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

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

ATP-dependent proton transport in membrane vesicles prepared from the medullary bone of egg-laying hens, a source rich in osteoclasts, was characterized. Proton transport was abolished by bafilomycin A1 (10 nM) and N-ethylmalemide (50 microM), but not by oligomycin (15 micrograms/ml), vanadate (100 microM) or SCH 28080 (100 microM), thereby differentiating this H(+)-ATPase from the F1F0- and phosphorylated-type of ATPases. Preincubation of the membrane vesicles at 0 degrees C for 1 h in the presence of KCl (0.3 M) and Mg-ATP (5 mM) resulted in almost complete loss of H(+)-transport activity (cold-inactivation). Preventing the formation of a membrane potential by voltage clamp (Kin+ = Kout+ + valinomycin) increased both the rate of H(+)-transport and the equilibrium delta pH, suggesting an electronic proton transport mechanism. Thus, the H(+)-ATPase in this bone-derived membrane vesicle preparation shows the characteristics of a vacuolar H(+)-ATPase in its inhibitor- and cold-sensitivity and its electrogenic mechanism. The anion sensitivity of the H(+)-ATPase was investigated by varying the intra- and/or extra-vesicular salt composition. The H(+)-ATPase had no absolute requirement for any specific anion, but membrane permeable anions were found to stimulate proton transport activity, presumably by acting as charge compensators for the electrogenic hydrogen ion transport. However, some anions, such as sulfate, acetate and nitrate were directly inhibitory to the ATPase. The results are in agreement with the recently proposed mechanism of osteoclast acidification: a vacuolar H(+)-ATPase working in parallel with a Cl(-)-channel resulting in electroneutral HCl secretion.
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PMID:Characterization of proton transport in bone-derived membrane vesicles. 844 16

An understanding of the fluid and electrolyte transport properties of any epithelium requires knowledge of the direction, rate, and regulation of fluid transport and the composition of the fluid. Although human airway epithelial likely play a key role in controlling the quantity and composition of the respiratory tract fluid, evidence for such a role is not available. To obtain such knowledge, we measured fluid and electrolyte transport by cultured human nasal epithelia. Under basal conditions we found that epithelia absorbed Na+ and fluid; both processes were inhibited by addition of amiloride to the mucosal surface. These data suggest that active Na+ absorption is responsible for fluid absorption. Interestingly, Na+ absorption was not accompanied by the net absorption of Cl-; some other anion accompanied Na+. The combination of cAMP agonists and mucosal amiloride stimulated the secretion of NaCl-rich fluid. But surprisingly, the response to cAMP agonists in the absence of amiloride showed substantial intersubject variability: cAMP stimulated fluid secretion across some epithelia, for others, cAMP stimulated fluid absorption. The explanation for the differences in response is uncertain, but we speculate that the magnitude of apical membrane Na+ conductance may modulate the direction of fluid transport in response to cAMP. We also found that airway epithelial secrete H+ and absorb K+ under basal conditions; both processes were inhibited by cAMP agonists. Because the H+/K(+)-ATPase inhibitor, SCH 28080, inhibited K+ absorption, an apical membrane H+/K(+)-ATPase may be at least partly responsible for K+ and H+ transport. However, H+/K+ exchange could not entirely account for the luminal acidification. The finding that cAMP agonists inhibited luminal acidification may be explained by the recent finding that cAMP increases apical HCO3- conductance. These results provide new insights into how the intact airway epithelium may modify the composition of the respiratory tract fluid.
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PMID:Fluid and electrolyte transport by cultured human airway epithelia. 847 2

Renal proximal tubular Na,K-ATPase plays an important role in the maintenance of sodium homeostasis and it is known that dopamine (DA) exerts an inhibitory effect on the activity of this enzyme. We have found that DA-induced inhibition of Na,K-ATPase is abolished in the spontaneously hypertensive rats (SHR) in comparison with age-matched Wistar-Kyoto (WKY) rats. Dopamine inhibits Na,K-ATPase via phospholipase C coupled protein kinase C pathway. The enzyme protein kinase C subsequently causes inhibition of Na,K-ATPase. In the SHR, DA-induced activation of phospholipase C is diminished, which in turn is responsible for the abolished inhibition of Na,K-ATPase. We have now shown that DA-induced activation of protein kinase C, which results from activation of DA-1 receptors is also abolished in the SHR which would account for the failure of DA to inhibit Na,K-ATPase in the hypertensive animals. Recently, we have examined the possibility that the failure of DA to inhibit Na,K-ATPase activity may be related to abnormal expression of DA receptors. In radioligand binding studies with [3H] SCH 23390 as a DA-1 receptor ligand and [3H] spiroperidol as a DA-2 receptor ligand we showed that both [3H] SCH 23390 and [3H] spiroperidol bindings are best fit to one site model in either WKY or SHR. Both Bmax and KD of either ligand binding to proximal tubule in the SHR were not statistically different from their WKY counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Dopaminergic modulation of Na,K-ATPase activity in the proximal tubules of normotensive and hypertensive rats. 852 73

The sensitivies of double-barrelled K(+)-selective micro-electrodes (KSMs) employing the low-impedance membrane cocktail based on the neutral K(+)-selective ion carrier valinomycin (Fluka, Cocktail B 60398) to the following 3 different classes of inhibitors of K+ transport were measured: (1) general metabolic inhibitors (dinitrophenol, potassium cyanide, sodium azide, rotenone, dicyclohexylcarbodiimide, salicylhydroxamic acid); (2) P-type ATPase inhibitors (vanadate, ouabain, amiloride, SCH 28080); and (3) anion-dependent K+ transport inhibitors (bumetanide, 4-acetamide-4-isothiocyanostilbene-2,2-disulphonic acid). Of the 12 inhibitors tested, only dinitrophenol had any significant effect on the response of KSMs to K+ activity. Comparison of the calibrations in solutions with and without 0.1 mM dinitrophenol showed that this inhibitor behaved as a 'classical' interferent whereby its contribution to the K+ activity signal was statistically significant at K+ activities of 36.0 mM and less. However, at higher K+ activities (97.0 mM), dinitrophenol interference was not significant. It was possible to correct for the DNP interference and to obtain measurements of intracellular K+ activity in insect muscles.
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PMID:Sensitivity of valinomycin-based K(+)-selective micro-electrodes to inhibitors of K+ transport. 853 96

Gastric H+,K(+)-ATPase was functionally expressed in the human kidney HEK293 cell line. The expressed enzyme catalyzed ouabain-resistant K(+)-dependent ATP hydrolysis. The K(+)-ATPase activity was inhibited by SCH 28090, a specific inhibitor of gastric proton pump, in a dose-dependent manner. By using this functional expression system in combination with site-directed mutagenesis, we investigated effects of mutations in the putative cation binding site and the catalytic center of the gastric H+,K(+)-ATPase. In Na+,K(+)-ATPase, the glutamic acid residue in the 4th transmembrane segment is regarded as one of the residues responsible for the K(+)-induced conformational change (Kuntzweiler, T. A., Wallick, E. T., Johnson, C. L., and Lingrel, J. B. (1995) J. Biol. Chem. 270, 2993-3000). When the corresponding glutamic acid (Glu-345) of H+,K(+)-ATPase was mutated to aspartic acid, lysine, or valine, the SCH 28080-sensitive K(+)-ATPase activity was abolished. However, when this residue was replaced by glutamine, about 50% of the activity was retained. This mutant showed a 10-fold lower affinity for K+ (Km = 2.6 mM) compared with the wild-type enzyme (Km = 0.24 mm). Thus, Glu-345 is important in determining the K+ affinity of H+,K(+)-ATPase. When the aspartic acid residue in the phosphorylation site was mutated to glutamic acid, this mutant showed no SCH 28080-sensitive K(+)-ATPase activity. Thus, amino acid replacement of the phosphorylation site is not tolerated and a stringent structure appears to be required for enzyme activity. When the lysine residue in the fluorescein isothiocyanate binding site (part of ATP binding site) was mutated to arginine, asparagine, or glutamic acid, the SCH 28080-sensitive K(+)-ATPase activity was eliminated. However, the mutant in which this residue was changed to glutamine had about 30% of the activity, suggesting that amino acid replacement of this site is tolerated to a certain extent.
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PMID:Functional expression of gastric H+,K(+)-ATPase and site-directed mutagenesis of the putative cation binding site and catalytic center. 857 49

Employing a simple one-step sucrose gradient fractionation method, gastric mucosal membrane of Syrian hamster was prepared and demonstrated to be specifically enriched in H+,K+-ATPase activity. The preparation is practically devoid of other ATP hydrolyzing activity and contains high K+-stimulated ATPase activity of at least 4-5 fold compared to basal ATPase activity. The H+,K+-ATPase showed hydroxylamine-sensitive phosphorylation and K+-dependent dephosphorylation of the phosphoenzyme, characteristic inhibition by vanadate, omeprazole and SCH 28080, and nigericin-reversible K+-dependent H+-transport--properties characteristic of gastric proton pump. One notable difference with H+,K+-ATPase of other species has been the observation of valinomycin-independent H+ transport in such membrane vesicles. It is proposed that such H+,K+-ATPase-rich hamster gastric mucosal membrane preparation might provide a unique model to study physiological aspects of H+,K+-ATPase function in relation to HCl secretion.
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PMID:Characterization of Syrian hamster gastric mucosal H+,K+-ATPase. 859 24

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+.
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PMID:Ontogeny of K+ transport in rat distal colon. 877 42

The effect of H+-K+ ATPase inhibitors on airway smooth muscle tone was investigated in vitro. Four H+-K+ ATPase inhibitors, SCH 28080 (2-methyl-8-(phenylmethoxy)-imidazo[1,2-a] pyridine-3-acetonitrile), SK&F 96067 (3-butyryl-4-(2-methylphenylamino)-8-methoxy-quinoline), omaprezole (5-methoxy-2-(((4-methoxy-3,5-dimethyl-2-pyridinyl)-methyl)-sulfinyl)-1H -benzimidazole) and NC-1300-B (2-(2-dimethylaminobenzylsulfiny)-5-methoxybenzimidazole), induced concentration-dependent relaxation of guinea pig trachea with spontaneous tone, with IC50 values of 5.9, 7.1, 155 and 79 microM, respectively, SCH 28080 and omeprazole also relaxed airways precontracted with carbachol or histamine in the presence of indomethacin. Relaxation was similar in intact and epithelium-denuded tracheal preparations, suggesting that the airway epithelium neither mediates or modulates relaxation induced by H+-K+ ATPase inhibitors. SCH 28080-induced relaxation was not influenced by tetrodotoxin, suggesting that it is not neurogenically mediated. Bafilomycin A1 and concanamycin A had no effect on guinea pig tracheal spontaneous tone, suggesting that relaxation induced by H+-K+ ATPase inhibitors is not due to an interaction with a vacuolar H+ ATPase. SCH 28080 also induced concentration-dependent relaxation of human bronchi precontracted with histamine. These results demonstrate that several structurally and/or mechanistically distinct H+-K+ ATPase inhibitors cause relaxation of airway smooth muscle in vitro, and suggest that a H+-K+ ATPase or similar pathway may play a role in the maintenance of airway smooth muscle tone.
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PMID:H+-K+ ATPase inhibitors cause relaxation of guinea pig and human airway smooth muscle in vitro. 878 67

We investigated the effect of dopamine on Na+,K(+)-ATPase activity in cultured aortic smooth muscle cells. Na+,K(+)- ATPase activity was measured by a coupled enzyme assay. Our results demonstrate that dopamine and dopamine receptor agonists, SKF-38393 (a D1 receptor agonist) and quinpirole (a D2 receptor agonist) produced 62%, 50% and 49% inhibition of Na+,K(+)-ATPase activity in aortic smooth muscle cells, respectively. The combination of the two agonists produced inhibition similar to that of dopamine. Dopamine- and the agonist-induced Na+,K(+)-ATPase inhibition was blocked by selective receptor antagonists. The Na+,K(+)-ATPase inhibition by SKF-38393 but not by quinpirole was abolished by pertussis toxin. Na+,K(+)-ATPase inhibition was also achieved by guanosine triphosphate analog GTP-gamma-S. SKF-38393 but not quinpirole stimulated phosphoinositide hydrolysis rate in rat aortic slices. SKF-38393-induced phosphoinositide hydrolysis stimulation was reversed by SCH-23390, a dopamine D1 receptor antagonist, and attenuated by pertussis toxin. In conclusion, our observations indicate that dopamine and dopamine receptor agonists inhibit Na+,K(+)-ATPase activity through specific vascular receptors. Dopamine D1 receptors are linked to pertussis toxin sensitive-mechanism(s) and a GTP-binding protein appears to be coupled to the enzyme inhibition. Finally, the inhibition of Na+,K(+)-ATPase activity in response to dopamine D1 receptor activation may be mediated by the phospholipase C signaling pathway.
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PMID:Regulation of Na+,K(+)-ATPase activity by dopamine in cultured rat aortic smooth muscle cells. 881 57

The human ATP1AL1 gene encodes a protein expressed in brain, kidney, and skin and that is highly homologous to the recently cloned nongastric isoforms of H-K-adenosinetriphosphatase H-K-ATPase). We have generated polyclonal antibodies against the protein encoded by ATP1AL1 and used them to monitor the protein's expression and distribution in transfection studies. The protein was retained in the endplasmic reticulum when it was transiently expressed alone in COS cells. In COS cells cotransfected with ATP1AL1 plus gastric H-K-ATPase beta-subunit cDNAs (ATP1AL1-gH-K beta), both proteins reached the surface. Stably transfected lines of HEK 293 cells expressing both of these proteins demonstrate a 86Rb+ uptake activity sensitive to both 2-methyl,8-(phenylmeoxy)imidazo(1,2-a)pyridine 3-acetonitrile (SCH-28080) and ouabain (inhibitory constants of approximately 131 and 42 microM, respectively). Outward proton fluxes were measured in the same cells as the spontaneous intracellular pH (pHi) recovery in Cells loaded with a pH-sensitive dye [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] and subjected to acid loading through an NH4Cl pulse. The cells expressing both the ATP1AL1-encoded protein and the gastric H-K-ATPase beta-subunit possess a net acid extrusion activity that can be inhibited by 1 mM ouabain. Comparison of the 86Rb+ influx and proton efflux, however, does not support equal H+/Rb+ exchange mediated by this pump under the conditions of pHi-monitoring experiments. Moreover, whereas the acid extrusion activity mediated by the pump shows a marked pH dependence, the 86Rb+ uptake activity present in the cells expressing the ATP1AL1-gH-K beta complex cannot be stimulated by acute lowering of pHi. These data suggest that the ATP1AL1-encoded protein is the catalytic alpha-subunit of a human K(+)-dependent ATPase. The possible implications of the discrepancy between 86Rb+ uptake and pHi monitoring data are discussed.
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PMID:Functional expression of the cDNA encoded by the human ATP1AL1 gene. 885 15


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