Sodium-dependent high-affinity uptake of taurine by isolated rat brain capillaries.

Transport of taurine has been demonstrated in capillary preparations from adult rat brains using [3H]taurine. Taurine transport is mediated by a saturable high-affinity system which is entirely dependent on sodium ions. The apparent maximal influx (Vmax) and half-saturation concentration (Km) corresponded to 1.06.10(-4) mumol/min per mg protein and 27.5 microM, respectively. Competition experiments in the presence of sodium ion showed that [3H]taurine uptake was strongly inhibited by 0.1 mM unlabeled structural analogues of taurine such as beta-alanine and hypotaurine as well as unlabeled taurine. gamma-Aminobutyric acid (GABA) (0.1 mM) inhibited the uptake of labeled taurine by 30%, whereas isethionic acid, L-methionine, L-2,4-diaminobutyric acid, glycine, L-cysteinesulfonic acid and cystamine did not exhibit any inhibitory effect. The results suggest that the Na+ gradient is the principal source of energy for taurine transport into isolated brain capillaries. This transport system may play an active role in the regulation of taurine concentration in the brain extracellular space.

Regressive or lethal lead encephalopathy in the suckling rat. Correlation of lead levels and morphological findings.

Lead encephalopathy was produced in immature Sprague-Dawley rats with an intraperitoneal (IP) injection of 60 micrograms/g body weight of lead acetate administered daily from the fifth day after birth. Macroscopic and light microscopic study of the nervous system, estimations of the blood-brain barrier permeability to proteins and brain water content were performed every two days thereafter. Lead levels in total blood, plasma, and several brain areas were measured at the same intervals by flameless atomic absorption spectrometry. Electron microscopic study of the cerebellum was done 2, 6, and 12 days after beginning lead administration. After two days of lead administration and before any pathological change occurred the increase in lead level was greater in the cerebellum than in other brain areas. After four to six days, hemorrhagic lead encephalopathy developed and was most prominent in regions with higher lead levels. From day 11 to 14, there were two possible courses: a) improvement of the clinical status and morphological findings in 25% of the animals, or b) progression of abnormal clinical signs and death. Cerebral edema, both intra- and extracellular, may have contributed to the fatal evolution. The mechanism of this edema appeared complex and may have involved resorption failure. Good correlations were observed among progression of the clinical signs, high water content in the brain, morphological evidence of cerebral edema, and a high cerebellar lead level. In contrast, high blood lead levels could be associated with clinical improvement, normal brain water content, and regression of the pathological findings. These data suggest that differences in evolution are more likely related to differences in the development of resistance of the cerebral capillary to lead, or in the efflux of lead, rather than to the blood lead concentrations.

Lipid synthesis by rat brain microvessel endothelial cells in tissue culture.

Lipid synthesis and its regulation by serum lipoproteins at the microvascular blood-brain barrier were studied using primary cultures of microvascular endothelial cells from rat brain. These cells are capable of synthesizing all their lipids (neutral lipids, phospholipids, glycolipids) from the water-soluble compounds, glucose, acetate, acetoacetate and beta-hydroxybutyrate. The ketone bodies, especially acetoacetate, are the preferred substrates for lipid synthesis. The incorporation patterns of acetate, acetoacetate and beta-hydroxybutyrate are very similar, indicating that these precursors contribute to lipid synthesis via the same metabolic route. However, the metabolic pathway is different for glucose, which is preferentially incorporated into phospholipids. The existence of an inverse relationship between lipid synthesis and the serum lipoprotein concentration suggests that cultured cerebral endothelial cells are capable of taking up lipids, principally cholesterol, contained in the serum lipoproteins. Cellular lipids would thus be supplied both by intracellular lipid synthesis and by serum lipoproteins. The difference between cholesterol synthesis rates in cultured cerebral endothelial cells and in isolated brain microvessel cells could be partly explained by the fact that the lipoprotein concentration is much lower in the culture medium than in rat serum.

Cerebral blood flow and blood-brain influx of some neutral amino acids in control and hypothyroid 16-day-old rats.

Rats were made hypothyroid by a daily subcutaneous injection of propylthiouracil beginning the first day after birth. CBF, brain plasma volume, blood-brain extraction, and influx of some neutral amino acids were studied in 16-day-old animals. In hypothyroid rats, the brain plasma volume was decreased by approximately 30%. CBF was decreased by greater than 50%. This decrease was the highest in cerebellum. Blood-brain extraction of small neutral amino acids (alanine, serine, cysteine) was greatly enhanced. This greater extraction compensated for the decreased supply of alanine brought about by its decreased plasma concentration and the lower CBF. In contrast, the extraction of the large amino acids tested (leucine, phenylalanine) was hardly increased, and the influx of phenylalanine was slightly decreased. These results suggest an alteration in the maturation of the brain capillary bed and capillary transport for neutral amino acids in hypothyroidism. The differential effect of hypothyroidism on some small and large amino acids is an additional argument for the existence of two systems of transport for neutral amino acids at the luminal membrane of brain capillary endothelial cells of immature rats.

Evidence for an alanine, serine, and cysteine system of transport in isolated brain capillaries.

Brain capillaries isolated from 2-month-old male and female Sprague-Dawley rats were used to study the transport of neutral amino acids. The uptake of alanine, leucine, and alpha-methylaminoisobutyric acid (MeAIB) was a linear function of time for the first minute of incubation. Based on these observations, an incubation time of 1 min was used to measure transport activities. The intracellular water volume of the isolated capillaries was 2.2 microliters/mg protein. This value was significantly lower (1.8 microliter/mg protein) when measured in the absence of sodium. L-Alanine, L-serine, and L-cysteine were taken up from the abluminal surface of brain capillaries by a sodium- and energy-dependent, carrier-mediated system. This uptake, for the most part, was not inhibited by MeAIB. System ASC (alanine-serine-cysteine) appeared to be of primary importance for the transport of these amino acids in isolated brain capillaries. The apparent Km and Vmax values for L-alanine uptake by ASC transport, based on the Hofstee plot presentation, were 1.3 mM and 0.975 nmol/microliter water content/min, respectively. The results also indicate that the transport of MeAIB and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) was limited to the sodium-dependent system A (alanine) and the sodium-independent system L (leucine), respectively.

Age-dependent alteration in regional cerebrovascular permeability during drug-induced epilepsy.

Age-related changes in blood-brain barrier permeability were investigated during pentylenetetrazol-induced seizures in rats aged from 15 days to 120 days. Tracers such as [14C]sucrose and [3H]inulin which diffuse very slowly across the intact endothelium were simultaneously injected i.v. in rats treated with pentylenetetrazol (PTZ) or in control animals. Permeability-surface area products (PA) were determined in 9 brain regions. Pentylenetetrazol-induced seizures caused a significant increase in PA for both sucrose and inulin in all brain regions studied. Blood-brain barrier dysfunction was present only in animals in which the mean arterial blood pressure rose at seizure onset. Although increased blood-brain barrier permeability was found partly in similar areas in both young and adult rat brains, in adults the increase was the highest in the preoptic area, septum, colliculus inferior, hypothalamus and in the cerebellum while the increase was comparatively much smaller in the same areas of young brains. The increase in blood-brain barrier permeability was extremely high in the hippocampus, hypothalamus and cerebellum of 15-day-old rat brain and, was least affected in the corpus striatum and cerebral cortex in contrast to older rats. From the results obtained it may be concluded that the increased cerebrovascular permeability induced by pentylenetetrazol differs markedly in localization in young and adult rats. The age-dependent increased blood-brain barrier integrity is not over all dependent on variations in the blood pressure, but rather on progressive maturation of capillaries and changes in their internal structure, and local phenomena in neuronal activity during the seizures.

Passage of spermidine across the blood-brain barrier in short recirculation periods following global cerebral ischemia: effects of mild hyperthermia.

Transport of a polyamine (PA), spermidine (SPMD) into rat brain at various early postischemic periods was studied. Rats underwent 20 min of four-vessel occlusion (4VO) followed by 5, 10, 30 and 60 min of recirculation (RC) periods with natural brain temperature. 3H-aminoisobutyricacid (AIB) and 14C-SPMD were utilised to search dual functions of the blood-brain barrier (BBB); barrier and carrier functions, respectively. Unidirectional blood-to-brain transfer constant (Kin) was calculated for AIB and SPMD in four brain regions-parieto-temporal cortex, striatum, hippocampus and cerebellum. Kin for SPMD ranged between 1.2+/-0.3 x 10(3) ml g(-1) min(-1) (for striatum) and 2.2+/-0.4 x 10(3) ml g(-1) min(-1) (for cerebellum) in controls. Kin for AIB showed similar values. At 5 and 10 min RC periods, Kin for both substances increased in a non-specific manner in all brain regions studied. In the cortex, Kin for SPMD at 5 and 10 min RC periods were 3.2+/-0.4 x 10(3) and 2.9+/-0.3 x 10(3) ml g(-1) min(-1), respectively, and found to be maximum with respect to other brain regions studied. 30 and 60 min RC groups showed specific transport for SPMD, whilst there were no changes for Kin for AIB, in all brain regions studied. Hippocampus showed the maximum increase in Kin SPMD at 60 min RC (2.7+/-0.3 x 10(3) ml g(-1) min(-1)), corresponding to a percentage rise of 121%. Intraischemic mild brain hyperthermia (39 degrees C) gave rise to a striking increase in Kin at 60 min postischemia for both substances. These results suggest that there is a specific transport of SPMD into brain at 30 and 60 min RC periods following 20 min of forebrain ischemia. Moreover, dual functions of the BBB were perturbed with intracerebral mild hyperthermia during ischemia.

Effects of respiratory acidosis and alkalosis on the distribution of cyanide into the rat brain.

The aim of this study was to determine whether respiratory acidosis favors the cerebral distribution of cyanide, and conversely, if respiratory alkalosis limits its distribution. The pharmacokinetics of a nontoxic dose of cyanide were first studied in a group of 7 rats in order to determine the distribution phase. The pharmacokinetics were found to best fit a 3-compartment model with very rapid distribution (whole blood T(1/2)alpha = 21.6 +/- 3.3 s). Then the effects of the modulation of arterial pH on the distribution of a nontoxic dose of intravenously administered cyanide into the brains of rats were studied by means of the determination of the permeability-area product (PA). The modulation of arterial blood pH was performed by variation of arterial carbon dioxide tension (PaCO2) in 3 groups of 8 anesthetized mechanically ventilated rats. The mean arterial pH measured 20 min after the start of mechanical ventilation in the acidotic, physiologic, and alkalotic groups were 7.07 +/- 0.03, 7.41 +/- 0.01, and 7.58 +/- 0.01, respectively. The mean PAs in the acidotic, physiologic, and alkalotic groups, determined 30 s after the intravenous administration of cyanide, were 0.015 +/- 0.002, 0.011 +/- 0.001, and 0.008 +/- 0.001 s(-1), respectively (one-way ANOVA; p < 0.0087). At alkalotic pH the mean permeability-area product was 43% of that measured at acidotic pH. This effect of pH on the rapidity of cyanide distribution does not appear to be limited to specific areas of the brain. We conclude that modulation of arterial pH by altering PaCO2 may induce significant effects on the brain uptake of cyanide.

Developmental changes in the pattern of amino acid transport at the blood-brain barrier in rats.

Oldendorf's method has been widely used to estimate and characterize the transport of amino acids across the blood-brain barrier in rats. However, it cannot be used with very young animals. A modification of this method (retrograde injection into the right brachial artery, instead of orthograde injection into the common carotid artery) allowed the estimation of the brain uptake index of some amino acids in 5-, 12- and 19-day-old rats, as well as the study of self- and cross-inhibition and of sodium dependency. The results obtained showed that the pattern of transport of amino acids was different in 5-day-old and in 19-day-old rats. In young rats, besides the presence of the L-system, which transported large neutral amino acids as in adult rats, the presence of another system of transport for neutral amino acids was strongly suggested. The activity of this system which transported alanine, serine, cysteine and threonine, decreased during development and it had many of the characteristics of the ASC system described by Christensen. In addition, the presence of a system of transport for beta-amino acids at the blood-brain barrier is suggested.

Free amino acids and related substances in human glial tumours and in fetal brain: comparison with normal adult brain.

An ion exchange automatic chromatographic analysis of the free amino acid concentrations of 18 human glial tumours and of 4 human fetal brains was carried out and the concentrations were compared to those of 13 biopsy specimens of normal adult brain. In addition, the concentrations of the amino acids of the glial tumours were compared to those of 7 intracerebral metastases of various origin. The chromatograms of several tumour specimens showed an unidentified peak overlapping proline. As far as the amino acid concentrations are concerned they varied depending upon the origin of the sample. The concentrations of most amino acids were higher in fetal brain than in adult brain with the exception of aspartic acid, glutamic acid, glutamine, cystathionine and GABA. Two peptides: glutathione and homocarnosine were absent in fetal brain and were present in adult brain. In glial tumours, homocarnosine and some amino acids, namely aspartic acid, glutamic acid and GABA, showed lower concentrations than in normal brain. Some amino acids were in the same concentration as in normal brain: taurine, phosphoethanolamine, glutamine and cystathionine. Most of the others were in higher concentrations than in normal brain, mainly proline. The results suggest that the concentrations of 5 compounds: taurine, proline, cystathionine, GABA and homocarnosine, taken as a whole, provide information on the origin of the sample.

Taurine transport at the blood-brain barrier: an in vivo brain perfusion study.

Taurine transport into six brain regions of equithesin-anesthetized rats was studied by the in situ brain perfusion technique. This technique gives both accurate measurements of cerebrovascular amino acid transport and allows complete control of the perfusate amino acid composition. Final wash procedure showed that taurine efflux occurred rapidly from endothelial cells. The taurine influx into endothelial cells was sodium and chloride dependent suggesting that the sodium and chloride gradients are the principal source of energy for taurine transport into endothelial cells. Taurine transport could be fitted by a model with saturable components. The kinetic constants in the parietal cortex were 1.4 x 10(-4) mumol/s/g for the apparent Vmax and 0.078 mM for the apparent Km. Competition experiments in the presence of sodium ions showed that [14C]taurine uptake was strongly inhibited by the structural analogs of taurine, hypotaurine and beta-alanine.

Glutamate is transported across the rat blood-brain barrier by a sodium-independent system.

Transport of L-glutamate from blood to brain in equithesin-anesthetized rats was examined using in situ brain perfusion combined with multiple-time/graphical analysis. In situ perfusion allowed precise control of the composition of the perfusate, which was necessary for a detailed investigation of glutamate transport, while multiple time/graphical analysis permitted evaluation of the rapidly reversible volume and the period when the influx was unidirectional. Glutamate had no reversible volume and efflux from brain occurred after 30 s of perfusion. The in situ transfer coefficient (Kin) ranged from 0.74 +/- 0.07 mul/s per g in parietal cortex to 0.44 +/- 0.07 mul/s per g in hippocampus. L-Glutamate uptake was unaffected by removal of sodium from the perfusate, reduced by 5 mM L-glutamate, L-homocysteate, L-aspartate, plasma and 0.1 mM L-glutamate, while L-cystine did not reduce its uptake. These results suggest that the transport system for glutamate is saturated mainly by L-glutamate at physiological conditions and that it is not the sodium-independent x-C system since glutamate transport was not reduced by L-cystine except in hippocampus and that it was responsive to L-aspartate.

Initial process of diffusion of small molecules from blood vessels to the meninges in the young rat.

Pathways taken by peripherally administered small molecules when entering the brain were investigated in 6-day-old rats by radioautography and fluorescence microscopy after intravenous administration and rapid freezing. L-[U-14C]Phenylalanine, [U-14C]sucrose and sodium fluorescein reached the brain within less than 5 seconds. These blood-borne molecules were found in the subarachnoid cisterns and the superficial parenchyma. These results suggest a special permeability of the arachnoid layer and/or the pial vessels. Phenylalanine alone reached the deep parenchyma because of the existence of a specific endothelial carrier.

Evaluation of anesthetic effects on parameters for the in situ rat brain perfusion technique.

Studies of drug distribution to brain should be controlled for the experimental method used. Numerous methods have been employed to ascertain brain distribution and many of these approaches use anesthetic agents. The in situ rat brain perfusion method is one of the most sensitive and widely used methods for evaluating brain distribution profiles. There has been no evaluation of the effects of anesthetic agents on parameters associated with this method (i.e. cerebral perfusion fluid flow, brain vascular volume and blood-brain barrier permeability). We evaluated the effects of the anesthetic agents pentobarbital and ketamine combinations on these baseline parameters. The results suggest that the anesthetic agent has no effect on these parameters and anesthetic selection is open to the choice of the investigator when using the perfusion method.

Hormonal influence on the permeability of the blood-brain barrier: effect of an analog of adrenocorticotropic hormone, beta 1-24 corticotrophin.

Regional unidirectional transport of alpha-aminoisobutyric acid (AIB) (mol. wt.: 104) and sucrose (mol wt.: 342) which have a low permeability across the intact endothelium was investigated in brain of rats either treated with synacthène: an analog of ACTH, tetracosactide retard (beta-1-24 corticotrophin) or in brain of placebo-treated controls. Three days treatment with synacthène, reduced the rate of influx of AIB and sucrose in most of the brain regions studied especially in thalamus, hypothalamus, cortex, and caudate nucleus without affecting the vascular compartment. The brainstem, cerebellum and white matter were less affected. These experimental findings may suggest that ACTH exhibits significant influence on hormonal regulation of blood-brain barrier permeability. Thereby such a regulation may involve the entry of polar compounds into the CNS and may influence the central effects of diffusion-limited drugs.

Effect of dimethyl sulfoxide on blood-to-brain transfer of alpha-aminoisobutyric acid: examination of regional blood-brain barrier function.

The effect of dimethyl sulfoxide (DMSO) on brain capillary permeability has been controversial. We have studied the effect of DMSO on unidirectional transport of alpha-aminoisobutyric acid (AIB) across the blood-brain barrier (BBB) in rats. Rats were treated with 15% DMSO intraperitoneally (i.p.), intravenously (i.v.) or by an i.p. injection in combination with an i.v. injection, or in some cases intra-arterially by rapid infusion into left external carotid artery. The unidirectional blood-to-brain transfer constant (Ki) for AIB was measured in each group after the animals were killed. DMSO administration did not significantly increase Ki as compared to control Kj. These results show that it is unlikely that DMSO increases the permeability of BBB and therefore do not support the proposal that DMSO can act as a carrier at the BBB for compounds with restricted vascular permeability.

Blood-brain barrier permeability: regional alterations after acute and chronic administration of ethinyl estradiol.

In this study we measured the effect of acute and chronic estrogen treatment on cerebrovascular permeability to sucrose and inulin. Animals were subcutaneously injected once with 0.1 micrograms/rat of ethinyl estradiol or injected daily with the same drug dose for 3 weeks. Control rats received the same amount of arachis oil vehicle. Three weeks treatment but not the single injection of ethinyl estradiol produced significant increases in the cerebrovascular permeability-surface area product for sucrose and inulin in almost all brain regions.

Effect of dietary n-3 fatty acid deficiency on blood-to-brain transfer of sucrose, alpha-aminoisobutyric acid and phenylalanine in the rat.

Possible alterations in blood-to-brain unidirectional transport of sucrose (mol. wt., 342), alpha-aminoisobutyric acid (mol. wt., 104), and L-phenylalanine (mol. wt., 165) induced by a diet deficient in n-3 polyunsaturated fatty acids were studied with respect to blood-brain barrier function. Two groups of rats were for to two generations with a semisynthetic diet. One group of rats was fed a peanut oil+rapeseed oil diet which contained both essential fatty acids: linoleic acid (18:2 n-6) and alpha-linolenic acid, (18:3 n-3). Another group was fed a diet of peanut oil, this diet (containing 18:2 n-6) was deficient in alpha-linolenic acid. The experiments were performed at 6 months of age. Unidirectional transfer rate constants (Ki) of sucrose, alpha-aminoisobutyric acid and L-phenylalanine were measured. The diet based on peanut oil (deficient in n-3) caused a greater blood-to-brain transport of sucrose but not of alpha-aminoisobutyric acid or L-phenylalanine. These observations indicate that regardless of the mechanisms involved, alterations in essential fatty acids induced by diet can modulate to some extent the blood-brain transport of hydrophilic molecules without a carrier.

The effect of mercurials on amino acid transport and rubidium uptake by isolated rat brain microvessels.

The present studies were undertaken to investigate the in vitro effects of mercuric chloride (HgCl2) and methylmercury chloride (CH3HgCl) on the uptake of [3H]alanine, [3H]phenylalanine, [3H]glutamic acid, [14C]alpha-methylamino-isobutyric acid, and Rubidium (86Rb), as well as on the activity of gamma-glutamyl transpeptidase (gamma-GTP) in rat brain microvessels. Similar patterns of inhibition of uptake by capillaries of amino acids and 86Rb were observed in the presence of both mercury compounds. Marked inhibition of uptake was seen at concentrations of 10(-5) and 10(-4) M of both compounds, but the uptake was not inhibited at concentrations less than 10(-5) M. Capillary gamma-GTP activity was not influenced by either HgCl2 or CH3HgCl, suggesting that this enzyme does not play any role in the transport of the amino acids studied in these experiments. The results indicate that the main toxic effect of mercury compounds is inhibition of amino acid transport. The concentration at which mercury exerts its inhibitory effect in this study is similar to that estimated to have been present in the brains of victims during the Minamata epidemic.

Effects of lead acetate on cerebral glutathione peroxidase and catalase in the suckling rat.

Exposure of immature rats to lead acetate results in hemorrhagic encephalopathy of variable evolution. As the maintenance of adequate protection against peroxides may be critical in this condition, the activities of selenium-glutathione peroxidase and catalase in cerebrum and cerebellum of suckling rats poisoned with lead acetate were studied from day six to day sixteen post-exposure. Age-related decreases of glutathione peroxidase and catalase activities in both controls and lead poisoned animals were observed. An increase in catalase activity was observed in cerebrum and cerebellum of lead-treated rats compared to controls. Glutathione peroxidase activity did not change significantly in cerebrum over the period studied. By contrast, glutathione peroxidase activity in cerebellum of lead-treated rats remained at about twice the control level over most of the study period. This apparent increase in glutathione peroxidase activity may be due either to a slower ontogenic decrease of its specific activity or to enzyme induction in response to oxidant stress in cerebellum.