Organic cation/carnitine transporter OCTN2 (Slc22a5) is responsible for carnitine transport across apical membranes of small intestinal epithelial cells in mouse

The organic cation/carnitine transporter OCTN2 is responsible for renal tubular reabsorption of its endogenous substrate, carnitine, although its physiological role in small intestine remains controversial. Here we present direct evidence for a predominant role of OCTN2 in small intestinal absorption of carnitine based on experiments with juvenile visceral steatosis (jvs) mice, which have a hereditary deficiency of the octn2 gene. Uptake of carnitine, assessed with an Ussing-type chamber system, from the apical surface of the small intestine was saturable and higher than that from the basal surface in wild-type mice, whereas carnitine uptake having these characteristics was almost absent in jvs mice. Saturable uptake of carnitine was also confirmed in isolated enterocytes obtained from wild-type mice, and the Km value obtained (approximately 20 microM) was close to that reported for carnitine uptake by human embryonic kidney 293 cells stably expressing mouse OCTN2 (Slc22a5). The carnitine uptake by enterocytes was decreased in the presence of various types of organic cations, and this inhibition profile was similar to that of mouse OCTN2, whereas uptake of carnitine was quite small and unsaturable in enterocytes obtained from jvs mice. Immunohistochemical and immunoprecipitation analyses suggested colocalization of OCTN2 with PDZK1, an adaptor protein that functionally regulates OCTN2. Immunoelectron microscopy visualized both OCTN2 and PDZK1 in microvilli of absorptive epithelial cells. These findings indicate that OCTN2 is predominantly responsible for the uptake of carnitine from the apical surface of mouse small intestinal epithelial cells, and it may therefore be a promising target for oral delivery of therapeutic agents that are OCTN2 substrates.     

Molecular and physiological evidence for multifunctionality of carnitine/organic cation transporter OCTN2

OCTN2 is an Na(+)-dependent transporter for carnitine, which is essential for fatty acid metabolism, and its functional defect leads to fatal systemic carnitine deficiency (SCD). It also transports the organic cation tetraethylammonium (TEA) in an Na(+)-independent manner. Here, we studied the multifunctionality of OCTN2, by examining the transport characteristics in cells transfected with mouse OCTN2 and in juvenile visceral steatosis (jvs) mice that exhibit a SCD phenotype owing to mutation of the OCTN2 gene. The physiological significance of OCTN2 as an organic cation transporter was confirmed by using jvs mice. The embryonic fibroblasts from jvs mice exhibited significantly decreased transport of [(14)C]TEA. Pharmacokinetic analysis of [(14)C]TEA disposition demonstrated that jvs mice showed decreased tissue distribution and renal secretory clearance. In transport experiments using OCTN2-expressing cells, TEA and carnitine showed mutual trans-stimulation effects in their transport, implying a carnitine/TEA exchange mechanism. In addition, Na(+) affected the affinity of carnitine for OCTN2, whereas Na(+) is unlikely to be involved in TEA transport. This is the first molecular and physiological demonstration of the operation of an organic cation transporter in renal apical membrane. The results are consistent with the physiological coupling of carnitine reabsorption with the secretion of organic cations.     

Activities of cytochrome p450 enzymes in liver and kidney microsomes from systemic carnitine deficiency mice with a gene mutation of carnitine/organic cation transporter

Juvenile visceral steatosis (jvs) mice, isolated from the C3H-H-2 degrees strain, exibit a systemic carnitine deficiency (SCD) phenotype and develop fatty liver, hyperammonemia and hypoglycemia. This phenotype is caused by a missense mutation (Leu352Arg) of a sodium-dependent carnitine/organic cation transporter, Octn2 (Slc22a5). The jvs mouse could be a useful model for pharmacokinetics and drug metabolism studies concerning Octn2 substrate drugs. In the present study, the effects of the SCD phenotype on the cytochrome P450 (P450 or CYP) dependent activities of four endobiotic and seven xenobiotic oxidations catalyzed by liver and kidney microsomes from jvs mice were investigated. The jvs-type mutation was genotyped by PCR-RFLP. The contents of total P450 and NADPH-P450 reductase were similar in the the liver microsomes from male or female mice of the wild-type and those heterozygous or homozygous for the jvs-type mutation. The 6beta-hydroxylation activities of testosterone and progesterone (marker for Cyp3a) based on the protein contents were 1.2- to 2.0-fold higher in liver microsomes from jvs/jvs-type mice compared to jvs/wt- or wt/wt-type mice. Coumarin 7-hydroxylation activities (marker for Cyp2a) were decreased to 0.7-fold in the male jvs/jvs-type mice. The activities of lauric acid 12-hydroxylation (a marker for Cyp4a) and aniline p-hydroxylation (a marker for Cyp2e1) in liver microsomes were increased 1.4- to 1.9-fold in female jvs/jvs-type mice. Genotoxic activation of 2-aminofluorene (a marker for Cyp4b1) by male and female mouse kidney microsomes were not affected by the SCD phenotype. These results demonstrated that the SCD phenotype affected the P450-dependent catalytic activities in liver microsomes. The jvs mouse could provide valuable information in drug interaction and drug metabolism studies of OCTN2 substrate drugs and new compounds in development.     

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.     

Acetyl-L-carnitine permeability across the blood-brain barrier and involvement of carnitine transporter OCTN2

OCTN2 (SLC22A5), an organic cation/carnitine transporter, is widely distributed throughout the body, including the brain. In the present study, the involvement of OCTN2 in acetyl-L-carnitine (ALCAR) permeation across the blood-brain barrier (BBB) was examined using a microdialysis method in mouse. OCTN2 function was examined by comparison of wild-type mice with jvs mice, which express defective OCTN2 and are considered a model for primary systemic carnitine deficiency. Zero-net-flux method analysis indicated higher in vivo recovery of ALCAR and lower physiological ALCAR concentration in thalamus extracellular fluid (ECF) in jvs mice compared with wild-type mice. Externally added ALCAR showed significantly slower initial uptake across the BBB in jvs mouse. These results indicated that OCTN2 is functionally involved in ALCAR transfer across the BBB. Total radioactivity in ECF after i.v. administration of radiolabelled ALCAR remained constant for the rest of the experimental period. Accordingly, our results indicate that ALCAR is transported from blood to brain ECF by OCTN2 at least in part, and its concentration in brain ECF is regulated by other events such as protein binding and anabolic reactions in the brain, as well as by transport across the BBB.     

Characteristics of L-carnitine transport in cultured human hepatoma HLF cells.

The recently cloned organic cation transporter, OCTN2, isolated as a homologue of OCTN1, has been shown to be of physiological importance in the renal tubular reabsorption of filtered L-carnitine as a high-affinity Na+ carnitine transporter in man. Although the mutation of the OCTN2 gene has been proved to be directly related to primary carnitine deficiency, there is little information about the L-carnitine transport system in the liver. In this study, the characteristics of L-carnitine transport into hepatocytes were studied by use of cultured human hepatoma HLF cells, which expressed OCTN2 mRNA to a greater extent than OCTN1 mRNA. The uptake of L-carnitine into HLF cells was saturable and the Eadie-Hofstee plot showed two distinct components. The apparent Michaelis constant and the maximum transport rate were 6.59+/-1.85 microM (mean+/-s.d.) and 78.5+/-21.4 pmol/5 min/10(6) cells, respectively, for high-affinity uptake, and 590+/-134 microM and 1507+/-142 pmol/5 min/10(6) cells, respectively, for low-affinity uptake. The high affinity L-carnitine transporter was significantly inhibited by metabolic inhibitors (sodium azide, dinitrophenol, iodoacetic acid) and at low temperature (4 degrees C). Uptake of [3H]L-carnitine also required the presence of Na+ ions in the external medium. The uptake activity was highest at pH 7.4, and was significantly lower at acidic or basic pH. L-Carnitine analogues (D-carnitine, L-acetylcarnitine and gamma-butyrobetaine) strongly inhibited uptake of [3H] L-carnitine, whereas beta-alanine, glycine, choline, acetylcholine and an organic anion and cation had little or no inhibitory effect. In conclusion, L-carnitine is absorbed by hepatocytes from man by an active carrier-mediated transport system which is Na+-, energy- and pH-dependent and has properties very similar to those of the carnitine transporter OCTN2.     

Functional characterization of ergothioneine transport by rat organic cation/carnitine transporter Octn1 (slc22a4)

It has been reported that organic cation/carnitine transporter 1 (OCTN1) is associated with rheumatoid arthritis and Crohn's disease. Additionally, we reported that OCTN1 is expressed in hematopoietic cells, and is associated with proliferation and differentiation of erythroid cells. However, physiological role of OCTN1 is still unclear. Ergothioneine, an anti-oxidant, was recently reported to be a good substrate of human OCTN1. However, the transport characteristics of ergothioneine in rat remains to be clarified. The present study, is to further investigate the role of rat Octn1 on transport of ergothioneine in rat Octn1 transfected cells and natively expressing cell line PC12 derived from rat adrenal pheochromocytoma. [(3)H]Ergothioneine uptake by rat Octn1 stably transfected HEK293 cells was saturable, sodium dependent with 1 : 1 stoichiometry of ergothioneine, and pH dependent. Since ergothioneine was reported to presumably play a protective role against oxidative stress-induced apoptosis in PC12 cells, its transport in this cell line was investigated. The expression of rat Octn1 and a saturable and Na(+)-dependent transport of ergothioneine were observed in PC12 cells, suggesting that ergothioneine transport in this cell line may be mediated by rat Octn1. These findings suggested that rat Octn1 may act as a survival factor by taking up ergothioneine to suppress oxidative stress in this cell line. In conclusion, functional characteristics of ergothioneine transport by rat Octn1 is similar to that of human OCTN1 and it is suggested that rat Octn1 is important by transporting anti-oxidant ergothioneine in PC12 cells, though its role in vivo is to be investigated.     

Genetic deficiency of carnitine/organic cation transporter 2 (slc22a5) is associated with altered tissue distribution of its substrate pyrilamine in mice

Carnitine/organic cation transporter 2 (OCTN2) recognizes various cationic compounds as substrates in vitro, but information on its pharmacokinetic role in vivo is quite limited. This paper demonstrates altered tissue distribution of the OCTN2 substrate pyrilamine in juvenile visceral steatosis (jvs) mice, which have a hereditary defect of the octn2 gene. At 30 min after intravenous injection of pyrilamine, the tissue‐to‐plasma concentration ratio (K_p) in the heart and pancreas was higher, whereas the K_p in kidney and testis was lower in jvs mice compared with wild‐type mice. Pyrilamine transport studies in isolated heart slices confirmed higher accumulation, together with lower efflux, of pyrilamine in the heart of jvs mice. The higher accumulation in heart slices of jvs mice was abolished by lowering the temperature, by increasing the substrate concentration, and in the presence of other H₁ antagonists or another OCTN2 substrate, carnitine, suggesting that OCTN2 extrudes pyrilamine from heart tissue. On the other hand, the lower distribution to the kidney of jvs mice was probably due to down‐regulation of a basolateral transporter coupled with OCTN2, because, in jvs mice, (i) the K_p of pyrilamine in kidney assessed immediately after intravenous injection (～1 min) was also lower, (ii) the urinary excretion of pyrilamine was lower, and (iii) the uptake of pyrilamine in kidney slices was lower. The renal uptake of pyrilamine was saturable (K_m～236 µM ) and was strongly inhibited by cyproheptadine, astemizole, ebastine and terfenadine. The present study thus indicates that genetic deficiency of octn2 alters pyrilamine disposition tissue‐dependently. Copyright © 2009 John Wiley & Sons, Ltd.     

Maternal and Fetal Tissue Distribution of L-Carnitine in Pregnant Mice: Low Accumulation in the Brain

The distribution of L-carnitine was studied by whole-body autoradiographyin pregnant CD-1 mice at 1, 2, 3, and 6 hr after receiving L-[¹⁴C]carnitine.Highest concentrations of carnitine were found in maternal tissuesincluding liver, placenta, kidney, myocardium, and choroid plexus.High retention of tissue carnitine in excess of blood levelssuggests the existence of concentrative uptake mechanism. Labeledcarnitine was not detectable in either maternal or fetal brain.This suggests that the brain barrier systems limit the accessof L-carnitine to the brain. In fetus, the level of carnitinewas less than that seen in the maternal tissues, however, thetissue distribution was similar. The fetal tissue carnitineconcentration increased with time. These findings suggest thatrelief of encephalopathy due to toxic organic anions in metabolicdisorders following L-carnitine supplementation appears to beperipheral metabolic effects rather than direct access to thecentral nervous system. However, the physiological role forthe concentrative uptake of L-carnitine by the choroid plexusremains to be determined. Transport of carnitine into fetaltissues via placenta further suggests the possibility of prenataltherapy in pregnancies at risk for certain inherited metabolicdisorders.     

OCTN2-mediated carnitine uptake in a newly discovered human proximal tubule cell line (Caki-1)

The proximal tubular reabsorption of carnitine in the human kidney is significant because more than 95% of the carnitine filtered in the kidney is reabsorbed by the proximal tubules therefore maintaining the homeostatic balance of carnitine in the body. Objectives of this study include the characterization of OCTN2 function in the Caki-1 cell line and the potential interactions of carnitine uptake with renally secreted drugs, including drugs of quaternary ammonium structure. Caki-1 cells were additionally characterized to be of proximal tubule nature, and an apical membrane expression pattern of OCTN2 in Caki-1 cells was discovered. Uptake studies with radiolabeled L-carnitine in Caki-1 cells revealed a Na+- and temperature-dependent carrier-mediated process (K(m) = 15.90 microM) which was unaffected by pH in a range from 6.5 to 8.5. All drugs tested were able to inhibit the carnitine uptake process to different degrees. The quaternary ammonium compounds ciclotropium bromide and ipratropium bromide were strong inhibitors with IC(50) values of 30 microM and 95 microM, respectively. The observed kinetics, immunohistolocalization, and inhibition studies indicate that the high-affinity uptake of carnitine in the Caki-1 cell line is most likely mediated by OCTN2. The interaction of drugs at the renal level with OCTN2 indicates a possible pathway for the final step of cationic secretion into the urine.     

Species specificity profiling of rat and human organic cation/carnitine transporter Slc22a5/SLC22A5 (Octn2/OCTN2)

The purpose of this study was to characterize the uptake of carnitine, the physiological substrate, and the uptake of 3-(2,2,2-trimethylhydrazinium)propionate, a consensus substrate by rat Octn2 and human OCTN2 transporters as well as to characterize drug-mediated inhibition of l-carnitine uptake by the rat and human orthologs overexpressed in CHO-K1 cells. l-carnitine and 3-(2,2,2-trimethylhydrazinium)propionate were found to be a lower affinity substrate for rat Octn2 (K_M = 32.66 ± 5.11 μM and 23.62 ± 4.99 μM respectively) than for human OCTN2 (K_M = 3.08 ± 0.74 μM and 7.98 ± 0.63 μM). The intrinsic clearance (CL_int) value for carnitine was higher for the human than for the rat transporter (22.82 ± 5.57 ml/min*mg vs 4.008 ± 0.675 ml/min*mg). For 3-(2,2,2-trimethylhydrazinium)propionate, in contrast, the CL_int value for rat Octn2 was higher than for human OCTN2 (323.9 ± 72.8 ml/min*mg vs 65.11 ± 5.33 ml/min*mg).Furthermore, many pharmacologically important drugs were shown to affect l-carnitine transport by Octn2/OCTN2. The correlation between the IC₅₀ datasets for the rat and human transporter resulted in an r value of 0.47 (p > 0.05). However, the greatest difference was less than seven-fold and 13 of 15 compounds yielded a difference less than 3-fold.Thus, the transporters from these two species showed an overlapping but somewhat different substrate and inhibitor specificity.     

Expression, localization, and function of the carnitine transporter octn2 (slc22a5) in human placenta.

L-carnitine is assumed to play an important role in fetal development, and there is evidence that carnitine is transported across the placenta. The protein involved in this transfer, however, has not been identified on a molecular level. We therefore characterized localization and function of the carnitine transporter OCTN2 in human placenta. Significant expression of OCTN2 mRNA was detected in human placenta applying real-time polymerase chain reaction technology. Confocal immunofluorescence microscopy using an antibody directed against the carboxy terminus of OCTN2 protein revealed that it is predominantly expressed in the apical membrane of syncytiotrophoblasts. This was confirmed by the costaining of organic anion-transporting polypeptide B and MRP2, which are known to be expressed mainly in the basal and apical syncytiotrophoblasts membrane, respectively. To further support this finding, we performed transport studies using basal and apical placenta membrane vesicles. We could demonstrate that the carnitine uptake into the apical vesicles was about eight times higher compared with the basal ones. Moreover, this uptake was sodium- and pH-dependent with an apparent K(m) value of 21 muM and inhibited by verapamil, which is in line with published data for recombinant OCTN2. Finally, experiments using trophoblasts in cell culture revealed that expression of OCTN2 paralleled human choriogonadotropin production and thus is modulated by cellular differentiation. In summary, we show expression and function of OCTN2 in human placenta. Moreover, several lines of evidence indicate that OCTN2 is localized in the apical membrane of syncytiotrophoblasts, thereby suggesting a major role in the uptake of carnitine during fetal development.     

Functional regions of organic cation/carnitine transporter OCTN2 (SLC22A5): roles in carnitine recognition

The organic cation/carnitine transporter OCTN2 transports carnitine in a sodium-dependent manner, whereas it transports organic cations sodium-independently. To elucidate the functional domain in OCTN2, we constructed chimeric proteins of human OCTN2 (hOCTN2) and mouse OCTN3 (mOCTN3) and introduced mutations at several amino acids conserved among human, rat and mouse OCTN2. We found that transmembrane domains (TMD) 1-7 are responsible for organic cation transport and for sodium dependence in carnitine transport. Within TMD1-7, Q180 and Q207 of hOCTN2 are the critical amino acids for the sodium dependence, and double mutation of Q180 and Q207 resulted in minimal change in transport activity when sodium was removed from the uptake medium. We propose that sodium-dependent affinity for carnitine is dependent on sodium recognition by these critical amino acids in hOCTN2, whereas carnitine transport by OCTN2 requires functional linkage between TMD1-7 and TMD11.     

Plasma and urine pharmacokinetics of free and of short-chain carnitine after administration of carnitine in man

To 6 healthy volunteers 30 mg/kg of L-carnitine (1,3-hydroxy-4-N-trimethylamino-butyrate) were injected intravenously and plasma levels (mumol/l) of free and short-chain carnitine were determined at different times between 0.033 and 24 h. The urinary excretion of L-carnitine and short-chain carnitine in 24 h was also measured. After a period of wash-out the subjects received 100 mg/kg of L-carnitine orally and plasma levels were determined between 0.5 and 24 h. The urinary excretion of L-carnitine was measured for a period of 18.5-33 h after treatment. 3 of the volunteers also received 30 mg/kg of L-carnitine orally. Carnitine plasma levels were determined at different times between 0.5 and 18 h, while the urinary excretion of L-carnitine was measured for 48 h following the treatment. The results could indicate the presence of saturation phenomena in the absorption process for the oral doses used; specific research is required to ascertain this phenomena. The transfer of carnitine from central to extravascular volume is relatively rapid, as is its urinary excretion. The short half-life of carnitine and acetyl-carnitine can suggest the use of new forms of administration (slow-release).     

In Vivo Evidence of Organic Cation Transporter-Mediated Tracheal Accumulation of the Anticholinergic Agent Ipratropium in Mice

Ipratropium bromide (IPR) is an anticholinergic used to treat chronic obstructive pulmonary disease (COPD), and is a substrate of organic cation transporters. The present study aimed to assess the contribution of organic cation transporters to tracheobronchial absorption of IPR in vivo by directly injecting [³H]IPR into the tracheal lumen of mice and measuring its accumulation in tracheal tissue. RT-PCR and immunohistochemical analysis showed that Octnl, Octn2, and Oct2 were localized at epithelial cells in the respiratory tract. Electron-microscopic immunohistochemistry indicated that Octnl and Octn2 were localized at the apical portions of ciliated epithelial cells of trachea. In vitro uptake studies in HEK293 cells expressing these transporters demonstrated that IPR is a preferred substrate of Octn2. Inhibition of mouse tracheal accumulation of [³H]IPR by carnitine was concentration-dependent, reaching a maximum of 42% at 1 mM, whereas inhibition by 0.1 mM MPP + amounted to 62%. Tracheal accumulation of [³H]IPR was unchanged when mice were simultaneously injected with Octnl substrate ergothioneine and organic anion transporter substrate estrone sulfate. These results suggest that Octn2 is involved in membrane permeation of IPR in the respiratory tract in vivo. Targeting organic cation transporters may be an effective strategy for delivery of cationic anti-COPD drugs to patients.     

A substrate pharmacophore for the human organic cation/carnitine transporter identifies compounds associated with rhabdomyolysis

The human organic cation/carnitine transporter (hOCTN2) is a high affinity cation/carnitine transporter expressed widely in human tissues and is physiologically important for the homeostasis of L-carnitine. The objective of this study was to elucidate the substrate requirements of this transporter via computational modeling based on published in vitro data. Nine published substrates of hOCTN2 were used to create a common feature pharmacophore that was validated by mapping other known OCTN2 substrates. The pharmacophore was used to search a drug database and retrieved molecules that were then used as search queries in PubMed for instances of a side effect (rhabdomyolysis) associated with interference with L-carnitine transport. The substrate pharmacophore was composed of two hydrogen bond acceptors, a positive ionizable feature and ten excluded volumes. The substrate pharmacophore also mapped 6 out of 7 known substrate molecules used as a test set. After searching a database of ~800 known drugs, thirty drugs were predicted to map to the substrate pharmacophore with L-carnitine shape restriction. At least 16 of these molecules had case reports documenting an association with rhabdomyolysis and represent a set for prioritizing for future testing as OCTN2 substrates or inhibitors. This computational OCTN2 substrate pharmacophore derived from published data partially overlaps a previous OCTN2 inhibitor pharmacophore and is also able to select compounds that demonstrate rhabdomyolysis, further confirming the possible linkage between this side effect and hOCTN2.     

PDZ adaptor protein PDZK2 stimulates transport activity of organic cation/carnitine transporter OCTN2 by modulating cell surface expression.

A part of the organic cation transporter families (OCT3, OCTN1, and OCTN2) has recently been identified to physically interact with PDZ (PSD95, Dlg, and ZO1) domain-containing proteins, although the physiological relevance of such interaction has not yet been fully examined. Here we have examined the stimulatory effect of PDZK2 [also named NaPi-Cap2 and intestinal and kidney-enriched PDZ protein (IKEPP)] on those cation transporters. In HEK293 cells, coexpression with PDZK2 increased the uptake of carnitine by OCTN2 with minimal effect on its substrate recognition specificity, but not for transport activity of OCT3 or OCTN1. The stimulatory effect of PDZK2 on OCTN2 was compatible with an approximately 2 times increase in transport capacity and can be accounted for by the increase in cell surface expression of OCTN2. Coexpression of PDZK2 did not affect carnitine transport activity of OCTN2 with deletion of the last four amino acids, which were found to be important for the interaction, suggesting involvement of physical interaction of the two proteins in the increase of cell surface expression of OCTN2. In mouse kidney, colocalization of PDZK2 and OCTN2 occurred predominantly in the region that was close to, but not the same as, the surface of apical membranes where OCTN2 alone was observed, suggesting the existence of OCTN2 in the subapical compartment that interacts with PDZK2. The present data have thus proposed an "intracellular pool" for OCTN2 that may be relevant to the stabilization of cell surface expression of OCTN2, thereby increasing transport activity for carnitine.     

Rat Kidney Slices for Evaluation of Apical Membrane Transporters in Proximal Tubular Cells

Kidney slice has been often used as a tool reflecting basolateral transport in renal tubular epithelial cells. Recently, we reported that several important apical reabsorptive transporters such as Octn1/2, Sglt1/2, and Pept1/2 were functional in mouse kidney slices as well as transporter activities in basolateral side, which have been well accepted. Because rats are often used for preclinical pharmacodynamic and pharmacokinetic studies as well as mice, it is important to confirm applicability of rat kidney slices for evaluation of apically expressed transporters. The present study investigates usefulness of kidney slices from rats for evaluation of apical membrane transporters for efflux (multidrug resistance 1a, mdr1a) as well as influx (Octn1/2, Sglt1/2, Pept1/2). Na⁺-dependent uptake of ergothioneine (Octn1), carnitine (Octn2), and methyl-α-D-glucopyranoside (Sglt1/2) by rat kidney slices was observed, and the uptake was decreased by selective inhibitors. In addition, uptake of glycyl-sarcosine (Pept1/2) showed H⁺-dependence and was decreased by selective inhibitor. Furthermore, accumulation of mdr1a substrate azasetron was increased in the presence of zosuquidar, an mdr1a inhibitor, while strain differences existed. In conclusion, rat kidney slices should be useful for evaluation of renal drug disposition regulated by transporters in apical as well as basolateral membranes of rat renal proximal tubule cells.     

Recent advances in drug delivery via the organic cation/carnitine transporter 2 (OCTN2/SLC22A5)

Introduction: Transporters in the plasma membrane have been exploited successfully for the delivery of drugs in the form of prodrugs and nanoparticles. Organic cation/carnitine transporter 2 (OCTN2, SLC22A5) has emerged as a viable target for drug delivery. OCTN2 is a Na⁺-dependent high-affinity transporter for L-carnitine and a Na⁺-independent transporter for organic cations. OCTN2 is expressed in the blood-brain barrier, heart, liver, kidney, intestinal tract and placenta and plays an essential role in L-carnitine homeostasis in the body.

Areas covered: In recent years, several studies have been reported in the literature describing the utility of OCTN2 to enhance the delivery of drugs, prodrugs and nanoparticles. Here we summarize the salient features of OCTN2 in terms of its role in the cellular uptake of its physiological substrate L-carnitine in physiological and pathological context; the structural requirements for recognition and the recent advances in OCTN2-targeted drug delivery systems, including prodrugs and nanoparticles, are discussed.

Expert opinion: This transporter has great potential to be utilized as a target for drug delivery to improve oral absorption of drugs in the intestinal tract. It also has potential to facilitate the transfer of drugs across the biological barriers such as the blood-brain barrier, blood-retinal barrier, and maternal-fetal barrier.     

Contributions of phosphorylation to regulation of OCTN2 uptake of carnitine are minimal in BeWo cells

Physiological functions of organic cation transporters (OCTs) in the placenta include transporting essential nutrients from the maternal to fetal circulations. OCTN2 transports carnitine with high affinity, and the transport of several drugs has also been shown to be mediated by this transporter. In this work, the role of phosphorylation and dephosphorylation mechanisms in regulating OCTN2 was investigated by observing the effects of various activators and inhibitors of kinases and phosphatases on the uptake of carnitine in BeWo cells, a human choriocarcinoma trophoblast cell line frequently used as an in vitro model of the rate-limiting barrier for maternal-fetal exchange. Preincubation with genistein resulted in significant increases in both alkaline phosphatase (ALP) activity and carnitine uptake. Levamisole, an ALP inhibitor, caused a more substantial decrease in carnitine uptake than expected from its corresponding decrease in ALP activity. It was determined that levamisole competitively inhibits carnitine uptake, with a K(i) value of 1.01+/-0.05mM, and this effect has a greater role in decreasing carnitine uptake than any indirect effects of ALP inhibition upon OCTN2 function. Progesterone also competitively inhibited carnitine uptake (K(i)=48.6+/-5.0muM), but had no effect on ALP activity in BeWo cells.