Saturable Transport Research Articles

Leptin signaling pathways in the central nervous system: interactions between neuropeptide Y and melanocortins

No other hormone has drawn more attention than leptin in recent studies on the control of appetite, body weight and obesity. This hormone is produced by adipose tissue and enters the brain via a saturable specific transport mechanism. Leptin acts in the hypothalamus to modulate food intake and heat production as well as several other neuroendocrine pathways. The mechanisms through which leptin exerts its central nervous effects are now better understood. Proopiomelanocortin- and neuropeptide Y-containing neurons in the hypothalamus have emerged as potent candidate mediators of leptin action. These two neuropeptides have been shown to exert opposing effects using different pathways. Recently, Cowley et al. (2001) described a new circuit in the regulation of neuronal activity by leptin with an interaction between these two pathways. These data add complexity to the mechanisms by which leptin achieves its effects in the central nervous system, but they also offer potential mechanisms to explain the phenomenon of leptin resistance observed in obesity.

Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide

We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large (∼1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9) 8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin–PEG-3400 (0.6–100 μM) and the bioconjugate (0.1–30 μM) was studied using Caco-2 cell monolayers. Inhibition of biotin–PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05–12.5 μM and 0.003–30 μM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9) 8, were used in the control studies. Biotin–PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells ( K m=6.61 μM) and CHO/hSMVT cells ( K m=1.26 μM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9) 8 also showed saturable transport kinetics in Caco-2 cells ( K m=6.13 μM) and CHO/hSMVT cells ( K m=8.19 μM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower ( P<0.001) in all cases. The present results demonstrate that biotin–PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9) 8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential for enhancing the cellular uptake and transport of small peptide therapeutic agents.

Characterization of spermine uptake by Ehrlich tumour cells in culture.

Spermine is taken up by Ehrlich ascites tumour cells through a specific, saturable, temperature and energy-dependent transport system with a remarkably low affinity constant for spermine (around 1 microM). In the absence of a potassium ion gradient through the plasma membrane, spermine uptake remains saturable but the value of the Km for spermine is much higher (153 microM). Difluormethylornithine treatment (3 mM for 48h) induces significant increases in Vmax values (up to 9-fold) and changes in the Km values with scarce statistical significance. Among the biogenic amines tested, only spermidine and, partly, agmatine seem to share the same transport system with spermine. No difference is observed in the rate of spermine transport when assays are carried out in the presence of 50-fold excess of ornithine or calcium, or 100-fold excess of glutamine.

Regional differences in peptide degradation by rat cerebral microvessels: A potential novel regulatory mechanism for communication between blood and brain

The blood-brain barrier (BBB), composed of the microvessels of cerebral capillary endothelial cells, regulates the passage of peptides into the brain in several ways, mainly by saturable transport or passive diffusion. Here we describe an additional mechanism by which this regulatory function can occur. Cerebral microvessels were isolated from different regions of the brain and incubated with the mu-opiate selective endomorphin-1 (Tyr-Pro-Trp-Phe-NH 2) or the opiate-modulating Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH 2), both tetrapeptides selectively tritiated at the Pro. Degradation was determined by HPLC. For both peptides, the metabolism by microvessels from the cerebral cortex was much greater than that by microvessels from the hypothalamus or pons. For endomorphin-1, the least degradation was in the pons; for Tyr-MIF-1 there was no difference in metabolism by microvessels from the pons or hypothalamus. The results show a novel mechanism at the BBB by which the BBB can selectively regulate the activity of different peptides in different regions of the brain.

Saturable transport of the neurokinin-1 non-peptide antagonist LY303870 across the rat blood-brain barrier after intravenous administration

LY303870 (LY) is a non-peptide neurokinin-1 receptor antagonist that has effects on the brain after peripheral administration. We determined whether LY given by intravenous (iv) injection can cross the blood-brain barrier (BBB). Multiple-time regression analysis showed the unidirectional influx rate (Ki) from blood to brain for LY labeled with tritium to be 6.41 ± 0.85 μl/g-min and influx was inhibited by unlabeled LY. HPLC and mass spectrometry showed LY was stable in blood and brain. LY reached a brain/serum ratio of 190 ± 12 μl/g with about 0.07% of the injected dose entering each gram of brain. These results show that LY is transported across the BBB from serum into brain by a saturable system.

Saturable brain-to-blood transport of endomorphins.

Opiate-modulating tetrapeptides such as tyrosine-melanocyte-stimulating hormone-release inhibiting factor-1 (Tyr-MIF-1; Tyr-Pro-Leu-Gly-NH2) and Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) are saturably transported from brain to blood. We examined whether two recently described endogenous opiate tetrapeptides with similar structures, the mu-specific endomorphins, also are transported across the blood-brain barrier (BBB). We found that the efflux rates of endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) were each self-inhibited by an excess of the respective endomorphin, thereby defining saturable transport. Cross-inhibition of the transport of each endomorphin by the other indicated shared transport. By contrast, no inhibition of the efflux of either endomorphin resulted from coadministration of Tyr-MIF-1, indicating that peptide transport system-1 (PTS-1) was not involved. Tyr-W-MIF-1, which is partially transported by PTS-1, significantly (P<0.01) decreased the transport of endomorphin-1 and tended (P=0.051) to decrease the transport of endomorphin-2, consistent with its role as both an opiate and antiopiate. Although involved in modulation of pain, coinjection of calcitonin gene-related peptide or constriction of the sciatic nerve did not appear to inhibit endomorphin efflux. Thus, the results demonstrate the existence of a new efflux system across the BBB which saturably transports endomorphins from brain to blood.

Friedrich Miescher Prize awardee lecture review. A conserved family of nuclear export receptors mediates the exit of messenger RNA to the cytoplasm.

The distinguishing feature of eukaryotic cells is the segregation of RNA biogenesis and DNA replication in the nucleus, separate from the cytoplasmic machinery for protein synthesis. As a consequence, messenger RNAs (mRNAs) and all cytoplasmic RNAs from nuclear origin need to be transported from their site of synthesis in the nucleus to their final cytoplasmic destination. Nuclear export occurs through nuclear pore complexes (NPCs) and is mediated by saturable transport receptors, which shuttle between the nucleus and cytoplasm. The past years have seen great progress in the characterization of the mRNA export pathway and the identification of proteins involved in this process. A novel family of nuclear export receptors (the NXF family), distinct from the well-characterized family of importin beta-like proteins, has been implicated in the export of mRNA to the cytoplasm.

Role of the System L permease LAT1 in amino acid and iodothyronine transport in placenta.

The feto-placental unit relies on a maternal supply of indispensable amino acids and iodothyronines for early development and normal growth. We examined the role of the System L transporter in placental uptake of these substances, using the human placental choriocarcinoma cell line BeWo as a model experimental system. BeWo cells express both heavy (4F2hc) and light (LAT1, LAT2) chains of the System L holotransporter. Saturable transport of both L-[(3)H]tryptophan and [(125)I]tri-iodo-L-thyronine in BeWo cells includes components sensitive to inhibition by the System-L-specific substrate 2-endoamino-bicycloheptane-2-carboxylic acid; kinetic properties of these components indicate that the 4F2hc-LAT1 transporter isoform is likely to predominate for the carriage of both substances at physiologically relevant concentrations. Both 4F2hc and LAT1 proteins are also expressed in human placental membranes and LAT1 at least is localized largely to the syncytiotrophoblast layer of the term human placenta. The 4F2hc-LAT1 transporter might therefore serve a vital role in supplying the developing fetus and the placenta with both thyroid hormones and indispensable amino acids from the maternal circulation.

Role of the System L permease LAT1 in amino acid and iodothyronine transport in placenta

The feto-placental unit relies on a maternal supply of indispensable amino acids and iodothyronines for early development and normal growth. We examined the role of the System L transporter in placental uptake of these substances, using the human placental choriocarcinoma cell line BeWo as a model experimental system. BeWo cells express both heavy (4F2hc) and light (LAT1, LAT2) chains of the System L holotransporter. Saturable transport of both l-[3H]tryptophan and [125I]tri-iodo-l-thyronine in BeWo cells includes components sensitive to inhibition by the System-L-specific substrate 2-endoamino-bicycloheptane-2-carboxylic acid; kinetic properties of these components indicate that the 4F2hc-LAT1 transporter isoform is likely to predominate for the carriage of both substances at physiologically relevant concentrations. Both 4F2hc and LAT1 proteins are also expressed in human placental membranes and LAT1 at least is localized largely to the syncytiotrophoblast layer of the term human placenta. The 4F2hc-LAT1 transporter might therefore serve a vital role in supplying the developing fetus and the placenta with both thyroid hormones and indispensable amino acids from the maternal circulation.

Collisionless kinetic-fluid closure and its application to the three-mode ion temperature gradient driven system

A novel closure model is presented to give a set of fluid equations which describe a collisionless kinetic system. In order to take account of the time reversal symmetry of the collisionless kinetic equation, the new closure model relates the parallel heat flux to the temperature and the parallel flow in terms of the real-valued coefficients in the unstable wave number space. Effects of the closure model on turbulence saturation and anomalous transport are investigated based on kinetic and fluid entropy balances. When the closure model is applied to the three-mode ion temperature gradient (ITG) driven system, the fluid system of equations reproduces the exact nonlinear kinetic solution found by Watanabe, Sugama, and Sato [Phys. Plasmas 7, 984 (2000)]. Oscillatory behaviors and initial amplitude dependence of other numerical kinetic solutions of the three-mode ITG problem can also be accurately described by the fluid system.

In Vivo Measurement of Blood‐Brain Barrier Permeability

This unit describes various protocols for the in vivo quantitation of drug permeability across the rodent blood - brain barrier. Methods for the measurement of drug influx or efflux are described, and support protocols are provided for determining intravascular capillary volume and cerebral perfusion flow. An in situ perfusion technique is also provided for assessing whether transport of a test compound occurs by carrier-mediated or saturable transport.

In vitro demonstration of a saturable transport system for leptin across the blood-brain barrier

A saturable blood-to-brain transport system for leptin across the blood-brain barrier (BBB) has been observed in vivo. Since the main component of the non-fenestrated microvessels of the BBB is the endothelial cell, we established an in vitro culture system of these cerebrovascular cells to study leptin transport and to determine whether the self-inhibition of leptin transport characteristic of a saturable system occurs at this level. The results show that 125I-leptin crossed from the luminal to abluminal side of a monolayer of cerebral microvessel cells significantly faster than the albumin and lactalbumin controls. This transport of 125I-leptin across an in vitro BBB was significantly faster than in the opposite direction and was dose-relatedly inhibited by the addition of unlabeled leptin. Thus, the results establish that the saturable transport system for leptin across the BBB occurs at the level of the endothelial cells of the BBB.

Pretreatment with glucose increases entry of urocortin into mouse brain

Although urocortin is a potent inhibitor of food ingestion after peripheral administration, it was recently shown that under normal conditions this peptide crosses the blood-brain barrier (BBB) at a very slow rate. We examined whether hyperglycemia could stimulate the rate of entry (K i) of 125I-urocortin into the mouse brain. In euglycemic mice, 125I-urocortin injected iv entered the brain at a rate similar to that of the vascular marker 99mTc-albumin. However, injection of glucose (3 g/kg, ip) 0.5, 1, or 2 h before the 125I-urocortin greatly increased the influx of urocortin. Without the glucose, the self-inhibition characteristic of a saturable transport system was not apparent. Self-inhibition could be demonstrated after the glucose injection, indicating activation of a transport system for urocortin that was saturable. Injection of insulin (10 U/kg, ip) 1 or 2 h before the 125I-urocortin decreased the K i. Thus, the entry of urocortin into brain can be activated by changes in the concentration of blood glucose, illustrating the responsiveness of the BBB to regulatory influences.

Glucose Tolerance and Peripheral Glucose Utilization in Rainbow Trout (Oncorhynchus mykiss), American Eel (Anguilla rostrata), and Black Bullhead Catfish (Ameiurus melas)

This study tests the hypothesis that glucose tolerance in fish is related to nutrient preference and is correlated with white muscle glucose transporter and phosphorylation (hexokinase) activities. Glucose clearance was investigated in the carnivorous rainbow trout (Oncorhynchus mykiss) and American eel (Anguilla rostrata) (feeding and fasting) and the omnivorous black bullhead catfish (Ameiurus melas). Glucose tolerance was assessed by an intravenous glucose tolerance test, injecting 250 mg glucose/kg body weight and tracking blood glucose concentrations over 24 h. Both feeding eel and feeding catfish returned plasma glucose levels to baseline within 60 min of glucose injection. Glucose values remained elevated for more than 360 min in both the food-deprived eel and the feeding rainbow trout. Glucose transport studies in white muscle membrane vesicles provided evidence for the presence of a stereospecific, saturable glucose transporter in all three species. Affinity constants (Km) ranged from 8 to 14 mM while Vmax values ranged from 75 to 150 pmol/s/mg protein. Neither kinetic parameter differed significantly between species. Cytochalasin B and phloretin did not significantly inhibit glucose transport, implying that these transporters are unlike the mammalian muscle glucose transporters (GLUT). In fact, Northern and Western blot analyses of mRNA and protein from white and red muscles and heart did not detect a mammalian-type GLUT-1 or -4 in any of the species examined. Glucose phosphorylation indicated the presence of a hexokinase activity (low Km enzyme) but again there were no differences in kinetic parameters between species. These studies demonstrate that glucose tolerance in fish is species-dependent but none of the parameters examined clearly differentiate between the species examined. Certainly a stereospecific glucose transporter exists in white skeletal muscle of the fish studied but no molecular or kinetic similarities to the mammalian GLUTs were found. Whether these transporters are insulin-sensitive or contribute to glucose tolerance requires further molecular characterization.

Enhanced leptin transport across the blood–brain barrier by α1-adrenergic agents

Leptin is a 16 kDa protein secreted by fat cells which regulates body weight and thermogenesis at sites within the brain. Blood-borne leptin reaches those brain sites because of a saturable transport system located at the blood–brain barrier (BBB). Impaired transport occurs in obese rodents and likely underlies the resistance to the actions of peripheral leptin seen in obesity. Here, we show that leptin transport into the brain is enhanced 2–3-fold by epinephrine and other agents which are more specific for the α1 adrenergic receptor. Epinephrine had no effect on the transport across the BBB of insulin or tumor necrosis factor, on BBB integrity, or on the size of the vascular space of the brain. Dopamine, acetylcholine, histamine, serotonin, thyroid hormones, and phentolamine were without effect. Of several amino acids tested, only the catecholamine precursor tyrosine had an effect on leptin transport. Epinephrine was effective after intravenous or intraperitoneal injection, but neither epinephrine nor any of the other monoamines given by intracerebroventricular injection had an effect on leptin transport. These results show that epinephrine likely acts at a site on the luminal surface of the BBB. In conclusion, epinephrine works at an α1-like adrenergic, luminal side to enhance the transport of leptin across the BBB.

Uptake mechanism of valproic acid in human placental choriocarcinoma cell line (BeWo)

Valproic acid is an anticonvulsant widely used for the treatment of epilepsy. However, valproic acid is known to show fetal toxicity, including teratogenicity. In the present study, to elucidate the mechanisms of valproic acid transport across the blood–placental barrier, we carried out transcellular transport and uptake experiments with human placental choriocarcinoma epithelial cells (BeWo cells) in culture. The permeability coefficient of [ 3H]valproic acid in BeWo cells for the apical-to-basolateral flux was greater than that for the opposite flux, suggesting a higher unidirectional transport in the fetal direction. The uptake of [ 3H]valproic acid from the apical side was temperature-dependent and enhanced under acidic pH. In the presence of 50 μM carbonyl cyanide p-trifluoromethoxylhydrazone, the uptake of [ 3H]valproic acid was significantly reduced. A metabolic inhibitor, 10 mM sodium azide, also significantly reduced the uptake of [ 3H]valproic acid. Therefore, valproic acid is actively transported in a pH-dependent manner on the brush-border membrane of BeWo cells. Kinetic analysis of valproic acid uptake revealed the involvement of a non-saturable component and a saturable component. The Michaelis constant for the saturable transport ( K t) was smaller under acidic pH, suggesting a proton-linked active transport mechanism for valproic acid in BeWo cells. In the inhibitory experiments, some short-chain fatty acids, such as acetic acid, lactic acid, propanoic acid and butyric acid, and medium-chain fatty acids, such as hexanoic acid and octanoic acid, inhibited the uptake of [ 3H]valproic acid. The uptake of [ 3H]valproic acid was also significantly decreased in the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, salicylic acid and furosemide, which are well-known inhibitors of the anion exchange system. Moreover, p-aminohippuric acid significantly reduced the uptake of [ 3H]valproic acid. These results suggest that an active transport mechanism for valproic acid exists on the brush-border membrane of placental trophoblast cells and operates in a proton-linked manner.

Multiple transport pathways for dibasic amino acids in the larval midgut of the silkworm Bombyx mori

The transport pathways for dibasic amino acids were investigated in brush border membrane vesicles (BBMV) from the anterior–middle (AM) and posterior (P) regions of Bombyx mori midgut. In the absence of K +, a low-affinity saturable transport of arginine in both AM- and P-BBMV ( K m 1.01 mM, V max 4.07 nmol/7s/mg protein and K m 1.38 mM, V max 2.26 nmol/7s/mg protein, respectively) was detected. Arginine influx was dependent on the membrane electrical potential (Δ ψ) and increased raising the alkalinity of the external medium from pH 7.2 to 10.6. Competition experiments indicated the following order of substrate affinity: arginine, homoarginine, N G-monomethylarginine, N G-nitroarginine>lysine≫ornithine>cysteine>methionine. Leucine, valine and BCH (2-amino-2-norbornanecarboxylic acid) did not inhibit arginine influx. In the presence of external K +, the influx of arginine as a function of arginine concentration fitted to a complex saturation kinetics compatible with both a low-affinity and a high-affinity component. The latter ( K m 0.035 mM, V max 2.54 nmol/7s/mg protein) was fully characterized. The influx rate had an optimum at pH 8.8, was strongly affected by Δ ψ and was homogeneous along the midgut. The substrate affinity rank was: homoarginine>arginine, N G-monomethylarginine≫cysteine, lysine≫ N G-nitroarginine>ornithine>methionine. Leucine and amino acids with a hydrophobic side chain were not accepted. This system is also operative in the absence of potassium, with the same order of specificity but a very low activity. Lysine influx is mediated by two more transport systems, the leucine uniport and the K +/leucine symport specific for amino acids with a hydrophobic side chain that recognizes lysine at extravesicular pH values (pH out) exceeding 9. Both the uniport and the symport differ from the cationic transport systems so far identified in mammals because they are unaffected by N-ethylmaleimide, have no significant affinity for neutral amino acids in the presence of the cation and show a striking difference in their optimum pH.

Effect of LPS on the permeability of the blood–brain barrier to insulin

Insulin has emerged as an important neuropeptide. Central actions of insulin appear to oppose those in the periphery. Insulin is transported across the blood–brain barrier (BBB) by a saturable transport system. The permeability of the BBB to insulin is altered by various events, but no studies exist that have examined the permeability of the BBB to insulin during infection or inflammation, states which can induce peripheral insulin resistance. We looked at the effects of lipopolysaccharide (LPS), a bacterial endotoxin and a powerful cytokine releaser, on the permeability of the BBB to human insulin in CD-1 mice. Intraperitoneal injections of LPS significantly increased the uptake by the brain of 131I–insulin and disrupted the BBB to 125I–albumin. After subtraction of the brain/serum ratio for 125I–albumin, brain/serum ratios for insulin were increased: 10.38±0.70 μl/g (LPS) vs. 3.62±0.27 μl/g (no LPS), P<0.0001, showing that LPS increased the uptake of insulin independent of BBB disruption. This increase in insulin uptake was due to enhanced saturable transport. Pretreatment with indomethacin 10 min before LPS injections enhanced BBB disruption, but not insulin transport. Pretreatment with the nitric oxide (NO) synthase inhibitor aminoguanidine had no effect on insulin or albumin uptake, but pretreatment with NG-nitro- l-arginine methyl ester ( l-NAME) enhanced insulin transport, but not BBB disruption. We conclude that LPS increases the saturable transport of insulin across the BBB independent of disruption and prostaglandins with potentiation by NO inhibition. Such increased transport could potentiate the central effects of insulin and so contribute to the peripheral insulin resistance seen with infection and inflammation.

Leptin transport at the blood–cerebrospinal fluid barrier using the perfused sheep choroid plexus model

Leptin is secreted by adipose tissue and thought to regulate appetite at the central level. Several studies have explored the central nervous system (CNS) entry of this peptide across the blood–brain and blood–cerebrospinal fluid (CSF) barriers in parallel, but this is the first to explore the transport kinetics of leptin across the choroid plexus (blood–CSF barrier) in isolation from the blood–brain barrier (BBB). This is important as the presence of both barriers can lead to ambiguous results from transport studies. The model used was the isolated Ringer perfused sheep choroid plexus. The steady-state extraction of [ 125I]leptin (7.5 pmol l −1) at the blood face of the choroid plexus was 21.1±5.7%, which was greater than extraction of the extracellular marker, giving a net cellular uptake for [ 125I]leptin (14.0±3.7%). In addition, trichloroacetic acid precipitable [ 125I] was detected in newly formed CSF, indicating intact protein transfer across the blood–CSF barrier. Human plasma concentrations of leptin are reported to be 0.5 nM. Experiments using 0.5 nM leptin in the Ringer produced a concentration of leptin in the CSF of 12 pM (similar to that measured in humans). [ 125I]Leptin uptake at the blood–plexus interface using the single-circulation paired tracer dilution technique (uptake in <60 s) indicated the presence of a saturable transport system, which followed Michaelis–Menten-type kinetics ( K m=16.3±1.8 nM, V max=41.2±1.4 pmol min −1 g −1), and a non-saturable component ( K d=0.065±0.002 ml min −1 g −1). In addition, secretion of new CSF by the choroid plexuses was significantly decreased with leptin present. This study indicates that leptin transport at the blood–CSF barrier is via saturable and non-saturable mechanisms and that the choroid plexus is involved in the regulation of leptin availability to the brain.

Characterization of the blood-brain barrier choline transporter using the in situ rat brain perfusion technique.

Choline enters brain by saturable transport at the blood-brain barrier (BBB). In separate studies, both sodium-dependent and passive choline transport systems of differing affinity have been reported at brain capillary endothelial cells. In the present study, we re-examined brain choline uptake using the in situ rat brain perfusion technique. Saturable brain choline uptake from perfusion fluid was best described by a model with a single transporter (V:(max) = 2.4-3.1 nmol/min/g; K(m) = 39-42 microM) with an apparent affinity (1/Km)) for choline five to ten-fold greater than previously reported in vivo, but less than neuronal 'high-affinity' brain choline transport (K(m) = 1-5 microM). BBB choline uptake from a sodium-free perfusion fluid using sucrose for osmotic balance was 50% greater than in the presence of sodium suggesting that sodium is not required for transport. Hemicholinium-3 inhibited brain choline uptake with a K(i) (57 +/- 11 microM) greater than that at the neuronal choline system. In summary, BBB choline transport occurs with greater affinity than previously reported, but does not match the properties of the neuronal choline transporter. The V:(max) of this system is appreciable and may provide a mechanism for delivering cationic drugs to brain.