Effects of 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) and chlorpromazine on NO3- transport via anion exchanger in erythrocytes: inertness of DIDS in whole blood

We examined the effects of chlorpromazine on NO(3)(-) transport between erythrocytes (RBCs) and extracellular fluid. Chlorpromazine (10 microg/ml) did not influence NO(3)(-) movement in both whole blood and RBC suspension. Though an anion exchanger (AE1) inhibitor DIDS (4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, 100 microM) did not alter NO(3)(-) movement in whole blood, it inhibited the movement in a concentration-dependent manner in the RBC suspension. The inhibition was abrogated by plasma and albumin concentration-dependently. Our results indicated that chlorpromazine had no effect on NO(3)(-) transport through AE1 and that the inertness of DIDS on AE1 in whole blood is due to interference by albumin in plasma.     

Different mechanisms mediate uptake of lead in a rat astroglial cell line.

The mechanism by which lead (Pb) enters astrocytes was examined in a rat astroglial cell line in order to characterize specific pathways for transport. Pb uptake was saturable at pH 5.5 and 7.4, although quantitative differences existed in the Michaelis-Menten constants. At pH 7.4, the Vmax and Km were 2700 fmoles/mg protein/min and 13.4 microM, respectively, whereas the Vmax and Km were 329 fmoles/mg and 8.2 microM in the buffer at pH 5.5, respectively. The presence of extracellular iron inhibited uptake in a buffer at pH 5.5 but not at pH 7.4. Cells treated with the iron chelator deferoxamine displayed higher levels of the iron transporter divalent metal transporter 1 (DMT1) mRNA and protein, and consistent with increased DMT1 expression, the treated cells displayed greater uptake of Pb in the buffer at pH 5.5 but not at pH 7.4. Alternatively, at pH 7.4, the transport of Pb was blocked by the anion transporter inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (DIDS), which bound to cell surface proteins at concentrations that were similar to those that blocked Pb uptake. DIDS did not inhibit uptake of Pb in the buffer at pH 5.5. Greater uptake of Pb was observed in a buffer containing sodium bicarbonate, which was abrogated in the presence of DIDS. In summary, the astroglial cell line displays two distinct pH-sensitive transport mechanisms for Pb.     

Double-transfected MDCK cells expressing human OCT1/MATE1 or OCT2/MATE1: determinants of uptake and transcellular translocation of organic cations

BACKGROUND AND PURPOSE

The organic cation transporters 1 (OCT1) and 2 (OCT2) mediate drug uptake into hepatocytes and renal proximal tubular cells, respectively. Multidrug and toxin extrusion protein 1 (MATE1) is a major component of subsequent export into bile and urine. However, the functional interaction of OCTs and MATE1 for uptake and transcellular transport of the oral antidiabetic drug metformin or of the cation 1-methyl-4-phenylpyridinium (MPP⁺) has not fully been characterized.

EXPERIMENTAL APPROACH

Single-transfected Madin-Darby canine kidney (MDCK) cells as well as double-transfected MDCK-OCT1-MATE1 and -OCT2-MATE1 cells were used to study metformin and MPP⁺ uptake into and transcellular transport across cell monolayers, along with their concentration and pH dependence.

KEY RESULTS

Cellular accumulation of MPP⁺ and metformin was significantly reduced by 31% and 46% in MDCK-MATE1 single-transfected cells compared with MDCK control cells (10 µM; P < 0.01). Over a wide concentration range (10–2500 µM) metformin transcellular transport from the basal into the apical compartment was significantly higher in the double-transfected cells compared with the MDCK control and MDCK-MATE1 monolayers. This process was not saturated up to metformin concentrations of 2500 µM. In MDCK-OCT2-MATE1 cells basal to apical MPP⁺ and metformin transcellular translocation decreased with increasing pH from 6.0 to 7.5.

CONCLUSIONS AND IMPLICATIONS

Our data demonstrate functional interplay between OCT1/OCT2-mediated uptake and efflux by MATE1. Moreover, MATE1 function in human kidney might be modified by changes in luminal pH values.     

Quantitative assessment of the cell penetrating properties of RI-Tat-9: evidence for a cell type-specific barrier at the plasma membrane of epithelial cells

Penetration of epithelial cells represents the rate-determining step for the absorption of many drugs and pharmaceutical macromolecules such as proteins and nucleic acid therapeutics. While the potential of using cell-penetrating peptides (CPPs) to facilitate absorption has been increasingly recognized, the mechanism of cell penetration and the uptake into certain cells have recently been called into question due to methodological artifacts. Therefore, the objective of this study was to quantitatively assess the ability of RI-Tat-9, a proteolytically stable CPP, to penetrate epithelial cell monolayers. The permeability of RI-Tat-9 with two epithelial cell lines, Madin-Darby canine kidney (MDCK) and Caco-2 cells, was comparable to the leakiness of the respective intact monolayers. Microscopic imaging showed that fluorescence-tagged RI-Tat-9 did not enter these cells, further supporting a paracellular transport mechanism. Although insufficient data were generated in these studies to generalize the observed phenomenon, the entry of RI-Tat-9 into nonepithelial T lymphocytic MT2 cells, possibly by endocytosis, suggested that a cell type-specific barrier might exist that controlled uptake of RI-Tat-9 by cells. Compared to that in MT2 and HeLa cells, the active uptake of the peptide into MDCK monolayers was much slower and showed no dependence of cell energy. Furthermore, the equilibrium binding of RI-Tat-9 to MDCK cells at 0 degrees C was indicative of an interaction with a nonspecific receptor. A correlation between binding density and concentration difference across a leaky separation barrier suggested that repulsion of free peptide molecules by bound peptide molecules at the MDCK monolayer surface may be significant at micromolar concentrations. The results of this study quantitatively show that Tat CPP uptake into two commonly used epithelial cell types is minimal and possibly cell type-specific. Implications for Tat CPP-assisted drug delivery are discussed.     

Parathyroid hormone-stimulated cadmium accumulation in Madin-Darby canine kidney cells.

Although most renal cadmium transport occurs in proximal tubules indirect evidence suggests that distal tubules may also transport this heavy metal. Since the distal nephron is the site at which parathyroid hormone (PTH) regulates calcium absorption, we evaluated the effects of PTH on Cd2+ accumulation in Madin-Darby canine kidney (MDCK) cells. MDCK cells express a distal-like phenotype including PTH-sensitive adenylyl cyclase and stimulation of calcium transport. MDCK cells were grown to confluence in phenol red-free Dulbecco's modified Eagle's medium. PTH increased 109CdCl2 accumulation in a concentration-dependent manner over the range of 10(-11)-10(-9) M bPTH[1-34]. At 10(-9) M, PTH increased Cd2+ accumulation maximally by 205%. The PTH antagonist, bPTH[3-34], failed to augment 109Cd2+ accumulation. The dihydropyridine agonist, Bay k 8644, in the presence of PTH, increased 109Cd2+ uptake by 200% over vehicle-treated controls and by approximately 100% over PTH or Bay k 8644 alone. The apparent Km for Bay k 8644 activation was 1.3 microM. Bay k 8644-augmented 109Cd2+ uptake was competitively inhibited by the calcium channel antagonist nifedipine. No voltage dependence of Bay k 8644-amplified 109Cd2+ uptake could be detected. Based on these observations we conclude: (1) MDCK cells accumulate Cd2+; (2) PTH increases Cd2+ uptake into MDCK cells; and (3) Cd2+ entry in kidney epithelial cells is mediated, at least in part, by dihydropyridine-sensitive calcium channels.     

The development of tolerance to Clostridium perfringens type D epsilon-toxin in MDCK and G-402 cells

The epithelial Madin Darby canine kidney (MDCK) cell line, Caucasian renal leiomyoblastoma (G-402) cells, human small airways epithelial (HSAE) cells, human bronchial epithelial (HBE) cells and human renal proximal tubule (HRPT) epithelial cells were examined for sensitivity to Clostridium perfringens biotype D epsilon-toxin. MDCK and G-402 cells were confirmed as being the only established cell lines that are sensitive to the toxin. HSAE, HBE and HRPT epithelial cells were only found to be sensitive to the toxin at concentrations of > 1 mg/ mL. Cultures of MDCK and G-402 cells, with increased resistance (tolerance) to the cytotoxic effects of epsilon-toxin, were developed by exposing these cultures to progressively higher concentrations of toxin. The greatest relative increase in tolerance to epsilon-toxin was developed in MDCK cells, in which the LC50 in control cultures was 2 microg/mL as determined by the MTS/PMS assay system; after selection for tolerance, this was raised to 100 microg/mL. This represents a 50-fold increase in tolerance as measured by this index. Using G-402 cells, it was possible to increase the LC50 by twofold from 290 to 590 microg/mL. Subsequent 2-D electrophoresis of membrane preparations from tolerant and control MDCK cells revealed that the expression of a discrete group of proteins found in control cells with a range of molecular weights from 32-36 kDa, all with acidic isoelectric points (IEPs), were either not expressed in epsilon-toxin tolerant cells or had undergone a shift in IEP to a more alkaline pH in tolerant cells. This suggests that epsilon-toxin lethality in MDCK cells may be mediated by membrane-located proteins. Their absence or alteration in toxin-resistant cells would, at least partly, explain the failure of most cell lines to demonstrate sensitivity to this toxin, despite being derived from tissues that are damaged by epsilon-toxin. This approach may have utility in the study of other toxin-cell interactions and could be used in the development of novel medical countermeasures by identifying cellular targets which mediate toxin lethality.     

Polarized efflux of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein from cultured epithelial cell monolayers.

We have investigated the polarity of the efflux of the intracellular pH fluorochrome 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) from layers of epithelial Madin-Darby canine kidney (MDCK, Strains I and II) and human intestinal (Caco-2, HCT-8 and T84) cells grown on porous membranes. In Strain I MDCK cells, BCECF efflux was effectively reduced by indomethacin (50% inhibition with 100 microM) and 5-nitro-2-(3-phenylpropyl-amino)-benzoate (NPPB; 50% inhibition with 10 microM). Replacement of external Cl- with bromide, iodide or nitrate did not alter BCECF efflux, while substitution with methanesulphonate resulted in a small but significant reduction. All five cell lines form confluent epithelial layers when grown on porous membranes. Efflux of BCECF from Strain I MDCK epithelial layers into the apical solution was approximately three times greater than into the basal solution. Addition of indomethacin to the apical solution attenuated efflux into the apical but not the basal solution, while basal indomethacin was effective against basal efflux. NPPB has a similar specificity of action. Adrenaline, a stimulant of electrogenic Cl- secretion, did not alter the pattern of BCECF efflux. BCECF efflux was also polarized, with apical efflux greater than basal efflux, in MDCK Strain II and Caco-2 epithelial layers. In contrast, BCECF efflux into the basal and apical media was equivalent in layers formed from HCT-8 and T84 cells. However, indomethacin reduced efflux in all five epithelial lines, although the relative sensitivities of the apical and basal efflux rates to indomethacin varied, as did the sensitivity to the sidedness of application of indomethacin. In MDCK and HCT-8 epithelial layers, transepithelial vinblastine secretion mediated by P-glycoprotein was not inhibited by indomethacin. The data are consistent with the hypothesis that BCECF efflux is a manifestation of a novel ATP-dependent xenobiotic secretory efflux mechanism in renal and gastrointestinal epithelia. The factors regulating the polarity of BCECF efflux, both the indomethacin-sensitive and -insensitive components, have yet to be elucidated.     

Determination of epithelial magnesium transport with stable isotopes

The inability to adequately determine Mg(2+) flux rates with radiotracer studies has stymied our efforts to understand how magnesium is transported by epithelial cells. To evaluate epithelial Mg(2+) transport, a stable 25Mg isotope was used to measure magnesium uptake into normal and Mg(2+)-depleted Madin-Darby canine kidney (MDCK) cells. 25Mg entry rates were significantly increased in Mg(2+)-depleted cells relative to those cultured in normal magnesium media, 0.5 mM. 25Mg uptake was inhibited by external La(3+) but not Ca(2+) in both normal and Mg(2+)-depleted cells suggesting a specific entry pathway. These results with 25Mg were the same as with microfluorescence determinations using mag-fura-2. We have shown that Mg(2+) entry into epithelial cells reflects transepithelial transport; accordingly, increased Mg(2+) uptake in Mg(2+)-depleted cells provides an important intrinsic control of renal magnesium absorption. Furthermore, these studies indicate that cellular Mg(2+) transport may be quantitated with the use of stable isotopes that may be successfully applied to cells other than epithelia.     

Renal vectorial transport of berberine mediated by organic cation transporter 2 (OCT2) and multidrug and toxin extrusion proteins 1 (MATE1) in rats

Berberine, a well‐known plant alkaloid derived from Rhizoma coptidis, has potential applications as a therapeutic drug for diabetic nephropathy. However, the transporter‐mediated renal transport of berberine remains largely unclear. This study aimed to investigate the renal transport mechanism of berberine using transfected cells, kidney slices and animal experiments. In Madin‐Darby canine kidney (MDCK) cells stably expressing rat OCT2 (MDCK‐rOCT2) and kidney slices, saturable and non‐saturable uptake of berberine was observed, and corticosterone could inhibit the uptake of berberine, with IC₅₀ values of 0.1 μm and 147.9 μm , respectively. In double‐transfected cells, the cellular accumulation of berberine into MDCK‐rOCT2 and MDCK‐rOCT2‐rMATE1 (MDCK cells stably expressing rOCT2 and rMATE1) cells was significantly higher than the uptake into MDCK cells. Meanwhile, berberine transcellular transport was considerably higher in double‐transfected MDCK‐rOCT2‐rMATE1 cells than in MDCK and MDCK‐rOCT2 cells. Corticosterone for MDCK‐rMATE1 and MDCK‐MDR1 and pyrimethamine for MDCK‐rMATE1 at high concentrations could inhibit the efflux of berberine. In animal experiments, compared with the berberine alone group, the cumulative urinary excretion of berberine significantly decreased in the corticosterone or pyrimethamine pretreatment groups. In the rat kidney, pyrimethamine increased, and a low dose of corticosterone (5 mg/kg) decreased, the berberine concentration. However, there was no apparent change in the renal concentration of berberine in rats pretreated with corticosterone (10 or 20 mg/kg). Thus, berberine is not only a substrate of OCT2 and P‐glycoprotein, but is also a substrate of MATE1. Both OCT2 and MATE1 mediate the renal vectorial transport of berberine.     

Slc39a14 gene encodes ZIP14, a metal/bicarbonate symporter: similarities to the ZIP8 transporter

The mouse and human genomes contain 14 highly conserved SLC39 genes. Viewed from an evolutionary perspective, SLC39A14 and SLC39A8 are the most closely related, each having three noncoding exons 1. However, SLC39A14 has two exons 4, giving rise to Zrt- and Irt-related protein (ZIP)ZIP14A and ZIP14B alternatively spliced products. C57BL/6J mouse ZIP14A expression is highest in liver, duodenum, kidney, and testis; ZIP14B expression is highest in liver, duodenum, brain, and testis; and ZIP8 is highest in lung, testis, and kidney. We studied ZIP14 stably retroviral-infected mouse fetal fibroblast cultures and transiently transfected Madin-Darby canine kidney (MDCK) polarized epithelial cells. Our findings include: 1) ZIP14-mediated cadmium uptake is proportional to cell toxicity, but manganese is not; 2) ZIP14B has a higher affinity than ZIP14A toward Cd(2+) (K(m) = 0.14 versus 1.1 microM) and Mn(2+) uptake (K(m) = 4.4 versus 18.2 microM); 3) ZIP14A- and ZIP14B-mediated Cd(2+) uptake is most inhibited by Zn(2+), and next by Mn(2+) and Cu(2+); 4) like ZIP8, ZIP14A- and ZIP14B-mediated Cd(2+) uptake is dependent on extracellular HCO(3)(-); 5) like ZIP8, ZIP14 transporters are localized on the apical surface of MDCK-ZIP cells; and 6) like ZIP8, ZIP14 proteins are glycosylated. Tissues such as intestine and liver, located between the environment and the animal, show high levels of ZIP14; given the high affinity for ZIP14, Cd(2+) is likely to act as a rogue hitchhiker-displacing Zn(2+) or Mn(2+) and entering the body to cause unwanted cell damage and disease.     

Mobilization of intracellular calcium in kidney epithelial cells is inhibited by lead

The effect of lead (Pb) on intracellular calcium (Cai) after stimulation with agonists was studied in Madin-Darby canine kidney (MDCK) cells. In response to the agonist ADP, the levels of Cai increased by approximately threefold in MDCK cells bathed in a buffer with calcium (Ca) or in a buffer with nominal Ca. Pb inhibited the response to ADP in MDCK cells bathed in either buffer. The inhibition by Pb was observed after a 5 and 20-min exposure to Pb, but not after 2-min. Very high concentrations of ADP did not reverse the effects of Pb. Concentrations of Pb of 1 microM or more inhibited the response to ADP. Similarly, the response to bradykinin was also inhibited by Pb. Protein kinase C did not play a role since the protein kinase C inhibitor GF 109203X did not reverse the effects of Pb. Interestingly, MDCK cells treated with Pb at concentrations above 1 microM, for periods of 5-20 min, displayed elevated levels of inositol 1,4,5-trisphosphate. In conclusion, Pb inhibits mobilization of Cai after agonist stimulation by a mechanism that is unrelated to the type of agonist used. Evidence is presented suggesting that the inhibition is due to increases in levels of inositol 1,4,5-trisphosphate, which possibly decreases the amount of Cai available for mobilization.     

Role of the sodium-dependent phosphate co-transporters and of the phosphate complexes of uranyl in the cytotoxicity of uranium in LLC-PK1 cells

Although uranium is a well-characterized nephrotoxic agent, very little is known at the cellular and molecular level about the mechanisms underlying the uptake and toxicity of this element in proximal tubule cells. The aim of this study was thus to characterize the species of uranium that are responsible for its cytotoxicity and define the mechanism which is involved in the uptake of the cytotoxic fraction of uranium using two cell lines derived from kidney proximal (LLC-PK(1)) and distal (MDCK) tubule as in vitro models. Treatment of LLC-PK(1) cells with colchicine, cytochalasin D, concanavalin A and PMA increased the sodium-dependent phosphate co-transport and the cytotoxicity of uranium. On the contrary, replacement of the extra-cellular sodium with N-methyl-D-glucamine highly reduced the transport of phosphate and the cytotoxic effect of uranium. Uranium cytotoxicity was also dependent upon the extra-cellular concentration of phosphate and decreased in a concentration-dependent manner by 0.1-10 mM phosphonoformic acid, a competitive inhibitor of phosphate uptake. Consistent with these observations, over-expression of the rat proximal tubule sodium-dependent phosphate co-transporter NaPi-IIa in stably transfected MDCK cells significantly increased the cytotoxicity of uranium, and computer modeling of uranium speciation showed that uranium cytotoxicity was directly dependent on the presence of the phosphate complexes of uranyl UO(2)(PO(4))(-) and UO(2)(HPO(4))(aq). Taken together, these data suggest that the cytotoxic fraction of uranium is a phosphate complex of uranyl whose uptake is mediated by a sodium-dependent phosphate co-transporter system.     

Possible Role of Anion Exchanger AE2 as the Intestinal Monocarboxylic Acid/Anion Antiporter

Purpose. The purpose of the present study was to investigate the transport of organic monocarboxylic acids mediated by the anion exchanger AE2, which has been already reported to be present at several tissue cell membranes, including intestinal brush border membrane in rabbit. Methods. Membrane transport of organic monocarboxylic acids by AE2 was investigated by transient AE2-gene expression in HEK 293 cells and subsequent uptake studies by the cells. Results. Functional transfection of AE2 was confirmed by the enhanced ³⁶C1^– efflux from the cells. When preloaded with chloride anion, AE2-transfected cells demonstrated a significantly enhanced [¹⁴C]benzoic acid transport activity compared with mock-transfected cells. The AE2-mediated uptake was saturable with kinetic parameters of Km = 0.26 ± 0.08 mM and Vmax = 6.14 ± 0.52 nmol/mg protein/ 3 min, and the uptake of [¹⁴C]benzoic acid was pH-dependent with a maximal uptake at pH 6.5. AE2-mediated [¹⁴C]benzoic acid uptake was inhibited by Cl^–, HCO₃ ^–, and DIDS. AE2-transfected cells demonstrated significantly enhanced transport activity for nicotinic acid, propionic acid, butyric acid, and valproic acid as well as benzoic acid compared with mock-transfected cells. Conclusions. AE2 is functionally involved in the anion antiport for organic monocarboxylic acids as well as inorganic anions and is supposed to play a partial role in the intestinal transport of organic acids.     

Metformin transport by a newly cloned proton-stimulated organic cation transporter (plasma membrane monoamine transporter) expressed in human intestine.

Metformin is a widely used oral antihyperglycemic drug for the treatment of type II diabetes mellitus. The intestinal absorption of metformin is dose-dependent and involves an active, saturable uptake process. Metformin has been shown to be transported by the human organic cation transporters 1 and 2 (hOCT1-2). We recently cloned and characterized a novel proton-activated organic cation transporter, plasma membrane monoamine transporter (PMAT). We previously showed that PMAT transports many classic organic cations (e.g., monoamine neurotransmitters, 1-methyl-4-phenylpyridinium) in a pH-dependent manner and its mRNA is expressed in multiple human tissues. The goal of this study is to investigate whether metformin is a substrate of PMAT and whether PMAT plays a role in the intestinal uptake of metformin. Using Madin-Darby canine kidney cells stably expressing human PMAT, we showed that metformin is avidly transported by PMAT, with an apparent affinity (K(m) = 1.32 mM) comparable to those reported for hOCT1-2. Interestingly, the concentration-velocity profile of PMAT-mediated metformin uptake is sigmoidal, with a Hill coefficient of 2.64. PMAT-mediated metformin transport is greatly stimulated by acidic pH, with the uptake rate being approximately 4-fold higher at pH 6.6 than at pH 7.4. Using a polyclonal antibody against PMAT, we showed that the PMAT protein (58 kDa) was expressed in human small intestine and concentrated on the tips of the mucosal epithelial layer. Taken together, our results suggest that PMAT transports metformin, is expressed in human intestine, and may play a role in the intestinal absorption of metformin and possibly other cationic drugs.     

Multidrug resistance-associated protein 2 (MRP2) enhances 4-hydroxynonenal-induced toxicity in Madin-Darby canine kidney II cells

4-Hydroxy-trans-2,3-nonenal (HNE) is a toxic end product of lipid peroxidation. This multifunctional aldehyde reacts with proteins, phospholipids, and nucleic acids, consequently activating/inactivating enzymes, affecting signal transduction and gene expression. HNE is mainly detoxified by glutathione (GSH) conjugation. In our previous report, we showed that GSH conjugates of 4-hydroxynonenal (HNE-SG) are substrates of multidrug resistance-associated protein 2 (MRP2). MRP2 has been shown to export HNE-SG conjugates into the extracellular space. In the present study, the role of MRP2 in the detoxification of HNE was studied using Madin-Darby canine kidney II (MDCK II) cells expressing human MRP2. MRP2 reduced the intracellular accumulation of HNE-SG conjugate but unexpectedly increased the susceptibility of cells to HNE. The viability of cells was reduced to approximately 70% in the presence of 62.5 microM HNE in MDCK II cells expressing MRP2, whereas MDCK II cells remained unaffected. MRP2 accelerated the elimination of intracellular GSH via a conjugation reaction with HNE (half-life of GSH was 30.1 and 12.2 min for MDCK II cells and MDCK II cells expressing MRP2, respectively). Moreover, the consumption of GSH was unlimited in MDCK II cells expressing MRP2, finally resulting in necrosis. These results indicate that MRP2 has an adverse effect during the detoxification of HNE in MDCK II cells and suggest that expression of MRP2 may enhance the damage caused by oxidative stress.     

Agonist-dependent potentiation of vanilloid receptor transient receptor potential vanilloid type 1 function by stilbene derivatives

Transient receptor potential vanilloid type 1 (TRPV1) is a nonselective cation channel activated by capsaicin, low pH, and noxious heat and plays a key role in nociception. Understanding mechanisms for functional modulation of TRPV1 has important implications. One characteristic of TRPV1 is that channel activity induced by either capsaicin or other activators can be sensitized or modulated by factors involving different cell signaling mechanisms. In this study, we describe a novel mechanism for the modulation of TRPV1 function: TRPV1 function is modulated by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and its analogs. We found that, in rat dorsal root ganglion neurons, although DIDS did not induce the activation of TRPV1 per se but drastically increased the TRPV1 currents induced by either capsaicin or low pH. DIDS also blocked the tachyphylaxis of the low pH-induced TRPV1 currents. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), a DIDS analog, failed to enhance the capsaicin-evoked TRPV1 current but increased the low pH-evoked TRPV1 currents, with an effect comparable with that of DIDS. SITS also blocked the low pH-induced tachyphylaxis. DIDS also potentiated the currents of TRPV1 channels expressed in human embryonic kidney 293 cells, with an effect of left-shifting the concentration-response curve of the capsaicin-induced TRPV1 currents. This study demonstrates that DIDS and SITS, traditionally used chloride channel blockers, can modify TRPV1 channel function in an agonist-dependent manner. The results provide new input for understanding TRPV1 modulation and developing new modulators of TRPV1 function.     

Transport of dietary phenethyl isothiocyanate is mediated by multidrug resistance protein 2 but not P-glycoprotein

We demonstrated recently that phenethyl isothiocyanate (PEITC), a potent anticarcinogen present in cruciferous vegetables, inhibited P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) and that MRP1 can transport PEITC and/or its metabolites. In this study, we have examined whether PEITC is transported by P-gp and MRP2, two transporters with high expression in human intestine, liver and kidney. Using (14)C-PEITC, no significant difference was observed for the intracellular accumulation of PEITC in human breast cancer MCF-7/sensitive (control) and MCF-7/ADR (P-gp overexpressing) cells at PEITC concentrations of 1, 10 and 50 microM. Moreover, the presence of verapamil or PSC833, two P-gp inhibitors, had no significant effect on the intracellular accumulation of PEITC in P-gp overexpressing MCF-7/ADR and MDA435/LCC6MDR1 cells, indicating that PEITC may not be a substrate for P-gp. In contrast, (14)C-PEITC intracellular accumulation in the kidney epithelial MDCK II/MRP2 cells (transfected with human MRP2) was significantly lower than in the wild-type MDCK II/wt cells at PEITC concentrations of 1, 5, 10 and 50 microM. The presence of MK571, an MRP inhibitor, significantly enhanced (14)C-PEITC accumulation in MDCK II/MRP2 but not MDCK II/wt cells. Furthermore, depletion of intracellular glutathione (GSH) following treatment with buthionine sulphoximine, an inhibitor of GSH biosynthesis, significantly increased (14)C-PEITC intracellular accumulation in a concentration-dependent manner. Transcellular transport studies also demonstrated that depletion of intracellular GSH reduced the mean ratio of basal-to-apical transport to apical-to-basal transport of PEITC in MDCK II/MRP2, but not MDCK II/wt cell monolayers. These results indicate that GSH plays an important role in the MRP2-mediated transport of PEITC. The findings provide new information concerning the interactions between PEITC and membrane transporters and suggest the possibility of PEITC interactions with xenobiotics that are MRP2 substrates.     

Involvement of Rat Organic Cation Transporter 2 in the Renal Uptake of Jatrorrhizine

Jatrorrhizine is a protoberberine alkaloid derived from Coptis chinensis concentrated extremely in rat kidney. In the present study, the involvement of rat organic cation transporter 2 (rOCT2) in the renal uptake of jatrorrhizine in rat was investigated through in vitro and in vivo experiments. Saturable and nonsaturable uptakes of jatrorrhizine were observed in rat kidney slices and rOCT2–Madin–Darby canine kidney (MDCK) cells. Michaelis–Menten constants of 677.8 and 21.0 μM, maximum uptake rate of 123 (pmol/min)/mg kidney and 13.7 (pmol/min)/mg protein, and nonsaturable uptake clearance of 0.054 (μL/min)/mg kidney and 0.032 (μL/min)/mg protein were observed in rat kidney slices and rOCT2–MDCK cells, respectively. As inhibitors of rOCT2, corticosterone, verapamil, and cimetidine can inhibit jatrorrhizine uptake in rat kidney slices and rOCT2–MDCK cells. Their median inhibitory concentration in rat kidney slices was 7, 78, and 538 μM, whereas that in rOCT2–MDCK cells was 1.07, 86.5, and 151.8 μM. Coadministration with 20 mg/kg corticosterone, a selective inhibitor of rOCT2, reduced the jatrorrhizine concentration in the cortex and medulla in the in vivo experiment. Thus, rOCT2 is mainly responsible for the renal uptake of jatrorrhizine in rat and in the regulation of jatrorrhizine concentration in the kidney.     

The endoplasmic reticulum stress response is involved in apoptosis induced by aloe-emodin in HK-2 cells

Aloe-emodin (AE; 1,8-dihydroxy-3-hydroxymethyl-9,10-anthracenedione) is one of the primary active compounds in total rhubarb anthraquinones (TRAs), which induce nephrotoxicity in rats. However, it is still not known whether AE has a similar effect on human kidney cells. In this study, 3-(4,5,-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that AE decreases the viability of HK-2 cells (a human proximal tubular epithelial cell line) in a dose- and time-dependent manner. AE induced G2/M arrest of cell cycle in HK-2 cells, which was detected with propidium iodide (PI) staining. This apoptosis was further investigated by Hoechst staining, transmission electron microscopy (TEM), DNA fragmentation, and Annexin V/PI staining. Apoptosis of the cells was associated with caspase 3 activation, which was detected by Western blot analysis and a caspase activity assay. In addition, changes in the endoplasmic reticulum (ER) ultrastructure as observed by TEM showed the effects of AE on ER. Treatment with AE also resulted in an increase in eukaryotic initiation factor-2α (eIF-2α) phosphorylation, X-box binding protein 1 (XBP1) mRNA splicing, c-Jun N-terminal kinase (JNK) phosphorylation, glucose-regulated protein (GRP) 78 and CAAT/enhancer-binding protein-homologous protein (CHOP) accumulation. These results suggest that AE induces ER stress in HK-2 cells, which is involved in AE-induced apoptosis. In conclusion, AE induces apoptosis in HK-2 cells, and the ER stress is involved in AE-induced apoptosis in vitro. The implications of the toxic effects of AE for clinical use are unclear and these findings should be taken into account in the risk assessment for human exposure.