Capillaries Of Cortex Research Articles

Large-depth three-photon fluorescence microscopy imaging of cortical microvasculature on nonhuman primates with bright AIE probe In vivo

Multiphoton microscopy has been a powerful tool in brain research, three-photon fluorescence microscopy is increasingly becoming an emerging technique for neurological research of the cortex in depth. Nonhuman primates play important roles in the study of brain science because of their neural and vascular similarity to humans. However, there are few research results of three-photon fluorescence microscopy on the brain of nonhuman primates due to the lack of optimized imaging systems and excellent fluorescent probes. Here we introduced a bright aggregation-induced emission (AIE) probe with excellent three-photon fluorescence efficiency as well as facile synthesis process and we validated its biocompatibility in the macaque monkey. We achieved a large-depth vascular imaging of approximately 1 mm in the cerebral cortex of macaque monkey with our lab-modified three-photon fluorescence microscopy system and the AIE probe. Functional measurement of blood velocity in deep cortex capillaries was also performed. Furthermore, the comparison of cortical deep vascular structure parameters across species was presented on the monkey and mouse cortex. This work is the first in vivo three-photon fluorescence microscopic imaging research on the macaque monkey cortex reaching the imaging depth of ∼1 mm with the bright AIE probe. The results demonstrate the potential of three-photon microscopy as primate-compatible method for imaging fine vascular networks and will advance our understanding of vascular function in normal and disease in humans.

Pathomorphological changes of capillaries in the cerebral cortex in type 2 diabetes mellitus

The aim of the study was to determine microscopic, immunohistochemical, electron microscopic, morphometric parameters of capillaries in the cerebral cortex in deceased patients with type 2 diabetes mellitus (DM) in comparison with the conditional control group and the group of deceased patients with dyscirculatory ischemic encephalopathy. Materials and methods. Microscopic, immunohistochemical, electron microscopic and morphometric studies of the cerebral cortex capillaries were performed in 3 groups: group I – 20 deceased patients with type 2 DM, group II conditional control (CC) – 20 deceased patients without clinical morphological signs of DM and cerebrovascular pathology, group III – 20 patients with dyscirculatory ischemic encephalopathy (DIEP). Results. It was found that in type 2 DM, the capillaries in the cerebral cortex lose pericytes due to their apoptosis: the number of pericytes in the cortical capillaries in type 2 DM was statistically significantly lower by 57.14 % compared to CC group and 50.00 % lower compared to DIEP group, the level of caspase-3 expression in the cortical microvessels in type 2 DM was significantly higher by 230.85 % compared with CC group and 81.67 % higher than in DIEP group. According to electron microscopy in type 2 DM, apoptosis of pericytes and single endothelial cells in the cerebral cortex capillaries was determined as well as significant expansion of basement membranes with the accumulation of electron-dense amorphous material and collagen fibrils. According to the results of morphometry, the outer diameter of the cortical capillaries in type 2 DM group was 4.90 % significantly larger, the inner diameter was 9.78 % smaller and the walls were 66.62 % thicker (compared with CC group) due to the accumulation of PAS-positive substances of blood serum and fibrosis, confirmed by 22.96 % greater area of type IV collagen expression in the microvessel walls. Conclusions. The pathomorphological changes of microvessels identified in deceased patients with type 2 diabetes mellitus are signs of diabetic cerebral microangiopathy.

Therapeutic development of group B Streptococcus meningitis by targeting a host cell signaling network involving EGFR

Group B Streptococcus (GBS) remains the most common Gram‐positive bacterium causing neonatal meningitis and GBS meningitis continues to be an important cause of mortality and morbidity. In this study, we showed that GBS penetration into the brain occurred initially in the meningeal and cortex capillaries, and exploits a defined host cell signaling network comprised of S1P2, EGFR, and CysLT1. GBS exploitation of such network in penetration of the blood–brain barrier was demonstrated by targeting S1P2, EGFR, and CysLT1 using pharmacological inhibition, gene knockout and knockdown cells, and gene knockout animals, as well as interrogation of the network (up‐ and downstream of each other). More importantly, counteracting such targets as a therapeutic adjunct to antibiotic therapy was beneficial in improving the outcome of animals with GBS meningitis. These findings indicate that investigating GBS penetration of the blood–brain barrier provides a novel approach for therapeutic development of GBS meningitis.

Targeting E. coli invasion of the blood-brain barrier for investigating the pathogenesis and therapeutic development of E. coli meningitis.

Escherichia coli is the most common Gram-negative bacillary organism causing neonatal meningitis. Escherichia coli meningitis remains an important cause of mortality and morbidity, but the pathogenesis of E. coli penetration of the blood-brain barrier remains incompletely understood. Escherichia coli entry into the brain occurs in the meningeal and cortex capillaries, not in the choroid plexus, and exploits epidermal growth factor receptor (EGFR) and cysteinyl leukotrienes (CysLTs) for invasion of the blood-brain barrier. The present study examined whether EGFR and CysLTs are inter-related in their contribution to E. coli invasion of the blood-brain barrier and whether counteracting EGFR and CysLTs is a beneficial adjunct to antibiotic therapy of E. coli meningitis. We showed that (a) meningitis isolates of E. coli exploit EGFR and CysLTs for invasion of the blood-brain barrier, (b) the contribution of EGFR is upstream of that of CysLTs, and (c) counteracting EGFR and CysLTs as an adjunctive therapy improved the outcome (survival, neuronal injury and memory impairment) of animals with E. coli meningitis. These findings suggest that investigation of host factors contributing to E. coli invasion of the blood-brain barrier will help in enhancing the pathogenesis and development of new therapeutic targets for E. coli meningitis in the era of increasing resistance to conventional antibiotics.

Nr4a1-Dependent Ly6Clow Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal

SummaryThe functions of Nr4a1-dependent Ly6Clow monocytes remain enigmatic. We show that they are enriched within capillaries and scavenge microparticles from their lumenal side in a steady state. In the kidney cortex, perturbation of homeostasis by a TLR7-dependent nucleic acid “danger” signal, which may signify viral infection or local cell death, triggers Gαi-dependent intravascular retention of Ly6Clow monocytes by the endothelium. Then, monocytes recruit neutrophils in a TLR7-dependent manner to mediate focal necrosis of endothelial cells, whereas the monocytes remove cellular debris. Prevention of Ly6Clow monocyte development, crawling, or retention in Nr4a1−/−, Itgal−/−, and Tlr7host−/−BM+/+ and Cx3cr1−/− mice, respectively, abolished neutrophil recruitment and endothelial killing. Prevention of neutrophil recruitment in Tlr7host+/+BM−/− mice or by neutrophil depletion also abolished endothelial cell necrosis. Therefore, Ly6Clow monocytes are intravascular housekeepers that orchestrate the necrosis by neutrophils of endothelial cells that signal a local threat sensed via TLR7 followed by the in situ phagocytosis of cellular debris.

Selective increase of two ABC drug efflux transporters at the blood–spinal cord barrier suggests induced pharmacoresistance in ALS

ATP-binding cassette (ABC) drug efflux transporters in the CNS are predominantly localized to the luminal surface of endothelial cells in capillaries to impede CNS accumulation of xenobiotics. Inflammatory mediators and cellular stressors regulate their activity. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of upper and lower motor neurons characterized by extensive neuroinflammation. Here we tested the hypothesis that disease-driven changes in ABC transporter expression and function occur in ALS. Given the multitude of ABC transporters with their widespread substrate recognition, we began by examining expression levels of several ABC transporters. We found a selective increase in only two transporters: P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) both at mRNA and protein levels, in the SOD1-G93A mouse model of ALS, specifically in disease-affected CNS regions. Detailed analysis revealed a similar disease-driven increase in P-gp and BCRP levels in spinal cord microvessels, indicating that their altered expression occurs at the blood spinal cord barrier. Transport activity of P-gp and BCRP increased with disease progression in spinal cord and cerebral cortex capillaries. Finally, P-gp and BCRP protein expression also increased in spinal cords of ALS patients. Preclinical drug trials in the mouse model of ALS have failed to decisively slow or arrest disease progression; pharmacoresistance imparted by ABC transporters is one possible explanation for these failures. Our observations have large implications for ALS therapeutics in humans and suggest that the obstacle provided by these transporters to drug treatments must be overcome to develop effective ALS pharmacotherapies.

The effects of short-term enriched environment on capillaries of the middle-aged rat cortex

There has been no study investigating the effects of enriched environment on the capillaries of cortex with new stereological methods. In the present study, both 14 month female and male Sprague-Dawley rats were randomly divided into enriched environment (EE) rats and standard environment (SE) rats. EE rats were reared in enriched environment and SE rats were reared in standard environment for 4 months. The effects of short-term enriched environment on the cortex volume and on the total volume, total length, total surface area and mean diameter of the capillaries in the cortex of mid-aged Sprague-Dawley rats were quantitatively investigated with immunohistochemistry technique and unbiased stereological methods. There were no significant differences in the cortex volume, the total length and total surface area of the capillaries in the cortex between EE rats and SE rats. The total volume of the capillaries in the cortex of female EE rats and male EE rats was significantly increased when compared to female SE rats and male SE rats. The mean diameter of the capillaries in the cortex of female EE rats was significantly decreased when compared to that in female SE rats, but there was no significant difference in the mean diameter of the capillaries in the cortex between male EE rats and male SE rats. The present results indicate that enriched environment had a positive effect on the capillaries in the cortex of middle-aged rats. The present study might provide an important morphological basis for searching the ethology strategy to delay the progress of brain aging in the future.

Frequency distribution function of red blood cell velocities in single capillaries of the rat cerebral cortex using intravital laser-scanning confocal microscopy with high- speed camera

Background: Brain metabolism depends largely on oxygen and therefore constant delivery of oxygen to the brain is more important than to any other organ. Previously we reported a newly developed method to automatically calculate red blood cell (RBC) flow and its temporal modulations at the layer 1 of the rat cerebral cortex. Objective: To examine a general tendency of frame-rate dependency of RBC velocities and heterogeneity of RBC movements in single capillaries identified by fluorescein isothiocyanate (FITC)-dextran staining. Methods: In urethane-anesthetized rats with a closed cranial window, intravenously administered FITC-labeled RBCs were traced at 125, 250 or 500 frames/s (fps) with a laser-scanning confocal fluorescence microscope and their velocities were automatically calculated with home-made software KEIO-IS2. Results: RBC velocities in single capillaries were not constant but variable, and were dependent on frame rate, with average values of 0.85 ± 0.43 mm/s at 125 fps, 1.34 ± 0.73 mm/s at 250 fps, and 2.05�1.59 mm/s at 500 fps. When all capillary RBCs were plotted against their velocities (frequency distribution function of RBC velocities), RBC velocities were clustered at around 1.0 mm/s, smearing at higher velocities up to 9.4 mm/s. High RBC velocity was only detected from frame analysis with high frame rates since such high-flow RBCs were uncounted at low frame rates. RBC velocities higher than 6.5 mm/s were statistically significantly outlined from the main population (p<0.01). Such a group of capillaries was considered to belong to thoroughfare channels, although their diameter was almost the same as that of ordinary ones. Conclusion: Extra-high flow capillaries are confirmed in the cerebral cortex and these may be thoroughfare channels or non-nutritional capillaries carrying 42 % of the blood in reserve.

Prolonged exercise induces angiogenesis and increases cerebral blood volume in primary motor cortex of the rat

Plastic changes in motor cortex capillary structure and function were examined in three separate experiments in adult rats following prolonged exercise. The first two experiments employed T-two-star (T 2*)-weighted and flow-alternating inversion recovery (FAIR) functional magnetic resonance imaging to assess chronic changes in blood volume and flow as a result of exercise. The third experiment used an antibody against the CD61 integrin expressed on developing capillaries to determine if motor cortex capillaries undergo structural modifications. In experiment 1, T 2*-weighted images of forelimb regions of motor cortex were obtained following 30 days of either repetitive activity on a running wheel or relative inactivity. The proton signal intensity was markedly reduced in the motor cortex of exercised animals compared with that of controls. This reduction was not attributable to alterations of vascular iron levels. These results are therefore most consistent with increased capillary perfusion or blood volume of forelimb regions of motor cortex. FAIR images acquired during experiment 2 under normocapnic and hypercapnic conditions indicated that resting cerebral blood flow was not altered under normal conditions but was elevated in response to high levels of CO 2, suggesting that prolonged exercise increases the size of a capillary reserve. Finally, the immunohistological data indicated that exercise induces robust growth of capillaries (angiogenesis) within 30 days from the onset of the exercise regimen. Analysis of other regions failed to find any changes in perfusion or capillary structure suggesting that this motor activity-induced plasticity may be specific to motor cortex. These data indicate that capillary growth occurs in motor areas of the cerebral cortex as a robust adaptation to prolonged motor activity. In addition to capillary growth, the vascular system also experiences heightened flow under conditions of activation. These changes are chronic and observable even in the anesthetized animal and are measurable using noninvasive techniques.

Microvascular changes associated with postischaemic hypoperfusion in rats.

The present study was undertaken to explore the cause of postischaemic hypoperfusion through morphological observations of the microstructure of brain cortex capillaries in rats with postischaemic hypoperfusion. Sixteen rats were used. The left middle cerebral artery of each animal was occluded for one hour (n = 8) or 2 hours (n = 8) and was followed by reperfusion for 2 hours. The regional cerebral blood flow (rCBF) of the ischaemia induced brain cortex was monitored continuously during the experiment and the microstructure of the brain cortex capillaries was then observed under electron microscope. Postischaemic hypoperfusion was observed in both ischaemia groups. The rCBF after (1.5 hours of reperfusion was significantly lower in the 2-hour ischaemia group than in the one-hour ischaemia group. The number of endothelial microvilli (MV) per capillary in the one-hour ischaemia group did not differ significantly from the control (the right cortex capillaries), whereas it was significantly higher in the 2-hour ischaemia group (p < 0.05). The ratio of the inner diameter to the outer diameter of capillaries decreased significantly in both ischaemia groups, and the ratio in the 2-hour ischaemia group was significantly lower than that in the one-hour ischaemia group (p < 0.05). Thus the present study statistically revealed that as postischaemic perfusion of the brains decreases, the number of MV increases, and endothelial cells swell more markedly. These microvascular changes seem to represent morphological factors associated with postischaemic hypoperfusion.

Blood Flow Structuring and Its Alterations in Capillaries of the Cerebral Cortex

Various manifestations of blood flow structuring were investigated in rabbit cerebral cortex capillaries, which possess the most narrow lumina of all parts of the body. Blood flow structuring in the capillaries was characterized by the presence of a stable and comparatively large parietal plasma layer, which changed insignificantly under control and ischemic conditions, but disappeared when blood stasis developed inside the capillaries. The axial core of the blood flow in the capillaries, which occupied almost two-thirds of the intracapillary volume under normal conditions, consisted of significantly deformed (stretched along the microvessels’ axes) and nonaggregated erythrocytes. During ischemia the shape of the erythrocytes did not change appreciably; only the blood plasma intervals between them increased significantly, demonstrating reduction of the local hematocrit. During primary blood stasis caused by enhanced intravascular erythrocyte aggregation, typical blood flow structuring became significantly disturbed: red cells filled the whole, or almost the whole, capillary lumina and did not leave visible space for plasma inside the microvessel lumina. We concluded that normal blood flow structuring is a deciding factor in the blood rheological properties of microvessels. Its disturbance, caused by fast accumulation of erythrocytes in the capillary lumina, results in blood rheological disorders and in a slow down to a full stop of the blood flow, despite a preserved arteriolovenular pressure difference.

Laser-Doppler measurements of concentration and velocity of moving blood cells in rat cerebral circulation.

In brain cortex all capillaries are perfused with plasma at anyone time while the flow of blood cells is heterogeneous. Increased blood flow is associated with increased number of moving erythrocytes in the microcirculation, while capillary recruitment in its classical anatomical sense appears not to exist in the brain. Modulation of the concentration of flowing erythrocytes may influence the oxygen supply to the tissue. Therefore, we examined the possibility that laser-Doppler flowmetry (LDF) could be used to quantify changes in the microvascular concentration of moving blood cells (CMBC) and blood cell velocity (< v >) by comparing LDF measurements with electromagnetic flow measurements in vitro, and confocal laser-scanning microscopy in vivo in the brain of anaesthetized male Wistar rats. In vitro measurements showed that CMBC was affected by changes in haematocrit, while < v > correlated almost linearly with blood cell velocity measured electromagnetically within a relevant physiological range. In vivo studies during hypercapnia (PaCO2 from 39 +/- 4 to 66 +/- 5 mmHg) with confocal laser scanning microscopy disclosed a 39 +/- 10% increase of cortical capillary erythrocytes, while CMBC measured with LDF increased by 37 +/- 5%. Erythrocyte flow velocity in brain cortex capillaries increased by 65 +/- 17% with confocal microscopy as compared to 72 +/- 8% with LDF. Local electrical stimulation of cerebellar cortex, and application of adenosine or sodium-nitroprusside, increased CMBC and < v > simultaneously, while during hypercapnia the < v > increase preceded the CMBC increase by 30 s. The CMBC rise rapidly reached a steady state in response to all types of stimulation, while < v > continued to increase during the major part, or the entire stimulation period. In conclusion, our data support the hypothesis that LDF may be useful for haemodynamic studies of brain microcirculation.

Periendothelial Acetylcholine Synthesis and Release in Bovine Cerebral Cortex Capillaries

Choline acetyltransferase (ChAT) activity is present in isolated cerebral capillaries, where it has been considered to be a marker for perivascular cholinergic nerve terminals. However, ChAT-like immunoreactivity has been visualized in endothelial cells. This finding raised the possibility that at least part of the biochemically detected ChAT has a nonneuronal origin. To evaluate the relative contribution of endothelial cells and nerve fibers to the total acetylcholine (ACh)-synthesizing capacity of cerebral capillaries, ChAT activity and ACh release were measured in capillaries and in purified endothelial cells isolated from bovine cerebral cortex. Isolated capillaries showed ChAT activity, which was inhibited by 2-benzoylethyl trimethylammonium to the same extent as cerebral ChAT. When preincubated with [3H]choline, these capillaries presented a calcium-dependent enhancement in tritium release upon electrical field stimulation. Purified endothelial cells had minor ChAT activity and lacked the ability to release tritium in response to electrical stimulation, although the endothelial markers alkaline phosphatase, gamma-glutamyltranspeptidase, and 1,1'-dioctadecyl-1,3,3',3'-tetramethyl-iodocarbocyanide perchlorate-labeled acetylated low-density lipoprotein uptake were fully preserved. These data indicate that, within isolated cerebral capillaries, ACh is synthesized and released by a periendothelial structure. The fact that ACh release is provoked by electrical stimulation and by a calcium-dependent mechanism strongly suggests that cerebrovascular ACh has a neuronal origin.

Experimental cerebral infarction in the dog: electron microscopic studies of the microvasculature.

Ultramicromorphological changes of the cerebral vessels were studied by observation of ultrathin section specimens under the transmission electron microscope, vascular cast specimens under the scanning electron microscope, and freeze-fracture replica specimens under the transmission electron microscope. Extensive infarction was found in areas supplied by the middle cerebral artery, but the disturbances in the microvasculature of the lesion are not simple. Bleeding came from arterioles in the basal ganglia, a perforating arterial terminal zone, and from venules in the corticomedullary border, a cortical arterial terminal zone. The number of pinocytotic vesicles was 3 times normal in the capillaries of ischemic cortex, and vesicle size increased 1.3 times. The tight junctions revealed discontinuity of strand-composing intramembranous particles, but no obvious disruption. It is concluded, therefore, that vesicular transport plays a major role in the increase of capillary permeability during experimental infarction.

Synthesis of acetylcholine by endothelial cells isolated from rat brain cortex capillaries

Choline acetyltransferase (ChAT) activity was measured in a fraction of endothelial cells obtained by collagenase digestion from previously isolated rat brain cortex capillaries. ChAT specific activity in the dissociated cells (0.264 nmol·mg protein −1·min −1) was nearly 7 times higher than the activity measured in ‘capillary-forming’ cells (0.038 nmol·mg protein −1·min −1). It is now known whether this increase was caused by ChAT enrichment of the former cellular fraction, and/or by a change in the specific activity of the enzyme during the isolation procedure, but the finding would be in favor of the intrinsic localization of cerebrovascular ChAT in endothelial cells. This would support the hypothesis of a role for locally synthesized ACh in the vasodilating response to noxious stimuli.

Corpus luteum angiogenic factor is related to fibroblast growth factor.

An angiogenic growth factor present in bovine corpus luteum (CL) has been purified to apparent homogeneity by a combination of differential salt precipitation, ion exchange chromatography, and heparin-Sepharose chromatography. It is a single chain polypeptide with an apparent mol wt of 15,000 and an amino acid composition similar to that previously reported for pituitary and brain fibroblast growth factor (FGF). Sequence analysis of the first 17 residues of the CL-derived growth factor identified the sequence; His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-X-Lys-Asn-Gly-Gly-X-Phe-Leu. This sequence is identical to residues 16-33 of bovine pituitary and brain FGF, indicating that the CL-derived growth factor is an amino-terminally truncated form of FGF and is otherwise similar, if not identical, to FGF. The biological activity of CL FGF is indistinguishable from that of pituitary or brain FGF. It is highly active in triggering the proliferation of cultured bovine vascular endothelial cells derived either from large vessels (aortic arch) or from corpus luteum and adrenal cortex capillaries (half-maximal stimulation at 20-40 pg/ml and saturation at 400-600 pg/ml). In vivo implants containing 50 ng to 1 microgram CL-derived growth factor stimulate neovascularization in the chorioallantoic membrane of the chick embryo. In addition to being mitogenic for vascular endothelial cells, CL FGF also stimulates the proliferation of a wide variety of mesoderm- and neuroectoderm-derived cells, including vascular smooth muscle cells, granulosa and adrenal cortex cells, rabbit costal chondrocytes, and corneal endothelial cells.

Decrease of choline acetyltransferase activity of rat cortex capillaries with aging.

Choline acetyltransferase (ChAT) activity was estimated in brain cortex capillaries isolated from 3-, 12-, 18-, and 24-month-old rats. Maximum enzymatic activity was found at 12 months (55 +/- 0.3 pmol X mg-1 protein X min-1; mean +/- SEM) and then it decreased to reach a minimum at 24 months (34 +/- 3.1 pmol X mg-1 protein X min-1). A less marked decrease of enzymatic activity was also found in cortex homogenate and in a synaptosomal fraction obtained from the same groups of rats. Loss of ChAT of brain capillaries with aging could be related to a general phenomenon of cortical cholinergic deficit in that condition.

DNA Damage in Irradiated Endothelial Cells of the Rat Cerebral Cortex. Protective Action of Cysteamine in Vivo

The induction and repair of DNA damage in single endothelial cells of rat cerebral cortex capillaries were studied in vivo and in vitro. Capillaries from the cerebral cortex were prepared in suspension, embedded in agarose on microscope slides, and treated with alkaline solution (unwinding of DNA). After neutralization the slides were stained with the fluorescent dye acridine orange and endothelial cell nuclei were evaluated in a microscope photometer. The intensity of the red fluorescence (from single-stranded DNA) divided by the green fluorescence (from double-stranded DNA) was used as a measured of DNA strand breaks. The results showed that most DNA strand breaks were repaired within 30 min postirradiation. A linear dose-effect relationship was found up to 18 Gy. Similar results were obtained from in vitro and in vivo experiments. Cysteamine administered 20 min before irradiation in vivo gave a protective effect on the cells studied. An EMF of 1.3 was determined.

Ultrastructural and biochemical studies of the brain and other organs in rats after chronic ethanol administration: I. Electronmicroscopic investigations of the morphologic elements of the blood-brain barrier in the rat after ethanol intoxication

The aim in view was to establish whether chronic administration of ethanol causes ultrastructura changes in the brain of rats, particularly in the structures through which alcohol penetrates to the brain, that is the cerebral cortex capillaries. The experiments were performed with 5 Wistar rats, three of which received ethanol according to RATCLIFFE'S model for 8 weeks in increasing concentrations from 2.5 to 25 per cent. Two rats served as control. In the endothelium of some capillaries of the brain cortex in the rats ingesting ethanol an enlargement of the cell nuclei was observed. The number of mitochondria in the cytoplasm and of micropinocytic vesicles was found to increase, and proliferation of the smooth endoplasmic reticulum and Golgi system were noted. Considerable oedema was observed in the astrocytic processes surrounding the vessels, with the presence of numerous mitochondria of abnormal shape and huge size. Oedema of perivascular astrocytic processes and enhanced pinocytosis seem to indicate an increased permeability of the blood-brain barrier as the result of the toxic effect of ethanol.

Comportement de la γ-glutamyl transférase des capillaires cérébraux de mouton vis-à-vis des aminoacides et du glutathion

gamma-glutamyl Transferase fron Sheep brain cortex capillaries was studied from the point of view of transport of aminoacids across blood brain barrier. Excess substrate inhibition was competitive and observed both with donor (glutathione) and various acceptors (methionine, alanine, tryptophan) but not with arginine. Excess glutathione inhibition of transfer reaction is concomitant with an increase of total reaction (transfer + hydrolysis + autotranspeptidation). With regard to aminoacids, the greater the K'm the stronger the inhibition. This inhibition is the result of formation of a dead complex. Lineweaver-Burk plots 1/v versus 1/[acceptor] give straight lines meeting at the same point, whereas 1/v verus 1/[donor] plots are roughly parallel for high aminoacid concentrations and become secant for the low ones. Replots of slopes vs. 1/[acceptor] are not linear: the lower the aminoacid affinity the more pronounced the slope replot curvature. Thus kinetic patterns are consistent with a branched ping-pong mechanism including a ternary complex (Enzyme-acceptor-H2O) at high or low relative concentration, which balances the two branches. The estimated value of kinetic parameters does not support the hypothesis of major implication of the enzyme in brain uptake of aminoacids.