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)

MDCK cells display several acid-base transport systems found in intercalated cells, such as Na+-H+ exchange, H+-K+ ATPase and Cl-/HCO-3 exchange. In this work we studied the functional activity of a vacuolar H+-ATPase in MDCK cells and its chloride dependence. We measured intracellular pH (pHi) in monolayers grown on glass cover slips utilizing the pH sensitive probe BCECF. To analyze the functional activity of the H+ transporters we observed the intracellular alkalinization in response to an acute acid load due to a 20 mm NH+4 pulse, and calculated the initial rate of pHi recovery (dpHi/dt). The cells have a basal pHi of 7.17 +/- 0.01 (n = 23) and control dpHi/dt of 0.121 +/- 0.006 (n = 23) pHi units/min. This pHi recovery rate is markedly decreased when Na+ was removed, to 0.069 +/- 0.004 (n = 16). It was further reduced to 0.042 +/- 0. 005 (n = 12) when concanamycin 4.6 x 10(-8) m (a specific inhibitor of the vacuolar H+-ATPase) was added to the zero Na+ solution. When using a solution with zero Na+, low K+ (0.5 mm) plus concanamycin, pHi recovery fell again, significantly, to 0.023 +/- 0.006 (n = 14) as expected in the presence of a H+-K+-ATPase. This result was confirmed by the use of 5 x 10(-5) m Schering 28080. The Na+ independent pHi recovery was significantly reduced from 0.069 +/- 0.004 to 0.042 +/- 0.004 (n = 12) when NPPB 10(-5) m (a specific blocker of Cl- channels in renal tubules) was utilized. When the cells were preincubated in 0 Cl-/normal Na+ solution for 8 min. before the ammonium pulse, the pHi recovery fell from 0.069 +/- 0.004 to 0.041 +/- 0.007 (n = 12) in a Na+ and Cl- free solution. From these results we conclude that: (i) MDCK cells have two Na+-independent mechanisms of pHi recovery, a concanamycin sensitive H+-ATPase and a K+ dependent, Schering 28080 sensitive H+-K+ ATPase; and, (ii) pHi recovery in Na+-free medium depends on the presence of a chloride current which can be blocked by NPPB and impaired by preincubation in Cl--free medium. This finding supports a role for chloride in the function of the H+ ATPase, which might be electrical shunting or a biochemical interaction.
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PMID:H+ ATPase and Cl- interaction in regulation of MDCK cell pH. 959 78

Two mechanisms of H+ ion secretion in the proximal tubule that mediate bicarbonate reabsorption have been identified: the brush border Na/H exchanger and electrogenic H+ ion secretion. Angiotensin II (AII) has been shown to be a regulator of the luminal Na+/H+ exchanger and the basolateral Na+/HCO3- cotransporter. In the present study, we examined the effects of AII on H+-ATPase activity in isolated proximal tubule fragments. H+-ATPase activity was assessed by monitoring intracellular pH after Na+ removal from the bath. In addition, we investigated the effects on pH recovery of the proton pump inhibitor bafilomycin A1, removal of Cl-, and of colchicine. pH was continuously measured with the pH-sensitive fluorescent dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Recovery of cell pH was observed in the absence of external Na+ and was significantly accelerated by AII. The AII-stimulated pH recovery was completely abolished by bafilomycin A1, by removal of Cl-, by NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate; a potent Cl- channel blocker], and by colchicine. We conclude from these studies that AII stimulates proton extrusion via H+-ATPase by a Cl--dependent process involving brush border insertion of vesicles. This process may contribute to up-regulation of HCO3- reabsorption along the proximal tubule when tubules are exposed to AII.
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PMID:Angiotensin II stimulates vesicular H+-ATPase in rat proximal tubular cells. 968 38

The effect of Cd(2+) on chloride secretion was examined in A6 renal epithelia cells by chloride-sensitive fluorescence (SPQ probe) and by the short-circuit-current (I(sc)) technique. Depleting the cells of Cl(-) suggests that the Cd(2+)-activated I(sc) (DeltaI(sc(Cd))) is dependent on the presence of Cl(-) ions. Among the Cl(-)-channel inhibitors the fenemates, flufenamic acid (FFA) and niflumic acid (NFA), and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) significantly lowered DeltaI(sc(Cd)) compared with control level. In SPQ-loaded A6 cells, Cd(2+) evoked an increase in Cl(-) secretion ([DeltaCl(-)](Cd)), which significantly exceeded the basal Cl(-) transport and was blockable by FFA and NFA. The closely related metals, Zn(2+) or Ni(2+), were also able to activate Cl(-) secretion. Preexposure of Zn(2+) or Ni(2+) completely prevented [DeltaCl(-)](Cd), suggesting that Zn(2+) and Ni(2+) probably use similar mechanisms. Like Cd(2+), thapsigargin (TG), an inhibitor of intracellular Ca(2+)-ATPase and the Ca(2+)-ionophore A23187, induced an increase in I(sc). Moreover, TG and Cd(2+) were able to neutralize the responses of the counterparts as also observed in I(sc) measurements, which indicates that Cd(2+) activates Cl(-) secretion in a Ca(2+)-dependent manner. Hence, this study supports the idea that basolateral Cd(2+) (possibly also Zn(2+) and Ni(2+)), probably through a Ca(2+)-sensing receptor, causes calcium mobilization that activates apical fenemate-sensitive chloride channels leading to chloride secretion in A6 cells.
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PMID:Chloride secretion in kidney distal epithelial cells (A6) evoked by cadmium. 1070 66

The role of H(+)-ATPase in proximal tubule cell pH regulation was studied by microperfusion techniques and by confocal microscopy. In a first series of experiments, proximal S3 segments of rabbit kidney were perfused "in vitro" while their cell pH was measured by fluorescence microscopy after loading with BCECF. In Na(+)- and Cl(-)-free medium, cell pH fell by a mean of 0.37+/-0.051 pH units, but after a few minutes started to rise again slowly. This rise was of 0.17 +/-0.022 pH units per min, and was significantly reduced by bafilomycin and by the Cl(-) channel blocker NPPB, but not by DIDS. In a second series of experiments, subcellular vesicles of proximal tubule cells of S3 segments of mouse kidney were studied by confocal microscopy after visualization by acridine orange or by Lucifer yellow. After superfusion with low Na(+) solution, which is expected to cause cell acidification, vesicles originally disposed in the basolateral and perinuclear cell areas, moved toward the apical area, as detected by changes in fluorescence density measured by the NIH Image program. The variation of apical to basolateral fluorescence ratios during superfusion with NaCl Ringer with time was 0.0018+/- 0.0021 min(-1), not significantly different from zero (P>0.42). For superfusion with Na(+)0 Ringer, this variation was 0.081+/-0.015 min(-1), P<0.001 against 0. These slopes were markedly reduced by the Cl(-) channel blocker NPPB, and by vanadate at a concentration that has been shown to disrupt cytoskeleton function. These data show that the delayed alkalinization of proximal tubule cells in Na(+)-free medium is probably due to a vacuolar H(+)-ATPase, whose activity is stimulated in the presence of Cl(-), and dependent on apical insertion of subcellular vesicles. The movement of these vesicles is also dependent on Cl(-) and on the integrity of the cytoskeleton.
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PMID:Cell pH and H(+) secretion by S3 segment of mammalian kidney: role of H(+)-ATPase and Cl(-). 1108

By using Western blot and RT-PCR analyses, the expression of ClC-5, a member of the ClC family of voltage-gated chloride channels, and its mRNA was detected in OK cells. The effect of chloride channel inhibitors on receptor-mediated endocytosis of albumin was examined in OK cells and compared to that of vacuolar H(+)-ATPase inhibitors. Accumulation of fluorescein-isothiocyanate (FITC)-albumin, a receptor-mediated endocytosis marker, was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a chloride channel inhibitor, in a concentration-dependent fashion. In contrast, uptake of FITC-inulin, a fluid-phase endocytosis marker, was not affected by NPPB. Other chloride channel inhibitors, 4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid and diphenylamine-2-carboxylic acid, also inhibited FITC-albumin uptake. NPPB, as well as a vacuolar H(+)-ATPase inhibitor bafilomycin A(1), caused a decrease in the affinity and in the maximal velocity of FITC-albumin uptake. These results suggest that chloride channel, most likely ClC-5, plays an important role in the receptor-mediated endocytosis of albumin in OK cells.
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PMID:Expression of chloride channel, ClC-5, and its role in receptor-mediated endocytosis of albumin in OK cells. 1126 94

In several tissues ammonium ions are able to use the transport pathways of other ions, particularly of K+. We investigated this possibility in the C11 clone of MDCK cells, thought to represent intercalated cells, in control and 0 Cl- conditions. Cell pH was measured by ratiometric fluorescence microscopy using the pH indicator BCECF. After preincubating the cells for 10 min in control or 0 Cl- (substituted by gluconate) Ringer, an ammonium pulse was applied to induce cell acidification. The magnitude of the initial alkalinization (DeltapH) was 0.24+/-0.03 ( n=28) pH units in controls, which fell to 0.023+/-0.01 ( n=12) in 0 Cl-, suggesting uptake of NH4+ balancing the alkalinization by NH3. Addition of 10(-3) M bumetanide or furosemide to the 0 Cl- medium, or 10(-4 )M hexamethylene amiloride, did not alter DeltapH. However, with 5 mM Ba+, DeltapH increased to 38% of control. When 2.5x10(-4) M ouabain, an inhibitor of Na+-K+ ATPase, was used, DeltapH increased to 46% of control. Inhibition of H+-K+ ATPase by SCH28080 or by omeprazol caused significant increase in DeltapH. In 0 Cl- solution, these cells underwent a mean volume reduction (-d V) of -10.24+/-1.96% per 10 min as measured by confocal microscopy. To investigate if NH4+ influx was regulated by cell volume or by cell Cl-, volume reduction was avoided by two procedures. When preincubating with NPPB, a Cl- channel blocker, in 0 Cl-, volume reduction was inhibited (d V=-2.12% per 10 min), and DeltapH was 0.24+/-0.04 ( n=5). When the cells were preincubated in hypotonic 0 Cl- (260 mosmol/l), cell volume reduction was abolished (d V=+2.6% per 10 min) and DeltapH was 0.52+/-0.07 ( n=7). Thus, activation of NH4+ influx by several transporters was due to volume reduction rather than to [Cl-] alteration.
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PMID:Factors affecting ammonium uptake by C11 clone of MDCK cells. 1245 40

The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na+-Cl- cotransporter located at the apical membrane, and Na+, K+ ATPase at the basolateral side. Cl- transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl- channels on microdissected mouse DCTs using the patch-clamp technique. A channel of approximately 9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NPo in cell-attached patches was not modified when tubules were preincubated in the presence of 10-5 M forskolin, but the channel was inhibited by phorbol ester (10-6 M). In addition, NPo was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM (NPo increased threefold), or pH increased from 6.4 to 8.0 (NPo increased 15-fold). Selectivity experiments conducted on inside-out patches showed that the Na+ to Cl- relative permeability was 0.09, and the anion selectivity sequence Cl(-)--I(-) > Br(-)--NO3(-) > F(-). Intracellular NPPB (10-4 M) and DPC (10-3 M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl- channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family.
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PMID:A chloride channel at the basolateral membrane of the distal-convoluted tubule: a candidate ClC-K channel. 1266 33

Hypertonicity induced by NaCl, but not by urea or mannitol, up-regulates expression of the gamma subunit of Na/K-ATPase in cells of the murine inner medullary collecting duct line (IMCD3) by activation of the Jun kinase 2 (JNK2) pathways. We examined the ionic mediators of the osmosensitive response. An increase in osmolality to 550 milliosmoles per kg of water (mosmol/kgH2O) for 48 h by replacement of NaCl with choline chloride did not prevent the up-regulation of the gamma subunit. Neither Na+ ionophores nor inhibitors of cellular Na+ uptake altered the up-regulation of the gamma subunit or JNK activation. Changes in cell cation concentrations driven by incubation in low-K+ medium were effective in up-regulating the alpha1 subunit of Na/K-ATPase but did not have any effect on the gamma subunit. The replacement of NaCl with choline chloride did not down-regulate gamma-subunit expression in cells adapted to hypertonicity. In contrast, the replacement of NaCl with sodium acetate, or pretreatment of cells with the Cl- channel inhibitor 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB) completely blocked gamma-subunit up-regulation, inhibited JNK activation, and caused a significant decrement in cell survival in hypertonic but not isotonic conditions. In adapted cells, replacement of 300 mosmol/kgH2O NaCl with sodium acetate resulted in down-regulation of the gamma subunit. In conclusion, we describe a Na+-independent, Cl--dependent mechanism for hypertonicity-mediated activation of the JNK and the subsequent synthesis of the gamma subunit of Na/K-ATPase, which are necessary for cellular survival in these anisotonic conditions.
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PMID:Chloride, not sodium, stimulates expression of the gamma subunit of Na/K-ATPase and activates JNK in response to hypertonicity in mouse IMCD3 cells. 1274 99

Iono- and osmoregulation by the blood-feeding hemipteran Rhodnius prolixus involves co-ordinated actions of the upper and lower Malpighian tubules. The upper tubule secretes ions (Na(+), K(+), Cl(-)) and water, whereas the lower tubule reabsorbs K(+) and Cl(-) but not water. The extent of KCl reabsorption by the lower tubule in vitro was monitored by ion-selective microelectrode measurement of Cl(-) and/or K(+) concentration in droplets of fluid secreted by Malpighian tubules isolated under oil. An earlier study proposed that K(+) reabsorption involves an omeprazole-sensitive apical K(+)/H(+) ATPase and Ba(2+)-sensitive basolateral K(+) channels. This paper examines the effects acetazolamide and of compounds that inhibit chloride channels, Cl(-)/HCO(3)(-) exchangers and Na(+)/K(+)/2Cl(-) or K(+)/Cl(-) co-transporters. The results suggest that Cl(-) reabsorption is inhibited by acetazolamide and by Cl(-) channel blockers, including diphenylamine-2-carboxylate(DPC) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), but not by compounds that block Na(+)/K(+)/Cl(-) and K(+)/Cl(-) co-transporters. Measurements of transepithelial potential and basolateral membrane potential during changes in bathing saline chloride concentration indicate the presence of DPC- and NPPB-sensitive chloride channels in the basolateral membrane. A working hypothesis of ion movements during KCl reabsorption proposes that Cl(-) moves from lumen to cell through a stilbene-insensitive Cl(-)/HCO(3)(-) exchanger and then exits the cell through basolateral Cl(-) channels.
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PMID:KCl reabsorption by the lower malpighian tubule of rhodnius prolixus: inhibition by Cl(-) channel blockers and acetazolamide. 1276 77

The photoreceptors lie between the inner retina and the retinal pigment epithelium (RPE). The release of glutamate by the phototoreceptors can signal changes in light levels to inner retinal neurons, but the role of glutamate in communicating with the RPE is unknown. Since RPE cells are known to release ATP, we asked whether glutamate could trigger ATP release from RPE cells and whether this altered cell signalling. Stimulation of the apical face of fresh bovine RPE eyecups with 100 mum NMDA increased ATP levels more than threefold, indicating that both receptors for NMDA and release of ATP occurred across the apical membrane of fresh RPE cells. NMDA increased ATP levels bathing cultured human ARPE-19 cells more than twofold, with NMDA receptor inhibitors MK-801 and d-AP5 preventing this release. Blocking the glycine site of the NMDA receptor with 5,7-dichlorokynurenic acid prevented ATP release from ARPE-19 cells. Release was also blocked by channel blocker NPPB and Ca(2+) chelator BAPTA, but not by cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide or vesicular release inhibitor brefeldin A. Glutamate produced a dose-dependent release of ATP from ARPE-19 cells that was substantially inhibited by MK-801. NMDA triggered a rise in cell Ca(2+) that was blocked by MK-801, by the ATPase apyrase, by the P2Y(1) receptor antagonist MRS2179 and by depletion of intracellular Ca(2+) stores with thapsigargin. These results suggest that glutamate stimulates NMDA receptors on the apical membrane of RPE cells to release ATP. This secondary release can amplify the glutaminergic signal by increasing Ca(2+) inside RPE cells, and might activate Ca(2+)-dependent conductances. The interplay between glutaminergic and purinergic systems may thus be important for light-dependent interactions between photoreceptors and the RPE.
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PMID:Glutamate acts at NMDA receptors on fresh bovine and on cultured human retinal pigment epithelial cells to trigger release of ATP. 1680 61


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