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 transporters responsible for apical proton secretion were examined in neonatal and adult proximal convoluted tubules (PCT). Transporter activity was assayed from the rate of recovery of cell pH after cell acidification following exposure to NH4Cl. Cell pH was monitored in in vitro perfused tubules using the pH sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Recovery from an acid load in adult PCT occurred at 0.52 +/- 0.09 pH units/min in the presence of sodium and 0.25 +/- 0.05 in the absence of sodium (p less than 0.05). One mmol/L N-ethylmaleimide, an inhibitor of the H(+)-ATPase, inhibited the sodium-independent pH recovery from an acid load consistent with a H(+)-ATPase on the apical membrane. In neonatal PCT, recovery from an acid load was 0.39 +/- 0.08 pH units/min in the presence of sodium and only 0.08 pH units/min in the absence of sodium (p less than 0.05). Studies using 4 mmol/L luminal amiloride, an inhibitor of the Na+/H+ antiporter, were consistent with a larger fraction of pH recovery from an acid load in neonatal PCT being due to the Na+/H+ antiporter compared with adult PCT. Thus, maturation of the PCT involves an increase in activity of a sodium-independent proton secretory mechanism, presumably the H(+)-ATPase.
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PMID:Developmental changes in rabbit juxtamedullary proximal convoluted tubule acidification. 131 22

An electrokinetic model was developed to calculate the time course of electrical parameters, ion fluxes, and intracellular ion activities for experiments performed in airway epithelial cells. Model variables included cell [Na], [K], [Cl], volume, and membrane potentials. The model contained apical membrane Cl, Na, and K conductances, basolateral membrane K conductance, Na/K/2 Cl and Na/Cl symport, and 3 Na/2 K ATPase, and a paracellular conductance. Transporter permeabilities and ion saturabilities were determined from reported ion flux data and membrane potentials in intact canine trachea. Without additional assumptions, the model predicted accurately the measured short-circuit current (Isc), cellular conductances, voltage-divider ratios, open-circuit potentials, and the time course of cell ion composition in ion substitution experiments. The model was used to examine quantitatively: (a) the effect of transport inhibitors on Isc and membrane potentials, (b) the dual role of apical Cl and basolateral K conductance in cell secretion, (c) whether the basolateral symporter requires K, and (d) the regulation of apical Cl conductance by cAMP and Ca-dependent signaling pathways. Model predictions gave improved understanding of the interrelations among transporting systems and in many cases gave surprising predictions that were not obvious without a detailed model. The model developed here has direct application to secretory or absorptive epithelial cells in the kidney thick ascending limb, cornea, sweat duct, and intestine in normal and pathophysiological states such as cystic fibrosis and cholera.
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PMID:Model of ion transport regulation in chloride-secreting airway epithelial cells. Integrated description of electrical, chemical, and fluorescence measurements. 169 71

Functional and immunocytochemical studies indicate that intercalated cells in the adult rabbit cortical collecting duct (CCD) possess an H-K-adenosinetriphosphatase (H-K-ATPase). Because growing subjects must retain K+ and excrete H+, we sought to determine whether H-K-ATPase is present in the CCD early in life and, if so, to assess its activity and polarity. H-K-ATPase activity was defined as the initial rate of Sch-28080-inhibitable K+-dependent cell pH (pHi) recovery observed, in the absence of Na+, in response to an in vitro acid load. Transporter activity was assayed in intercalated cells labeled with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and apical cell surface marker rhodamine peanut lectin (PNA) in split-open CCDs isolated from neonatal and adult New Zealand White rabbits. In Na+-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions (nominal absence of CO2/HCO3-), the rate of K+-dependent pH(i) recovery from a NH4Cl-induced acid load was similar in newborn (0.056 +/- 0.015 pH U/min, n = 9) and adult (0.060 +/- 0.019 pH U/min; n = 9, P = not significant) cells. This rate of K+-dependent pH(i) recovery was significantly reduced by 10-20 pM Sch-28080, an inhibitor of gastric H-K-ATPase, in both newborns (0.009 +/- 0.003 pH U/min, n = 7) and adults (0.013 +/- 0.007 pH U/min, n = 9) (P < 0.05 compared with rates in absence of inhibitor). To determine whether the location of the transporter is consistent with a role in K+ absorption and H+ secretion, pH(i) recovery of acutely acid-loaded intercalated cells in neonatal CCDs (n = 7) microperfused and bathed in the absence of Na+ and K+ was monitored after selective addition of K+ to either the luminal or basolateral membrane. Addition of 5 mM K+ led to a significantly greater rate of pH(i) recovery when it was added to the luminal rather than the peritubular solution (0.049 +/- 0.005 vs. 0.018 +/- 0.005 pH U/min, P < 0.05). We conclude that PNA-binding intercalated cells of the neonatal CCD possess H-K-ATPase activity, predominantly located in the apical membrane. This provides a mechanism for H secretion and K+ retention, processes required for growth.
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PMID:H-K-ATPase activity in PNA-binding intercalated cells of newborn rabbit cortical collecting duct. 912 92

Null mutations in genes encoding V-ATPase subunits in Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations. Treatment of these null mutants with ethylmethanesulfonate causes mutations that suppress the V-ATPase null phenotype, and the mutant cells are able to grow at pH 7.5. The suppressor mutants were denoted as svf (suppressor of V-ATPase function). The frequency of svf is relatively high, suggesting a large target containing several genes for the ethylmethanesulfonate mutagenesis. The suppressors' frequency is dependent on the individual genes that were inactivated to manifest the V-ATPase null mutation. The svf mutations are recessive, because crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that are unable to grow at pH 7.5. A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or sensitivity and distribution of membrane proteins in different targets was discovered. The family was defined as VTC (Vacuolar Transporter Chaperon) and it contains four genes in the S. cerevisiae genome. Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in svf phenotype manifested by growth at pH 7.5. Deletion of the VTC1 gene (DeltaVTC1) results in a reduced amount of V-ATPase in the vacuolar membrane. These mutant cells fail to accumulate quinacrine into their vacuoles, but they are able to grow at pH 7.5. The VTC1 null mutant also results in a reduced amount of the plasma membrane H(+)-ATPase (Pma1p) in membrane preparations and possibly mis-targeting. This observation may provide an explanation for the svf phenotype in the double disruptant mutants of DeltaVTC1 and DeltaVMA subunits.
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PMID:A novel family of yeast chaperons involved in the distribution of V-ATPase and other membrane proteins. 1048 Aug 97

The vacuolar H(+)-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force, V-ATPases function exclusively as ATP-dependent proton pumps. The proton-motive force generated by V-ATPases in organelles and across plasma membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. The enzyme is also vital for the proper functioning of endosomes and the Golgi apparatus. In contrast to yeast vacuoles, which maintain an internal pH of approximately 5. 5, it is believed that the vacuoles of lemon fruit may have a pH as low as 2. Similarly, some brown and red algae maintain an internal pH as low as 1 in their vacuoles. It was yeast genetics that allowed the identification of the special properties of individual subunits and the discovery of the factors that are involved in V-ATPase biogenesis and assembly. Null mutations in genes encoding V-ATPase subunits of Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations. Treatment of these null mutants with ethyl methanesulphonate causes mutations that suppress the V-ATPase null phenotype, and these cells are able to grow at pH 7.5. The suppressor mutants were denoted as svf (Suppressor of V-ATPase Function). The svf mutations are recessive: crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that were not able to grow at pH 7.5. A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or on the sensitivity and distribution of membrane proteins in different targets was discovered. We termed this gene family VTC (Vacuolar Transporter Chaperon) and discovered four genes in S. cerevisiae that belong to the family. Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in an svf phenotype that was able to grow at pH 7.5. Apparently, Vtc1p is one of a few membrane organizers that determine the relative amounts of different membrane proteins in the various cellular membranes. We utilize the numerous yeast mutants generated in our laboratory to identify the specific organelle whose acidification is vital. The interaction between V-ATPase and the secretory pathway is investigated.
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PMID:The cellular biology of proton-motive force generation by V-ATPases. 1060 Jun 77

Nephrotoxicity in humans and experimental animals due to chronic exposure to cadmium (Cd) is manifested by defects in the reabsorptive and secretory functions of proximal tubules (PT). The main symptoms of Cd nephrotoxicity, including polyuria, phosphaturia, aminoaciduria, glucosuria, and proteinuria, suggest that various brush-border membrane (BBM) transporters are the main targets of Cd. Specific transporters may be either directly inhibited by Cd or lost from the BBM after Cd treatment, or both. We have recently proposed that Cd may impair the vesicle-dependent recycling of BBM transporters by inhibiting vacuolar H+-ATPase (V-ATPase) activity and endocytosis in PT cells (Herak-Kramberger CM, Sabolic I, and Brown D. Kidney Int 53: 1713-1726, 1998). The mechanism underlying the Cd effect was further explored in an in vivo model of experimental Cd nephrotoxicity induced by Cd-metallothionein (Cd-MT; 0.4 mg Cd/kg body mass; a single dose sc) in rats. The time-dependent redistribution of various BBM transporters was examined in this model by fluorescence and gold-labeling immunocytochemistry on tissue sections and by immunoblotting of isolated renal cortical BBM. In PT cells of Cd-MT-treated rats, we observed 1) shortening and loss of microvilli; 2) time-dependent loss of megalin, V-ATPase, aquaporin-1 (AQP1), and type 3 Na+/H+ exchanger (NHE3) from the BBM; 3) redistribution of these transporters into vesicles that were randomly scattered throughout the cell cytoplasm; and 4) redistribution of NHE3, but not megalin, into the basolateral plasma membrane. The internalization of BBM transporters was accompanied by fragmentation and loss of microtubules and by an increased abundance of alpha-tubulin monomers in PT cells. Transporter redistribution was detectable as early as 1 h after Cd-MT treatment and increased in magnitude over the next 12 h. We conclude that the early mechanism of Cd toxicity in PT cells may include a colchicine-like depolymerization of microtubules and impaired vesicle-dependent recycling of various BBM proteins. These processes may lead to a time-dependent loss of cell membrane components, resulting in reabsorptive and secretory defects that occur in Cd-induced nephrotoxicity.
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PMID:Cd-MT causes endocytosis of brush-border transporters in rat renal proximal tubules. 1242 37

The renal collecting duct is the primary site for the ammonia secretion necessary for acid-base homeostasis. Recent studies have identified the presence of putative ammonia transporters in the collecting duct, but whether the collecting duct has transporter-mediated ammonia transport is unknown. The purpose of this study was to examine basolateral ammonia transport in the mouse collecting duct cell (mIMCD-3). To examine mIMCD-3 basolateral ammonia transport, we used cells grown to confluence on permeable support membranes and quantified basolateral uptake of the radiolabeled ammonia analog [14C]methylammonia ([14C]MA). mIMCD-3 cell basolateral MA transport exhibited both diffusive and transporter-mediated components. Transporter-mediated uptake exhibited a Km for MA of 4.6 +/- 0.2 mM, exceeded diffusive uptake at MA concentrations below 7.0 +/- 1.8 mM, and was competitively inhibited by ammonia with a Ki of 2.1 +/- 0.6 mM. Transporter-mediated uptake was not altered by inhibitors of Na+-K+-ATPase, Na+-K+-2Cl(-) cotransporter, K+ channels or KCC proteins, by excess potassium, by extracellular sodium or potassium removal or by varying membrane potential, suggesting the presence of a novel, electroneutral ammonia-MA transport mechanism. Increasing the outwardly directed transmembrane H+ gradient increased transport activity by increasing Vmax. Finally, mIMCD-3 cells express mRNA and protein for the putative ammonia transporter Rh B-glycoprotein (RhBG), and they exhibit basolateral RhBG immunoreactivity. We conclude that mIMCD-3 cells express a basolateral electroneutral NH4+/H+ exchange activity that may be mediated by RhBG.
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PMID:Basolateral ammonium transport by the mouse inner medullary collecting duct cell (mIMCD-3). 1514 71

The collecting duct is the primary site of urinary ammonia secretion; the current study determines whether apical ammonia transport in the mouse inner medullary collecting duct cell (mIMCD-3) occurs via nonionic diffusion or a transporter-mediated process and, if the latter, presents the characteristics of this apical ammonia transport. We used confluent cells on permeable support membranes and examined apical uptake of the ammonia analog [(14)C]methylammonia ([(14)C]MA). mIMCD-3 cells exhibited both diffusive and saturable, transporter-mediated, nondiffusive apical [(14)C]MA transport. Transporter-mediated [(14)C]MA uptake had a K(m) of 7.0 +/- 1.5 mM and was competitively inhibited by ammonia with a K(i) of 4.3 +/- 2.0 mM. Transport activity was stimulated by both intracellular acidification and extracellular alkalinization, and it was unaltered by changes in membrane voltage, thereby functionally identifying an apical, electroneutral NH(4)(+)/H(+) exchange activity. Transport was bidirectional, consistent with a role in ammonia secretion. In addition, transport was not altered by Na(+) or K(+) removal, not inhibited by luminal K(+), and not mediated by apical H(+)-K(+)-ATPase, Na(+)-K(+)-ATPase, or Na(+)/H(+) exchange. Finally, mIMCD-3 cells express the recently identified ammonia transporter family member Rh C glycoprotein (RhCG) at its apical membrane. These studies indicate that the renal collecting duct cell mIMCD-3 has a novel apical, electroneutral Na(+)- and K(+)-independent NH(4)(+)/H(+) exchange activity, possibly mediated by RhCG, that is likely to mediate important components of collecting duct ammonia secretion.
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PMID:Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD-3). 1579 90

To date three potential candidates for parietal cell basolateral Cl(-) entry have been described: the highly 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive Cl(-)/HCO(3)(-) exchanger AE2, the HCO(3)(-) and lowly DIDS-sensitive SLC26A7 protein, and the Na(+)-2Cl(-)K(+) cotransporter (NKCC1). In this study we investigate the contribution of these pathways to secretagogue stimulated acid secretion. Individually hand-dissected rat gastric glands were microfluorimetrically monitored for Cl(-) influx and pH(i) changes. Transporter activity was determined by varying ion content and through the use of pharmacological inhibitors. Expression of SLC26A7 in rat parietal cells was shown by immunohistochemistry and Western blot. SLC26A7 was inhibited by 5-Nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB) (100 microM) in the Xenopus laevis oocyte expression system. Cl(-) influx in parietal cells was enhanced by histamine, depended partially on endogenous HCO(3)(-) synthesis and completely on extracellular Na(+). Removal and subsequent readdition of Cl(-) revealed a low and a high DIDS-sensitive HCO(3)(-) extrusion system contributing to Cl(-) uptake. At acidic pH(i), however, H(+) extrusion via the H(+),K(+)-ATPase depending on Cl(-) uptake was abolished only in the presence of 100 microM (NPPB) and at high (250 microM) DIDS concentration. There was no effect of the NKCC inhibitor bumetanide on stimulated H(+) extrusion. These results would be compatible with SLC26A7 as a Cl(-) uptake system under histamine stimulation.
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PMID:SLC26A7 can function as a chloride-loading mechanism in parietal cells. 1740 55

Chloroacetanilide herbicides are among the most commonly used herbicides in agriculture. Several studies have demonstrated a number of them to be carcinogenic. ATP binding cassette (ABC) transporters are efflux pumps expressed in cell membranes, which form an important wall of defense against xenobiotics from different sources. We tested the interaction of the herbicides acetochlor, alachlor, dimetachlor, metazachlor, metolachlor, propachlor and prynachlor with human multidrug resistance transporters MDR1, MRP1, MRP2 and BCRP. A number of metabolites were studied for interaction with MRP1, MRP2 and MRP3. Transporter interactions were studied by measuring ATPase activity, inhibition of fluorescent dye efflux and vesicular transport. Also inhibition of MDR1 was monitored by measuring digoxin transport on Caco-2 monolayers and paclitaxel toxicity on K562-MDR cells. Acetochlor, alachlor, metolachlor and metazachlor showed specific interactions with MDR1. Digoxin permeability and paclitaxel cytotoxicity studies revealed that these herbicides are potent inhibitors of MDR1 that can modulate drug absorption and cause chemosensitization of cells. MRP1 was demonstrated to transport an important intermediate of the acetochlor detoxification pathway. Several specific interactions were shown when studying the interaction of chloroacetanilides with human transporter proteins. This study suggests an important role for transporter proteins in hazard prediction of agrochemicals and demonstrates how transporter interactions can be easily detected using in vitro screening methods.
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PMID:Specific interactions of chloroacetanilide herbicides with human ABC transporter proteins. 1843 74


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