RESUMO
This study examined the effect of estrogen (17beta-estradiol) on renal handling of organic cation, tetraethylammonium (TEA), both in vivo and in vitro. Clearance of TEA in ovariectomized (OVX) mice was increased by 38% above intact animals, which was able to be returned to control level by estrogen supplementation. The mechanism of this effect was examined in isolated mouse renal proximal tubules (mRPT), showing that [(3)H]-TEA uptake was higher in OVX mice than control, and estrogen supplementation returned uptake to normal level. Kinetics analysis of [(3)H]-TEA uptake indicated an increase in numbers of organic cation transporters in OVX mice but no change in substrate affinity. However, mRNA levels determined by quantitative real time polymerase chain reaction of the relevant transporters at basolateral (organic cation transporter (OCT)1, OCT2 and OCT3) and apical (organic cation/carnitine transporter (OCTN)1, OCTN2 and multidrug and toxin extrusion (MATE)1) membranes of OVX mice were not significantly changed, with only MATE2 mRNA of OVX mice being significantly decreased. The realization that estrogen status affects renal clearance of organic cations will be of importance when assessing the susceptibility of an individual to drug-induced toxicity.
Assuntos
Estradiol/metabolismo , Estrogênios/metabolismo , Rim/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Bloqueadores dos Canais de Potássio/farmacocinética , Tetraetilamônio/farmacocinética , Animais , Transporte Biológico , Cátions/farmacocinética , Regulação para Baixo , Estradiol/farmacologia , Estrogênios/farmacologia , Feminino , Inativação Metabólica , Camundongos , Proteínas de Transporte de Cátions Orgânicos/genética , Ovariectomia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Increasing evidence suggests a key role of transport proteins in the pharmacokinetics of drugs. Within the solute carrier (SLC) family, various organic cation transporters (OCTs), organic anion transporters (OATs), and organic anion transporting polypeptides (OATPs) that interact with drug molecules have been identified. Traditionally, cellular uptake assays require multiple steps and provide low experimental throughput. We here demonstrate the use of a scintillation proximity approach to detect substrate uptake by human drug transporters in real time. HEK293 cells stably transfected with hOCT1, hOATP1B1, or hOAT3 were grown directly in Cytostar-T scintillating microplates. Confluent cell monolayers were incubated with 14C- or 3H-labeled transporter substrates. Cellular uptake brings the radioisotopes into proximity with the scintillation plate base. The resulting light emission signals were recorded on-line in a microplate scintillation counter. Results show time- and concentration-dependent uptake of 14C-tetraethylammonium, 3H-methylphenylpyridinium (HEK-hOCT1), 3H-estradiol-17beta-D-glucuronide (HEK-hOATP1B1), and 3H-estrone-3-sulfate (HEK-hOAT3), while no respective uptake was detected in empty vector-transfected cells. Km of 14C-tetraethylammonium and 3H-estrone-3-sulfate uptake and hOAT3 inhibition by ibuprofen and furosemide were similar to conventional dish uptake studies. The scintillation proximity approach is high throughput, amenable to automation and allows for identification of SLC transporter substrates and inhibitors in a convenient and reliable fashion, suggesting its broad applicability in drug discovery.
Assuntos
Fator 1 de Transcrição de Octâmero/fisiologia , Transportadores de Ânions Orgânicos Sódio-Independentes/fisiologia , Transportadores de Ânions Orgânicos/fisiologia , Contagem de Cintilação , Anti-Inflamatórios não Esteroides/farmacocinética , Anti-Inflamatórios não Esteroides/farmacologia , Transporte Biológico/efeitos dos fármacos , Isótopos de Carbono/farmacocinética , Linhagem Celular , Furosemida/farmacocinética , Furosemida/farmacologia , Glucuronídeos/farmacocinética , Humanos , Ibuprofeno/farmacocinética , Ibuprofeno/farmacologia , Cinética , Modelos Lineares , Transportador 1 de Ânion Orgânico Específico do Fígado , Fator 1 de Transcrição de Octâmero/genética , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Plasmídeos/genética , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacocinética , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Tetraetilamônio/farmacocinética , Transfecção , Trítio/farmacocinéticaRESUMO
Human organic cation transporter 2 (hOCT2) is essential for the renal tubular secretion of many toxic organic cations. Previously, of the cysteines (C437, C451, C470, and C474) that occur within transmembrane helices that comprise the hydrophilic cleft (proposed site of substrate binding), only C474 was accessible to maleimide-PEO(2)-biotin (hydrophilic thiol-reactive reagent), and covalent modification of this residue caused lower transport rates (Pelis RM, Zhang X, Dangprapai Y, Wright SH, J Biol Chem 281: 35272-35280, 2006). Thus it was hypothesized that the environmental contaminant Hg(2+) (as HgCl(2)) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [(3)H]tetraethylammonium (TEA) into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by HgCl(2), with an IC(50) of 3.9 +/- 0.11 microM. Treatment with 10 microM HgCl(2) caused a sixfold reduction in the maximal rate of TEA transport but did not alter the affinity of hOCT2 for TEA. To determine which cysteines interact with Hg(2+), a mutant with all four cleft cysteines converted to alanines (quadruple mutant), and four variants of this mutant, each with an individual cysteine restored, were created. The quadruple mutant was less sensitive to HgCl(2) than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the HgCl(2) effect on transport, MTSEA-biotin only interacted with C451 and C474. These data indicate that C451 and C474 of hOCT2 reside in the aqueous milieu of the cleft and that interaction of Hg(2+) with these residues causes reduced TEA transport activity.
Assuntos
Cisteína/efeitos dos fármacos , Cloreto de Mercúrio/farmacologia , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Biotina/análogos & derivados , Biotina/farmacologia , Células CHO , Cricetinae , Cricetulus , Cisteína/química , Humanos , Cinética , Proteínas de Transporte de Cátions Orgânicos/efeitos dos fármacos , Transportador 2 de Cátion Orgânico , Especificidade por Substrato , Tetraetilamônio/farmacocinéticaRESUMO
OCTN1 (SLC22A4) transports cationic compounds such as tetraethylammonium in a pH-sensitive and sodium-independent manner in cultured cells, and is expressed in wide variety of tissues, including kidney, muscle, placenta, heart, and others. This study focused on the clarification of its subcellular distribution in kidney and on its driving force to throw light on the pharmacological and physiological roles of OCTN1. Uptake of [14C]tetraethylammonium by membrane vesicles prepared from HEK293 cells stably transfected with human OCTN1 cDNA was osmolarity-sensitive, and the Km of tetraethylammonium was 1.28 mM at intravesicular and extravesicular pH values of 6.0 and 7.4, respectively. Tetraethylammonium uptake was pH-dependent, and overshoot uptake was observed in the presence of an outwardly directed proton gradient. A protonophore and membrane potential affected the overshoot uptake. Furthermore, preloading tetraethylammonium in the vesicles significantly increased the rate of uptake of [14C]tetraethylammonium. In mouse kidney, OCTN1 was expressed predominantly at the apical membrane of cortical proximal tubular epithelial cells. It was concluded that OCTN1 is involved in renal excretion of organic cations across the apical membrane in a pH-dependent, membrane potential-sensitive manner and is affected significantly by the organic cations on the trans side, showing counter transport activity.
Assuntos
Rim/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Tetraetilamônio/farmacocinética , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Sódio/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Membro 5 da Família 22 de Carreadores de Soluto , Frações Subcelulares/metabolismo , SimportadoresRESUMO
Recently, we cloned a human organic cation transporter, hOCT1, which is expressed primarily in the liver. hOCT1 plays an important role in the cellular uptake and elimination of various xenobiotics including therapeutically important drugs. HIV protease inhibitors are a new class of therapeutic agents. The purpose of this study was to elucidate the interactions of HIV protease inhibitors with hOCT1 and to determine whether hOCT1 is involved in the elimination of these compounds. We studied the interactions of HIV protease inhibitors with hOCT1 in a transiently transfected human cell line, HeLa. Uptake studies were carried out 40 h post-transfection using the radiolabeled model organic cation, [(14)C]tetraethylammonium (TEA), under different experimental conditions. In cis-inhibition studies, all of the HIV protease inhibitors tested, i.e., indinavir (IC(50) of 62 microM), nelfinavir (IC(50) of 22 microM), ritonavir (IC(50) of 5.2 microM), and saquinavir (IC(50) of 8.3 microM) inhibited TEA uptake in HeLa cells expressing hOCT1. However, none of the HIV protease inhibitors trans-stimulated [(14)C]TEA uptake, suggesting that they are poorly translocated by hOCT1. Nelfinavir, ritonavir, and saquinavir demonstrated an apparent "trans-inhibition" effect. No enhanced uptake of [(14)C]saquinavir was observed in hOCT1 DNA-transfected cells versus empty vector-transfected cells. These data suggest that HIV protease inhibitors are potent inhibitors, but poor substrates, of hOCT1. Some HIV protease inhibitors may potently inhibit the uptake and elimination of cationic drugs that are substrates for hOCT1, leading to potential drug-drug interactions. Other transporters, e.g., MDR1 and MRP1, in HIV-targeted cells may control the intracellular concentrations of HIV protease inhibitors.
Assuntos
Proteínas de Transporte/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Radioisótopos de Carbono , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Relação Dose-Resposta a Droga , Expressão Gênica , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Humanos , Indinavir/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Nelfinavir/farmacologia , Transportador 1 de Cátions Orgânicos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ritonavir/farmacologia , Saquinavir/farmacologia , Tetraetilamônio/farmacocinética , Células Tumorais CultivadasRESUMO
Renal secretion of organic cations and anions are pleiotropic, active processes in mammals. Some nucleosides such as deoxyadenosine (dAdo), 2-chlorodeoxyadenosine, and azidothymidine are secreted by human and rodent kidneys. Previous work (J. A. Nelson, J. F. Kuttesch, Jr., and B. H. Herbert. Biochemical Pharmacology 32: 2323-2327, 1983) indicated a role for the classic organic cation transporter (OCT) in the secretion of the dAdo analog, 2'-deoxytubercidin, by mouse kidney. Using [14C]tetraethylammonium bromide ([14C]TEA) as a substrate, we tested several renal cell lines for a nucleoside-sensitive OCT. American opossum kidney proximal tubule cells (OK) express a cimetidine-sensitive and metabolic-dependent ability to efflux TEA. Other classic OCT inhibitors and several nucleosides also inhibit TEA efflux by these cells in a manner reflecting structural specificity for the carrier. Inhibition of OCT by nucleosides is not a universal feature of OCTs, since TEA transport mediated by cloned rat kidney OCT2 in the Xenopus laevis oocyte system was not inhibited by the same nucleosides. In conclusion, OK cells appear to possess an OCT that may also transport some nucleosides by a novel carrier.
Assuntos
Proteínas de Transporte/metabolismo , Rim/metabolismo , Proteínas de Membrana/metabolismo , Nucleosídeos/farmacologia , Proteínas de Transporte de Cátions Orgânicos , Animais , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/genética , Linhagem Celular , Clonagem Molecular , Humanos , Rim/citologia , Células LLC-PK1 , Proteínas de Membrana/efeitos dos fármacos , Oócitos/metabolismo , Transportador 1 de Cátions Orgânicos , Transportador 2 de Cátion Orgânico , Suínos , Tetraetilamônio/antagonistas & inibidores , Tetraetilamônio/metabolismo , Tetraetilamônio/farmacocinética , Tubercidina/análogos & derivados , Tubercidina/farmacocinética , Xenopus laevisRESUMO
The polyspecific drug transporter OCT1 is a plasma transmembrane protein involved in the uptake of cationic drugs into hepatocytes. In order to determine whether hepatic OCT1 levels, like those of the other cationic drug transporter P-glycoprotein, may be altered during hepatocarcinogenesis, we have investigated OCT1 expression and activity in rat liver carcinoma cells. Similar levels of OCT1 mRNAs were evident in both normal liver and diethylnitrosamine-induced hepatocarcinomas by Northern blot analysis. In contrast, five hepatoma cell lines (Fao, Faza, H5, HTC and RHC1) showed either a decrease or an absence of OCT1 expression compared to normal hepatocytes; these hepatoma cells also displayed lower intracellular accumulation of tetraethylammonium (TEA), a well-known substrate for OCT1. However, among the hepatoma cell lines, the well-differentiated Fao cell line was found to retain substantial levels of OCT1 expression and of intracellular TEA uptake. Therefore, these data provide the first evidence that OCT1 expression is well-preserved in chemically-induced rat malignant neoplastic liver lesions, whereas it is either decreased or undetectable in hepatoma cell lines, which may be related to the loss of various liver functions usually occurring in these cell lines.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Transporte Biológico , Carcinógenos , Carcinoma Hepatocelular/induzido quimicamente , Dietilnitrosamina , Feminino , Neoplasias Hepáticas/induzido quimicamente , Masculino , Transportador 1 de Cátions Orgânicos , RNA Mensageiro/metabolismo , Ratos , Tetraetilamônio/farmacocinéticaRESUMO
Intestinal absorption and renal tubular secretion are transport processes determining the availability and the disposition of drugs in the body. In this review, our studies on the molecular and cell biological analyses of intestinal absorption and renal secretion of drugs are described. We evaluated the transepithelial transport and the cellular accumulation of peptide-like drugs such as beta-lactam antibiotics and bestatin (a dipeptide-like antineoplastic agent) in the human adenocarcinoma colon cell line, Caco-2, as an in vitro model for studying absorption mechanisms of these drugs. We have found that the transcellular transport of these peptide-like drugs is mediated by both the apically- and basolaterally-localized peptide transporters. To characterize molecular aspects of absorption of the peptide-like drugs, we studied cDNA cloning of H+/peptide cotransporters, PEPT1 and PEPT2, expressed in rats. The rat PEPT1 has been shown to mediate the H- coupled uphill transport of beta-lactam antibiotics across the brush-border membranes of the intestinal and renal epithelia. The rat PEPT2 is expressed predominantly in the kidney, but not in the intestine, mediating tubular reabsorption of the peptide-like drugs. We examined the transcellular transport of organic cations across monolayers of the kidney epithelial cell line, LLC-PK1. We have found that LLC-PK1 cells possess the H+/organic cation antiporter and the membrane potential-sensitive organic cation transporter in the apical and basolateral membranes, respectively, thereby tetraethylammonium (TEA) being transported unidirectionally from the basolateral to the apical side of the monolayers. We have isolated a cDNA encoding a rat kidney-specific organic cation transporter, OCT 2, which transports TEA in a H(+)-gradient independent manner, suggesting that OCT2 is localized to the basolateral membranes of renal tubular cells. In addition, a cDNA encoding a novel rat organic anion transporter, OAT-K1, has been cloned. OAT-K1 is expressed exclusively in the renal proximal tubules, and mediates the transport of methotrexate. Analyses of the molecular and cell biological mechanisms for drug absorption and secretion will provide information for the understanding of organ specific drug transport systems and for the development of drug design and/or drug delivery system.
Assuntos
Absorção Intestinal , Rim/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/farmacocinética , Transporte Biológico , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Clonagem Molecular , DNA , Humanos , Lactamas , Dados de Sequência Molecular , Ratos , Tetraetilamônio/farmacocinéticaRESUMO
Muscle potassium content and supplementation with potassium gluconate were evaluated in normokalemic cats with chronic renal failure (CRF). Affected cats received standard medical therapy for renal failure and either placebo (sodium gluconate) or potassium gluconate. At the beginning of the study and after 6 months of supplementation, glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were estimated using 3H-inulin and 14C-tetraethylammonium bromide (TEA) clearances. Muscle potassium content was determined in biopsy specimens using atomic absorption spectroscopy. Muscle biopsy samples obtained from cats with CRF before treatment had significantly lower muscle potassium content than did those from normal control cats. Over the 6-month period of supplementation, muscle potassium content increased both in cats with CRF that received potassium gluconate and in those that received placebo (sodium gluconate). Serum potassium concentration and fractional excretion of potassium remained relatively unchanged in both groups of cats throughout the treatment period. There were no significant differences in the percentage change in GFR and ERPF between treatment groups over the 6-month time period. Median values for pH, HCO3-, and total CO2 at 6 months were higher than baseline in the potassium gluconate group but lower than baseline in the sodium gluconate group.