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.     

Blood–Brain Barrier Transport of Valproic Acid

Valproic acid distribution in brain is less than that of other anticonvulsants such as phenytoin or phenobarbital. Possible mechanisms for this decreased distribution space in brain include (a) increased plasma protein binding of valproate relative to the other anticonvulsants and (b) asymmetric blood-brain barrier (BBB) transport of valproate such that the brain-to-blood flux exceeds the blood-to-brain flux. These mechanisms are investigated in the present studies using the intracarotid injection technique in rats and rabbits. In the rat, the brain uptake index (BUI) of [14C]valproate relative to [3H]water is 51 +/- 6%, indicating the blood-to-brain transport of water is twofold greater than that of valproate. However, the BUI of [14C]valproate relative to [3H]water decreased with time after carotid injection during a 4-min washout period, which indicates that brain-to-blood transport of valproate is greater than that of water. This suggests that the permeability of the BBB to valproate is polarized, with antiluminal permeability being much greater than luminal permeability. In rabbits, the BUI of [14C]valproate is 47 +/- 7% in newborns and 17 +/- 6% in adult animals. However, the high drug extraction in newborns may be attributed to decreased cerebral blood flow in the neonate as the BBB permeability-surface area (PS) products are unchanged (e.g., PS = 0.13 and 0.11 ml min-1 X g-1 in the newborn and adult rabbit, respectively). With regard to plasma protein binding effects on valproate transport, brain valproate uptake was also measured in the presence of human, lamb, pig, rat, horse, goat, hamster, dog, and mouse sera. Higher brain uptakes were observed when the unbound fraction of drug increased. However, our data indicate that a fraction of the valproic acid entering the capillaries bound to plasma proteins had the capacity to equilibrate with brain because of enhanced drug dissociation from albumin in the brain microcirculation. Since plasma protein-bound valproate is available for uptake by brain, the major factor underlying the diminished distribution of the drug in brain appears to be the asymmetric transport properties of the BBB to valproic acid.     

Kinetics of Regional Blood–Brain Barrier Transport and Brain Phosphorylation of Glucose and 2-Deoxyglucose in the Barbiturate–Anesthetized Rat

Abstract: Recent studies indicate the lumped constant (LC), which defines the relative rates of brain utilization of glucose and 2-deoxyglucose (2-DG), doubles to values > 1.0 under conditions of hypoglycemia. Since changes in the LC should be predictable given the kinetic parameters of blood-brain barrier (BBB) transport and brain phosphorylation of glucose and 2-DG, the present studies were designed to measure the necessary kinetic parameters. The carotid injection technique was used to determine cerebral blood flow and the K_m , V_max, and K _D of glucose and 2-DG transport through the BBB in seven brain regions in rats anesthetized with 50 mg/kg i.p. pentobarbital. Regional glucose transport through the BBB was characterized by an average K_m = 6.3 m m , average V_max = 0.53 μmol min⁻¹g⁻¹, and average K _D= 0.022 ml min⁻¹g⁻¹. The nonsaturable route of transport of glucose represented on the average 40% of the total glucose influx into brain regions at an arterial glucose concentration of 10 m m . In addition, the rate constants of phosphorylation of glucose and 2-DG were measured for each region. Substitutions of the measured kinetic parameters for sugar transport and phosphorylation into equations defining the LC confirm the observation that the LC would be expected to vary under extreme conditions such as hypoglycemia and to exceed values of 1.0 under these conditions.     

Blood–Brain Barrier Transport of Kynurenines: Implications for Brain Synthesis and Metabolism

To evaluate the potential contribution of circulating kynurenines to brain kynurenine pools, the rates of cerebral uptake and mechanisms of blood-brain barrier transport were determined for several kynurenine metabolites of tryptophan, including L-kynurenine (L-KYN), 3-hydroxykynurenine (3-HKYN), 3-hydroxyanthranilic acid (3-HANA), anthranilic acid (ANA), kynurenic acid (KYNA), and quinolinic acid (QUIN), in pentobarbital-anesthetized rats using an in situ brain perfusion technique. L-KYN was found to be taken up into brain at a significant rate [permeability-surface area product (PA) = 2-3 x 10(-3) ml/s/g] by the large neutral amino acid carrier (L-system) of the blood-brain barrier. Best-fit estimates of the Vmax and Km of saturable L-KYN transfer equalled 4.5 x 10(-4) mumol/s/g and 0.16 mumol/ml, respectively. The same carrier may also mediate the brain uptake of 3-HKYN as D,L-3-HKYN competitively inhibited the brain transfer of the large neutral amino acid L-leucine. For the other metabolites, uptake appeared mediated by passive diffusion. This occurred at a significant rate for ANA (PA, 0.7-1.6 x 10(-3) ml/s/g), and at far lower rates (PA, 2-7 x 10(-5) ml/s/g) for 3-HANA, KYNA, and QUIN. Transfer for KYNA, 3-HANA, and ANA also appeared to be limited by plasma protein binding. The results demonstrate the saturable transfer of L-KYN across the blood-brain barrier and suggest that circulating L-KYN, 3-HKYN, and ANA may each contribute significantly to respective cerebral pools. In contrast, QUIN, KYNA, and 3-HANA cross the blood-brain barrier poorly, and therefore are not expected to contribute significantly to brain pools under normal conditions.     

Measurement of Solute Transport Across the Blood–Brain Barrier in the Perfused Guinea Pig Brain: Method and Application to N-Methyl-α-Aminoisobutyric Acid

A technique for the vascular perfusion of the guinea pig head in vivo, suitable for measurements of blood-to-brain transport under controlled conditions of arterial inflow, has been developed. With a perfusion pressure ranging between 13 and 18 kPa and PCO2 in the arterial inflow of 5 and 5.5 kPa, cerebral blood flow, measured with [14C]butanol, was about 1 ml min-1 g-1 in the cerebral cortex, hippocampus, and caudate-putamen of the ipsilateral hemisphere; in the cerebellum and pontine white matter it was considerably less, and much higher perfusion pressures were required to establish equal blood flow throughout the whole brain. Regional water content, Na+/K+ ratio, ATP, energy charge potential, and lactate content of the ipsilateral side of perfused and nonperfused brain were not significantly different after 10 min perfusion. The D-[3H]mannitol space did not exceed 1% after 30 min of perfusion, indicating the integrity of the barrier. Over this period, EEG, ECG, and respiratory waveform remained normal. When [14C]N-methyl-alpha-aminoisobutyric acid (MeAIB), and D-[3H]mannitol were perfused together over periods extending to 30 min progressive uptakes of both solutes by the parietal cortex could be measured, and the unidirectional transfer constants estimated from multiple time-uptake data. The Kin for MeAIB (0.75 X 10(-3) ml min-1 g-1) was some three times that for mannitol. It is concluded that the technique provides a stable, well-controlled environment in the cerebral microvasculature of the ipsilateral perfused brain hemisphere suitable for examining the transport of slowly penetrating solutes into the brain.     

An Easier, Reproducible, and Mass-Production Method to Study the Blood–Brain Barrier In Vitro

To provide an "in vitro" system for studying brain capillary function, we have developed a process of coculture that closely mimics the "in vivo" situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. Under these conditions, endothelial cells retain all the endothelial cell markers and the characteristics of the blood-brain barrier, including tight junctions and gamma-glutamyl transpeptidase activity. The average electric resistance for the monolayers was 661 omega cm2. The system is impermeable to inulin and sucrose but allows the transport of leucine. Arabinose treatment increases transcellular transport flux by 70%. The relative ease with which such monolayers can be produced in large quantities would facilitate the "in vitro" study of brain capillary functions.     

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.     

Blood–Brain Glucose Transport in the Conscious Rat: Comparison of the Intravenous and Intracarotid Injection Methods

Abstract: The unidirectional transfer of d -glucose from blood to parietal cortex tissue of the brain of awake rats was measured by single intravenous injection of tracer glucose, as well as by single intracarotid injection according to the method of Oldendorf. The maximal unidirectional blood–brain glucose transfer rate (T_max) was 407 μ mol (100 g)^–1 min^–1 when measured by intravenous injection, and 352 μ mol (100 g)^–1 min^–1 when measured by intracarotid injection. The half–saturation constants (K_m) were 7.8 mm and 16.8 HIM, respectively. The comparison shows that the two methods give similar results when cerebral perfusion is assessed accurately.     

Kinetics of Regional Blood-Brain Barrier Glucose Transport and Cerebral Blood Flow Determined with the Carotid Injection Technique in Conscious Rats

Anesthetics, particularly barbiturates, have depressive effects on cerebral blood flow and metabolism and likely have similar effects on blood-brain barrier (BBB) transport. In previous studies utilizing the carotid injection technique, it was necessary to anesthetize the animals prior to performing the experiment. The carotid injection technique was modified by catheter implantation in the external carotid artery at the bifurcation of the common carotid artery. The technique was used to determine cerebral blood flow, the Km, Vmax, and KD of glucose transport in hippocampus, caudate, cortex, and thalamus-hypothalamus in conscious rats. Blood flow increased two to three times from that seen in the anesthetized rat. The Km in the four regions ranged between 6.5 and 9.2 mM, the Vmax ranged between 1.15 and 2.07 mumol/min/g, and the KD ranged between 0.015 and 0.035 ml/min/g. The Km and KD in the conscious rat did not differ from the values seen in the barbiturate anesthetized rat. The Vmax, on the other hand, increased two- to three-fold from that seen in the anesthetized rat and was nearly proportional to the increase in blood flow seen in the conscious rat. The development of the external carotid catheter technique now allows for determination of BBB substrate transport in conscious animals.     

Blood–Brain Transfer of Glucose and Glucose Analogs in Newborn Rats

Little is known of the selectivity of the blood-brain barrier at birth. Hexoses are transported through the barrier by a facilitating mechanism. To study the capacity of this mechanism to distinguish between analogs of D-glucose, we compared the transport of fluorodeoxyglucose, deoxyglucose, glucose, methylglucose, mannose, galactose, mannitol, and iodoantipyrine across the cerebral capillary endothelium in newborn Wistar rats. Cerebral blood flow, glucose consumption, and the blood-brain permeabilities of the hexoses were 25-50% of the adult values but the ratios between the permeabilities of the individual hexoses were similar to the ratios observed in adult rats. The mannitol clearance into brain was considerably higher than in adult rats (about 10-fold), indicating a higher endothelial permeability to small polar nonelectrolytes. The brain water content was higher in newborn than in adult rats and was associated with a higher steady-state distribution of labeled methylglucose between brain and blood. Hexose concentrations were determined relative to whole blood because the apparent erythrocyte membrane permeability to glucose was as high as in humans and thus considerably higher than in adult rats. The half-saturation concentration of glucose transport across the blood-brain barrier was considerably higher than in adult rats, about three-fold, suggesting that net blood-brain glucose transfer is less sensitive to blood glucose fluctuation in newborn than in adult rats.     

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.     

Kinetics of Neutral Amino Acid Transport Across the Blood-Brain Barrier

Neutral amino acid (NAA) transport across the blood-brain barrier was examined in pentobarbital-anesthetized rats with an in situ brain perfusion technique. Fourteen of 16 plasma NAAs showed measurable affinity for the cerebrovascular NAA transport system. Values of the transport constants (Vmax, Km, KD) were determined for seven large NAAs from saturation studies, whereas Km values for five small NAAs were estimated from inhibition studies. These data, together with our previous work, provide a complete set of constants for prediction of NAA influx from plasma. Among the NAAs, Vmax varied at least fivefold and Km varied approximately 700 fold. The apparent affinity (1/Km) of each NAA was related linearly (r = 0.910) to the octanol/water partition coefficient, a measure of NAA side-chain hydrophobicity. Predicted influx values from transport constants and average plasma concentrations agree well with values measured using plasma perfusate. These results provide accurate new estimates of the kinetic constants that determine NAA transport across the blood-brain barrier. Furthermore, they suggest that affinity of a L-alpha-amino acid for the transport system is determined primarily by side-chain hydrophobicity.     

Cysteine and Cystine Transport at the Blood-Brain Barrier

Abstract: The nature of cysteine and cystine uptake from the cerebral capillary lumen was studied in the rat using the carotid injection technique. [³⁵S]-Cysteine uptake was readily inhibited by the synthetic amino acid 2-amino-bicyclo(2,2,1)heptane-2-carboxylic acid (BCH), the defining substrate for the leucine-preferring (L) system in the Ehrlich ascites cell. The addition of non-radioactive alanine or serine, representatives of the alanine, serine, and cysteine-preferring (ASC) system, produced no significant decrease in the uptake of cysteine after cysteine transport by the L system was blocked with BCH. This indicated that the major component of cysteine's transport from the brain capillary lumen was by the L system with no detectable uptake of cysteine by the ASC system. No carrier-mediated transport of cystine, the disulfide form of the amino acid, was detected, nor was there any inhibition by cystine of the transport of the neutral amino acid methionine or the basic amino acid arginine. These results suggest that the ASC system, if present, is not quantitatively important for the transport of neutral amino acids from the brain capillary lumen.     

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.     

血脑屏障与脑血管疾病的相关研究

血脑屏障（blood brain barrier,BBB）的主要结构包括：脑毛细血管内皮细胞及其间的紧密连接（tight junction,TJ）、基底膜、基 底膜下星型胶质细胞终足。血脑屏障是存在于血液和脑组织之间的一层屏障系统,在许多大脑疾患的病理过程中,BBB 的破坏导 致通透性增高都是不可避免的一个环节。BBB是保证中枢神经系统的正常生理功能的重要屏障系统。目前已有大量关于血脑屏 障通透性在脑血管疾病中的变化研究。本文分别从血脑屏障的结构和功能,药物通过血脑屏障的方法和功能,脑缺血损伤、阿尔 茨海默病、帕金森病和多发性硬化症等不同的脑病变与血脑屏障通透性的变化及中医药应用等方面做一综述。有针对性地对 BBB和大脑疾病进行进一步的研究与探索,将会为临床治疗相关疾病带来新的视角与机遇。     

Kinetics of Neutral Amino Acid Transport Through the Blood-Brain Barrier of the Newborn Rabbit

Abstract: Since protein synthesis in the developing brain may, under certain conditions, be limited by amino acid availability, the present studies were undertaken to characterize the kinetics of large neutral amino acid transport through the blood-brain barrier (BBB) of the newborn rabbit. The K_m, V_max, and K_D of the transport of eight amino acids were determined by a nonlinear regression analysis of data obtained with the carotid injection technique. Compared with kinetic parameters observed for the adult rat, the K_m, V_max, and K_D of amino acid transport were all two- to threefold higher in the newborn. Albumin was found to bind tryptophan actively in vitro , but had no inhibitory effect on tryptophan transport through the newborn BBB. Glutamine was transported through the BBB of the newborn at rates severalfold higher than are seen in the adult rat. However, glutamine transport was not inhibited by high concentrations of N -methylaminoisobutyric acid (NMAIB), a model amino acid that is specific for the alanine-preferring or A-system present in peripheral tissues. In conclusion, these studies show that the BBB neutral amino acid transport system of the newborn rabbit has a lower affinity and higher capacity than does the BBB of the adult rat. Under conditions of high plasma amino acids, the increased capacity of the newborn transport system allows for a higher rate of amino acid transport into brain than would occur via the lower capacity system present in the adult rat brain.     

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.     

Fatty Acid Transport Through the Blood-Brain Barrier

Across the cerebral capillaries, the anatomical locus of the blood-brain barrier, the unidirectional influxes of the saturated fatty acids, octanoic and myristic acids, and the unsaturated essential fatty acid, linoleic acid, were measured. Employing an in situ rat brain perfusion technique that allows control of perfusate composition and accurate measurement of perfusate-to-brain fatty acid transport, we found that both [¹⁴C]octanoic and [¹⁴C]myristic acids were transported through the blood-brain barrier in vivo, in large part, by a specific, probenecid-sensitive transport system. However, the transport of [¹⁴C]linoleic acid was not probenecid sensitive. With 0.5 μM fatty acid but no plasma proteins in the perfusate, the permeability-surface area constant was higher for myristic acid (4.8 × 10^--²× s^-1) than for octanoic and linoleic acids (1.5 and 1.2 × 10^--²× s^-1, respectively). Approximately 70, 30, and 25% of the [¹⁴C]myristic, [¹⁴C]octanoic, or [¹⁴C]linoleic acids, respectively, were extracted from the perfusate.     

Blood–Brain Barrier Pericytes Are the Main Source of γ-Glutamyltranspeptidase Activity in Brain Capillaries

Cerebral endothelial cells form the selective permeability barrier between brain and blood by virtue of their impermeable tight junctions and the presence of specific carrier systems. These specialized properties of brain capillaries are reflected in the presence of proteins that are not found in other capillaries of the body. gamma-Glutamyltranspeptidase (GGT) has been widely used as a marker for brain capillaries and differentiated properties of brain endothelial cells. By using histochemical and biochemical methods we have investigated the expression of GGT in isolated capillaries, cultured brain endothelial cells and pericytes, and cocultures of astrocytes and brain endothelial cells. It was surprising that the majority of GGT activity was associated with pericytes, but not endothelial cells, suggesting that GGT is a specific marker for brain pericytes. The remaining GGT activity that was associated with endothelial cells rapidly disappeared from cultured cells but was reinduced in cocultures with astrocytes. Our results emphasize the need for pure endothelial cells for the investigation of blood-brain barrier characteristics.     

Effect of Diabetes Mellitus on the Permeability of the Blood–Brain Barrier to Insulin

Banks, W. A., J. B. Jaspan and A. J. Kastin. Effect of diabetes mellitus on the permeability of the blood–brain barrier to insulin. Peptides 18(10) 1577–1584, 1997.—Insulin derived from the peripheral circulation has been shown to exert various effects on the brain due to its ability to cross the blood–brain barrier (BBB). The relation between diabetes mellitus and insulin has been extensively studied for peripheral tissues but not for central nervous system tissues. We examined the effects that streptozotocin- or alloxan-induced diabetes have on the transport of insulin across the murine BBB. We used multiple-time regression analysis to measure the unidirectional influx rate constant (K_i) and vascular association (V_i) of intravenously injected, radioactively labeled human insulin (I-Ins). Treatment with streptozotocin induced an enhancement of both the K_i and V_i of I-Ins that correlated with the onset of diabetes. Brain perfusion showed that the enhanced uptake was not due to altered vascular space or levels of insulin in the serum. Alloxan enhanced K_i and V_i after 5 days but the early phase of diabetes was associated with a decreased K_i. Hyperglycemia induced by the intraperitoneal injection of glucose elevated the V_i but abolished the K_i. Furthermore, altered I-Ins uptake by brain was not associated with changes in brain or body weight. These results show that there is an increased uptake of I-Ins by the brain in the diabetic state that is not due to acute changes in the serum levels of glucose or insulin, altered vascular space, or catabolic events. Chronic changes in levels of glucose, insulin or other hormone or neuroendocrine agents are likely to underlie the altered rate of transport of insulin across the BBB of diabetic mice.