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Albendazole (ABZ) is an anthelmintic frequently used to treat haemonchosis, a common parasitosis of ruminants caused by the gastrointestinal nematode Haemonchus contortus. This parasite is able to protect itself against ABZ via the formation of inactive ABZ-glycosides. The present study was designed to deepen the knowledge about the role of UDP-glycosyltransferases (UGTs) in ABZ glycosylation in H. contortus. The induction effect of phenobarbital, a classical inducer of UGTs, as well as ABZ and ABZ-sulphoxide (ABZSO, the main active metabolite of ABZ) on UGTs expression and UGT activity toward ABZ was studied ex vivo in isolated adult nematodes. The effect of three potential UGT inhibitors (5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine and sulfinpyrazone) on ABZ glycosylation was tested. Pre-incubation of nematodes with ABZ and ABZSO led to increased expression of several UGTs as well as ABZ-glycosides formation in subsequent treatment. Phenobarbital also induced UGTs expression, but did not affect ABZ biotransformation. In the nematode's subcellular fraction, sulfinpyrazone inhibited UGT activity toward ABZ, although no effect of other inhibitors was observed. The inhibitory potential of sulfinpyrazone on the formation of ABZ-glycosides was also proved ex vivo in living nematodes. The obtained results confirmed the role of UGTs in ABZ biotransformation in H. contortus adults and revealed sulfinpyrazone as a potent inhibitor of ABZ glycosylation in this parasite. The possible use of sulfinpyrazone with ABZ in combination therapy merits further research., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The discovery of drugs capable of inhibiting SARS-CoV-2 is a priority for human beings due to the severity of the global health pandemic caused by COVID-19. To this end, repurposing of FDA-approved drugs such as NSAIDs against COVID-19 can provide therapeutic alternatives that could be utilized as an effective safe treatment for COVID-19. The anti-inflammatory activity of NSAIDs is also advantageous in the treatment of COVID-19, as it was found that SARS-CoV-2 is responsible for provoking inflammatory cytokine storms resulting in lung damage. In this study, 40 FDA-approved NSAIDs were evaluated through molecular docking against the main protease of SARS-CoV-2. Among the tested compounds, sulfinpyrazone 2 , indomethacin 3 , and auranofin 4 were proposed as potential antagonists of COVID-19 main protease. Molecular dynamics simulations were also carried out for the most promising members of the screened NSAID candidates ( 2 , 3 , and 4 ) to unravel the dynamic properties of NSAIDs at the target receptor. The conducted quantum mechanical study revealed that the hybrid functional B3PW91 provides a good description of the spatial parameters of auranofin 4 . Interestingly, a promising structure-activity relationship (SAR) was concluded from our study that could help in the future design of potential SARS-CoV-2 main protease inhibitors with expected anti-inflammatory effects as well. NSAIDs may be used by medicinal chemists as lead compounds for the development of potent SARS-CoV-2 (M pro ) inhibitors. In addition, some NSAIDs can be selectively designated for treatment of inflammation resulting from COVID-19.

Lymphatic filariasis (LF), a morbid disease caused by the tissue-invasive nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori, affects millions of people worldwide. Global eradication efforts have significantly reduced worldwide prevalence, but complete elimination has been hampered by limitations of current anti-filarial drugs and the lack of a vaccine. The goal of this study was to evaluate B. malayi intestinal UDP-glucuronosyltransferase (Bm-UGT) as a potential therapeutic target. To evaluate whether Bm-UGT is essential for adult filarial worms, we inhibited its expression using siRNA. This resulted in a 75% knockdown of Bm-ugt mRNA for 6 days and almost complete suppression of detectable Bm-UGT by immunoblot. Reduction in Bm-UGT expression resulted in decreased worm motility for 6 days, 70% reduction in microfilaria release from adult worms, and significant reduction in adult worm metabolism as detected by MTT assays. Because prior allergic-sensitization to a filarial antigen would be a contraindication for its use as a vaccine candidate, we tested plasma from infected and endemic normal populations for Bm-UGT-specific IgE using a luciferase immunoprecipitation assay. All samples (n = 35) tested negative. We then tested two commercially available medicines known to be broad inhibitors of UGTs, sulfinpyrazone and probenecid, for in vitro activity against B. malayi. There were marked macrofilaricidal effects at concentrations achievable in humans and very little effect on microfilariae. In addition, we observed that probenecid and sulfinpyrazone exhibit a synergistic macrofilaricidal effect when used in combination with albendazole. The results of this study demonstrate that Bm-UGT is an essential protein for adult worm survival. Lack of prior IgE sensitization in infected and endemic populations suggest it may be a feasible vaccine candidate. The finding that sulfinpyrazone and probenecid have in vitro effects against adult B. malayi worms suggests that these medications have promise as potential macrofilaricides in humans., Competing Interests: The authors have declared that no competing interests exist.

Background: Ixodes scapularis organic anion transporting polypeptides (OATPs) play important roles in tick-rickettsial pathogen interactions. In this report, we characterized the role of these conserved molecules in ticks infected with either Lyme disease agent Borrelia burgdorferi or tick-borne Langat virus (LGTV), a pathogen closely related to tick-borne encephalitis virus (TBEV)., Results: Quantitative real-time polymerase chain reaction analysis revealed no significant changes in oatps gene expression upon infection with B. burgdorferi in unfed ticks. Synchronous infection of unfed nymphal ticks with LGTV in vitro revealed no significant changes in oatps gene expression. However, expression of specific oatps was significantly downregulated upon LGTV infection of tick cells in vitro. Treatment of tick cells with OATP inhibitor significantly reduced LGTV loads, kynurenine amino transferase (kat), a gene involved in the production of tryptophan metabolite xanthurenic acid (XA), levels and expression of several oatps in tick cells. Furthermore, bioinformatics characterization of OATPs from some of the medically important vectors including ticks, mosquitoes and lice revealed the presence of several glycosylation, phosphorylation and myristoylation sites., Conclusions: This study provides additional evidence on the role of arthropod OATPs in vector-intracellular pathogen interactions.

Background: UDP-glycosyltransferases (UGTs) are phase II detoxification enzymes widely distributed within living organisms. Their involvement in the biotransformation of various lipophilic endogenous compounds and phytoalexins in insects has been documented. However, the roles of this enzyme family in insecticide resistance have rarely been reported. Here, the functions of UGTs in chlorantraniliprole resistance in Plutella xylostella were investigated., Results: Treatment with sulfinpyrazone and 5-nitrouracil (both inhibitors of UGT enzymes) significantly increased the toxicity of chlorantraniliprole against the third instar larvae of P. xylostella. Among the 23 UGT transcripts examined, only UGT2B17 was found to be over-expressed (with a range from 30.7- to 77.3-fold) in all four chlorantraniliprole-resistant populations compared to the susceptible one (CHS). The knock-down of UGT2B17 by RNA interference (RNAi) dramatically increased the toxicity of chlorantraniliprole by 27.4% and 29.8% in the CHS and CHR (resistant) populations, respectively. In contrast, exposure to phenobarbital significantly increased the relative expression of UGT2B17 while decreasing the toxicity of chlorantraniliprole to the larvae by 14.0%., Conclusion: UGT2B17 is involved in the detoxification of chlorantraniliprole, and its over-expression may play an important role in chlorantraniliprole resistance in P. xylostella. These results shed some light upon and further our understanding of the mechanisms of diamide insecticide resistance in insects. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

We used an enzyme induction approach to study the role of detoxification enzymes in the interaction of the anthelmintic compound naphthalophos with Haemonchus contortus larvae. Larvae were treated with the barbiturate phenobarbital, which is known to induce the activity of a number of detoxification enzymes in mammals and insects, including cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UDPGTs), and glutathione (GSH) S-transferases (GSTs). Cotreatment of larvae with phenobarbital and naphthalophos resulted in a significant increase in the naphthalophos 50% inhibitory concentration (IC50) compared to treatment of larvae with the anthelmintic alone (up to a 28-fold increase). The phenobarbital-induced drug tolerance was reversed by cotreatment with the UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, probenecid, and sulfinpyrazone. Isobologram analysis of the interaction of 5-nitrouracil with naphthalophos in phenobarbital-treated larvae clearly showed the presence of strong synergism. The UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, and probenecid also showed synergistic effects with non-phenobarbital-treated worms (synergism ratio up to 3.2-fold). This study indicates that H. contortus larvae possess one or more UDPGT enzymes able to detoxify naphthalophos. In highlighting the protective role of this enzyme group, this study reveals the potential for UDPGT enzymes to act as a resistance mechanism that may develop under drug selection pressure in field isolates of this species. In addition, the data indicate the potential for a chemotherapeutic approach utilizing inhibitors of UDPGT enzymes as synergists to increase the activity of naphthalophos against parasitic worms and to combat detoxification-mediated drug resistance if it arises in the field., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Goals: The available scientific data indicate that the pathomechanism of Parkinson's disease (PD) involves the accumulation of endogenous and exogenous toxic substances. The disruption of the proper functioning of certain transporters in the blood-brain barrier and in the blood-cerebrospinal fluid barrier in PD would accompany to that accumulation. Although there is an emerging role of the dysfunction of multidrug resistance-associated proteins (MRPs), members of ATP-b nding cassette (ABC) transporter superfamily, in neurodegenerative disorders, there is only a few available data as regards PD. So the aim of our study was the assessment of the role of certain MRPs (1 ,2, 4 and 5) in neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, Methods: Following the intraperitoneal administration of silymarin (with MRP1, 2, 4 and 5 inhibitory effects), naringenin (with MRP1, 2 and 4 stimulatory effects), sulfinpyrazone (with MRP1, 4 and 5 inhibitory and MRP2 stimulatory effects) and allopurinol (with MRP4 stimulatory effect in doses of 100 mg/kg, 100 mg/kg, 100 mg/kg and 60 mg/kg, respectively, for one week before and after the administration of MPTP in C57B/6 mice in acute dosing regimen the striatal concentrations of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid has been measured using high-performance liquid chromatography., Results: Although the results of these experiments showed that neither of these substances exerted significant influence on MPTP-induced striatal depletion of dopamine and its metabolites, naringenin exerted a slight prevention of dopamine decrease, while allopurinol considerably enhanced the MPTP-induced lethality in mice. The explanation of these findings would be that the stimulation of MRP1- and MRP2-mediated transport of glutathione conjugates of toxic substances may have slight beneficial effects, while stimulation of MRP4-mediated efflux of brain urate, which has an important antioxidant potency, may worsen the effects of oxidative stress.

The transient receptor potential channel subtype A member 1 (TRPA1) is a nonselective cation channel widely viewed as having therapeutic potential, particularly for pain-related indications. Realization of this potential will require potent, selective modulators; however, currently the pharmacology of TRPA1 is poorly defined. As TRPA1 is calcium permeable, calcium indicators offer a simple assay format for high-throughput screening. In this report, we show that probenecid, a uricosuric agent used experimentally in screening to increase loading of calcium-sensitive dyes, activates TRPA1. Prolonged probenecid incubation during the dye-loading process reduces agonist potency upon subsequent challenge. When Chinese Hamster Ovary (CHO)-hTRPA1 or STC-1 cells, which endogenously express TRPA1, were dye loaded in the presence of 2 mM probenecid TRPA1, agonists appeared less potent; EC(50) for allyl isothiocyante agonists in CHO-hTRPA1 was increased from 1.5±0.19 to 7.32±1.20 μM (P<0.01). No significant effect on antagonist potency was observed when using the agonist EC(80) concentration determined under the appropriate dye-loading conditions. We suggest an alternative protocol for calcium imaging using another blocker of anion transport, sulfinpyrazone. This blocker significantly augments indicator dye loading and the screening window, but is not a TRPA1 agonist and has no effect on agonist potency.

Aim: Hyperuricaemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. The kidney plays a dominant role in maintaining plasma urate levels through the excretion process. Human renal urate transporter URAT1 is thought to be an essential molecule that mediates the reabsorption of urate on the apical side of the proximal tubule. In this study the pharmacological characteristics and clinical implications of URAT1 were elucidated., Methods: Madin-Darby canine kidney (MDCK) cells stably expressing URAT1 (MDCK-URAT1) were established and examined the interactions of URAT1 with various drugs such as benzbromarone and its metabolites including 6-hydroxybenzbromarone, angiotensin-converting enzyme inhibitors, non-steroidal anti-inflammatory drugs and urate transport inhibitors including E3040 and probenecid., Results: MDCK-URAT1 cells exhibited a time- and dose-dependent increase in urate uptake, with a Km value of 570.7 µmol/L. When an URAT1-green fluorescent protein fusion protein construct was expressed in MDCK cells, the protein was sorted mainly to the apical side of the membrane. The drugs except for captoril dose-dependently inhibited urate uptake mediated by URAT1, with half maximal inhibitory concentration (IC(50) ) values ranging 0.05-716 µmol/L., Conclusion: Comparing these IC(50) values with intratubular concentrations of unbound drugs in humans, it is thought that URAT1 is a target molecule of uricosuric drugs, including 6-hydroxybenzbromarone, probenecid, indomethacin and salicylate, to inhibit urate reabsorption in vivo. In addition, a cell line that stably expressing URAT1 could be a useful tool for the development of uricosuric drugs., (© 2010 The Authors. Nephrology © 2010 Asian Pacific Society of Nephrology.)

We used the paclitaxel-resistant human small cell lung cancer subline PC-6/TAX1-1, selected from PC-6 cells by paclitaxel, to test whether MRP7/ABCC10 (ABCC10) confers paclitaxel resistance. We found that gene expression of both ABCB1/MDR1 (ABCB1) and ABCC10 was higher in PC-6/TAX1-1 cells than in PC-6 cells. The expression levels of ABCC10 showed a significant inverse correlation with paclitaxel sensitivity (r = 0.574; P < 0.05) in 17 non-small cell lung cancer (NSCLC) cells unlike the expression levels of ABCB1. Pretreatment with the ABCC10 inhibitor sulfinpyrazone altered the sensitivity to paclitaxel in ABCC10-expressing NSCLC cells, concomitant with increased intracellular paclitaxel accumulation. These findings suggest that expression of the ABCC10 gene is induced by paclitaxel and that ABCC10 confers paclitaxel resistance by enhancing the efflux for paclitaxel. To confirm this hypothesis, we tested the effect on paclitaxel cytotoxicity of decreasing the expression of ABCC10 by small interfering RNA and found that this enhanced paclitaxel cytotoxicity in NCI-H23 cells concomitant with increased intracellular paclitaxel accumulation. These data indicate that ABCC10 may be one of the biomarkers for paclitaxel resistance in NSCLC.

In vitro pyrimethamine response of Plasmodium falciparum isolates and dihydrofolate reductase (dhfr) gene sequences were analyzed in 2004-2005 and compared with our previous data. Most isolates (n = 103, all dhfr mutants) had 50% inhibitory concentrations (IC(50)s) > or = 119 nM, and six isolates had low IC(50)s (five wild-type or mixed dhfr, 0.04-1.37 nM; one triple mutant, 6.4 nM). Of 194 isolates, only 7 had the wild-type dhfr and 187 were mutants. The results of the two methods were highly concordant and indicated a significant increase (P < 0.05) in the prevalence of mutant, pyrimethamine-resistant P. falciparum between 1994 and 2005. The addition of probenecid or sulfinpyrazone to pyrimethamine resulted in a slight-to-moderate decrease in the level of in vitro pyrimethamine resistance without rendering the parasites susceptible to pyrimethamine. Analysis of molecular markers may be useful for the long-term surveillance of antifolate-resistant malaria.

In allopurinol-allergic patients, uricosuric agents are often used in the treatment of hyperuricemia. The existing uricosuric agents are not without problems and the availability of better and safer alternatives is highly desirable. Our previous study (J Pharmacol Exp Ther (2006) 316:169-175) has demonstrated that morin (3,5,7,2',4'-pentahydroxyflavone), which occurs in the twigs of Morus alba L. documented in traditional Chinese medicinal literature for treatment of conditions akin to gout, is a potent inhibitor of urate uptake in rat renal brush-border membrane vesicles. It is also effective in lowering uric acid level in a hyperuricemic rat model in vivo. Whether morin is an equally effective uricosuric agent in human requires verification. The human urate anion transporter (hURAT1) has recently been cloned and identified to be the organic anion transporter that mediates renal urate reabsorption in the human kidney. In the present investigation, human embryonic kidney cells were transfected with hURAT1 and the expression was validated by reverse transcription-polymerase chain reaction and subcellular distribution of the exogenously introduced transporter by confocal microscopy. The inhibitory actions of morin on human renal urate reabsorption were demonstrated using this system. The IC50 value of the inhibition by morin was determined to be 2.0 microM, compared with 50 microM for probenecid, 100 microM for sulfinpyrazone, and 0.3 microM for benzbromarone. Kinetic analysis of the uptake inhibition by morin indicates that this compound is a competitive inhibitor of urate uptake on the human urate transporter with a K(i) value of 5.74 microM.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that belongs to the same family as multidrug resistance-associated proteins whose main function is to expel xenobiotics and physiological organic anions from the cell interior. Despite the overall structural similarity with these membrane proteins, CFTR is not an active transporter but is instead a Cl- channel. We have tested the ability of known inhibitors of multidrug resistance-associated proteins to affect CFTR Cl- currents. We have found that sulfinpyrazone, probenecid, and benzbromarone are also inhibitors of CFTR activity, with a mechanism involving blockage of the channel pore.

Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.

Cytochrome P4503A4 (CYP3A4) is the principal drug-metabolizing enzyme in human liver. Drug-drug interactions (DDIs) caused by induction of CYP3A4 can result in decreased exposure to coadministered drugs, with potential loss of efficacy. Immortalized hepatocytes (Fa2N-4 cells) have been proposed as a tool to identify CYP3A4 inducers. The purpose of the current studies was to characterize the effect of known inducers on CYP3A4 in Fa2N-4 cells, and to determine whether these in vitro data could reliably project the magnitude of DDIs caused by induction. Twenty-four compounds were chosen for these studies, based on previously published data using primary human hepatocytes. Eighteen compounds had been shown to be positive for induction, and six compounds had been shown to be negative for induction. In Fa2N-4 cells, all 18 positive controls produced greater than 2-fold maximal CYP3A4 induction, and all 6 negative controls produced less than 1.5-fold maximal CYP3A4 induction. Subsequent studies were conducted to determine the relationship between in vitro induction data and in vivo induction response. The approach was to relate in vitro induction data (E(max) and EC(50) values) with efficacious free plasma concentrations to calculate a relative induction score. This score was then correlated with decreases in area under the plasma concentration versus time curve values for coadministered CYP3A4 object drugs (midazolam or ethinylestradiol) from previously published clinical DDI studies. Excellent correlations (r(2) values >0.92) were obtained, suggesting that Fa2N-4 cells can be used for identification of inducers as well as prediction of the magnitude of clinical DDIs.

To test whether ascorbic acid might be involved in the antioxidant defenses of inflammatory cells, we studied ascorbate uptake and recycling by quiescent and lipopolysaccharide-activated RAW264.7 murine macrophages. These cells concentrated ascorbate 100-fold in overnight culture, achieving steady-state concentrations of more than 10 mM at extracellular concentrations of 20-100 muM. This steep gradient was generated by high-affinity sodium-dependent ascorbate transport. The latter likely reflects function of the SVCT2 (SLC23A2), since this protein was detected on immunoblots. Dehydroascorbate, the two-electron oxidized form of ascorbate, was also taken up and reduced to ascorbate by the cells. Dehydroascorbate reduction required rapid recycling of GSH from GSSG by glutathione reductase. Activation of ascorbate-containing macrophages with lipopolysaccharide transiently depleted intracellular ascorbate without affecting GSH. Recovery of intracellular ascorbate required function of the SVCT2 transporter, the activity of which was modestly enhanced by lipopolysaccharide. Lipopolysaccharide treatment nearly doubled intracellular GSH concentrations over 2 h. Despite lipopolysaccharide-induced oxidant stress, this GSH increase was associated with a comparable increase in reduction of dehydroascorbate to ascorbate. These results show that macrophages maintain millimolar concentrations of ascorbate through function of the SVCT2 and that activated cells have an enhanced ability to transport and recycle ascorbate, possibly reflecting its role as an intracellular antioxidant.

Collagen II is the major protein component of articular cartilage and forms the collagen fibril network, which provides the tensile strength of cartilage. Collagen II synthesis is enhanced by ascorbic acid (vitamin C) at both a transcriptional and post-transcriptional level. While the importance of ascorbic acid in the synthesis of collagen has been established, the mechanism by which this essential nutrient is transported into chondrocytes has not been investigated previously. We have characterized the transport of the reduced form of ascorbic acid in passaged primary human chondrocytes to discern the physiologically relevant pathways of ascorbic acid transport in cartilage. We have found that chondrocytes are robust concentrators of ascorbic acid, capable of transporting the reduced form, and concentrating total ascorbic acid, in the reduced form and its metabolites, 960-fold over the concentration in the extracellular milieu. Chondrocyte transport of ascorbic acid was sodium and temperature dependent, stereoselective for the L-forms, and inhibited by the anion transport inhibitor, sulfinpyrazone. Chondrocytes preferentially expressed the full-length and functional isoform of sodium-dependent vitamin C transporter 2 (SVCT2). When this transcript was suppressed with sequence-specific siRNAs, the active transport component of ascorbic acid was abolished. Thus, we provide the first evidence that SVCT2 mediates the secondary active and concentrative transport of ascorbic acid in human chondrocytes.

Although smooth muscle and endothelial cells in pig coronary artery are morphologically and functionally distinct, ascorbate uptake has been characterized only in smooth muscle cells. Ascorbate transporters in kidney and intestinal epithelial cells differ from those in smooth muscle. We examined ascorbate transport and mRNA expression of sodium-dependent vitamin C transporters (SVCT) by RT-PCR in the pig coronary artery endothelial cell cultures. When 14C-ascorbate uptake in endothelial cells was examined as 14C or by HPLC, the two values did not differ from each other. 14C-ascorbate uptake was Na(+)-dependent, stereoselective for L-ascorbate and inhibited by sulfinpyrazone. The kinetic characteristics of the uptake were: Km = 27 +/- 3 microM (Hill coefficient = 1) for ascorbate and Km = 73 +/- 14 mM (Hill coefficient = 2) for Na+. Surprisingly, endothelial cells had similar kinetic parameters as smooth muscle cells, except for a slightly lower uptake velocity in endothelial cells. Comparison with the smooth muscle showed that both tissue types expressed mRNA for SVCT2. Endothelial cells differ from epithelial cells which express mainly SVCT1 but resemble smooth muscle cells in this respect.

p-Aminohippurate (PAH) is the classical substrate used in the characterization of organic anion transport in renal proximal tubular cells. Although basolateral transporters for PAH uptake from blood into the cell have been well characterized, there is still little knowledge on the apical urinary efflux transporters. The multidrug resistance protein 2 (MRP2/ABCC2) is localized to the apical membrane and mediates ATP-dependent PAH transport, but its contribution to urinary PAH excretion is not known. In this report, we show that renal excretion of PAH in isolated perfused kidneys from wild-type and Mrp2-deficient (TR(-)) rats is not significantly different. Uptake of [(14)C]PAH in membrane vesicles expressing two different MRP2 clones isolated from Sf9 and MDCKII cells exhibited a low affinity for PAH (Sf9, 5 +/- 2 mM; MDCKII, 2.1 +/- 0.6 mM). Human MRP4 (ABCC4), which has recently been localized to the apical membrane, expressed in Sf9 cells had a much higher affinity for PAH (K(m) = 160 +/- 50 microM). Various inhibitors of MRP2-mediated PAH transport also inhibited MRP4. Probenecid stimulated MRP2 at low concentrations but had no effect on MRP4; but at high probenecid concentrations, both MRP2 and MRP4 were inhibited. Sulfinpyrazone only stimulated MRP2, but inhibited MRP4. Real-time PCR and Western blot analysis showed that renal cortical expression of MRP4 is approximately fivefold higher as compared with MRP2. MRP4 is a novel PAH transporter that has higher affinity for PAH and is expressed more highly in kidney than MRP2, and may therefore be more important in renal PAH excretion.

Purpose: The objective of the study was to determine the effect of a potent and selective CYP2C9 inhibitor, sulfinpyrazone (Anturane), on the pharmacokinetics of nateglinide (Starlix), a novel antidiabetic drug which is primarily (approximately 70%) metabolized via CYP2C9., Methods: This was a randomized, open-label, two-period, crossover study in 18 healthy volunteers. Nateglinide was administered as a single 120-mg oral dose alone (reference) on day 1 or in combination with sulfinpyrazone (test) on day 7, following twice-daily 200-mg oral doses (i.e., 400 mg/day) of sulfinpyrazone for 7 days. Pharmacokinetic parameters of nateglinide were determined following the administration of nateglinide alone, and when administered in combination with sulfinpyrazone. Plasma nateglinide concentrations were determined using a validated high-performance liquid chromatography method., Results: The administration of nateglinide in combination with sulfinpyrazone resulted in approximately 28% higher mean AUC of nateglinide (90% CI for test-reference ratio: 1.20-1.39) with no differences in mean peak plasma concentration (Cmax; 90% CI test-reference ratio: 0.86-1.12) compared with nateglinide-alone treatment. The time to reach Cmax (tmax) and the elimination half-life of nateglinide were similar between the two treatments. Both treatments were safe and well tolerated., Conclusions: Sulfinpyrazone increased the mean exposure of nateglinide by 28% when both drugs were administered in combination. Nateglinide, given as a single dose or co-administered with multiple doses of sulfinpyrazone, was safe and well tolerated in healthy subjects.

Skeletal muscle and brain are major sites of glucose transport and ascorbate (vitamin C) storage. Ascorbate is oxidized to dehydroascorbic acid (DHAA) when used as an enzyme cofactor or free radical scavenger. We evaluated the hypothesis that glucose regulates DHAA uptake and reduction to ascorbate (i.e., recycling) by skeletal muscle cells and cerebral astrocytes. DHAA uptake was inhibited partially by glucose added simultaneously with DHAA. Comparison of wild type L6 skeletal muscle cells with an L6-derived cell line (D23) deficient in facilitative hexose transporter isoform 3 (GLUT3), indicated that both GLUT3 and facilitative hexose transporter isoform 1 (GLUT1) mediate DHAA uptake. Preincubation of muscle cells with glucose inhibited the rates of glucose and DHAA uptake, and decreased the intracellular concentration of ascorbate derived from recycling of DHAA. In contrast, glucose preincubation did not depress GLUT1 protein and activity levels or DHAA recycling in astrocytes. These results establish that glucose downregulates subsequent recycling of DHAA by skeletal muscle cells but not astrocytes.

This study examined the accumulation and transport of two related systemic opioids used as antidiarrhoeal drugs and compared their rates of transport with known P-glycoprotein (P-gp) substrates used in our in vitro environment. Cellular uptake and efflux and transcellular transport were all determined using Caco-2 cells after exposure to loperamide or diphenoxylate, with or without a range of efflux inhibitors. Bidirectional transport studies of 5 microM loperamide showed efflux to be fivefold higher than influx (42 x 10(-6) compared to 8 x 10(-6) cm/s); however, this decreased to twofold at 10 microM and was abolished using 100 microM loperamide. An uptake pathway was also discovered when P-gp was inhibited which, in the presence of Ca(2+) channel blockers, was amplified, providing a potential mechanism for central nervous system effects to be increased upon blockage of L-type calcium channels, quite separate from any P-gp inhibition. Diphenoxylate transport, however, showed little sign of P-gp-mediated efflux. Diphenoxylate accumulated readily within cells, yet transport through cells was very low. Additionally, efflux inhibitors had little impact on transport or accumulation, suggesting that diphenoxylate was not a substrate for an efflux mechanism.

Purpose: Previous studies indicated that ATP-binding cassette (ABC) membrane transporters protect against UV-induced apoptosis. We investigated the effect of UVB and X-ray irradiation on the export function of these ABC transporters in primary lymphocytes and various cancer cell lines., Material and Methods: We used rhodamine accumulation assays in various human malignant cell lines and peripheral blood lymphocytes (PBL). Cells were irradiated with up to 960 mJ/cm2 and up to 50 Gy of UVB and X-ray, respectively., Results: We demonstrated that UVB as well as X-ray irradiation inhibit the export function of the ABC transporters in a dose-dependent fashion. For PBL, this effect did not correlate with an apoptotic phenotype. In the case of the tumor cell lines, even though the irradiation-induced inhibition of membrane transporters was accompanied by phosphatidylserine exposure, only a minority of cells had lost their mitochondrial membrane potential during the observation period. Furthermore, we demonstrated that the inhibition of membrane transporters is not a general feature of apoptosis., Conclusion: Irradiation inhibits the export function of ABC transporters. Although some of the irradiated cells undergo apoptosis following irradiation, the inhibition is an unique feature accompanying irradiation and not a general hallmark of apoptotic cell death. The inhibition of drug export by irradiation may offer new potential for reverting multidrug resistance of cancer cells.

Primary rat astrocyte cultures absorbed dehydroascorbic acid from the medium and reduced it to intracellular ascorbate. Uptake of dehydroascorbic acid (5-200 microM) was inhibited only partially by glucose (10 mM). The remaining glucose-insensitive component of dehydroascorbic acid uptake was inhibited reversibly by sulfinpyrazone (IC(50) = 80 microM). Dehydroascorbic acid uptake was not mediated by Na(+)-ascorbate cotransporters or volume-sensitive anion channels because it was neither Na(+)-dependent nor blocked by the channel antagonist, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Oxidative stress, induced in astrocytes by the lipophilic radical generator tert-butyl hydroperoxide, decreased intracellular glutathione concentration and inhibited accumulation of intracellular ascorbate from dehydroascorbic acid. Subsequent administration of either the native antioxidant alpha-tocopherol (200 microM) or anesthetic concentrations of the antioxidant sedative propofol (1-8 microM, administered 30 min after tert-butyl hydroperoxide), did not change glutathione concentration but restored the ability of astrocytes to accumulate intracellular ascorbate from dehydroascorbic acid. These results are consistent with a novel mechanism of astrocytic ascorbate accumulation that is inhibited by lipophilic radicals and protected by lipophilic antioxidants such as propofol., (Copyright 2002 Wiley-Liss, Inc.)

Sulfinpyrazone, a potent uricosuric drug, was tested in vitro for its scavenging action against oxygen free radicals. In this study, sulfinpyrazone was able to scavenge 1,1-diphenyl-2-picrylhydrazyl radical with IC50 value of 29.82 micrograms/ml compared to butylated hydroxytoluene (BHT, IC50 value = 20.15 micrograms/ml) and Trolox (IC50 value = 16.01 micrograms/ml). It was able to scavenge superoxide anion with IC50 value of 27.72 micrograms/ml compared to Trolox (IC50 value = 22.08 micrograms/ml) and ascorbic acid (IC50 value = 14.65 micrograms/ml). The hydroxyl radical scavenging activity of sulfinpyrazone is in a concentration-dependent fashion. In the range of concentrations used, sulfinpyrazone was not a scavenger toward H2O2. However, the intracellular H2O2-induced 2',7'-dichlorofluorescin diacetate (DCF-DA) fluorescence in HL-60 cells was significantly reduced by sulfinpyrazone during 30-60 min of incubation. Finally, phorbol-12-myristate-13-acetate induced-lucigenin chemiluminescence in whole blood was markedly inhibited by sulfinpyrazone. Our results suggest a new direction for the pharmacological actions of sulfinpyrazone in free radical scavenging properties.

Recent studies in our laboratory have shown that the loop diuretic, furosemide, is actively secreted by Caco-2 cells and rat jejunal tissue. This active secretion could be the result of efflux transporters such as P-gp, MRP1 or MRP2 (cMOAT). To determine if any of these transporters is responsible for the secretion of furosemide, we compared directional permeability in the wild-type cell lines, MDCK strains I and II, and LLC-PK1, vs. cell lines that overexpress a single transporter, in both the presence and absence of various inhibitors, for furosemide as compared to vinblastine. Sulfinpyrazone significantly inhibited the transport of vinblastine in MRP2 expressing cells, but not the wild-type controls. Vinblastine could not be confirmed as a substrate of MRP1. We were also unable to demonstrate that any particular transporter affected furosemide in excess of the background effects of endogenous transporters in the parental cell lines. Furosemide secretion from these kidney-derived cell lines is probably not the primary result of any of the well characterized efflux transporters (P-gp, MRP1 or MRP2), although they may still play a role in the observed Caco-2 secretion. This equivocal result acknowledges the difficulty in trying to determine the effect of a single protein in a complicated expression system., (Copyright 2002 S. Karger AG, Basel)

The ATP-binding cassette (ABC) proteins comprise a large superfamily of transmembrane transporters that utilize the energy of ATP hydrolysis to translocate their substrates across biological membranes. Multidrug resistance protein (MRP) 2 (ABCC2) belongs to subfamily C of the ABC superfamily and, when overexpressed in tumor cells, confers resistance to a wide variety of anticancer chemotherapeutic agents. MRP2 is also an active transporter of organic anions such as methotrexate (MTX), estradiol glucuronide (E217betaG), and leukotriene C4 and is located on the apical membrane of polarized cells including hepatocytes where it acts as a biliary transporter. We recently identified a highly conserved tryptophan residue in the related MRP1 that is critical for the substrate specificity of this protein. In the present study, we have examined the effect of replacing the analogous tryptophan residue at position 1254 of MRP2. We found that only nonconservative substitutions (Ala and Cys) of Trp1254 eliminated [3H]E217betaG transport by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effect. In addition, only the most conservatively substituted mutant (W1254Y) transported [3H]leukotriene C4, whereas all other substitutions eliminated transport of this substrate. On the other hand, all substitutions of Trp1254 eliminated transport of [3H]MTX. Finally, we found that sulfinpyrazone stimulated [3H]E217betaG transport by wild-type MRP2 4-fold, whereas transport by the Trp1254 substituted mutants was enhanced 6-10-fold. In contrast, sulfinpyrazone failed to stimulate [3H]MTX transport by either wild-type MRP2 or the MRP2-Trp1254 mutants. Taken together, our results demonstrate that Trp1254 plays an important role in the ability of MRP2 to transport conjugated organic anions and identify this amino acid in the putative last transmembrane segment (TM17) of this ABC protein as being critical for transport of MTX.

We examined the role of multidrug resistance protein (MRP) 1 (ABCC1) in the emergence of mitoxantrone (MX) cross-resistance in a MCF7 breast cancer cell line selected for resistance to etoposide. The resistant cell line, MCF7/VP, expresses high levels of MRP1, whereas the parental cell line, MCF7/WT, does not. MCF7/VP cells are 6-10-fold cross-resistant to MX when compared with MCF7/WT cells. Drug transport studies in intact MCF7/VP cells revealed that MX resistance is associated with reduced MX accumulation due to enhanced MX efflux. MX efflux is ATP dependent and inhibited by sulfinpyrazone and cyclosporin A. Inhibition of MX efflux with these agents sensitizes cells to MX cytotoxicity and partially reverses MX resistance in MCF7/VP cells. Whereas resistance is partially attributable to increased MX efflux in MRP1-expressing MCF7/VP cells, we found no evidence for glutathione or other conjugates of MX in these cells. Moreover, glutathione depletion with buthionine sulfoximine had no effect on MX transport or sensitivity in MCF7/VP cells. MRP1 substrates are generally amphiphilic anions such as glutathione conjugates or require the presence of physiological levels of glutathione for MRP1-mediated transport. Therefore we conclude that MRP1 overexpression is unlikely to be responsible for increased MX efflux and resistance in MCF7/VP cells. In considering the potential involvement of other MRP family isoforms, a 3-fold increase in the expression of MRP5 was observed in MCF7/VP cells. However, stable expression of a transduced MRP5 expression vector in MCF7/WT cells failed to confer MX resistance. Because other transporters known to be associated with MX resistance, including P-glycoprotein and BCRP/MXR (ABCG2), are not expressed in MCF7/VP cells, we conclude that increased MX efflux and resistance in MCF7/VP cells is attributable to a novel transport mechanism or that MX represents a novel class of cationic, glutathione-independent MRP1 substrates.

Background: Management of cyclosporine (CsA)-associated hyperuricemia in heart transplantation (HT) is difficult. Because of the myelotoxicity of combined allopurinol and azathioprine, we tested sulfinpyrazone., Methods: We studied 120 HT recipients (109 men; mean age at HT, 52+/-10 years). All had received allopurinol for at least 6 months, which was stopped for 1 month before initiation of sulfinpyrazone. Mean follow-up from HT to onset of sulfinpyrazone (200 mg/day) was 59+/-41 months. We stopped the drug after 6+/-2 months. We compared CsA level and daily dose, serum creatinine, blood urea, and uric acid at onset and before interruption of sulfinpyrazone and, as control, in the last 6 months of allopurinol., Results: Mean uricemia decreased with allopurinol (0.58+/-0.12 vs. 0.41+/-0.07 mmol/liter, p = 0.0001) as well as with sulfinpyrazone (0.51+/-0.13 vs. 0.40+/-0.12 mmol/liter, p = 0.0001). Mean creatinine increased (171+/-42 and 164+/-35 micromol/liter, p = 0.01) with allopurinol, whereas it tended to decrease with sulfinpyrazone (160+/-35 and 154+/-48 micromol/liter, p = NS). Mean urea did not change with allopurinol (14+/-5 vs. 15+/-7 mmol/liter, p = NS), but fell with sulfinpyrazone (14.01+/-5 vs. 12.60 +/-5 mmol/liter, p = 0.0004). Mean CsA levels were constant with allopurinol (193+/-73 vs. 188+/-65 ng/ml, p = NS), although CsA dose was slightly reduced (2.7+/-0.8 vs. 2.6+/-0.8 mg/kg/day, p = 0.007). Conversely, CsA levels dropped with sulfinpyrazone (183+/-89 vs. 121 +/-63 ng/ml, p = 0.0001) despite an increase in CsA daily dose (2.6 +/-0.9 vs. 2.8+/-0.9 mg/kg/day, p = 0.0001). Two subjects were treated for acute rejection. We observed no other side effects. In HT recipients sulfinpyrazone, as an alternative to allopurinol, is effective in achieving metabolic control of hyperuricemia. However, this drug reduced CsA levels, thus the risk of rejection is present.

Previous studies in our laboratory have shown that the phase III efflux transporter multidrug-resistance protein (MRP)1 can act synergistically with the phase II conjugating glutathione S-transferases (GST) to confer resistance to the toxicities of some electrophilic drugs and carcinogens. To determine whether the distinct efflux transporter MRP2 could also potentiate GST-mediated protection from electrophilic toxins, we examined the effect of regulatable GSTP1-1 expression in MRP2-rich HepG2 cells on 4-nitroquinoline 1-oxide (4NQO)-induced cytotoxicity and genotoxicity (nucleic-acid adduct formation). Expression of GSTP1-1 was associated with a fourfold to tenfold protection from 4NQO-induced cytotoxicity. Inhibition of MRP2-mediated efflux activity by sulfinpyrazone or cyclosporin A completely reversed GSTP1-1-associated resistance-a result indicating that GSTP1-1-mediated cytoprotection is absolutely dependent on MRP2 efflux activity. Moreover, MRP2 efflux activity also augmented GSTP1-1-mediated protection from 4NQO-induced nucleic-acid adduct formation. We conclude that MRP2-mediated efflux of the glutathione conjugate of 4NQO and/or another toxic derivative of 4NQO is required to support GSTP1-1-associated protection from 4NQO toxicities in HepG2 cells.

Pig deendothelialized coronary artery rings and smooth muscle cells cultured from them accumulated ascorbate from medium containing Na(+). The accumulated material was determined to be ascorbate using high-performance liquid chromatography. We further characterized ascorbate uptake in the cultured cells. The data fitted best with a Hill coefficient of 1 for ascorbate (K(asc) = 22 +/- 2 microM) and 2 for Na(+) (K(Na) = 84 +/- 10 mM). The anion transport inhibitors sulfinpyrazone and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) inhibited the uptake. Transferring cultured cells loaded with (14)C-ascorbate into an ascorbate-free solution resulted in a biphasic loss of radioactivity - an initial sulfinpyrazone-insensitive faster phase and a late sulfinpyrazone-sensitive slower phase. Transferring loaded cells into a Na(+)-free medium increased the loss in the initial phase in a sulfinpyrazone-sensitive manner, suggesting that the ascorbate transporter is bidirectional. Including peroxide or superoxide in the solution increased the loss of radioactivity. Thus, ascorbate accumulated in coronary artery smooth muscle cells by a Na(+)-dependent transporter was lost in an ascorbate-free solution, and the loss was increased by removing Na(+) from the medium or by oxidative stress., (Copyright 2000 S. Karger AG, Basel)

Cytosolic calcium modulates the activity of osteoclasts, large multinucleate cells that resorb bone. Nuclear events, such as gene transcription, are also calcium-regulated in these cells, and fluorescence imaging has suggested that calcium signals produced by some stimuli are specifically targeted to, or amplified within, osteoclast nuclei. We used two alternative techniques of dye loading to examine the changes of intracellular calcium induced in rat osteoclasts by three stimuli. Osteoclasts loaded with the calcium indicator Fura-2 by the acetoxymethyl (AM) ester technique appeared to display marked nuclear calcium amplification. During stimulation with integrin-binding peptides, ATP, or high extracellular calcium, fluorescence ratios recorded from the nuclei rose higher than did ratios recorded from extranuclear regions. In contrast, nuclear calcium amplification was not observed after AM loading in the presence of the anion transport inhibitor sulfinpyrazone, nor in osteoclasts injected with Fura-2 conjugated to a high MW dextran. In these cells, nuclear fluorescence ratios were equal to the extranuclear values at all times: upon stimulation by an agonist, the nuclear and cytosolic calcium concentrations increased by the same amount. The calcium changes seen in stimulated osteoclasts can no longer be taken as evidence for the general validity of the phenomenon of nuclear calcium amplification.

To examine the role of multidrug resistance protein 1 (MRP1) and glutathione S-transferases (GSTs) in cellular resistance to antineoplastic drugs, derivatives of MCF7 breast carcinoma cells were developed that express MRP1 in combination with one of three human cytosolic isozymes of GST. Expression of MRP1 alone confers resistance to several drugs representing the multidrug resistance phenotype, drugs including doxorubicin, vincristine, etoposide, and mitoxantrone. However, co-expression with MRP1 of any of the human GST isozymes A1-1, M1-1, or P1-1 failed to augment MRP1-associated resistance to these drugs. In contrast, combined expression of MRP1 and GST A1-1 conferred approximately 4-fold resistance to the anticancer drug chlorambucil. Expression of MRP1 alone failed to confer resistance to chlorambucil, showing that the observed protection from chlorambucil cytotoxicity was absolutely dependent upon GST A1-1 protein. Moreover, using inhibitors of GST (dicumarol) or MRP1 (sulfinpyrazone), it was shown that in MCF7 cells resistance to chlorambucil requires both intact MRP1-dependent efflux pump activity and, for full protection, GST A1-1 catalytic activity. These results are the first demonstration that GST A1-1 and MRP1 can act in synergy to protect cells from the cytotoxicity of a nitrogen mustard, chlorambucil.

Two membrane glycoproteins acting as energy-dependent efflux pumps, mdr-encoded P-glycoprotein (P-gp) and the more recently described multidrug resistance-associated protein (MRP), are known to confer cellular resistance to many cytotoxic hydrophobic drugs. In the brain, P-gp has been shown to be expressed specifically in the capillary endothelial cells forming the blood-brain barrier, but localization of MRP has not been well characterized yet. Using RT-PCR and immunoblot analysis, we have compared the expression of P-gp and Mrp1 in homogenates, isolated capillaries, primary cultured endothelial cells, and RBE4 immortalized endothelial cells from rat brain. Whereas the mdr1a P-gp-encoding mRNA was specifically detected in brain microvessels and mdr1b mRNA in brain parenchyma, mrp1 mRNA was present both in microvessels and in parenchyma. However, Mrp1 was weakly expressed in microvessels. Mrp1 expression was higher in brain parenchyma, as well as in primary cultured brain endothelial cells and in immortalized RBE4 cells. This Mrp1 overexpression in cultured brain endothelial cells was less pronounced when the cells were cocultured with astrocytes. A low Mrp activity could be demonstrated in the endothelial cell primary monocultures, because the intracellular [3H]vincristine accumulation was increased by several MRP modulators. No Mrp activity was found in the cocultures or in the RBE4 cells. We suggest that in rat brain, Mrp1, unlike P-gp, is not predominantly expressed in the blood-brain barrier endothelial cells and that Mrp1 and the mdr1b P-gp isoform may be present in other cerebral cells.

The fluorescent dye 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) has been widely used as an indicator of cytosolic pH. Here we report that BCECF localizes to hydrogenosomes (hydrogen-generating organelles found in several phylogenetically separate groups of anaerobic protists) in Trichomonas vaginalis, where it was observable by fluorescence microscopy. Its cellular location was confirmed by treatment of BCECF-loaded cells with diaminobenzidine and hydrogen peroxide together with UV illumination. This produced an osmiophilic precipitate in the matrix of hydrogenosomes, observable by electron microscopy. Use of a short (7.5 min) loading period, loading on ice, use of concentrations of BCECF (acetoxymethyl ester) down to 10 nM, and inclusion of the anion channel blockers probenicid or sulfinpyrazone, or the K+/H+ ionophore nigericin in the loading buffer all failed to prevent hydrogenosomal accumulation of BCECF. This uptake was best observed when intact cells were loaded with the ester form of BCECF, but could also be seen using free BCECF following either incubation with ruptured cells or electroporation of intact cells. Hydrogenosomal BCECF loading was also obtained with washed cell lysates, without cytoplasm or metabolic substrates. We tested a range of other fluorogenic dyes designed for cytosolic labeling, and found that the calcium indicator fura-2 (acetoxymethyl ester) and the cell viability marker fluorescein diacetate also labeled hydrogenosomes. The results illustrate a novel use for BCECF as a fluorescent marker for hydrogenosomes (the first such marker), but present a warning against the indiscriminate use of fluorogenic ester dyes to measure properties of the cytosol in hydrogenosome-containing organisms - the dyes may also be indicating the properties of the hydrogenosome.

Inhibition of [14C]-urate uptake by uricosuric and antiuricosuric agents was investigated in human brush-border membrane vesicles, urate being transported either by anion exchange mechanisms or by voltage sensitive pathway. The IC50 for drugs on [14C]-urate uptake in vesicles loaded with 1 mM cold urate or with 5 mM lactate was, respectively: 0.7 and 0.3 microM for benzbromarone; 6 and 4 microM for salicylate; 133 and 13 microM for losartan; 520 and 190 microM for sulfinpyrazone and 807 and 150 microM, for probenecid. The IC50 ratio for [14C]-urate uptake in exchange for cold urate or for lactate varied from about 1 for salicylate to 10 for losartan, supporting the hypothesis that two distinct anion exchangers are involved in urate transport. Application of Hill equation revealed that urate/anion exchangers have more than one binding site, possibly two binding sites with high cooperativity, for benzbromarone and sulfinpyrazone, but only one for probenecid, salicylate and losartan. The uricosuric diuretic, tienilic acid was 10 to 50 times more potent than hydrochlorothiazide, chlorothiazide and furosemide, for inhibiting [14C]-urate uptake in exchange for cold urate. This higher potency is the reason of its uricosuric properties. All uricosuric agents, as well as the antiuricosuric agents, pyrazinoate and ethambutol, had a much lower potency for inhibiting [14C]-urate uptake through the voltage sensitive pathway (apical secretory step) than through the urate/anion exchangers. This suggests that antiuricosuria, induced by pyrazinoate and ethambutol, as well as by low concentrations of uricosuric agents, does not result from an inhibition of the apical voltage sensitive pathway.

Background: Fluorescent indicators that show alterations in excitation and/or emission spectra in response to changes in [Ca2+] are widely used for quantitative cytosolic [Ca2+] measurements. There are several reports of changes in apparent [Ca2+] due only to illumination, however. These results have been attributed either to photodamage to the cells or to photodegradation of the indicator. Light-induced alteration in the behavior of the dye or cells would severely hamper the interpretation of experimental data. We examined this phenomenon in indo-1 loaded cells using confocal laser scanning microscopy., Results: Illumination of indo-1 loaded GH3 cells leads to a decrease in apparent basal [Ca2+] and decreased peaks after depolarization with KCl. When cells were double loaded with indo-1 and fluo-3, the effect of UV illumination was noticed only with the former dye. UV irradiation of indo-1 in simple buffers caused overall photobleaching and conversion to a fluorescent but Ca2+-insensitive species. The latter effect cannot be canceled by ratiometric calibration and is due to loss of carboxymethyl groups from the anilino nitrogens. This photodegradation was inhibited by extracellular administration of 10 to 100 microM Trolox, a water-soluble vitamin E analog., Conclusions: Photodegradation processes like that observed for indo-1 are likely to be possible for all cation indicators that contain bis(carboxymethyl)anilino moieties, which include essentially all fluorescent indicators for Ca2+ and Mg2+ currently in biological use. If unrecognized, this photochemical dealkylation leads to an underestimation of the analyte concentrations depending on the intensity and duration of illumination. The problem can be avoided by including cell-permeant antioxidants such as Trolox in the bathing solution. The ultimate solution would be to redesign the indicators to minimize photodegradation in the absence of antioxidants.

The multidrug resistance protein MRP has been shown to mediate the transport of glutathione S-conjugates across membranes. In this study we demonstrate that the glutathione S-conjugate of the diuretic drug ethacrynic acid, which is an efficient inhibitor of glutathione S-transferases, is a high-affinity substrate and inhibitor of the glutathione S-conjugate pump associated with MRP. This implies that ethacrynic acid may modulate drug resistance of tumor cells not only by inhibiting glutathione S-transferase activity, but also by inhibiting the export of drug conjugates from the cell by MRP.

The objective of this study was to develop an analytical approach to determine whether the illegal application of clenbuterol in cattle as an anabolic agent can be concealed by co-treatment with substances that affect urinary excretion. Female veal calves were dosed orally with 0.8 microgram clenbuterol per kg of body weight twice daily for 28 days, as licensed for the therapeutic use which is registered in most European countries. On the eighth day of clenbuterol treatment each calf was additionally dosed orally either with probenecid, sulfinpyrazone or pyrazinamide at three different doses that were increased in weekly intervals. During the treatment blood and urine samples were obtained and analysed for clenbuterol by enzyme immunoassay and by high performance liquid chromatography/ enzyme immunoassay to determine whether these drugs or their metabolites interfered with the immunological detection of clenbuterol. Clenbuterol could be in plasma (approximately 200 pg ml-1) and urine (1-40 ng ml-1) 5 h after the initial intake and throughout the whole treatment. None of the drugs reduced urinary excretion of clenbuterol to concentrations below the limit of detection. There was no prevention of clenbuterol detection in urine samples from calves that were co-treated with the drugs tested in this study. Our results demonstrate the uselessness of applying these drugs in order to conceal the illegal use of clenbuterol in meat production.

Objective: In several systems, exogenous ascorbate (reduced vitamin C) has been shown to protect against microvascular injury induced by reactive oxygen species. Since skeletal muscle is relatively resistant to oxidative injury, it is possible that under physiological conditions endogenous ascorbate in the muscle microvasculature affords such protection. To examine the ability of microvascular endothelium to accumulate ascorbate, we aimed (1) to develop an in vitro model of microvascular endothelial cells derived from rat hindlimb skeletal muscles and (2) to investigate the uptake and steady-state concentration of ascorbate in these cells., Methods: Microvascular cells were enzymatically dissociated, isolated on a density gradient, and grown in serum-supplemented medium. After passaging, they were tested for formation of tube-like structures, coagulation factor VIII antigen expression, Griffonia simplicifolia lectin I-isolectin B4 binding, and acetylated low-density lipoprotein (LDL) uptake. Concentrations of reduced ascorbate were measured by high-performance liquid chromatography (HPLC) with electrochemical detection. Transport activity was assessed on the basis of the initial rate [14C]ascorbate uptake., Results: The cultured cells tested positively for factor VIII antigen expression, lectin binding, LDL uptake, and tube formation. Although these cells did not synthesize ascorbate de novo, they accumulated reduced vitamin C when it was added to the culture medium. The initial rate of [14C]ascorbate uptake was 0.9 mumol/g cell protein 10 min when cells were incubated with 10 microM of the radiolabeled vitamin. This uptake was Na+-dependent and was blocked by the organic anion transport inhibitor sulfinpyrazone, but was not acutely affected by glucose. Following incubation with a physiological concentration of vitamin C (100 microM L-ascorbate), cells accumulated a high concentration of ascorbate within 6 h (approximately 16 mM at steady-state). Steady-state cellular ascorbate concentration was also dependent on extracellular Na+ and sensitive to sulfinpyrazone., Conclusions: Microvascular cells derived from rat hindlimb muscles demonstrated endothelial characteristics. These cells accumulated reduced vitamin C by means of Na+-dependent ascorbate transporters, which are distinct from hexose carriers. The high endothelial ascorbate concentration at steady-state is consistent with the role of ascorbate as a major antioxidant in the skeletal muscle microvasculature.

Sulfinpyrazone markedly potentiates the anticoagulant effect of warfarin. The increased clotting time is accompanied by a marked decrease in the clearance of (S)-warfarin by virtue of a decrease in the P4502C9-catalyzed formation clearance to its major and inactive metabolite (S)-7-hydroxywarfarin. These data suggested that the mechanism of the drug interaction might be mediated through the inhibition of the catalytic activity of P4502C9 by sulfinpyrazone. However, initial human liver microsomal studies indicated that the in vitro Ki, for inhibition of (S)-7-hydroxywarfarin formation by sulfinpyrazone is at least 25-fold higher than the therapeutic concentration of sulfinpyrazone in vivo. This result implied that other inhibitors probably contribute to the interaction. Kinetic studies conducted on sulfinpyrazone and two major metabolites, sulfinpyrazone sulfide and sulfinpyrazone sulfone, in microsomes prepared from three human livers give mean Ki's of 230 microM, 17 microM, and 73 microM respectively. Because sulfinpyrazone and its sulfide metabolite attain comparable plasma concentrations during the course of therapy, our inhibition results suggest that the sulfide metabolite is likely to be the primary species responsible for the inhibition of P4502C9-catalyzed formation of (S)-7-hydroxywarfarin and the decrease in (S)-warfarin clearance in vivo.

A method is described to facilitate flow cytometric analysis of calcium mobilization upon stimulation of murine T cell hybrids. In these transformed cell lines, the accuracy of cytometric measurement of free cytoplasmic calcium with Fluo-3 is compromised by the rapid loss of the intracellular dye. We have found that the addition of sulfinpyrazone, a known organic-anion transporter inhibitor in epithelial cells and in macrophages, severely impairs the leakage of the Fluo-3 probe from the cytoplasmic matrix. Under appropriate conditions, sulfinpyrazone has little effect on the cell physiology and permits the detection of calcium influx in a variety of murine T cell hybrids.

Purpose: To investigate the mechanism(s) by which intracellular cAMP levels are elevated to induce pigment granule aggregation in teleost retinal pigment epithelium (RPE)., Methods: Pigment granule migration was studied in vitro using RPE sheets isolated from dark-adapted green sunfish, Lepomis cyanellus. After preculture to allow pigment granule dispersion, RPE sheets were incubated with various agents to test their ability to induce pigment granule aggregation. RPE sheets were then fixed, and pigment granule position was assessed microscopically., Results: Pigment granule aggregation was induced by nonderivatized cAMP. At maximally effective concentration (1 mM), cAMP was as effective as its more membrane-permeant analogs dbcAMP and 8-Br-cAMP. Forskolin (1 or 10 microM), a stimulator of adenylyl cyclase, was also effective at inducing pigment aggregation. Two inhibitors of organic anion transport, probenecid and sulfinpyrazone, inhibited cAMP-induced aggregation by approximately 80% but had no effect on forskolin-induced aggregation. Several agents shown to stimulate RPE adenylyl cyclase in other species failed to induce pigment aggregation in isolated RPE sheets., Conclusions: Our observations strongly suggest that exogenously applied, nonderivatized cAMP can gain access to the cytoplasm of isolated RPE cells via organic anion transporters. Thus, if cAMP were secreted by retinal cells into the subretinal space, it could be taken up by RPE cells and subsequently act as an intracellular messenger to activate dark-adaptive physiological processes such as pigment granule aggregation.

Intravenous injection of platelet-activating factor (PAF) (0.36 mumol/kg b.w.) in mice induced severe hemoconcentration, leucopenia, thrombocytopenia and finally the death of 85% of the tested animals. Combined inhibition of histamine and serotonin by promethazine and chlorpromazine, 6.24 and 3.12 mg/kg b.w. subcutaneously, protected the mice from PAF in part, reducing the death rate to 43%. These drugs did not protect the mice against the PAF-induced hemoconcentration, leucopenia and thrombocytopenia. Sulfinpyrazone (100 mg/kg b.w.) intravenously was the most effective both in protecting mice from PAF-induced death, reducing the death rate to 17%, and from thrombocytopenia, although hemoconcentration persisted. These results indicated that an important component of the PAF-induced systemic effects is mediated by reactions which can be inhibited by sulfinpyrazone. Furthermore, PAF-induced thrombocytopenia is not a direct PAF effect since it can be inhibited by sulfinpyrazone.

Macrophages possess organic anion transporters that carry membrane-impermeant fluorescent dyes, such as lucifer yellow (LY) and carboxy-fluorescein, from the cytoplasm into endosomes and out of the cells. Probenecid, an organic anion transport inhibitor, blocks these processes. Prolonged incubation of J774 cells in medium containing 2.5 mM probenecid eventually kills most of these cells. To identify J774 variants that express increased organic anion transport activity, we selected probenecid-resistant (PBR) J774 cells by growing them in medium containing increasing concentrations of probenecid. When PBR and unselected J774 cells were loaded with LY by ATP4- permeabilization, the amount of LY accumulated by the PBR cells was about half that in the unselected cells. This difference was abolished by adding 10 mM probenecid to the medium in which the cells were loaded, suggesting that the diminished LY accumulation in PBR cells was due to enhanced LY secretion and that the PBR cells expressed increased organic anion transport activity. Direct comparison of LY efflux from J774 and PBR J774 cells showed a faster initial rate of secretion of LY from PBR J774 cells than from unselected J774 cells. To determine whether LY efflux is mediated by P-glycoprotein, we compared LY efflux in unselected J774 cells, PBR J774 cells, and multidrug-resistant J774 cells (J7.C1). LY efflux from J7.C1 cells was not sensitive to verapamil, which inhibits multidrug-resistance transporters, and reverses the multidrug-resistant phenotype of J7.C1 cells. The rates of LY efflux from unselected J774 and J7.C1 cells were virtually identical.(ABSTRACT TRUNCATED AT 250 WORDS)

Suspecting that platelet thromboemboli could play a role in the pathogenesis of myocardial ischemia, we have done a random-order, double-blind, crossover study of the effect of the platelet-active drug sulfinpyrazone on treadmill exercise-induced angina pectoris in 30 men with coronary artery disease. The mean duration of exercise before onset of angina was 43 s longer after taking sulfinpyrazone than before and 11 s shorter after taking placebo than before. Analysis of variance for crossover design showed that the mean difference between the values obtained before and after sulfinpyrazone was significantly different (p < 0.01) from the mean difference between the values before and after placebo. Sulfinpyrazone had no effect on the mean heart rate-blood pressure product at onset of angina, change in ST segment during exercise, or preexercise platelet aggregate ratio and bleeding time. Exercise until angina occurred did not affect the platelet aggregate ratio.

Organic anion transport in polarized epithelia and macrophages has previously been studied by monitoring the efflux of fluorescent organic anion dyes from cells. We adapted this strategy to the study organic anion transport in lymphocytes. Cloned lymphoma cells and normal and activated human T cells were loaded with a membrane-impermeant, organic anion dye (Lucifer Yellow) by electroporation. Dye efflux in lymphocytes was rapid, energy-dependent, and inhibitable by organic anion transporter inhibitors. Dye efflux could not be attributed to the effects of electroporation. In addition, electroporated, dye-loaded T helper cells retained the ability to properly respond to specific antigen. Thus, dye loss occurred in viable, functionally competent cells. These experiments demonstrate that electroporation is an effective means of loading cells with Lucifer Yellow, and that lymphocytes possess organic anion transporters that are functionally similar to those previously described for secretory epithelia and macrophages.

Monoisoamyl meso-2,3-dimercaptosuccinate (Mi-ADMS) and the unesterified 2,3-dimercaptosuccinic acid (DMSA) were evaluated for relative reactivities against metallothionein (MT)-bound cadmium (Cd) in vitro by elution of the reaction products through Sephadex G-75 gel. After 3 hr of incubation, Mi-ADMS removed about 70% of the Cd from Cd-MT, and a new peak emerged which corresponded to that obtained by elution of a 2:1 molar mixture of Mi-ADMS and Cd. Only about 15% of the Cd was removed from Cd-MT by DMSA. After 24 hr of incubation with Mi-ADMS, no evidence remained of the presence of Cd-MT; all of the Cd was recovered in a very high molecular weight fraction and in a fraction corresponding to Cd ion. In contrast, after 24 hr of incubation with DMSA, 25% of the Cd was still present as Cd-MT, while the remainder eluted in a fraction corresponding to a 2:1 molar complex of DMSA and Cd. When Mi-ADMS was administered to Cd-bearing mice which had received an inhibitor of organic anion transport, probenecid (PBC) or sulfinpyrazone (SPZ), prior to administration of the monoester, there was a marked attenuation of the Cd mobilizing actions of Mi-ADMS as reflected in whole body Cd levels. Analysis of organ Cd concentrations revealed that PBC blocked primarily the mobilization of renal Cd by Mi-ADMS, while the principal action of SPZ in antagonizing the action of Mi-ADMS was on hepatic Cd mobilization. It was concluded that Mi-ADMS has a higher affinity for Cd in Cd-MT than does DMSA, and that the access of Mi-ADMS to intracellular Cd is, at least in part, mediated by the organic anion transport system.