpighian tubules and the apical projections of the auxiliary cells in antennal sensilla. The functional mechanism of a primary H+-pumping V-ATPase and a secondary H+-dependent K+ transport postulated for K+-transporting insect epithelia may be further applicable to active Na+ or Cl- transport and would provide a unifying concept for all ouabain-insensitive electrogenic ion transport in insects. The findings from the midgut investigations, however, are the first instance in which a V-ATPase provides an alternative to the Na+/K+-ATPase in energizing secondary active transport in animal plasma membranes.
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Michea, Luis, Verónica Irribarra, I. Annelise Goecke, and Elisa T. Marusic. "Reduced Na-K pump but increased Na-K-2Cl cotransporter in aorta of streptozotocin-induced diabetic rat." American Journal of Physiology-Heart and Circulatory Physiology 280, no. 2 (February 1, 2001): H851—H858. http://dx.doi.org/10.1152/ajpheart.2001.280.2.h851.
Full textAbstract:
The activities of Na-K-ATPase and Na-K-2Cl cotransporter (NKCC1) were studied in the aorta, heart, and skeletal muscle of streptozotocin (STZ)-induced diabetic rats and control rats. In the aortic rings of STZ rats, the Na-K-ATPase-dependent 86Rb/K uptake was reduced to 60.0 ± 5.5% of the control value ( P < 0.01). However, Na-K-ATPase activity in soleus skeletal muscle fibers of STZ rats and paired control rats was similar, showing that the reduction of Na-K-ATPase activity in aortas of STZ rats is tissue specific. To functionally distinguish the contributions of ouabain-resistant (α1) and ouabain-sensitive (α2 and α3) isoforms to the Na-K-ATPase activity in aortic rings, we used either a high (10−3 M) or a low (10−5M) ouabain concentration during 86Rb/K uptake. We found that the reduction in total Na-K-ATPase activity resulted from a dramatic decrement in ouabain-sensitive mediated 86Rb/K uptake (26.0 ± 3.9% of control, P < 0.01). Western blot analysis of membrane fractions from aortas of STZ rats demonstrated a significant reduction in protein levels of α1- and α2-catalytic isoforms (α1 = 71.3 ± 9.8% of control values, P < 0.05; α2 = 44.5 ± 11.3% of control, P < 0.01). In contrast, aortic rings from the STZ rats demonstrated an increase in NKCC1 activity (172.5 ± 9.5%, P < 0.01); however, in heart tissue no difference in NKCC1 activity was seen between control and diabetic animals. Transport studies of endothelium-denuded or intact aortic rings demonstrated that the endothelium stimulates both Na-K-ATPase and Na-K-2Cl dependent 86Rb/K uptake. The endothelium-dependent stimulation of Na-K-ATPase and Na-K-2Cl was not hampered by diabetes. We conclude that abnormal vascular vessel tone and function, reported in STZ-induced diabetic rats, may be related to ion transport abnormalities caused by changes in Na-K-ATPase and Na-K-2Cl activities.
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Bertorello, A., and A. Aperia. "Regulation of Na+-K+-ATPase activity in kidney proximal tubules: involvement of GTP binding proteins." American Journal of Physiology-Renal Physiology 256, no. 1 (January 1, 1989): F57—F62. http://dx.doi.org/10.1152/ajprenal.1989.256.1.f57.
Full textAbstract:
This study evaluates the involvement of GTP-dependent regulatory proteins (G-proteins) in the regulation of Na+-K+-ATPase activity in proximal convoluted tubule (PCT) segments. Single PCT segments were dissected from rat kidney and permeabilized to allow nucleotides and medium free access to the interior of the cell. A GDP analogue that blocks GTP-dependent activation of the G-protein, GDP beta S (400 microM) significantly inhibited PCT Na+-K+-ATPase activity when Na in the medium (Nam) was greater than or equal to 70 mM. The inhibition was attenuated when Nam was 55 and 35 mM and was no longer significant when Nam was 25 mM. GDP beta S had no inhibitory effect on the activity of purified Na+-K+-ATPase. A nonhydrolyzable GTP analogue, GppNHp (50 microM) significantly increased Na+-K+-ATPase activity when Nam was 25 and 35 mM, but not when Nam was 55-140 mM. Dopamine (DA) and DA1 plus DA2 agonists significantly inhibit Na+-K+-ATPase activity. DA inhibition was competitively abolished by GppNHp. In PCT segments from rats pretreated with pertussis toxin, DA and DA1 plus DA2 agonist inhibition of Na+-K+-ATPase activity was abolished. In PCT segments from rats pretreated with cholera toxin, basal Na+-K+-ATPase activity was increased, but DA significantly inhibited Na+-K+-ATPase activity. Na+-K+-ATPase activity in PCT segments is regulated via a G-protein that stimulates Na+-K+-ATPase activity and a DA-activated pertussis toxin-sensitive G-protein that inhibits Na+-K+-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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Dong, J., N. A. Delamere, and M. Coca-Prados. "Inhibition of Na(+)-K(+)-ATPase activates Na(+)-K(+)-2Cl- cotransporter activity in cultured ciliary epithelium." American Journal of Physiology-Cell Physiology 266, no. 1 (January 1, 1994): C198—C205. http://dx.doi.org/10.1152/ajpcell.1994.266.1.c198.
Full textAbstract:
Inhibition of Na(+)-K(+)-ATPase activates Na(+)-K(+)-2Cl- cotransporter activity in cultured ciliary epithelium. Am. J. Physiol. 266 (Cell Physiol. 35): C198-C205, 1994.--86Rb uptake experiments were conducted to measure Na(+)-K(+)-ATPase activity and Na(+)-K(+)-2Cl- cotransporter activity in a cell line derived from rabbit nonpigmented ciliary epithelium. The presence of a Na(+)-K(+)-2Cl- cotransporter was supported by the observation of a bumetanide-sensitive 86Rb uptake component that was dependent on the extracellular concentration of both sodium and chloride. Potassium influx mediated by the Na(+)-K(+)-2Cl- cotransporter and Na(+)-K(+)-ATPase accounted for approximately 46 and 33% of total potassium uptake, respectively, whereas both ouabain- and bumetanide-resistant uptake accounted for 9%. Inhibition of the Na(+)-K(+)-ATPase had a stimulatory effect on Na(+)-K(+)-2Cl- cotransporter activity, which was dependent on the extent and duration of Na(+)-K(+)-ATPase inhibition. Ouabain treatment stimulated the potassium (86Rb) efflux rate and reduced intracellular potassium ([K]i). Potassium channel blockers suppressed the ouabain-activated potassium efflux and inhibited the ouabain-induced activation of the Na(+)-K(+)-2Cl- cotransporter. We conclude that Na(+)-K(+)-ATPase inhibition leads to the opening of potassium channels, which exacerbates the depletion of cellular potassium; Na(+)-K(+)-2Cl- cotransporter stimulation caused by the fall of [K]i overrides the tendency of increased cellular sodium to inhibit the cotransporter.
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Koenderink, Jan B., Herman G. P. Swarts, H. Christiaan Stronks, Harm P. H. Hermsen, Peter H. G. M. Willems, and Jan Joep H. H. M. De Pont. "Chimeras of X+,K+-ATPases." Journal of Biological Chemistry 276, no. 15 (January 16, 2001): 11705–11. http://dx.doi.org/10.1074/jbc.m010804200.
Full textAbstract:
In this study we reveal regions of Na+,K+-ATPase and H+,K+-ATPase that are involved in cation selectivity. A chimeric enzyme in which transmembrane hairpin M5-M6 of H+,K+-ATPase was replaced by that of Na+,K+-ATPase was phosphorylated in the absence of Na+and showed no K+-dependent reactions. Next, the part originating from Na+,K+-ATPase was gradually increased in the N-terminal direction. We demonstrate that chimera HN16, containing the transmembrane segments one to six and intermediate loops of Na+,K+-ATPase, harbors the amino acids responsible for Na+specificity. Compared with Na+,K+-ATPase, this chimera displayed a similar apparent Na+affinity, a lower apparent K+affinity, a higher apparent ATP affinity, and a lower apparent vanadate affinity in the ATPase reaction. This indicates that theE2K form of this chimera is less stable than that of Na+,K+-ATPase, suggesting that it, like H+,K+-ATPase, de-occludes K+ions very rapidly. Comparison of the structures of these chimeras with those of the parent enzymes suggests that the C-terminal 187 amino acids and the β-subunit are involved in K+occlusion. Accordingly, chimera HN16 is not only a chimeric enzyme in structure, but also in function. On one hand it possesses the Na+-stimulated ATPase reaction of Na+,K+-ATPase, while on the other hand it has the K+occlusion properties of H+,K+-ATPase.
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Bełtowski, Jerzy, Andrzej Marciniak, Grazyna Wójcicka, and Dionizy Górny. "Regulation of renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase by the cyclic AMP-protein kinase A signal transduction pathway." Acta Biochimica Polonica 50, no. 1 (March 31, 2003): 103–14. http://dx.doi.org/10.18388/abp.2003_3717.
Full textAbstract:
We investigated the effect of the cyclic AMP-protein kinase A (PKA) signalling pathway on renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase. Male Wistar rats were anaesthetized and catheter was inserted through the femoral artery into the abdominal aorta proximally to the renal arteries for infusion of the investigated substances. Na(+),K(+)-ATPase activity was measured in the presence of Sch 28080 to block ouabain-sensitive H(+),K(+)-ATPase and improve specificity of the assay. Dibutyryl-cyclic AMP (db-cAMP) administered at a dose of 10(-7) mol/kg per min and 10(-6) mol/kg per min increased Na(+),K(+)-ATPase activity in the renal cortex by 34% and 42%, respectively, and decreased it in the renal medulla by 30% and 44%, respectively. db-cAMP infused at 10(-6) mol/kg per min increased the activity of cortical ouabain-sensitive H(+),K(+)-ATPase by 33%, and medullary ouabain-sensitive H(+),K(+)-ATPase by 30%. All the effects of db-cAMP were abolished by a specific inhibitor of protein kinase A, KT 5720. The stimulatory effect on ouabain-sensitive H(+),K(+)-ATPase and on cortical Na(+),K(+)-ATPase was also abolished by brefeldin A which inhibits the insertion of proteins into the plasma membranes, whereas the inhibitory effect on medullary Na(+),K(+)-ATPase was partially attenuated by 17-octadecynoic acid, an inhibitor of cytochrome p450-dependent arachidonate metabolism. We conclude that the cAMP-PKA pathway stimulates Na(+),K(+)-ATPase in the renal cortex as well as ouabain-sensitive H(+),K(+)-ATPase in the cortex and medulla by a mechanism requiring insertion of proteins into the plasma membrane. In contrast, medullary Na(+),K(+)-ATPase is inhibited by cAMP through a mechanism involving cytochrome p450-dependent arachidonate metabolites.
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Kjeldsen, K. "Complete quantification of the total concentration of rat skeletal-muscle Na+ + K+-dependent ATPase by measurements of [3H]ouabain binding." Biochemical Journal 240, no. 3 (December 15, 1986): 725–30. http://dx.doi.org/10.1042/bj2400725.
Full textAbstract:
In the standard [3H]ouabain-binding assay for quantification of the Na,K-ATPase (Na+ + K+-dependent ATPase) concentration in rat skeletal muscles, samples are incubated for 2 × 60 min in 1 microM-[3H]ouabain at 37 degrees C followed by a wash-out for 4 × 30 min at 0 degree C. To obtain accurate determinations, values determined by this standard assay should be corrected for non-specific uptake and retention of [3H]ouabain (11% overestimation), loss of specifically bound [3H]ouabain during wash-out (21% underestimation), evaporation from muscle samples during weighing (4% overestimation), impurity of [3H]ouabain (5% underestimation) and incomplete saturation of [3H]ouabain binding sites (6% underestimation). Thus corrected the standard [3H]ouabain-binding assay determines the total Na,K-ATPase concentration. Hence, in the soleus muscle of 12-week-old rats the total [3H]ouabain-binding-site concentration is 278 +/- 20 pmol/g wet wt. This is at variance with the evaluation of the Na,K-ATPase concentration from Na,K-ATPase activity measurements in muscle membrane fractions, where the recovery of Na,K-ATPase is only 2-18%. Quantification of the total Na,K-ATPase concentration is of particular importance since it is a prerequisite for the discussion of quantitative aspects of the Na,K-ATPase.
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Del Castillo, J. R., M. C. Sulbaran-Carrasco, and L. Burguillos. "K+ transport in isolated guinea pig colonocytes: evidence for Na(+)-independent ouabain-sensitive K+ pump." American Journal of Physiology-Gastrointestinal and Liver Physiology 266, no. 6 (June 1, 1994): G1083—G1089. http://dx.doi.org/10.1152/ajpgi.1994.266.6.g1083.
Full textAbstract:
K+ transport mechanisms in epithelial cells isolated from guinea pig distal colon have been studied using 86Rb as a tracer. A transport pathway has been identified that is proposed to be identical to the mechanism mediating transepithelial K+ absorption. Guinea pig colonocytes take up K+ through at least three separate mechanisms: 1) a Na(+)-dependent, ouabain-sensitive influx that is consistent with the Na(+)-K+ pump, 2) a Na(+)-dependent bumetanide-sensitive influx consistent with the Na(+)-K(+)-2Cl- cotransporter, and 3) a Na(+)-independent ouabain-sensitive influx, consistent with an apical colonic K+ pump. These transport mechanisms are sensitive to metabolic inhibition by rotenone and to vanadate, a blocker of type P adenosinetriphosphatase (ATPases). SCH-28080, an inhibitor of gastric K(+)-H(+)-ATPase, was without effect. Measurements of net K+ fluxes revealed that isolated colonocytes concentrated K+ by two processes: 1) a Na(+)-dependent ouabain-sensitive mechanism, which is compatible with the Na(+)-K+ pump and 2) a Na(+)-independent ouabain-sensitive mechanism consistent with the proposed absorptive K+ pump. These concentrative mechanisms were also inhibited by rotenone and vanadate, but not by SCH-28080. The Na(+)-independent ouabain-sensitive K+ pump was present in the distal colon, but absent in the proximal colon and the small intestine of guinea pig. It is proposed that this Na(+)-independent ouabain-sensitive K+ pump mediates K+ absorption and is related to the luminal K(+)-ATPase.
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Singh, H., and S. L. Linas. "Role of protein kinase C in beta 2-adrenoceptor function in cultured rat proximal tubule epithelial cells." American Journal of Physiology-Renal Physiology 273, no. 2 (August 1, 1997): F193—F199. http://dx.doi.org/10.1152/ajprenal.1997.273.2.f193.
Full textAbstract:
Renal sodium excretion is regulated by the adrenergic system. We recently demonstrated the presence of functional beta 2-adrenoceptors (beta 2-AR) in cultured rat proximal tubule epithelial cells beta 2-AR activation resulted in increases in Na-K-adenosinetriphosphatase (Na-K-ATPase) activity and transcellular sodium transport as a consequence of increased apical sodium entry. The purpose of this study was to determine the role of protein kinase C (PKC) on beta 2-AR-dependent increases in Na-K-ATPase activity and sodium transport in proximal tubules. To determine the effect of PKC on basal function, cultured rat proximal tubule cells were exposed to phorbol 12-myristate 13-acetate (PMA). PMA increased apical Na entry (+/-80%), decreased Na-K-ATPase activity (+/-25%), and prevented increases in Na-K-ATPase activity after sodium entry facilitation with monensin. Decreases in Na-K-ATPase activity were associated with decreases in sodium transport (+/-30%). To determine whether beta 2-AR function was transduced by PKC, PKC activity was measured in cells exposed to the selective beta 2-AR agonist metaproterenol. Metaproterenol caused increases in PKC activity, which were blocked by a beta 2-AR but not by a beta 1-AR-receptor antagonist. beta 2-AR-dependent increases in apical Na entry, Na-K-ATPase activity, and sodium transport were blocked by calphostin C or staurosporine. To determine whether PKC had additional effects on beta 2-AR function, cells were exposed to metaproterenol and PMA. Metaproterenol-induced increases in Na-K-ATPase activity and sodium transport were blocked by PMA. In conclusion, beta 2-AR-mediated increases in Na-K-ATPase activity and sodium flux are transduced by PKC acting through increases in apical Na entry. However, activation of PKC by phorbol esters inhibits beta 2-AR-dependent increases in Na-K-ATPase activity and sodium transport.
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Tamiya, Shigeo, Mansim C. Okafor, and Nicholas A. Delamere. "Purinergic agonists stimulate lens Na-K-ATPase-mediated transport via a Src tyrosine kinase-dependent pathway." American Journal of Physiology-Cell Physiology 293, no. 2 (August 2007): C790—C796. http://dx.doi.org/10.1152/ajpcell.00579.2006.
Full textAbstract:
The Na-K-ATPase is vital for maintenance of lens transparency. Past studies using intact lens suggested the involvement of tyrosine kinases in short-term regulation of Na-K-ATPase. Furthermore, in vitro phosphorylation of a lens epithelial membrane preparation by Src family kinases (SFKs), a family of nonreceptor tyrosine kinases, resulted in modification of Na-K-ATPase activity. Here, the effect of purinergic agonists, ATP and UTP, on Na-K-ATPase function and SFK activation was examined in the rabbit lens. Na-K-ATPase function was examined using two different approaches, measurement of ouabain-sensitive potassium (86Rb) uptake by the intact lens, and Na-K-ATPase activity in lens epithelial homogenates. ATP and UTP caused a significant increase in ouabain-sensitive potassium (86Rb) uptake. Na-K-ATPase activity was increased in the epithelium of lenses pretreated with ATP. Lenses treated with ATP or UTP displayed activation of SFKs as evidenced by increased Western blot band density of active SFK (phosphorylated at the active loop Y416) and decreased band density of inactive SFKs (phosphorylated at the COOH terminal). A single PY416-Src immunoreactive band at ∼60 kDa was observed, suggesting not all Src family members are activated. Immunoprecipitation studies showed that band density of active Src, and to a lesser extent active Fyn, was significantly increased, while active Yes did not change. Preincubation of the lenses with SFK inhibitor PP2 abolished the ATP-induced increase in ouabain-sensitive potassium (86Rb) uptake. The results suggest selective activation of Src and/or Fyn is part of a signaling mechanism initiated by purinergic agonists that increases Na-K-ATPase-mediated transport in the organ-cultured lens.
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Zhou, Xiaoming, I. Jeanette Lynch, Shen-Ling Xia, and Charles S. Wingo. "Activation of H+-K+-ATPase by CO2 requires a basolateral Ba2+-sensitive pathway during K restriction." American Journal of Physiology-Renal Physiology 279, no. 1 (July 1, 2000): F153—F160. http://dx.doi.org/10.1152/ajprenal.2000.279.1.f153.
Full textAbstract:
We studied the activation of H+-K+-ATPase by CO2 in the renal cortical collecting duct (CCD) of K-restricted animals. Exposure of microperfused CCD to 10% CO2 increased net total CO2 flux ( J t CO2 ) from 4.9 ± 2.1 to 14.7 ± 4 pmol · mm−1· min−1 ( P < 0.05), and this effect was blocked by luminal application of the H+-K+-ATPase inhibitor Sch-28080. In the presence of luminal Ba, a K channel blocker, exposure to CO2 still stimulated J t CO2 from 6.0 ± 1.0 to 16.8 ± 2.8 pmol · mm−1 · min−1 ( P < 0.01), but peritubular application of Ba inhibited the stimulation. CO2substantially increased 86Rb efflux (a K tracer marker) from 93.1 ± 23.8 to 249 ± 60.2 nm/s ( P < 0.05). These observations suggest that during K restriction 1) the enhanced H+-K+-ATPase-mediated acidification after exposure to CO2 is dependent on a basolateral Ba-sensitive mechanism, which is different from the response of rabbits fed a normal-K diet, where activation of the H+-K+-ATPase by exposure to CO2 is dependent on an apical Ba-sensitive pathway; and 2) K/Rb absorption via the apical H+-K+-ATPase exits through a basolateral Ba-sensitive pathway. Together, these data are consistent with the hypothesis of cooperation between H+-K+-ATPase-mediated acidification and K exit pathways in the CCD that regulate K homeostasis.
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Juel, Carsten, Nikolai B. Nordsborg, and Jens Bangsbo. "Exercise-induced increase in maximal in vitro Na-K-ATPase activity in human skeletal muscle." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 304, no. 12 (June 15, 2013): R1161—R1165. http://dx.doi.org/10.1152/ajpregu.00591.2012.
Full textAbstract:
The present study investigated whether maximal in vitro Na-K-ATPase activity in human skeletal muscle is changed with exercise and whether it was altered by acute hypoxia. Needle biopsies from 14 subjects were obtained from vastus lateralis before and after 4 min of intense muscle activity. In addition, six subjects exercised also in hypoxia (12.5% oxygen). The Na-K-ATPase assay revealed a 19% increase ( P < 0.05) in maximal velocity ( Vmax) for Na+-dependent Na-K-ATPase activity after exercise and a tendency ( P < 0.1) toward a decrease in Km for Na+ (increased Na+ affinity) in both normoxia and hypoxia. In contrast, the in vitro Na-K-ATPase activity determined with the 3- O-MFPase technique was 11–32% lower after exercise in normoxia ( P < 0.05) and hypoxia ( P < 0.1). Based on the different results obtained with the Na-K-ATPase assay and the 3- O-MFPase technique, it was suggested that the 3- O-MFPase method is insensitive to changes in Na-K-ATPase activity. To test this possibility, changes in Na-K-ATPase activity was induced by protein kinase C activation. The changes quantified with the Na-K-ATPase assay could not be detected with the 3- O-MFPase method. In addition, purines stimulated Na-K-ATPase activity in rat muscle membranes; these changes could not be detected with the 3- O-MFPase method. Therefore, the 3- O-MFPase technique is not sensitive to changes in Na+ sensitivity, and the method is not suited to detecting changes in Na-K-ATPase activity with exercise. In conclusion, muscle activity in humans induces an increased in vitro Na+-dependent Na-K-ATPase activity, which contributes to the upregulation of the Na-K-ATPase in association with exercise both in normoxia and hypoxia.
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Nepal, Niraj, Subha Arthur, and Uma Sundaram. "Unique Regulation of Na-K-ATPase during Growth and Maturation of Intestinal Epithelial Cells." Cells 8, no. 6 (June 15, 2019): 593. http://dx.doi.org/10.3390/cells8060593.
Full textAbstract:
Na-K-ATPase on the basolateral membrane provides the favorable transcellular Na gradient for the proper functioning of Na-dependent nutrient co-transporters on the brush border membrane (BBM) of enterocytes. As cells mature from crypts to villus, Na-K-ATPase activity doubles, to accommodate for the increased BBM Na-dependent nutrient absorption. However, the mechanism of increased Na-K-ATPase activity during the maturation of enterocytes is not known. Therefore, this study aimed to determine the mechanisms involved in the functional transition of Na-K-ATPase during the maturation of crypts to villus cells. Na-K-ATPase activity gradually increased as IEC-18 cells matured in vitro from day 0 (crypts) through day 4 (villus) of post-confluence. mRNA abundance and Western blot studies showed no change in the levels of Na-K-ATPase subunits α1 and β1 from 0 to 4 days post-confluent cells. However, Na-K-ATPase α1 phosphorylation levels on serine and tyrosine, but not threonine, residues gradually increased. These data indicate that as enterocytes mature from crypt-like to villus-like in culture, the functional activity of Na-K-ATPase increases secondary to altered affinity of the α1 subunit to extracellular K+, in order to accommodate the functional preference of the intestinal cell type. This altered affinity is likely due to increased phosphorylation of the α1 subunit, specifically at serine and tyrosine residues.
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Hayhurst, R. A., and R. G. O'Neil. "Time-dependent actions of aldosterone and amiloride on Na+-K+-ATPase of cortical collecting duct." American Journal of Physiology-Renal Physiology 254, no. 5 (May 1, 1988): F689—F696. http://dx.doi.org/10.1152/ajprenal.1988.254.5.f689.
Full textAbstract:
The actions of aldosterone on Vmax Na+-K+-ATPase activity and length of latent period were assessed for the rabbit cortical collecting duct (CCD). Initially, animals were moderately aldosterone depleted and then treated with a constant infusion of physiological doses of aldosterone. Aldosterone administration had no influence after 3 h but caused a detectable increase with 6 (borderline significance) or more hours. An apparent plateau was reached between 24 and 48 h at twice the initial activity. This aldosterone-induced stimulation could be abolished by simultaneous treatment of the animals with amiloride, demonstrating a Na+-dependent modulation of the Vmax Na+-K+-ATPase activity. The aldosterone-stimulated enzyme had kinetic properties similar to those reported by others, but the latent period for aldosterone action on the Vmax Na+-K+-ATPase activity averaged near 6 h in the present study, as opposed to the highly variable period (from 1 h to several days) seen by others. This latent period variability was shown to be directly related to the initial Vmax Na+-K+-ATPase activity in the CCD and could be likened to an "end product dependent" latent period, i.e., the lower the initial end product (Vmax Na+-K+-ATPase activity) the shorter the latent period. Hence aldosterone's actions on the Na+-K+-ATPase of the CCD would be consistent with a single mechanism of action, i.e., increased synthesis, but with a variable modulation of this synthesis, which is dependent on the initial Vmax Na+-K+-ATPase activity of the CCD cells and/or the initial aldosterone status of the animal.
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Mordasini, David, Mauro Bustamante, Martine Rousselot, Pierre-Yves Martin, Udo Hasler, and Eric Féraille. "Stimulation of Na+ transport by AVP is independent of PKA phosphorylation of the Na-K-ATPase in collecting duct principal cells." American Journal of Physiology-Renal Physiology 289, no. 5 (November 2005): F1031—F1039. http://dx.doi.org/10.1152/ajprenal.00128.2005.
Full textAbstract:
Arginine-vasopressin (AVP) stimulates Na+ transport and Na-K-ATPase activity via cAMP-dependent PKA activation in the renal cortical collecting duct (CCD). We investigated the role of the Na-K-ATPase in the AVP-induced stimulation of transepithelial Na+ transport using the mpkCCDc14 cell model of mammalian collecting duct principal cells. AVP (10−9 M) stimulated both the amiloride-sensitive transepithelial Na+ transport measured in intact cells and the maximal Na pump current measured by the ouabain-sensitive short-circuit current in apically permeabilized cells. These effects were associated with increased Na-K-ATPase cell surface expression, measured by Western blotting after streptavidin precipitation of biotinylated cell surface proteins. The effects of AVP on Na pump current and Na-K-ATPase cell surface expression were dependent on PKA activity but independent of increased apical Na+ entry. Time course experiments revealed that in response to AVP, the cell surface expression of both endogenous Na-K-ATPase and hybrid Na pumps containing a c- myc-tagged wild-type human α1-subunit increased transiently. Na-K-ATPase cell surface expression was maximal after 30 min and then declined toward baseline after 60 min. Immunoprecipitation experiments showed that PKA activation did not alter total phosphorylation levels of the endogenous Na-K-ATPase α-subunit. In addition, mutation of the PKA phosphorylation site (S943A or S943D) did not alter the time course of increased cell surface expression of c- myc-tagged Na-K-ATPase in response to AVP or to dibutyryl-cAMP. Therefore, stimulation of Na-K-ATPase cell surface expression by AVP is dependent on PKA but does not rely on α1-subunit phosphorylation on serine 943 in the collecting duct principal cells.
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Nepal, Niraj, Subha Arthur, Molly R. Butts, Soudamani Singh, Balasubramanian Palaniappan, and Uma Sundaram. "Molecular Mechanism of Stimulation of Na-K-ATPase by Leukotriene D4 in Intestinal Epithelial Cells." International Journal of Molecular Sciences 22, no. 14 (July 15, 2021): 7569. http://dx.doi.org/10.3390/ijms22147569.
Full textAbstract:
Na-K-ATPase provides a favorable transcellular Na gradient required for the functioning of Na-dependent nutrient transporters in intestinal epithelial cells. The primary metabolite for enterocytes is glutamine, which is absorbed via Na-glutamine co-transporter (SN2; SLC38A5) in intestinal crypt cells. SN2 activity is stimulated during chronic intestinal inflammation, at least in part, secondarily to the stimulation of Na-K-ATPase activity. Leukotriene D4 (LTD4) is known to be elevated in the mucosa during chronic enteritis, but the way in which it may regulate Na-K-ATPase is not known. In an in vitro model of rat intestinal epithelial cells (IEC-18), Na-K-ATPase activity was significantly stimulated by LTD4. As LTD4 mediates its action via Ca-dependent protein kinase C (PKC), Ca levels were measured and were found to be increased. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, also mediated stimulation of Na-K-ATPase like LTD4, while BAPTA-AM (Ca chelator) and calphostin-C (Cal-C; PKC inhibitor) prevented the stimulation of Na-K-ATPase activity. LTD4 caused a significant increase in mRNA and plasma membrane protein expression of Na-K-ATPase α1 and β1 subunits, which was prevented by calphostin-C. These data demonstrate that LTD4 stimulates Na-K-ATPase in intestinal crypt cells secondarily to the transcriptional increase of Na-K-ATPase α1 and β1 subunits, mediated via the Ca-activated PKC pathway.
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Féraille, Eric, Pascal Béguin, Maria-Luisa Carranza, Sandrine Gonin, Martine Rousselot, Pierre-Yves Martin, Hervé Favre, and Käthi Geering. "Is Phosphorylation of the α1 Subunit at Ser-16 Involved in the Control of Na,K-ATPase Activity by Phorbol Ester–activated Protein Kinase C?" Molecular Biology of the Cell 11, no. 1 (January 2000): 39–50. http://dx.doi.org/10.1091/mbc.11.1.39.
Full textAbstract:
The α1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufoα1 subunits. In cells incubated at 37°C, phorbol 12,13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally (∼20–30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A2, and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18°C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing α1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase α1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.
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Schmidt, T. A., S. Hasselbalch, P. A. Farrell, H. Vestergaard, and K. Kjeldsen. "Human and rodent muscle Na(+)-K(+)-ATPase in diabetes related to insulin, starvation, and training." Journal of Applied Physiology 76, no. 5 (May 1, 1994): 2140–46. http://dx.doi.org/10.1152/jappl.1994.76.5.2140.
Full textAbstract:
As determined by vanadate-facilitated [3H]ouabain binding to intact samples, semistarvation and untreated streptozotocin- or partial pancreatectomy-induced diabetes reduced rat soleus muscle Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) concentration by 12–21% (P < 0.05). Conversely, insulin treatment of rats with streptozotocin-induced diabetes induced an increase of 18-26% above control (P < 0.05). Treadmill training diminished the reduction in muscle [3H]ouabain binding site concentration induced by untreated diabetes to only 2–5%. No significant variation was observed in rat cerebral cortex Na(+)-K(+)-ATPase concentration as a result of diabetes, semistarvation, or insulin treatment. In human subjects, Na(+)-K(+)-ATPase concentration in vastus lateralis muscle biopsies was 17 and 22% greater (P < 0.05), respectively, in patients with treated non-insulin-dependent diabetes mellitus (n = 24) and insulin-dependent diabetes mellitus (n = 7) than in control subjects (n = 8). A positive linear correlation between muscle Na(+)-K(+)-ATPase and plasma insulin concentrations was observed (r = 0.50, P = 0.006; n = 29). Thus, insulin seems a regulator of muscle Na(+)-K(+)-ATPase concentration, reduction of muscle Na(+)-K(+)-ATPase concentration with untreated diabetes bears similarities with undernourishment, and physical conditioning may ameliorate the muscle Na(+)-K(+)-ATPase concentration decrease induced by diabetes.
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Tomita, K., A. Owada, Y. Iino, N. Yoshiyama, and T. Shiigai. "Effect of vasopressin on Na+-K+-ATPase activity in rat cortical collecting duct." American Journal of Physiology-Renal Physiology 253, no. 5 (November 1, 1987): F874—F879. http://dx.doi.org/10.1152/ajprenal.1987.253.5.f874.
Full textAbstract:
Vasopressin (V) causes a sustained increase in Na reabsorption and K secretion in isolated cortical collecting ducts (CCD) from rats. Because increased Na reabsorption may be associated with increased Na+-K+-ATPase activity, we investigated effects of V, given either in vivo or in vitro, on Na+-K+-ATPase activity in isolated nephron segments of rats. Na+-K+-ATPase activities were measured by coupling the hydrolysis of ATP to the production of a fluorescent nucleotide. In addition to CCD, other microdissected structures were medullary thick ascending limbs of Henle's loop, cortical thick ascending limbs of Henle's loop, and outer medullary collecting duct. To determine the time course of the response, Na+-K+-ATPase activities were measured in CCD 1 h, 3 h, 1 day, 3 days, and 7 days after intramuscular administrations of V. There was a significant increase in Na+-K+-ATPase activity in CCD after in vivo V administration for 7 days but not in any other segment. The activities increased after 3 days of administration of V. For in vitro experiments, CCD were incubated with 10(-6) M V for 1-3 h. Na+-K+-ATPase activities did not change after 1- or 3-h exposure of V in CCD in vitro. We conclude that prolonged V administration in vivo increases Na+-K+-ATPase activity in CCD. Because, in vitro exposure to V does not increase Na+-K+-ATPase activity, we conclude that rapid V-dependent increases in Na and K transport previously demonstrated in isolated perfused tubules are not dependent on a change in maximal Na+-K+-ATPase activity.
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Sarkar, Pradip K., Avijit Biswas, Arun K. Ray, and Joseph V. Martin. "Mechanisms of L-Triiodothyronine-Induced Inhibition of Synaptosomal Na+-K+-ATPase Activity in Young Adult Rat Brain Cerebral Cortex." Journal of Thyroid Research 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/457953.
Full textAbstract:
The role of thyroid hormones (TH) in the normal functioning of adult mammalian brain is unclear. Our studies have identified synaptosomal Na+-K+-ATPase as a TH-responsive physiological parameter in adult rat cerebral cortex. L-triiodothyronine (T3) and L-thyroxine (T4) both inhibited Na+-K+-ATPase activity (but not Mg2+-ATPase activity) in similar dose-dependent fashions, while other metabolites of TH were less effective. Although both T3and theβ-adrenergic agonist isoproterenol inhibited Na+-K+-ATPase activity in cerebrocortical synaptosomes in similar ways, theβ-adrenergic receptor blocker propranolol did not counteract the effect of T3. Instead, propranolol further inhibited Na+-K+-ATPase activity in a dose-dependent manner, suggesting that the effect of T3on synaptosomal Na+-K+-ATPase activity was independent ofβ-adrenergic receptor activation. The effect of T3on synaptosomal Na+-K+-ATPase activity was inhibited by theα2-adrenergic agonist clonidine and by glutamate. Notably, both clonidine and glutamate activateGi-proteins of the membrane second messenger system, suggesting a potential mechanism for the inhibition of the effects of TH. In this paper, we provide support for a nongenomic mechanism of action of TH in a neuronal membrane-related energy-linked process for signal transduction in the adult condition.
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Cai, Ting, Haojie Wang, Yiliang Chen, Lijun Liu, William T. Gunning, Luis Eduardo M. Quintas, and Zi-Jian Xie. "Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase." Journal of Cell Biology 182, no. 6 (September 15, 2008): 1153–69. http://dx.doi.org/10.1083/jcb.200712022.
Full textAbstract:
Here, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase α1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase–depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.
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Vinciguerra, Manlio, Georges Deschênes, Udo Hasler, David Mordasini, Martine Rousselot, Alain Doucet, Alain Vandewalle, Pierre-Yves Martin, and Eric Féraille. "Intracellular Na+ Controls Cell Surface Expression of Na,K-ATPase via a cAMP-independent PKA Pathway in Mammalian Kidney Collecting Duct Cells." Molecular Biology of the Cell 14, no. 7 (July 2003): 2677–88. http://dx.doi.org/10.1091/mbc.e02-11-0720.
Full textAbstract:
In the mammalian kidney the fine control of Na+ reabsorption takes place in collecting duct principal cells where basolateral Na,K-ATPase provides the driving force for vectorial Na+ transport. In the cortical collecting duct (CCD), a rise in intracellular Na+ concentration ([Na+]i) was shown to increase Na,K-ATPase activity and the number of ouabain binding sites, but the mechanism responsible for this event has not yet been elucidated. A rise in [Na+]i caused by incubation with the Na+ ionophore nystatin, increased Na,K-ATPase activity and cell surface expression to the same extent in isolated rat CCD. In cultured mouse mpkCCDcl4 collecting duct cells, increasing [Na+]i either by cell membrane permeabilization with amphotericin B or nystatin, or by incubating cells in a K+-free medium, also increased Na,K-ATPase cell surface expression. The [Na+]i-dependent increase in Na,K-ATPase cell-surface expression was prevented by PKA inhibitors H89 and PKI. Moreover, the effects of [Na+]i and cAMP were not additive. However, [Na+]i-dependent activation of PKA was not associated with an increase in cellular cAMP but was prevented by inhibiting the proteasome. These findings suggest that Na,K-ATPase may be recruited to the cell membrane following an increase in [Na+]i through cAMP-independent PKA activation that is itself dependent on proteasomal activity.