Shin-ichi Akanuma

Lipophilicity and Transporter Influence on Blood-Retinal Barrier Permeability: A Comparison with Blood-Brain Barrier Permeability

ABSTRACTPurposeTo determine the lipophilicity trend line from the relationship between the blood-retinal barrier (BRB) permeability and the lipophilicity of permeants and compare it with that of the blood-brain barrier (BBB).MethodsThe retinal (RUI) and brain uptake index (BUI) of 26 radiolabeled compounds across the rat BRB and BBB, respectively, were measured using the carotid artery injection method.ResultsRUI was determined using 13 compounds expected to be transported from blood to the retina by passive diffusion and with a log n-octanol/Ringer distribution coefficient (DC) ranging from −2.56 to 2.48. The RUI values were correlated with the log of the DC [RUI = 46.2 × exp (0.515 × log DC), r2 = 0.807]. A similar trend was obtained between BUI and lipophilicity. The RUI value for substrates of the influx transporters and P-glycoprotein (P-gp) was greater and smaller than the lipophilicity trend line, respectively. In contrast, [3H]verapamil, which is a substrate of P-gp, has a greater RUI value than the lipophilicity trend line, but not for BUI, suggesting that the BRB has an influx transport system for verapamil.ConclusionsThe lipophilicity trend line constructed from the RUI and DC values is considered to reflect the transport properties of drugs undergoing passive diffusion across the BRB.

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/(ming brain), and this value was twofold greater than the brain-to-blood efflux clearance (137 μL/(ming 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. [(3)H]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.

Blood-to-retina transport of biotin via Na+-dependent multivitamin transporter (SMVT) at the inner blood-retinal barrier

The purpose of this study was to elucidate the mechanisms of biotin transport across the inner blood-retinal barrier (inner BRB). [(3)H]Biotin transport in the retina across the inner BRB was examined using an in vivo integration plot and retinal uptake index analyses in rats. The transport mechanism was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells) as an in vitro inner BRB model. The apparent influx permeability clearance (K(in)) per gram retina of [(3)H]biotin was found to be 5.55 microL/(min g retina). The K(in) of [(3)H]biotin was 8.9-fold greater than that of [(3)H]D-mannitol, a non-permeable paracellular marker. [(3)H]Biotin uptake by the retina was found to be significantly inhibited by biotin and pantothenic acid, supporting carrier-mediated influx transport of biotin at the inner BRB. [(3)H]Biotin uptake by TR-iBRB2 cells was Na(+)-, temperature-, and concentration-dependent with a K(m) of 146 microM. These forms of transport were significantly inhibited by Na(+)-dependent multivitamin transporter (SMVT) substrates such as biotin, pantothenic acid, lipoic acid, and desthiobiotin. These transport properties are consistent with those of biotin transport by SMVT. SMVT mRNA was expressed in TR-iBRB2 cells and isolated rat retinal vascular endothelial cells. Our findings suggest that SMVT is involved in the transport of biotin from the circulating blood to the retina across the inner BRB.

Involvement of LAT1 and LAT2 in the high- and low-affinity transport of L-leucine in human retinal pigment epithelial cells (ARPE-19 cells)

System L, which is encoded by LAT1 and LAT2, is an amino acid transport system that transports neutral amino acids, including several essential amino acids in an Na+-independent manner. Due to its broad substrate selectivity, system L has been proposed to mediate the transport of amino-acid-related drugs across the blood-tissue barriers. We characterized L-leucine transport and its corresponding transporter in a human retinal pigment epithelial cell line (ARPE-19 cells) as an in vitro model of the outer blood-retinal barrier. [3H]L-leucine uptake by ARPE-19 cells took place in an Na+-, Cl(-)-independent and saturable manner with K(m) values of 8.71 and 220 microM. This process was more potently cis-inhibited by substrates of LAT1 than those of LAT2. [3H]L-leucine efflux from ARPE-19 cells was trans-stimulated by substrates of LAT1 and LAT2 through the obligatory exchange mechanism of system L. Although RT-PCR analysis demonstrated that LAT1 and LAT2 mRNA are expressed in ARPE-19 cells, the LAT1 mRNA concentration is 42-fold higher than that of LAT2. Moreover, immunoblot analysis demonstrated that LAT1 is expressed in ARPE-19 cells. In conclusion, although the transport function of LAT1 is greater than that of LAT2, LAT1 and LAT2 are involved in L-leucine transport in ARPE-19 cells.

Role of the blood–cerebrospinal fluid barrier transporter as a cerebral clearance system for prostaglandin E2 produced in the brain

An increasing level of prostaglandin (PG) E2 is involved in the progression of neuroinflammation induced by ischemia and bacterial infection. Although an imbalance in the rates of production and clearance of PGE2 under these pathological conditions appears to affect the concentration of PGE2 in the cerebrospinal fluid (CSF), the regulatory system remains incompletely understood. The purpose of this study was to investigate the cellular system of PGE2 production via microsomal PGE synthetase‐1 (mPGES‐1), the inducible PGE2‐generating enzyme, and PGE2 elimination from the CSF via the blood–CSF barrier (BCSFB). Immunohistochemical analysis revealed that mPGES‐1 was expressed in the soma and perivascular sheets of astrocytes, pia mater, and brain blood vessel endothelial cells, suggesting that these cells are local production sites of PGE2 in the CSF. The in vivo PGE2 elimination clearance from the CSF was eightfold greater than that of d‐mannitol, which is considered to reflect CSF bulk flow. This process was inhibited by the simultaneous injection of unlabeled PGE2 and β‐lactam antibiotics, such as benzylpenicillin, cefazolin, and ceftriaxone, which are substrates and/or inhibitors of organic anion transporter 3 (OAT3). The characteristics of PGE2 uptake by the isolated choroid plexus were at least partially consistent with those of OAT3. OAT3 was able to mediate PGE2 transport with a Michaelis–Menten constant of 4.24 μM. These findings indicate that a system regulating the PGE2 level in the CSF involves OAT3‐mediated PGE2 uptake by choroid plexus epithelial cells, acting as a cerebral clearance pathway via the BCSFB of locally produced PGE2.

Propranolol transport across the inner blood-retinal barrier: potential involvement of a novel organic cation transporter.

The influx transport of propranolol across the inner blood-retinal barrier (BRB) was investigated. In the in vivo analysis of carotid artery single-injection method, [(3) H]propranolol uptake by the retina was greater than that of an internal reference compound, and was reduced by several organic cations. In the in vitro uptake study, TR-iBRB2 cells, an in vitro model of the inner BRB, showed a time-, concentration-, pH- and temperature-dependent [(3) H]propranolol uptake, suggesting the involvement of a carrier-mediated transport process in the influx of propranolol across the inner BRB. In the inhibition study, various organic cations, including drugs and candidates for the treatment of the retinal diseases, inhibited the [(3) H]propranolol uptake by TR-iBRB2 cells with no significant effects by the substrates and inhibitors of well-characterized organic cation transporters, suggesting that the influx transport of propranolol is performed by a novel transporter at the inner BRB. An analysis of the relationship between the inhibitory effect and the lipophilicity of inhibitors suggests a lipophilicity-dependent inhibitory effect of amines on the [(3) H]propranolol uptake by TR-iBRB2 cells. These results showed that influx transport of propranolol across the inner BRB is performed by a carrier-mediated transport process, suggesting the involvement of a novel organic cation transporter.

Characteristics of glycine transport across the inner blood–retinal barrier

Although glycine plays a pivotal role in neurotransmission and neuromodulation in the retina and is present in high concentration in the retina, the source of retinal glycine is still unclear. The purpose of the present study was to investigate glycine transport across the inner blood-retinal barrier (inner BRB). [(14)C]Glycine transport at the inner BRB was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells) as an in vitro model of the inner BRB and in vivo vascular injection techniques. [(14)C]Glycine uptake by TR-iBRB2 cells was Na(+)- and Cl(-)-dependent, and concentration-dependent with Michaelis-Menten constants of 55.4 microM and 8.02 mM, and inhibited by glycine transporter 1 (GlyT1) and system A inhibitors. These uptake studies suggest that GlyT1 and system A are involved in [(14)C]glycine uptake by TR-iBRB2 cells. RT-PCR analysis demonstrated that GlyT1 and system A (encoding ATA 1 and ATA2) mRNA are expressed in TR-iBRB2 cells. An in vivo study suggested that [(14)C]glycine is transported from blood to the retina whereas [(14)C]alpha-methylaminoisobutyric acid, a selective substrate for system A, is not. In conclusion, GlyT1 most likely mediates glycine transport at the inner BRB and is expected to play an important role in regulating the glycine concentration in the neural retina.

γ-Aminobutyric Acid Transporter 2 Mediates the Hepatic Uptake of Guanidinoacetate, the Creatine Biosynthetic Precursor, in Rats

Guanidinoacetic acid (GAA) is the biosynthetic precursor of creatine which is involved in storage and transmission of phosphate-bound energy. Hepatocytes readily convert GAA to creatine, raising the possibility that the active uptake of GAA by hepatocytes is a regulatory factor. The purpose of this study is to investigate and identify the transporter responsible for GAA uptake by hepatocytes. The characteristics of [14C]GAA uptake by hepatocytes were elucidated using the in vivo liver uptake method, freshly isolated rat hepatocytes, an expression system of Xenopus laevis oocytes, gene knockdown, and an immunohistochemical technique. In vivo injection of [14C]GAA into the rat femoral vein and portal vein results in the rapid uptake of [14C]GAA by the liver. The uptake was markedly inhibited by γ-aminobutyric acid (GABA) and nipecotinic acid, an inhibitor of GABA transporters (GATs). The characteristics of Na+- and Cl−-dependent [14C]GAA uptake by freshly isolated rat hepatocytes were consistent with those of GAT2. The Km value of the GAA uptake (134 µM) was close to that of GAT2-mediated GAA transport (78.9 µM). GABA caused a marked inhibition with an IC50 value of 8.81 µM. The [14C]GAA uptake exhibited a significant reduction corresponding to the reduction in GAT2 protein expression. GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. This distribution pattern was consistent with that of the creatine biosynthetic enzyme, S-adenosylmethionine∶guanidinoacetate N-methyltransferase. GAT2 makes a major contribution to the sinusoidal GAA uptake by periportal hepatocytes, thus regulating creatine biosynthesis in the liver.

Function and regulation of taurine transport in Müller cells under osmotic stress

In the retina, taurine works as an osmolyte to exert a neuroprotective function, and it has been proposed that Müller cells, a major type of retinal glial cells, are involved in the osmolarity regulation of retinal neural cells by controlling the taurine concentration in retinal extracellular fluid (ECF). However, the detailed mechanism of taurine transport in Müller cells has not fully examined, and we investigated this using a conditionally immortalized rat Müller cell line (TR-MUL5 cells). In the uptake study, TR-MUL5 cells exhibited Na(+)-, Cl(-)-dependent [(3)H]taurine uptake with a K(m) of 37.9μM. The [(3)H]taurine uptake by TR-MUL5 cells was strongly inhibited by β-alanine and hypotaurine, substrates of taurine transporter TAUT (SLC6A6), and RT-PCR and immunoblot analyses demonstrated the expression of TAUT in Müller cells, suggesting the involvement of TAUT in taurine uptake by Müller cells. In the efflux study, [(3)H]taurine efflux by TR-MUL5 cells under hypotonic conditions was significantly greater than that under isotonic conditions, and significantly enhanced by sphingosine 1-phosphate (S1P), suggesting that the volume-sensitive taurine release is enhanced via G protein-coupled receptors (GPCRs) in Müller cells. Furthermore, [(3)H]taurine efflux by TR-MUL5 cells under hypotonic conditions was significantly inhibited in the presence of the volume-sensitive organic osmolyte and anion channel (VSOAC) inhibitor, suggesting a major contribution of VSOAC to the volume-sensitive taurine release by Müller cells. This is the first description of the detailed mechanism of taurine transport in Müller cells, indicating a possible function of Müller cells in retinal neuroprotection by regulating osmolarity of retinal ECF.

β-Alanine and l-histidine transport across the inner blood-retinal barrier: Potential involvement in l-carnosine supply

The supply of L-carnosine, a bioactive dipeptide of β-alanine and l-histidine, to the retina across the blood-retinal barrier (BRB) was studied. The in vivo and in vitro studies revealed low uptake activities for [(3)H]Gly-Sar, a representative dipeptide, suggesting that l-carnosine transport plays only a minor role at the BRB. The in vivo study using rats showed approximately 18- and 23-fold greater retinal uptake indexes (RUI) for [(3)H]β-alanine and [(3)H]l-histidine compared with that of a paracellular marker, respectively. The RUI of [(3)H]β-alanine was taurine- and γ-aminobutyric acid-sensitive, and the in vitro uptake by TR-iBRB2 cells showed time- concentration- and temperature-dependent [(3)H]β-alanine uptake, suggesting that a carrier-mediated process was involved in β-alanine transport across the inner BRB. [(3)H]β-Alanine uptake was inhibited by taurine and β-guanidinopropionic acid, suggesting that taurine transporter (TAUT/SLC6A6) is responsible for the influx transport of β-alanine across the inner BRB. Regarding l-histidine, the l-leucine-sensitive RUI of [(3)H]l-histidine was identified, and the in vitro [(3)H]l-histidine uptake by TR-iBRB2 cells suggested that a carrier-mediated process was involved in l-histidine transport across the inner BRB. The inhibition profile suggested that L-type amino acid transporter (LAT1/SLC7A5) is responsible for the influx transport of l-histidine across the inner BRB. These results show that the influx transports of β-alanine and l-histidine across the inner BRB is carried out by TAUT and LAT1, respectively, suggesting that the retinal l-carnosine is supplied by enzymatic synthesis from two kinds of amino acids transported across the inner BRB.