Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.3.44 (P-glycoprotein)
 13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated the polarity of the efflux of the intracellular pH fluorochrome 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) from layers of epithelial Madin-Darby canine kidney (MDCK, Strains I and II) and human intestinal (Caco-2, HCT-8 and T84) cells grown on porous membranes. In Strain I MDCK cells, BCECF efflux was effectively reduced by indomethacin (50% inhibition with 100 microM) and 5-nitro-2-(3-phenylpropyl-amino)-benzoate (NPPB; 50% inhibition with 10 microM). Replacement of external Cl- with bromide, iodide or nitrate did not alter BCECF efflux, while substitution with methanesulphonate resulted in a small but significant reduction. All five cell lines form confluent epithelial layers when grown on porous membranes. Efflux of BCECF from Strain I MDCK epithelial layers into the apical solution was approximately three times greater than into the basal solution. Addition of indomethacin to the apical solution attenuated efflux into the apical but not the basal solution, while basal indomethacin was effective against basal efflux. NPPB has a similar specificity of action. Adrenaline, a stimulant of electrogenic Cl- secretion, did not alter the pattern of BCECF efflux. BCECF efflux was also polarized, with apical efflux greater than basal efflux, in MDCK Strain II and Caco-2 epithelial layers. In contrast, BCECF efflux into the basal and apical media was equivalent in layers formed from HCT-8 and T84 cells. However, indomethacin reduced efflux in all five epithelial lines, although the relative sensitivities of the apical and basal efflux rates to indomethacin varied, as did the sensitivity to the sidedness of application of indomethacin. In MDCK and HCT-8 epithelial layers, transepithelial vinblastine secretion mediated by P-glycoprotein was not inhibited by indomethacin. The data are consistent with the hypothesis that BCECF efflux is a manifestation of a novel ATP-dependent xenobiotic secretory efflux mechanism in renal and gastrointestinal epithelia. The factors regulating the polarity of BCECF efflux, both the indomethacin-sensitive and -insensitive components, have yet to be elucidated.
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PMID:Polarized efflux of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein from cultured epithelial cell monolayers. 151 Jun 94

The characteristics of volume-activated chloride currents, drug transport function and levels of P-glycoprotein (PgP) expression were compared between two human chronic erythroleukemia cell lines: a parental (K562) cell line and a derivative obtained by vinblastine selection (K562 VBL400). Parental K562 cells showed no detectable P-glycoprotein expression, measured at the protein level (immunofluorescence labeling with monoclonal antibodies), and had very low levels of MDR-1 mRNA expression (RT-PCR analysis), when compared with levels measured in K562 VBL400. Differences in Pgp-mediated transport were estimated by comparing the rates of Fluo3 accumulation. The higher drug-transport function of K562 VBL400 cells (e.g., lower Fluo3 accumulation) correlated with their elevated levels of MDR-1. The rate of dye transport was sensitive to verapamil but was not affected by the tonicity of the extracellular medium. In contrast to the clear differences in transport function, the characteristics of chloride currents induced by cell swelling were indistinguishable between the two cell lines. Currents measured in the whole-cell configuration were outwardly rectifying, had a higher permeability to iodide than to chloride (SCN- > I- > Cl- > gluconate), were potently blocked by NPPB and were unresponsive to verapamil. The percentage of responding cells and the mean current density were nearly identical in both cell lines. In addition, activation of the volume-sensitive current was not prevented during whole-cell recordings obtained with pipettes containing high concentration of cytotoxic drugs (vincristine or vinblastine). These results do not lend support to the previously reported association between Pgp expression and volume-sensitive chloride channels, and suggest that a different protein is responsible for this type of chloride channel in K562 cells.
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PMID:Drug-transport and volume-activated chloride channel functions in human erythroleukemia cells: relation to expression level of P-glycoprotein. 763 88

Resistance to chemotherapeutic agents in neoplastic cells is often mediated by expression of P-glycoprotein, which functions as a drug-efflux pump for a broad range of substrates. We have used a combination of patch clamp and video-imaging techniques to examine the expression and drug-efflux function of P-glycoprotein and to determine the possible correlation with swelling-activated chloride channels in drug-sensitive and -resistant cell lines. Two pairs of cell lines were used in these experiments: (a) control NIH-3T3 cells and a corresponding MDR1-transfectant; and (b) control 8226 myeloma cells and a derivative cell line selected for resistance to chemotherapeutic agents. Control cells lacked detectable P-glycoprotein expression based on Western blotting, immunofluorescence staining with a specific monoclonal antibody, and a functional assay of rhodamine-123 (R123) efflux. Resistant cells expressed P-glycoprotein at high levels and rapidly exported R123. During whole-cell recording using either hyperosmotic pipette solution or hypoosmotic Ringer solution, cell swelling was accompanied by Cl- channel opening in all four cell lines. The rates of induction, biophysical properties and magnitudes of Cl conductance (gCl) were indistinguishable between control and corresponding multidrug-resistant cells: gCl reached 0.96 +/- 0.31 (n = 14) and 0.83 +/- 0.31 nS/pF (mean +/- SD; n = 31) in NIH-3T3 and NIH-3T3/MDR cells, respectively; and 0.31 +/- 0.20 (n = 9) and 0.37 +/- 0.22 nS/pF (n = 7) in 8226 and 8226/Dox40 cells, respectively. gCl exhibited moderate outward rectification in symmetrical Cl- solutions, with a rectification ratio of 1.4 at +/- 50 mV. Cl- channels slowly closed during strong depolarization beyond +60 mV. Using video-imaging techniques with SPQ as a fluorescent probe, we monitored Cl(-)-channel opening in intact drug-sensitive and -resistant cells. gCl, measured either with whole-cell recording or SPQ imaging, was blocked by DIDS (voltage-dependent Kd < 50 microM at +40 mV), NPPB (Kd approximately 30 microM), and tamoxifen (complete and irreversible block approximately 10 microM). None of these blockers inhibited R123 efflux. NPPB accelerated R123 efflux, an effect that was mimicked by CCP, a mitochondrial uncoupler. In contrast, verapamil selectively blocked R123 efflux (Kd = 0.3 to 0.5 microM); 10 microM left gCl unaltered. Induction of gCl was not affected by vincristine or doxorubicin in the pipette solution. Moreover, the rate of R123 efflux did not change during cell swelling. We conclude that P-glycoprotein and swelling-activated chloride channels function independently and are separable by expression and by pharmacological sensitivities.
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PMID:Swelling-activated chloride channels in multidrug-sensitive and -resistant cells. 769 67

1. We have studied the permeation and pharmacological properties of a recently described volume-activated, calcium-insensitive, small-conductance Cl(-)-channel in endothelial cells from human umbilical vein. 2. The relative permeability for various anions was I- > Cl- approximately Br- > F- > gluconate- (1.63 +/- 0.36: 1:0.95 +/- 0.16:0.46 +/- 0.04:0.19 +/- 0.07, n = 10). 3. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) induced a fast and reversible block of the current (Ki = 29 mumol l-1). 4. Extracellular ATP induced a low-affinity block of the current, that showed a small voltage-dependence (K1 = 4.9 mmol l-1 at +80 mV and K1 = 8.2 mmol l-1 at -80 mV). 5. Extracellularly applied arachidonic acid (10 mumol l-1) irreversibly blocked the current in 5 out of 9 cells. This block seems to be non-specific, because other ionic currents, e.g. inwardly rectifying K+ currents, were blocked as well. 6. Tamoxifen induced a high affinity block of the current (K1 = 2.9 mumol l-1). Block and reversal of block were however much slower than with NPPB. 7. Cytotoxic compounds, which are substrates of the P-glycoprotein multidrug transporter, loaded into endothelial cells via the patch pipette, exerted only minor effects on the volume-activated current. Vinblastine and colcemid did not affect the volume-activated current, whereas daunomycin and vincristine induced a slow 'run-down' of the current. 8. The similarity between permeation and pharmacological properties of volume-activated Cl--currents in endothelial cells and those in many other cell types may suggest that they all belong to the same family of volume-activated small-conductance Cl--channels. Evidence that they belong to the class of P-glycoprotein associated Cl--channels is however only marginal, whereas their biophysical characteristics differ significantly from those of the CIC-2 volume-activated Cl--channels.
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PMID:Permeation properties and modulation of volume-activated Cl(-)-currents in human endothelial cells. 795 63

1. The whole-cell patch clamp technique was used to investigate the swelling-activated currents in bovine non-pigmented ciliary epithelial (NPCE) cells. 2. Exposure to hypotonic solution activated a current that was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The I-V relationship was shifted in the direction expected for a Cl- current when the external Cl- was replaced by gluconate (permeability ratio P(gluconate)/PCl = 0.17). The inhibition of the current evoked by voltage clamp steps of +80 mV yielded an IC50 for NPPB of 13.4 microM. 3. The current was found to be dependent on ATP. With ATP in the patch pipette the current could be repeatedly activated by exposure to hypotonic solution but when ATP was omitted the current ran down with time. 4. The development of this current was associated with visible cell swelling and inhibitors of regulatory volume decrease in these cells, e.g. tamoxifen, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), also inhibited this current. 5. The volume-activated current was additionally blocked by NPPB, verapamil, quinidine and dideoxyforskolin. 6. The current was independent of external calcium and exhibited slight outward rectification and time-dependent inactivation at strong depolarizing potentials. 7. Disrupting the cytoskeleton and microtubules with cytochalasin B and colchicine had no effect on the activation of the Cl- current. 8. An antibody (C219) to the MDR1 gene product, P-glycoprotein, caused a functional block of the swelling-activated Cl- current when added to the patch pipette. 9. Immunofluorescence studies using the monoclonal antibodies C219 and JSB-1 demonstrated the presence of P-glycoprotein in the ciliary epithelial cells. The immunofluorescence was stronger on the non-pigmented than on the pigmented cells. 10. It is concluded that swelling in NPCE cells activates a Ca(2+)-independent, ATP-dependent Cl- current and that the activity of this current is associated with P-glycoprotein. 11. It is suggested that this Cl- current contributes to regulatory volume decrease and may participate in the secretory activity of these cells.
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PMID:P-glycoprotein regulates a volume-activated chloride current in bovine non-pigmented ciliary epithelial cells. 881 8

Iodide efflux, an index of anion permeability, has been monitored in cultured rat brain endothelial cells. Following hypotonicity-induced swelling, large, rapid increases in permeability occur, the extent of these increases depending on the degree of hypotonicity. Such large responses are not observed with rat aortic endothelial cells. Results of anion substitution experiments suggest that iodide efflux is via a chloride channel rather than an exchanger. The efflux increase is blocked by NPPB (100 microM) but not by DIDS or DPC at 100 microM. It is dependent on intracellular ATP but unaffected by removal of external calcium. Increasing internal calcium using A23187 does not produce a change in efflux, but depletion of calcium reduces or eliminates the response to hypotonicity. The response is reduced by pimozide (2-50 microM) that inhibits the actions of calmodulin and by pBPB (10 microM) that affects phospholipase A2 activity. It is eliminated by 5-lipoxygenase inhibitors (L-656,224 and ETH615, 10 microM) but is unaffected by cyclo-oxygenase inhibitors (indomethacin and piroxicam, 1-100 microM). It is blocked by some modulators of P-glycoprotein activity, e.g., verapamil (100 microM), tamoxifen (50 microM), and progesterone (100 microM) but not by others, e,g., forskolin (40 microM), dideoxyforskolin (40 microM), quinidine (100 microM) and cyclosporin A (10 microM).
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PMID:Hypotonicity-induced changes in anion permeability of cultured rat brain endothelial cells. 910 87

Purines can modify ciliary epithelial secretion of aqueous humor into the eye. The source of the purinergic agonists acting in the ciliary epithelium, as in many epithelial tissues, is unknown. We found that the fluorescent ATP marker quinacrine stained rabbit and bovine ciliary epithelia but not the nerve fibers in the ciliary bodies. Cultured bovine pigmented and nonpigmented ciliary epithelial cells also stained intensely when incubated with quinacrine. Hypotonic stimulation of cultured epithelial cells increased the extracellular ATP concentration by 3-fold; this measurement underestimates actual release as the cells also displayed ecto-ATPase activity. The hypotonically triggered increase in ATP was inhibited by the Cl--channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in both cell types. In contrast, the P-glycoprotein inhibitors tamoxifen and verapamil and the cystic fibrosis transmembrane conductance regulator (CFTR) blockers glybenclamide and diphenylamine-2-carboxylate did not affect ATP release from either cell type. This pharmacological profile suggests that ATP release is not restricted to P-glycoprotein or the cystic fibrosis transmembrane conductance regulator, but can proceed through a route sensitive to NPPB. ATP release also was triggered by ionomycin through a different NPPB-insensitive mechanism, inhibitable by the calcium/calmodulin-activated kinase II inhibitor KN-62. Thus, both layers of the ciliary epithelium store and release ATP, and purines likely modulate aqueous humor flow by paracrine and/or autocrine mechanisms within the two cell layers of this epithelium.
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PMID:A release mechanism for stored ATP in ocular ciliary epithelial cells. 961 58

The ability to maintain cellular volume is an important general physiological function. Swelling induced by hypotonic stress results in the opening of channels, through which ions exit with accompanying water loss (regulatory volume decrease, RVD). RVD has been shown to occur in mammalian sperm, primarily through the opening of quinine-sensitive potassium channels. However, as yet, direct evidence for the participation of anion channels in sperm RVD has been lacking. The chloride channel type ClC-3 is believed to be involved in RVD in other cell types. Using electronic cell sizing for cell volume measurement, the following results were obtained. (i) The anion channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), tamoxifen and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) increased hypotonic swelling in concentration-dependent fashion, whereas verapamil (P-glycoprotein inhibitor) had little effect. The most potent, NPPB and DIDS, blocked RVD without affecting cell membrane integrity at effective concentrations. (ii) When gramicidin was included to dissipate Na+/K+ gradients, major secondary swelling was observed under hypotonic conditions. This secondary swelling could be reduced by NPPB, and suppressed completely by replacing chloride in the medium with sulphate, an ion which does not pass through chloride channels. It was deduced that the initial hypotonic swelling activated an anion channel through which chloride ions could then enter freely down a concentration gradient, owing to the lack of a counter-gradient of potassium. (iii) Taurine, an osmolyte often involved in RVD, does not appear to play a role in sperm RVD because lengthy preincubation with taurine did not alter sperm RVD response. Our observations provide direct evidence that a chloride channel (possibly ClC-3) is involved in the process of volume regulation in mammalian sperm.
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PMID:Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm. 1536 53

Studies in the human, transgenic mice, and cattle indicate that sperm cell volume regulation plays an important role in male fertility as spermatozoa encounter a hypo-osmotic challenge upon ejaculation into the female tract. Physiological regulatory volume decrease (RVD) was examined using flow cytometry in murine sperm released into incubation medium mimicking uterine osmolality and including putative channel inhibitors. The involvement of K+ channels was indicated by the recovery of volume regulation by the K+ ionophore valinomycin in defective sperm from infertile transgenic mice, and from blockage of RVD by quinine in normal sperm. However, in neither case was the recovery complete. The involvement of volume-sensitive osmolyte and anion channels (VSOAC) were investigated using blockers effective in other cell types. NPPB (5-nitro-2(3-phenylpropylamino) benzoic acid) and tamoxifen inhibited RVD but SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid) at 0.4 and 1 mM had no effect whereas DIDS (di-isothiocyanato-stilbene-2,2'-disulphonic acid) at 1 mM enhanced RVD. Verapamil, but not another P-glycoprotein antagonist cyclosporin, caused sperm swelling which persisted in the presence of valinomycin, in Ca2+-free medium and in the presence of thapsigargin, but swelling was abolished by the Ca2+ ionophore A23187. Nifedipine was slightly effective in blocking RVD. Analysis by Western blotting failed to reveal ClC-2 and ClC-3 members of the chloride channel family in murine or rat sperm proteins despite signal bands in positive tissue controls. These findings implicate the involvement of some unidentified VSOAC in sperm volume regulation, which is probably Ca+-dependent.
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PMID:The role of anion channels and Ca2+ in addition to K+ channels in the physiological volume regulation of murine spermatozoa. 1580 61