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)

Epithelial cell height was measured in order to estimate the cell volume of dark cells from the ampullae of the semicircular canal of the gerbil. Under control conditions, addition of 10(-4) mol/l piretanide, 10(-5) mol/l 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB), 5 mmol/l barium or 10(-3) mol/l quinidine had no significant effect on cell height. Addition of 10(-4) mol/l NPPB or 10(-3) mol/l ouabain led to a small significant decrease in cell height which was not reversible. Substitution of Na+ by N-methyl-D-glucamine or of Cl- by gluconate led to a significant and reversible reduction in cell height. Isotonic elevation of [K+] from 3.6 to 25 mmol/l in a PO4-buffered, HCO3-free solution led to an increase in cell height from 5.8 +/- 0.1 (SEM) to 8.7 +/- 0.2 microns (n = 62) during the first 40 s. During prolonged exposure to elevated [K+] (3-5 min; n = 19), some tissue samples underwent a regulatory volume decrease. K(+)-induced swelling was absent in both isotonic Cl(-)-free and isotonic Na(+)-free solutions and was inhibited by the loop diuretic piretanide (10(-5) and 10(-4) mol/l) or by the (Na+ + K+) ATPase inhibitor ouabain (10(-3) mol/l) or by 10(-4) mol/l NPPB. After the removal of ouabain or 10(-4) mol/l NPPB, K(+)-induced swelling under control conditions was enhanced and was less reversible as compared to control conditions before the experiment. K(+)-induced swelling was not altered by NPPB (10(-5) mol/l) or barium (5 mmol/l); however, barium slowed shrinking upon return of [K+] to control level. In the presence of 10(-3) mol/l quinidine, K(+)-induced swelling was enhanced and not reversible. These data suggest that dark cells from the semicircular canal possess an Na+2Cl-K+ cotransporter as a solute uptake mechanism and a solute efflux mechanism which is sensitive to barium and inhibited by quinidine.
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PMID:K(+)-induced swelling of vestibular dark cells is dependent on Na+ and Cl- and inhibited by piretanide. 169 72

The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is characterized by its high content of Na+/K(+)-ATPase and a Cl- conductance, which function in parallel in salt reabsorption. In order to reconstitute the Cl- channels, TALH membrane vesicles were solubilized in 1% sodium cholate in buffer containing 200 mM KCl, followed by dilution with soybean lipids (final ratio of protein/detergent/lipid of 1:3:15 in mg) and removal of the detergent by gel filtration on Sephadex G-50. Cl- channel activity in the liposomes was determined by a 36Cl- uptake assay where the accumulation of the radioactive tracer against its chemical gradient is driven by the membrane potential (positive inside) generated by an outward Cl- gradient. The 36Cl- uptake by the KCl-loaded liposomes was dependent on the inclusion of membrane protein and was abolished by valinomycin, indicating the involvement of a conductive pathway. It was also inhibited by 36% by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Solubilization of the Cl- channels in cholate was optimal in the presence of 200 mm KCl, but was found to decrease markedly at low ionic strength. SDS-PAGE analysis of the proteins extracted by cholate at high and low salt concentrations showed that the Cl- channel-containing high KCl extract was enriched in the 96 and 55 kDa alpha- and beta-subunits of the Na+/K(+)-ATPase (the major proteins in the membrane preparation) and several minor protein bands. Treatment of the membrane vesicles with the radioactive analogue of DIDS, [3H]2DIDS, labeled primarily a 65 and a 31 kDa protein. The solubilization of the 31 kDa protein by cholate depended markedly on the ionic strength and thus paralleled the solubilization pattern of Cl- channel activity. Furthermore, the labeling of the 31 kDa protein was prevented by nonradioactive DIDS and by NPPB but not by other compounds, indicating that it may be a Cl- channel component.
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PMID:Reconstitution of a kidney chloride channel and its identification by covalent labeling. 215 22

1. Necturus gastric mucosa secretes Cl- actively across the gastric glands which are composed almost entirely of acid- and enzyme-secreting oxynticopeptic cells. Single channel studies on Necturus oxynticopeptic cells have shown that the basolateral membrane possesses multiple K(+)-selective channels but no observable Cl- channels while the apical membrane has Cl- channels but no observable K+ channels. To relate these channel properties to the conductance of the whole cell we have investigated the macroscopic membrane currents with conventional whole-cell patch-clamp techniques. 2. When bathed in amphibian Ringer solution, gastric oxynticopeptic cells had a membrane resistance of 47.8 +/- 2.8 M omega and a membrane capacitance of 75.5 +/- 2.7 pF (n = 82). This gave a specific membrane resistance of 3260 +/- 160 omega cm2 (n = 82). Reversal potentials of the oxynticopeptic cells were -13.8 +/- 1.2 mV (n = 45) for an intracellular Cl- concentration ([Cl-]i) of 42 mM and were significantly more negative -24.4 +/- 3.1 mV (n = 31, P < 0.001) for [Cl-]i = 22 mM. 3. In the absence of ATP in the pipette solution, there was an 80% reduction of the whole-cell current with a typical half-time (t1/2) of 5 min. The run-down was not observed when the pipette solution contained 4 mM ATP. 4. A slow and voltage-independent inhibition of 80% of the whole-cell currents occurred after addition of NPPB (35 microM). Ba2+ (10 mM) produced a reversible inhibition of 20% of the total current. Together, 35 microM NPPB and 10 mM Ba2+ eliminated 95% of the whole-cell currents. These data suggest that in the resting oxynticopeptic cells Cl- carried the major fraction of the current while K+ ions carried only a small fraction. 5. Total replacement of Cl- in the pipette and bath solution by gluconate- increased the membrane resistance to 751 +/- 104 M omega (n = 53) and shifted the reversal potential to -38.1 +/- 2.8 mV (n = 53). 6. Increasing the bath K+ concentration from 6 to 91 mM activated a current which had a high selectivity for K+ over choline+, Li+, Na+, Rb+ and Cs+ and was independent of Cl-. The activation of this K+ current (IK*) by high external K+ was not seen with ATP-free pipette solution. 7. Ba2+ or Cs+ had a voltage-dependent blocking effect of this inward K+ current. Ouabain (1 mM) or SCH 28080 (200 microM), specific inhibitors of the Na+,K(+)-ATPase and H+,K(+)-ATPase, had no effect.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Whole-cell currents in isolated resting Necturus gastric oxynticopeptic cells. 750 8

Agonists which stimulate the inositol 1,4,5 trisphosphate ([1,4,5]-IP3)-dependent mobilization of Ca2+ from intracellular stores also stimulate entry of divalent cations across the cell membrane. Under appropriate experimental conditions, divalent cation entry across the cell membrane can be monitored as the rate at which the intracellular fluorescence of divalent cation indicators is quenched by the addition of Mn2+ to the extracellular medium. We report that addition of vasopressin to fura-2-loaded glomerular mesangial cells in culture markedly accelerated the rate at which Mn2+ quenched fura-2 fluorescence at its Ca(2+)-insensitive wavelength in the presence of extracellular NaCl, but that this quench response was attenuated when Cl- was removed from the extracellular medium by equimolar substitution with impermeant anions (gluconate, methanesulfonate, acetate, lactate). Similarly, loss of agonist-induced quench also occurred when Cl- was substituted with gluconate in K(+)-containing media. Addition of the Cl- channel inhibitor, 5-nitro-2-(3-phenylpropylaminobenzoic acid) (NPPB), also inhibited Mn(2+)-induced quench of fura-2 fluorescence following vasopressin addition. In contrast, in the presence of gramicidin to provide an alternate conductance pathway to accompany divalent cation entry, agonist-dependent Mn2+ quench occurred even in the absence of extracellular Cl-, indicating that the requirement for Cl- was not the result of cotransport on a common transporter nor the result of Cl- serving as a necessary cofactor for divalent cation entry. A similar dependence on extracellular Cl- was observed for other Ca(2+)-mobilizing agonists such as endothelin, as well as the intracellular Ca2+ ATPase inhibitor, thapsigargin. Extracellular Cl- dependence for agonist-induced divalent cation entry was also reflected in a corresponding extracellular Cl- dependence for agonist-induced mesangial cell contraction. It has been previously shown by ourselves (Kremer et al., 1992a, Am. J. Physiol., 262:F668-F678) and others that agonist-stimulated calcium mobilization in mesangial cells is accompanied by inhibition of K+ conductance and increased Cl- conductance. Accordingly, we conclude that the current findings suggest that activation of Cl- conductance provides regulated charge compensation for receptor-mediated divalent cation entry in response to Ca(2+)-mobilizing vasoconstrictor agonists in mesangial cells.
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PMID:Chloride is required for receptor-mediated divalent cation entry in mesangial cells. 752 36

Heavy endosomes were isolated from proximal tubules using a combination of magnesium precipitation and wheat-germ agglutinin negative selection techniques. Two small GTPases (Rab4 and Rab5) known to be specifically present in early endosomes were identified in our preparations. Endosomal acidification was followed fluorimetrically using acridine orange. In presence of chloride ions and ATP, the formation of a proton gradient (delta pH) was observed. This process is due to the activity of an electrogenic V-type ATPase present in the endosomal membrane since specific inhibitors bafilomycin and folimycin effectively prevented or eliminated endosomal acidification. In presence of chloride ions (K(m) = 30 mM) the formation of the proton gradient was optimal. Inhibitors of chloride channel activity such as DIDS and NPPB reduced acidification. The presence of sodium ions stimulated the dissipation of the proton gradient. This effect of sodium was abolished by amiloride derivative (MIA) but only when loaded into endosomes, indicating the presence of a physiologically oriented Na+/H(+)-exchanger in the endosomal membrane. Monensin restored the gradient dissipation. Thus three proteins (V-type ATPase, Cl(-)-channel, Na+/H(+)-exchanger) present in early endosomes isolated from proximal tubules may regulate the formation, maintenance and dissipation of the proton gradient.
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PMID:Proton gradient formation in early endosomes from proximal tubules. 891 81

Biliary epithelial cells (cholangiocytes) modulate bile fluidity and alkalinity absorbing and/or secreting fluid and electrolytes, particularly HCO3- and Cl-. Mechanisms responsible for transepithelial H+/HCO3- secretion in human cholangiocytes are largely unknown. Human cholangiocytes isolated by enzymatic digestion and immunomagnetic purification from normal liver tissue obtained from reduced grafts used for pediatric liver transplantation were cultured in the presence of human hepatocyte growth factor. Maintenance of cholangiocyte phenotypic features was assessed using markers such as cytokeratin 19, gamma-glutamyltranspeptidase, vimentin, factor VIII-related antigen, desmin, epithelial membrane antigen (EMA), and human epithelial antigen (HEA) 125. Intracellular pH (pHi) transients were measured microfluorimetrically 2'7'-Bis(2-carboxyethyl)-5,6, carboxyfluorescein-acetossimethylester (BCECF). In the absence of HCO3-, pHi recovery from an intracellular acid load (ammonia pre-pulse technique) was Na(+)-dependent and amiloride-inhibitable. No Na(+)-independent recovery was recorded even after stimulation with agents raising intracellular cyclic adenosine monophosphate (cAMP) concentrations. In the presence of HCO3-, recovery from an intracellular acid load required Na+, but was only partly inhibited by amiloride. In these conditions H+ extrusion was inhibited by 4,4-diisothiocyan atostilben-2,2-disulfonic acid (DIDS) and by intracellular Cl- depletion. Acute removal of extracellular Cl induced a pHi alkalinization that was inhibited by DIDS. pHi recovery from an intracellular alkaline load (isohydric CO2 changes) was Cl(-)-dependent and DIDS-inhibitable. Administration of agents raising intracellular cAMP concentrations increased both Na(+)-dependent and Na(+)-independent Cl-/HCO-3 exchange activity. Stimulation of Cl-/HCO3- exchange activity was not prevented by the Cl- channel inhibitor 5'-nitro-2(2)-phenylpropyl-amino-benzoate(NPPB). In conclusion, human cholangiocytes possess two acid extruders (Na+/H+exchanger and Na(+)-dependent Cl-/HCO3- exchange) and an acid loader (Cl-/HCO3- exchange), whereas no evidence was found for cAMP activated H(+)-ATPase. Bicarbonate influx is thus mainly mediated by Na-dependent Cl-/HCO3- exchange, whereas Na+:HCO-3 cotransport is not active in the physiological range of pHi. Stimulation of Na(+)-independent Cl-/HCO3- exchanger by cAMP does not require activation of Cl- conductances. These mechanisms may underlay hormone-regulated biliary HCO3- secretion in the human biliary tree.
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PMID:Na(+)-dependent and -independent Cl-/HCO-3 exchange mediate cellular HCO3- transport in cultured human intrahepatic bile duct cells. 909 7

The presence of an electrogenic H+-ATPase has been described in the late distal tubule, a segment which contains intercalated cells. The present paper studies the electrogenicity of this transport mechanism, which has been demonstrated in turtle bladder and in cortical collecting duct. Transepithelial PD (Vt) was measured by means of Ling-Gerard microelectrodes in late distal tubule of rat renal cortex during in vivo microperfusion. The tubules were perfused with electrolyte solutions to which 2 x 10(-7) M bafilomycin or 4.6 x 10(-8) M concanamycin were added. No significant increase in lumen-negative Vt upon perfusion with these inhibitors as compared to control, was observed as well as when 10(-3) m amiloride, 10(-5) M benzamil or 3 mM Ba2+ were perfused alone or in combination. The effect of an inhibition of electrogenic H+ secretion, i.e., increase in lumen-negative Vt by 2-4 mV, was observed only when Cl- channels were blocked by 10(-5) M 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). This blocker also reduced the rate of bicarbonate reabsorption in this segment from 1.21 +/- 0.14 (n = 8) to 0.62 +/- 0.03 (8) nmol.cm-2.sec-1 as determined by stationary microperfusion and pH measurement by ion-exchange resin microelectrodes. These results indicate that: (i) the participation of the vacuolar H+ ATPase in the establishment of cortical late distal tubule Vt is minor in physiological conditions, but can be demonstrated after blocking Cl- channels, thus suggesting a shunting effect of this anion; and, (ii) the rate of H+ secretion in this segment is reduced by a Cl- channel blocker, supporting coupling of H+-ATPase with Cl- transport.
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PMID:Role of Cl- in electrogenic H+ secretion by cortical distal tubule. 915 60

The mechanism of acidification in the cortical distal tubule of mammalian kidney was analysed by "in vivo" microperfusion and using MDCK cells in culture, by electrophysiological and by cell pH microfluorescence techniques. An electrogenic effect of the vacuolar H(+)-ATPase, which has been localized to the intercalated cells of the cortical distal tubule (connecting segment and initial collecting duct) was only observed after blocking Cl- channels by NPPB. In MDCK cells, the recovery of cell pH after an acid pulse in Na(+)-free medium was also depressed by NPPB, indicating that Cl- ions have an important role in the function of H+ ATPase. The regulation by hormonal agents of distal H+ transport due to Na+/H+ exchange and to vacuolar H+ ATPase, was also studied by microperfusion and cell pH techniques. Angiotensin and vasopressin at picomolar concentrations stimulated both transport mechanisms in late distal tubule, and only Na+/H+ exchange in the early segment. In MDCK cells, cell pH recovery in the presence of Na+ was stimulated by picomolar concentrations of angiotensin and vasopressin, and inhibited by micromolar levels, both effects being reverted by micromolar ANP. Studies with specific antagonists suggest that the luminal effect of angiotensin is mediated by AT1 receptors, and of vasopressin by V1 receptors. There is evidence that cell Ca2+ may have an important regulatory role in the action of these hormones.
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PMID:Mechanisms and regulation of H+ transport in distal tubule epithelial cells. 926 82

Mechanisms of active NaCl uptake across the posterior gills of the shore crab Carcinus maenas were examined using radiochemical and electrophysiological techniques. In order to measure short-circuit current (Isc), transepithelial conductance (Gte) and area-related unidirectional fluxes of Na+ and Cl-, single split gill lamellae (epithelium plus cuticle) of hyperregulating shore crabs were mounted in a modified Ussing chamber. The negative short-circuit current measured with haemolymph-like NaCl saline on both sides of the epithelium could be inhibited by application of basolateral ouabain (ouabain inhibitor constant KOua=56&plusmn;10 &micro;mol l-1), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; KNPPB=7.5&plusmn;2.5 mmol l-1) or Cs+ (10 mmol l-1). From the apical side, Isc was nearly completely blocked by Cs+ (10 mmol l-1) or Ba2+ (15 &micro;mol l-1), whereas apical addition of furosemide (1 mmol l-1) resulted in only a small current decrease. Cl- influxes were linearly related to negative Isc. The ratio between net influxes of Cl- and Na+ was found to be approximately 2:1. With a single membrane preparation, achieved by permeabilizing the basolateral membrane with amphotericin B, Cl- influxes which were driven by a concentration gradient were shown to depend on the presence of apical Na+ and K+. On the basis of these observations, we propose that active and electrogenic absorption of NaCl across the gill epithelium of hyperregulating shore crabs proceeds as in the thick ascending limb of Henle's loop in the mammalian nephron. Accordingly, branchial NaCl transport is mediated by apical K+ channels in cooperation with apical Na+/K+/2Cl- cotransporters and by the basolateral Na+/K+-ATPase and basolateral Cl- channels.
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PMID:Active absorption of Na+ and Cl- across the gill epithelium of the shore crab Carcinus maenas: voltage-clamp and ion-flux studies 931 45

Marginal cells constitute the endolymph-facing epithelium responsible for the secretion of endolymph by the stria vascularis in the inner ear. We have studied the possible involvement of Cl- conductance and Na+-K+-Cl- cotransport in the mechanism of changes in cell volume upon isotonic Cl- depletion/restoration. Changes in cell volume were estimated from video-microscopic images with the aid of an image processor. Marginal cells shrank to approximately 80% of their original volume in 30 s and to 65-70% in 90 s upon total replacement of [Cl]o (approximately 150 mM) by gluconate-, and the original volume of the shrunken cells was restored within 2 min after restoration of Cl-. The order of potency of anions to induce isotonic shrinkage was gluconate > I- > F- > Br-. The cell shrinkage caused by Cl- depletion was partially inhibited by 5-Nitro-2-(3-phenyl-propylamino)-benzoic acid (NPPB, 0.2 mM), but not by either 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (SITS, 0.5 mM), bumetanide (10 microM) or ouabain (1 mM). The cell shrinkage caused by a reduction of [Cl]o from approximately 150 mM to 7.5 mM was not affected by [K]o in the range of 3.6 mM to 72 mM. These results suggest that the main efflux pathway(s) responsible for the 'Cl removal'-induced shrinkage depends on volume-correlated Cl- conductance (Takeuchi and Irimajiri, J. Membrane Biol. 150, 47-62, 1996) and that this pathway(s) is essentially independent of the Na+-K+-Cl- cotransporter, the Na+,K+-ATPase, and the K+-Cl- cotransporter. With regard to volume recovery after isotonic shrinkage, its critical dependence on the simultaneous presence of Na+, K+ and Cl- in the bath and its substantial inhibition by bumetanide (10 microM) both indicate a major role for Na+-K+-Cl- cotransport. The strong influence on cell volume of solute fluxes working through the Cl- channel and the Na+-K+-Cl- cotransporter implies an essential role for these pathways in the ion transport mechanism(s) of the marginal cell.
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PMID:Changes in the volume of marginal cells induced by isotonic 'Cl- depletion/restoration': involvement of the Cl- channel and Na+-K+-Cl- cotransporter. 938 89


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