Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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).
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PMID:K+ reabsorption by the lower Malpighian tubule of Rhodnius prolixus: inhibition by Ba2+ and blockers of H+/K+-ATPases 931 3

Using a comparative approach, the mechanisms involved in maintenance of the transmembrane K+ activity gradients in the larval body-wall muscles of two insects, Phormia terraenovae (Diptera) and Spodoptera exigua (Lepidoptera), have been investigated. Double-barrelled K+-selective microelectrodes were used to obtain simultaneous measurements of intracellular K+ activity and membrane potential, whilst ordinary microelectrodes were used to monitor input resistance. By application of a variety of general metabolic blockers, the K+ gradients in both P. terraenovae and S. exigua muscle were found to be maintained, at least in part, by a metabolic component. Differences in sensitivity to dinitrophenol of the two insects suggested that the ATP-dependence of maintenance of the K+ gradient was significantly higher in P. terraenovae than in S. exigua. Vanadate sensitivity suggested that both insects possess P-type ATPases. The K+ activity gradient in P. terraenovae muscles was also found to be ouabain-sensitive, indicating the involvement of a Na+/K+-ATPase. In contrast, the K+ gradient in S. exigua muscles proved to be totally insensitive to ouabain but sensitive to amiloride. Application of the H+/K+-ATPase-specific inhibitor SCH 28080 suggested the presence of an H+/K+ pump similar to the mammalian gastric H+/K+-ATPase in the lepidopteran muscles. P. terraenovae muscles, however, were found to be totally insensitive to this inhibitor. Using the anion (Cl-)-dependent transport inhibitors bumetanide and SITS (4-acetamide-4-isothiocyanostilbene-2,2-disulphonic acid), P. terraenovae muscles were shown not to possess a Cl--dependent K+ transport mechanism. In contrast, a bumetanide-sensitive K+/Cl- cotransporter was likely to be involved in maintenance of the K+ gradient in S. exigua muscle. An additional SITS-sensitive Cl-/HCO3- exchanger could also have some indirect involvement in K+ maintenance through regulation of the inward Cl- gradient. The results are integrated in two ionic models, one for each insect, which could account for the bulk of K+ transport in the body-wall muscles of these insects.
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PMID:Maintenance of the K+ activity gradient in insect muscle compared in diptera and lepidoptera: contributions of metabolic and exchanger mechanisms 931 70

To characterize and localize a K+/H+ antiport mechanism in the renal medullary thick ascending limb (MTAL), membrane vesicles were isolated from a rat MTAL homogenate. K+/H+ antiport (in > out H+ gradient-stimulated 86Rb+ uptake) was abolished by barium and verapamil (apparent Ki of 55 microM) but unaffected by other K+ channel blockers such as quinidine and high amiloride concentrations. SCH 28080, a H+/K+-ATPase blocker, did not affect K+/H+ antiport. K+/H+ antiport activity was correlated positively with the enrichment factor of the membranes in the apical marker enzyme alkaline phosphatase (r = 0.875, p < 0.01) and negatively correlated with the enrichment factor in basolateral Na+/K+-ATPase (r = -0.665, p < 0.05). Moreover, a functional interaction occurred with Na+/H+ exchange (NHE) consistent with colocation of K+/H+ antiport and apical NHE-3, not basolateral NHE-1. K+/H+ antiport was shown by intracellular pH measurements to be inhibited by arginine vasopressin and 8-bromo-cAMP through cAMP-dependent protein kinase (protein kinase A) activation. These results demonstrate the presence of a K+/H+ antiport mechanism, which is inhibited by arginine vasopressin via protein kinase A, in the apical membrane of the MTAL.
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PMID:Apical location and inhibition by arginine vasopressin of K+/H+ antiport of the medullary thick ascending limb of rat kidney. 932 90

K-dependent H+ extrusion was investigated using fluorescence techniques in rabbit cortical collecting tubules (CCTs). Experiments were performed in split-open tubules from normal animals exposed to the intracellular pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). This preparation permitted the study of individual intercalated cells (ICs). In the ICs, partial recovery of pH(i) was observed in response to an acute acid load upon readdition of 5 mM K to the superfusate. This recovery was SCH 28080-inhibitable (10(-5) M) and ouabain-insensitive suggesting the process is mediated by a gastric-type H-K ATPase. To see if H-K ATPase plays a role in acid secretion its function was evaluated under chronic metabolic acidosis (CMA) conditions. CMA was induced by replacing drinking water with 75 mM NH4Cl in 5% sucrose for 10-14 days. The SCH 28080-inhibitable K-dependent pH(i) recovery rate was three-fold higher in CMA ICs compared to controls. To determine the location of the H-K ATPase, CCTs were microperfused and individual peanut lectin binding (PNA) ICs studied. K-dependent pH(i) recovery was measured in response to an NH4Cl pulse. An apical SCH 28080-inhibited K-dependent pH(i) recovery process was observed in control and CMA ICs. Taken together these data confirm the existence of a gastric-type H-K ATPase in ICs of rabbit CCT. Based on our findings the H-K ATPase is found on the apical side of the cell and is stimulated under conditions of CMA.
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PMID:Characterization and regulation of H-K-ATPase in intercalated cells of rabbit cortical collecting duct. 939 65

The alpha subunit of eukaryotic P-type ATPases has ten experimentally defined transmembrane or membrane inserted segments. The fifth and sixth of these are short, not predicted by hydropathy analysis, do not insert independently into microsomal membranes, and are readily removed after tryptic digestion and therefore may be membrane inserted sequences. Acid transport by the gastric H, K-ATPase is covalently inhibited by several substituted pyridyl methylsulfinyl benzimidazoles, such as omeprazole. These act as probes of accessible extracytoplasmic thiols because they are accumulated in the acid transporting gastric vesicles and then convert to thiol reactive, cationic tetracyclic sulfenamides. Inhibition is due mainly to disulfide formation with Cys813 or Cys822 in M5/6 and perhaps with a contribution from Cys892 in the loop between transmembrane segment (TM) 7 and TM8. Identification of the specific cysteine responsible for inhibition should be able to define the turn between M5 and M6. The gastric H,K-ATPase alpha-beta heterodimer was expressed as a fusion protein in HEK 293 cells. Transient transfection resulted in most of the protein being retained in the endoplasmic reticulum with only core glycosylation and minor activity of the ATPase evident. Stable transfection resulted in plasma membrane localization of the protein and complex glycosylation. The transfected but not the control cells displayed cation-stimulated, SCH 28080-inhibited ATPase activity and SCH 28080- and omeprazole-inhibited 86Rb uptake. The two cysteines in M5/6 and Cys892 in the TM7/8 loop were mutated to the amino acids found in the Na,K-ATPase in order to determine which of the three cysteine residues were important for benzimidazole inhibition. Mutation of one, two, or all three cysteines did not alter enzyme activity, 86Rb transport, or SCH 28080 inhibition. Only removal of Cys822 blocked omeprazole inhibition of 86Rb transport. These data suggest that Cys822 is present in a region of the enzyme most easily accessed by the cationic sulfenamide formed by omeprazole, presumably the turn between M5 and M6.
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PMID:Identification of the site of inhibition by omeprazole of a alpha-beta fusion protein of the H,K-ATPase using site-directed mutagenesis. 959 13

In the reaction cycle of P-type ATPases, an acid-stable phosphorylated intermediate is formed which is present in an intracellularly located domain of the membrane-bound enzymes. In some of these ATPases, such as Na+,K+-ATPase and gastric H+, K+-ATPase, extracellular K+ ions stimulate the rate of dephosphorylation of this phosphorylated intermediate and so stimulate the ATPase activity. The mechanism by which extracellular K+ ions stimulate the dephosphorylation process is unresolved. Here we show that three mutants of gastric H+,K+-ATPase lacking a negative charge on residue 820, located in transmembrane segment six of the alpha-subunit, have a high SCH 28080-sensitive, but K+-insensitive ATPase activity. This high activity is caused by an increased 'spontaneous' rate of dephosphorylation of the phosphorylated intermediate. A mutant with an aspartic acid instead of a glutamic acid residue in position 820 showed hardly any ATPase activity in the absence of K+, but K+ ions stimulated ATPase activity and the dephosphorylation process. These findings indicate that the negative charge normally present on residue 820 inhibits the dephosphorylation process. K+ ions do not stimulate dephosphorylation of the phosphorylated intermediate directly, but act by neutralizing the inhibitory effect of a negative charge in the membrane.
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PMID:Constitutive activation of gastric H+,K+-ATPase by a single mutation. 960 85

1. Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N-formyl-Met-Leu-Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pHi) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pHi and migration of fMLP stimulated PMNs. 2. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling. 3. Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling. 4. Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP. 5. H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV. 6. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H+ exchanger. In fMLP-stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited. 7. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole. 8. Western analysis with a monoclonal anti gastric H+/K+ ATPase beta-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs. 9. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+-dependent H+-extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.
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PMID:Effect of inhibitors of Na+/H+-exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes. 969 Aug 53

Dopamine (DA) and fencamfamine (FCF) modulatory action on Na,K-ATPase and Mg-ATPase activity were evaluated in rat striatum. DA and FCF induced a decrease in Na,K-ATPase, without affecting Mg-ATPase activity. The effect of FCF was dose-dependent from 10 to 100 microM, with an IC50 of 4.7 x 10(-5) M. Furthermore, the effect of FCF (100 microM) increasing AMPc levels, but not GMPc, was nonadditive with that of DA (10 microM), which is consistent to a common site of action. The 8-bromo-cyclic AMP also induced a specific reduction in the Na,K-ATPase activity. The reduction of Na,K-ATPase induced by FCF (100 microM) was blocked by either SCH 23390 or sulpiride, which are D1 and D2 receptor antagonists. The decrease in striatal NA,K-ATPase activity induced by FCF was blocked by KT 5720, a selective inhibitor of cyclic AMP-dependent protein kinase (PKA), but not by KT 5823, a selective inhibitor of cyclic GMP-dependent protein kinase (PKG). Otherwise, KT 5720 or KT 5823 did not produce any change in Na,K-ATPase or Mg-ATPase activity. These data suggest that FCF reduces Na,K-ATPase activity through cyclic AMP-dependent changes in protein phosphorylation via a PKA mechanism.
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PMID:Fencamfamine modulates sodium, potassium-ATPase through cyclic AMP and cyclic AMP-dependent protein kinase in rat striatum. 982 1

We recently demonstrated that the ratio between colonic K+ absorptive and K+ secretive pathways was higher in infant than in adult rats. To test the hypothesis that hormones selectively affect these pathways during ontogeny we examined the effect of adrenergic agonists on cellular K+ uptake in distal colon from infant (10-day-old) and adult (50-day-old) rats. Here we describe that adrenaline (10(-5) M) increased total and ouabain-insensitive 86Rb uptake in both age groups, but it did not affect ouabain-sensitive 86Rb uptake. This stimulation was more pronounced in adult than in infant rats. The effect of adrenaline was mediated via beta-adrenergic receptors. Incubation in vitro with beta-agonist, isoproterenol, stimulated SCH-28080-sensitive, i.e. H+, K(+)-ATPase-dependent, 86Rb uptake in adult but not in infant rats. The threshold dose of beta-agonist was at 10(-7) M, and the maximal activation was observed at 10(-5) M. In vivo inhibition of beta-adrenergic system with propranolol caused a significant decrease in H+, K(+)-ATPase-dependent 86Rb uptake in infant but not in adult colon. In conclusion, this study suggests that the higher colonic K+ absorption in infant rats may be as a result of a selective beta-adrenergic up-regulation leading to stimulation of the apical H+, K(+)-ATPase.
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PMID:Beta-adrenergic stimulation of cellular K+ uptake in rat distal colon. 985 19

Gastric H+,K+-ATPase can be inhibited by imidazo pyridines like 2-methyl-8-[phenylmethoxy] imidazo-(1,2a) pyridine 3-acetonitrile (SCH 28080). The drug shows a high affinity for inhibition of K+-activated ATPase and for prevention of ATP phosphorylation. The inhibition by SCH 28080 can be explained by assuming that SCH 28080 binds to both the E2 and the phosphorylated intermediate (E2-P) forms of the enzyme. We observed recently that some mutants, in which glutamic acid 820 present in transmembrane domain six of the catalytic subunit had been replaced (E820Q, E820N, E820A), lost their K+-sensitivity and showed constitutive ATPase activity. This ATPase activity could be inhibited by similar SCH 28080 concentrations as the K+-activated ATPase of the wild-type enzyme. SCH 28080 also inhibited ATP phosphorylation at 21 degrees C of the mutants E820D, E820N, and E820A, although with varying efficacy and affinity. ATP-phosphorylation of mutant E820Q was not inhibited by SCH 28080; in contrast, the phosphorylation level at 21 degrees C was nearly doubled. These findings can be explained by assuming that mutation of Glu820 favors the E1 conformation in the order E820Q >E820A >E820N >wild-type = E820D. The increase in the phosphorylation level of the E820Q mutant can be explained by assuming that during the catalytic cycle the E2-P intermediate forms a complex with SCH 28080. This intermediate hydrolyzes considerably slower than E2-P and thus accumulates. The high tendency of the E820Q mutant for the E1 form is further supported by experiments showing that ATP phosphorylation of this mutant is rather insensitive towards vanadate, inorganic phosphate, and K+.
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PMID:Conformation-dependent inhibition of gastric H+,K+-ATPase by SCH 28080 demonstrated by mutagenesis of glutamic acid 820. 1005 39


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