Human organic cation transporter (OCT1 and OCT2) gene polymorphisms and therapeutic effects of metformin

Organic cation transporters (OCTs) are responsible for the hepatic and renal transport of metformin. In this study we analyzed variants of OCT1 and OCT2 genes in 33 patients (24 responders and nine non-responders) based on the hypothesis that polymorphisms in both genes contribute to large inter-patient variability in the clinical efficacy of metformin. The sequences of the 5′-flanking and coding regions of the two genes of interest were screened by single-strand conformation polymorphism (SSCP) analysis. To compare the causative factors between responders and non-responders, we performed stepwise discriminant functional analysis. Age, body mass index (BMI) and treatment with lipid-lowering agents were demonstrated as positive predictors, and two mutations in the OCT1 gene, −43T > G in intron 1 and 408Met > Val (1222A > G) in exon 7, were negative and positive predictors, respectively, for the efficacy of metformin; the predictive accuracy was 55.5% (P < 0.05). Subsequent study indicated that OCT1 mRNA levels tended to be lower in human livers with the 408Met (1222A) variant, though the differences did not reach the level of significance. In this study it is suggested that OCT1 and OCT2 gene polymorphisms have little contribution to the clinical efficacy of metformin.     

Mouse organic cation transporter 1 determines properties and regulation of basolateral organic cation transport in renal proximal tubules

The proximal tubule of mouse kidney expresses mouse organic cation transporter 1 (mOCT1), mOCT2, and much less mOCT3. Therefore, mOCT-mediated transport across the basolateral membrane of proximal tubules reflects properties of at least mOCT1 and mOCT2. Here, we unraveled substrate affinities and modulation of transport activity by acute regulation by protein kinases on mOCT1 and mOCT2 separately and compared these findings with those from isolated proximal tubules of male and female mOCT2^?/? mice. These data are also compared to our recent reports on isolated tubules from wild-type and mOCT1/2 double knockout (mOCT1/2^?/?) mice. OCT-mediated transport in proximal tubules of mOCT2^?/? mice was only 20 % lower compared to those isolated from wild-type mice. While mOCT1 was regulated by all five pathways examined [protein kinase A (PKA), protein kinase C (PKC), p56^lck, phosphoinositide 3-kinase (PI3K), and calmodulin (CaM)], mOCT2 activity was modulated by PKA, p56^lck, and CaM only, however, in the same direction. As mOCT-mediated transport across the basolateral membrane of mOCT2^?/? mice expressing only mOCT1 and to a small amount mOCT3 was identical to that observed for tubules isolated from wild-type mice and to that observed for human embryonic kidney 293 (HEK293) cells stably expressing mOCT1, mOCT1 represents the relevant paralog for OCT-dependent organic cation transport in the mouse kidney. Gender does not play a major role in expression and activity of renal OCT-mediated transport in the mouse. Properties of mouse OCT considerably differ from those of rat or human origin, and thus, observations made in these rodents cannot directly be transferred to the human situation.     

Selective regulation of cardiac organic cation transporter novel type 2 (OCTN2) in dilated cardiomyopathy

Organic cation transporters (OCT1-3 and OCTN1/2) facilitate cardiac uptake of endogenous compounds and numerous drugs. Genetic variants of OCTN2, for example, reduce uptake of carnitine, leading to heart failure. Whether expression and function of OCTs and OCTNs are altered by disease has not been explored in detail. We therefore studied cardiac expression, heart failure–dependent regulation, and affinity to cardiovascular drugs of these transporters. Cardiac transporter mRNA levels were OCTN2>OCT3>OCTN1>OCT1 (OCT2 was not detected). Proteins were localized in vascular structures (OCT3/OCTN2/OCTN1) and cardiomyocytes (OCT1/OCTN1). Functional studies revealed a specific drug-interaction profile with pronounced inhibition of OCT1 function, for example, carvedilol [half maximal inhibitory concentration (IC₅₀), 1.4 μmol/L], diltiazem (IC₅₀, 1.7 μmol/L), or propafenone (IC₅₀, 1.0 μmol/L). With use of the cardiomyopathy model of coxsackievirus-infected mice, Octn2mRNA expression was significantly reduced (56% of controls, 8 days after infection). Accordingly, in endomyocardial biopsy specimens OCTN2 expression was significantly reduced in patients with dilated cardiomyopathy, whereas the expression of OCT1-3 and OCTN1 was not affected. For OCTN2 we observed a significant correlation between expression and left ventricular ejection fraction (r = 0.53, P < 0.0001) and the presence of cardiac CD3⁺ T cells (r = −0.45, P < 0.05), respectively. OCT1, OCT3, OCTN1, and OCTN2 are expressed in the human heart and interact with cardiovascular drugs. OCTN2 expression is selectively reduced in dilated cardiomyopathy patients and predicts the impairment of cardiac function.Cardiovascular diseases are the leading cause of death. Consequently, numerous drugs targeting the cardiovascular system are given to a large number of patients. Little is known about drug concentrations at their target sites, which can be modulated by local factors, among them drug efflux and uptake transporters.¹ In particular, with regard to tissues such as skeletal or cardiac muscle, knowledge about transporter expression as a prerequisite for intracellular drug concentrations is still limited. Yet, several transport proteins, among them members of the ATP-binding cassette proteins (ABC transporter), have been identified within the cardiovascular system.² The potential relevance of transport proteins in these structures is highlighted by a recent study on transporter expression in skeletal muscle cells, indicating an impact of uptake and efflux transporters on local statin concentrations and hence on statin-mediated myotoxicity.³ Although several publications address expression of efflux-mediating ABC transport proteins in the human heart, the mode of cardiac drug uptake is unclear. In this context, we attempted to demonstrate that the cardiac expression of two uptake transporters in humans and the expression of one of those, the carnitine transporter OCTN2, were affected by cardiac disease.^4,5In the present study, we investigated the cardiac expression of uptake transporters for organic cation transporters [OCT(N)s], namely, the OCT1-3 (SLC22A1-3) and OCTN1 and OCTN2 (SLC22A4 and 5). Recent studies indicate an important pharmacologic role of these transporters.⁶ For example, in a functional study of the ubiquitously expressed OCT3 in knockout mice, the distribution of the OCT standard substrate 1-methyl-4-phenylpyridinium (MPP⁺) was selectively altered in cardiac tissue.⁷ Furthermore, there is evidence of an interaction of the physiologically important carnitine transporters OCTN1 and OCTN2^8,9 with certain drugs, thereby acting as an uptake transporter or being inhibited in their physiologic function by these compounds.⁶ Such interactions may have consequences for systemic and cardiac carnitine homeostasis as already discussed for the valproic acid–induced carnitine depletion.¹⁰ On the other hand, these transporters may modify drug action itself by controlling local concentrations, which has recently been shown for OCTN1 and the hERG (KCNH2) channel blocker quinidine.¹¹Although the tissue-specific mRNA expression of these transporters has been investigated in general studies,¹² knowledge about their cardiac localization and disease-dependent regulation is still limited. In addition to drugs modifying these proteins on a functional level (eg, inhibiting l-carnitine uptake), changes in cardiac expression will result in an altered uptake of both endogenous substrates and drugs. Therefore, this study focused on disease-dependent expression of cardiac OCT(N)s and possible functional interactions with cardiovascular drugs. Taken together, we demonstrate selective regulation of OCTN2 by impaired cardiac function and studied the interaction profile of OCT(N)s and cardiovascular drugs.     

Properties and regulation of organic cation transport in freshly isolated mouse proximal tubules analyzed with a fluorescence reader-based method

The main elimination site of organic cations (OCs) is the renal proximal tubule (PT). OC transporters (OCT) accept endogenous and exogenous substances and xenobiotics. As transgenic mouse models are increasingly used in translational medicine, functional properties with special focus on regulation of OCT of isolated mouse PTs were studied with a new fluorescence reader-based method, which allows studying larger numbers of tubules per kidney. OC transport across the basolateral membrane of PTs from male mice was measured as initial uptake of the fluorescent dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP). A microtiter plate fluorescence reader was used to semi-automatically analyze OC transport in freshly isolated tubules. Relative mRNA expression of OCT1/OCT2/OCT3 in PTs was 1/0.3/0.01 and did not vary from S1 to S3 segments. ASP was transported by PTs with a K _m of 6 μM. It was inhibited by TEA, TPA, or cimetidine (IC₅₀ = 5, 19, or 53 μM, respectively). Angiotensin II stimulated ASP uptake (+63%), while stimulation of PKC reduced (−37%) OC transport. Inhibition of p56^lck tyrosine kinase (−60%), of PI3K (−36%), of Ca²⁺/calmodulin (−25%), or of PKA (−33%) reduced OC transport. In PTs from OCT1/2^−/− mice ASP uptake was reduced to ~20%. Using this fluorescence reader-based method, we report substrate specificities and a complex pattern of acute regulation of OC transport in isolated mouse PTs. Compared to isolated human PTs or rat and human OCT isoforms expressed in HEK293-cells, OC transport across the basolateral membrane of freshly isolated mouse PTs shows similarities but also specific differences.     

Inhibitory effect of selective cyclooxygenase-2 inhibitor etoricoxib on human organic anion transporter 3 (hOAT3)

It is well known that nonsteroidal anti-inflammatory drugs (NSAIDs) delay the elimination of methotrexate. One of the mechanisms is thought to be inhibition of methotrexate uptake via human organic anion transporter 3 (hOAT3, SLC22A8) in the renal proximal tubule by NSAIDs. In this study, we evaluated the inhibitory effects of selective cyclooxygenase-2 inhibitor etoricoxib on hOAT3 by uptake experiments using Xenopus laevis oocytes. The injection of hOAT3 cRNA stimulated the uptake of methotrexate into the oocytes, and its transport was inhibited by etoricoxib. Etoricoxib inhibited estrone sulfate uptake by hOAT3 dose dependently, and the 50% inhibitory concentration was estimated to be 9.8 μM. Eadie-Hofstee plot analysis showed that etoricoxib inhibited hOAT3 in a competitive manner. These findings show that etoricoxib has inhibitory effect on hOAT3, and that the potential is comparable to that of traditional NSAIDs.     

Glycerol-3-phosphate transporter of Escherichia coli: structure, function and regulation

Glycerol-3-phosphate (G3P) plays a major role in glycolysis and phospholipid biosynthesis in the cell. Escherichia coli uses a secondary membrane transporter protein, GlpT, to uptake G3P into the cytoplasm. The crystal structure of the protein was recently determined to 3.3 A resolution. The protein consists of an N- and a C-terminal domain, each formed by a compact bundle of six transmembrane alpha-helices. The substrate-translocation pore is found at the domain interface and faces the cytoplasm. At the closed end of the pore is the substrate binding site, which is formed by two arginine residues. In combination with biochemical data, the crystal structure suggests a single binding site, alternating access mechanism for substrate translocation, namely, the substrate bound at the N- and C-terminal domain interface is transported across the membrane via a rocker-switch type of movement of the domains. Furthermore, GlpT may serve as a structural and mechanistic paradigm for other secondary active membrane transporters.     

Lymphoid source and target of murine leukocyte adherence inhibition factor (LAIF)

The cellular source of murine LAIF detected by an indirect leukocyte adherence inhibition (LAI) assay using capillary tubes was investigated using non-induced peritoneal cells (PC) from normal and spindle-cell sarcoma-bearing A/J mice. Cell populations containing >95% T-cells, B-cells or macrophages were prepared from PC using a series of elimination and enrichment procedures. The in vitro incubation of enriched T-cell populations (<0.05% macrophages) from tumor-bearing mice with the specific soluble tumor antigen did not result in the release of detectable levels of LAIF; however, the addition of 10% normal isologous macrophages to the T-cells resulted in a significant release of LAIF, Enriched B-cell populations did not release LAIF either by themselves or when 10% normal isologous macrophages were added. Cell populations containing both T-cells and B-cells produced significant levels of LAIF, but only when suitable numbers of macrophages were present. Treatment with anti-Thy-1.2 alloantiserum and complement resulted in the abrogation of LAIF production by mixed cell populations. Using Lyt-1.2 and Lyt-2.2 alloantisera and complement to prepare either Lyt-1.2 or Lyt-2.2 depleted T-cell populations, it was found that the Lyt-1.2 subpopulation was responsible for the release of LAIF in this test system. LAIF was found to be effective in reducing the glass adherence of macrophages but not of T-cells or B-cells.     

Heteroduplex analysis of cloned rat endogenous replication-defective (30 S) retrovirus and Harvey murine sarcoma virus

DNA representing endogenous rat replication-defective (30 S) retrovirus has been cloned at the SacI sites of the EK2 vector λgtWes.λB. Heteroduplex analysis of this cloned 30 S DNA to cloned Harvey murine sarcoma virus (Ha-MuSV) DNA suggests that the region of Ha-MuSV which codes for the p21 “src” protein is not found in the rat endogenous replication-defective (30 S) virus and that the p21 gene represents a 1.1-kb (kilobase) substitution of genetic material in the 30 S genome prior to or during the formation of Ha-MuSV. Additionally, a small region (0.56 kb) of nonhomology, representing either a deletion or an insertion, has been found near the middle of the 30 S viral genome and a large region of nonhomology (1.02 kb), representing that portion of Ha-MuSV which is mouse in origin, has been observed at the 3′ end of the respective genomes. These results are in good agreement with the localization of the p21 gene as determined by restriction enzyme analysis and transfection studies of both 30 S and Harvey murine and sarcoma virus DNAs (Ellis et al., submitted for publication).     

长托宁治疗有机磷农药中毒的应用研究

目的探讨在急性有机磷农药中毒抢救治疗中,使用长托宁治疗急性有机磷农药中毒患者的疗效评价。方法65例急性有机磷农药中毒患者随机分为对照组(30例)和治疗组(35例)。结果治疗组应用长托宁、氯磷定联合治疗,对照组采用传统疗法。结论在急性有机磷农药中毒抢救治疗中,使用长托宁在较短时间内能明显缓解患者中毒症状,提高胆碱脂酶活性恢复,减少呼吸衰竭的发生。     

Molecular cloning, functional characterization and genomic organization of four alternatively spliced isoforms of the human organic cation transporter 1 (hOCT1/SLC22A1)

Ann. Hum. Genet . (1999), 63 , 473–482
Correction
The authors wish to add the following correction to this paper:
The genomic organization of the human organic cation transporter (hOCT1/SLC22A1) has recently been described by us to consist of 7 exons [Molecular cloning, functional characterization and genomic organization of four alternatively spliced isoforms of the human organic cation transporter 1( hOCT1 / SLC22A1 ); Ann. Hum. Genet . 63 : 473–482]. A reexamination revealed 11 exons instead of 7. The mistake occurred through cDNA contamination. The corrected gene structure of the hOCT1 gene is available at EMBL under the following accession numbers:
AJ243995 (Exon 1), AJ243996 (Exon 2), AJ276051 (Exon 3), AJ276052 (Exon 4), AJ276053 (Exon 5 and 6), AJ245460 (Exon 7), AJ243998 (Exon 8), AJ243999 (Exon 9 and 10) and AJ244000 (Exon 11).     

Molecular cloning, functional characterization and genomic organization of four alternatively spliced isoforms of the human organic cation transporter 1 (hOCT1/SLC22A1)

In this study we report the cloning of four human OCT1 ( hOCT1 / SLC22A1 ) isoforms: a long form, hOCT1G/L554, and three shorter forms (hOCT1G/L506, hOCT1G483 and hOCT1G353). All four variants could be identified in the human glioma cell line SK-MG-1, whereas only two isoforms (hOCT1G/L554 and hOCT1G/L506) were found in human liver cDNA. The hOCT1G/L554 represents the full length hOCT1 since the sequence of this clone is more than 99% identical to previously cloned hOCT1 cDNAs. Elucidation of the gene structure of human OCT1 demonstrated that the other isolated isoforms are alternatively spliced variants. The hOCT1 gene consists of 7 exons and 6 introns. When stably expressed in human embryonic kidney (HEK293) cells, only the full length hOCT1 cDNA mediated decynium-22 (D22)-sensitive uptake of tritiated 1-methyl-4-phenylpyridinium ([³H]-MPP⁺).     

The mitochondrial transporter family (SLC25): physiological and pathological implications

The mitochondrial carriers (MCs) shuttle a variety of metabolites across the inner mitochondrial membrane (i.m.m.). In man they are encoded by the SLC25 genes. Some MCs have isoforms encoded by different SLC25 genes, whereas the phosphate carrier has two variants arising from an alternative splicing of SLC25A3. Six MCs have been sequenced after purification, and many more have been identified from their transport and kinetic properties following heterologous over-expression and reconstitution into liposomes. All MCs of known function belong to the same protein family, since their polypeptide chains consist of three tandemly related sequences of about 100 amino acids, and the repeats of the different carriers are homologous. They probably function as homodimers, each monomer being folded in the membrane into six transmembrane segments. The functional information obtained in studies with mitochondria and/or the reconstituted system has helped to gain an insight into the physiological role of the MCs in cell metabolism, as have tissue distribution, the use of knock-out mice (and/or yeast) and over-expression in human cell lines (or yeast) of individual carriers and isoforms. At the same time, the cloning and functional identification of many SLC25 genes has made it possible (i) to identify the genes (and their defects) responsible for some diseases, e.g. Stanley syndrome and Amish microcephaly, and (ii) where the genes were already known, to characterize the function of the gene products and hence understand the molecular basis and the symptoms of the diseases, e.g. hyperornithinaemia, hyperammonaemia and homocitrullinuria (HHH) syndrome and type II citrullinemia. It is likely that further extension and functional characterization of the SLC25 gene family will elucidate other diseases caused by MC deficiency.Abbreviations AAC ADP/ATP carrier - AGC aspartate/glutamate carrier - ANC peroxisomal adenine nucleotide carrier - BKA bongkrekic acid - CAC carnitine/acylcarnitine carrier - CATR carboxyatractyloside - CoA coenzyme A - CIC citrate carrier - DIC dicarboxylate carrier - DNC deoxynucleotide carrier - GC glutamate carrier - GDC Graves disease carrier - i.m.m. inner mitochondrial membrane - MC mitochondrial carrier - MCF mitochondrial carrier family - MTSEA (2-aminoethyl)-methanethiosulphonate hydrobromide - OAA oxaloacetate - ODC oxodicarboxylate carrier - OGC oxoglutarate carrier - OMIM Online Mendelian Inheritance in Man (database) - ORC ornithine carrier - PEP phosphoenolpyruvate - PiC phosphate carrier - SLC25 name of the human mitochondrial solute carrier gene family, assigned by the Human Genome Organisation (HUGO) nomenclature committee - TMS transmembrane segment - UCP uncoupling protein This revised version was published in December 2003 because additional corrections were requested by the guest editor.