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)

Ouabain is a plant-derived cardiac glycoside that inhibits the catalytic activity of Na(+),K(+)-ATPase (sodium pump; NKA). Dihydroouabain, a derivative of ouabain with a reduced lactone ring, is commonly used as a sodium pump antagonist. It has been assumed that commercially available dihydroouabain is homogeneous. We now report that preparations of dihydroouabain contain two components each with a different potency for inhibition of sodium pump activity. We used reverse-phase HPLC chromatography, UV spectrophotometry, electrospray ionization-mass spectrometry (ESI-MS), nuclear magnetic resonance (NMR) spectroscopy and two independent bioassays to characterize these compounds. The two dihydroouabain fractions (Dho-A and Dho-B) resolved by 3 min chromatographically, had UV absorbance maxima at 196 nm, and comprised 37% and 63% of the stock dihydroouabain, respectively. The molar potency of each component for inhibition of NKA from porcine cerebral cortex differed by 4. 4-fold (Dho-A, IC(50) = 7.13 +/- 0.8 microM; Dho-B, IC(50) = 1.63 +/- 0.12 microM). The relative potencies were 9% and 40% of those of ouabain, respectively. A similar pattern for phosphorylation of NKA was observed. Mass spectrometry (ESI-MS) and fragmentation patterns are consistent with Dho-A and Dho-B being isomers of identical molecular mass (587 Da) and each with six hydroxyl groups, a deoxyhexose sugar moiety and a lactone ring. Furthermore, NMR spectroscopy revealed structural differences between Dho-A and Dho-B by displaying noticeably different chemical shifts at only two groups of proton resonances assigned to H-21 and H-22. The ESI-MS and NMR results confirm the presence of the isomerism at C20 of the lactone ring. Our results demonstrate the existence of two molecular forms of dihydroouabain, each with a different biological potency. These findings underscore the importance of characterizing the purity of dihydroouabain commercial preparations. It also provides possible molecular models for investigating the metabolism of endogenous ouabain-like factors recently reported in mammals.
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PMID:Two biologically active isomers of dihydroouabain isolated from a commercial preparation. 1056 63

Inhibition of Na(+),K(+)-ATPase (NKA) activity in renal epithelial cells by activation of G protein-coupled receptors is mediated by phosphorylation of the catalytic alpha-subunit followed by endocytosis of active molecules. We examined whether agonists that counteract this effect do so by dephosphorylation of the alpha-subunit or by preventing its internalization through a direct interaction with the endocytic network. Oxymetazoline counteracted the action of dopamine on NKA activity, and this effect was achieved not by preventing alpha-subunit phosphorylation, but by impaired endocytosis of alpha-subunits into clathrin vesicles and early and late endosomes. Dopamine-induced inhibition of NKA activity and alpha-subunit endocytosis required the interaction of adaptor protein 2 (AP-2) with the catalytic alpha-subunit. Phosphorylation of the alpha-subunit is essential because dopamine failed to promote such interaction in cells lacking the protein kinase C phosphorylation residue (S18A). Confocal microscopy confirmed that oxymetazoline prevents incorporation of NKA molecules into clathrin vesicles by inhibiting the ability of dopamine to recruit clathrin to the plasma membrane. Dopamine decreased the basal levels of inositol hexakisphosphate (InsP(6)), whereas oxymetazoline prevented this effect. Similar increments (above basal) in the concentration of InsP(6) induced by oxymetazoline prevented AP-2 binding to the NKA alpha-subunit in response to dopamine. In conclusion, inhibition of NKA activity can be reversed by preventing its endocytosis without altering the state of alpha-subunit phosphorylation; increased InsP(6) in response to G protein-coupled receptor signals blocks the recruitment of AP-2 and thereby clathrin-dependent endocytosis of NKA.
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PMID:G protein-coupled receptors regulate Na+,K+-ATPase activity and endocytosis by modulating the recruitment of adaptor protein 2 and clathrin. 1071 25

Na,K-ATPase (NKA, Na-pump), an alphabeta heteromer, is the receptor for cardiac glycosides (CG) which exert a positive inotropic effect by inhibiting enzyme activity, decreasing the driving force for Na,Ca-exchange (NCX) and increasing cellular content and release of Ca2+ during depolarization. Our previous study of regional distribution of NKA in non-failing human hearts demonstrated that Na-pump alpha2-, alpha3- and beta1-isoforms were 30-50% lower in right atrium (RA) compared with left ventricle (LV), resulting in overall lower NKA activity and CG binding site number and increased sensitivity to inotropic stimulation. In failing human heart LV Na-pump alpha1, alpha3 and beta1 proteins were reduced 30-40%, with no change in alpha2 or NCX; NKA activity and CG binding sites decreased 40%, and sensitivity to inotropic stimulation increased, all compared to LV from non-failing hearts. In this study we investigated the influence of region specific factors (e.g. hemodynamics) on the regulation of NKA isoform and NCX expression in heart failure by comparing the pattern of change in right atrial myocardium during heart failure with that previously determined for LV. In RA samples from failing hearts, alpha1-, alpha2- and beta1-isoform protein expression were decreased by 40, 50 and 25%, respectively, with no significant change in alpha3 or NCX levels relative to non-failing hearts (both n= 12). Thus, alphabeta1 decreases in both RA and LV during heart failure, while alpha2beta1 is reduced only in RA and alpha3beta1 only in LV. This indicates that there are not only regional differences in normal cardiac Na-pump isoform expression but also regional differences in the pattern of isoform expression as a function of failure that may have distinct functional consequences in the adaptive process of heart failure. The mechanisms underlying Na,K-ATPase regulation and effect of hemodynamics remain to be investigated.
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PMID:Region specific regulation of sodium pump isoform and Na,Ca-exchanger expression in the failing human heart--right atrium vs left ventricle. 1135 98

Elevated arterial blood pressure is a common heritable susceptibility in the human population. The high penetrance of this trait in industrialized societies may be influenced by the interactions of environmental factors and common genetic variants. This review examines the role of the renal sodium pump (sodium, potassium-ATPase, NKA) in hypertension and its integration into mechanisms of body sodium balance. In particular, renal NKA provides an appealing target by which inherited factors caninfluence renal sodium reabsorption. Recent work has indicated how some such genetic mechanisms may function. In this paper, the capacity of renal NKA to integrate environmental and heritable factors to increase blood pressure are examined.
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PMID:Abnormalities of sodium pump function in hypertension and the role of endogenous cardiotonic steroids. 1135

Several hydroindenic derivatives (7a-methyl-2,3,5,6,7,7a-hexahydro-1H-indenes), bearing an amidinohydrazone at C-5 and different moieties at C-1, have been synthesized and evaluated for their inotropic and chronotropic effects on right- and left-guinea-pig-atria activity. Three of them showed the same profile as digoxin, although with lower potency. The effect on Na(+),K(+)-ATPase (NKA) was also evaluated for these three compounds, observing that two of them, with the same absolute configuration as natural cardenolides, are also NKA inhibitors, while the compound with the opposite configuration lacks such an effect. More interestingly, both active compounds act without affecting the cardiac rhythm. This could be related to the selective inhibition of the human alpha2beta1 isozyme (associated with the inotropic effect) with respect to the alpha1beta1 isozyme (associated with the maintenance of basal ionic levels in the cell and the toxic effect of cardenolides).
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PMID:Inotropic activity of hydroindene amidinohydrazones. 1175 84

Several vasoconstrictor agents can regulate the phosphorylation status of the Na(+)-K(+) ATPase (NKA). We have recently demonstrated that mammalian tissues contain an endogenous bufadienolide, digitalis-like alpha(1)-NKA-selective ligand, marinobufagenin (MBG). Protein kinase C induces phosphorylation of the alpha(1)-NKA isoform, the major isoform in vascular smooth muscle, kidney, and heart cells. We hypothesized that protein kinase C-induced phosphorylation of NKA can potentiate the effect of endogenous digitalis-like ligands, and that such potentiation can occur in an NKA isoform-specific fashion. A protein kinase C activator, phorbol 12,13-diacetate (PDA, 50 nmol/L), induced phosphorylation of the alpha1-NKA from human mesenteric artery (HMA) sarcolemma and rat kidney but not that of the alpha(3)-NKA from rat fetal brain. In HMA sarcolemma, which predominantly contains alpha(1)-NKA, PDA (50 nmol/L) potentiated the NKA-inhibitory effect of MBG at the level of high-affinity binding sites (0.05 +/- 0.03 nmol/L versus 4.0 +/- 1.7 nmol/L, P<0.05). In contrast, PDA did not affect the NKA inhibition by ouabain, an alpha(3)-NKA ligand. In isolated endothelium-denuded HMA artery rings, 50 nmol/L PDA potentiated the MBG-induced vasoconstriction (EC(50), 17 +/- 6 nmol/L versus 150 +/- 40 nmol/L; P<0.01). Our results suggest that alpha(1)-isoform-specific NKA inhibition by the endogenous digitalis-like ligand, MBG, is substantially enhanced via NKA phosphorylation by protein kinase C. Thus, an interaction of protein kinase C-dependent phosphorylation and MBG on NKA activity may underlie the synergistic vasoactive effects of MBG and other endogenous vasoconstrictors in hypertension.
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PMID:Phorbol diacetate potentiates na(+)-k(+) ATPase inhibition by a putative endogenous ligand, marinobufagenin. 1184 1

Our laboratory has shown that dopamine D(2)-like receptor activation causes stimulation of Na(+), K(+)-ATPase (NKA) activity in the proximal tubules of the rat kidney. The present study was designed to investigate the cellular signaling mechanisms mediating this response to D(2)-like receptor activation. We measured the stimulation of NKA activity by bromocriptine (D(2)-like receptor agonist) in the absence and presence of PD-98059 [p44/42 mitogen-activated protein kinase (MAPK) kinase inhibitor] and genistein (tyrosine kinase inhibitor) in renal proximal tubules. Both agents inhibited bromocriptine-mediated stimulation of NKA, suggesting the involvement of p44/42 MAPK and tyrosine kinase in this response. Additionally, we found that bromocriptine increased the phosphorylation of p44/42 MAPK in the proximal tubules, which was blocked by PD-98059 and genistein. These results show that D(2)-like receptor activation causes stimulation of NKA activity by means of a tyrosine kinase-p44/42 MAPK pathway in the proximal tubules of the kidney.
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PMID:Role of tyrosine kinase and p44/42 MAPK in D(2)-like receptor-mediated stimulation of Na(+), K(+)-ATPase in kidney. 1188 Mar 31

The dynamics of branchial Na(+),K(+),2Cl(-) cotransporter (NKCC) and Na(+),K(+)-ATPase (NKA) expression were investigated in brown trout and Atlantic salmon during salinity shifts and the parr-smolt transformation, respectively. In the brown trout, Western blotting revealed that NKCC and NKA abundance increased gradually and in parallel (30- and ten-fold, respectively) after transfer to seawater (SW). The NKA hydrolytic activity increased ten-fold after SW-transfer. Following back-transfer to fresh water (FW), the levels of both proteins and NKA activity decreased. The NKCC immunostaining in the gill of SW-acclimated trout was strong, and mainly localized in large cells in the filament and around the bases of the lamellae. In FW-acclimated trout, immunostaining was less intense and more diffuse. Partial cDNAs of the secretory NKCC1 isoform were cloned and sequenced from both brown trout and Atlantic salmon gills. Two differently sized transcripts were detected by Northern blotting in the gill but not in other osmoregulatory tissues (kidney, pyloric caeca, intestine). The abundance in the gill of these transcripts and of the associated NKCC protein increased four- and 30-fold, respectively, during parr-smolt transformation. The abundance of NKA alpha-subunit protein also increased in the gill during parr-smolt transformation though to a lesser extent than enzymatic activity (2.5- and eight-fold, respectively). In separate series of in vitro experiments, cortisol directly stimulated the expression of NKCC mRNA in gill tissue of both salmonids. The study demonstrates the coordinated regulation of NKCC and NKA proteins in the gill during salinity shifts and parr-smolt transformation of salmonids.
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PMID:Dynamics of Na(+),K(+),2Cl(-) cotransporter and Na(+),K(+)-ATPase expression in the branchial epithelium of brown trout (Salmo trutta) and Atlantic salmon (Salmo salar). 1211 7

Postischaemic acute renal failure (ARF) is influenced by sex. Na(+), K(+)-ATPase (NKA) plays a crucial role in the pathogenesis of postischaemic ARF. We tested the impact of sex on mRNA, protein expression, cellular distribution and enzyme activity of NKA following renal ischaemia-reperfusion (I-R) injury. The left renal pedicle of uninephrectomized female (F) and male (M) Wistar rats was clamped for 55 min followed by 2 h (T2) and 16 h (T16) of reperfusion. Uninephrectomized, sham-operated F and M rats served as controls (n= 6 per group). Blood urea nitrogen, serum creatinine and renal histology were evaluated to detect the severity of postischaemic ARF. mRNA expression of NKA alpha1 and beta1 subunits were detected by RT-PCR. The effect of I-R on cellular distribution was compared by Triton X-100 extraction. Cellular proteins were divided into Triton-insoluble and Triton-soluble fractions and assessed by Western blot. NKA enzyme activity was also determined. After the ischaemic insult blood urea nitrogen and serum creatinine were higher and renal histology showed more rapid progression in M versus F (P < 0.05). mRNA expression of the NKA alpha1 subunit decreased in I-R groups versus controls, but was higher in F versus M both in control and I-R groups (P < 0.05). However, protein levels of the NKA alpha1 subunit in total tissue homogenate did not differ in controls, but were higher in F versus M in I-R groups (P < 0.05). Triton X-100 extractability was lower in F versus M at T16 (P < 0.05). NKA enzyme activity was the same in controls, but was higher in F versus M in I-R groups (T2: 14.9 +/- 2.3 versus 9.15 +/- 2.21 U) (T16: 11.7 +/- 4.1 versus 5.65 +/- 2.3 U; P < 0.05). mRNA and protein expression of the NKA beta1 subunit did not differ between F and M in any of the protocol. We concluded that NKA is more protected from the detrimental effects of postischaemic injury in females. Higher mRNA and protein expression of the NKA alpha1 subunit and higher enzyme activity might be additional contributing factors to the improved postischaemic renal function of female rats.
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PMID:Sex differences in the alterations of Na(+), K(+)-ATPase following ischaemia-reperfusion injury in the rat kidney. 1467 89

Patients treated with glucocorticoids have elevated skeletal muscle ouabain binding sites. The major Na(+)-K(+)-ATPase (NKA) isoform proteins found in muscle, alpha2 and beta1, are increased by 50% in rats treated for 14 days with the synthetic glucocorticoid dexamethasone (DEX). This study addressed whether the DEX-induced increase in the muscle NKA pool leads to increased insulin-stimulated cellular K+ uptake that could precipitate hypokalemia. Rats were treated with DEX or vehicle via osmotic minipumps at one of two doses: 0.02 mg.kg(-1).day(-1) for 14 days (low DEX; n = 5 pairs) or 0.1 mg.kg(-1).day(-1) for 7 days (high DEX; n = 6 pairs). Insulin was infused at a rate of 5 mU.kg(-1).min(-1) over 2.5 h in conscious rats. Insulin-stimulated cellular K+ and glucose uptake rates were assessed in vivo by measuring the exogenous K+ infusion (K+(inf)) and glucose infusion (Ginf) rates needed to maintain constant plasma K+ and glucose concentrations during insulin infusion. DEX at both doses decreased insulin-stimulated glucose uptake as previously reported. Ginf (in mmol.kg(-1).h(-1)) was 10.2 +/- 0.6 in vehicle-treated rats, 5.8 +/- 0.8 in low-DEX-treated rats, and 5.2 +/- 0.6 in high-DEX-treated rats. High DEX treatment also reduced insulin-stimulated K+) uptake. K+(inf) (in mmol.kg(-1).h(-1)) was 0.53 +/- 0.08 in vehicle-treated rats, 0.49 +/- 0.14 in low-DEX-treated rats, and 0.27 +/- 0.08 in high-DEX-treated rats. DEX treatment did not alter urinary K+ excretion. NKA alpha2-isoform levels in the low-DEX-treated group, measured by immunoblotting, were unchanged, but they increased by 38 +/- 15% (soleus) and by 67 +/- 3% (gastrocnemius) in the high-DEX treatment group. The NKA alpha1-isoform level was unchanged. These results provide novel evidence for the insulin resistance of K+ clearance during chronic DEX treatment. Insulin-stimulated cellular K+ uptake was significantly depressed despite increased muscle sodium pump pool size.
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PMID:Dexamethasone treatment causes resistance to insulin-stimulated cellular potassium uptake in the rat. 1521 56


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