ATPase assay

Accumulation/efflux studies can be performed on different cell systems or membrane vesicle preparations. In the accumulation assays, uptake of a probe over time, typically either fluorescent (e.g. calcein-AM (CAM) [25-27]) or radiolabeled, into the cell or membrane vesicles is measured in the presence or absence of a known P-gp inhibitor. As P-gp transports substrates out of the cells, the inhibition of the protein would result in an increase in the amount of the probe in the cell. Accumulation studies in cells that overexpress P-gp can be compared to those obtained in the parental cell line that does not have as high a level of P-gp expression. The probe in the absence of inhibitors shows lower accumulation in P-gp expressing cells than in P-gp deficient cells. Similarly, probe accumulation is increased under conditions where P-gp is inhibited such that the difference in accumulation in P-gp deficient and overexpressing cells, respectively, becomes smaller. Accumulation assays poorly distinguish substrates and inhibitors of P-gp and, as far as transport assays are concerned, are also influenced by a passive diffusion property of molecules [20]. In contrast to transport assays, both accumulation (i.e. calcein-AM assay) and ATPase assays tend to fail in the identification ofrelatively low permeable compounds as P-gp active compounds [20]. 

Based on several years of experience and analyses, some exceptions have been introduced. The Na+/K+ ATPase assay is screened at 3 x 10 M because of a low hit rate. The cutoff for IC50 follow-up has been raised to 40% inhibition for CCKb, GABAa, GABAB(ib), Kainate, Glycine (strychnine-insensitive), N (neuronal alpha bungarotoxin insensitive) and PAF assays. 

However, in two recently published studies from Roche (Schwab et al. 2003) and Glaxo (Polli et al. 2001) our limitations in studying MDR1 became obvious several compounds known as substrates or inhibitors of MDR1 were not clearly identified, others were identified only by one or two of the test methods (either ATPase assay or calcein-efflux or rhodamine efflux or cellular efflux studies). 

Among the membrane-based assay systems, the ATPase assay can be used to identify ABC substrates, since ABC transporters require ATP to transport substrates across the cell membrane. Using isolated membranes containing the ABC transporter of interest or reconstituted ABC protein preparations, ABC substrates would be revealed by an observed increase in ATPase activity (colorimetric detection of inorganic phosphate). In an alternative, inhibition-type (indirect) setup, the test compound is added to a well-established ABC substrate, which creates high ATPase activity. If the test compound is also an ABC substrate, the increased ATPase activity will decrease. 

Different assays are used to monitor the function of P-gp such as (i) ATPase assays (ii) drug transport assays across confluent, polarized cell monolayers and (iii) competition assays with reference substrates. The different assays address different functional aspects of P-gp. 

Quetiapine Antipsychotic drug MDRl ATPase assay, LLC-PKl cells [78, 150] ... 

Fig. 31. Reversible inactivation of Fi (p Asp-380->Cys) by formation of a specific p y intersubunit disulfide bond. Wild-type (w.t.) and mutant F, samples were treated with DTNB and aliquots were taken for SDS-PAGE and ATPase assays. See text for other details. Figure source Duncan, Bulygin, Zhou, Hutcheon and Cross (1995) Rotation of subunits during catalysis by Escherichia coli F,-ATPase. Proc Nat Acad Sci, USA 92 10966.

Experimental methods will not be described since these are detailed in the literature P2X purinoceptors [26-30] P2Y-purinoceptors [29-31] P2U purinoceptors [30] P2T-purinoceptors [32] ecto-ATPase assay [31]. 

Pluronic P85 was also reported to cause a higher degree of alteration in the P-gp functions than MRP2 and MRPl in ATPase assay of P-gp, MRPl, and MRP2 and inhibition assays with their substrates, vinblastine, and leucotriene-C4 (Batrakova et al., 2004). Cremophor EL (cremophor) is a nonionic solubilizer and emulsifier used for some hydrophobic drugs and fat-soluble vitamins. In a Phase I trial of cremophor as a 6-h infusion every 3 weeks performed with bolus doxorubicin (50mg/m ), the AUC of doxorubicin increased from 1448 (CV of 24%) to 1786 h ng/mL (CV of 15%) in the presence of cremophor, whereas the AUC of doxorubicinol increased from 252 (CV of 42%) to 486 (CV of 22%) h ng/mL. Such interactions can be due to decreased clearance of doxorubicin 612 (CV of 29%) to 477 (CV of 15%)... 

Membrane-Based Assays Membranes prepared from cells expressing transporters have been widely used to study the function of ABC efflux pumps and to identify their substrates or inhibitors. Currently, there are two major membrane-based assays the ATPase assay and the membrane vesicular transport (uptake) assay. Compared to the cell-based assay, the membrane-based assay has several advantages including (1) the assay can be used to characterize the effect of a xenobiotic on one specific efflux transporter (2) the assay can be easily employed in a high throughput mode (3) membranes are easy to be maintained after preparation and (4) the assay is easy to conduct. 

The rate of phosphorylation is much faster than the overall turnover rate of the enzyme, suggesting that the phosphorylation step is not rate-limiting [71], Surprisingly, Wallmark and Mirdh [74] found that at low substrate concentration (5 /rM ATP) Na" decreases the rate of phosphorylation, the steady-state level of phosphorylation and the (K" -l-H )-ATPase activity. In the absence of, Na+ has a stimulating effect on the ATPase activity at this ATP concentration. Since at ATP concentrations normally used in the ATPase assay (1-5 mM) Na does not affect the ATPase activity, these findings are difficult to interpret. 

An ATPase assay is performed by monitoring ADP formation using [ot- P] ATP (400 Ci/mmol, Amersham Pharmacia Biotech) on polyethyleneimine (PEI) cellulose thin-layer sheets (TLC sheet, Polygram cel 300 PEI, Macherey-Nagel, Germany), based on the procedure reported previously. ... 

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