Lyme borreliosis in the Netherlands: strong increase in GP consultations and hospital admissions in past 10 years.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
P R Wielinga
Publications and source records attributed to P R Wielinga.
Online fluorescent method to assess BCRP/ABCG2 activity in suspension cells.
An online method was developed to monitor BCRP mediated efflux of fluorescent substrates in suspension cells. To this end, a 2-compartment system consisting of a transwell cup and a cuvette was used. In this system we were able to observe differences in efflux kinetics between BCRP overexpressing RPMI 8226/MR cells and parental myeloid RPMI 8226(s) cells using only 50,000 cells per experiment. 8226/MR cells displayed a larger cellular efflux rate of the BCRP substrate Hoechst 33342, as compared to the wildtype cells. This difference in efflux rate was completely decreased in the presence of the BCRP inhibitor Ko143.
ATP Binding Cassette Transporter, Subfamily G, Mem↗
Thiopurine metabolism and identification of the thiopurine metabolites transported by MRP4 and MRP5 overexpressed in human embryonic kidney cells.
Mercaptopurines have been used as anticancer agents for more than 40 years, and most acute lymphoblastic leukemias are treated with 6-mercaptopurine (6MP) or 6-thioguanine (TG). Overexpression of the two related multidrug resistance proteins MRP4 and MRP5 has been shown to confer some resistance against mercaptopurines, which has been attributed to extrusion of mercaptopurine metabolites by these transporters. We have analyzed the mercaptopurine metabolites formed in human embryonic kidney cells and determined which metabolites are extruded by MRP4 and MRP5. Incubation with 6MP led to the formation of thioinosine and thioxanthosine metabolites and we found that thio-IMP was transported by both MRP4 and MRP5; MRP5 showed the highest transport rate. In contrast, only MRP5 transported thioxanthosine monophosphate (tXMP). During incubation with TG, the monophosphorylated form of thioguanosine was transported by both MRP4 and MRP5; the highest transport rate was for MRP4. Similarly, only 6-methyl-thio-IMP was formed during incubation with 6-methyl mercaptopurine riboside. This compound was a substrate for both MRP4 and MRP5; MRP4 showed the highest transport rate. Our results show that all major thiopurine monophosphates important in the efficacy of mercaptopurine treatment are transported by MRP4 and MRP5, although the substrate specificity of the two transporters differs in detail.
Biological Transport↗
The relative importance of passive and P-glycoprotein mediated anthracycline efflux from multidrug-resistant cells.
For the four anthracyclines idarubicin, daunorubicin, epirubicin and doxorubicin the passive and active efflux rates in intact multidrug resistant cells were compared. Although highly similar structurally, these anti-tumor agents differ in lipophilicity and membrane permeability (k). The method we used was based on the continuous measurement of the cellular efflux and determination of the ratio (RVp) of transport rates just before and just after inhibition of the active transport with verapamil (Vp). Hence, RVp - 1 should reflect the active transport rate relative to the passive transport rate. If cells were single, well-stirred compartments, RVp - 1 should equal Vmax/(k.Km), where Vmax is the maximal pumping rate and Km is the Michaelis constant. However, using the plasma membrane permeabilizing agent digitonin, we found an effective intracellular anthracycline store. Particularly, when the efflux was fast, e.g. with idarubicin or in intensively pumping cells, the intracellular transport began to control the cellular efflux. Under these conditions, k underestimated the true plasma membrane permeability (k0) and RVp - 1 underestimated Vmax/(k.Km). Based on the effects of digitonin on the efflux rates in pumping and nonpumping cells, we developed an index (RVp,corrected - 1) which should equal Vmax/(k0. Km). The term Vmax/(k0.Km) varied substantially between the drugs. It appears that differences in lipophilicity between the drugs do not affect passive efflux and pumping equally. This demonstrates that passive permeation plays a substantial and independent role in determining the drug resistance for these anthracyclines. The methods developed here enable dissection of this role from that of drug pumping and intracellular subcompartmentation.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export.
The multidrug resistance proteins MRP1 and MRP2 are members of the same subfamily of ATP-binding cassette transporters. Besides organic molecules conjugated to negatively charged ligands, these proteins also transport cytotoxic drugs for which no negatively charged conjugates are known to exist. In polarized MDCKII cells, MRP1 routes to the lateral plasma membrane, and MRP2 to the apical plasma membrane. In these cells MRP1 transports daunorubicin, and MRP2 vinblastine; both transporters export reduced glutathione (GSH) into the medium. We demonstrate that glutathione transport in MDCKII-MRP1 cells is inhibited by the inhibitors of organic anion transporters sulfinpyrazone, indomethacin, probenecid and benzbromarone. In MDCKII-MRP2 cells, GSH export is stimulated by low concentrations of sulfinpyrazone or indomethacin, whereas export is inhibited down to control levels at high concentrations. We find that unmodified sulfinpyrazone is a substrate for MRP2, also at concentrations where GSH export is inhibited. We also show that GSH export in MDCKII-MRP2 cells increases in the presence of vinblastine, and that the stoichiometry between drug and GSH exported is between two and three. Our data indicate that transport of sulfinpyrazone and vinblastine is associated with GSH export. However, at high sulfinpyrazone concentrations this compound is transported without GSH. Models of MRP action are discussed that could explain these results.
Anion Transport Proteins↗
In vitro transepithelial drug transport by on-line measurement: cellular control of paracellular and transcellular transport.
Studies on transcellular transport across epithelial cell layers are performed mostly by discontinuous sampling of the transported compound. This has several drawbacks, e.g., it gives disturbances in volume, it limits the time-resolution, and is often laborious. In this report we introduce a method to measure transepithelial transport of fluorescent compounds continuously. The time-resolution is at the (sub)minute scale, allowing the measurement of the change in transport rate before and after transport modulation. We will describe how we used the method to measure transcellular and paracellular transport. For highly membrane-impermeable compounds, the paracellular transport and the regulation of the tight junctions was studied in wild-type and MDR1 cDNA transfected epithelial canine kidney cells (MDCKII). The effect of the multidrug transporter P-glycoprotein (Pgp) on the transepithelial transport was studied. Addition of the Pgp inhibitor SDZ PSC 833 showed a modulation of the idarubicin (IDA) and daunorubicin (DNR) transport, which was larger during transport from the basolateral to the apical side than in the reverse direction. By modeling the transepithelial transport, we found that in these cells Pgp had more effect on the basolateral to apical transport than vice versa, which can be attributed to a relatively large passive permeation coefficient for the cellular basolateral plasma membrane.
Algorithms↗
A method for studying plasma membrane transport with intact cells using computerized fluorometry.
A new method is presented for measuring rapid efflux of fluorescent compounds from monolayer cells. Cells grown on a glass coverslip were loaded with a fluorescent substrate. Thereafter, the coverslip was installed outside the light path in a stirred and thermostated cuvette of a fluorometer. The efflux was recorded by measuring the changes of fluorescence in the extracellular medium. The method was used to study the kinetics of active and passive plasma membrane transport of the P-glycoprotein substrates rhodamine 123 and daunorubicin. The method has advantages over other methods: (1) no radioactively labeled substrate is needed, (2) fluorescence of the transported substrate is not compromised by the cells, (3) changes in the extracellular concentration of the substrate can be monitored continuously and therefore a substantial improvement of the kinetic resolution is obtained, and (4) the measurement setup is relatively simple and a standard fluorometer can be used. From the efflux data, cellular transport parameters could be calculated, such as passive permeation coefficients and active transport rates.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
P-glycoprotein-independent decrease in drug accumulation by phorbol ester treatment of tumor cells.
The effect of a change in the phosphorylation state of the drug transporter P-glycoprotein (P-gp) on its drug transport activity was studied for the substrates daunorubicin (DNR), etoposide (VP-16), and calcein acetoxymethyl ester (Cal-AM). Phorbol ester (PMA), added to stimulate phosphorylation of P-gp by protein kinase C (PKC), caused a decrease in the cellular accumulation of DNR and VP-16, both in multidrug-resistant (MDR) P-gp-overexpressing cells and in wild-type cells. Since treatment of cells with kinase inhibitor staurosporine (ST) reversed this effect of PMA and the non-PKC-stimulating phorbol ester 4alpha-phorbol, 12,13-didecanoate (4alphaPDD) did not result in a decreased DNR accumulation, we conclude that this effect is the result of kinase activity. The concentration dependence of the inhibition of P-gp by verapamil (Vp) was not influenced by PMA. Accumulation of the P-gp substrate Cal-AM was not influenced by PMA in wild-type cells. Therefore, Cal-AM was used to study the effect of PMA-induced phosphorylation of P-gp on its transport activity. Activation of PKC with PMA or inhibition of protein phosphatase 1/2A (PP1/PP2A) with okadaic acid (OA) did not affect the accumulation of Cal-AM in the MDR cells or wild-type cells. The kinase inhibitor ST increased the Cal-AM accumulation only in the MDR cells. Neither stimulating PKC with PMA nor inhibiting PP1/PP2A with OA led to a decreased inhibition of P-gp by ST, indicating that ST inhibits P-gp directly. From these experiments, we conclude that PKC and PP1/PP2A activity do not regulate the drug transport activity of P-gp. However, these studies provide evidence that PMA-induced PKC activity decreases cellular drug accumulation in a P-gp-independent manner.
ATP Binding Cassette Transporter, Subfamily B, Mem↗