Organic Cation/carnitine Transporters Research Articles

Urinary Excretion of Tetrodotoxin Modeled in a Porcine Renal Proximal Tubule Epithelial Cell Line, LLC-PK₁.

This study examined the urinary excretion of tetrodotoxin (TTX) modeled in a porcine renal proximal tubule epithelial cell line, LLC-PK1. Time course profiles of TTX excretion and reabsorption across the cell monolayers at 37 °C showed that the amount of TTX transported increased linearly for 60 min. However, at 4 °C, the amount of TTX transported was approximately 20% of the value at 37 °C. These results indicate that TTX transport is both a transcellular and carrier-mediated process. Using a transport inhibition assay in which cell monolayers were incubated with 50 µM TTX and 5 mM of a transport inhibitor at 37 °C for 30 min, urinary excretion was significantly reduced by probenecid, tetraethylammonium (TEA), l-carnitine, and cimetidine, slightly reduced by p-aminohippuric acid (PAH), and unaffected by 1-methyl-4-phenylpyridinium (MPP+), oxaliplatin, and cefalexin. Renal reabsorption was significantly reduced by PAH, but was unaffected by probenecid, TEA and l-carnitine. These findings indicate that TTX is primarily excreted by organic cation transporters (OCTs) and organic cation/carnitine transporters (OCTNs), partially transported by organic anion transporters (OATs) and multidrug resistance-associated proteins (MRPs), and negligibly transported by multidrug and toxic compound extrusion transporters (MATEs).

Multiple SLC and ABC Transporters Contribute to the Placental Transfer of Entecavir.

Entecavir (ETV), a nucleoside analog with high efficacy against hepatitis B virus, is recommended as a first-line antiviral drug for the treatment of chronic hepatitis B. However, scant information is available on the use of ETV in pregnancy. To better understand the safety of ETV in pregnant women, we aimed to demonstrate whether ETV could permeate placental barrier and the underlying mechanism. Our study showed that small amount of ETV could permeate across placenta in mice. ETV accumulation in activated or nonactivated BeWo cells (treated with or without forskolin) was sharply reduced in the presence of 100 µM of adenosine, cytidine, and in Na+ free medium, indicating that nucleoside transporters possibly mediate the uptake of ETV. Furthermore, ETV was proved to be a substrate of concentrative nucleoside transporter (CNT) 2 and CNT3, of organic cation transporter (OCT) 3, and of breast cancer resistance protein (BCRP) using transfected cells expressing respective transporters. The inhibition of ETV uptake in primary human trophoblast cells further confirmed that equilibrative nucleoside transporter (ENT) 1/2, CNT2/3, OCT3, and organic cation/carnitine transporter (OCTN) 2 might be involved in ETV transfer in human placenta. Therefore, ETV uptake from maternal circulation to trophoblast cells was possibly transported by CNT2/3, ENT1/2, and OCTN2, whereas ETV efflux from trophoblast cells to fetal circulation was mediated by OCT3, and efflux from trophoblast cells to maternal circulation might be mediated by BCRP, multidrug resistance-associated protein 2, and P-glycoprotein. The information obtained in the present study may provide a basis for the use of ETV in pregnancy.

Secondary Carnitine Deficiency in Dialysis Patients: Shall We Supplement It?

Carnitine, essential for fatty acid β-oxidation, is obtained from diet and through de novo biosynthesis. The organic cation/carnitine transporter 2 (OCTN2) facilitates carnitine cellular transport and kidney resorption. Carnitine depletion occurs in OCTN2-deficient patients, with serious clinical complications including cardiomyopathy, myopathy, and hypoketotic hypoglycaemia. Neonatal screening can detect OCTN2 deficiency. OCTN2-deficiency is also known as primary carnitine deficiency. Carnitine deficiency may result from fatty acid β-oxidation disorders, which are diagnosed via plasma acylcarnitine profiling, but also under other conditions including haemodialysis. Given the importance of the kidney in maintaining carnitine homeostasis, it is not unexpected that longterm haemodialysis treatment is associated with the development of secondary carnitine deficiency, characterised by low endogenous L-carnitine levels and accumulation of deleterious medium and long- chain acylcarnitines. These alterations in carnitine pool composition have been implicated in a number of dialysis-related disorders, including erythropoietin-resistant renal anaemia. The association between erythropoietin resistance and carnitine levels has been demonstrated, with the proportion of medium and long-chain acylcarnitines within the total plasma carnitine pool positively correlated with erythropoietin resistance. Recent research has demonstrated that carnitine supplementation results in a significant reduction in erythropoietin dose requirements in patients with erythropoietin-resistant anaemia. Few studies have been conducted assessing the treatment of carnitine deficiency and haemodialysisrelated cardiac complications, particularly in children. Thus, a study was recently conducted which showed that intravenous carnitine in children receiving haemodialysis significantly increased plasma carnitine.

Luteolin potentiates the sensitivity of colorectal cancer cell lines to oxaliplatin through the PPARγ/OCTN2 pathway.

Oxaliplatin is a chemotherapeutic agent used in the treatment of colorectal cancers. However, the mechanism controlling the cellular uptake and efflux of oxaliplatin is not completely understood. Organic cation/carnitine transporter 2 (OCTN2) is a member of the solute carrier superfamily and is a determinant of oxaliplatin uptake. OCTN2 is regulated by peroxisome proliferator-activated receptor γ (PPARγ) binding to the PPAR-response element within the first intron. Luteolin is a naturally occurring flavonoid and an agonist of PPARγ. Thus, we hypothesize that luteolin-mediated OCTN2 expression and activity potentiate the sensitivity of cancer cells to oxaliplatin. In this study, luteolin increased mRNA and protein expression of OCTN2 in a time-dependent and dose-dependent manner in colorectal cancer SW480 cells. This induction was attenuated by PPARγ antagonist GW9662 as well as by PPARγ knockdown, suggesting that the induction by luteolin is dependent on PPARγ. In uptake studies, luteolin increased the binding affinity of OCTN2 toward oxaliplatin and enhanced intracellular concentration of oxaliplatin. This finding is likely because of the increase of PDZ domain containing 1 (PDZK1) and PDZ domain containing 3 (PDZK2), which are known to facilitate the expression of OCTN2 on the cell surface and/or enhance transporter activity. Moreover, cell viability and cell apoptosis assays showed that luteolin increased oxaliplatin uptake and intracellular accumulation through OCTN2. Thus, our study showed that luteolin increased the sensitivity of colorectal cancer SW480 cells to oxaliplatin, likely through the PPARγ/OCTN2 pathway.

Different Involvement of Promoter Methylation in the Expression of Organic Cation/Carnitine Transporter 2 (OCTN2) in Cancer Cell Lines

Organic cation/carnitine transporter 2 (OCTN2) is responsible for the cellular uptake of the antineoplastic agent, oxaliplatin. Epigenetic modification is a possible mechanism of altered drug-transporter expression in cancers, leading to altered efficacy of chemotherapeutic drugs. However, the mechanisms governing OCTN2 regulation are not completely understood. In this study, the low levels of OCTN2 in HepG2 and LS174T cells were elevated by the demethylating reagent, decitabine (DCA). To further reveal the epigenetic mechanism of down-regulation of OCTN2, we found that Region-1 within the OCTN2 promoter (spanning −354 to +85) was a determinant of OCTN2 expression in a luciferase reporter assay. Moreover, methylation-specific PCR (MSP) and bisulfite genomic sequencing showed that the degree of individual methylated CpG sites within this region was inversely correlated with the levels of OCTN2 in different cancer cells. Application of DCA to HepG2 and LS174T cells reversed the hypermethylation status of the OCTN2 promoter and increased OCTN2 expression, enhancing cellular uptake of oxaliplatin. Thus, we identified that promoter methylation is responsible for epigenetic down-regulation of OCTN2 in HepG2 and LS174T cells. Given the essential role of OCTN2 in cancer cell uptake of chemotherapeutics, and thus treatment efficacy, pretreatment with a demethylating reagent is a possible strategy for optimizing pharmacotherapies against cancers.

Characterisation of H441 cells as an in vitro model of distal lung epithelial barrier

No continuously growing cell line of human distal lung epithelial phenotype and with the ability to form polarised, electrically tight monolayers has been described to date. Here, we investigated, if the human lung adenocarcinoma cell line H441 has potential to overcome this issue.Epithelial barrier properties were studied by immunocytochemistry (ICC) against ZO‐1 and measurement of RT. The expression of MDR1 and organic cation/carnitine transporters (OCT/Ns) was also investigated by ICC and immunoblot. Uptake of [14C]‐TEA and [3H]‐acetylcarnitine was carried out to assess OCT/N function. Furthermore, the effects of OCT/N modulators on organic cation uptake were studied.H441 cells formed confluent, electrically tight monolayers with peak RT values of 600–1000 Ω·cm2, after 8 days in culture. Presence of P‐gp, OCT1–3, OCTN1 and 2 was confirmed by Western blot and ICC. Uptake of organic cations was time and temperature‐dependent. In the case of [14C]‐TEA, verapamil and MPP+ most efficiently inhibited uptake, whilst amantadine and HC‐3 did not show an effect. D‐carnitine and ergothioneine significantly attenuated [3H]‐acetylcarnitine uptake into H441 cells.H441 cells have the ability to form monolayers with appreciable barrier properties. Moreover, their drug transporter expression and activity is consistent with what has been reported for human alveolar epithelial cells in primary culture.

Transport of organic cations across human bronchial and bronchiolar epithelial barriers in vitro

In this study, we aimed to characterise the function of organic cation/carnitine transporters (OCT/Ns) in human bronchial (Calu‐ 3) and bronchiolar (H441) epithelial cells by using radiolabelled model substrates. The effect of time on uptake of [14C]‐TEA (TEA) and [3H]‐acetylcarnitine (Ac‐Car) was studied. In addition, bi‐directional transport studies were carried out. TEA uptake was found to be time‐dependent in both cell lines, however, H441 cells exhibited a higher level of uptake. Transport experments revealed net‐absorption of both compounds investigated. The apparent permeability coefficient (Papp) for apical‐to‐basolateral transport was 1.93±0.03×10−6 cm/s (TEA) and 1.36±0.01×10−5 cm/s (Ac‐ Car) across Calu‐3 cells. The corresponding basolateral‐to‐apical values were 1.05±0.02×10−6 cm/s (TEA) and 2.32±0.05×10−6 cm/s (Ac‐Car), respectively. In the case of H441 cells, Papp values for TEA were 2.09±0.03×10−5 cm/s (a‐to‐b) and 8.99±0.06×10−6 cm/s (b‐to‐a), and for Ac‐Car 2.71±0.02×10−5 cm/s (a‐to‐b) and for Ac‐ Car 1.14±0.02×10−5 (b‐to‐a), respectively. These data further support the hypothesis that organic cations are actively transported at the human air‐blood barrier, and that the involved transporter sites might play a role in processing of positively charged pharmaceuticals. This work is supported by SFI grant 07/SRC/B1154.

Transport of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) in human respiratory epithelial cells

Organic cation/carnitine transporters (OCT/N) mediate uptake of positively charged molecules. Their role in lung epithelium; however, is not well understood. OCT/N expression and activity was studied in cell lines of human alveolar (A549), bronchial (16HBE14o- and Calu-3) and intestinal (Caco-2) epithelium. Protein levels were largely comparable for all OCT/Ns in the respiratory epithelial cell lines studied; however, OCT2 was exclusively observed in A549 cells. OCT1 and -2 were present at significantly higher levels in Caco-2 cells, compared with the pulmonary epithelial cell types. OCTN1 and -2 were also more abundant in Caco-2. Only OCT3 was expressed evenly across all cell lines investigated. Uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) was dependent on concentration, temperature, membrane potential and pH. In 16HBE14o-, Calu-3 and Caco-2 monolayers, substrate saturation of ASP+ uptake was not reached. Alveolar A549 cells showed saturable ASP+ uptake via two transporter sites with Km values of 12.5±4.0μM and 456.9±164.5μM, respectively. This uptake was sensitive to organic cations, but insensitive to carnitine and lysine. We conclude that uptake of organic cations is facilitated by distinct pathways in different regions of lung mucosa. Luminally localised OCT2 appears to be exclusively involved in the alveolar epithelium, whereas basolateral localised OCT3 might play a role in alveolar as well as in bronchial epithelial cells.

The Acetylcholinesterase Inhibitors Competitively Inhibited an Acetyl l-Carnitine Transport Through the Blood–Brain Barrier

We investigated the interaction of acetylcholinesterase (AChE) inhibitors with acetyl-L-carnitine (ALCAR) transporter at the blood-brain barrier (BBB). ALCAR uptake by conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB cells), as an in vitro model of BBB, were characterized by cellular uptake study using [(3)H]ALCAR. In vivo brain uptake of [(3)H]ALCAR was determined by brain uptake index after carotid artery injection in rats. In results, the transport properties for [(3)H]ALCAR by TR-BBB cell were consistent with those of ALCAR transport by the organic cation/carnitine transporter 2 (OCTN2). Also, OCTN2 was confirmed to be expressed in the cells. The uptake of [(3)H]ALCAR by TR-BBB cells was inhibited by AChE inhibitors such as donepezil, tacrine, galantamine and rivastigmine, which IC(50) values are 45.3, 74.0, 459 and 800 μM, respectively. Especially, donepezil and galantamine inhibited the uptake of [(3)H]ALCAR competitively, but tacrine and rivastigmine inhibited noncompetitively. Furthermore, [(3)H]ALCAR uptake by the rat brain was found to be significantly decreased by quinidine, donepezil and galantamine. Our results suggest that transport of AChE inhibitors such as donepezil and galantamine through the BBB is at least partly mediated by OCTN2 which is involved in transport of ALCAR.

Overview of SLC22A and SLCO Families of Drug Uptake Transporters in the Context of Cancer Treatments

The effectiveness of many anticancer agents is dependent on their disposition to the intracellular space of cancerous tissue. Accumulation of anticancer drugs at their sites of action can be altered by both uptake and efflux transport proteins, however the majority of research on the disposition of anticancer drugs has focused on drug efflux transporters and their ability to confer multidrug resistance. Here we review the roles of uptake transporters of the SLC22A and SLCO families in the context of cancer therapy. The many first-line anticancer drugs that are substrates of organic cation transporters (OCTs) organic cation/carnitine transporters (OCTNs) and organic anion- transporting polypeptides (OATPs) are summarized. In addition, where data is available a comparison of the localization of drug uptake transporters in healthy and cancerous tissues is provided. Expression of drug uptake transporters increases the sensitivity of cancer cell lines to anticancer substrates. Furthermore, early observational studies have suggested a causal link between drug uptake transporter expression and positive outcome in some cancers. Quantification of drug transporters by mass spectrometry will provide an essential technique for generation of expression data during future observational clinical studies. Screening of drug uptake transporter expression in primary tumors may help differentiate between susceptible and resistant cancers prior to therapy.

Activity assessment of organic cation/carnitine transporters in A549 alveolar epithelial cells

Uptake and release of reported substrates via organic cation/carnitine transporters (OCT/Ns) was studied in A549 monolayers in an attempt to investigate the role of OCT/Ns in alveolar cell function. The OCT/N probes, ASP+, [14C]‐TEA and [3H]‐acetylcarnitine (Ac‐Car) were used in the experiments. [3H]‐verapamil, generally considered a substrate for P‐gp/MDR1, was also studied. The effect of time, temperature and various pharmacological agents on uptake and release of the compounds was investigated. All compounds exhibited temperature‐sensitive and time‐ and concentration‐dependent uptake and release. The involvement of both non saturable and saturable uptake carriers was observed in the case of the OCT/N probes. Km values were 27.8 μM (ASP+), 744.4 μM (TEA) and 17.7 μM (Ac‐Car), respectively. Uptake was significantly inhibited by MPP+, Decynium 22 and amantadine. Verapamil uptake was found to be saturable, mediated by a single transporter (Km 57.9 μM) and not inhibited by organic cations. The efflux of ASP+ from A549 monolayers was also saturable (Km 32.9 μM). L‐carnitine and amantadine had no significant effect on ASP+ efflux. Our data suggest that OCT/Ns are involved in membrane transport of organic cations in the alveolar epithelium. In addition, OCTs, but not OCTNs contributed to ASP+ efflux from A549 monolayers.

Competitive inhibition of [14C]‐TEA uptake in A549 cell monolayers by formoterol and salbutamol

Formoterol (For) and salbutamol (Sal), two β2‐receptor agonistic drugs, are positively charged at physiological pH. Here, we investigated, if organic cation/carnitine transporters (OCT/Ns) are involved in the translocation of the drugs in human alveolar epithelial cells (A549). A549 cells were grown to confluent monolayers for 5 days. Investigation of OCT/N‐mediated transport was performed using [14C]‐tetraethylammonium (TEA, 10 μM), [3H]‐acetylcarnitine (5.5 nM, Ac‐Car) and the β2‐agonists (both at 500 μM) in KRB. A549 monolayers were incubated for 30 min (TEA) or 20 min (Ac‐Car) at 37°C. Competitive inhibition studies were also performed in which TEA uptake (0.25 – 2 mM) was studied in the presence and absence of For and Sal for 30 min. The β2‐agonists showed pronounced inhibition of TEA uptake into A549 monolayers. TEA uptake was decreased to 15.4% (For) and 54.5% (Sal) of control, respectively. Neither β2‐receptor agonist had a significant effect on Ac‐Car uptake. Eadie‐Hofstee analysis of TEA uptake revealed linearity and Km values of 858.2 μM and 1050.6 μM in the presence of For and Sal, respectively, which was a 6.5‐fold (For) and 4.5‐fold (Sal) increase compared to control values. No significant change in Vmax was observed. Ki values were 115 μM (For) and 271 μM (Sal). β2‐agonists competitively inhibited TEA uptake via OCTs, but no interaction between the drugs and OCTNs was found.

Xenobiotic, bile acid, and cholesterol transporters: function and regulation.

Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting beta polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) alpha and beta] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of regulatory factors that influence transporter expression and function, including transcriptional activation and post-translational modifications as well as subcellular trafficking. Sex differences, ontogeny, and pharmacological and toxicological regulation of transporters are also addressed. Transporters are important transmembrane proteins that mediate the cellular entry and exit of a wide range of substrates throughout the body and thereby play important roles in human physiology, pharmacology, pathology, and toxicology.

Involvement of OCTN2 in the Transport of Acetyl-l-Carnitine across the Inner Blood–Retinal Barrier

To elucidate the mechanisms of acetyl-L-carnitine transport across the inner blood-retinal barrier (inner BRB). In vivo integration plot and retinal uptake index (RUI) analyses were used to examine acetyl-L-[(3)H]carnitine transport in the retina across the inner BRB in rats. RUI was determined from the ratio of acetyl-L-[(3)H]carnitine and [(14)C]n-butanol, a freely diffusible internal reference, in the retina divided by the same ratio in the solution injected in the carotid artery. The transport mechanism was characterized in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), as an in vitro inner BRB model. The apparent influx permeability clearance (K(in)) per gram retina of acetyl-L-[(3)H]carnitine was found to be 2.31 microL/(minute . g retina). The K(in) of acetyl-L-[(3)H]carnitine was 3.7-fold greater than that of [(3)H]D-mannitol, a nonpermeable paracellular marker. Acetyl-L-[(3)H]carnitine uptake by the retina was found to be significantly inhibited by L-carnitine and acetyl-L-carnitine, supporting a carrier-mediated influx transport of acetyl-L-carnitine at the inner BRB. L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake by TR-iBRB2 cells was Na(+)- and concentration-dependent, with a K(m) of 29 and 26 microM, respectively. These forms of transport were significantly inhibited by organic cation/carnitine transporter (OCTN) substrates and inhibitors such as L-carnitine and acetyl-L-carnitine, tetraethylammonium, quinidine, and betaine. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was predominantly expressed in TR-iBRB2 cells and isolated rat retinal vascular endothelial cells. The findings suggest that OCTN2 is involved in the transport of acetyl-L-carnitine from the circulating blood to the retina across the inner BRB.

Role of Estrogen in Renal Handling of Organic Cation, Tetraethylammonium: in Vivo and in Vitro Studies

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.

Acute Administration of Cefepime Lowers L-Carnitine Concentrations in Early Lactation Stage Rat Milk 3

Our study investigated the potential for important in vivo drug-nutrient transport interactions at the lactating mammary gland using the L-carnitine transporter substrates, cefepime and L-carnitine, as proof-of-concept. On d 4 (n = 6/treatment) and d 10 (n = 6/treatment) of lactation, rats were administered cefepime (250 mg/h) or saline by continuous i.v. infusion (4 h). Serum and milk L-carnitine and cefepime concentrations were quantified by HPLC-UV. In whole mammary gland, organic cation/carnitine transporter (OCTN)1, OCTN2, OCTN3, amino acid transporter B 0,+ (ATB 0,+), and L-carnitine transporter 2 expression were determined by quantitative RT-PCR and by western blot and immunohistochemistry when possible. Cefepime caused a 56% decrease in milk L-carnitine concentrations on lactation d 4 (P = 0.0048) but did not affect milk L-carnitine at lactation d 10 or serum L-carnitine concentrations at either time. The mean L-carnitine and cefepime milk:serum ratios (M/S) decreased from 9.1 ± 0.4 to 4.9 ± 0.6 (P < 0.0001) and 0.89 ± 0.3 to 0.12 ± 0.02 (P = 0.0473), respectively, between d 4 and d 10 of lactation. In both groups, OCTN2 (P < 0.0001), OCTN3 (P = 0.0039), and ATB 0,+ (P = 0.004) mRNA expression and OCTN2 protein (P < 0.0001) were higher in mammary glands at d 4 of lactation compared with d 10. Immunohistochemistry revealed OCTN1 and OCTN2 localization in the mammary alveolar epithelium and OCTN3 expression in the interstitial space and blood vessel endothelium. In conclusion, cefepime significantly decreased milk L-carnitine concentrations only at d 4 of lactation. Relative to d 10, enhanced expression of OCTN2 and ATB 0,+ in mammary glands at d 4 of lactation and higher M/S (L-carnitine and cefepime) suggests cefepime competes with L-carnitine for L-carnitine transporters expressed in the lactating mammary gland to adversely affect L-carnitine milk concentrations and these effects depend upon lactation stage.

Regulation of Octn2 Transporter (SLC22A5) by Peroxisome Proliferator Activated Receptor Alpha

The tissue distribution and disposition of carnitine, which plays an important role in the transport of long-chain fatty acids across the mitochondrial inner membrane for beta-oxidation, are well controlled by carnitine transporter organic cation/carnitine transporter 2 (OCTN2). Since little information is available on regulation of the expression of the OCTN2 gene, we examined the factors that affect the expression level of rat Octn2 (rOctn2), focusing on nuclear receptor peroxisome proliferator activated receptor alpha (PPARalpha), which regulates expression of genes associated with beta-oxidation of fatty acids. mRNA of rOctn2 was induced by the PPARalpha ligand fenofibrate in primary-cultured rat hepatocytes. Further, the PPARalpha ligand Wy14643 increased the expression of Octn2 in wild-type mice, but not in PPARalpha knockout mice. Analysis of the rOctn2 promoter region suggested the presence of putative cis elements of PPARalpha. Wistar rats treated with intraperitoneal fenofibrate administration showed increased expression of rOctn2 mRNA in liver, and uptake of [3H]carnitine by freshly isolated hepatocytes derived from those rats was also increased. In conclusion, our results indicate that the nuclear receptor PPARalpha directly up-regulates the expression of rOctn2 and increases the hepatic uptake of carnitine via rOctn2.

Localization of organic cation/carnitine transporter (OCTN2) in cells forming the blood–brain barrier

Carnitine beta-hydroxy-gamma-(trimethylammonio)butyrate - a compound necessary in the peripheral tissues for a transfer of fatty acids for their oxidation within the cell, accumulates in the brain despite low beta-oxidation in this organ. In order to enter the brain, carnitine has to cross the blood-brain barrier formed by capillary endothelial cells which are in close interaction with astrocytes. Previous studies, demonstrating expression of mRNA coding two carnitine transporters - organic cation/carnitine transporter 2 (OCTN2) and B(0,+) in endothelial cells, did not give any information on carnitine transporters polarity in endothelium. Therefore more detailed experiments were performed on expression and localization of a high affinity carnitine transporter OCTN2 in an in vitro model of the blood-brain barrier by real-time PCR, western blot analysis, and immunocytochemistry. The amount of mRNA was comparable in endothelial cells and kidney, when referred to house-keeping genes, it was, however, significantly lower in astrocytes. Polarity of OCTN2 localization was further studied in an in vitro model of the blood-brain barrier with use of anti-OCTN2 antibodies. Z-axis analysis of the confocal microscope pictures of endothelial cells, with anti-P-glycoprotein antibodies as the marker of apical membrane, showed OCTN2 localization at the basolateral membrane and in the cytoplasmic region in the vicinity of nuclei. Localization of OCTN2 suggest that carnitine can be also transported from the brain, playing an important role in removal of certain acyl esters.

Disruption of AtOCT1, an organic cation transporter gene, affects root development and carnitine‐related responses in Arabidopsis

In animals, organic cation/carnitine transporters (OCTs) are involved in homeostasis and distribution of various small endogenous amines (e.g. carnitine, choline) and detoxification of xenobiotics such as nicotine. Here, we describe the characterization of AtOCT1, an Arabidopsis protein that shares most of the conserved features of mammalian plasma membrane OCTs. Transient expression of an AtOCT1::GFP fusion protein in onion epidermal cells and Arabidopsis protoplasts supported localization in the plasmalemma. AtOCT1 functionally complemented the Deltacit2/Deltaagp2p yeast strain that is defective in plasma membrane carnitine transport. Disruption of AtOCT1 in an Arabidopsis oct1-1 knockout mutant affected both the expression of carnitine-related genes and the developmental defects induced by exogenous carnitine. RT-PCR and promoter-uidA fusion analysis showed that AtOCT1 was expressed in vascular tissues of various organs and at sites of lateral root formation. Correlating with this expression pattern, oct1-1 seedlings grown in vitro exhibited a higher degree of root branching than the wild-type, showing that the disruption of AtOCT1 affected root development under certain conditions.

Functional expression of the organic cation/carnitine transporter 2 in rat astrocytes

In this study, we sought to identify the transporters that mediate the uptake of L-carnitine and acetyl-L-carnitine in cultured rat cortical astrocytes. L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake were both saturable, and mediated by a single Na(+)-dependent transport system. Uptake of both was inhibited by L-carnitine, D-carnitine, acetyl-L-carnitine and various organic cations. Acylcarnitines (acetyl-, butyryl-, hexanoyl-, octanoyl- and palmitoyl-L-carnitine) also interacted with L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine transport. 2-Amino-2-norbornane carboxylic acid, a known inhibitor of amino acid transporter B(0,+) (ATB(0,+)), did not cause any significant inhibition. A highly significant correlation was found between the potencies of acylcarnitines in the inhibition of L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake and the acyl chain length of acylcarnitines. The expression of mRNA for organic cation/carnitine transporters (OCTNs), carnitine transporter 2 (CT2) and ATB(0,+) in astrocytes was investigated by reverse transcription (RT)-PCR. OCTN2 mRNA was expressed in astrocytes, whereas the expression of OCTN1, OCTN3 and CT2 mRNA could not be detected. ATB(0,+) mRNA was expressed at very low levels in astrocytes. Western blotting analysis indicated that anti-OCTN2 polyclonal antibody recognized a band of 70 kDa in both kidney and astrocyte preparations. OCTN2 immunoreactivity was detected in rat astrocytes by immunocytochemical staining. Inhibition of OCTN2 expression by RNA interference significantly inhibited L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake into astrocytes. These results suggest that OCTN2 is functionally expressed in rat astrocytes, and is responsible for L-carnitine and acetyl-L-carnitine uptake in these cells.