Brain-to-blood elimination of 24S-hydroxycholesterol from rat brain is mediated by organic anion transporting polypeptide 2 (oatp2) at the blood-brain barrier

24S-Hydroxycholesterol (24S-OH-chol), a major cerebral cholesterol metabolite, is an endogenous ligand for the liver X receptor and is a potential stimulant of cholesterol release from glial cells. The elimination mechanism of 24S-OH-chol from the brain is one of the key issues for understanding cerebral cholesterol homeostasis. The purpose of the present study was to clarify the molecular mechanism of the elimination process of 24S-OH-chol across the blood–brain barrier (BBB). After an intracerebral injection in rats, [³H]24S-OH-chol was eliminated from the brain and the radioactivity derived from [³H]24S-OH-chol was detected in the plasma, while [³H]cholesterol was not significantly eliminated from the brain. Co-administration of unlabeled 24S-OH-chol significantly inhibited the [³H]24S-OH-chol elimination, while no inhibitory effect was seen at the same concentration of cholesterol. The [³H]24S-OH-chol elimination was inhibited by co-administration of probenecid, but not by benzylpenicillin. Pre-administration of digoxin completely inhibited the elimination. Xenopus laevis oocytes expressing rat oatp2 exhibited significant transport of [³H]24S-OH-chol, and this was inhibited by unlabeled 24S-OH-chol and digoxin, indicating that rat oatp2 transports 24S-OH-chol. These results are the first direct demonstration that 24S-OH-chol undergoes elimination from the brain to blood across the BBB via a carrier-mediated process, which involves oatp2 expressed at the BBB in rats.     

Blood-brain barrier transport of pramipexole, a dopamine D2 agonist

The blood-brain barrier (BBB) transport of pramipexole, a potent dopamine receptor agonist with high efficacy for Parkinson's disease, was mainly characterized using immortalized rat brain capillary endothelial cells (RBEC)1 as an in vitro BBB model. [(14)C]Pramipexole uptake by RBEC1 was dependent on temperature and pH, but not sodium ion concentration or membrane potential. The uptake was inhibited by several organic cations including pyrilamine. Mutual inhibition was observed between pramipexole and pyrilamine. In addition, [(14)C]pramipexole uptake was stimulated by preloading unlabeled pramipexole. RT-PCR analysis for organic cation transporters (rOCT1-3, rOCTN1-2) in RBEC1 was performed. The mRNA level of rOCTN2 was the highest, followed by rOCTN1, while expression of rOCT1, rOCT2 and rOCT3 was negligible. The brain uptake of [(14)C]pramipexole, which was measured by the in situ rat brain perfusion technique, was significantly inhibited by unlabeled pramipexole. These results suggest that pramipexole is, at least in part, transported across the BBB by an organic cation-sensitive transporter. The pramipexole transport in RBEC1 was pH-dependent, but sodium- and membrane potential-independent.     

Localization of organic anion transporting polypeptide 3 (oatp3) in mouse brain parenchymal and capillary endothelial cells

Organic anion transporting polypeptide 3 (oatp3) transports various CNS-acting endogenous compounds, including thyroid hormones and prostaglandin E2, between extra- and intracellular spaces, suggesting a possible role in CNS function. The purpose of this study was to clarify the expression and localization of oatp3 in the mouse brain. RT-PCR analysis revealed that oatp3 mRNA is expressed in brain capillary-rich fraction, conditionally immortalized brain capillary endothelial cells, choroid plexus, brain and lung, but not in liver or kidney, where oatp1, 2 and 5 mRNAs were detected. Immunohistochemical analysis with anti-oatp3 antibody suggests that oatp3 protein is localized at the brush-border membrane of mouse choroid plexus epithelial cells. Furthermore, intense immunoreactivity was detected in neural cells in the border region between hypothalamus and thalamus, and in the olfactory bulb. Immunoreactivity was also detected in brain capillary endothelial cells in the cerebral cortex. These localizations in the mouse brain suggest that oatp3 plays roles in blood-brain and -cerebrospinal fluid barrier transport of organic anions and signal mediators, and in hormone uptake by neural cells.     

Blood-brain barrier transport of a novel micro 1-specific opioid peptide,H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA)

The purpose of this study was to clarify the mechanism of the blood-brain barrier (BBB) transport of H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA), which is a novel dermorphin analog with high affinity for the micro 1-opioid receptor. The in vivo BBB permeation influx rate of [125I]TAPA after an i.v. bolus injection (7.3 pmol/g body weight) into mice was estimated to be 0.265 +/- 0.025 microL/(min.g of brain). The influx rate of [125I]TAPA was reduced 70% by the coadministration of unlabeled TAPA (33 nmol/g of brain), suggesting the existence of a specific transport system for TAPA at the BBB. In order to elucidate the BBB transport mechanism of TAPA, a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4) was used as an in vitro model of the BBB. The acid-resistant binding of [125I]TAPA, which represents the internalization of the peptide into cells, was temperature- and concentration-dependent with a half-saturation constant of 10.0 +/- 1.7 microm. The acid-resistant binding of TAPA was significantly inhibited by 2,4-dinitrophenol, dansylcadaverine (an endocytosis inhibitor) and poly-l-lysine and protamine (polycations). These results suggest that TAPA is transported through the BBB by adsorptive-mediated endocytosis, which is triggered by binding of the peptide to negatively charged sites on the surface of brain capillary endothelial cells. Blood-brain barrier transport via adsorptive-mediated endocytosis plays a key role in the expression of the potent opioid activity of TAPA in the CNS.     

Organic anion transporter 3 is involved in the brain-to-blood efflux transport of thiopurine nucleobase analogs

Thiopurines are used as antileukemic drugs. However, during chemotherapy CNS relapses occur due to the proliferation of leukemic cells in the CNS resulting from restricted drug distribution in the brain. The molecular mechanism for this limited cerebral distribution remains unclear. The purpose of this study was to identify the transporter responsible for the brain-to-blood transport of thiopurines across the blood-brain barrier (BBB) using the brain efflux index method. [14C]6-Mercaptopurine (6-MP) and [3H]6-thioguanine were eliminated from rat brain in a time-dependent manner. The elimination of [14C]6-MP was inhibited by substrates of rat organic anion transporters (rOATs), including indomethacin and benzylpenicillin. rOAT1 and rOAT3 exhibited 6-MP uptake, while benzylpenicillin inhibited rOAT3-mediated uptake, but not that by rOAT1. rOAT3-mediated [14C]6-MP uptake was also inhibited by other thiopurine derivatives. Although methotrexate inhibited rOAT3-mediated [14C]6-MP uptake, the Ki value was 17.5-fold greater than the estimated brain concentration of methotrexate in patients receiving chemotherapy. Accordingly, 6-MP would undergo efflux transport by OAT3 from the brain without any inhibitory effect from coadministered methotrexate in the chemotherapy. In conclusion, rOAT3 is involved in the brain-to-blood transport of thiopurines at the BBB and is one mechanism of limited cerebral distribution.     

Involvement of organic anion transporters in the efflux of uremic toxins across the blood-brain barrier

Renal failure causes multiple physiological changes involving CNS dysfunction. In cases of uremia, there is close correlation between plasma levels of uremic toxins [e.g. 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), hippurate (HA) and indoleacetate (IA)] and the degree of uremic encephalopathy, suggesting that uremic toxins are involved in uremic encephalopathy. In order to evaluate the relevance of uremic toxins to CNS dysfunction, we investigated directional transport of uremic toxins across the blood-brain barrier (BBB) using in vivo integration plot analysis and the brain efflux index method. We observed saturable efflux transport of [(3)H]CMPF, [(14)C]HA and [(3)H]IA, which was inhibited by probenecid. For all uremic toxins evaluated, apparent efflux clearance across the BBB was greater than apparent influx clearance, suggesting that these toxins are predominantly transported from the brain to blood across the BBB. Saturable efflux transport of [(3)H]CMPF, [(14)C]HA and [(3)H]IA was completely inhibited by benzylpenicillin, which is a substrate of rat organic anion transporter 3 (rOat3). Taurocholate and digoxin, which are common substrates of rat organic anion transporting polypeptide (rOatp), partially inhibited the efflux of [(3)H]CMPF. Transport experiments using a Xenopus laevis oocyte expression system revealed that CMPF, HA and IA are substrates of rOat3, and that CMPF (but not HA or IA) is a substrate of rOap2. These results suggest that rOat3 mediates brain-to-blood transport of uremic toxins, and that rOatp2 is involved in efflux of CMPF. Thus, conditions typical of uremia can cause inhibition of brain-to-blood transport involving rOat3 and/or rOatp2, leading to accumulation of endogenous metabolites and drugs in the brain.     

Role of blood-brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate,a uremic toxin: its involvement in neurotransmitter metabolite clearance from the brain

Renal impairment is associated with CNS dysfunctions and the accumulation of uremic toxins, such as indoxyl sulfate, in blood. To evaluate the relevance of indoxyl sulfate to CNS dysfunctions, we investigated the brain-to-blood transport of indoxyl sulfate at the blood-brain barrier (BBB) using the Brain Efflux Index method. [(3)H]Indoxyl sulfate undergoes efflux transport with an efflux transport rate of 1.08 x 10(-2)/min, and the process is saturable with a Km of 298 microm. This process is inhibited by para-aminohippuric acid, probenecid, benzylpenicillin, cimetidine and uremic toxinins, such as hippuric acid and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid. RT-PCR revealed that an OAT3 mRNA is expressed in conditionally immortalized rat brain capillary endothelial cell lines and rat brain capillary fraction. Xenopus oocytes expressing OAT3 were found to exhibit [(3)H]indoxyl sulfate uptake, which was significantly inhibited by neurotransmitter metabolites, such as homovanillic acid and 3-methoxy-4-hydroxymandelic acid, and by acyclovir, cefazolin, baclofen, 6-mercaptopurine, benzoic acid, and ketoprofen. These results suggest that OAT3 mediates the brain-to-blood transport of indoxyl sulfate, and is also involved in the efflux transport of neurotransmitter metabolites and drugs. Therefore, inhibition of the brain-to-blood transport involving OAT3 would occur in uremia and lead to the accumulation of neurotransmitter metabolites and drugs in the brain.     

The intracellular NPxY motif is critical in maintaining the function and expression of human organic anion transporting polypeptide 1B1

Organic anion transporting polypeptides (OATPs, gene symbol SLCO) mediate sodium-independent transport of endogenous compounds such as bile salts, hormones and their conjugates as well as toxins and drugs. OATP1B1 is the major OATP specifically expressed at the basolateral membrane of human hepatocytes and many clinically important drugs have been shown to be substrates of the transporter. According to the computer-based hydropathy analysis, a large intracellular loop 3 (IL3) is situated between transmembrane domain 6 and 7 of OATPs, in which a conserved NPxY motif is found. In the current study, HEK293 cells expressing the HA-tagged OATP1B1 was utilized to investigate the role of the NPxY motif for the function and expression of the transporter. Alanine replacement of N335 or P336 retained substantial uptake function; while simultaneous mutation of these residues resulted in a double mutant that lost almost all the transport activity. On the other hand, Y338A showed >80% reduction for estrone-3-sulfate uptake. Plasma membrane protein analysis revealed that N335/P336A completely lost its cell surface protein expression; while that of Y338A is dramatically reduced. Further investigation with pharmacological inhibitors and immunocytochemistry demonstrated that N335/336A is detained in the Golgi apparatus and Y338A exhibited accelerated protein degradation rate compared to that of the wild-type. Conservative replacement of Y338 with phenylalanine fully recovered uptake and expression of the transporter. In summary, a new role was observed for the NPxY motif located in the IL3 of OATP1B1, which may affect processing and stability of the transporter.     

Effects of perfluoroalkyl carboxylic acids on the uptake of sulfobromophthalein via organic anion transporting polypeptides in human intestinal Caco-2 cells

We performed a detailed investigation of the uptake of sulfobromophthalein (BSP) from the apical membrane of Caco-2 cells, which is a substrate for organic anion transporting polypeptides (OATPs), and calculated the kinetic parameters of BSP uptake as follows: K_m = 13.9 ± 1.3 μM, V_max = 1.15 ± 0.07 nmol (mg protein)^?1 (5 min)^?1, and k_d = 38.2 ± 0.53 μL (mg protein)^?1 (5 min)^?1. Coincubation with medium-chain (C7–C11) perfluoroalkyl carboxylic acids (PFCAs), such as perfluoroheptanoic acid (PFHpA, C7), perfluorooctanoic acid (PFOA, C8), perfluorononanoic acid (PFNA, C9), perfluorodecanoic acid (PFDA, C10) and perfluoroundecanoic acid (PFUnDA, C11), significantly decreased BSP uptake by 27–55%, while coincubation with short- (C3–C6) and long-chain (C12–C14) PFCAs decreased the uptake only slightly. Dixon plotting suggested that PFOA, PFNA and PFDA competitively inhibited the BSP uptake with inhibition constant (K_i) values of 62.2 ± 1.3 μM, 35.3 ± 0.1 μM and 43.2 ± 0.3 μM, respectively. PFCAs with medium-chains could be substrates for OATPs, probably OATP2B1, which is the most abundantly expressed OATP isoform in Caco-2 cells.     

Functional and molecular characterization of adenosine transport at the rat inner blood-retinal barrier

The purpose of the present study was to characterize the adenosine transport system(s) at the inner blood-retinal barrier (inner BRB). A conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2), used as an in vitro model of the inner BRB, expresses equilibrative nucleoside transporter 1 (ENT1), ENT2, concentrative nucleoside transporter 2 (CNT2), and CNT3 mRNAs. TR-iBRB2 cells exhibited an Na⁺-independent and concentration-dependent [³H]adenosine uptake with a Michaelis-Menten constant of 28.5 μM and a maximum uptake rate of 814 pmol/(min mg protein). [³H]Adenosine uptake by TR-iBRB2 cells was strongly inhibited by 2 mM adenosine, inosine, uridine, and thymidine. On the other hand, this process was not inhibited by 100 nM nitrobenzylmercaptopurine riboside and dipyridamole. These uptake studies suggest that ENT2 is involved in [³H]adenosine uptake by TR-iBRB2 cells. Quantitative real-time PCR revealed that the expression of ENT2 mRNA is 5.5-fold greater than that of ENT1 mRNA. An in vivo study suggested that [³H]adenosine is transported from the blood to the retina and significantly inhibited by adenosine and thymidine. The results of this study show that ENT2 most likely mediates adenosine transport at the inner BRB and is expected to play an important role in regulating the adenosine concentration in the retina.