Inhibitor Probenecid Research Articles

Evidence of Oatp and Mdr1 in cryopreserved rat hepatocytes

Transport proteins control uptake of drugs into the liver (e.g., organic anion transporting polypeptide (Oatp)) and excretion of drugs from the liver (e.g., multidrug resistance protein 1 (Mdr1)). In this study, cryopreserved rat hepatocytes were used to investigate the effect of different culture conditions (suspension, conventional culture and sandwich culture) on the uptake of [ 3H]-taurocholate ± probenecid and the efflux of [ 3H]-vinblastine ± ketoconazole; mRNA levels of Oatp1a1, Oatp1a4, Mdr1a and Mdr1b were determined using real-time reverse transcription polymerase chain reaction (RT-PCR) and protein expression of Mdr was assessed by immunocytochemistry. The uptake of [ 3H]-taurocholate was higher in cryopreserved rat hepatocytes maintained in suspension as compared to hepatocytes in culture. A significant time dependent decline in the uptake of [ 3H]-taurocholate was noticed from day 2 to day 4 in conventional and sandwich cultures. [ 3H]-taurocholate uptake was significantly reduced using the inhibitor probenecid. Oatp mRNA expression in hepatocytes in suspension was similar to that of liver, whereas much lower levels were detected in the cultures; this was in accordance with [ 3H]-taurocholate uptake results. Mdr1 activity was assessed by accumulation of the Mdr1 selective substrate, [ 3H]-vinblastine, in hepatocytes using ketoconazole as an inhibitor. The results showed Mdr1 activity in cryopreserved rat hepatocytes in conventional and sandwich cultures. A time dependent increase in Mdr1 activity was noticed from day 2 to day 4. Mdr1 activity was not found using hepatocytes in suspension. Mdr1 mRNA expression was high in cryopreserved hepatocytes from both culture systems. Immunocytochemistry showed the Mdr protein in membranes of hepatocytes in culture as well as in that of hepatocytes in liver sections. In conclusion, the present study showed that cryopreserved rat hepatocytes maintained canalicular transport activity (Mdr1) and basolateral transport activity. Hepatocytes in suspension had a higher uptake of taurocholate with a high Oatp (1a1 and 1a4) mRNA expression as compared to hepatocytes in culture. The presence of Mdr1 in both conventional and sandwich culture was confirmed at mRNA level, by protein expression as well as transport activity.

Uptake of Chemically Reactive, DNA-Damaging Sulfuric Acid Esters into Renal Cells by Human Organic Anion Transporters

The procarcinogen 1-methylpyrene is activated by hepatic enzymes via 1-hydroxymethylpyrene to 1-sulfooxymethylpyrene (1-SMP), a highly reactive and mutagenic metabolite. Previously, high levels of 1-SMP DNA adducts were observed in rat kidneys after intraperitoneal administration of 1-hydroxymethylpyrene or 1-SMP. This study examined whether organic anion transporters (OAT) that are expressed at the basolateral membrane of proximal tubule cells are involved in uptake of SMP. Human epithelial kidney (HEK293) cells that stably express human OAT1 (hOAT1) and hOAT3 were used. Stable isomers of 1-SMP, (2-SMP and 4-SMP) competitively inhibited the uptake of characteristic substrates p-aminohippurate for hOAT1 and estrone sulfate for hOAT3. Both inhibitors exhibited high affinity for hOAT1 (K(i) = 4.4 microM for 2-SMP; K(i) = 5.1 microM for 4-SMP) as well as hOAT3 (K(i) = 1.9 microM for 2-SMP; K(i) = 2.1 microM for 4-SMP). The uptake rate of 4-SMP (at a concentration of 10 microM) by hOAT1- and hOAT3-expressing cells was 3.0 and 1.6 times higher, respectively, than in control cells. Uptake of the reactive isomer 1-SMP was investigated using as the end point the level of DNA adducts that were formed in the cells. After exposure to 1-SMP (10 microM), the DNA adduct level was 4.6 and 3.0 times higher in hOAT1- and hOAT3-expressing cells, respectively, than in control cells. The enhanced DNA adduct formation in hOAT-expressing cells was abolished in the presence of the OAT inhibitor probenecid. This study indicates that OAT can mediate the basolateral uptake of reactive sulfuric acid esters into proximal tubule cells and thereby participate in kidney cell damage by these compounds.

Acquired resistance to chloroquine in human CEM T cells is mediated by multidrug resistance–associated protein 1 and provokes high levels of cross‐resistance to glucocorticoids

To explore the onset and molecular mechanism of resistance to the antimalarial disease-modifying antirheumatic drug (DMARD) chloroquine (CQ) in human CEM T cells. Human CEM cells were used as an in vitro model system to study the development of CQ resistance by growing cells in stepwise increasing concentrations of CQ. Over a period of 6 months, CEM cell lines developed 4-5-fold resistance to CQ. CQ resistance was associated with the specific overexpression of multidrug resistance-associated protein 1 (MRP-1), an ATP-driven drug efflux pump. This was illustrated by 1) overexpression of MRP-1 by Western blotting and 2) the complete reversal of CQ resistance by the MRP-1 transport inhibitors MK571 and probenecid. Importantly, CQ-resistant CEM cells retained full sensitivity to other DMARDs, including methotrexate, leflunomide, cyclosporin A, and sulfasalazine, but exhibited a high level of cross-resistance (>1,000-fold) to the glucocorticoid dexamethasone. The mechanistic basis for the latter was associated with aberrant signaling via the cAMP-protein kinase A pathway, since the cAMP-inducing agent forskolin reversed dexamethasone resistance. Finally, CQ-resistant CEM cells displayed a markedly reduced capacity to release proinflammatory cytokines (tumor necrosis factor alpha) and chemokines (interleukin-8). Induction of overexpression of the multidrug resistance efflux transporter MRP-1 can emerge after long-term exposure to CQ and results in CQ resistance and collateral resistance to dexamethasone. These findings warrant further detailed investigations into the possible role of MRP-1 and other members of the superfamily of drug efflux pumps in diminishing the efficacy of DMARDs in rheumatoid arthritis treatment.

Transepithelial transport of fluorescent p-glycoprotein and MRP2 substrates by insect Malpighian tubules: confocal microscopic analysis of secreted fluid droplets

Transport of fluorescent substrates of p-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) by insect Malpighian tubules was examined using confocal laser scanning microscopy (CLSM). Isolated tubules of the cricket Teleogryllus commodus accumulated the MRP2 substrate Texas Red in the cells and lumen at concentrations up to 20 and 40 times, respectively, those in the bathing medium. Quantitative CLSM analysis of fluorochrome transport in some cricket tubules and all Drosophila tubules was not practical because of interfering effects of concretions in the cells and lumen. Samples of fluid secreted by tubules set up in Ramsay assays were therefore collected in hollow rectangle glass capillaries. Transepithelial dye flux was calculated as the product of fluid secretion rate (measured in the Ramsay assay) and dye concentration (measured by CLSM of the fluid samples). Dose-response curves for transport and the ratio of dye concentration in the secreted fluid to that in the bathing medium (S/M) were determined for Texas Red as well as for P-gp substrates (rhodamine 123, daunorubicin), the organic anion fluorescein and the organic cation quinacrine. Transepithelial transport of Texas Red was reduced by the MRP2 inhibitors MK571 and probenecid. Transport of daunorubicin was reduced by the P-gp inhibitors verapamil and quinacrine and also by the organic cation tetraethylammonium. The results indicate the presence of P-gp-like and MRP2-like transporters in the Malpighian tubules of both species.

Multidrug resistance-associated protein 2 (MRP2) affects hepatobiliary elimination but not the intestinal disposition of tenofovir disoproxil fumarate and its metabolites

The role of multidrug resistance-associated protein 2 (MRP2) on the intestinal disposition and hepatobiliary elimination of tenofovir disoproxil fumarate (DF) and its metabolites [tenofovir (mono)ester and tenofovir] was studied in the Caco-2 system, Ussing chambers and rat in-situ efflux experiments. In the Caco-2 model and Ussing chambers, no statistically significant differences in transport could be observed when the MRP inhibitor probenecid was included. In Ussing chambers, transport was also similar when using intestinal tissue from MRP2-deficient rats. After intravenous administration of tenofovir DF, the excretion of tenofovir [(mono)ester] in bile was significantly decreased in MRP2-deficient rats and in rats treated with probenecid. The area under the blood concentration–time curve was increased in MRP2-deficient rats [1.0 ± 0.1 and 0.36 ± 0.03 µM.min−1 for tenofovir and tenofovir (mono)ester, respectively] and rats treated with probenecid (1.42 ± 0.04 and 0.36 ± 0.02 µM.min−1) compared with control rats (0.64 ± 0.05 and 0.15 ± 0.06 µM.min−1). The appearance of tenofovir [(mono)ester] in intestinal perfusate was similar in control rats upon co-administering probenecid or when using MRP2-deficient rats. In conclusion, MRP2 appeared to have no modulatory effect on the intestinal disposition of tenofovir and tenofovir (mono)ester. However, inhibition (probenecid) or the total absence of MRP2 (MRP2-deficient rats) significantly reduced hepatobiliary elimination, which was accompanied by increased systemic exposure.

Expression of Multidrug Transporters MRP1, MRP2, and BCRP Shortly after Status Epilepticus, during the Latent Period, and in Chronic Epileptic Rats

Overexpression of multidrug transporters may play a role in the development of pharmacoresistance by decreasing extracellular drug levels in the brain. However, it is not known whether overexpression is due to an initial insult or evolves more gradually because of recurrent spontaneous seizures. In the present study, we investigated the expression of different multidrug transporters during epileptogenesis in the rat. In addition, we determined whether these transporters affected phenytoin (PHT) distribution in the brain. Expression of multidrug resistance-associated proteins MRP1 and MRP2 and breast cancer-resistance protein (BCRP) was examined after electrically induced status epilepticus (SE) by immunocytochemistry and Western blot analysis. Brain/blood PHT levels were determined by high-performance liquid chromatography (HPLC) analysis in the presence and absence of the MRP inhibitor probenecid. Shortly after SE, MRP1, MRP2, and BCRP were upregulated in astrocytes within several limbic structures, including hippocampus. In chronic epileptic rats, these proteins were overexpressed in the parahippocampal cortex, specifically in blood vessels and astrocytes surrounding these vessels. Overexpression was related to the occurrence of SE and was present mainly in rats with a high seizure frequency. Brain PHT levels were significantly lower in epileptic rats compared with control rats, but pharmacologic inhibition of MRPs increased the PHT levels. Overexpression of MRP and BCRP was induced by SE as well as recurrent seizures. Moreover, overexpression was associated with lower PHT levels in the brain, which was reversed through inhibition of MRPs. These data suggest that administration of antiepileptic drugs in combination with specific inhibitors for multidrug transporters may be a promising therapeutic strategy in pharmacoresistant patients.

Specificity of the fluorescein transport process in Malpighian tubules of the cricketAcheta domesticus

We demonstrate the presence of an efficient, multispecific transport system for excretion of organic anions in the Malpighian tubules of the cricket Acheta domesticus using fluorescein (FL) as a model substrate. Malpighian tubules rapidly accumulated FL via a high affinity process (Km = 7.75 micromol l(-1)); uptake was completely eliminated by the prototypical organic anion transport inhibitor probenecid (1 mmol l(-1)), but not by p-aminohippuric acid (3 mmol l(-1)). FL uptake was inhibited by monocarboxylic acids at a high concentration (3 mmol l(-1)), and inhibition was more effective with an increase in the carbon chain of the monocarboxylic acid (37% inhibition by 5-carbon valeric acid, and 89% inhibition by 7-carbon caprylic acid). Likewise, tests using a series of aliphatic glutathione conjugates indicated that only the compound with the longest side-chain (decyl-glutathione) significantly inhibited FL uptake (81% inhibition). FL uptake was inhibited by a number of xenobiotics, including a plant alkaloid (quinine), herbicides (2,4-dichlorophenoxyacetic acid and 4-(2,4-dichlorophenoxy)-butyric acid), and the insecticide metabolites malathion monocarboxylic acid (MMA) and 3-phenoxybenzoic acid (PBA), suggesting that this transport system plays an active role in excretion of xenobiotics from Acheta by Malpighian tubules. HPLC quantification of MMA and PBA accumulation into Malpighian tubules verified that MMA accumulation was via a mediated transport process, but suggested that PBA accumulation was by nonspecific binding. The presence of a transport system in Malpighian tubules that handles at least one pesticide metabolite (MMA) suggests that transport processes could be a mechanism conferring resistance to xenobiotic exposure in insects.

THE DIETARY POLYPHENOL ELLAGIC ACID IS A POTENT INHIBITOR OF hOAT1

Ellagic acid (EA), a polyphenol present in berries, has been demonstrated to be preventive of esophageal and colon cancer in animals. Here, we have studied the ability of organic anion transporters (OATs) and organic anion-transporting polypeptides (OATPs) to transport EA. The accumulation of radiolabeled (14)C]EA, [(3)H]p-aminohippuric acid (PAH), [(14)C]glutarate, [(3)H]estrone sulfate, [(3)H]ochratoxin A, and [(3)H]taurocholic acid +/- inhibitor(s) was tested in OAT- and OATP-expressing oocytes. Oocytes expressing human (h)OAT1, rat (r)Oat1, and hOAT4 accumulated 6.5-, 7.1-, and 8.9-fold more EA, respectively, than did water-injected oocytes. This accumulation was prevented by the prototype OAT inhibitors bromosulfophthalein and probenecid. rOatp1, mouse (m)Oat2, hOAT3, and mOat5 showed no EA transport. The uptake of the prototype OAT substrate PAH in hOAT1-expressing oocytes was dose dependently and potently inhibited by EA with an IC(50)of 207 nM. In conclusion, we have demonstrated that the OAT family members hOAT1, rOat1, and hOAT4 mediate transport of EA, with a very high affinity for hOAT1.

Factors Influencing [F-18] 2-Fluoro-2-Deoxy-d-Glucose (F-18 FDG) Accumulation in Melanoma Cells: Is FDG a Substrate of Multidrug Resistance (MDR)?

In order to specify the influence of multidrug-resistance (MDR) on the accumulation of the PET tracer, F-18 FDG ([Fluorine-18] 2-fluoro-2-deoxy-D-glucose, in melanoma cells, both the MDR function and expression of two human melanoma cell lines SK-MEL 23 and 24, were evaluated. The effects of MDR modulators on FDG accumulation and efflux were also investigated. A functional analysis using representative MDR fluorescent substrates and inhibitors clarified the following characteristics: 1) SK-MEL 23 possesses a highly active function of MRP, but not P-gp. 2) SK-MEL 24 possesses weak functions of both MRP and P-gp. Western blot analysis using monoclonal antibodies for MDR expression demonstrated an exceedingly high MRP expression of SK-MEL 23 and only slight P-gp and MRP expression of SK-MEL 24, corresponding to the functional data. The efflux inhibition assay using F-18 FDG revealed a considerable retention of FDG in SK-MEL 23 in the presence of the MRP inhibitor probenecid. It was also found that the P-gp inhibitor verapamil depressed the FDG efflux of SK-MEL 24. Our present in vitro study suggests that FDG may be a substrate of MDR in some melanoma cells and further MDR may be one of the important factors affecting FDG-PET melanoma imaging.

Regional Intestinal Absorption and Biliary Excretion of Fluvastatin in the Rat: Possible Involvement of mrp2

The first purpose of this study was to investigate the in vivo absorption, biliary secretion, and first-pass effect of fluvastatin following regional intestinal dosing in the rat. We also examined the membrane transport mechanisms and made in silico predictions of the relative importance of various intestinal regions to the human absorption of fluvastatin. Fluvastatin was administered intravenously (2, 10, and 20 micromol/kg) and into the duodenum (1.46, 2.92, 7.32, and 14.6 micromol/kg), jejunum (14.6 micromol/kg), ileum (1.46 and 14.6 mciromol/kg), and colon (1.46 and 14.6 micromol/kg) as a solution to conscious rats. In a separate group of rats, bile was collected after an i.v. dose of fluvastatin (2 micromol/kg). In the Caco-2 model the bidirectional transport of fluvastatin (16 microM) was investigated with and without various efflux inhibitors (verapamil, vinblastine, probenecid, and indomethacin, 160 microM). The human in vivo absorption of fluvastatin from an oral immediate release tablet and that from an oral extended release tablet (both 40 mg) were simulated in GastroPlus. Neither the dose nor the intestinal region influenced the bioavailability of fluvastatin significantly. The rate of absorption was, however, affected by both the dose and the site of administration; duodenum = jejunum > colon > ileum, and higher following the high dose. Increasing the i.v. dose from 2 to 20 micromol/kg decreased the clearance (26 +/- 3 to 12 +/- 1 mL/min/kg), the hepatic extraction (66 +/- 8 to 30 +/- 2%), and the volume of distribution (7.3 +/- 0.3 to 2.1 +/- 0.7 L/kg) for fluvastatin (p < 0.05). Neither bile cannulation nor bile sampling affected the pharmacokinetics. Fluvastatin was secreted into the bile, probably by active transport. The in vitro permeability for fluvastatin was high (>10 x 10(-6) cm/s). Indomethacin, but not the other inhibitors, affected the transport in both directions suggesting mrp2 to be involved. In silico, 93% of the dose was absorbed from the small intestine and 6% from the colon when given as an immediate release formulation. The corresponding values for an extended release formulation were 21% and 74%, respectively. In conclusion, fluvastatin exhibits dose-dependent pharmacokinetics in the rat. The rate of absorption (Cmax, Tmax, and Cmax/AUC(lqc)) from the intestinal tract is both region and dose-dependent in the rat. This may be due to the involvement of mrp2 in the intestine and/or in the liver. These absorption properties have to be considered in the development of an extended release formulation of fluvastatin.

Inhibition of multidrug transporters by verapamil or probenecid does not alter blood-brain barrier penetration of levetiracetam in rats

Overexpression of multidrug efflux transporters such as P-glycoprotein (Pgp; ABCB1) or multidrug resistance proteins (MRPs; ABCC) in the blood-brain barrier has recently been suggested to explain, at least in part, pharmacoresistance in epilepsy, which affects about 30% of all patients with this common brain disorder. The novel antiepileptic drug (AED) levetiracetam (LEV) is an effective and well tolerated drug in many patients with otherwise AED-refractory epilepsy. One explanation for the favorable efficacy of LEV in pharmacoresistant patients would be that LEV is not a substrate for Pgp or MRPs in the BBB. In the present study, we used in vivo microdialysis in rats to study whether the concentration of LEV in the extracellular fluid of the cerebral cortex can be modulated by inhibition of Pgp or MRPs, using the Pgp inhibitor verapamil and the MRP1/2 inhibitor probenecid. Local perfusion with verapamil or probenecid via the microdialysis probe did not increase the extracellular brain concentration of LEV, which is in contrast to various other AEDs which have been studied previously by the same experimental protocol in this model. The data indicate that brain uptake of LEV is not affected by Pgp or MRP1/2 which may be an important reason for its antiepileptic efficacy in patients whose seizures are poorly controlled by other AEDs.

MRP4- and MRP5-mediated efflux of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) by microglia

The pathogenesis of HIV-associated dementia has been primarily linked to the infection of microglia cells, the immune cells of the brain. We have recently confirmed the expression of several ATP-dependent efflux transporters in microglia, namely MRP1 and P-glycoprotein. In this study we investigate whether microglia functionally express two additional MRP family members: MRP4 and MRP5. Methods. Bis(pivaloyloxymethyl) PMEA (Bis-POM-PMEA), a lipophilic ester pro-drug of the well characterized MRP4 and 5 substrate PMEA, was chosen to examine transport characteristics in a microglia cell line (MLS-9). Conversion of Bis-POM-PMEA to PMEA in MLS-9 was verified using Thin Layer Chromatography. Results. mRNA and protein expression of MRP4/5 was confirmed by RT-PCR and Western blot analysis, respectively. PMEA efflux by MLS-9 monolayers was saturable and ATP-dependent, but glutathione independent. Sulfinpyrazone and the nucleoside-analog drug zidovudine-monophosphate decreased PMEA efflux significantly, as did the MRP inhibitors genistein, indomethacin, and probenecid. Conclusions. Our results demonstrate that microglia express multiple subfamilies of ABC transporters. Considering the differing substrate specificities of each of the transport proteins, these results imply that the overall distribution and accumulation of several classes of antiretroviral drugs can be highly regulated in cellular targets of HIV infection in the brain. Supported by CIHR and OHTN. Clinical Pharmacology & Therapeutics (2004) 75, P64–P64; doi: 10.1016/j.clpt.2003.11.241

Effect of probenecid and quinidine on the transport of alovudine (3'-fluorothymidine) to the rat brain studied by microdialysis.

Microdialysis was used to sample extracellular unbound concentrations of alovudine in order to study the influence of well-known transport inhibitors (probenecid and quinidine) on the transport of alovudine between the blood and the brain extracellular fluid or whole brain tissue. The AUC (area under the time versus concentration curve) ratio brain extracellular fluid/serum was 0.17+/-0.036 after a subcutaneous injection of alovudine 25 mg/kg in rats treated with probenecid 25 mg/kg subcutaneous (n=5), which was not significantly different from the control group (AUC ratio 0.24+/-0.039). Perfusion through the microdialysis probe with probenecid 100 microM (n=4) also had no effect on the brain extracellular fluid/serum AUC ratio after alovudine 25 mg/kg subcutaneous. The AUC ratio brain extracellular fluid/serum was 0.085+/-0.009 after subcutaneous injection of alovudine 25 mg/kg in rats treated with quinidine 25 mg/kg intraperitoneally (n=8), which was significantly lower than the control group. However, the whole brain tissue concentration was not significantly different between control rats (n=5) and rats treated with quinidine (n=4) 1 hr after subcutaneous injection of alovudine 25 mg/kg (brain to serum ratios being 0.11+/-0.006 and 0.10+/-0.005 respectively). Finally, the microdialysis recovery of alovudine increased with increasing concentrations (10, 50, 250, 1250 microM) of alovudine in the perfusion fluid. The recovery of alovudine was increased in quinidine-treated rats but not in those given probenecid. Thus, probenecid does not significantly influence the concentration gradient of alovudine over the blood-brain barrier in the rat after systemic or after local administration, while quinidine lowered brain extracellular fluid concentration of alovudine, but not total brain tissue concentration. The mechanism behind this phenomenon is not yet known.

Uremic toxins of organic anions up-regulate PAI-1 expression by induction of NF-κB and free radical in proximal tubular cells

Uremic toxins have been suggested to promote progression of chronic renal failure. We have shown that organic anion transporter-mediated uptake of uremic toxins induces oxidative stress in opossum kidney renal tubular cells overexpressing the transporter. Plasminogen activator inhibitor-1 (PAI-1) and nuclear factor-kappa B (NF-kappaB) are major factors known to promote tubulointerstitial fibrosis. The present study examined the signaling pathway that is activated by uremic toxins to induce PAI-1 and activate NF-kappaB in human renal proximal tubular cells (HK-2). Uremic toxins in the form of organic anion were examined their ability to induce oxidative stress, PAI-1 gene expression, and NF-kappaB activation in HK-2. PAI-1 expression was measured by enzyme-linked immunosorbent assay (ELISA) and the Northern blotting. Human PAI-1 promoter activity was estimated by luciferase reporter gene (NKkappaB-luc) assay. NF-kappaB activation was measured by the pNFkappaB-luc reporter gene and electrophretic gel mobility shift assay. Among organic anion species tested, indoxyl sulfate and indoleacetic acid induced free radical production in HK-2. A nonspecific transporter inhibitor (probenecid) suppressed the IS-stimulated radical production. Indoxyl sulfate and indoleacetic acid dose dependently increased the expressions of PAI-1 mRNA and protein in these cells. The luciferase reporter gene assay revealed that indoxyl sulfate and indoleacetic acid dose dependently activated NF-kappaB and PAI-1 promoter. Activation of NF-kappaB was also confirmed by an electrophoretic gel mobility shift assay. Both antioxidant and NF-kappaB inhibitors dose dependently inhibited the activation of PAI-1 promoter by indoxyl sulfate. Uremic toxins induce free radical production by renal tubular cells and activate NF-kappaB which, in turn, up-regulates PAI-1 expression. Thus, progression of chronic renal failure may be promoted by PAI-1 up-regulation induced by uremic toxins.

Multidrug resistance protein MRP2 contributes to blood-brain barrier function and restricts antiepileptic drug activity.

The blood-brain barrier (BBB) is a physical and metabolic barrier between the brain and the systemic circulation, which functions to protect the brain from circulating drugs, toxins, and xenobiotics. ATP-dependent multidrug transporters such as P-glycoprotein (Pgp; ABCB1), which are found in the apical (luminal) membranes of brain capillary endothelial cells, are thought to play an important role in BBB function by limiting drug penetration into the brain. More recently, the multidrug resistance protein MRP2 (ABCC2) has been found in the luminal surface of brain capillary endothelium of different species, including humans. In endothelial cells from patients with drug-resistant epilepsy, MRP2 was shown to be overexpressed, indicating that it may be critically involved in multidrug resistance of such patients. However, the role of MRP2 in drug disposition into the brain is defined poorly. Herein, we used different strategies to study the contribution of MRP2 to BBB function. First, the MRP inhibitor probenecid was shown to increase extracellular brain levels of the major antiepileptic drug phenytoin in rats, indicating that phenytoin is a substrate of MRP2 in the BBB. This was substantiated by using MRP2-deficient TR- rats, in which extracellular brain levels of phenytoin were significantly higher compared with the normal background strain. In the kindling model of epilepsy, coadministration of probenecid significantly increased the anticonvulsant activity of phenytoin. In kindled MRP2-deficient rats, phenytoin exerted a markedly higher anticonvulsant activity than in normal rats. These data indicate that MRP2 substantially contributes to BBB function.

Regulation of intracellular cyclic AMP in skeletal muscle cells involves the efflux of cyclic nucleotide to the extracellular compartment.

(1) This report analyses the intracellular and extracellular accumulation of cyclic AMP in primary rat skeletal muscle cultures, after direct and receptor-dependent stimulation of adenylyl cyclase (AC). (2) Isoprenaline, calcitonin gene-related peptide (CGRP) and forskolin induced a transient increase in the intracellular cyclic AMP that peaked 5 min after onset stimulation. (3) Under stimulation with isoprenaline or CGRP, the intracellular cyclic AMP initial rise was followed by an exponential decline, reaching 46 and 52% of peak levels in 10 min, respectively. (4) Conversely, the forskolin-dependent accumulation of intracellular cyclic AMP decreased slowly and linearly, reaching 49% of the peak level in 30 min. (5) The loss of intracellular cyclic AMP from peak levels, induced by direct or receptor-induced activation of AC, was followed by an increase in the extracellular cyclic AMP. (6) This effect was independent on PDEs, since it was obtained in the presence of 3-isobutyl-1-methylxanthine (IBMX). (7) Besides, in isoprenaline treated cells, the beta-adrenoceptor antagonist propranolol reduced both intra- and extracellular accumulation of cyclic AMP, whereas the organic anion transporter inhibitor probenecid reduced exclusively the extracellular accumulation. (8) Together our data show that direct or receptor-dependent activation of skeletal muscle AC results in a transient increase in the intracellular cyclic AMP, despite the continuous presence of the stimulus. The temporal declining of intracellular cyclic AMP was not dependent on the cyclic AMP breakdown but associated to the efflux of cyclic nucleotide to the extracellular compartment, by an active transport since it was prevented by probenecid.

Loss of Multidrug Resistance Protein 1 Expression and Folate Efflux Activity Results in a Highly Concentrative Folate Transport in Human Leukemia Cells

We studied the molecular basis of the up to 46-fold increased accumulation of folates and methotrexate (MTX) in human leukemia CEM-7A cells established by gradual deprivation of leucovorin (LCV). CEM-7A cells consequently exhibited 10- and 68-fold decreased LCV and folic acid growth requirements and 23-25-fold hypersensitivity to MTX and edatrexate. Although CEM-7A cells displayed a 74-86-fold increase in the reduced folate carrier (RFC)-mediated influx of LCV and MTX, RFC overexpression per se cannot induce a prominently increased folate/MTX accumulation because RFC functions as a nonconcentrative anion exchanger. We therefore explored the possibility that folate efflux activity mediated by members of the multidrug resistance protein (MRP) family was impaired in CEM-7A cells. Parental CEM cells expressed substantial levels of MRP1, MRP4, poor MRP5 levels, whereas MRP2, MRP3 and breast cancer resistance protein were undetectable. In contrast, CEM-7A cells lost 95% of MRP1 levels while retaining parental expression of MRP4 and MRP5. Consequently, CEM-7A cells displayed a 5-fold decrease in the [(3)H]folic acid efflux rate constant, which was identical to that obtained with parental CEM cells, when their folic acid efflux was blocked (78%) with probenecid. Furthermore, when compared with parental CEM, CEM-7A cells accumulated 2-fold more calcein fluorescence. Treatment of parental cells with the MRP1 efflux inhibitors MK571 and probenecid resulted in a 60-100% increase in calcein fluorescence. In contrast, these inhibitors failed to alter the calcein fluorescence in CEM-7A cells, which markedly lost MRP1 expression. Replenishment of LCV in the growth medium of CEM-7A cells resulted in resumption of normal MRP1 expression. These results establish for the first time that MRP1 is the primary folate efflux route in CEM leukemia cells and that the loss of folate efflux activity is an efficient means of markedly augmenting cellular folate pools. These findings suggest a functional role for MRP1 in the maintenance of cellular folate homeostasis.

The Role of Drug-resistance Proteins in Medically Refractory Epilepsy.

Drug Resistance in Epilepsy: Expression of Drug-resistance Proteins in Common Causes of Refractory Epilepsy Sisodiya SM, Lin WR, Harding BN, Squier MV, Thom M. Brain 2002;125(Pt 1):22–31 Epilepsy is resistant to drug treatment in about one third of cases, but the mechanisms underlying this drug resistance are not understood. In cancer, drug resistance has been studied extensively. Among the various resistance mechanisms, overexpression of drug-resistance proteins, such as multidrug resistance gene-1 P-glycoprotein (MDR1) and multidrug resistance–associated protein 1 (MRP1), has been shown to correlate with cellular resistance to anticancer drugs. Previous studies in human epilepsy have shown that MDR1 and MRP1 also may be overexpressed in brain tissue from patients with refractory epilepsy; expression has been shown in glia and neurons, which do not normally express these proteins. We examined expression of MDR1 and MRP1 in refractory epilepsy from three common causes, dysembryoplastic neuroepithelial tumors (DNTs; eight cases), focal cortical dysplasia (FCD; 14 cases), and hippocampal sclerosis (HS; eight cases). Expression was studied immunohistochemically in lesional tissue from therapeutic resections and compared with expression in histologically normal adjacent tissue. With the most sensitive antibodies, in all eight DNT cases, reactive astrocytes within tumor nodules expressed MDR1 and MRP1. In five of eight HS cases, reactive astrocytes within the gliotic hippocampus expressed MDR1 and MRP1. Of 14 cases of FCD, MDR1 and MRP1 expression was noted in reactive astrocytes in all cases. In five FCD cases, MRP1 expression also was noted in dysplastic neurons. In FCD and DNTs, accentuation of reactivity was noted around lesional vessels. Immunoreactivity was always more frequent and intense in lesional reactive astrocytes than in glial fibrillary acidic protein–positive reactive astrocytes in adjacent histologically normal tissue. MDR1 is able to transport some antiepileptic drugs (AEDs), and MRP1 also may do so. The overexpression of these drug-resistance proteins in tissue from patients with refractory epilepsy suggests one possible mechanism for drug resistance in patients with these pathologies. We propose that overexpressed resistance proteins reduce the interstitial concentration of AEDs in the vicinity of the epileptogenic pathology and thereby render the epilepsy caused by these pathologies resistant to treatment with AEDs. P-Glycoprotein and Multidrug Resistance–associated Protein Are Involved in the Regulation of Extracellular Levels of the Major Antiepileptic Drug Carbamazepine in the Brain Potschka H, Fedrowitz M, Loscher W. Neuroreport 2001;12:3557–3560 Despite considerable advances in the pharmacotherapy of epilepsy, about 30% of epilepsy patients are refractory to antiepileptic drugs (AEDs). In most cases, a patient who is resistant to one major AED also is refractory to other AEDs, although these drugs act by different mechanisms. The mechanisms that lead to drug resistance in epilepsy are not known. Recently, overexpression of multidrug transporters, such as P-glycoprotein (PGP) and multidrug resistance–associated protein (MRP), has been reported in surgically resected epileptogenic human brain tissue and suggested to contribute to the drug resistance of epilepsy. However, it is not known to what extent multidrug transporters such as PGP or MRP are involved in transport of AEDs. In the present study, we used in vivo microdialysis in rats to study whether the concentration of carbamazepine in the extracellular fluid of the cerebral cortex can be enhanced by inhibition of PGP or MRP, using the PGP inhibitor verapamil and the MRP inhibitor probenecid. Local perfusion with verapamil or probenecid via the microdialysis probe increased the extracellular concentration of carbamazepine. The data indicate that both PGP and MRP participate in the regulation of extracellular brain concentrations of the major AED carbamazepine.

The Potential for an Interaction between MRP2 (ABCC2) and Various Therapeutic Agents: Probenecid as a Candidate Inhibitor of the Biliary Excretion of Irinotecan Metabolites

Irinotecan hydrochloride (CPT-11) is an anticancer agent with unpredictable bouts of diarrhea as a dose-limiting toxic side-effect. Since the biliary excretion of its active metabolite (SN-38) and SN-38 glucuronide (SN38-Glu), which are mediated by the multidrug resistance associated protein-2 (MRP2/ABCC2), has been proposed to be related to this gastrointestinal toxicity, we have attempted here to examine the potential of various therapeutic agents to interact with the biliary excretion in order to identify MRP2 inhibitors to prevent this toxicity. The inhibition constants (K(i)) of 26 compounds were examined for the transport of a typical MRP2 substrate in isolated canalicular membrane vesicles. Of these, 13 compounds inhibited the transport with K(i) values from 0.0461 to 281 microM. Three inhibitors (probenecid, sulfobromophthalein and glycyrrhizin) were also found to inhibit the biliary excretion of SN-38 and SN38-Glu in rats in vivo, and the degrees of inhibition were compatible with the estimated values based on the ratios of K(i) and unbound concentrations in circulating plasma. A similar estimation of the potential inhibitory effect in human was also examined by considering both the K(i) of each therapeutic agent and its unbound concentration both in circulating plasma and the inlet to the liver. The predicted degrees of inhibition by most compounds were minimal whereas approximately 75% inhibition was predicted for probenecid. Thus, probenecid may be a candidate which can be used clinically to inhibit the biliary excretion of CPT-11 metabolites, whereas an interaction between most of the other compounds and MRP2 is more unlikely.

Interleukin-1β stimulates cyclic GMP efflux in brain astrocytes

In rat brain astroglia-enriched cultures long-term treatment with interleukin-1β induces NO release and stimulation of soluble guanylyl cyclase. The cGMP formed is recovered in the extracellular medium but not in the cell monolayer. The interleukin-1β effect is mediated by type I receptor and potentiated by interferon-γ. In cells treated with bacterial endotoxin a larger NO-dependent cGMP accumulation occurs only intracellularly, however a significant cGMP egression is observed when cells are co-treated with interleukin-1β. The non-selective anion transport inhibitors probenecid and verapamil block cGMP efflux, indicating that interleukin-1β stimulates a cGMP transporter.