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.     

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). [¹⁴C]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. [¹⁴C]Glycine uptake by TR-iBRB2 cells was Na⁺- and Cl⁻-dependent, and concentration-dependent with Michaelis–Menten constants of 55.4 μM 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 [¹⁴C]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 [¹⁴C]glycine is transported from blood to the retina whereas [¹⁴C]α-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.     

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). [¹⁴C]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. [¹⁴C]Glycine uptake by TR-iBRB2 cells was Na⁺- and Cl⁻-dependent, and concentration-dependent with Michaelis–Menten constants of 55.4 μM 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 [¹⁴C]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 [¹⁴C]glycine is transported from blood to the retina whereas [¹⁴C]α-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.     

Cationic amino acid transporter 1-mediated l-arginine transport at the inner blood–retinal barrier

The purpose of this study was to identify the transporter mediating l -arginine transport at the inner blood–retinal barrier (BRB). The apparent uptake clearance of [³H] l -arginine into the rat retina was found to be 118 μL/(min·g retina), supporting a carrier-mediated influx transport of l -arginine at the BRB. [³H] l -Arginine uptake by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), used as an in vitro model of the inner BRB, was primarily an Na⁺-independent and saturable process with Michaelis-Menten constants of 11.2 μM and 530 μM. This process was inhibited by rat cationic amino acid transporter (CAT) 1-specific small interfering RNA as well as substrates of CATs, l -arginine, l -lysine, and l -ornithine. The expression of cationic amino acid transporter (CAT) 1 mRNA was 25.9- and 796-fold greater than that of CAT3 in TR-iBRB2 and magnetically isolated rat retinal vascular endothelial cells, respectively. The expression of CAT1 protein was detected in TR-iBRB2 cells and immunostaining of CAT1 was observed along the rat retinal capillaries. In conclusion, CAT1 is localized in retinal capillary endothelial cells and at least in part mediates l -arginine transport at the inner BRB. This process seems to be closely involved in visual functions by supplying precursors of biologically important molecules like nitric oxide in the neural retina.     

Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells

The purpose of this study was to identify the uptake mechanism of γ-aminobutyric acid (GABA) via taurine transporter (Slc6a6/TauT) and its relationship with GABA transport at the inner BRB. Rat Slc6a6/TauT-transfected HeLa cells exhibited Na⁺-, Cl⁻-, and concentration-dependent [³H]GABA uptake with a K_m of 1.5 mM. Taurine, β-alanine, and GABA markedly inhibited Slc6a6/TauT-mediated uptake of [³H]GABA. The uptake of [³H]GABA by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) was Na⁺-, Cl⁻-, and concentration-dependent with a K_m of 2.0 mM. This process was more potently inhibited by substrates of Slc6a6/TauT, taurine and β-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a K_i of 74 μM. [³H]Taurine also exhibited competitive inhibition with a K_i of 1.8 mM in the presence of GABA. In conclusion, rat Slc6a6/TauT has the ability to use GABA as a substrate and Slc6a6/TauT-mediated GABA transport appears to be present at the inner BRB.     

Blood-to-retina transport of creatine via creatine transporter (CRT) at the rat inner blood-retinal barrier

The purpose of this study was to elucidate the mechanisms of blood-to-retina creatine transport across the blood-retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The creatine transport across the BRB in vivo and creatine uptake in an in vitro model of the inner BRB (TR-iBRB2 cells) were examined using [(14)C]creatine. Identification and localization of the creatine transporter (CRT) were carried out by RT-PCR, western blot, and immunoperoxidase electron microscopic analyses. An in vivo intravenous administration study suggested that [(14)C]creatine is transported from the blood to the retina against the creatine concentration gradient that exists between the retina and blood. [(14)C]Creatine uptake by TR-iBRB2 cells was saturable, Na(+)- and Cl(-)-dependent and inhibited by CRT inhibitors, suggesting that CRT is involved in creatine transport at the inner BRB. RT-PCR and western blot analyses demonstrated that CRT is expressed in rat retina and TR-iBRB2 cells. Moreover, using an immunoperoxidase electron microscopic analysis, CRT immunoreactivity was found at both the luminal and abluminal membranes of the rat retinal capillary endothelial cells. In conclusion, CRT is expressed at the inner BRB and plays a role in blood-to-retina creatine transport across the inner BRB.     

Stereospecificity of High-and Low-Affinity Transport of Choline Analogues into Rat Cortical Synaptosomes

The present experiments used methylcholines to examine the stereoselectivity of choline transport into rat synaptosomes. R(+)-alpha-methylcholine and S(+)-beta-methylcholine were significantly better inhibitors of the high-affinity choline transport system than were their enantiomers. Although both enantiomers of alpha- and of beta-methylcholine inhibited [3H]choline transport, only R(+)-alpha-methylcholine and S(+)-beta-methylcholine could be transported by the high-affinity choline uptake mechanism. Therefore, we conclude that the chiral requirements for recognition of and for transport by the high-affinity transporter are clearly different. In addition to high-affinity choline transport, Na(+)-independent low-affinity transport was measured. This process transported R(+)-alpha-methylcholine, but not S(-)-alpha-methylcholine; however, it showed no stereoselectivity for the enantiomers of beta-methylcholine. Thus, high- and low-affinity choline transport mechanisms exhibit distinct differences in their substrate selectivities. We suggest that the stereoselective properties of choline transport might present a unique opportunity to study choline uptake and metabolism.     

Cation Transport Specificity at the Blood–Brain Barrier

The molecular identification, expression and cloning of membrane-bound organic cation transporters are being completed in isolated in vitro membranes. In vivo studies, where cation specificity overlaps, need to complement this work. Method: Cross-inhibition of [³H]choline and [³H]thiamine brain uptake by in situ rat brain perfusion. Results: [³H]Choline brain uptake was not inhibited by thiamine at physiologic concentrations (100 nM). However, choline ranging from 100 nM to 250 M inhibited [³H]thiamine brain uptake, though not below levels observed at thiamine concentrations of 100 nM. Conclusion: (1) The molecular family of the blood–brain barrier (BBB) choline transporter may be elucidated in vitro by its interaction with physiologic thiamine levels, and (2) two cationic transporters at the BBB may be responsible for thiamine brain uptake.     

Molecular and functional characterization of choline transporter in rat renal tubule epithelial NRK-52E cells

Homeostatic regulation of the plasma choline concentration depends on the effective functioning of a choline transporter in the kidney. However, the nature of the choline transport system in the kidney is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake in the rat renal tubule epithelial cell line NRK-52E. Choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (K_m) of 16.5 μM and a maximal velocity (V_max) of 133.9 pmol/mg protein/min. The V_max value of choline uptake was strongly enhanced in the absence of Na⁺ without any change in K_m values. The increase in choline uptake under Na⁺-free conditions was inhibited by Na⁺/H⁺ exchanger (NHE) inhibitors. Choline uptake was inhibited by the choline uptake inhibitor hemicholinium-3 (HC-3) and organic cations, and was decreased by acidification of the extracellular medium and by intracellular alkalinization. Collapse of the plasma membrane H⁺ electrochemical gradient by a protonophore inhibited choline uptake. NRK-52E cells mainly express mRNA for choline transporter-like proteins (CTL1 and CTL2), and NHE1 and NHE8. CTL1 protein was recognized in both plasma membrane and mitochondria. CTL2 protein was mainly expressed in mitochondria. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in NRK-52E cells and is responsible for choline uptake. This choline transport system uses a directed H⁺ gradient as a driving force, and its transport functions in co-operation with NHE8. Furthermore, the presence of CTL2 in mitochondria provides a potential site for the control of choline oxidation.     

Regulation of Rat Brain Synaptosomal [3H]Hemicholinium-3 Binding and [3H]Choline Transport Sites Following Exposure to Choline Mustard Aziridinium Ion

Abstract: Choline uptake by cholinergic nerve terminals is increased by depolarization; the literature suggests that this results from either the appearance of occult transporters or the increased activity of existing ones. The present experiments attempt to clarify the mechanism by which choline transport is regulated by testing if the preexposure of synaptosomes to choline mustard aziridinium ion prevents the stimulation-induced appearance of hemicholinium-3 binding sites and/or choline transport activity. Choline mustard inhibited irreversibly most of the “ground-state” (basal) high-affinity choline transport but only 50% of “ground-state” hemicholinium-3 binding sites. Exposure of both striatal and hippocampal synaptosomes to the mustard, before stimulation, inhibited K⁺-stimulated increases in choline transport and of [³H]hemicholinium-3 binding. We conclude that the mechanism by which choline transport is regulated involves the increased activity of a pool of transport sites that are occluded to hemicholinium-3 but are available to choline mustard aziridinium ion, and presumably to choline, before stimulation. However, the concentration of mustard needed to inhibit the stimulation-induced increase of [³H]hemicholinium-3 binding and choline transport was lower for striatal synaptosomes than for hippocampal synaptosomes. In the absence of extracellular Ca²⁺ or presence of high Mg²⁺ levels, the choline mustard did not prevent the appearance of extra striatal hemicholinium-3 binding sites. Also, high Mg²⁺ levels removed the ability of the mustard to inhibit K⁺-stimulated increases of either [³H]hemicholinium-3 binding or choline transport by hippocampal synaptosomes. In contrast, the preexposure of hippocampal synaptosomes to the mustard in the presence of a calcium ionophore (A23187) reduced the concentration of inhibitor needed to prevent the activation of [³H]hemicholinium-3 binding and choline uptake. Thus, we conclude that the ability of the choline mustard to alkylate the pool of choline transporters that are activated by stimulation appears dependent on the entry of extracellular Ca²⁺.     

Sodium-dependent and -independent Choline Uptake by Type II Epithelial Cells from Rat Lung

The uptake of ³H-labeled choline by a suspension of isolated type II epithelial cells from rat lung has been studied in a Ringer medium. Uptake was linear for 4 min at both 0.1 μm and 5.0 μm medium choline; at 5 μm, only 10% of the label was recovered in a lipid fraction. Further experiments were conducted at the low concentration (0.1 μm), permitting characterization of the properties of high-affinity systems. Three fractions of choline uptake were detected: (i) a sodium-dependent system that was totally inhibited by hemicholinium-3 (HC-3); (ii) a sodium-independent uptake, when Na⁺ was replaced by Li⁺, K⁺ or Mg²⁺, inhibited by HC-3; (iii) a residual portion persisting in the absence of Na⁺ and unaffected by HC-3. Choline uptake was sigmoidally related to the medium Na⁺ concentration. Kinetic properties of the uptake of 0.1 μm ³H-choline in the presence and absence of medium Na⁺ were examined in two ways. (a) Inhibition by increasing concentrations of unlabeled choline (0.5–100 μm) was consistent with the presence of two Michaelis-Menten-type systems in the presence of Na⁺; a Na⁺-dependent portion (a mean of 0.52 of the total) had a K _m for choline of 1.5 μm while K _m in the absence of Na⁺ (Li⁺ substituting) was 18.6 μm. (b) Inhibition by HC-3 (0.3–300 μm) gave K_i values of 1.7 μm and 5.0 μm HC-3 for the Na⁺-dependent and -independent fractions. The apparent K _m of the Na⁺-dependent uptake is lower than that reported previously for lung-derived cells and is in the range of the K _m values reported for high-affinity, Na⁺-dependent choline uptake by neuronal cells. Received: 18 February 1997/Revised: 7 December 1997     

Inhibition of ATP-Induced Ca<Superscript>2+</Superscript> Influx by Corticosterone in Dorsal Root Ganglion Neurons

In addition to the classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomic effects on neurons. In the present study, the effect of corticosterone (CORT) on ATP-induced Ca²⁺ mobilization in cultured dorsal root ganglion (DRG) neurons were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of intracellular calcium concentration ([Ca²⁺]i). ATP, an algesic agent, caused [Ca²⁺]i increase in DRG neurons by activation of P2X receptor. Pretreatment with CORT (1 nM–1 μM for 5 min) inhibited ATP-induced [Ca²⁺]i increase in DRG neurons. The rapid inhibition of ATP-induced Ca²⁺ response by CORT was concentration-dependent, reversible and could be blocked by glucocorticoid receptor antagonist RU38486 (10 μM). Furthermore, the inhibitory effect of CORT was abolished by protein kinase A inhibitor H89 (10 μM), but was not influenced by protein kinase C inhibitor Chelerythrine chloride (10 μM). On the other hand, membrane-impermeable bovine serum albumin-conjugated corticosterone had no effect on ATP-induced [Ca²⁺]i transients. These observations suggest that a nongenomic pathways may be involved in the effect of CORT on ATP-induced [Ca²⁺]i transients in cultured DRG neurons.     

Effect of Cyclanilide on Auxin Activity

Cyclanilide is a plant growth regulator that is registered for use in cotton at different stages of growth, to either suppress vegetative growth (in combination with mepiquat chloride) or accelerate senescence (enhance defoliation and boll opening, used in combination with ethephon). This research was conducted to study the mechanism of action of cyclanilide: its potential interaction with auxin (IAA) transport and signaling in plants. The activity of cyclanilide was compared with the activity of the auxin transport inhibitors NPA and TIBA. Movement of [³H]IAA was inhibited in etiolated corn coleoptiles by 10 μM cyclanilide, NPA, and TIBA, which demonstrated that cyclanilide affected polar auxin transport. Although NPA inhibited [³H]IAA efflux from cells in etiolated zucchini hypocotyls, cyclanilide had no effect. NPA did not inhibit the influx of IAA into cells in etiolated zucchini hypocotyls, whereas cyclanilide inhibited uptake 25 and 31% at 10 and 100 μM, respectively. Also, NPA inhibited the gravitropic response in tomato roots (85% at 1 μM) more than cyclanilide (30% at 1 μM). Although NPA inhibited tomato root growth (30% at 1 μM), cyclanilide stimulated root growth (165% of control at 5 μM). To further characterize cyclanilide action, plasma membrane fractions from etiolated zucchini hypocotyls were obtained and the binding of NPA, IAA, and cyclanilide studied. Cyclanilide inhibited the binding of [³H]NPA and [³H]IAA with an IC₅₀ of 50 μM for both. NPA did not affect the binding of IAA, nor did IAA affect the binding of NPA. Kinetic analysis indicated that cyclanilide is a noncompetitive inhibitor of both NPA and IAA binding, with inhibition constants (K _i) of 40 and 2.3 μM, respectively. These data demonstrated that cyclanilide interacts with auxin-regulated processes via a mechanism that is distinct from other auxin transport inhibitors. This research identifies a possible mechanism of action for cyclanilide when used as a plant growth regulator.     

Effects of zinc ex vivo and intracellular zinc chelator in vivo on taurine uptake in goldfish retina

Taurine and zinc exert neurotrophic effects. Zinc modulates Na⁺/Cl⁻-dependent transporters. This study examined the effect of zinc (ZnSO₄) ex vivo and zinc chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) in vivo on [³H]taurine transport in goldfish retina. The effect of TPEN in vivo on taurine and zinc levels was determined. Isolated cells were incubated in Ringer with zinc (0.1–100 μM). Taurine transport was done with taurine (0.001–1 mM) and 50 nM [³H]taurine. Zinc (100 μM) noncompetitively inhibited taurine transport. TPEN was administered intraocularly and retinas extracted 3, 5 and 10 days later. Taurine was determined by HPLC (nmol/mg protein) and zinc by spectrophotometry ICP (mg/mg protein). Taurine and zinc levels decreased at 3 days and increased at 10 days after TPEN administration. At 10 days after intraocular TPEN, taurine transport affinity increased (K _s = 0.018 ± 0.006 vs. 0.028 ± 0.008 mM). Apparently, zinc deficiency affects the taurine–zinc complex and taurine availability. The increased taurine uptake affinity by TPEN was possibly associated with a response to maximize retinal taurine content at low zinc concentration.     

Molecular and functional characterization of choline transporter in human colon carcinoma HT-29 cells

We examined the molecular and functional characterization of choline uptake in human colon carcinomas using the cell line HT-29. Furthermore, we explored the possible correlation between choline uptake and cell proliferation. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na⁺ from the uptake buffer strongly enhanced choline uptake. This increase in component of choline uptake under Na⁺-free conditions was inhibited by a Na⁺/H⁺ exchanger 1 (NHE1) inhibitor. Collapse of the plasma-membrane H⁺ electrochemical gradient by a protonophore inhibited choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium and by intracellular alkalinization. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), CTL2, CTL4 and NHE1 mRNA are mainly expressed in HT-29 cells. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in HT-29 cells and is responsible for choline uptake in these cells. We conclude that choline transporters, especially CTL1, use a directed H⁺ gradient as a driving force, and its transport functions in co-operation with NHE1. Finally, cell proliferation was inhibited by HC-3 and tetrahexylammonium chloride (THA), which strongly inhibits choline uptake. Identification of this novel CTL1-mediated choline uptake system provides a potential new target for therapeutic intervention.     

Regulation of [<Superscript>3</Superscript>H] <Emphasis Type="SmallCaps">d</Emphasis>-Aspartate Release from Mammalian Isolated Retinae by Hydrogen Sulfide

Hydrogen sulfide (H₂S), can produce pharmacological effects on neural and non-neural tissues from several mammalian species. The present study investigates the pharmacological action of H₂S, (using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na₂S as donors) on amino acid neurotransmission (using [³H] d-aspartate as a marker for glutamate) from isolated, superfused bovine and porcine retinae. Isolated neural retinae were incubated in Krebs solution containing [³H] d-aspartate at 37°C. Release of [³H] d-aspartate was elicited by high potassium (K⁺ 50 mM) pulse. Both NaHS and Na₂S donors caused an inhibition of K⁺-evoked [³H] d-aspartate release from isolated bovine retinae without affecting basal [³H] d-aspartate efflux yielding IC₅₀ values of 0.006 and 6 μm, respectively. Furthermore, NaHS inhibited depolarization-evoked release of [³H] d-aspartate from isolated porcine retinae with an IC₅₀ value of 8 μM. The inhibitory action of NaHS on [³H] d-aspartate release from porcine retinae was blocked by propargyglycine, a selective inhibitor of cystathionine γ-lyase (CSE). Our results indicate that H₂S donors can inhibit amino acid neurotransmission from both isolated bovine and porcine retinae, an effect that is dependent, at least in part, on intramural biosynthesis of H₂S.     

Some Operational Characteristics of Glycine Release in Rat Retina: The Role of Reverse Mode Operation of Glycine Transporter Type-1 (GlyT-1) in Ischemic Conditions

Rat posterior eyecups containing the retina were prepared, loaded with [³H]glycine and superfused in order to determine its release originated from glycinergic amacrine cells and/or glial cells. Deprivation of oxygen and glucose from the Krebs-bicarbonate buffer used for superfusion evoked a marked increase of [³H]glycine release, an effect that was found to be external Ca²⁺-independent. Whereas oxygen and glucose deprivation increased [³H]glycine release, its uptake was reduced suggesting that energy deficiency shifts glycine transporter type-1 operation from normal to reverse mode. The increased release of [³H]glycine evoked by oxygen and glucose deprivation was suspended by addition of the non-competitive glycine transporter type-1 inhibitor NFPS and the competitive inhibitor ACPPB further suggesting the involvement of this transporter in the mediation of [³H]glycine release. Oxygen and glucose deprivation also evoked [³H]glutamate release from rat retina and the concomitantly occurring release of the NMDA receptor agonist glutamate and the coagonist glycine makes NMDA receptor pathological overstimulation possible in hypoxic conditions. [³H]Glutamate release was suspended by addition of the excitatory amino acid transporter inhibitor TBOA. Sarcosine, a substrate inhibitor of glycine transporter type-1, also increased [³H]glycine release probably by heteroexchange shifting transporter operation into reverse mode. This effect of sarcosine was also external Ca²⁺-independent and could be suspended by NFPS. Energy deficiency in retina induced by ouabain, an inhibitor of the Na⁺–K⁺-dependent ATPase, and by rotenone, a mitochondrial complex I inhibitor added with the glycolytic inhibitor 2-deoxy-d-glucose, led to increase of retinal [³H]glycine efflux. These effects of ouabain and rotenone/2-deoxy-d-glucose could also be blocked by NFPS pointed to the preferential reverse mode operation of glycine transporter type-1 as a consequence of impaired cellular energy homeostasis. Immunohistochemical studies revealed that glycine transporter type-1, of which reverse mode operation assures [³H]glycine release, is expressed in amacrine cells in the inner nuclear and plexiform layers of the retina and also in Müller macroglia cells. We conclude that disruption of the balanced normal/reverse mode operation of glycine transporter type-1 is likely a significant factor contributing to neurotoxic processes of the retina. The possibility to inhibit glycine transporter type-1 mediated glycine efflux by drugs more potently than glycine uptake might offer some therapeutic potential for the treatment of various neurodegenerative disorders of the retina.     

Molecular and functional characterization of an Na+-independent choline transporter in rat astrocytes

In this study, we examined the molecular and functional characterization of choline uptake into cultured rat cortical astrocytes. Choline uptake into astrocytes showed little dependence on extracellular Na+. Na+-independent choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (Km) of 35.7 +/- 4.1 microm and a maximal velocity (Vmax) of 49.1 +/- 2.0 pmol/mg protein/min. Choline uptake was significantly decreased by acidification of the extracellular medium and by membrane depolarization. Na+-independent choline uptake was inhibited by unlabeled choline, acetylcholine and the choline analogue hemicholinium-3. The prototypical organic cation tetrahexylammonium (TEA), and other n-tetraalkylammonium compounds such as tetrabutylammonium (TBA) and tetrahexylammonium (THA), inhibited Na+-independent choline uptake, and their inhibitory potencies were in the order THA > TBA > TEA. Various organic cations, such as 1-methyl-4-tetrahydropyridinium (MPP+), clonidine, quinine, quinidine, guanidine, N-methylnicotinamide, cimetidine, desipramine, diphenhydramine and verapamil, also interacted with the Na+-independent choline transport system. Corticosterone and 17beta-estradiol, known inhibitors of organic cation transporter 3 (OCT3), did not cause any significant inhibition. However, decynium22, which inhibits OCTs, markedly inhibited Na+-independent choline uptake. RT-PCR demonstrated that astrocytes expressed low levels of OCT1, OCT2 and OCT3 mRNA, but the functional characteristics of choline uptake are very different from the known properties of these OCTs. The high-affinity Na+-dependent choline transporter, CHT1, is not expressed in astrocytes as evidenced by RT-PCR. Furthermore, mRNA for choline transporter-like protein 1 (CTL1), and its splice variants CTL1a and CTL1b, was expressed in rat astrocytes, and the inhibition of CTL1 expression by RNA interference completely inhibited Na+-independent choline uptake. We conclude that rat astrocytes express an intermediate-affinity Na+-independent choline transport system. This system seems to occur through a CTL1 and is responsible for the uptake of choline and organic cations in these cells.     

Complex of Amyloid β Peptides with 24-Hydroxycholesterol and Its Effect on Hemicholinium-3 Sensitive Carriers

Brains of Alzheimer disease patients in early stages of dementia contain an increased 24(S)-hydroxycholesterol (cerebrosterol)/cholesterol ratio when compared to controls. In this study, effects of amyloid β peptides and of racemic 24-hydroxycholesterol were evaluated in vitro on undepleted or cholesterol-depleted hippocampal synaptosomes of young and old rats via a high-affinity choline transport and membrane anisotropy measurements. Depletion of membrane cholesterol decreased the transport of [3H]choline, increased the specific binding of [3H]hemicholinium-3 and decreased membrane anisotropy. However, less alterations were found in old when compared to young brains. 500 nM nonaggregated peptides were ineffective but aggregated fragment 1–42 evoked marked drops in the transport and anisotropy values on depleted synaptosomes. 50 μM 24-hydroxycholesterol inhibited choline transport on depleted synaptosomes but it did not influence membrane anisotropy. Peptides eliminated the actions of oxysterol on choline carriers in young but not in old rats. On the other hand, oxysterol eliminated the effects of peptides on membrane anisotropy. Our study suggests a possible role of membrane cholesterol in the regulation of choline carriers and supports data reporting a protective role of membrane cholesterol against toxic effects of amyloid β peptides. Moreover, via Raman spectroscopy we demonstrate for the first time that peptides form a complex with 24-hydroxycholesterol.     

Identification of Sodium-Dependent, High-Affinity Choline Uptake Sites in Rat Brain with [3H]Hemicholinium-3

[3H]Hemicholinium-3 (HC-3) was used to label sodium-dependent, high-affinity choline uptake sites in regions of rat brain. Autoradiography revealed a high density of [3H]HC-3 binding sites in brain regions with a high density of cholinergic terminals, such as the interpeduncular nucleus, caudate-putamen, and olfactory tubercle. This distribution of [3H]HC-3 binding sites was in close agreement with the amounts of choline acetyltransferase in specific nuclei and subregions of rat brain. Destruction of presynaptic cholinergic projections in the cerebral cortex and the basal ganglia by injection of excitotoxins reduced [3H]HC-3 binding by 40-50%. These data indicate that sodium-dependent [3H]HC-3 binding sites are related to the choline transport system present in cholinergic neurons.