Eain M. Cornford

Independent blood-brain barrier transport systems for nucleic acid precursors

The blood-brain barrier permeability to certain 14-C-labelled purine and pyrimidine compounds was studied by simultaneous injection in conjunction with two reference isotopes into the rat common carotid artery and decapitation 15s later. The amount of 14-C-labelled base or nucleoside remaining in brain was expressed in relation to 3-H2O (a highly diffusible internal standard) and 113m-In-labelled EDTA (an essentially non-diffusible internal standard). Of the 17 compounds tested, measurable, saturable uptakes were established for adenine, adenosine, guanosine, inosine and uridine. Two independent transport systems in the rat blood-brain barrier were defined. One transported adenine (Km equals 0.027 mM) and could be inhibited with hypoxanthine. Adenosine (Km equals 0.018 mM), guanosine, inosine and uridine all cross-inhibit, defining a second independent nucleoside carrier system. Adenosine inhibited [14-D]uridine uptake more effectively than did uridine, suggesting a weaker affinity of uridine for this nucleoside carrier.

Carrier mediated blood-brain barrier transport of choline and certain choline analogs.

Blood‐brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a Km= 442 ± 60 μM and Vmax= 10.0 ± 0.6 nmol min‐1 g‐1. In 14–15‐day‐old suckling forebrains a similar Km (= 404 ± 88 μM) but higher Vmax (= 12.5 ± 1.5 nmol min‐1 g‐1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis‐Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex Km= 310 ± 103 μM and Vmax= 12.6 ± 2.8 nmol min‐1g‐1; paleocortex Km= 217 ± 76 μM and Vmax= 7.2 ± 1.5 nmol min‐1 g‐1).

Dose dependent reduction of glucose utilization by pentobarbital in rat brain.

A new method of determining the rate of glucose utilization in brain regions of individual rats has been used to measure the dose dependency of the reduction of the metabolic activity of the cerebral cortex by pentobarbital. Cerebral cortical glucose utilization is depressed to a basal level of 44% of the control rate when cerebral pentobarbital levels exceed 50 μg per g of tissue. The major portion of this effect occurs between the cerebral pentobarbital range of 10-20 μg per g, which can be achieved by 1/5 to 1/10 the normal anesthetic intraperitoneal dosage. If a depression of brain metabolism is responsible for the previously reported protection of the brain from ischemic damage, these data suggest a substantial reduction of brain metabolic rate is achieved in the rat at a barbiturate dosage which may be therapeutically relevant in the human after acute brain ischemia.

Newborn Rabbit Blood–Brain Barrier Is Selectively Permeable and Differs Substantially from the Adult

Abstract: Examination of blood‐brain barrier (BBB) function by the intracarotid injection technique has been utilized in studies of newborn (6–30 h) and adult rabbits. The exclusion of mannitol (mol. wt. 182), dextran (mol. wt. 60,000–90,000) and indium‐bound EDTA indicate that the newborn BBB has restrictive properties similar to the adult. At birth, saturable, carrier‐mediated transport mechanisms are present, regulating the entry of glucose, amino acids, organic acids, purines, nucleosides and choline. No difference in brain uptake of glucose was observed between adult and newborn, but considerably higher uptake rates for arginine, choline and adenine were seen in the newborn. In contrast to suggestions of an immature barrier in young animals, these studies indicate that a sophisticated, selective BBB is operative at birth. Furthermore, the specific selectivity and dramatic increases seen for certain metabolites imply a vital function in the newborn for these carrier systems.

The Human Brain GLUT1 Glucose Transporter: Ultrastructural Localization to the Blood—Brain Barrier Endothelia

Immunogold electron microscopy was used to examine human brain resections to localize the GLUT1 glucose transporter. The tissue examined was obtained from a patient undergoing surgery for treatment of seizures, and the capillary profiles examined had characteristics identical to those described previously for active, epileptogenic sites (confirmed by EEG analyses). A rabbit polyclonal antiserum to the full-length human erythrocyte glucose transporter (GLUT1) was labeled with 10-nm gold particle-secondary antibody conjugates and localized immunoreactive GLUT1 molecules in human brain capillary endothelia, with <0.25% of the particles beyond the capillary profile. Erythrocyte membranes were also highly immunoreactive, whereas macrophage membranes were GLUT1-negative. The number of immunoreactive sites per capillary profile was observed to be 10-fold greater in humans than in previous studies of rat and rabbit brain capillaries. In addition, half of the total number of immunoreactive gold particles were localized to the luminal capillary membrane. We suggest that the blood–brain barrier GLUT1 glucose transporter is up-regulated in seizures, and this elevated transporter activity is characterized by increased GLUT1 transporters, particularly on the luminal capillary membranes. In addition, acute modulation of glucose transporter activity is presumed to involve translocation of GLUT1 from cytoplasmic to luminal membrane sites, demonstrable with quantitative immunogold electron microscopy.

Localization of brain endothelial luminal and abluminal transporters with immunogold electron microscopy

SummaryImmunogold electron microscopy has identified a variety of blood-brain barrier (BBB) proteins with transporter and regulatory functions. For example, isoforms of the glucose transporter, protein kinase C (PKC), and caveolin-1 are BBB specific. Isoform 1 of the facilitative glucose transporter family (GLUT1) is expressed solely in endothelial (and pericyte) domains, and ∼75% of the protein is membrane-localized in humans. Evidence is presented for a water cotransport function of BBB GLUT1. A shift in transporter polarity characterized by increased luminal membrane GLUT1 is seen when BBB glucose transport is upregulated; but a greater abluminal membrane density is seen in the human BBB when GLUT1 is downregulated. PKC colocalizes with GLUT1 within these endothelial domains during up- and downregulation, suggesting that a PKC-mediated mechanism regulates human BBB glucose transporter expression. Occludin and claudin-5 (like other tight-junctional proteins) exhibit a restricted distribution, and are expressed solely within interendothelial clefts of the BBB. GFAP (glial fibrillary acidic protein) is uniformly expressed throughout the foot-processes and the entire astrocyte. But the microvascular-facing membranes of the glial processes that contact the basal laminae are also polarized, and their transporters may also redistribute within the astrocyte. Monocarboxylic acid transporter and water channel (Aquaporin-4) expression are enriched at the glial foot-process, and both undergo physiological modulation. We suggest that as transcytosis and efflux mechanisms generate interest as potential neurotherapeutic targets, electron microscopic confirmation of their site-specific expression patterns will continue to support the CNS drug discovery process.

Blood–brain barrier permeability to small and large molecules

The objective of this article is to provide the reader with an update of some of the BBB research highlights which have occurred in recent times, and to review the impact and contributions of immunogold electron microscopic studies on our understanding of the brain capillary endothelium. Glucose and monocarboxylic acids are two small molecules which this review will focus upon; and advances in immunogold characterization of the GLUT1 glucose transporter and the MCT1 and MCT2 monocarboxylic acid nutrient transporters will be discussed. Human serum albumin is chosen as a representative large molecule, and it has recently been shown that immunogold identification of this protein can serve as an indicator of compromised BBB function in a variety of pathophysiological conditions.

Developmental Modulations of Blood-Brain Barrier Permeability as an Indicator of Changing Nutritional Requirements in the Brain

Summary: The intracarotid injection technique has been utilized to examine blood-brain barrier function in studies of newborn (>24 h), 7, 14, 21 and 28 day-old, as well as adult rabbits. The age-related modulations in blood-brain barrier transport of adenine, arginine, choline, lactate and tryptophan were defined and demonstrated to be independent of each other. Lactic acid uptake was unusual in that the brain uptake index (BUI) was found to be greatest at 7 days postpartum. Elevated lactate uptake continues until 14 days and is then reduced. As indicated below, for all of the other metabolites examined, a maximal BUI was observed in the newborn brain and BUIs typically showed some sort of inverse relationship to animal age. The BUI of arginine is apparently halved in the first 7 days postnatally, and continues to decrease, reaching the value seen for the adult rabbit by an age of 21 days. In contrast, the brain uptake of adenine is unusual in that there appears to be a very gradual reduction in brain uptake occurring throughout the suckling period. A 3-fold decrease in the BUI of choline was observed during the first 2 wk postpartum. Tryptophan uptake undergoes a 4-fold reduction in the first 4 wk postnatally. Only minor variations in the uptakes of glucose and butanol (a reference substance which is completely cleared by brain over a wide range of blood flow rates) were observed over the range of ages examined. Therefore modulations in adenine, arginine, choline, lactate and tryptophan permeability are not attributable to blood flow alterations.Speculation: The brain uptake rates of the metabolites which gain access by way of specific carrier mediated transport mechanisms are presumed to reflect nutritional requirements of the developing brain. Thus the present study indicates that the inclusion of these metabolites (adenine, arginine, choline, tryptophan and monocarboxylic acids such as lactate) would be beneficial in clinical intravenous feeding of the neonate, to meet the apparent needs of the developing central nervous system.

Interictal Seizure Resections Show Two Configurations of Endothelial Glut1 Glucose Transporter in the Human Blood–Brain Barrier

Immunogold electron microscopy was used to analyze and quantify the Glut1 glucose transporter in brain tissue from five patients undergoing surgery for treatment of seizures. Samples were prepared from two different regions of each resection: (1) the most actively spiking epileptogenic site, and (2) the least actively spiking region, as indicated by intraoperative EEG monitoring. Two configurations of endothelial cell Glut1 were observed. About one half of the capillary profiles examined displayed abundant Glut1 immunoreactivity on both luminal and abluminal endothelial membranes. In the remainder of the profiles, reduced Glut1 labeling was seen, but adjacent erythrocyte membranes remained highly Glut1 immunoreactive, suggesting that reduced endothelial Glut1 reactivity was not attributable to method artifacts. Immunogold studies using antisera to human glial fibrillary acidic protein and human serum albumin demonstrated increased quantities of these two epitopes in the extravascular regions in which more EEG spiking activity had been demonstrated. These observations were consistent with the hypotheses that capillary integrity was more compromised, and gliosis was quantitatively increased, in the more actively spiking region of the resection. Altered glucose transporter activity in the blood–brain barrier was characterized by a bimodal Glut1 distribution in which the smaller (type B) endothelial cells displayed low Glut1 immunoreactivity, whereas adjacent (and even contiguous) larger (type A) endothelial cells showed 5- to 10-fold greater expression of membrane Glut1 transporter protein. Because this transporter facilitates glucose entry to the brain, small pericapillary volumes of brain tissue may have quite different concentrations of glucose. We hypothesize that in complex partial seizures and other forms of brain insult, an alteration of blood–brain barrier Glut1 glucose transporter activity is indicated by the appearance of these two subpopulations of endothelial cells. In comparison with previous studies of human brain capillaries in hemangioblastoma and brain injury, endothelial Glut1 density was apparently reduced (interictally) in affected temporal lobes of patients with complex partial seizures.

pH dependence of blood-brain barrier permeability to lactate and nicotine.

Brain uptake of radiolabeled D and L-lactate, D-glucose and nicotine, as measured by the intra-carotid bolus method, was examined over a range of pH of the injected solution. The uptake of L-lactate was highest at pH 6.1, and lowered significantly at pH 7.2, 7.5 and 8.4. In contrast, the uptake of the D-enantiomer was not as dramatically affected. Glucose uptake was not affected by alterations in pH. Nicotine uptake decreased with pH reduction through a range of 8.3-4.2. These data suggest that it is the uncharged molecule which penetrates the blood-brain barrier by both carrier and lipid mediation. A mechanism relating to these observations is postulated and possible relevance to lactate washout from ischemic brain discussed.