Involvement of a Novel Organic Cation Transporter in Verapamil Transport Across the Inner Blood-Retinal Barrier

Purpose

To clarify the transport and inhibition characteristics involved in verapamil transport across the inner blood-retinal barrier (inner BRB).

Methods

The transport of [³H]verapamil across the inner BRB was investigated using retinal uptake index and integration plot analyses in rats. The detailed transport characteristics were studied using TR-iBRB2 cells, a conditionally immortalized rat retinal capillary endothelial cell line that is an in vitro model of the inner BRB.

Results

The apparent influx permeability clearance of [³H]verapamil was 614 μL/(min·g retina), which is 4.7-fold greater than that of brain. The retinal uptake of [³H]verapamil was slightly increased by 3 mM verapamil and 10 mM qunidine and inhibited by 40 mM pyrilamine, supporting the carrier-mediated efflux and influx transport of verapamil across the inner BRB. TR-iBRB2 cells exhibited a concentration-dependent uptake of [³H]verapamil with a K _m of 61.9 μM, and the uptake was inhibited by several cations, such as pyrilamine, exhibiting a different profile from the identified transporters. These transport properties suggest that verapamil transport at the inner BRB takes place via a novel organic cation transporter.

Conclusions

Our findings suggest that a novel organic cation transporter is involved in verapamil transport from the blood to the retina across the inner BRB.     

Effects of Bisphosphonates on Glucose Transport in a Conditionally Immortalized Rat Retinal Capillary Endothelial Cell Line (TR-iBRB Cells)

The objective of the present study was to elucidate the effect of bisphosphonates, anti-osteoporosis agents, on glucose uptake in retinal capillary endothelial cells under normal and high glucose conditions. The change of glucose uptake by pre-treatment of bisphosphonates at the inner blood-retinal barrier (iBRB) was determined by measuring cellular uptake of [³H]3-O-methyl glucose (3-OMG) using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB cells) under normal and high glucose conditions. [³H]3-OMG uptake was inhibited by simultaneous treatment of unlabeled D-glucose and 3-OMG as well as glucose transport inhibitor, cytochalasin B. On the other hand, simultaneous treatment of alendronate or pamidronate had no significant inhibitory effect on [³H]3-OMG uptake by TR-iBRB cells. Under high glucose condition of TR-iBRB cells, [³H]3-OMG uptake was increased at 48 h. However, [³H]3-OMG uptake was decreased significantly by pre-treatment of alendronate or pamidronate compared with the values for normal and high glucose conditions. Moreover, geranylgeraniol (GGOH), a mevalonate pathway intermediate, increased the uptake of [³H]3-OMG reduced by bisphosphonates pre-treatment. But, pre-treatment of histamine did not show significant inhibition of [³H]3-OMG uptake. The glucose uptake may be down regulated by inhibiting the mevalonate pathway with pre-treatment of bisphosphonates in TR-iBRB cells at high glucose condition.     

Blood-to-retina transport of riboflavin via RFVTs at the inner blood-retinal barrier

Riboflavin (vitamin B₂) supply to the retina across the inner blood-retinal barrier (BRB) was investigated. In rats, the apparent influx permeability clearance of [³H]riboflavin (62.8 μL/(min·g retina)) was much higher than that of a non-permeable paracellular marker, suggesting the facilitative influx transport of riboflavin across the BRB. The retinal uptake index (RUI) of [³H]riboflavin was 59.0%, and significantly reduced by flavin adenine dinucleotide (FAD), but not by l-ascorbic acid, suggesting the substrate specificity of riboflavin transport. TR-iBRB2 cells, an in vitro model of the inner BRB, showed a temperature- and concentration-dependent [³H]riboflavin uptake with a K_m of 113 nM, suggesting that the influx transport of riboflavin across the inner BRB involves a carrier-mediated process. [³H]Riboflavin uptake by TR-iBRB2 cells was slightly altered by Na⁺- and Cl⁻-free buffers, suggesting that riboflavin transport at the inner BRB is preferentially Na⁺- and Cl⁻-independent. [³H]Riboflavin uptake by TR-iBRB2 cells was significantly reduced by riboflavin analogues while the uptake remained unchanged by other vitamins. The function and inhibition profile suggested the involvement of riboflavin transporters (SLC52A/RFVT) in riboflavin transport at the inner BRB, and this is supported by expression and knockdown analysis of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3) in TR-iBRB2 cells.     

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, [³H]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 [³H]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 [³H]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 [³H]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.     

Effects of mazindol on rat brain synaptosomal monoamine uptake

The effects of the anorectic drug mazindol on the uptake of [³H]NA and (³H]5HT by rat hypothalamic synaptosomes and the uptake of [³H]DA by rat striatal synaptosomes were investigated. In in vitro studies drugs were added to the incubation medium. In ex vivo experiments drugs were injected i.p. at various times prior to death and synaptosomal [³H] monoamine uptake subsequently determined. Two other anorectics (d-amphetamine and dl-fenfluramine) and two inhibitors of monoamine uptake (chlorimipramine and desipramine) were included for comparative purposes. Mazindol was a potent inhibitor of [³H]NA and [³H]DA uptake in vitro being approx. 0.5 times as potent as desipramine and d-amphetamine respectively. The abilities of mazindol, fenfluramine and desipramine to block the in vitro uptake of [³H]5HT were comparable and all three drugs were appreciably less potent than chlorimipramine. Following 1hr pretreatment, d-amphetamine was the most potent of the five drugs at inhibiting synaptosomal [³H]NA and [³H]DA uptake. Mazindol was approx. 2.5 times more potent than desipramine at blocking [³H]NA uptake. In contrast to the other drugs, pretreatment with large doses of mazindol had essentially no effect on hypothalamic synaptosomal [³H]5HT uptake. Results of ex vivo studies thus confirm in vivo findings that mazindol is a selective inhibitor of rat brain catecholamine uptake.     

Effects of ORG 6582 on monoamine uptake in vitro

In vivo studies have indicated that Org 6582 is a potent long acting selective inhibitor of 5HT uptake. The objective of this study was to investigate the effects of Org 6582 on the uptake of [³H]DA into synaptosome-rich homogenates of rat corpus striatum and on the uptake of [³H]NA or [³H]5HT into synaptosome-rich homogenates of rat hypothalamus. Two experimental approaches were adopted. In one, drugs were directly added to the incubation medium. In the other, rats were injected interperitoneally (i.p.) with the drugs under study at various times prior to death and synaptosomal [³H]monoamine uptake subsequently determined. Org 6582 was a competitive inhibitor of [³H]5HT uptake with a K_i value of 8.9 × 10^?8M. The ability of Org 6582 to inhibit [³H]5HT uptake was sixteen and seventy-two times greater than its effect on[³H]NA and [³H]DA uptake respectively. At 1 hr after injection, Org 6582 equalled fluoxetine and was more potent than chlorimipramine at blocking [³H]5HT uptake. The duration of inhibition of [³H]5HT uptake by chlorimipramine was appreciably shorter than that of either Org 6582 or fluoxetine. In contrast to both desipramine and chlorimipramine, Org 6582 was essentially devoid of effect on [³H]NA uptake at 1 hr after injection. [³H]DA uptake was also unaffected by 1 hr pretreatment with Org 6582. The findings of this study confirm that Org 6582 is a potent long acting selective inhibitor of 5HT uptake.     

Monophosphorylation by deoxycytidine kinase affects apparent cellular uptake of decitabine in HCT116 colon cancer cells

Decitabine (DAC), a nucleoside-related DNA methylation inhibitor, is taken up into cancer cells via equilibrative nucleoside transporter 1 (ENT1), and is then monophosphorylated by deoxycytidine kinase (dCK). In the present study, we examined the contribution of dCK to the uptake of DAC in HCT116 colon cancer cells. Irinotecan and etoposide inhibited the uptake of [³H]-uridine and [³H]-DAC at 10 s and 5 min, while cytarabine and gemcitabine only inhibited that of [³H]-DAC at 5 min. Irinotecan and etoposide inhibited [³H]-DAC uptake in negative control small interfering RNA (siRNA)- or dCK siRNA-transfected cells at 10 s, whereas cytarabine and gemcitabine did not. Cytarabine and gemcitabine inhibited DAC monophosphate generation by the cytosolic proteins of HCT116 cells and recombinant human dCK protein, assessed using polyethylenimine cellulose thin-layered chromatography. Simulations using simple kinetic models showed that apparent DAC uptake in dCK and ENT1 siRNA-treated cells was attributed to its conversion to monophosphates or a decrease in the cellular flux, respectively, and that the apparent uptake of DAC in dCK-knockdown and ENT1-knockdown cells was similar at longer times, but differed at a very short time. These results suggest that the apparent uptake of DAC is affected by ENT1 and dCK in HCT116 cells.     

Riboflavin transport mediated by riboflavin transporters (RFVTs/SLC52A) at the rat outer blood-retinal barrier

The previous in vivo study revealed the carrier-mediated transport of riboflavin (vitamin B₂) across the blood-retinal barrier (BRB). In the present study, the blood-to-retina supply of riboflavin across the outer BRB was assessed in RPE-J cells, a rat-derived in vitro cell model of the outer BRB that is formed by the retinal pigment epithelial cells. In the directional uptake analysis on collagen-coated Transwell® inserts, RPE-J cells showed higher basal-to-cell (B-to-C) uptake (22.8 μL/mg protein) of [³H]riboflavin than apical-to-cell (A-to-C) uptake (13.5 μL/mg protein). RPE-J cells showed concentration- and temperature-dependent uptake of [³H]riboflavin with a K_m of 297 nM, suggesting the involvement of carrier-mediated process in the blood-to-retina transport of riboflavin across the outer BRB. In RPE-J cells, [³H]riboflavin uptake was affected under a K⁺-replacement condition while no effect was observed under a choline-replacement condition and at different pH values. Uptake of [³H]riboflavin by RPE-J cells was markedly reduced by riboflavin, flavin adenine dinucleotide (FAD), and lumichrome with no significant effect noted for other vitamins. The obtained results suggested the involvement of riboflavin transporters (SLC52A/RFVT) at the outer BRB, and this is supported by the expression and knockdown analyses of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3).     

A comparison of the inhibitory effects of new non-tricyclic amine uptake inhibitors on the uptake of norepinephrine and 5-hydroxytryptamine into synaptosomes of the rat brain

The effects of new non-tricyclic amine uptake inhibitors, FS32 and FS97, on the uptake of [³H]-norepinephrine (NE) into the hypothalamic synaptosomes and [³H]-5-hydroxytryptamine (5-HT) into whole brain synaptosomes were studied. Their effects were compared with those of tricyclic antidepressants. The uptake of [³H]-NE was inhibited competitively by FS32 and FS97 with a respective K_i value of 6.5 × 10^?7 M and 3.8 × 10^?7 M. The potency of FS32 and FS97 to inhibit this uptake was almost comparable to that of clomipramine and imipramine, respectively. In the case of [³H]-5-HT uptake, FS32 and FS97 also showed competitive inhibition with a respective K_i value of 2.9 × 10^?6 M and 5.9 × 10^?6M. The ability of FS32 to inhibit [³H]-5-HT uptake was almost equal to that of nortriptyline, while FS97 was two times more potent than iprindole in inhibiting this uptake.     

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 µM. 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. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2475–2482, 2010     

Effects of pyroglutamic acid on corticostriatal glutamatergic transmission

The effects of l-pyroglutamic acid, a molecule structurally derived from l-glutamic acid (Glu), were measured on the high affinity of uptake of glutamic acid from striatal synaptosomes of the rat and on the binding of [l-³H]glutamic acid to striatal membranes. The results showed a competitive inhibition of the high affinity transport of glutamic acid by l-pyroglutamic acid in vitro with no effect on the uptake of γ-aminobutyric acid (GABA). An inhibition of the binding of [l-³H]glutamic acid to striatal membranes was also detected. Significant high affinity uptake of [l-³H]pyroglutamic acid was evident in synaptosomes from the striatum. A regional distribution study of the uptake processes for [l-³H]glutamic acid and [l-³H]pyroglutamic acid in different areas of the brain showed a similar distribution, suggesting that an uptake of [l-³H]pyroglutamic acid, although weak, occurs in glutamatergic nerve terminals. This proposal was further reinforced by measuring the effects of a large cortical lesion involving frontal and parietal areas on the uptake of [l-³H]glutamic acid and [l-³H]pyroglutamic acid in synaptosomes from the striatum. The results showed a large decrease in the uptake processes of both labelled molecules showing that the uptake of [l-³H]pyroglutamic acid, as for glutamic acid mainly occurred in corticostriatal nerve terminals, although other uptake sites are not excluded.     

A note on the blockade of uptake of noradrenaline by 4-chloro-alpha,alpha-dimethylphenethylaminopropan-2-one in rodents

4-Chloro-αα-dimethylphenethylaminopropan-2-one blocked uptake of [³H]noradrenaline in the heart, had no effect on the endogenous levels of catecholamines or 5-hydroxytryptamine in the heart, brain or adrenals and decreased the [³H]noradrenaline-depleting activity of metaraminol, but not reserpine, in rodents. The compound appears to act by interfering with the active transport of noradrenaline through the nerve cell membrane.     

Effect of morphine in vivo on uptake of [3H]choline and release of [3H]acetylcholine from rat striatal synaptosomes

The effect of morphine on the rat striatal cholinergic system was investigated in vitro by measuring the rates of [³H]choline uptake and [³H]acetylcholine release in striatal synaptosomes after in vivo injections of morphine sulfate. Morphine caused a 50 per cent increase in the V_max of [³H]choline uptake. Although a concomitant increase was also measured in the amount of [³H] acetylcholine released, it could be explained by the previous increase in uptake. It is suggested that morphine had an overall stimulatory effect on the striatal cholinergic system which may be a transynaptic phenomenon rather than a direct effect on the cholinergic cell.     

Differential mechanistic investigation of protective effects from imperatorin and sec-O-glucosylhamaudol against arsenic trioxide-induced cytotoxicity in vitro

Background and purposeThe clinical use of arsenic trioxide (As₂O₃) for treating acute promyelocytic leukemia (APL) is limited due to its severe cardiotoxicity. The possible mechanisms of As₂O₃-induced cardiotoxicity include DNA fragmentation, reactive oxygen species (ROS) generation, cardiac ion channel changes and apoptosis. The present study is designed to investigate the protective effects of imperatorin and sec-O-glucosylhamaudol and to explore their mechanistic involvement in As₂O₃-induced cytotoxicity.Experimental methodsCell viability assay, Lactate dehydrogenase (LDH) release, Acridine orange/ethidium bromide (AO/EB) double staining, Caspase-3 activity assay, ROS generation, cellular calcium levels, mRNA expression levels by qRT-PCR and protein expression levels by Western blotting were measured in H9c2 cells in combination with As₂O₃ and imperatorin or sec-O-glucosylhamaudol.Key resultsWe observed that H9c2 cells treated with imperatorin or sec-O-glucosylhamaudol were more resistant to As₂O₃-induced cell death. Both imperatorin and sec-O-glucosylhamaudol reduced H9c2 cell apoptosis, but both imperatorin and sec-O-glucosylhamaudol had no effects on Caspase-3 activity and intracellular calcium accumulation. Furthermore, imperatorin was capable of suppressing ROS generation, while sec-O-glucosylhamaudol did not show this effect. Moreover, imperatorin and sec-O-glucosylhamaudol triggered Nrf2 activation, which resulted in upregulation of downstream phase II metabolic enzymes and antioxidant protein/enzyme, probably offering cellular protection to As₂O₃-induced cardiotoxicity via the Nrf2 signal pathway.Conclusions and implicationsImperatorin and sec-O-glucosylhamaudol can ameliorate As₂O₃-induced cytotoxicity and apoptosis in H9c2 cells, the mechanisms probably related to antioxidation. As₂O₃ in combination with imperatorin or sec-O-glucosylhamaudol could be considered as a novel strategy to expand the clinical application of As₂O₃.     

Effects of phencyclidine on [3H]catecholamine and [3H]serotonin uptake in synaptosomal preparations from rat brain

Phencyclidine inhibited uptake in vitro of [³H]norepinephrine (ic₅₀ 0.52 μM), [³H]dopamine (ic₅₀ 0.73 μM) and [³H]serotonin (ic₅₀ 0.80 μM) in crude synaptosomal preparations from rat brain through a competitive mechanism. Phencyclidine was fairly similar in potency to d-amphetamine and methylphenidate in inhibiting catecholamine uptake but was 8 times more potent than d-amphetamine and 34 times more potent than methylphenidate in inhibiting [³H]serotonin uptake.     

Clobenpropit,a histamine H3 receptor antagonist/inverse agonist,inhibits [3H]-dopamine uptake by human neuroblastoma SH-SY5Y cells and rat brain synaptosomes

Background

Clobenpropit, a potent antagonist/inverse agonist at the histamine H₃ receptor (H₃R), reduced the cytotoxic action of 6-hydroxydopamine (6-OHDA) in neuroblastoma SH-SY5Y cells transfected with the human H₃R. We therefore set out to study whether this effect involved a receptor-independent action on dopamine transport.

6-Hydroxyindole is an endogenous long-lasting OATP1B1 inhibitor elevated in renal failure patients

The hepatic uptake transporter organic anion transporting polypeptide (OATP) 1B1 is inhibited by some uremic toxins; however, direct inhibition can only partially explain the delayed systemic elimination of substrate drugs in renal failure patients. This study aimed to examine the long-lasting inhibition of OATP1B1 by uremic toxins and their metabolites. Preincubation of HEK293/OATP1B1 cells with 21 uremic toxins resulted in almost no change in the uptake of a typical substrate [³H]estrone-3-sulfate (E₁S), although some directly inhibited [³H]E₁S uptake. In contrast, preincubation with an indole metabolite, 6-hydroxyindole, reduced [³H]E₁S uptake, even after the inhibitor was washed out before [³H]E₁S incubation. Such long-lasting inhibition by 6-hydroxyindole was time-dependent and recovered after a 3-h incubation without 6-hydroxyindole. Preincubation with 6-hydroxyindole increased the Km for [³H]E₁S uptake with minimal change in Vmax. This was compatible with no change in the cell-surface expression of OATP1B1, as assessed by a biotinylation assay. Preincubation with 6-hydroxyindole reduced [³H]E₁S uptake in human hepatocytes without changes in OATP1B1 mRNA. Plasma concentration of 6-hydroxyindole in renal failure patients increased as renal function decreased, but might be insufficient to exhibit potent OATP1B1 inhibition. In conclusion, 6-hydroxyindole is an endogenous long-lasting OATP1B1 inhibitor with elevated plasma concentrations in renal failure patients.     

Oxybutynin and trospium are substrates of the human organic cation transporters

The muscarinic antagonists oxybutynin and trospium are used as spasmolytic agents for the treatment of overactive urinary bladder disease. Recently, it has been shown that trospium, but not oxybutynin, is a substrate of the multidrug efflux carrier P-glycoprotein, but carrier-mediated drug uptake has not been directly analysed for both drugs. However, trospium has been previously shown to exhibit inhibitory potency for the organic cation transporters (OCTs). The aim of the present study was to examine whether trospium and oxybutynin are substrates, i.e. are transported by the human OCTs (hOCT₁, hOCT₂ and hOCT₃). Therefore, we measured total and specific (decynium-22-sensitive) uptake, and saturation kinetics of the uptake for [³H]oxybutynin and [³H]trospium in human embryonic kidney (HEK293) cells transiently transfected with the cDNA of hOCT₁, hOCT₂ or hOCT₃. In addition, we determined IC₅₀ values for inhibition of hOCT-mediated [³H]MPP⁺ uptake by unlabelled trospium and oxybutynin. Total uptake of [³H]oxybutynin was very high in all transfected HEK293 cells and only a small portion was due to specific, decynium-22-sensitive hOCT-mediated uptake. Oxybutynin inhibited [³H]MPP⁺ uptake by the three hOCTs with IC₅₀ values between 20 and 130 μM. Direct determination of transport kinetics was measurable only at hOCT₁ with K _m of 8 μM and V _max of 484 pmol/mg protein/min. The rank order of affinity (1/IC₅₀ or 1/K _m) of specific oxybutynin uptake was hOCT₁?>?hOCT₂?= hOCT₃. The observed high non-specific uptake is obviously a consequence of the high lipophilicity of this uncharged drug. Thus, hOCTs may not play a significant role for the overall pharmacokinetics and tissue distribution of oxybutynin. However, and in contrast to oxybutynin, uptake of [³H]trospium, an organic cation, was mainly due to carrier-mediated uptake by the three hOCTs. With IC₅₀ values of 18, 1.4 and 710 μM (at hOCT₁, hOCT₂ and hOCT₃, respectively) and K _m values of 17 and 8 μM and about identical V _max values of about 90 pmol/mg protein/min at hOCT₁ and hOCT₂, respectively; the rank order of affinity (1/IC₅₀ or 1/K _m) of specific uptake of trospium was hOCT₂?>?hOCT₁?>?>?hOCT₃. Thus, hOCTs very probably contribute to the active tubular and hepatobiliary secretion of trospium. Furthermore, hOCT₁ and hOCT₃ may be involved in the tissue uptake of this drug in the urinary bladder.