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1.
Anal Biochem ; 366(2): 117-25, 2007 Jul 15.
Article in English | MEDLINE | ID: mdl-17555702

ABSTRACT

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.


Subject(s)
Octamer Transcription Factor-1/physiology , Organic Anion Transporters, Sodium-Independent/physiology , Organic Anion Transporters/physiology , Scintillation Counting , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Biological Transport/drug effects , Carbon Isotopes/pharmacokinetics , Cell Line , Furosemide/pharmacokinetics , Furosemide/pharmacology , Glucuronides/pharmacokinetics , Humans , Ibuprofen/pharmacokinetics , Ibuprofen/pharmacology , Kinetics , Linear Models , Liver-Specific Organic Anion Transporter 1 , Octamer Transcription Factor-1/genetics , Organic Anion Transporters/genetics , Organic Anion Transporters, Sodium-Independent/genetics , Plasmids/genetics , Sodium Potassium Chloride Symporter Inhibitors/pharmacokinetics , Sodium Potassium Chloride Symporter Inhibitors/pharmacology , Tetraethylammonium/pharmacokinetics , Transfection , Tritium/pharmacokinetics
3.
J Cell Sci ; 116(Pt 22): 4521-32, 2003 Nov 15.
Article in English | MEDLINE | ID: mdl-14576346

ABSTRACT

In the cell nucleus, precursors to mRNA, pre-mRNAs, associate with a large number of proteins and are processed to mRNA-protein complexes, mRNPs. The mRNPs are then exported to the cytoplasm and the mRNAs are translated into proteins. The mRNAs containing in-frame premature stop codons are recognized and degraded in the nonsense-mediated mRNA decay process. This mRNA surveillence may also occur in the nucleus and presumably involves components of the translation machinery. Several translation factors have been detected in the nucleus, but their functional relationship to the dynamic protein composition of pre-mRNPs and mRNPs in the nucleus is still unclear. Here, we have identified and characterized the translation initiation factor eIF4H in the dipteran Chironomus tentans. In the cytoplasm, Ct-eIF4H is associated with poly(A+) RNA in polysomes. We show that a minor fraction of Ct-eIF4H enters the nucleus. This fraction is independent on the level of transcription. CteIF4H could not be detected in gene-specific pre-mRNPs or mRNPs, nor in bulk mRNPs in the nucleus. Our immunoelectron microscopy data suggest that Ct-eIF4H associates with mRNP in the cytoplasmic perinuclear region, immediately as the mRNP exits from the nuclear pore complex.


Subject(s)
Cell Nucleus/metabolism , Chironomidae/metabolism , Cytoplasm/metabolism , Eukaryotic Initiation Factors/metabolism , RNA, Messenger/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Eukaryotic Initiation Factors/genetics , Female , Microscopy, Immunoelectron , Molecular Sequence Data , Nuclear Pore/metabolism , Nucleocytoplasmic Transport Proteins , Oocytes/metabolism , Polyribosomes/metabolism , Protein Biosynthesis , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , Sequence Homology, Amino Acid , Transcription, Genetic , Xenopus/metabolism
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