Organic Anion Transporting Polypeptide 1A2 Research Articles

PH-dependent transport kinetics of the human organic anion-transporting polypeptide 1A2

Organic anion-transporting polypeptide (OATP) 1A2 is expressed on the apical sides of intestinal and renal epithelial cells and considered to be involved in the intestinal absorption and renal reabsorption of drugs. Although the transport activity of OATP1A2 is considered to be pH-dependent, the effects of pH on its kinetic parameters and on the potency of OATP1A2 inhibitors are yet to be elucidated. Some OATP are known to have multiple binding sites (MBS), but it remains unclear whether OATP1A2 has MBS. In the present study, we evaluated the influence of pH on the OATP1A2-mediated uptake of estrone 3-sulfate using OATP1A2-expressing HEK293 cells. The uptake of 0.3 μM estrone 3-sulfate by HEK293-OATP1A2 cells was pH-dependent. OATP1A2 exhibited bimodal saturation kinetics at pH 6.3 and 7.4. Compared with that seen at pH 6.3 (5.62 μM), the Km value of the high-affinity site was 8-fold higher at pH 7.4 (43.2 μM). In addition, the influence of pH on the potency of inhibitors varied among the examined inhibitors. These results suggest that the transport properties of OATP1A2 under lower pH conditions, such as those found in the microenvironments of the small intestinal mucosa and distal tubules, differ from those seen under neutral pH conditions.

A novel fluorescence-based functional assay for human OATP1A2 and OATP1C1 identifies interaction between third-generation P-gp inhibitors and OATP1A2.

Organic anion-transporting polypeptide 1A2 (OATP1A2), expressed in the human blood-brain barrier, promotes drug uptake from the blood and hence can be exploited for central nervous system-targeted drug delivery. The thyroid transporter OATP1C1, expressed in the choroid plexus and in astrocytes, is also a potential pharmacological target. Based on their established pharmacological relevance, screening the drug interaction profile of OATP1A2 and OATP1C1 is highly desirable. However, drug interaction screens require suitable model systems and functional assays. In the current study, uptake of a set of cell-impermeable fluorescent dyes was screened in HEK-293 and A431 cell lines overexpressing OATP1A2 and OATP1C1. Based on the uptake of fluorescent dye substrates, a functional assay was developed, which was used to characterize OATP inhibitors/substrates. We identify Live/Dead Green (LDG), Live-or-Dye 488, and sulforhodamines 101, G, and B as novel fluorescent substrates of OATP1A2 and OATP1C1. We show that LDG uptake is proportional to OATP1A2/1C1 expression, allowing the isolation of cells expressing high transporter levels. Additionally, dye uptake can be used to characterize the drug interaction pattern of OATP1A2 and OATP1C1. We demonstrate that third-generation P-glycoprotein inhibitors elacridar, tariquidar, and zosuquidar inhibit OATP1A2 function. Increased toxicity of elacridar in OATP1A2-expressing cells suggests that OATP1A2 may modulate the distribution of this compound. The fluorescence-based assays developed in the current study are a good alternative of radioligand-based tests and pave the way toward high-throughput screens for OATP1A2/1C1 drug interaction studies.

Organic anion transporting polypeptide1A2 mediates fentanyl uptake in cultured cells.

Individual differences in the response to fentanyl, which may be caused by different concentrations of the drug in the central nervous system, can complicate analgesic treatment. It has been reported that the organic anion transporting polypeptide(OATP) at the blood‑brain barrier(BBB) in Sprague‑Dawley rats may serve an important role in the transport of fentanyl across the BBB. However, whether human OATP can transport fentanyl has thus far not been reported. The present study aimed to establish a 293cell line stably overexpressing OATP1A2, and to determine whether OATP1A2 is able to transport fentanyl across the plasma membrane. Initially, 293cells were transfected with an OATP1A2‑expressing plasmid (referred to as 293‑OATP1A2 cells), and single colonies were selected and characterized following geneticin treatment. Subsequently, reverse transcription‑quantitative polymerase chain reaction and western blot analyses were conducted to verify the transfection efficiency. Furthermore, treatment of 293‑OATP1A2 cells with different concentrations of fexofenadine (FEX) and fentanyl was performed to investigate the transport function of OATP1A2 in 293cells. FEX and fentanyl uptake experiments were also performed with naringenin, an inhibitor of OATP1A2. The results indicated that FEX and fentanyl uptake was significantly increased in 293‑OATP1A2 cells compared with that in the control‑transfected cells. The 293‑OATP1A2‑mediated uptake of FEX at concentration of 100nM FEX was ~10‑fold higher than that of 293‑VCcells. The 293‑OATP1A2‑mediated uptake of fentanyl (100nM) was 5.1‑fold higher compared with that in 293‑VCcells. In 293‑OATP1A2 cells, the uptake of FEX without OATP1A2 inhibitor naringenin (100µg/ml) was 2.8‑fold higher compared with that in the presence of naringenin, and the uptake of fentanyl without naringenin was 7.3‑fold higher compared with that in the presence of naringenin (100µg/ml). In conclusion, 293cells that overexpressed OATP1A2 were successfully constructed, and OATP1A2 was revealed to mediate fentanyl uptake in the cultured cells.

Transporters in the Mammary Gland—Contribution to Presence of Nutrients and Drugs into Milk

A large number of nutrients and bioactive ingredients found in milk play an important role in the nourishment of breast-fed infants and dairy consumers. Some of these ingredients include physiologically relevant compounds such as vitamins, peptides, neuroactive compounds and hormones. Conversely, milk may contain substances—drugs, pesticides, carcinogens, environmental pollutants—which have undesirable effects on health. The transfer of these compounds into milk is unavoidably linked to the function of transport proteins. Expression of transporters belonging to the ATP-binding cassette (ABC-) and Solute Carrier (SLC-) superfamilies varies with the lactation stages of the mammary gland. In particular, Organic Anion Transporting Polypeptides 1A2 (OATP1A2) and 2B1 (OATP2B1), Organic Cation Transporter 1 (OCT1), Novel Organic Cation Transporter 1 (OCTN1), Concentrative Nucleoside Transporters 1, 2 and 3 (CNT1, CNT2 and CNT3), Peptide Transporter 2 (PEPT2), Sodium-dependent Vitamin C Transporter 2 (SVCT2), Multidrug Resistance-associated Protein 5 (ABCC5) and Breast Cancer Resistance Protein (ABCG2) are highly induced during lactation. This review will focus on these transporters overexpressed during lactation and their role in the transfer of products into the milk, including both beneficial and harmful compounds. Furthermore, additional factors, such as regulation, polymorphisms or drug-drug interactions will be described.

Transcriptome Profiling of Placenta through Pregnancy Reveals Dysregulation of Bile Acids Transport and Detoxification Function.

Placenta performs the function of several adult organs for the fetus during intrauterine life. Because of the dramatic physiological and metabolic changes during pregnancy and the strong association between maternal metabolism and placental function, the possibility that variation in gene expression patterns during pregnancy might be linked to fetal health warrants investigation. Here, next-generation RNA sequencing was used to investigate the expression profile, including mRNAs and long non-coding RNAs (lncRNAs) of placentas on day 60 of gestation (G60), day 90 of gestation (G90), and on the farrowing day (L0) in pregnant swine. Bioinformatics analysis of differentially expressed mRNAs and lncRNAs consistently showed dysregulation of bile acids transport and detoxification as pregnancy progress. We found the differentially expressed mRNAs, particularly bile salt export pump (ABCB11), organic anion-transporting polypeptide 1A2 (OATP1A2), carbonic anhydrase II (CA2), Na+-HCO3− cotransporter (NBC1), and hydroxysteroid sulfotransferases (SULT2A1) play an important role in bile acids transport and sulfation in placentas during pregnancy. We also found the potential regulation role of ALDBSSCG0000000220 and XLOC_1301271 on placental SULT2A1. These findings have uncovered a previously unclear function and its genetic basis for bile acids metabolism in developing placentas and have important implications for exploring the potential physiological and pathological pathway to improve fetal outcomes.

Epigallocatechin-3-Gallate Reduces Hepatic Oxidative Stress and Lowers CYP-Mediated Bioactivation and Toxicity of Acetaminophen in Rats.

Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea. To investigate the effects of dietary EGCG on oxidative stress and the metabolism and toxicity of acetaminophen in the liver, rats were fed diets with (0.54%) or without EGCG supplementation for four weeks and were then injected intraperitoneally with acetaminophen (1 g/kg). The results showed that EGCG lowered hepatic oxidative stress and cytochrome P450 (CYP) 1A2, 2E1, and 3A, and UDP-glucurosyltransferase activities prior to acetaminophen injection. After acetaminophen challenge, the elevations in plasma alanine aminotransferase activity and histological changes in the liver were ameliorated by EGCG treatment. EGCG reduced acetaminophen-induced apoptosis by lowering the Bax/Bcl2 ratio in the liver. EGCG mildly increased autophagy by increasing the LC3B II/I ratio. Lower hepatic acetaminophen–glutathione and acetaminophen–protein adducts contents were observed after EGCG treatment. EGCG increased glutathione peroxidase and NAD(P)H quinone 1 oxidoreductase activities and reduced organic anion-transporting polypeptides 1a1 expression in the liver after acetaminophen treatment. Our results indicate that EGCG may reduce oxidative stress and lower the metabolism and toxicity of acetaminophen. The reductions in CYP-mediated acetaminophen bioactivation and uptake transporter, as well as enhanced antioxidant enzyme activity, may limit the accumulation of toxic products in the liver and thus lower hepatotoxicity.

Expression of components of the urothelial cholinergic system in bladder and cultivated primary urothelial cells of the pig

BackgroundPorcine urinary bladders are widely used for uro-pharmacological examinations due to their resemblance to the human organ. However, characterisations of the porcine urothelium at the molecular level are scarce up to now. As it has become clear over the last years that this tissue plays an important role in the signaling-pathways of the bladder, we examined whether the transporter and receptor pattern (with focus on the transmitter acetylcholine) is comparable to the human urothelium. With regard to in vitro studies, we also investigated if there is a difference between the native tissue and cultivated primary urothelial cells in culture.MethodsUrothelium from German Landrace and Göttingen Minipig bladders was collected. One part of the German Landrace tissue was used for cultivation, and different passages of the urothelial cells were collected. The actual mRNA expression of different transporters and receptors was examined via quantitative real-time PCR. These included the vesicular acetylcholine transporter (VAChT), the choline acetyl transferase (ChAT), organic cation transporters 1–3 (OCT1–3), organic anion transporting polypeptide 1A2 (OATP1A2), P-glycoprotein (ABCB1), the carnitine acetyl-transferase (CarAT), as well as the muscarinic receptors 1–5 (M1–5).ResultsThere is a strong qualitative resemblance between the human and the porcine urothelium with regard to the investigated cholinergic receptors, enzymes and transporters. CarAT, OCT1–3, OATP1A2 and ABCB1 could be detected in the urothelium of both pig races. Moreover, all 5 M-receptors were prominent with an emphasis on M2 and M3. VAChT and ChAT could not be detected at all. Cultures of the derived urothelial cells showed decreased expression of all targets apart from ABCB1 and CarAT.ConclusionsBased on the expression pattern of receptors, transporters and enzymes of the cholinergic system, the porcine urinary bladder can be regarded as a good model for pharmacological studies. However, cultivation of primary urothelial cells resulted in a significant drop in mRNA expression of the targets. Therefore, it can be concluded that the intact porcine urothelium, or the whole pig bladder, may be appropriate models for studies with anticholinergic drugs, whereas cultivated urothelial cells have some limitation due to significant changes in the expression levels of relevant targets.

Interaction of swine organic anion transporting polypeptide 1a2 with tetracycline, macrolide and β-lactam antibiotics

Organic anion-transporting polypeptides (human OATPs; animals Oatps; gene symbol SLCO/Slco) are integral membrane proteins that mediate the sodium-independent transport of a wide range of endogenous compounds as well as many xenobiotics. Antibiotics, antidiabetic drugs, anti-inflammatory drugs, antifungals, antivirals, antihistamines, antihypertensives, fibrates, statins, cardiac glycosides, immunosuppressants, and anticancer drugs are among the substrates transported by OATPs. Because of the broad substrate specificity, wide tissue distribution and the involvement of drug-drug interaction, human OATPs have been extensively recognized as key determinants for drug absorption, distribution and excretion. In a previous study, we cloned a functional orthologue of human OATP1A2 from the pig liver and designated it as swine Oatp1a2 (sOatp1a2) based on sequence analysis and phylogenic study. In the present study, transport capability of swine Oatp1a2 for tetracyclines, macrolides and β-lactam antibiotics was investigated. It was found that most of the tested antibiotics, including the tetracycline family members such as tetracycline, doxycycline, oxytetracycline and chlortetracycline as well as the β-lactam antibiotics such as penicillin, amoxicillin and cefquinome are directly transported by sOatp1a2; while macrolides such as tylosin, tilmicosin, clarithromycin and erythromycin may only inhibit uptake function of the transporter. As a group of well-known inhibitors of OATP family members, the effect of flavonoids on sOatp1a2 function was evaluated and it was found that all the flavonoids tested are inhibitors of the swine transporter as well.

Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection.

Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease in chickens. Due to the absence of a highly effective cell culture system, there are few reports about the interaction between avian HEV and host cells. In this study, organic anion-transporting polypeptide 1A2 (OATP1A2) from chicken liver cells was identified to interact with ap237, a truncated avian HEV capsid protein spanning amino acids 313 to 549, by a glutathione S-transferase (GST) pulldown assay. GST pulldown and indirect enzyme-linked immunosorbent assays (ELISAs) further confirmed that the extracellular domain of OATP1A2 directly binds with ap237. The expression levels of OATP1A2 in host cells are positively correlated with the amounts of ap237 attachment and virus infection. The distribution of OATP1A2 in different tissues is consistent with avian HEV infection in vivo Finally, when the functions of OATP1A2 in cells are inhibited by its substrates or an inhibitor or blocked by ap237 or anti-OATP1A2 sera, attachment to and infection of host cells by avian HEV are significantly reduced. Collectively, these results displayed for the first time that OATP1A2 interacts with the avian HEV capsid protein and can influence viral infection in host cells. The present study provides new insight to understand the process of avian HEV infection of host cells.IMPORTANCE The process of viral infection is centered around the interaction between the virus and host cells. Due to the lack of a highly effective cell culture system in vitro, there is little understanding about the interaction between avian HEV and its host cells. In this study, a total of seven host proteins were screened in chicken liver cells by a truncated avian HEV capsid protein (ap237) in which the host protein OATP1A2 interacted with ap237. Overexpression of OATP1A2 in the cells can promote ap237 adsorption as well as avian HEV adsorption and infection of the cells. When the function of OATP1A2 in cells was inhibited by substrates or inhibitors, attachment and infection by avian HEV significantly decreased. The distribution of OATP1A2 in different chicken tissues corresponded with that in tissues during avian HEV infection. This is the first finding that OATP1A2 is involved in viral infection of host cells.

Longitudinal Visualization of Viable Cancer Cell Intratumoral Distribution in Mouse Models Using Oatp1a1-Enhanced Magnetic Resonance Imaging.

Multimodality reporter gene imaging provides valuable, noninvasive information on the fate of engineered cell populations. To complement magnetic resonance imaging (MRI) measures of tumor volume and 2-dimensional reporter-based optical measures of cell viability, reporter-based MRI may offer 3-dimensional information on the distribution of viable cancer cells in deep tissues. Here, we engineered human and murine triple-negative breast cancer cells with lentivirus encoding tdTomato and firefly luciferase for fluorescence imaging and bioluminescence imaging (BLI). A subset of these cells was additionally engineered with lentivirus encoding organic anion transporting polypeptide 1a1 (Oatp1a1) for MRI. Oatp1a1 operates by transporting gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) into cells, and it concomitantly improves BLI substrate uptake. After orthotopic implantation of engineered cells expressing or not expressing Oatp1a1, longitudinal fluorescence imaging, BLI, and 3-Tesla MRI were performed. Oatp1a1-expressing tumors displayed significantly increased BLI signals relative to control tumors at all time points (P < 0.05). On MRI, post-Gd-EOB-DTPA T1-weighted images of Oatp1a1-expressing tumors exhibited significantly increased contrast-to-noise ratios compared with control tumors and precontrast images (P < 0.05). At endpoint, tumors expressing Oatp1a1 displayed intratumoral MR signal heterogeneity not present at earlier time points. Pixel-based analysis of matched in vivo MR and ex vivo fluorescence microscopy images revealed a strong, positive correlation between MR intensity and tdTomato intensity for Oatp1a1-expressing tumors (P < 0.05), but not control tumors. These results characterize Oatp1a1 as a sensitive, quantitative, positive contrast MRI reporter gene for 3-dimensional assessment of viable cancer cell intratumoral distribution and concomitant BLI enhancement. This multimodality reporter gene system can provide new insights into the influence of viable cancer cell intratumoral distribution on tumor progression and metastasis, as well as improved assessments of anticancer therapies.

Organic Anion-Transporting Polypeptide 1a4 (Oatp1a4/Slco1a4) at the Blood-Arachnoid Barrier is the Major Pathway of Sulforhodamine-101 Clearance from Cerebrospinal Fluid of Rats.

The blood-arachnoid barrier (BAB), which is formed by arachnoid epithelial cells linked by tight junctions, has generally been considered impermeable to water-soluble substances. However, we recently demonstrated that organic anion transporters 1 and 3 (Oat1 and Oat3) play roles in drug clearance at the BAB. Here, we examined whether an organic anion-transporting polypeptide (Oatp) also plays a role, using the fluorescent organic anion sulforhodamine-101 (SR-101) as a model substrate. SR-101 was injected into the cisterna magna of rats in order to minimize the contribution of choroid plexus transport. The in vivo cerebrospinal fluid (CSF) elimination clearance of SR-101 after intracisternal administration was ninefold greater than that of fluorescein-labeled inulin, a bulk flow marker. In the case of pre-administration of taurocholate, a broad-spectrum inhibitor of Oatps, or digoxin, a strong substrate/inhibitor for Oatp1a4 but not for Oatp1a1, Oat1, and Oat3, the CSF elimination of SR-101 was significantly reduced, becoming similar to that of inulin, and thus indicating complete inhibition of SR-101 clearance from the CSF. The distribution of SR-101 fluorescence was restricted to the arachnoid mater in the absence of inhibitor, whereas the fluorescence was increased in the parenchyma of the spinal cord after co-injection of taurocholate or digoxin. Immunostaining confirmed the localization of Oatp1a4 in the arachnoid mater. These results indicate that Oatp1a4 at the BAB acts as an avid clearance pathway of SR-101 in the CSF to the blood. Thus, Oatp1a4 appears to play a major role in CSF detoxification by limiting the distribution of organic anions to the brain and spinal cord.

Organic anion-transporting polypeptide 1a4–mediated heterogeneous distribution of sulforhodamine-101 in rat hepatic lobules

It has been known that organic anion-transporting polypeptides (Oatps) involve hepatic transports several organic anionic compounds and drugs. This study aimed to investigate sulforhodamine-101 (SR-101) distribution in the rat liver, determine the molecules responsible for the distribution, and delineate the manner of distribution. After intravenous SR-101 administration, its distribution in frozen rat hepatic sections was examined. SR-101-derived signals were detected in regions around the hepatic central vein (CV), where immunohistochemistry (IHC) indicated high Oatp1a4 expression. The signals decreased with treatment by digoxin, a specific substrate for Oatp1a4. In vitro studies using isolated rat hepatocytes and rat Oatp1a4-expressing Xenopus laevis oocytes have suggested that SR-101 is an Oatp1a4 substrate and is taken up into rat hepatocytes mainly via Oatp1a4. Therefore, results suggested SR-101 zonation because of Oatp1a4 involvement and that Oatp1a4 function is dominant in the region around the hepatic CV in rat hepatic lobules.

The 5'-AMP-Activated Protein Kinase Regulates the Function and Expression of Human Organic Anion Transporting Polypeptide 1A2.

The organic anion transporting polypeptides (OATPs) are important membrane proteins that mediate the cellular uptake of drugs and endogenous substances. OATP1A2 is widely distributed in many human tissues that are targeted in drug therapy; defective OATP1A2 leads to altered drug disposition influencing therapeutic outcomes. 5'-AMP-activated protein kinase (AMPK) signaling plays an important role in the pathogenesis of the metabolic syndrome characterized by an increased incidence of type II diabetes and nonalcoholic fatty liver disease. This study investigated the regulatory role of AMPK in OATP1A2 transport function and expression. We found that the treatment of AMPK-specific inhibitor compound C (dorsomorphin dihydrochloride) decreased OATP1A2-mediated uptake of estrone-3-sulfate in a concentration- and time-dependent manner. The impaired OATP1A2 function was associated with a reduced Vmax [154.6 ± 17.9 pmol × (μg × 4 minutes)-1 in compound C-treated cells vs. 413.6 ± 52.5 pmol × (μg × 4 minutes)-1 in controls]; the Km was unchanged. The cell-surface expression of OATP1A2 was decreased by compound C treatment, but total cellular expression was unchanged. The impaired cell-surface expression of OATP1A2 was associated with accelerated internalization and impaired targeting/recycling. Silencing of the AMPK α1-subunit using specific small interfering RNA corroborated the findings with compound C and revealed a role for AMPK in regulating OATP1A2 protein stability. Overall, this study implicated AMPK in the regulation of the function and expression of OATP1A2, which potentially impacts on the disposition of OATP1A2 drug substrates that may be used to treat patients with the metabolic syndrome and other diseases.

Functional Expression of Organic Anion Transporting Polypeptide 1a4 Is Regulated by Transforming Growth Factor-β/Activin Receptor-like Kinase 1 Signaling at the Blood-Brain Barrier.

Central nervous system (CNS) drug delivery can be achieved by targeting drug uptake transporters such as Oatp1a4. In fact, many drugs that can improve neurologic outcomes in CNS diseases [3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (i.e., statins)] are organic anion transporting polypeptide (OATP) transport substrates. To date, transport properties and regulatory mechanisms of Oatp1a4 at the blood-brain barrier (BBB) have not been rigorously studied. Such knowledge is critical to develop Oatp1a4 for optimization of CNS drug delivery and for improved treatment of neurological diseases. Our laboratory has demonstrated that the transforming growth factor-β (TGF-β)/activin receptor-like kinase 1 (ALK1) signaling agonist bone morphogenetic protein 9 (BMP-9) increases functional expression of Oatp1a4 in rat brain microvessels. Here, we expand on this work and show that BMP-9 treatment increases blood-to-brain transport and brain exposure of established OATP transport substrates (i.e., taurocholate, atorvastatin, and pravastatin). We also demonstrate that BMP-9 activates the TGF-β/ALK1 pathway in brain microvessels as indicated by increased nuclear translocation of specific Smad proteins associated with signaling mediated by the ALK1 receptor (i.e., pSmad1/5/8). Furthermore, we report that an activated Smad protein complex comprised of phosphorylated Smad1/5/8 and Smad4 is formed following BMP-9 treatment and binds to the promoter of the Slco1a4 gene (i.e., the gene that encodes Oatp1a4). This signaling mechanism causes increased expression of Slco1a4 mRNA. Overall, this study provides evidence that Oatp1a4 transport activity at the BBB is directly regulated by TGF-β/ALK1 signaling and indicates that this pathway can be targeted for control of CNS delivery of OATP substrate drugs.

Sex-specific differences in organic anion transporting polypeptide 1a4 (Oatp1a4) functional expression at the blood\u2013brain barrier in Sprague\u2013Dawley rats

BackgroundTargeting endogenous blood–brain barrier (BBB) transporters such as organic anion transporting polypeptide 1a4 (Oatp1a4) can facilitate drug delivery for treatment of neurological diseases. Advancement of Oatp targeting for optimization of CNS drug delivery requires characterization of sex-specific differences in BBB expression and/or activity of this transporter.MethodsIn this study, we investigated sex differences in Oatp1a4 functional expression at the BBB in adult and prepubertal (i.e., 6-week-old) Sprague–Dawley rats. We also performed castration or ovariectomy surgeries to assess the role of gonadal hormones on Oatp1a4 protein expression and transport activity at the BBB. Slco1a4 (i.e., the gene encoding Oatp1a4) mRNA expression and Oatp1a4 protein expression in brain microvessels was determined using quantitative real-time PCR and western blot analysis, respectively. Oatp transport function at the BBB was determined via in situ brain perfusion using [3H]taurocholate and [3H]atorvastatin as probe substrates. Data were expressed as mean ± SD and analyzed via one-way ANOVA followed by the post hoc Bonferroni t-test.ResultsOur results showed increased brain microvascular Slco1a4 mRNA and Oatp1a4 protein expression as well as increased brain uptake of [3H]taurocholate and [3H]atorvastatin in female rats as compared to males. Oatp1a4 expression at the BBB was enhanced in castrated male animals but was not affected by ovariectomy in female animals. In prepubertal rats, no sex-specific differences in brain microvascular Oatp1a4 expression were observed. Brain accumulation of [3H]taurocholate in male rats was increased following castration as compared to controls. In contrast, there was no difference in [3H]taurocholate brain uptake between ovariectomized and control female rats.ConclusionsThese novel data confirm sex-specific differences in BBB Oatp1a4 functional expression, findings that have profound implications for treatment of CNS diseases. Studies are ongoing to fully characterize molecular pathways that regulate sex differences in Oatp1a4 expression and activity.

Pharmacokinetic changes of cefdinir and cefditoren and its molecular mechanisms in acute kidney injury in rats.

Acute kidney injury (AKI) was a common organ damage that often occurred after cisplatin. This study was aimed at investigating the pharmacokinetic changes of cefdinir and cefditoren in AKI rats, and elucidating the possible molecular mechanisms. The renal injury model was established by intraperitoneal injection of cisplatin (12 mg/kg). Plasma creatinine, blood urea nitrogen, the mRNA expression of Kim-1, hematoxylin and eosin staining and Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay were used to measure the degree of renal damage. On this basis, the pharmacokinetic changes of cefdinir and cefditoren were investigated in normal and AKI rats. RT-PCR and Western blot were performed to clarify the molecular mechanisms for the changes in the related transporters expression. The cumulative urinary excretion of cefdinir was significantly decreased and the plasma concentration was remarkably increased in AKI rats. The expression of organic anion transporter 1 (Oat1) and Oat3 in kidney was decreased. However, pharmacokinetics of cefditoren was not influenced. The expression of organic anion-transporting polypeptide 1a1 (Oatp1a1), Oatp1a4, Oatp1b2 and multidrug resistance-associated protein 2 (Mrp2) in liver was unchanged in AKI rats. The molecular mechanism of decreased expression of Oat1 and Oat3 was achieved through activating p53, and then increasing the expression of Bax and Caspase-3 and down regulating Bcl-2 in AKI rats. On this basis, the cumulative urinary excretion of cefdinir was significantly decreased and the plasma concentration of cefdinir was remarkably increased in AKI rats. However, the pharmacokinetic changes of cefditoren were not observed. Accordingly, cephalosporin antibiotics such as cefditoren should be firstly selected for the treatment in patients with AKI in clinic.

Yinchenhao Decoction Ameliorates Alpha-Naphthylisothiocyanate Induced Intrahepatic Cholestasis in Rats by Regulating Phase II Metabolic Enzymes and Transporters.

Yinchenhao Decoction (YCHD), a famous traditional Chinese formula, has been used for treating cholestasis for 1000s of years. The cholagogic effect of YCHD has been widely reported, but its pharmacodynamic material and underlying therapeutic mechanism remain unclear. By using ultra-high-performance liquid chromatography (UHPLC)-quadrupole time-of-flight mass spectrometry, 11 original active components and eight phase II metabolites were detected in rats after oral administration of YCHD, including three new phase II metabolites. And it indicated that phase II metabolism was one of the major metabolic pathway for most active components in YCHD, which was similar to the metabolism process of bilirubin. It arouses our curiosity that whether the metabolism process of YCHD has any relationship with its cholagogic effects. So, a new method for simultaneous quantitation of eight active components and four phase II metabolites of rhein, emodin, genipin, and capillarisin has been developed and applied for their pharmacokinetic study in both normal and alpha-naphthylisothiocyanate (ANIT)-induced intrahepatic cholestasis rats. The results indicated the pharmacokinetic behaviors of most components of YCHD were inhibited, which was hypothesized to be related to different levels of metabolic enzymes and transporters in rat liver. So dynamic changes of intrahepatic enzyme expression in cholestasis and YCHD treated rats have been monitored by an UHPLC-tandem mass spectrometry method. The results showed expression levels of UDP-glucuronosyltransferase 1-1 (UGT1A1), organic anion-transporting polypeptide 1A4 (OATP1A4), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1, sodium-dependent taurocholate cotransporter, and organic anion-transporting polypeptide 1A2 were significantly inhibited in cholestasis rats, which would account for reducing the drug absorption and the metabolic process of YCHD in cholestatic rats. A high dose (12 g/kg) of YCHD remarkably increased the expression of UGT1A1, bile salt export pump, MRP2, OATP1A4 in cholestasis rats presented it exhibited the greatest ameliorative effect on cholestasis, also particularly in histopathological examination and reducing levels of alanine transaminase, aspartate transaminase, total bilirubin, direct bilirubin, and total bile acid. Considering the metabolic process of bilirubin in vivo, the choleretic effect of YCHD is proven to be related to its regulatory action on expression of metabolic enzymes and transporters in cholestatic liver.

Organic Anion Transporting Polypeptide 1a4 is Responsible for the Hepatic Uptake of Cardiac Glycosides in Mice.

Among organic anion transporting polypeptide (Oatp) family transporters expressed in the rodent liver, such as Oatp1a1, Oatp1a4, Oatp1b2, and Oatp2b1, Oatp1a4 has a unique character to recognize neutral cardiac glycosides as a substrate in addition to organic anions. The relative contribution of Oatp1a4 to the substrate uptake into hepatocytes has not been clarified. In this study, we investigated the importance of Oatp1a4 in the hepatic uptake of its substrate drugs using Slco1a4-/- mice. The hepatic mRNA expression of Slco1a4 was decreased significantly in Slco1a4-/- mice, whereas no differences were seen in other hepatic transporters between wild-type and Slco1a4-/- mice. We determined the plasma concentrations and liver-to-plasma concentration ratios (Kp,liver) of Oatp1a4 substrates, including ouabain, digoxin, BQ-123, fexofenadine, rosuvastatin, pravastatin, nafcillin, and telmisartan, after continuous intravenous infusion. The plasma concentrations of ouabain and rosuvastatin were 2.1-fold and 1.7-fold higher in Slco1a4-/- mice, and Kp,liver of ouabain and digoxin were 13.4-fold and 4.3-fold lower in Slco1a4-/- mice, respectively. Furthermore, the biliary clearance of ouabain and digoxin with regard to plasma concentration were 21.9-fold and 4.1-fold lower in Slco1a4-/- mice, respectively, accompanied with a marked reduction in their Kp,liver, whereas the systemic clearance of ouabain, but not digoxin, was reduced significantly in Slco1a4-/- mice. These results suggest that Oatp1a4 plays a major role in the hepatic accumulation of cardiac glycosides in mice.

Association between a microRNA binding site polymorphism in SLCO1A2 and the risk of delayed methotrexate elimination in Chinese children with acute lymphoblastic leukemia

Organic anion-transporting polypeptide 1A2 (OATP1A2) is involved in the cellular uptake of methotrexate (MTX). Genetic variation in solute carrier organic anion transporter family member 1A2 (SLCO1A2, the coding gene of OATP1A2) has important implications for the elimination of MTX. We investigated the association between a microRNA (miRNA) binding site polymorphism (rs4149009 G > A) in the 3′-untranslated region (3′-UTR) of SLCO1A2 with the serum MTX concentrations in Chinese children with acute lymphoblastic leukemia (ALL). Genotyping for SLCO1A2 rs4149009 G > A in 141 children with ALL was performed using the Sequenom MassARRAY system. Serum MTX concentrations were determined by fluorescence polarization immunoassay. The percentages of MTX level ≥1 μmol/L at 42 h were compared among the AA, GA, and GG genotypes. The minor allele frequency observed in this study (33.0%) was significantly lower than that in the African samples reported in the 1000 Genomes Project (57.4%, P = 0.00). The incidence rate of delayed MTX elimination was significantly higher in patients with the GG genotype (23.1%) compared with the AA genotype (0.0%, P = 0.03). Bioinformatics tools predicted that the rs4149009 A allele would disrupt the putative binding sites of hsa-miR-324-3p and hsa-miR-1913. These results indicate that the rs4149009 G > A polymorphism might affect MTX pharmacokinetics by interfering with the function of miRNAs.

Determinants of postprandial plasma bile acid kinetics in human volunteers.

Bile acids (BA) are signaling molecules with a wide range of biological effects, also identified among the most responsive plasma metabolites in the postprandial state. We here describe this response to different dietary challenges and report on key determinants linked to its interindividual variability. Healthy men and women (n = 72, 62 ± 8 yr, mean ± SE) were enrolled into a 12-wk weight loss intervention. All subjects underwent an oral glucose tolerance test and a mixed-meal tolerance test before and after the intervention. BA were quantified in plasma by liquid chromatography-tandem mass spectrometry combined with whole genome exome sequencing and fecal microbiota profiling. Considering the average response of all 72 subjects, no effect of the successful weight loss intervention was found on plasma BA profiles. Fasting and postprandial BA profiles revealed high interindividual variability, and three main patterns in postprandial BA response were identified using multivariate analysis. Although the women enrolled were postmenopausal, effects of sex difference in BA response were evident. Exome data revealed the contribution of preselected genes to the observed interindividual variability. In particular, a variant in the SLCO1A2 gene, encoding the small intestinal BA transporter organic anion-transporting polypeptide-1A2 (OATP1A2), was associated with delayed postprandial BA increases. Fecal microbiota analysis did not reveal evidence for a significant influence of bacterial diversity and/or composition on plasma BA profiles. The analysis of plasma BA profiles in response to two different dietary challenges revealed a high interindividual variability, which was mainly determined by genetics and sex difference of host with minimal effects of the microbiota.NEW & NOTEWORTHY Considering the average response of all 72 subjects, no effect of the successful weight loss intervention was found on plasma bile acid (BA) profiles. Despite high interindividual variability, three main patterns in postprandial BA response were identified using multivariate analysis. A variant in the SLCO1A2 gene, encoding the small intestinal BA transporter organic anion-transporting polypeptide-1A2 (OATP1A2), was associated with delayed postprandial BA increases in response to both the oral glucose tolerance test and the mixed-meal tolerance test.