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.     

Brain distribution of 6-mercaptopurine is regulated by the efflux transport system in the blood-brain barrier

6-mercaptopurine (6-MP) has been used clinically for 40 years to maintain remission in patients with acute lymphoblastic leukemia (ALL). However, central nervous system (CNS) relapses frequently occur in patients with ALL who continuously receive anticancer drugs, including 6-MP, during remission maintenance therapy. The cause of such CNS relapse is not well understood. One possible reason may involve the restricted distribution of 6-MP in the brain. This study, therefore, investigates the blood-brain barrier (BBB) transport which largely regulates 6-MP distribution in the brain using a quantitative microdialysis technique and centers on the efflux transport of 6-MP across the BBB. The brain tissue, cerebrospinal fluid (CSF), or hippocampal interstitial fluid (ISF) concentration of 6-MP was very low compared with the unbound plasma concentration, suggesting that 6-MP distribution in the brain is highly restricted. Kinetic analyses of this BBB transport showed that the efflux clearance from brain ISF to plasma across the BBB (CLout) is approximately 20-times greater than the influx clearance from plasma to brain (CLin). The CLout was significantly reduced by 1mM N-ethylmaleimide (NEM), a sulfhydryl-modifying agent, suggesting the participation of transport protein in the efflux of 6-MP across the BBB. In addition, efflux transport was inhibited by an intracerebral infusion of probenecid (1.5 mM), p-aminohippuric acid (PAH, 3.0 mM), benzoate (3.6 mM), or salicylate (3.7 mM) administered through a microdialysis probe, but neither choline (0.8 mM) nor tetraethylammonium (TEA, 0.7 mM) had any effect. These data suggest that the restricted 6-MP brain distribution may be ascribed to efficient efflux from the brain, possibly via both the organic anion transport system, shared with probenecid and PAH, and the monocarboxylic acid transport system, shared with benzoate and salicylate.     

Blood–Brain Barrier Transport of Valproic Acid

Valproic acid distribution in brain is less than that of other anticonvulsants such as phenytoin or phenobarbital. Possible mechanisms for this decreased distribution space in brain include (a) increased plasma protein binding of valproate relative to the other anticonvulsants and (b) asymmetric blood-brain barrier (BBB) transport of valproate such that the brain-to-blood flux exceeds the blood-to-brain flux. These mechanisms are investigated in the present studies using the intracarotid injection technique in rats and rabbits. In the rat, the brain uptake index (BUI) of [14C]valproate relative to [3H]water is 51 +/- 6%, indicating the blood-to-brain transport of water is twofold greater than that of valproate. However, the BUI of [14C]valproate relative to [3H]water decreased with time after carotid injection during a 4-min washout period, which indicates that brain-to-blood transport of valproate is greater than that of water. This suggests that the permeability of the BBB to valproate is polarized, with antiluminal permeability being much greater than luminal permeability. In rabbits, the BUI of [14C]valproate is 47 +/- 7% in newborns and 17 +/- 6% in adult animals. However, the high drug extraction in newborns may be attributed to decreased cerebral blood flow in the neonate as the BBB permeability-surface area (PS) products are unchanged (e.g., PS = 0.13 and 0.11 ml min-1 X g-1 in the newborn and adult rabbit, respectively). With regard to plasma protein binding effects on valproate transport, brain valproate uptake was also measured in the presence of human, lamb, pig, rat, horse, goat, hamster, dog, and mouse sera. Higher brain uptakes were observed when the unbound fraction of drug increased. However, our data indicate that a fraction of the valproic acid entering the capillaries bound to plasma proteins had the capacity to equilibrate with brain because of enhanced drug dissociation from albumin in the brain microcirculation. Since plasma protein-bound valproate is available for uptake by brain, the major factor underlying the diminished distribution of the drug in brain appears to be the asymmetric transport properties of the BBB to valproic acid.     

Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.     

Blood-to-brain influx transport of nicotine at the rat blood–brain barrier: Involvement of a pyrilamine-sensitive organic cation transport process

Nicotine is the most potent neural pharmacological alkaloid in tobacco, and the modulation of nicotine concentration in the brain is important for smoking cessation therapy. The purpose of this study was to elucidate the net flux of nicotine transport across the blood–brain barrier (BBB) and the major contributor to nicotine transport in the BBB. The in vivo brain-to-blood clearance was determined by a combination of the rat brain efflux index method and a rat brain slice uptake study, and the blood-to-brain transport of nicotine was evaluated by in vivo vascular injection in rats and a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13 cells) as an in vitro model of the rat BBB. The blood-to-brain nicotine influx clearance was obtained by integration plot analysis as 272 μL/(min g brain), and this value was twofold greater than the brain-to-blood efflux clearance (137 μL/(min g brain)). Thus, it is suggested that the net flux of nicotine transport across the BBB is dominated by blood-to-brain influx transport. In vivo blood-to-brain nicotine transport was inhibited by pyrilamine. [³H]Nicotine uptake by TR-BBB13 cells exhibited time-, temperature-, and concentration-dependence with a K_m value of 92 μM. Pyrilamine competitively inhibited nicotine uptake by TR-BBB13 cells with a K_i value of 15 μM, whereas substrates and inhibitors of organic cation transporters had little effect. These results suggest that pyrilamine-sensitive organic cation transport process(es) mediate blood-to-brain influx transport of nicotine at the BBB, and this is expected to play an important role in regulating nicotine-induced neural responses.     

Efflux of a suppressive neurotransmitter, GABA, across the blood-brain barrier

In this study, GABA efflux transport from brain to blood was estimated by using the brain efflux index (BEI) method. [3H]GABA microinjected into parietal cortex area 2 (Par2) of the rat brain was eliminated from the brain with an apparent elimination half-life of 16.9 min. The blood-brain barrier (BBB) efflux clearance of [3H]GABA was at least 0.153 mL/min/g brain, which was calculated from the elimination rate constant (7.14 x 10(-2) x min(-1)) and the distribution volume in the brain (2.14 mL/g brain). Direct comparison of the apparent BBB influx clearance [3H]GABA (9.29 microL/min/g brain) and the apparent efflux clearance (153 microL/min/g brain) indicated that the efflux clearance was at least 16-fold greater than the influx clearance. In order to reduce the effect of metabolism in the neuronal cells following intracerebral microinjection, we determined the apparent efflux of [3H]GABA in the presence of nipecotic acid, a GABA transport inhibitor in parenchymal cells, using the BEI method. Under such conditions, the elimination of [3H]GABA across the BBB showed saturation and inhibition by probenecid in the presence of nipecotic acid. Furthermore, the uptake of [3H]GABA by MBEC4 cells was inhibited by GABA, taurine, beta-alanine and nipecotic acid in a concentration-dependent manner. It is likely that GABA inhibits the first step in the abluminal membrane uptake by brain endothelial cells, and that probenecid selectively inhibits the luminal membrane efflux transport process from the brain capillary endothelial cells based on the in vivo and in vitro evidence. The BBB acts as the efflux pump for GABA to reduce the brain interstitial fluid concentration.     

Brain-to-blood efflux transport of estrone-3-sulfate at the blood-brain barrier in rats

Efflux transport of estrogens such as estrone-3-sulfate (E1S), and estrone (E1) across the blood-brain barrier (BBB) was evaluated using the Brain Efflux Index (BEI) method. The apparent BBB efflux rate constant (Keff) of [3H]E1S, and [3H]E1 was 6.63 x 10(-2) +/- 0.77 x 10(-2) min(-1), and 6.91 x 10(-2) +/- 1.23 x 10(-2) min(-1), respectively. The efflux transport of [3H]E1S from brain across the BBB was a saturable process with Michaelis constant (Km) of 96.0 +/- 34.4 microM and 93.4 +/- 22.0 microM estimated by two different methods. By determining [3H]E1S metabolites using high performance liquid chromatography (HPLC) after intracerebral injection, significant amounts of [3H]E1S were found in the jugular venous plasma, providing direct evidence that most of [3H]E1S is transported from brain across the BBB in intact form. To compare the apparent efflux clearance across the BBB of E1S with that of E1, the brain distribution volume of E1S and E1 was estimated using the brain slice uptake method. The apparent efflux clearance of [3H]E1S was determined to be 74.9 +/- 3.8 microl/(min x g brain) due to the distribution volume of 1.13 +/- 0.06 ml/g brain. By contrast, the apparent efflux clearance of E1 was more than 227 +/- 3 microl/(min x g brain), since the distribution volume of [3H]E1 at 60 min was 3.28 +/- 0.13 ml/g. The E1S efflux transport process was inhibited by more than 40% by coadministration of bile acids (taurocholate, and cholate), and organic anions (sulfobromophthalein, and probenecid), whereas other organic anions did not affect the E1S efflux transport. The [3H]E1S efflux was significantly reduced by 48.6% after preadministration of 5 mM dehydroepiandrosterone sulfate. These results suggest that E1S is transported from brain to the circulating blood across the BBB via a carrier-mediated efflux transport system.     

ATP-binding cassette transporter A1 (ABCA1) deficiency does not attenuate the brain-to-blood efflux transport of human amyloid-beta peptide (1-40) at the blood-brain barrier

ATP-binding cassette transporter A1 (ABCA1) mediates apolipoprotein-dependent cholesterol release from cellular membranes. Recent studies using ABCA1 knockout mice have demonstrated that ABCA1 affects amyloid-beta peptide (A beta) levels in the brain and the production of senile plaque. Cerebral A beta(1-40) was eliminated from the brain to the circulating blood via the blood-brain barrier (BBB), which expresses ABCA1. Therefore, in the present study, we examined whether ABCA1 affects the brain-to-blood efflux transport of human A beta(1-40)(hA beta(1-40)) at the BBB. The apparent uptake of [125I]hA beta(1-40) into ABCA1-expressing HEK293 cells was not significantly different from that into parental HEK293 cells. In addition, the apparent uptake was not significantly affected even in the presence of apolipoprotein A-I as a cholesterol release acceptor. Moreover, [125I]hA beta(1-40) elimination from mouse brain across the BBB was not significantly different between ABCA1-deficient and wild-type mice 60 min after its administration into the cerebrum. These results suggest that ABCA1 does not directly transport hA beta(1-40) and a deficiency of ABCA1 does not attenuate the brain-to-blood efflux transport of hA beta(1-40) across the BBB.     

In vivo delivery of small interfering RNA targeting brain capillary endothelial cells

Brain capillary endothelial cells (BCECs) play an important role in blood-brain barrier (BBB) functions and pathophysiologic mechanisms in brain ischemia and inflammation. We try to suppress gene expression in BCECs by intravenous application of small interfering RNA (siRNA). After injection of large dose siRNA with hydrodynamic technique to mouse, suppression of endogenous protein and the BBB function of BCECs was investigated. The brain-to-blood transport function of organic anion transporter 3 (OAT3) that expressed in BCECs was evaluated by Brain Efflux Index method in mouse. The siRNA could be delivered to BCECs and efficiently inhibited endogenously expressed protein of BCECs. The suppression effect of siRNA to OAT3 is enough to reduce the brain-to-blood transport of OAT3 substrate, benzylpenicillin at BBB. The in vivo siRNA-silencing method with hydrodynamic technique may be useful for the study of BBB function and gene therapy targeting BCECs.     

Blood-Brain Barrier Produces Significant Efflux of L-Aspartic Acid but Not D-Aspartic Acid

The brain efflux index method has been used to clarify the mechanism of efflux transport of acidic amino acids such as L-aspartic acid (L-Asp), L-glutamic acid (L-Glu), and D-aspartic acid (D-Asp) across the blood-brain barrier (BBB). About 85% of L-[3H]Asp and 40% of L-[3H]Glu was eliminated from the ipsilateral cerebrum within, respectively, 10 and 20 min of microinjection into the brain. The efflux rate constant of L-[3H]Asp and L-[3H]Glu was 0.207 and 0.0346 min(-1), respectively. However, D-[3H]Asp was not eliminated from brain over a 20-min period. The efflux of L-[3H]Asp and L-[3H]Glu was inhibited in the presence of excess unlabeled L-Asp and L-Glu, whereas D-Asp did not inhibit either form of efflux transport. Aspartic acid efflux across the BBB appears to be stereospecific. Using a combination of TLC and the bioimaging analysis, attempts were made to detect the metabolites of L-[3H]Asp and L-[3H]Glu in the ipsilateral cerebrum and jugular vein plasma following a microinjection into parietal cortex, area 2. Significant amounts of intact L-[3H]Asp and L-[3H]Glu were found in all samples examined, including jugular vein plasma, providing direct evidence that at least a part of the L-Asp and L-Glu in the brain interstitial fluid is transported across the BBB in the intact form. To compare the transport of acidic amino acids using brain parenchymal cells, brain slice uptake studies were performed. Although the slice-to-medium ratio of D-[3H]Asp was the highest, followed by L-[3H]Glu and L-[3H]Asp, the initial uptake rate did not differ for both L-[3H]Asp and D-[3H]Asp, suggesting that the uptake of aspartic acid in brain parenchymal cells is not stereospecific. These results provide evidence that the BBB may act as an efflux pump for L-Asp and L-Glu to reduce the brain interstitial fluid concentration and act as a static wall for D-Asp.     

Brain to blood efflux transport of adenosine: blood-brain barrier studies in the rat

The blood-brain barrier (BBB) efflux transport of [(14)C] adenosine was studied using the brain efflux index (BEI) technique. BEI increased linearly over the first 2 min after injection, with deviation from linearity thereafter; 90.12 +/- 1.5% of the injected [(14)C] radioactivity remained within the brain after 20 min. The remaining tracer appears to be mainly intracellular, trapped by phosphorylation, as an almost linear increase of BEI over 20 min was observed after intracerebral injection of [(14)C] adenosine together with 5-iodo tubercidin. The BBB efflux clearance of [(14)C] radioactivity was estimated to be 27.62 +/- 5.2 micro L/min/g, almost threefold higher than the BBB influx clearance estimated by the brain uptake index technique. High-performance liquid chromatography (HPLC) analysis of blood plasma collected from the jugular vein after the intracerebral injection revealed metabolic breakdown of [(14)C] adenosine into nucleobases. The BBB efflux transport was saturable with apparent K(m) = 13.22 +/- 1.75 micro m and V(max) = 621.07 +/- 71.22 pmole/min/g, which indicated that BBB efflux in vivo is 6.2-12p mole/min/g, negligible when compared to the reported rate of adenosine uptake into neurones/glia. However, these kinetic parameters also suggest that under conditions of elevated ISF adenosine in hypoxia/ischaemia, BBB efflux transport could increase up to 25% of the uptake into neurones/glia and become an important mechanism to oppose the rise in ISF concentration. HPLC-fluorometry detected 93.6 +/- 5.25 nm of adenosine in rat plasma, which is 17- to 220-fold lower than the reported K(m) of adenosine BBB influx in rat. Together with the observed rapid degradation inside endothelial cells, this indicated negligible BBB influx of intact adenosine under resting conditions. Cross-inhibition studies showed that unlabelled inosine, adenine and hypoxanthine caused a decrease in BBB efflux of [(14)C] radioactivity in a concentration-dependent manner, with K(i) of 16.7 +/- 4.88, 65.1 +/- 14.1 and 71.1 +/- 16.9 micro m, respectively. This could be due to either competition of unlabelled molecules with [(14)C] adenosine or competition with its metabolites hypoxanthine and adenine for the same transport sites.     

Regulation by protein kinase C of organic anion transport driven by rat organic anion transporter 3 (rOAT3)

The organic anion transporter 3 (rOAT3) is a multispecific OAT localized at the basolateral membrane of the proximal tubule. The purpose of this study was to elucidate the role of protein kinase C (PKC) in the regulation of organic anion transport driven by rOAT3 and its mechanism of action. For this purpose, we established and utilized cells derived from the second segment of proximal tubule from mice stably expressing rOAT3 (S2 rOAT3). Phorbol 12-myristate 13-acetate (PMA), a PKC stimulator, attenuated the cellular uptake of estrone sulfate (ES), a prototype organic anion for rOAT3, in a dose- and time-dependent manner. PMA treatment resulted in a decrease in the Vmax, but not the Km of uptake of ES in S2 rOAT3. Treatment of S2 rOAT3 with other PKC stimulators or diacylglycerols also inhibited the uptake of ES, whereas that with an inactive phorbol ester did not. Chelerythrine chloride, a PKC inhibitor, reversed the PMA-induced decrease in uptake of ES in S2 rOAT3. These results suggest that PKC activation downregulates rOAT3-mediated organic anion transport. This down-regulation may be due to the inhibition of translocation or internalization of the rOAT3 protein, resulting in the decrease in the Vmax of rOAT3-mediated organic anion transport.     

Regional distribution of adenosine uptake in guinea-pig brain slices and the effect of some inhibitors: Evidence for nitrobenzylthioinosine-sensitive and insensitive sites?

The accumulation of [2-³H]adenosine was measured in slices prepared from 7 regions of the guinea-pig central nervous system. There was a similar level of uptake in forebrain regions (cerebral cortex, striatum, hippocampus and midbrain), a lower level in the cerebellum, with lowest uptake in the pons-medulla and spinal cord. Uptake in all regions was strongly inhibited by the nucleoside transport inhibitor dipyridamole and by 5-iodotubercidin, an adenosine kinase inhibitor. The activity of adenosine kinase was similar in crude supernatants prepared from 8 regions of the guinea-pig and rat brain, with the exception of the spinal cord (lower activity than other regions in the guinea-pig CNS) and olfactory bulb (higher activity than other regions in the rat CNS). 5-Nitrobenzylthioinosine (NBMPR) and related thiopurines produced about 50% inhibition of adenosine uptake into guinea-pig cerebral cortex slices at 200 nM but increasing the concentration did not produce significant further inhibition. [³H]NBMPR has been proposed as a useful tight-binding ligand for nucleoside transport sites in various tissues but it is suggested that the distribution of such binding sites in different regions of the CNS may not directly reflect the adenosine uptake capacity of these regions1. Data suggest that there may be NBMPR-sensitive and -insensitive sites. Results confirm those of previous studies which suggest that intracellular adenosine kinase plays an important part in the uptake of adenosine in guinea-pig brain. The relatively homogeneous distribution of adenosine uptake activity in the brain contrasts with the heterogeneous distribution of A1-adenosine receptors in the CNS.     

Evidence for an active transport of morphine-6-beta-d-glucuronide but not P-glycoprotein-mediated at the blood-brain barrier

Morphine-6-beta-d-glucuronide (M6G) is an active metabolite of morphine with high analgesic potency despite a low blood-brain barrier (BBB) permeability. The aim of the study was to elucidate its transport mechanism across the BBB. We first checked if M6G was effluxed by the P-glycoprotein (P-gp), as previously reported by others. Second, we investigated the role of anionic transporters like the multidrug resistance-associated protein mrp1 and the glucose transporter GLUT-1. The brain uptake of [14C]M6G was measured by the in situ brain perfusion technique in wild-type and deficient mice [mdr1a(-/-) and mrp1(-/-)], with and without probenecid, digoxin, PSC833 or d-glucose. No difference was found between P-gp and mrp1 competent and deficient mice. The brain uptake of [14C]M6G co-perfused with probenecid in wild-type mice was not significantly different from that found in group perfused with [14C]M6G alone. The co-perfusion of [14C]M6G with digoxin or PSC833 was responsible of a threefold decrease of its uptake in mdr1a competent and deficient mice, suggesting that another transporter than P-gp and sensitive to digoxin and PSC833, may be involved. The co-perfusion of [14C]M6G with d-glucose revealed a threefold decrease in M6G uptake. In conclusion, P-gp and mrp1 are not involved in the transport of M6G at the BBB level in contrast to GLUT-1 and a digoxin-sensitive transporter (probably oatp2), which can actively transport M6G but with a weak capacity.     

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.     

Involvement of rat organic anion transporter 3 (rOAT3) in cephaloridine-induced nephrotoxicity: in comparison with rOAT1

This study was performed to elucidate the possible involvement of organic anion transporter 3 (OAT3) in cephaloridine (CER)-induced nephrotoxicity and compare the substrate specificity between rOAT3 and rat OAT1 (rOAT1) for various cephalosporin antibiotics, using proximal tubule cells stably expressing rOAT3 (S2 rOAT3) and rOAT1 (S2 rOAT1). S2 rOAT3 exhibited a CER uptake and a higher susceptibility to CER cytotoxicity than did mock, which was recovered by probenecid. Various cephalosporin antibiotics significantly inhibited both estrone sulfate uptake in S2 rOAT3 and para-aminohippuric acid uptake in S2 rOAT1. The Ki values of CER, cefoperazone, cephalothin and cefazolin for rOAT3- and rOAT1-mediated organic anion transport ranged from 0.048 to 1.14 mM and from 0.48 to 1.32 mM, respectively. These results suggest that rOAT3, at least in part, mediates CER uptake and CER-induced nephrotoxicity as rOAT1. There was some difference of affinity between rOAT3 and rOAT1 for cephalosporin antibiotics.     

Peripheral inflammatory hyperalgesia modulates morphine delivery to the brain: a role for P-glycoprotein

P-glycoprotein (Pgp, ABCB1) is a critical efflux transporter at the blood-brain barrier (BBB) where its luminal location and substrate promiscuity limit the brain distribution of numerous therapeutics. Moreover, Pgp is known to confer multi-drug resistance in cancer chemotherapy and brain diseases, such as epilepsy, and is highly regulated by inflammatory mediators. The involvement of inflammatory processes in neuropathological states has led us to investigate the effects of peripheral inflammatory hyperalgesia on transport properties at the BBB. In the present study, we examined the effects of lambda-carrageenan-induced inflammatory pain (CIP) on brain endothelium regulation of Pgp. Western blot analysis of enriched brain microvessel fractions showed increased Pgp expression 3 h post-CIP. In situ brain perfusion studies paralleled these findings with decreased brain uptake of the Pgp substrate and opiate analgesic, [(3)H] morphine. Cyclosporin A-mediated inhibition of Pgp enhanced the uptake of morphine in lambda-carrageenan and control animals. This indicates that the CIP induced decrease in morphine transport was the result of an increase in Pgp activity at the BBB. Furthermore, antinociception studies showed decreased morphine analgesia following CIP. The observation that CIP modulates Pgp at the BBB in vivo is critical to understanding BBB regulation during inflammatory disease states.