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)

To determine the effects of oxidant injury on specialized functions of proximal tubular epithelial cells, we determined sodium-dependent uptake of glucose ([alpha-14C]methylglucoside) and phosphate (32Pi) in LLC-PK1 cells after exposure to 0-500 microM hydrogen peroxide. Oxidant stress resulted in significant (P < 0.01) inhibition of glucose and phosphate transport. Decreased transport of glucose and phosphate was associated with marked ATP depletion, decreased activity of the sodium pump as determined by 86Rb uptake, direct inhibition of Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity, and an increase in intracellular sodium content, whereas intracellular potassium content declined. Decreased glucose and phosphate transport, inhibition of 86Rb uptake and Na(+)-K(+)-ATPase activity, and altered intracellular ion content were prevented by catalase and partially prevented by the membrane-permeable iron chelator phenathroline, whereas the slowly membrane-permeable iron chelator deferoxamine had little or no effect. To determine whether oxidant injury could also inhibit transporter function at the membrane level, plasma membrane vesicles were isolated from LLC-PK1 cells exposed to 500 microM hydrogen peroxide. Such membrane vesicles exhibited decreased sodium-dependent glucose transport, whereas sodium-dependent phosphate transport was not altered. We conclude that oxidant injury results in ATP depletion and inactivation of Na(+)-K(+)-ATPase which leads to disruption of the normal ion gradients sufficient to interfere with glucose and phosphate transport. Glucose transport is also inhibited by disruption of transporter activity within the plasma membrane. These alterations are mediated in part by the intracellular generation of an iron-dependent radical.
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PMID:Oxidant-induced alterations in glucose and phosphate transport in LLC-PK1 cells: mechanisms of injury. 821 96

We have previously demonstrated using immunocytochemical, histochemical, and biochemical techniques that ischemia in vivo and ATP depletion in vitro result in dissociation of Na(+)-K(+)-adenosinetriphosphatase (ATPase) from the actin cytoskeleton and redistribution to the apical domain in renal proximal tubule cells. To directly evaluate whether apical Na(+)-K(+)-ATPase retained Na+ pumping activity, a rapidly reversible model of cellular ATP depletion in confluent LLC-PK1 cells grown on semipermeable membranes was utilized. Tight-junction integrity, monitored by electrical resistance, was lost during ATP depletion and reestablished during 2 h of ATP repletion. Total cellular Na(+)-K(+)-ATPase activity and total surface membrane [3H]ouabain binding remained constant, but specific apical [3H]ouabain binding increased (7 vs. 26 fmol/filter, P < 0.01). Apical [3H]ouabain binding returned to baseline during 5 h of ATP repletion. Apically applied ouabain was then used to selectively inhibit apical Na(+)-K(+)-ATPase. It had no effect on apical-to-basolateral Na+ flux under physiological conditions (1.3 +/- 0.61 vs. 1.27 +/- 0.46 meq.filter-1.30 min-1), but it increased the apical-to-basolateral flux in ATP-depleted and then repleted monolayers (0.39 +/- 0.12 vs. 0.83 +/- 0.27 meq.filter-1.30 min-1, P < 0.01), implying that apical Na(+)-K(+)-ATPase retained Na+ pumping activity. Together, these data imply that ATP depletion induces loss of surface membrane polarity resulting in redistribution of functional proteins to the alternate domain.
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PMID:Na(+)-K(+)-ATPase that redistributes to apical membrane during ATP depletion remains functional. 823 49

Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein consisting of an alpha-catalytic and a beta-glycoprotein subunit. Because these two subunits are derived from two separate genes, they may not be synthesized with stoichiometric equivalence. The aim of this study was to estimate relative rates of synthesis and degradation of nascent and mature Na(+)-K(+)-ATPase alpha- and beta-subunits to determine whether either of the nascent subunits accumulates in excess and, if so, the fate of the excess subunits. We studied a pig kidney cell line (LLC-PK1/Cl4) that expresses only alpha 1- and beta 1-subunits. Relative synthesis and degradation rates of nascent subunits were first estimated by pulsing cells for 10 min with [35S]methionine followed by chase periods of up to 120 min and by immunoprecipitation. We found that directly after labeling, beta-subunits were present in threefold excess over alpha-subunits and that nearly 50% of this beta-subunit pool was degraded by 60 min. Nascent alpha-subunits were not degraded during the chase period. In a second strategy to examine relative rates of nascent alpha- vs. beta-subunit accumulation, cells were pulsed for 5-60 min and immunoprecipitated directly (without chase). The rate of accumulation of labeled alpha was greater than that of beta between 5 and 60 min, consistent with the results of the pulse-chase strategy, demonstrating a significant component of degradation of beta during this period. Despite the very different degradation rates of newly synthesized alpha- vs. beta-subunits, the degradation rates of alpha- and beta-subunits beyond 4 h after synthesis were indistinguishable (t0.5 = 10-12 h).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Na(+)-K(+)-ATPase alpha 1- and beta 1-subunit degradation: evidence for multiple subunit specific rates. 838 85

Epithelial cells accumulate distinct populations of membrane proteins at their two plasmalemmal domains. We have examined the molecular signals which specify the differential subcellular distributions of two closely related ion pumps. The Na,K-ATPase is normally restricted to the basolateral membranes of numerous epithelial cell types, whereas the H,K-ATPase is a component of the apical surfaces of the parietal cells of the gastric epithelium. We have expressed full length and chimeric H,K-ATPase/Na,K-ATPase cDNAs in polarized renal proximal tubular epithelial cells (LLC-PK1). We find that both the alpha and beta subunits of the H,K-ATPase encode independent signals that specify apical localization. Furthermore, the H,K-ATPase beta-subunit possesses a sequence which mediates its participation in the endocytic pathway. The interrelationship between epithelial sorting and endocytosis signals suggested by these studies supports the redefinition of apical and basolateral as functional, rather than simply topographic domains.
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PMID:An ion-transporting ATPase encodes multiple apical localization signals. 838 70

This study investigates ischemia-induced degradation of the spectrin-based cytoskeleton in rat brain, heart, and kidney. Spectrin, in conjunction with ankyrin, structurally supports the plasma membrane and sequesters integral membrane proteins. After 60 and 120 min of ischemia, brain tissue displayed both spectrin and ankyrin breakdown. The spectrin fragmentation pattern is similar to previously reported ischemia-induced calpain I proteolysis of spectrin in N-methyl-D-aspartate receptor-containing neurons. Ischemic heart tissue displayed no spectrin or ankyrin degradation. Ischemic renal tissue showed minimal breakdown of spectrin but a major loss of ankyrin (25%/30 min of ischemia) that was essentially complete after 120 min of ischemia. Interestingly, this profound loss of ankyrin in the intact ischemic kidney was not mimicked in three renal cell lines (MDCK, LLC-PK1, and JTC cell lines) exposed to chemical anoxia. Immunocytochemistry showed ankyrin was concentrated in thick ascending limb (cTAL) cells and, although delayed by 30 min, was lost at the same rate as measured by immunoblot analysis. Spectrin and Na(+)-K(+)-ATPase, which complex with ankyrin, were essentially unaffected by ischemia. Ankyrin degradation in cTAL cells correlated with the loss of basal infolding organization. In conclusion, the spectrin-based cytoskeleton is differentially targeted by ischemia-induced degradative processes in different in vivo tissues.
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PMID:Degradation of spectrin and ankyrin in the ischemic rat kidney. 838 46

Na/K-ATPase in renal epithelium is expressed at the basolateral surface and thus is critical for vectorial solute transport. One potential mode of regulation of Na/K-ATPase involves the intracellular effector protein kinase C (PKC). In kidney cell lines, activation of PKC by the phorbol ester phorbol 12,13-dibutyrate (PDBu) (1 microM) inhibited Na/K-ATPase transport activity in OK cells (Vmax decreased 42%; p < 0.02), but not in LLC-PK1 cells. By immunoblot, both cell types expressed detectable levels of PKC alpha and PKC sigma. In response to PDBu, PKC alpha translocated from the cytosol to the membrane fractions of both cell lines. Phorbol ester treatment increased incorporation of 32PO4 in multiple substrates in both cell types, but a approximately 109-kDa substrate with neutral pI was detected only in the OK cell. Anti-LEAVE, directed against a highly conserved sequence in the H4-H5 loop of all known alpha isoforms of Na/K-ATPase, recognized a approximately 109-kDa membrane protein from both cell lines. Anti-LEAVE also identified a protein that comigrated with the large phosphoprotein which was only present in OK cells. Following 32PO4 loading and PDBu treatment, anti-LEAVE immunoprecipitated a approximately 109-kDa phosphoprotein in OK but not LLC-PK1 cells. These data support the notion that PKC is capable of phosphorylating the alpha subunit and inhibiting Na/K-ATPase transport activity in intact renal cells. Furthermore, they suggest that some forms of Na/K-ATPase in the kidney are not susceptible to PKC phosphorylation and that this heterogeneity may contribute to response diversity.
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PMID:Heterogeneity of protein kinase C-mediated rapid regulation of Na/K-ATPase in kidney epithelial cells. 839 56

Cadmium (Cd) uptake from the apical and basolateral membranes was investigated in LLC-PK1 cells grown as a monolayer on a permeable membrane. The cells were incubated at 37 degrees C for 1 hr with 1 microM CdCl2 from either the apical or the basolateral side. The accumulation of Cd from the apical side was 23% higher than that from the basolateral side. However, the translocation of Cd from the apical to the basolateral side and vice versa were similar. Cytotoxicity, as evaluated by transepithelial electrical resistance (TER), was undetectable at 1 microM Cd concentration. The preincubation of cells with carbonylcyanide p-(trifluoromethoxy)-phenylhydrazone (a metabolic inhibitor) and ouabain (a Na+/K+ - ATPase inhibitor) or coincubation with Cd and 2,4-dinitrophenol (a metabolic inhibitor) decreased both the accumulation (16-24%) and the translocation of Cd (22-25%) from the apical side, but not from the basolateral side. Incubation of cells with 50 and 75 microM CdCl2 for 1 hr resulted in 52-112% higher accumulation and translocation of Cd from the basolateral than from the apical side. The TER decreased at high Cd concentrations, suggesting that Cd concentrations of 10 microM and higher were cytotoxic. It is concluded that uptake of Cd from both the apical and the basolateral membranes represents passive diffusion. Additionally, under nontoxic conditions, about 20% of the Cd taken up at the apical membrane reflects carrier-mediated transport involving sodium ion- and energy-dependent processes; this accounts for higher accumulation through the apical membrane.
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PMID:Comparison of cadmium uptakes from apical and basolateral membranes of LLC-PK1 cells. 866 56

We have utilized polarized epithelial cells stably expressing neurotransmitter transporters to analyze the sorting behavior of these membrane proteins. The transporters for serotonin (5-HT), dopamine (DA), and norepinephrine (NE) are expected to be present in situ in the most distal extremities of axonal membranes, where they terminate the action of their biogenic amine substrates. Both Madin-Darby canine kidney (MDCK) and LLC-PK1 cells were stably transfected with cDNAs encoding either the rat 5-HT transporter (SERT), the human NE transporter (NET), or the rat or human DA transporter (DAT). These cells were grown on permeable filter supports, and the transporters were localized by three independent techniques. Confocal immunofluorescence microscopy indicated that each of the transporters expressed in LLC-PK1 cells was sorted to the basolateral membrane, co-localizing with the Na+/K+-ATPase. In MDCK cells, however, DAT was located primarily on the apical surface, while SERT and NET were found on the basolateral membranes. Cell surface biotinylation using an impermeant biotinylating reagent confirmed the immunocytochemistry results. Thus, SERT and NET in MDCK cells were labeled more efficiently from the basolateral medium than the apical medium, and DAT in MDCK cells was labeled more efficiently from the apical side than the basolateral side. Transport measurements in transfected MDCK cells agreed with the immunocytochemistry and biotinylation results. These results suggest the existence of cell-specific mechanisms that discriminate between neurotransmitter transporters for surface expression and render unlikely any simple hypothesis that sorting mechanisms in neurons and epithelia are identical.
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PMID:Cell-specific sorting of biogenic amine transporters expressed in epithelial cells. 866 73

Adenosine A1 receptor densities were increased in cultured LLC-PK1 and OK cells by chronic treatment with the adenosine receptor antagonists 1,3,7-trimethylxanthine (caffeine, 1 mM) and 1,3-dimethyl-8-cyclopentylxanthine [cyclopentyltheophylline (CPT), < or = 0.4 mM], respectively. The A1 receptor number per cell was increased twofold by 10-day treatments with 1 mM caffeine or 0.1 mM CPT, and the sodium-coupled glucose uptake was augmented twofold by 1 mM caffeine and sevenfold by 0.1 microM CPT (higher doses of CPT were progressively less stimulatory). Glucose uptake was blocked by acute (2-h) treatment with CPT, adenosine deaminase, or calphostin C. Caffeine (1 mM) or CPT (> or = 0.1 mM) inhibited cell proliferation for the first 10 days, then cell growth assumed a normal proliferative rate despite continued presence of antagonist. Cytosolic protein kinase C (PKC) beta-isoform immunoactivity and PKC-beta II mRNA were elevated at least twofold during 10 days of 0.1 mM CPT or 1 mM caffeine treatment. The sustained elevation in sodium-glucose symport and PKC activity observed with adenosine receptor antagonists was similar to acute (2-h) effects of the adenosine A1 agonist R(-)-N6-phenylisopropyladenosine (R-PIA, 0.1-1 microM). Moreover, cell proliferation was increased by adenosine (0.1 microM R-PIA), whereas Na-K-adenosinetriphosphatase activity was unaltered with chronic antagonist or acute adenosine treatments. Caffeine treatment may have some non-adenosine A1 receptor-mediated actions, because it slightly (30%) augmented protein kinase A activity. It is concluded that chronic exposure of proximal tubule cells to caffeine or CPT augments PKC and sodium-glucose transport but retards cell proliferation mainly via adenosine A1 receptor-mediated mechanisms.
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PMID:Upregulated renal adenosine A1 receptors augment PKC and glucose transport but inhibit proliferation. 877 86

All-trans-retinoic acid (atRA) is a regulator of cellular growth and differentiation. We investigated whether atRA can upregulate Na(+)-dependent cotransporters in opossum kidney (OK) cells and thus increase uptake from tubular fluid of several solutes needed for growth during early stages of ontogenesis. In OK cells, incubation with atRA for 24 h increased the Na+ gradient-dependent cotransports of phosphate, L-proline, L-glutamic acid, and SO(4)2- by a similar degree (approximately 40%) that was prevented by pretreatment with actinomycin D. In contrast, activities of other Na(+)-dependent transporters, Na(+)-K(+)-adenosinetriphosphatase, gamma-glutamyltranspeptidase, and leucine aminopeptidase, were unchanged by atRA. Cell proliferation determined by [3H]thymidine incorporation was not increased by atRA. The stimulatory effects of atRA and phosphate deprivation on Na(+)-Pi cotransport demonstrated additivity, whereas the combination of atRA and 3,5,3'-triiodothyronine did not. atRA stimulated Na(+)-Pi cotransport in LLC-PK1 cells with an analogous time course and to a similar extent as observed in OK cells. We conclude that atRA stimulates several Na(+)-dependent cotransporters via a genomic mechanism and may represent a synchronous adaptation to nutritional requirements of early phases of ontogenesis.
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PMID:Pleiotropic upregulation of Na(+)-dependent cotransporters by retinoic acid in opossum kidney cells. 932 17


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