OCT1 mRNA Research Articles

Regulatory Role of Testosterone in Organic Cation Transport: in Vivo and in Vitro Studies

The renal proximal tubule (RPT) plays a crucial role in organic cation (OC) secretion and has a major impact on pharmacokinetics of OC drugs. Secretory transport is vectorial. Thus, it involves transporters located at both basolateral and apical membranes. Although sex hormones have been shown to regulate OC transport, there is little data on the effect of testosterone on OC secretion in a whole animal. Therefore, we determined the clearance of tetraethylammonium (TEA), a model OC substrate, in intact and castrated male mice. Castration significantly decreased renal TEA secretion by 30%, and testosterone supplementation returned TEA secretion to control levels in castrated mice. The mechanism of this effect was further examined in isolated mouse renal proximal tubules (mRPT). TEA uptake in isolated mRPT from castrated mice was reduced by 36%. This effect was reversed in tubules from castrated mice supplemented with testosterone. Kinetic analysis of [(3)H]-TEA uptake in isolated mRPT showed a decreased V(max) with no change in K(m), implying that the decrease in transport rate was caused by lowering in the number of transporters in castrated mice rather than a change in transporter affinity. Quantitative real time polymerase chain reaction (real time PCR) revealed that organic cation transporter (OCT)2 is the major TEA transporter in male mice. Moreover, OCT2 mRNA level was significantly reduced after castration. Castrated mice also showed a modest increase in organic cation/carnitine transporter 1 (OCTN1) mRNA level, indicating that testosterone may also regulate apical OCTN1 expression. These data suggest that testosterone regulates transepithelial transport of OC through modulation of OCT2 expression in male mice.

Enhancing the Functional Activity of the OCT-1 Influx Pump May Overcome the Negative Impact of Low OCT-1 Activity in Imatinib Treated CML Patients

The human organic cation transporter-1 (hOCT-1) is the major active influx protein responsible for the transport of imatinib into blood cells 1,2. The functional activity of the OCT-1 protein is defined as the intracellular uptake and retention (IUR) of 14-C labelled imatinib into patient pre therapy mononuclear blood cells over a two hour period, which is inhibited by OCT-1 inhibitors such as prazosin or procainamide. The level of OCT-1 activity is a key determinant of the interpatient variation observed in intrinsic sensitivity to imatinib induced kinase inhibition (IC50imatinib3). We have previously demonstrated that a significantly greater proportion of de-novo CML patients with high functional activity of OCT-1, achieve a major molecular response (MMR: 3 log reduction in BCR-ABL mRNA from standardised baseline) when treated with imatinib, than patients with low OCT-1 Activity 4. We have also identified a link between dose and OCT-1 Activity, demonstrating that the negative impact of low OCT-1 Activity could be overcome to a variable extent by imatinib dose increase. However, not all patients can dose increase, largely because of tolerability issues. While the transport of second-generation ABL-kinase inhibitors (nilotinib and dasatinib5) is not OCT-1 mediated, the long term effect of these drugs is not yet known. Hence, we sought to identify strategies to increase OCT-1-mediated imatinib uptake. We queried the drug gene expression signatures in version 1 of the Connectivity Map (CMAP; Lamb J, Nat. Rev. Cancer 7; 54–60, 2007: http://www.broad.mit.edu/cmap) with 3 transporters including OCT-1. This identified the Rho kinase inhibitor fasudil and COX-2 inhibitor / celecoxib analogue LM1685 as potential up-regulators of OCT-1 mRNA. The impact of these drugs on OCT-1 mRNA expression and IC50imatinib (fasudil alone to date) has been analysed in two bcr-abl positive cell lines (K562 and KU812). The effect of these two candidate OCT-1 enhancers on OCT-1-mediated imatinib uptake was also assessed in 10 newly diagnosed chronic phase CML patients, previously demonstrated to have low OCT-1 Activity (4 with no demonstrable OCT-1 Activity), using the IUR assay.Table 1: Assessing the effects of fasudil and LM1685 on the intracellular transport of imatinib. These data demonstrate a statistically significant increase in OCT-1 Activity with LM1685, and show a strong trend towards significance with fasudil. Importantly, we show that patients with no demonstrable OCT-1 Activity (0ng/200,000 cells) have detectable Activity in the presence of both fasudil (Range 1.5 to 2ng/200,000 cells) and LM1685 (Range 1.5 to 4.5 ng/200,000 cells). We have previously demonstrated that patients with no detectable OCT-1 Activity universally fail to achieve imatinib therapeutic response milestones (imatinib failure), whereas 54% of patients with low, but detectable OCT-1 Activity achieve these milestones4. The ability to enhance the functional activity of the OCT-1 protein may therefore be of significant clinical relevance in this group. In addition we demonstrate an increase in imatinib IUR which, along with the increase in OCT-1 Activity, is likely associated with increased OCT-1 mRNA levels. Significantly, in the two CML cell lines tested we show a marked reduction in the IC50imatinib indicating that the observed increase in IUR and OCT-1 Activity translates to an increase in the kinase inhibitory activity of imatinib. Preliminary analysis in one patient analysed to date also indicates a reduction in IC50 from 0.48 to 0.35μM in the presence of fasudil. In the clinical scenario the use of such OCT-1 enhancers may improve the response of some imatinib treated patients to both standard and increased dose imatinib. Importantly, these findings validate the use of resources such as C-MAP to identify candidate drugs that may mediate desired changes in the levels of key proteins resulting in improved response to therapy.Fasudil (10μM) %increase from controlLM1685 (1μM) % increase from controlIUR of imatinibOCT-1 ActivityIC50imatinibIUR of imatinibOCT-1 ActivityK56276% (n=5)163% (n=5)51% reduction (n=3)41% (n=2)122% (n=2)KU81210.7% (n=2)75% (n=2)15% reduction (n=2)NANACML patients n=108%89%9%114%p value>0.050.08>0.050.03

Renal xenobiotic transporters are differentially expressed in mice following cisplatin treatment

The goal of this study was to identify alterations in mRNA and protein expression of various xenobiotic transport proteins in mouse kidney during cisplatin-induced acute renal failure. For this purpose, male C57BL/6J mice received a single dose of cisplatin (18 mg/kg, i.p.) or vehicle. Four days later, tissues were collected for assessment of plasma BUN, histopathological analysis of renal lesions, and mRNA and Western blot analysis of renal transporters including organic anion and cation transporters (Oat, Oct), organic anion transporting polypeptides (Oatp), multidrug resistance-associated proteins (Mrp), multidrug resistance proteins (Mdr), breast cancer resistance protein (Bcrp) and multidrug and toxin extrusion proteins (Mate). Cisplatin treatment caused necrosis of renal proximal tubules along with elevated plasma BUN and renal kidney injury molecule-1 mRNA expression. Cisplatin-induced renal injury increased mRNA and protein levels of the efflux transporters Mrp2, Mrp4, Mrp5, Mdr1a and Mdr1b. Uptake transporters Oatp2a1 and Oatp2b1 mRNA were also up-regulated following cisplatin. By contrast, expression of Oat1, Oat2, Oct2 and Oatp1a1 mRNA was reduced in cisplatin-treated mice. Expression of several uptake and efflux transporters was unchanged in cisplatin-treated mice. Apical staining of Mrp2 and Mrp4 proteins was enhanced in proximal tubules from cisplatin-treated mice. Collectively, these expression patterns suggest coordinated regulation of uptake and efflux pathways during cisplatin-induced renal injury. Reduced expression of basolateral and apical uptake transporters along with enhanced transcription of export transporters likely represents an adaptation to lower intracellular accumulation of chemicals, prevent their reabsorption and enhance urinary clearance.

Analysis of regulatory polymorphisms in organic ion transporter genes (SLC22A) in the kidney

Organic cation transporters (OCTs) and organic anion transporters (OATs) (SLC22A family) play crucial roles in the renal secretion of various drugs. Messengar ribonucleic acid (mRNA) expression of transporters can be a key factor regulating interindividual differences in drug pharmacokinetics. However, the source of variations in mRNA levels of transporters is unclear. In this study, we focused on single nucleotide polymorphisms (SNP) in the promoter region [regulatory SNPs (rSNPs)] as candidates for the factor regulating mRNA levels of SLC22A. We sequenced the promoter regions of OCT2 and OAT1-4 in 63 patients and investigated the effects of the identified rSNPs on transcriptional activities and mRNA expression. In the OCT2 promoter region, one deletion polymorphism (-578_-576delAAG) was identified; -578_-576delAAG significantly reduced OCT2 promoter activity (p < 0.05), and carriers of -578_-576delAAG tend to have lower OCT2 mRNA levels, but the difference is not significant. There was no rSNP in the OAT1 and OAT2 genes. The five rSNPs of OAT3 and one rSNP of OAT4 were unlikely to influence mRNA expression and promoter activity. This is the first study to investigate the influences of rSNPs on mRNA expression of SLC22A in the kidney and to identify a regulatory polymorphism affecting OCT2 promoter activity.

Interaction between nicotine and MPTP/MPP + in rat brain endothelial cells

To examine the interaction between nicotine and MPTP/MPP + in the blood–brain barrier, cellular uptake of MPTP and MPP + was studied in the presence of nicotine and several compounds, including MPTP/MPP + analogs and a specific inhibitor of organic cation transporter (OCT) in an adult rat brain microvascular endothelial cell line (ARBEC). The kinetic properties of the uptake of MPTP, MPP +, and nicotine were also examined. In addition, a microdialysis study was performed to evaluate the in vivo effect of nicotine (i.p.) on extracellular levels of MPTP and MPP + in the brain after intravenous administration of MPTP. The results showed that uptake of MPTP, MPP +, and nicotine was partly mediated by a carrier system that was sensitive to decynium22, a specific OCT inhibitor. RT-PCR showed the presence of OCT1 mRNA in ARBEC. Capacity for uptake of MPTP and nicotine was much higher than that for MPP + ( K m and V m values of 10.94 ± 1.44 μM and 0.049 ± 0.007 pmol/mg s, respectively, for MPP +, compared to values of 35.75 ± 0.85 μM and 40.95 ± 3.56 pmol/mg s for MPTP and 25.29 ± 6.44 μM and 51.15 ± 14.18 pmol/mg s for nicotine). In addition, nicotine competitively inhibited the uptake of both MPTP and MPP +, with inhibition constants ( K i) of 328 μM and 210 μM, respectively. In vivo microdialysis results showed that nicotine significantly reduced brain extracellular levels of MPTP in the first 30min (507.4 ± 8.5 ng/ml vs. 637.9 ± 30.8 ng/ml with and without nicotine pre-treatment, respectively), but did not have significant effect on those of MPP +. In conclusion, nicotine can inhibit in vitro cellular uptake and in vivo transfer of MPTP across the blood–brain barrier, which can be mediated by multiple pathways including OCT1.

The organic cation transporters (OCT1, OCT2, EMT) and the plasma membrane monoamine transporter (PMAT) show differential distribution and cyclic expression pattern in human endometrium and early pregnancy decidua

The non-neuronal monoamine transporters (OCT1, OCT2, EMT, and PMAT) play a key role in the clearance of monoamines from extracellular compartments. In a previous report we described endometrial distribution and cyclic variation of the vesicular monoamine transporter (VMAT2) mRNA and the neuronal norepinephrine transporter (NET) mRNA. In the present study we used in situ hybridization, real-time PCR and immunohistochemistry to reveal tissue distribution and cyclic variation of mRNA for the non-neuronal monoamine transporters in the human endometrium and early pregnancy decidua. We found that non-neuronal monoamine transporters are predominantly expressed in the stroma. The plasma membrane monoamine transporter (PMAT) mRNA expression peaked in the proliferative phase, whereas the extra-neuronal monoamine transporter (EMT) mRNA expression peaked in the secretory phase. The organic cation transporter 2 (OCT2) mRNA expression was exclusively detected in few scattered stromal cells and OCT1 mRNA was not detected at all. Our present results demonstrate that PMAT, EMT, and OCT2 transporters are expressed in the endometrial stroma and can potentially regulate reuptake of monoamines in general and histamine in particular. Taken together with our previous finding of VMAT2 mRNA in epithelial cells, we suggest a paracrine interaction between stromal and epithelial cells, which may modulate certain steps of the reproductive process.

A novel role for Oct-2 in the lipopolysaccharide-mediated induction of resistin gene expression in RAW264.7 cells

Although resistin was first suggested as a possible link between obesity and diabetes, we have demonstrated previously that expression of resistin is induced by LPS (lipopolysaccharide). In the present study, we showed that LPS increased levels of resistin mRNA and promoter activity in murine RAW264.7 macrophages. Investigation of cis-regulatory elements in the mouse resistin promoter required for LPS-mediated induction showed that an Octamer (ATTTGCAT) element, located at -914 to -907, was required for maximal promoter activity in response to LPS stimulation. Co-transfection of RAW264.7 cells with a resistin promoter-luciferase construct and an Oct-1 or Oct-2 expression plasmid (pCG-Oct-1 or pCG-Oct-2) showed that Oct-2, but not Oct-1, activated the resistin promoter upon LPS treatment. Binding of Oct-2 to the Octamer element was demonstrated by supershift DNA-affinity precipitation and chromatin immunoprecipitation assays. Reverse transcription-PCR and Western blot results showed that levels of Oct-2 mRNA and protein were both up-regulated by LPS in RAW264.7 cells. The LPS-induced increase in Oct-2 protein was inhibited by LY294002 (a phosphoinositide 3-kinase inhibitor) post-transcriptionally, and the inhibition also resulted in a lower response of both resistin mRNA and promoter activity to LPS treatment. Moreover, specific knockdown of Oct-2 by RNA interference impaired the LPS-induced increase in resistin mRNA and promoter activity. Together, these results indicate that Oct-2 is involved in the LPS-mediated induction of resistin gene expression in macrophages and suggest that activation of Oct-2 is a part of LPS signalling pathways in macrophages.

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.

Molecular Response to Imatinib Is Dependent on Dose in CML Patients with Low OCT-1 Influx Activity. Patients with High Activity May Respond Equally Well to Standard or Increased Dose Imatinib.

We have shown that IC50 is predictive of molecular response in newly diagnosed CML patients treated with 600mg of imatinib (TIDEL). OCT-1 is an influx transporter for imatinib and key determinant of interpatient variation in the IC50 assay. Using 14C imatinib we have performed an intracellular uptake and retention (IUR) assay and RQ-PCR for OCT-1 mRNA expression on blood cells from 42 patients at diagnosis, enrolled to TIDEL. Using the OCT-1 inhibitor Prazosin, we determined OCT-1 in-vitro activity to be the difference in IUR between assays performed with and without Prazosin. The median OCT-1 activity was 6.1ng (range of -3.6 to 27ng). Grouping patients about the median, into low and high OCT-1activity, we found a significant difference between the two groups in molecular response.Log Reduction BCR-ABL based on OCT-1 ActivityMonths6M12M18M24MLow OCT-1 Activity n=191.92.42.62.5High OCT-1 Activity n=232.072.93.63.8p value0.050.050.0030.007Low OCT-1 Activity<600mg n=101.41.71.72.4600mg n=92.72.62.93.6p value0.010.0230.0460.042High OCT-1 Activity<600mg n=112.42.93.33.7600mg n=122.42.83.33.9p value0.630.690.820.66Dividing groups into patients receiving 600mg imatinib and those receiving <600mg over the first 12 months of therapy revealed patients with high OCT-1 activity generally achieved excellent molecular response regardless of dose, whereas the response of patients with low OCT-1 activity was highly dose dependent. There was a significant difference in molecular response between the low and high OCT-1 activity groups on <600 mg (p=0.004 at 24 months) but no difference in the 600mg cohort, further indicating dose sensitivity of low OCT-1 activity patients. The level of OCT-1 mRNA (n=30) as determined by RQ-PCR correlated with the in-vitro OCT-1 activity (r=0.381 p=0.04). However, in this smaller cohort, OCT-1 mRNA level was not predictive of molecular response (low OCT-1 3.5 log(n=15); high OCT-1 3.9 log(n=16) p=0.169). The IC50 is predictive of molecular response to 12 months (low IC50 log reduction 3.0 (n=20) high IC50 2.5 (n=15) p=0.02), but not in this smaller cohort to 24 months (p=0.15). TIDEL mandated dose increase to 800mg where tolerable. We assessed the log reduction from 12 to 24 months based on dose.Log reduction from 12 to 24 months.Dose increaseYesNop valueLow OCT-1 activity0.33 n=120 n=70.002High OCT-1 activity0.59 n=140.46 n=90.637p value0.7030.034There was no statistical difference between the low and high OCT-1 activity groups when dose was escalated, however with constant dose, patients with low OCT-1 activity performed less well (Table 2). We conclude that OCT-1 influx activity is an important determinant of molecular response to imatinib, but its predictive value is closely linked to the dose received. This functional assay of OCT-1 activity performed at diagnosis may identify patients most likely to benefit from higher doses of imatinib or second generation ABL kinase inhibitors, and a cohort who are likely to respond well to standard dose imatinib.

Lead Exposure: Expression and Activity Levels of Oct-2 in the Developing Rat Brain

Lead is a highly neurotoxic metal, and the developing central nervous system is particularly vulnerable to the effects of lead. In this study, transcription factors (TFs) that are altered due to lead exposure were identified using macroarray analysis. Rat pups were lactationally exposed to 0.2% lead acetate from birth through weaning. Changes in the developmental profiles of 30 TFs were screened in hippocampal tissue on postnatal day (PND) 5, 15, and 30. The temporal patterns of some TFs were transiently upregulated or repressed following lead exposure in a stage-specific manner; however, Oct-2, which is involved in the regulation of key developmental processes, exhibited sustained elevations during the entire period of study. Lead-induced elevation of Oct-2 was validated by reverse transcriptase-polymerase chain reaction analysis; however, significant elevation of Oct-2 mRNA expression was detected only on PND 5. The DNA-binding activity and protein levels of Oct-2 were further evaluated and found to be consistently induced on PND 5. The elevations observed in Oct-2 mRNA and protein levels as well as DNA-binding activity on PND 5 suggest that developmental maintenance of Oct-2 DNA binding could be impacted through de novo synthesis. These findings identify Oct-2 as a potential molecular target for Pb and suggest that Oct-2 may be associated with lead-induced disturbances in gene expression.

Corticosterone-Sensitive Monoamine Transport in the Rat Dorsomedial Hypothalamus: Potential Role for Organic Cation Transporter 3 in Stress-Induced Modulation of Monoaminergic Neurotransmission

Glucocorticoid hormones act within the brain to alter physiological and behavioral responses to stress-related stimuli. Previous studies indicated that acute stressors can increase serotonin [5-hydroxytryptamine (5-HT)] concentrations in the dorsomedial hypothalamus (DMH), a midline hypothalamic structure involved in the integration of physiological and behavioral responses to stress. The current study tests the hypothesis that rapid, stress-induced accumulation of 5-HT is attributable to the inhibition of 5-HT transport via organic cation transporters (OCTs). OCTs are a family of high-capacity, bidirectional, multispecific transporters of organic cations (including 5-HT, dopamine, and norepinephrine) only recently described in brain. In peripheral tissues, organic cation transport via some OCTs is inhibited by corticosterone. We examined the expression and function of OCTs in the periventricular medial hypothalamus of male Sprague Dawley rats using reverse-transcriptase (RT)-PCR, immunohistochemistry, and in vitro transport assays. RT-PCR revealed expression of OCT3 mRNA, but not OCT1 or OCT2 mRNA, in the medial hypothalamus. OCT3-like immunoreactivity was observed in ependymal and glial-like cells in the DMH. Acutely prepared minces of rat medial hypothalamic tissue accumulated the OCT substrates [3H]-histamine and [3H]-N-methyl-4-phenylpyridinium ([3H]-MPP+). Consistent with the pharmacological profile of OCT3, corticosterone, 5-HT, estradiol, and the OCT inhibitor decynium22 dose-dependently inhibited histamine accumulation. Corticosterone and decynium22 also inhibited efflux of [3H]-MPP+ from hypothalamic minces. These data support the hypothesis that corticosterone-induced inhibition of OCT3 mediates stress-induced accumulation of 5-HT in the DMH and suggest that corticosterone may acutely modulate physiological and behavioral responses to stressors by altering serotonergic neurotransmission in this brain region.

Differential pharmacological in vitro properties of organic cation transporters and regional distribution in rat brain

Organic cation transporters (OCTs) are polyspecific carriers implicated in low-affinity, corticosteroid-sensitive extraneuronal catecholamine uptake in peripheral tissues. The three main OCT subtypes, OCT1, OCT2 and OCT3, are also present in the brain, but their central role remains unclear. In the present study, we investigated by comparative in situ hybridization analysis the regional distribution of these transporters in rat brain and compared their functional properties in stably transfected HEK293 cells expressing human or rat OCTs. In rat brain, OCT2 and OCT3 mRNAs are expressed predominantly in regions located at the brain-cerebrospinal fluid border, with OCT3 mRNA expression extending to regions that belong to monoaminergic pathways such as raphe nuclei, striatum and thalamus. After normalization with MPP + uptake, OCT2 and OCT3 subtypes share a similar monoamine preference profile, with higher transport efficacies for epinephrine and histamine than for the other monoamines. Interestingly, a significant level of epinephrine transport, previously only shown for rOCT2, is achieved by most OCTs subtypes. Finally, another novel finding was that OCTs are sensitive to 3,4-methylenedioxymetamphetamine (MDMA), phencyclidine (PCP), MK-801 and ketamine. Altogether, all our results suggest a functional specialization of OCT subtypes, based both on their intrinsic properties and their differential regional expression pattern in the brain.

Expression of osteopontin mRNA and OCT2 mRNA in human gastric carcinoma by tissue microarray and its significance

Expression of osteopontin mRNA and OCT2 mRNA in human gastric carcinoma by tissue microarray and its significance

TISSUE DISTRIBUTION AND ONTOGENY OF ORGANIC CATION TRANSPORTERS IN MICE

Organic cation transporters (Octs) play an important role in transporting cationic xeno- and endobiotics across biological membranes. Little is known about Octs in mice; therefore, the tissue distribution and developmental changes in the mRNA expression of Octs in mice were quantified. Oct1, Oct2, Oct3, Octn1, Octn2, and Octn3 mRNA expression was quantified in 14 tissues from male and female mice using the branched DNA signal amplification assay. Oct1 mRNA expression was highest in kidney, followed by liver. Oct2 mRNA was almost exclusively expressed in kidney, with male mice having twice that in female mice. The higher expression of Oct2 in male mice is due to testosterone. Oct3 mRNA was most highly expressed in placenta, ovary, and uterus, but was expressed at low levels in most tissues. Octn1 and Octn2 mRNA expression was similar, with the highest levels in kidney followed by small intestine. Octn3 mRNA was almost exclusively expressed in testes. The developmental expression of Oct1, Oct2, Octn1, and Octn2 mRNA in kidneys as well as Oct1 in liver was determined in young mice. Ontogenic expression data indicate that each of the Octs approached adult expression levels by about 3 weeks of age. The gender difference in Oct mRNA expression did not become apparent until day 30 after birth. The differences in tissue distribution of the Octs may play an important role in drug disposition to various tissues. Furthermore, low expression of the Octs in young animals may affect the pharmacokinetic behavior of drugs compared with that in adults.

Alternative Promoters and Tissue-Specific Regulation of Mouse oct-1 Gene Transcription

The gene encoding mouse transcription factor Oct-1 contains two exons, 1U and 1L, at the 5′ end. Oct-1 mRNA from all tissues contains exon 1U, whereas exon 1L is found only in Oct-1 mRNA from mouse and human lymphoid cells. Upstream of 1U and 1L exons, the respective specific promoters, U and L, are located at a considerable distance from each other (67 kb in the mouse otf-1 locus). These regions differ in their structure. The region upstream from exon 1U contains numerous Sp1 sites, whereas the region upstream from exon 1L contains homeospecific NTAATNN sites and two octa sites ATGCAAAT recognized by transcription factors Oct-1, Oct-2, and by other POU domain-containing proteins. The octa and homeospecific sites promote autoregulation of the oct-1 gene. Transfection of U and L promoter fragments within pGL3 Basic plasmid or pGL3 Enhancer vector into lymphoid NS/0 myeloma cells or 10(1) fibroblasts showed that the L promoter activity was many times higher in the myeloma cells than in fibroblasts. Between the sites of translation and transcription initiation from the L promoter, a nucleotide sequence was identified whose elimination resulted in a significantly higher efficiency of transcription initiation from this promoter. Thus, the oct-1 gene contains at least two alternative promoters: the U promoter apparently provides for the constitutive synthesis of Oct-1 protein, whereas the L promoter manifests tissue specificity, contains octa sites, and appears to be self-regulated.

Molecular and functional characterization of an Na+‐independent choline transporter in rat astrocytes

In this study, we examined the molecular and functional characterization of choline uptake into cultured rat cortical astrocytes. Choline uptake into astrocytes showed little dependence on extracellular Na+. Na+-independent choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (Km) of 35.7 +/- 4.1 microm and a maximal velocity (Vmax) of 49.1 +/- 2.0 pmol/mg protein/min. Choline uptake was significantly decreased by acidification of the extracellular medium and by membrane depolarization. Na+-independent choline uptake was inhibited by unlabeled choline, acetylcholine and the choline analogue hemicholinium-3. The prototypical organic cation tetrahexylammonium (TEA), and other n-tetraalkylammonium compounds such as tetrabutylammonium (TBA) and tetrahexylammonium (THA), inhibited Na+-independent choline uptake, and their inhibitory potencies were in the order THA > TBA > TEA. Various organic cations, such as 1-methyl-4-tetrahydropyridinium (MPP+), clonidine, quinine, quinidine, guanidine, N-methylnicotinamide, cimetidine, desipramine, diphenhydramine and verapamil, also interacted with the Na+-independent choline transport system. Corticosterone and 17beta-estradiol, known inhibitors of organic cation transporter 3 (OCT3), did not cause any significant inhibition. However, decynium22, which inhibits OCTs, markedly inhibited Na+-independent choline uptake. RT-PCR demonstrated that astrocytes expressed low levels of OCT1, OCT2 and OCT3 mRNA, but the functional characteristics of choline uptake are very different from the known properties of these OCTs. The high-affinity Na+-dependent choline transporter, CHT1, is not expressed in astrocytes as evidenced by RT-PCR. Furthermore, mRNA for choline transporter-like protein 1 (CTL1), and its splice variants CTL1a and CTL1b, was expressed in rat astrocytes, and the inhibition of CTL1 expression by RNA interference completely inhibited Na+-independent choline uptake. We conclude that rat astrocytes express an intermediate-affinity Na+-independent choline transport system. This system seems to occur through a CTL1 and is responsible for the uptake of choline and organic cations in these cells.

Donepezil, tacrine and α-phenyl-n-tert-butyl nitrone (PBN) inhibit choline transport by conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB)

In the present study, we have characterized the choline transport system and examined the influence of various amine drugs on the choline transporter using a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB) in vitro. The cell-to-medium (C/M) ratio of [3H]choline in TR-BBB cells increased time-dependently. The initial uptake rate of [3H]choline was concentration-dependent with a Michaelis-Menten value, Km, of 26.2 +/- 2.7 microM. The [3H]choline uptake into TR-BBB was Na+-independent, but was membrane potential-dependent. The [3H]choline uptake was susceptible to inhibition by hemicholinium-3, and tetraethylammonium (TEA), which are organic cation transporter substrates. Also, the uptake of [3H]choline was competitively inhibited with Ki values of 274 microM, 251 microM and 180 microM in the presence of donepezil hydrochloride, tacrine and alpha-phenyl-n-tert-butyl nitrone (PBN), respectively. These characteristics of choline transport are consistent with those of the organic cation transporter (OCT). OCT2 mRNA was expressed in TR-BBB cells, while the expression of OCT3 or choline transporter (CHT) was not detected. Accordingly, these results suggest that OCT2 is a candidate for choline transport at the BBB and may influence the BBB permeability of amine drugs.

Role of Soybean Oil Pat Emulsion in the Prevention of Hepatic Xenobiotic Transporter mRNA Up- and Down-Regulation Induced by Overdose of Pat-free Total Parenteral Nutrition in Infant Rats

Total parenteral nutrition (TPN) is associated with cholestasis and hepatic steatosis in human infants. The present study focused on the changes in hepatic xenobiotic transporters associated with overdose of fat-free or fat-containing TPN in infant rats. Three-week-old male Sprague-Dawley rats were divided into three groups: group 1 received an oral diet, group 2 received TPN without fat, and group 3 received TPN with 20% of its calories from fat (soybean oil emulsion). After TPN administration for 4 days, both serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, which are indicators of hepatic dysfunction, in group 2 were significantly higher (p<0.001) than those in the other groups, whereas there were no differences between groups 1 and 3 in either serum AST or ALT levels. The serum bilirubin concentration in group 2 was also markedly higher than that in the other groups. Mdr2, Bsep, Mrp2, Mrp6, Oct1, and Oat2 mRNA levels were decreased in group 2 (fat-free TPN) compared with those in group 1 (oral diet), whereas Mdr1b, Mrp1, and Mrp5 mRNA levels were increased. Specifically, the level of Mdr1b mRNA in group 2 was 16 times higher (p<0.001) than that in group 1. On the other hand, the changes in these mRNA expression levels in group 3 (fat-containing TPN) were smaller than those in group 2, and specifically, the expression levels of Mdr1b, Mrp1, Mrp5, Mrp6, and Oat2 mRNA in group 3 were not significantly different from those in group 1. The results of the present study indicate that including fat in the TPN regimen is very important in preventing the mRNA up- and down-regulation of xenobiotic transporters, which is considered to be the main factor responsible for the abnormal hepatic changes such as cholestasis associated with the excessive administration of fat-free TPN.

An organic cation transporter capable of transporting serotonin is upregulated in serotonin transporter-deficient mice

The serotonin (5HT) transporter (5HTT) regulates serotonergic neurotransmission by mediating the reuptake of 5HT from the synaptic cleft. The organic cation transporter (OCT) gene family physiologically transports a wide spectrum of organic cations. Although lacking the high affinity and selectivity of the 5HTT, OCT1 and OCT3 mediate low-affinity 5HT transport and therefore may participate in the clearance of excessive 5HT. Since concentrations of extracellular 5HT are increased in the brain of 5HTT-deficient mice, we investigated the role of OCTs in 5HT system homeostasis. We analyzed OCT1 and OCT3 gene expression in the brain of 5HTT knockout mice by semi-quantitative competitive PCR and in situ hybridization. We demonstrate that in 5HTT-deficient mice OCT3 mRNA concentrations were significantly increased in the hippocampus. In contrast, OCT1 expression was unchanged. Upregulation of OCT3 expression and enhanced low-affinity 5HT uptake may limit the adverse effects of elevated extracellular 5HT and may play a critical role in maintaining 5HT-dependent functions of the hippocampus in the absence of 5HTT.

Down-regulation of the organic cation transporter 1 of rat liver in obstructive cholestasis.

The liver plays a major role in biotransformation and elimination of various therapeutic agents and xenobiotics, many of which are organic cations and substrates of the organic cation transporter 1 (Oct1, Slc22a1). Oct1 is expressed at the basolateral membranes of hepatocytes and proximal renal tubules. Although Oct1 is the major uptake mechanism in hepatocytes for many pharmaceutical compounds, little is known about the effects of liver injury on this process. Our aim was to investigate the effects of obstructive cholestasis on Oct1 expression and function in liver and kidney. The effects of bile duct ligation (BDL) on Oct1 protein, messenger RNA (mRNA) expression, and tissue localization were determined in rat liver and kidney with Western analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunofluorescence. To assess Oct1 function, the model substrate tetraethylammonium ([(14)C]TEA) was administered intravenously to BDL and control rats and distribution of radioactivity was determined. Oct1 protein significantly decreased in cholestatic livers to 42.1 +/- 17.7% (P <.001), 15.5 +/- 4.7% (P <.05), and 8.6 +/- 2.7% (P <.05) of controls after 3, 7, and 14 days, respectively, but not in kidneys. Hepatic Oct1 mRNA decreased to 77.2 +/- 12.7%, 40.7 +/- 8.1% (P <.05), and 50.3 +/- 7.5% (P <.05) 3, 7, and 14 days after BDL, respectively. Tissue immunofluorescence corroborated these data. Hepatic accumulation of [(14)C]TEA in 14-day BDL rats was reduced to 29.6 +/- 10.9% of controls (P <.0005). In conclusion, obstructive cholestasis down-regulates Oct1 and impairs Oct1-mediated uptake in rat liver, suggesting that hepatic uptake of small cationic drugs may be impaired in cholestatic liver injury.