Phenylalanine Transport Across the Blood-Brain Barrier as Studied with the In Situ Brain Perfusion Technique

Unidirectional L-phenylalanine transport into six brain regions of pentobarbital-anesthetized rats was studied using the in situ brain perfusion technique. This technique allows both accurate measurements of cerebrovascular amino acid transport and complete control of perfusate amino acid composition. L-Phenylalanine influx into the brain was sodium independent and could be described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants in the parietal cortex equaled 6.9 X 10(-4) mumol/s/g for Vmax, 0.011 mumol/ml for Km, and 1.8 X 10(-4) ml/s/g for KD during perfusion with fluid that did not contain competing amino acids. D-Phenylalanine competitively inhibited L-phenylalanine transport with a Ki approximately 10-fold greater than the Km for L-phenylalanine. There were no significant regional differences in Km, KD, or Ki, whereas Vmax was significantly greater in the cortical lobes than in the other brain regions. L-Phenylalanine influx during plasma perfusion was only 30% of that predicted in the absence of competing amino acids. Competitive inhibition increased the apparent Km during plasma perfusion by approximately 20-fold, to 0.21 mumol/ml. These data provide accurate new estimates of the kinetic constants that describe L-phenylalanine transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer site affinity (1/Km) for L-phenylalanine is three- to 12-fold greater than previously estimated in either awake or anesthetized animals.     

Kinetic Analysis of Cerebrovascular Isoleucine Transport from Saline and Plasma

The concentration dependence of regional isoleucine transport across the blood-brain barrier was determined in anesthetized rats with the in situ brain perfusion technique of Takasato et al. [Am. J. Physiol. 247, H484-493 (1984)]. This technique allows, for the first time, accurate measurements of cerebrovascular amino acid transport in the absence of competing amino acids using saline perfusate, and in the presence of physiological concentrations of amino acids using plasma perfusate. Cerebrovascular isoleucine transport from saline perfusate followed Michaelis-Menten saturation kinetics where Vmax = 9 - 11 X 10(-4) mumol X s-1 X g-1 and Km = 0.054-0.068 mumol X ml-1 in six brain regions. A component of nonsaturable transport was not detected in any brain region even though perfusate isoleucine concentration was increased to greater than or equal to 150 times the normal plasma concentration. Isoleucine influx during plasma perfusion was only 8% of that predicted from the saline perfusion data due to transport inhibition by competing amino acids in plasma. Competitive inhibition increased the apparent Km for isoleucine transport from plasma by greater than or equal to 24-fold to 1.5-1.7 mumol X ml-1. These data provide accurate new estimates of the kinetic constants that describe amino acid transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer-site affinity (1/Km) for isoleucine is approximately fivefold greater than previously reported with the brain uptake index technique.     

Neutral amino acid transport at the human blood-brain barrier

The kinetics of human blood-brain barrier neutral amino acid transport sites are described using isolated human brain capillaries as an in vitro model of the human blood-brain barrier. Kinetic parameters of transport (Km, Vmax, and KD) were determined for eight large neutral amino acids. Km values ranged from 0.30 +/- 0.08 microM for phenylalanine to 8.8 +/- 4.6 microM for valine. The amino acid analogs N-methylaminoisobutyric acid and 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid were used as model substrates of the alanine- and leucine-preferring transport systems, respectively. Phenylalanine is transported solely by the L-system (which is sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid), and leucine is transported equally by the L- and ASC-system (which is sodium-dependent and N-methylaminoisobutyric acid-independent). Dose-dependent inhibition of the high affinity transport system by p-chloromercuribenzenesulfonic acid is demonstrated for phenylalanine, similar to the known sensitivity of blood-brain barrier transport in vivo. The Km values for the human brain capillary in vitro correlate significantly (r = 0.83, p less than 0.01) with the Km values for the rat brain capillary in vivo. The results show that the affinity of human blood-brain barrier neutral amino acid transport is very high, i.e. very low Km compared to plasma amino acid concentrations. This provides a physical basis for the selective vulnerability of the human brain to derangements in amino acid availability caused by a selective hyperaminoacidemia, e.g. hyperphenylalaninemia.     

Blood-Brain Barrier Transport of 1-Aminocyclohexanecarboxylic Acid, a Nonmetabolizable Amino Acid for In Vivo Studies of Brain Transport

Regional transport of 1-aminocyclohexanecarboxylic acid (ACHC), a nonmetabolizable amino acid, across the blood-brain barrier was studied in pentobarbital-anesthetized rats using an in situ brain perfusion technique. The concentration dependence of influx was best described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants of the frontal cortex equaled 9.7 X 10(-4) mumol/s/g for Vmax, 0.054 mumol/ml for Km, and 1.0 X 10(-4) ml/s/g for KD in the absence of competing amino acids. Saturable influx could be reduced by greater than 85% by either L-phenylalanine or 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with transport by the cerebrovascular neutral amino acid transport system. The transport Km for ACHC was one-fifth that for the more commonly used homologue, 1-aminocyclopentanecarboxylic acid, and was similar to values for several natural amino acids, such as L-methionine, L-isoleucine, and L-tyrosine. The results indicate that ACHC may be a useful probe for in vivo studies of amino acid transport into brain.     

Kinetic Constants for Blood—Brain Barrier Amino Acid Transport in Conscious Rats

The kinetic constants for large neutral amino acid (LNAA) transport across the blood-brain barrier (BBB) of conscious rats were determined in four brain regions: cortex, caudate-putamen, hippocampus, and thalamus-hypothalamus. Indwelling external carotid artery catheters allowed for single-bolus (200 microliters) injections directly into the arterial system of unanesthetized and lightly restrained animals. Our results showed lower brain uptake index values for conscious rats compared to previous reports for anesthetized animals which are consistent with higher rates of cerebral blood flow in the conscious animals. Km values were lower in the conscious animals and ranged from 29% to 87% of the Km values in pentobarbital-anesthetized animals whereas the KD values were about twofold higher in the conscious animals. No apparent regional differences were observed. Influx rates were determined which take into consideration flow rates and plasma amino acid concentrations. Our results showed an average amino acid influx value of 5.2 nmol/min/g, which is 53% higher than the average influx in pentobarbital-anesthetized animals. The present results in conscious animals regarding the low Km of LNAA transport across the BBB lend further support to the importance of fluctuations in plasma amino acid concentrations and LNAA transport competitive effects on brain amino acid availability.     

Phenylalanine transport at the human blood-brain barrier. Studies with isolated human brain capillaries

The exquisite sensitivity of brain amino acid availability to changes in plasma amino acid composition arises from the uniquely high affinity (low Km) of blood-brain barrier transport sites as compared to cell membrane transport systems in nonbrain tissues. The extension of this paradigm from rats to man assumes that the Km of blood-brain barrier amino acid transport in the human is low as in the rat. This hypothesis is tested in the present studies wherein isolated human brain capillaries are used as a model system for the human blood-brain barrier. Capillaries were obtained from autopsy brain between 20 and 45 h after death and were isolated in high yield and free of adjoining brain tissue. [3H]Phenylalanine transport into the isolated human, rabbit, or rat brain capillary was characterized by two saturable transport systems and a nonsaturable component. The Km values of phenylalanine transport into brain capillaries via the two saturable systems averaged 0.26 +/- 0.08 and 22.3 +/- 7.1 microM for five human subjects. These studies provide the first evidence for a very high affinity (Km = 0.26 microM) neutral amino acid transport system at the blood-brain barrier, and it is hypothesized that this system is selectively localized to the brain side of the blood-brain barrier. The results also show that the transport Km values for phenylalanine transport are virtually identical at both the rat and human blood-brain barrier.     

Changes in Regional Blood–Brain Transfer of l-Leucine Elicited by Arginine-Vasopressin

Arginine-vasopressin (AVP), injected into the carotid artery in physiological concentration together with L-leucine, changed kinetic constants of the blood-brain barrier (BBB) transport of this neutral amino acid without changing the cerebral blood flow (CBF). The maximum velocity of transport (Vmax), the half-saturation constant (Km), the nonsaturable transport constant (KD), and CBF were estimated in nine brain regions of male Wistar rats anesthetized with ether. In cerebral hemisphere, Vmax decreased from 21 nmol . min-1 . g-1 (control) to 7.6 nmol . min-1 . g-1 (AVP). Km decreased from 0.11 to 0.029 mM. Regional differences of the kinetic constants were found in controls as well as in AVP-treated animals. In all regions, the calculated constants Vmax and Km of animals coinjected with AVP were significantly decreased when compared to controls. A direct or indirect interaction of AVP with the transport system of large neutral amino acids is suggested.     

Pantothenic Acid Transport Through the Blood-Brain Barrier

The unidirectional influx of D-pantothenic acid (PA) across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique using [3H]D-PA (1.1 Ci/mmol). PA was transported across the blood-brain barrier by a saturable system that could be described by a Michaelis-Menten transport model with a half-saturation concentration and maximal influx rate of 19 microM and 0.21 nmol/g of brain/min, respectively. PA (0.3 microM) transport through the blood-brain barrier was significantly inhibited by probenecid, nonanoic acid, and biotin (all less than or equal to 0.25 mM), but not by penicillin G, pyruvate, beta-hydroxybutyrate, L-leucine (all 1 mM), or poly-L-lysine HBr (1 mg/ml). Probenecid (0.25 mM), nonanoic acid (0.5 mM), and PA (1.0 mM) did not inhibit [3H]L-leucine transport through the blood-brain barrier, whereas 30 microM-L-leucine inhibited [3H]leucine transport to 23% of control values. Thus, PA is transported through the blood-brain barrier by a low-capacity, saturable transport system with a half-saturation concentration approximately 10 times the plasma PA concentration. Although involved in the transfer of PA from blood into brain, this system does not play an important regulatory role in the synthesis of CoA from PA in brain.     

Site-directed mutagenesis of rabbit LAT1 at amino acids 219 and 234

The availability of amino acids in the brain is regulated by the blood-brain barrier (BBB) large neutral amino acid transporter type 1 (LAT1) isoform, which is characterized by a high affinity (low Km) for substrate large neutral amino acids. The hypothesis that brain amino acid transport activity can be altered with single nucleotide polymorphisms was tested in the present studies with site-directed mutagenesis of the BBB LAT1. The rabbit has a high Km LAT1 large neutral amino acid transporter, as compared to the low Km neutral amino acid transporter at the human or rat BBB. The rabbit LAT1 was cloned from a rabbit brain capillary cDNA library. Alignment of the amino acid sequences of rabbit, human, and rat LAT1 revealed two radical amino acid residues that differ in the rabbit relative to the rat or human LAT1. The G219D mutation had a modest effect on the Km and Vmax of tryptophan transport via cloned rabbit LAT1 in frog oocytes, but the W234L variant reduced the Km by 64% and the Vmax by 96%. Conversely, LAT1 transport of either tryptophan or phenylalanine was nearly normalized when the double mutation W234L/G219D variant was produced. These studies show that marked changes in the affinity and capacity of the LAT1 are caused by single nucleotide polymorphisms and that phenotype can be restored with a double mutation.     

Facilitated Transport of the Neurotoxin, β-N-Methylamino-l-Alanine, Across the Blood-Brain Barrier

beta-N-Methylamino-L-alanine (BMAA) is a neurotoxic plant amino acid that has been implicated in the pathogenesis of the high incidence amyotrophic lateral sclerosis and related parkinsonism dementia of the western Pacific. Previous studies have demonstrated that BMAA is taken up into brain following intravenous or oral administration. To examine the kinetics and mechanism of brain transfer, BMAA influx across the blood-brain barrier was measured in rats using an in situ brain perfusion technique. BMAA influx was found to be saturable with a maximal transfer rate (Vmax) of 1.6 +/- 0.3 x 10(-3) mumol/s/g and a half-saturation constant (Km) of 2.9 +/- 0.7 mM based on total perfusate BMAA concentration. Uptake was sodium independent and inhibitable by excess L-leucine, but not by L-lysine, L-glutamate, or methylaminoisobutyric acid, indicative of transfer by the cerebrovascular large neutral amino acid carrier. L-BMAA competitively reduced brain influx of L-[14C]leucine, as expected for cross-inhibition. The results demonstrate that BMAA is taken up into brain by the large neutral amino acid carrier of the blood-brain barrier and suggest that uptake may be sensitive to the same factors that affect neutral amino acid transport, such as diet, metabolism, disease, and age.     

KINETICS OF COMPETITIVE INHIBITION OF NEUTRAL AMINO ACID TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER

The transport of tryptophan across the blood-brain barrier is used as a specific example of a general approach by which rates of amino acid influx into brain may be predicted from existing concentrations of amino acids in plasma. The kinetics of inhibition of [¹⁴C]tryptophan transport by four natural neutral amino acids (phenylalanine, leucine, methionine, and valine) and one synthetic amino acid (α-methyl tyrosine) is studied with a tissue-sampling, single injection technique in the barbiturate-anesthetized rat. The equality of the K₁ (determined from cross-inhibition studies) and the K_m (determined from auto-inhibition data) for neutral amino acid transport indicate that these amino acids compete for a single transport site in accordance with the kinetics of competitive inhibition. Based on equations derived for competitive inhibition, apparent K_m values are computed for the essential neutral amino acids from known data on amino acid transport K_m and plasma concentrations. The apparent K_m values make possible predictions of the in vivo rates of amino acid influx into brain based on given plasma amino acid concentrations. Finally, a method is presented for determining transport constants from saturation data obtained with single injection techniques.     

Dibasic amino acid interactions with Na+-independent transport system asc in horse erythrocytes. Kinetic evidence of functional and structural homology with Na+-dependent system ASC

Amino acid transport in horse erythrocytes is regulated by three co-dominant allelomorphic genes coding for high-affinity transport activity (system asc1), low-affinity transport activity (system asc2) and transport-deficiency, respectively. The asc systems are selective for neutral amino acids of intermediate size, but unlike conventional system ASC, do not require Na+ for activity. In the present series of experiments we have used a combined kinetic and genetic approach to establish that dibasic amino acids are also asc substrates, systems asc1 and asc2 representing the only mediated routes of cationic amino acid transport in horse erythrocytes. Both transporters were found to exhibit a strong preference for dibasic amino acids compared with neutral amino acids of similar size. Apparent Km values (mM) for influx via system asc1 were L-lysine (9), L-ornithine (27), L-arginine (27), L-alanine (0.35). Corresponding Vmax estimates (mmol/l cells per h, 37 degrees C) were L-lysine (1.65), L-ornithine (2.15), L-arginine (0.54), L-alanine (1.69). Apparent Km values for L-lysine and L-ornithine influx via system asc2 were approximately 90 and greater than 100 mM, respectively, with Vmax values greater than 2 and greater than 1 mmol/l cells per h, respectively. Apparent Km and Vmax values for L-alanine uptake by system asc2 were 14 mM and 6.90 mmol/l cells per h. In contrast, L-arginine was transported by system asc2 with the same apparent Km as L-alanine (14 mM), but with a 77-fold lower Vmax. This dibasic amino acid was shown to cause cis- and trans-inhibition of system asc2 in a manner analogous to its interaction with system ASC, where the side-chain guanidinium group is considered to occupy the Na+-binding site on the transporter. Concentrations of extracellular L-arginine causing 50% inhibition of zero-trans L-alanine influx and half-maximum inhibition of L-alanine zero-trans efflux were 14 mM (extracellular L-alanine concentration 15 mM) and 3 mM (intracellular L-alanine concentration 15.5 mM), respectively. We interpret these observations as evidence of structural homology between the horse erythrocyte asc transporters and system ASC. Physiologically, intracellular L-arginine may function as an endogenous inhibitor of system asc2 activity.     

Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target size of system asc in red cells from Przewalski's horse. The transporter's in situ apparent molecular weight was 158,000 +/- 2500 (SE).     

Blood-Brain Barrier Transport of Basic Amino Acids Is Selectively Inhibited at Low pH

The transport of amino acids across the blood-brain barrier was measured with the single-pass carotid injection method. The pH of the injected bolus varied between 4.5 and 8.5. Arginine and lysine uptakes were inhibited 24% at pH 5.5 and 59% at pH 4.5. The uptakes of 2-aminobicyclo (2,2,1) heptane-2-carboxylic acid and phenylalanine were unaffected at this pH. There were also no changes observed in choline, glucose, or butanol transport. The Ki of arginine transport inhibition by H+ was 2.4 +/- 0.5 microM; i.e., pH 5.6 +/- 0.1. No change with pH occurred in the Km of arginine transport, while a significant decrease (p less than 0.01) was observed in the Vmax (10.2 +/- 2.3 nmol min-1 g-1 and 5.6 +/- 2.3 nmol min-1 g-1 at pH 7.5 and pH 5.5, respectively). This noncompetitive inhibition was found to be transient as arginine uptake at pH 7.5; it was measured by carotid injection 30 sec following a previous bolus which was buffered to pH 4.5, and was not significantly different from the control. This selective inhibition of the blood-brain barrier basic amino acid carrier demonstrates the advantage of the carotid injection approach in exposing the capillary exchange site to extreme alterations in chemical composition which could not be tolerated systemically.     

Limited Blood-Brain Barrier Transport of Polyamines

Transport of polyamines across the blood-brain barrier of adult rats was examined by measuring the amount of radioactivity that reached the forebrain 5 s after a "bolus" intracarotid injection. The values were expressed by the brain uptake index (BUI), which is the percentage of material transported in relation to freely diffusible water in a single passage through the brain. Transport was restricted as indicated by the respective BUI values, presented as means +/- SD (number of animals): putrescine, 5.3 +/- 0.8 (11); spermidine, 6.1 +/- 1.3 (7); and spermine, 5.8 +/- 0.5 (4). A kinetic study of the transport of [14C]putrescine showed that transport due to passive diffusion accounted for the majority of the observed influx (66% at 1 mM putrescine). However, a small saturable component exists with a Km value of 4-5 mM and a Vmax of 30 nmol X min-1 X g-1. This Km value is considerably higher than the circulating levels of the polyamine in the normal mature animal, and thus is unlikely to be of physiological significance. Competition studies indicated that putrescine does not interact with carriers for adenosine, arginine, choline, or leucine.     

Characterization of threonine transport into a kidney epithelial cell line (BSC-1). Evidence for the presence of Na(+)-independent system asc [corrected

The transport routes for threonine in a primate kidney epithelial cell line (BSC-1) grown as monolayer in continuous cell culture were studied. We discovered at least four different transport systems for threonine uptake. The Na(+)-dependent route shows biphasic kinetics with a low and high affinity parameter. The apparent kinetic constants for Km1 and Km2 were 0.3 and 36 mM with apparent Vmax values of 6.3 and 90 nmol/mg protein/min, respectively. The high affinity, low Km component resembles system ASC activity, with respect to substrate selectivity. The Na(+)-independent route also exhibits biphasic kinetics. A high affinity component (apparent Km of 1.0 mM, and apparent Vmax of 7.2 nmol/mg protein/min) is sensitive to inhibition by leucine and the aminoendolevo-rotatory isomer of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, suggesting participation by system L. The low affinity component (apparent Km of 10.2 mM, and apparent Vmax of 71 nmol/mg protein/min) was specifically inhibited by threonine, serine, and alanine and could be assigned to system asc. The discrimination between system L and asc is based upon differences in pH sensitivity, trans stimulation, and Ki values. In addition, the effects of harmaline, a suspected sodium transport site inhibitor, have been studied. Harmaline noncompetitively inhibited Na(+)-dependent threonine uptake but had no effect on Na(+)-independent transport of threonine. This report is the first to present evidence for the presence of system asc in renal epithelial cells. The physiological and biochemical significance of our findings are discussed.     

Blood–Brain Transport of Thiamine Monophosphate in the Rat: A Kinetic Study In Vivo

To calculate the kinetic parameters of thiamine monophosphate transport across the rat blood-brain barrier in vivo, different doses of a [35S]thiamine monophosphate preparation with a specific activity of 14.8 mCi.mmol-1 were injected in the femoral vein and the radioactivity was measured in arterial femoral blood and in the cerebellum, cerebral cortex, pons, and medulla 20 s after the injection. This short experimental time was used to prevent thiamine monophosphate hydrolysis. Thiamine monophosphate was transported into the nervous tissue by a saturable mechanism. The maximal transport rate (Jmax) and the half-saturation concentration (Km) equaled 27-39 pmol.g-1.min-1 and 2.6-4.8 microM, respectively. When compared with that of thiamine, thiamine monophosphate transport seemed to be characterized by a lower affinity and a lower maximal influx rate. At physiological plasma concentrations, thiamine monophosphate transport rate ranged from 2.06 to 4.90 pmol.g-1.min-1, thus representing a significant component of thiamine supply to nervous tissue.     

Enantiomeric selectivity of carbovir transport.

Carbovir (9-[4 alpha-(hydroxymethyl)cyclopent-2-ene-1 alpha-yl]guanine) (CBV) is a carbocyclic analogue of 2',3'-dideoxyguanosine that exhibits potent and selective in vitro activity against human immunodeficiency virus. Antiviral activity is associated with only the (-)-enantiomer. The transport characteristics of both (-)-CBV and (+)-CBV were investigated in human erythrocytes at 37 degrees C using a papaverine-stop assay. The influx of both enantiomers appeared saturable and was inhibited greater than 90% by a combination of adenine (a low Km permeant of the nucleobase carrier) and dilazep (a potent inhibitor of nucleoside transport). The influx of (-)-CBV and (+)-CBV proceeded primarily via the nucleobase carrier with Vmax (picomoles/second/5 microliters of cells)/Km (millimolar) values of 17/0.12 and 140/1.9, respectively. To a lesser extent, the influx of (-)-CBV and (+)-CBV also occurred via the nucleoside transporter. Although both compounds exhibited a similar low affinity for this latter carrier (Km approximately 2 mM), the Vmax for (-)-CBV influx was approximately 4-fold higher than the Vmax for (+)-CBV influx. We conclude that both CBV enantiomers enter human erythrocytes by two transporters that are enantiomerically selective.     

The complementary membranes forming the blood-brain barrier

Brain capillary endothelial cells form the blood-brain barrier. They are connected by extensive tight junctions, and are polarized into luminal (blood-facing) and abluminal (brain-facing) plasma membrane domains. The polar distribution of transport proteins allows for active regulation of brain extracellular fluid. Experiments on isolated membrane vesicles from capillary endothelial cells of bovine brain demonstrated the polar arrangement of amino acid and glucose transporters, and the utility of such arrangements have been proposed. For instance, passive carriers for glutamine and glutamate have been found only in the luminal membrane of blood-brain barrier cells, while Na-dependent secondary active transporters are at the abluminal membrane. This organization could promote the net removal of nitrogen-rich amino acids from brain, and account for the low level of glutamate penetration into the central nervous system. Furthermore, the presence of a gamma-glutamyl cycle at the luminal membrane and Na-dependent amino acid transporters at the abluminal membrane may serve to modulate movement of amino acids from blood-to-brain. Passive carriers facilitate amino acid transport into brain. However, activation of the gamma-glutamyl cycle by increased plasma amino acids is expected to generate oxoproline within the blood-brain barrier. Oxoproline stimulates secondary active amino acid transporters (Systems A and B(o)+) at the abluminal membrane, thereby reducing net influx of amino acids to brain. Finally, passive glucose transporters are present in both the luminal and abluminal membranes of the blood-brain barrier. Interestingly, a high affinity Na-dependent glucose carrier has been described only in the abluminal membrane. This raises the question whether glucose entry may be regulated to some extent. Immunoblotting studies suggest more than one type of passive glucose transporter exist in the blood-brain barrier, each with an asymmetrical distribution. In conclusion, it is now clear that the blood-brain barrier participates in the active regulation of brain extracellular fluid, and that the diverse functions of each plasma membrane domain contributes to these regulatory functions.     

Plasma membrane vesicles from isolated hepatocytes retain the increase of amino acid transport induced by dibutyryl cyclic AMP in intact cells

We have investigated the effect of cyclic AMP on hepatic amino acid transport by measuring the uptake of L-alanine in plasma membrane vesicles purified from hepatocytes incubated without or with dibutyryl cyclic AMP. The application of an Na+ gradient to vesicles from hepatocytes exposed to dibutyryl cyclic AMP, compared to control, resulted in a greater transient accumulation of L-alanine, whereas in the presence of a K+ gradient a similar slow equilibration of L-alanine was observed. Kinetic analysis of L-alanine influx revealed that the increased uptake resulted from an increased capacity (Vmax) with no change in the affinity (Km) of the carriers for L-alanine. These results strongly suggest that dibutyryl cyclic AMP induces stable changes at the membrane level probably by increasing the number of amino acid carrier molecules.