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)

The Na(+)-dependent hexose carrier, an endogenous apical marker, develops during differentiation of LLC-PK1, an established cell line with characteristics of the proximal tubule. This development was inhibited by the microtubule-disrupting drugs, colchicine and nocodazole, while it was insensitive to lumicolchicine. This strongly suggests that microtubules are involved in the plasma membrane expression of the Na(+)-dependent hexose carrier. We also analyzed the increase in activity of endogenous apical and basolateral membrane proteins during the polarization process. The development of three apical (Na(+)-dependent hexose carrier, gamma-glutamyltransferase and alkaline phosphatase) and one basolateral membrane protein (Na+/K(+)-ATPase) was studied during the reorganization of LLC-PK1 cells into a polarized epithelium. Colchicine inhibited the rapid, transient increase in the expression of the Na(+)-dependent hexose carrier during this polarization process. A similar result was observed for the development of the other apical proteins, while the development of Na+/K(+)-ATPase seemed to be largely insensitive to colchicine. Our results are in agreement with the model that the vesicles containing the apical membrane proteins use microtubules as tracks to reach the plasma membrane. The transport of vesicles containing basolateral membrane proteins clearly occurs by a different pathway which is independent on an intact microtubular network. Since the inhibition by the microtubule-disrupting drugs was complete, it can be concluded that after disruption of microtubules, the apical vesicles do not use the basolateral pathway by default.
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PMID:Development of the Na(+)-dependent hexose carrier in LLC-PK1 cells is dependent on microtubules. 197 53

In LLC-PK1 cells exposed to patulin (50 microM), lipid peroxidation, abrupt calcium influx, extensive blebbing, and total LDH release appeared to be serially connected events with each representing a step in the loss of structural integrity of the plasma membrane. The aforementioned patulin-induced events were prevented by concurrent incubation with butylated hydroxytoluene, deferoxamine, and cyclopiazonic acid, a fungal metabolite. Patulin also caused depletion of nonprotein sulfhydryls, increased 86Rb+ efflux, dome collapse, and eventually the loss of cell viability. These events were not prevented by antioxidants, results consistent with the hypothesis that they were also serially connected but occurring parallel to those previously mentioned. The earliest events observed in patulin-treated cells were the decrease in nonprotein sulfhydryls and increase in 86Rb+ efflux (5 min) which occurred before statistically significant alterations in protein-bound sulfhydryls. The increased potassium efflux (86Rb+ efflux) occurred via a pathway distinct from BaCl2, quinine, or tetraethylammonium sensitive potassium channels. This is the first published report of the antioxidant activity of indole tetramic acids (cyclopiazonic acid and cyclopiazonic acid imine). The protective effect of tetramic acids in LLC-PK1 cells was restricted to indole tetramic acids, and their prevention of lipid peroxidation did not involve iron chelation. The results of this study demonstrate that cyclopiazonic acid is a potent inhibitor of azide-insensitive, ATP-dependent, a23187-sensitive calcium uptake by the lysate of LLC-PK1 cells. This result is consistent with the hypothesis that the endoplasmic reticulum calcium transport ATPase is a sensitive target for cyclopiazonic acid in LLC-PK1 cells. These findings raise the interesting possibility that the antioxidant activity of indole tetramic acids may involve multiple novel mechanisms: surface charge alterations on the cytoplasmic surface of plasma membranes, alterations in calcium permeability in the plasma and endoplasmic reticulum membrane, and inhibition of the calcium-dependent ATPase of the endoplasmic reticulum.
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PMID:The mechanism of patulin's cytotoxicity and the antioxidant activity of indole tetramic acids. 203 42

In a previous study we compared the effects of patulin (PAT) and ouabain, a specific inhibitor of the Na(+)-K+ ATPase, and found significant differences with regard to the kinetics of Na+ influx and K+ efflux, and sulfhydryl reactivity in LLC-PK1 cells. The purpose of the present study was to determine the relationship between Na+ influx, K+ efflux, membrane potential ([3H]tetraphenylphosphonium accumulation), cellular viability [lactate dehydrogenase (LDH) release], and changes in cell morphology (blebs). The effects of PAT are concentration and time dependent. At concentrations of PAT above 10 microM there is a transient increase in intracellular electronegativity (less than 1 hr) followed by a sustained depolarization (greater than 1 hr) which is correlated with complete Na+ influx, K+ efflux, total LDH release, and bleb formation. However, at PAT concentrations of 5-10 microM there is a sustained increased intracellular electronegativity (4-8 hr) which is associated with partial Na+ influx and K+ efflux, no significant LDH release, and relatively few blebs. The hyperpolarizing effect may be a result of increased permeability to K+ relative to Na+. At times and concentrations which result in increased intracellular electronegativity, PAT has no effect on [3H]ouabain binding and thus increased Na+/K+ pump turnover does not seem to be the cause of the transient hyperpolarizing effect of PAT. These results are consistent with the hypothesis that PAT causes alterations in plasma membrane permeability which favor K+ efflux relative to Na+ influx. The toxic effects of PAT are irreversible in LLC-PK1 cells after even short pretreatment with PAT. The primary toxic lesion appears to be at some level other than that involving inhibition of macromolecular synthesis, perhaps the plasma membrane itself.
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PMID:Chronology of patulin-induced alterations in membrane function of cultured renal cells, LLC-PK. 215 17

In this review we have summarized the work of ourselves and others on ionic and hormonal regulation of synthesis of the sodium pump. No one central theme emerges from this summary. Rather, it appears that abundance can be regulated pre-translationally or posttranslationally. As reviewed recently, regulation of the expression of the beta glycoprotein subunit, which has no described enzymatic function, can regulate holoenzyme expression. In the kidney this is exemplified in our studies in LLC-PK1 cells and proximal tubule cells where pre-translational regulation of beta expression is key to increasing holoenzyme abundance, and also exemplified in the hypothyroid renal cortex where regulation of beta protein abundance post-translationally appears to impact the abundance of enzymatically active NaK-ATPase. Future studies in the field of ionic regulation of NaK-ATPase must be directed at elucidating the signals that mediate the response, and at how these signals alter the NaK-ATPase biosynthetic pathway from expression of alpha and beta genes, through to turnover of the mature NaK-ATPase heterodimer.
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PMID:Ionic regulation of the biosynthesis of NaK-ATPase subunits. 216 28

In this paper we establish the response of LLC-PK1/Cl4 cells, a pig kidney cell line, to incubation in medium containing 0.25 mM K+. The amounts of the Na,K-ATPase alpha and beta subunits, determined by Western blot, increase coordinately to greater than 2-fold over control by 24 h in low K+ and remained elevated for the duration of the study period (48 h). Na,K-ATPase activity, measured enzymatically, increased 1.4-fold by 24 h and remained elevated. In order to determine if this response was initiated pretranslationally, alpha and beta subunit mRNA levels were determined by Northern blot analysis. While there was no change in alpha-mRNA levels, beta levels increased significantly, to 1.9-fold over control by 6 h of treatment and remained elevated. This selective increase in beta-mRNA was accompanied by 1.6- and 3.1-fold increases in the respective rates of accumulation of newly synthesized alpha and beta subunits, assessed by immunoprecipitating subunits from pulse-labeled cells. The degradation rates of mature Na,K-ATPase subunits did not change during 16 h of exposure to low K+, but after 16 h there was a selective decrease in the alpha degradation rate, relative to control. These results suggest that increased pretranslational regulation of the beta subunit alone is sufficient to increase accumulation of both alpha and beta subunits. These findings support the notion that in LLC-PK1 cells newly synthesized beta is rate-limiting and thus regulates, through alpha beta assembly, the number of pumps transported to the plasma membrane.
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PMID:Low K+ increases Na,K-ATPase abundance in LLC-PK1/Cl4 cells by differentially increasing beta, and not alpha, subunit mRNA. 217 Apr 1

Freshly isolated rabbit proximal tubules (PT), confluent primary rabbit proximal tubule cultures (PTC) and LLC-PK1 cells were characterised. Brushborder enzyme activities were lower in PTC than in LLC-PK1: ratios were 0.026 for alkaline phosphatase (AP), 0.458 for alanine aminopeptidase (AAP) and 0.514 for gamma-glutamyl transpeptidase (GGT). PT/PTC ratios were 79.7 for AP, 7.96 for AAP and 3.45 for GGT. Specific activities of hexokinase (HK) and lactate dehydrogenase (LDH) were high in cultured cells as compared to PT: PT/PTC ratios were 0.063 and 0.033, while PTC/LLC-PK1 ratios were 0.406 and 1.19 for HK and LDH respectively. PTC/LLC-PK1 ratios were 2.21 for Na/K ATPase, 2.07 for succinate dehydrogenase, 1.12 for cathepsin B, 0.607 for N-acetyl-beta-D-glucosaminidase and 8.98 for glutathione-S-transferase. Adenylate cyclase response to parathormone (PTH), was similar in PTC and PT, but stimulated/basal ratios were higher in PT than in PTC. LLC-PK1 cells were stimulated by thyrocalcitonin (SCT), arginin-vasopressin (AVP) and PTH; stimulated/basal ratios ranked AVP greater than PTH greater than SCT. Differences between both types of cultures affect the choice of in vitro model for nephrotoxicity studies.
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PMID:Adenylate cyclase responses and biochemical characterization of primary rabbit proximal tubular cell cultures and LLC-PK1 cells. 228 70

Over a period of 2-3 wk after plating, cultured LLC-PK1 (pig kidney) cells develop a high capacity for Na+-dependent accumulation of alpha-methyl-D-glucoside. To further the analysis of this developmental process, we have developed a method for separating transporting from nontransporting cells on the basis of density changes accompanying hexose accumulation and the corresponding uptake of water. Volume regulation was prevented by suspending the cells in a K+-free, Cl(-)-free Na-gluconate medium. Na+-dependent transport was maintained at nearly control levels by addition of low concentrations of (NH4)2SO4, since NH+4 stimulates Na+-K+-ATPase at the K+ site and allows for the extrusion of accumulated Na+; NH+4-stimulated hexose uptake is ouabain sensitive. With volume regulation blocked but with transport near normal, transporting cells exhibited a phlorizin-sensitive density shift in methylglucoside-containing medium and could be separated from nontransporting cells on Percoll gradients.
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PMID:Separation of hexose-transporting from nontransporting LLC-PK1 cells on density gradients. 242 Jan 87

Patulin (PAT), a compound produced by certain species of Aspergillus, Penicillium, and Byssochlamys, is frequently found associated with agricultural commodities. PAT has many effects on membrane function, including the inhibition of the isolated Na+-K+ ATPase. In this study, a scanning electron microscope equipped with an energy dispersive spectroscopy X-ray microanalysis system was used to examine individual cultured renal epithelial cells (LLC-PK1) in order to determine the effects of PAT on the relative intracellular ion concentrations. The estimated EC50 (60 min) for both sodium influx and potassium efflux was between 10 and 50 microns for ouabain. For PAT, the EC50 (60 min) was 250 microns for sodium influx and 100 microns for potassium efflux. However, 1 mM patulin at 240 min caused complete reversal of the sodium and potassium content of cells, and 1 mM ouabain at 240 min did not. The effect of patulin on sodium and potassium flux was both concentration and time dependent and was reversed by dithiothreitol and glutathione. PAT (250 microM) but not ouabain (250 microM) induced massive blebbing of LLC-PK1 cells. Thus, the interaction of PAT with cellular membranes involves both alterations in the regulation of intracellular ion content and the cytoskeleton. We hypothesize that patulin alters intracellular ion content via Na+-K+ ATPase and non-Na+-K+ ATPase mechanisms.
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PMID:Patulin-induced ion flux in cultured renal cells and reversal by dithiothreitol and glutathione: a scanning electron microscopy (SEM) X-ray microanalysis study. 254 48

Bovine hypothalamus contains a high affinity, specific, reversible inhibitor of mammalian Na+-K+-ATPase. Kinetic analysis using isolated membrane fractions showed binding and dissociation rates of the hypothalamic factor (HF) to be (like ouabain) relatively long (off rate = 60 min). To determine whether the kinetics of inhibition in intact cells might be more consistent with regulation of physiological processes in vivo, binding and dissociation reactions of HF in intact renal epithelial cells (LLC-PK1) were studied using 86Rb+ uptake and [3H]ouabain binding. As with membranes, a 60-min incubation with HF inhibited Na+-K+-ATPase in LLC-PK1 cells. In contrast to membrane studies, no prolonged incubation with LLC-PK1 was needed to observe inhibition of Na+-K+-ATPase. HF caused a 33% inhibition of ouabain-sensitive 86Rb+ influx within 10 min. Incubation of cells with HF followed by washout showed rapid reversal of pump inhibition and a doubling of pump activity. The dose-response curve for HF inhibition of LLC-PK1 86Rb+ uptake showed a sigmoidal shape consistent with an allosteric binding reaction. Thus HF is a potent regulator of Na+-K+-ATPase activity in intact renal cells, with binding and dissociation reactions consistent with relevant physiological processes.
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PMID:Na+ pump in renal tubular cells is regulated by endogenous Na+-K+-ATPase inhibitor from hypothalamus. 284 5

We purified a polar digitalis-like factor to apparent homogeneity from human urine using inhibition of 3H-ouabain binding to intact human erythrocytes to monitor digitalis-like activity. Since endogenous digitalis-like factor may act as a natriuretic hormone, we tested the binding characteristics of this urine-derived ouabain-displacing compound to the sodium pump in intact renal epithelial cells, in order to assess its potential physiological significance. Cultured canine and porcine epithelial cell lines, MDCK and LLC-PK1, had a high sodium pump density as estimated from 3H-ouabain binding sites. Human urine-derived ouabain-displacing compound showed a dose-related inhibition of 3H-ouabain binding to both of these cells, similar to the inhibition of unlabelled ouabain. These findings indicate that the ouabain-displacing compound is capable of acting on the sodium pump in intact renal epithelial cells and may be the circulating regulator of Na+,K+-ATPase.
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PMID:Effects of human urine-derived digitalis-like factor on cultured renal tubular epithelial cells. 285 36


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