Determination of P-Glycoprotein ATPase Activity Using Luciferase

Matsunaga, Tamihide; Kose, Eiji; Yasuda, Sachiyo; Ise, Hirohiko; Ikeda, Uichi; Ohmori, Shigeru

doi:10.1248/bpb.29.560

Cited by 15 publications

(10 citation statements)

References 33 publications

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“…This value is close to the K m for digoxin transport in human Pgp from Caco-2 cells of 385 μM [83]. Although our K m value was in the general range of previously determined K m values for digoxin-induced ATPase activation of Pgp, the previously determined K m values vary widely in the literature [80][81][82]. A K m value of 1.2 μM for ATPase activation by digoxin was determined in Caco-2 membrane vesicles containing human Pgp [80], whereas a K m value of 83.7 μM for ATPase activation was reported for human Pgp-enriched insect cell membranes [82].…”

Section: Resultssupporting

confidence: 83%

“…The digoxin-induced activation of ATP hydrolysis kinetics was monophasic and reached a maximum ∼2-fold activation or ∼1300 nmol·min − 1 ·mg − 1 (Figure 2, open circles), which is in the range observed previously [80][81][82]. The kinetics were fit to the Michaelis-Menten equation (eqn 1) and gave values for V MAX and K m of 1344 + − 149.8 nmol·min − 1 ·mg − 1 and 240.4 + − 68.1 μM respectively.…”

Section: Resultssupporting

confidence: 81%

“…Although our K m value was in the general range of previously determined K m values for digoxin-induced ATPase activation of Pgp, the previously determined K m values vary widely in the literature [80][81][82]. A K m value of 1.2 μM for ATPase activation by digoxin was determined in Caco-2 membrane vesicles containing human Pgp [80], whereas a K m value of 83.7 μM for ATPase activation was reported for human Pgp-enriched insect cell membranes [82]. For CR1R12 cells containing Pgp, maximal activation of ATP hydrolysis was not even reached at 1000 μM digoxin [83] implying a K m that is considerably higher than 500 μM.…”

Section: Resultsmentioning

confidence: 46%

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Unravelling the complex drug–drug interactions of the cardiovascular drugs, verapamil and digoxin, with P-glycoprotein

2016

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SynopsisDrug-drug interactions (DDIs) and associated toxicity from cardiovascular drugs represents a major problem for effective co-administration of cardiovascular therapeutics. A significant amount of drug toxicity from DDIs occurs because of drug interactions and multiple cardiovascular drug binding to the efflux transporter P-glycoprotein (Pgp), which is particularly problematic for cardiovascular drugs because of their relatively low therapeutic indexes. The calcium channel antagonist, verapamil and the cardiac glycoside, digoxin, exhibit DDIs with Pgp through non-competitive inhibition of digoxin transport, which leads to elevated digoxin plasma concentrations and digoxin toxicity. In the present study, verapamil-induced ATPase activation kinetics were biphasic implying at least two verapamil-binding sites on Pgp, whereas monophasic digoxin activation of Pgp-coupled ATPase kinetics suggested a single digoxin-binding site. Using intrinsic protein fluorescence and the saturation transfer double difference (STDD) NMR techniques to probe drug-Pgp interactions, verapamil was found to have little effect on digoxin-Pgp interactions at low concentrations of verapamil, which is consistent with simultaneous binding of the drugs and non-competitive inhibition. Higher concentrations of verapamil caused significant disruption of digoxin-Pgp interactions that suggested overlapping and competing drug-binding sites. These interactions correlated to drug-induced conformational changes deduced from acrylamide quenching of Pgp tryptophan fluorescence. Also, Pgp-coupled ATPase activity kinetics measured with a range of verapamil and digoxin concentrations fit well to a DDI model encompassing non-competitive and competitive inhibition of digoxin by verapamil. The results and previous transport studies were combined into a comprehensive model of verapamil-digoxin DDIs encompassing drug binding, ATP hydrolysis, transport and conformational changes.

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Section: Resultssupporting

confidence: 83%

Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 46%

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Unravelling the complex drug–drug interactions of the cardiovascular drugs, verapamil and digoxin, with P-glycoprotein

2016

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“…This biphasic response for P-gp ATPase activation observed for oxycodone has also been reported for a variety of compounds, e.g., promethazine, propafenone, quinidine, dipridamole, and digoxin, and indicates that these compounds require threshold concentrations for measurable ATPase activation. 32,33 It should be noted that certain compounds stimulate P-gp ATPase activity, but are not transported by P-gp across cell membranes. 31 For this reason, a series of bidirectional transport studies were conducted using Caco-2 cells, a cell line known to express both P-gp and MRP.…”

Section: Discussionmentioning

confidence: 99%

Oxycodone induces overexpression of P‐glycoprotein (ABCB1) and affects paclitaxel's tissue distribution in Sprague Dawley rats

Hassan

Myers

Lee

et al. 2007

Journal of Pharmaceutical Sciences

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Previous studies suggest that P-glycoprotein (P-gp) modulates the PK/PD of many compounds including opioid agonists and chemotherapeutic agents. The objective of this study was to assess the P-gp affinity status of oxycodone, the P-gp expression, and the paclitaxel's tissue distribution in oxycodone-treated rats. P-gp ATPase assay, Caco-2 transepithelial permeability studies, and mdr1a/b (−/−) mice were used to assess the P-gp affinity status of oxycodone. P-gp expression was determined by Western blot analysis while [ 14 C] paclitaxel's distributions in the liver, kidney, brain, and plasma tissues were determined by liquid scintillation counter. Oxycodone stimulated the P-gp ATPase activity in a concentration-dependant manner. The Caco-2 secretory transport of oxycodone was reduced from 3.64 ×10 −5 to 1.96 × 10 −5 cm/s (p <0.05) upon preincubation with the P-gp inhibitor, verapamil. The brain levels of oxycodone in mdr1a/b (+/+) were not detectable (<15 ng/mL) while in mdr1a/b (−/−) the average levels were 115 ± 39 ng/mL. The P-gp protein levels were increased by 1.3-4.0 folds while paclitaxel's tissue distributions were decreased by 38-90% (p <0.05) in oxycodone-treated rats. These findings display that oxycodone is a P-gp substrate, induces overexpression of P-gp, and affects paclitaxel's tissue distribution in a manner that may influence its chemotherapeutic activity.

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“…P-gp ATPase activity measurement P-gp ATPase activity was determined by measuring the release of inorganic phosphate (P i ) from the consumption of ATP using the light-generating reaction of firefly luciferase [33] . According to the manufacturer's protocol for the Pgp-Glo TM assay system with recombinant human P-gp-containing membrane, the P-gp-containing membrane (25 μg/well) was preincubated in a 96-well with 100 nmol/L 4α-PMA and various concentrations of PMA for 5 min at 37 °C.…”

Section: Atp Assaymentioning

confidence: 99%

Phorbol 12-myristate 13-acetate inhibits P-glycoprotein-mediated efflux of digoxin in MDCKII-MDR1 and Caco-2 cell monolayer models

Huang

et al. 2013

Acta Pharmacol Sin

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Aim: To investigate the effects of phorbol 12-myristate 13-acetate (PMA), a PKC activator, on P-glycoprotein-mediated efflux of digoxin in two cell transport models. Methods: Caco-2 cells, wild MDCKII cells (MDCKII-WT) and MDCKII cells transfected stably with human MDR1-gene encoding P-gp (MDCKII-MDR1) were examined. Cell viability was evaluated with MTT assay. Bidirectional transport of digoxin was evaluated in these cells. Intracellular ATP level was measured using ATP assay. P-gp ATPase activity was analyzed using a Pgp-Glo TM assay. Results: PMA (10 μmol/L) did not reduce the viability of the 3 types of cells. In Caco-2 and MDCKII-MDR1 cell monolayers, PMA (1, 10 and 100 nmol/L) dose-dependently inhibited the basolateral to apical transport of digoxin, but did not change the apical to basolateral transport. In addition, PMA did not affect both the basolateral to apical and apical to basolateral transport of digoxin in MDCKII-WT cell monolayer. In agreement with the above results, PMA dose-dependently reduced intracellular ATP level and stimulated P-gp ATPase activity in both Caco-2 and MDCKII-MDR1 cells. Verapamil (a positive control, 100 μmol/L) caused similar inhibition on digoxin efflux as PMA did, whereas 4α-PMA (a negative control, 100 nmol/L) had no effect. Conclusion: PMA significantly inhibited P-gp-mediated efflux of digoxin in both Caco-2 and MDCKII-MDR1 cell monolayers via PKC activation.

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Determination of P-Glycoprotein ATPase Activity Using Luciferase

Cited by 15 publications

References 33 publications

Unravelling the complex drug–drug interactions of the cardiovascular drugs, verapamil and digoxin, with P-glycoprotein

Unravelling the complex drug–drug interactions of the cardiovascular drugs, verapamil and digoxin, with P-glycoprotein

Oxycodone induces overexpression of P‐glycoprotein (ABCB1) and affects paclitaxel's tissue distribution in Sprague Dawley rats

Phorbol 12-myristate 13-acetate inhibits P-glycoprotein-mediated efflux of digoxin in MDCKII-MDR1 and Caco-2 cell monolayer models

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