Notes: Blood–brain barrier (BBB) transport of large neutral amino acids is mediated by the large neutral amino acid transporter type 1 (LAT1 transporter). Although the gene encoding the Glut1 glucose transporter is up-regulated in hypoxia, the response of the LAT1 gene to hypoxia is not known. The present study investigates the changes in the LAT1 mRNA in cultured bovine brain capillary endothelial cells exposed to 1% O2 for 24–48 h. The LAT1 mRNA was initially down-regulated in hypoxia with reciprocal changes in the Glut1 mRNA. No changes in the 4F2hc mRNA in hypoxia were observed. Hypoxia caused an initial de-stabilization of the LAT1 mRNA, and the t1/2 of the LAT1 mRNA in control and hypoxic cells was 6.4 ± 0.5 and 2.4 ± 0.1 h, respectively. To further explore post-transcriptional regulation of LAT1 gene expression, the polysome and cytosol fractions of the control and hypoxic endothelial cells were isolated, and LAT1 mRNA binding proteins were detected by ultraviolet light cross-linking. Whereas the cytosol contained no LAT1 mRNA binding proteins, the cell polysome fraction expressed several LAT1 mRNA binding proteins, including principal 40-, 70- and 80-kDa proteins. These studies are consistent with post-transcriptional de-stabilization of the LAT1 large neutral amino acid transporter in hypoxia.

Notes: Abstract: Blood-brain barrier (BBB) function is endowed by the expression of unique proteins within the brain capillary endothelium. In the absence of knowing the function of BBB-specific proteins, one strategy for identification of these proteins is the purification and amino acid sequencing of proteins within the brain capillary that are not found in other cells. Earlier studies have shown that a 16-18K triplet of low-molecular-weight proteins in isolated brain capillaries is not found in either erythrocytes or in capillary-free preparations of synaptosomal proteins. Therefore, the present studies describe the purification of the 16-18K triplet of proteins as well as a 14K protein in isolated brain capillaries using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and C4 reverse-phase HPLC. Amino acid sequencing of the N-terminus of the 14K, 17K, and 18K proteins and of two tryptic peptides of the 16K protein showed that these proteins are α-globin, histone 2B, histone 3, and histone 2A, respectively. SDS-PAGE of subcellular fractions of bovine brain capillaries demonstrated that the 16-18K triplet of histone proteins migrated in the nuclear fraction. In addition, a 34K doublet and a 200K protein were localized in the nuclear pellet. Therefore, the present studies demonstrate that the predominant 14-18K proteins seen on SDS-PAGE of isolated brain capillaries are known proteins and provide a general scheme for purification of brain capillary proteins isolated following SDS-PAGE.

Notes: Abstract: The blood–brain barrier (BBB) transport of a highly lipid-soluble peptide, [3H]cyclosporin, was studied in ketamine-anesthetized rats using the carotid artery injection technique. For comparison, peptide transport into rat liver was also assessed with the portal vein injection technique. Despite the high lipid solubility of this peptide (1-octanol/Ringer's partition coefficient = 991 ± 55), the extraction by rat brain was only 2.9 ± 0.5% in the presence of 80% human serum, and this value approximated the extraction for a poorly diffusible substance such as [3H]inulin, 2.0 ± 0.1%. In contrast, the hepatic extraction of [3H]cyclosporin was high, 84 ± 2%, in the presence of 80% human serum. The BBB transport of cyclosporin is markedly restricted owing to the combined effects of binding by serum proteins and a paradoxically low permeability of the BBB to the peptide.

Notes: The study of the regulation of low-abundance blood-brain barrier (BBB) transcripts either in isolated brain microvessels or in endothelial cells in tissue culture (ECL cells) requires isolation of poly(A)+ mRNA. Therefore, we describe here a single-step method for isolation of polyfA)+ mRNA from brain capillaries or ECL cells using proteinase K/sodium dodecyl sulfate cell lysis and oligo-de-oxythymidine cellulose affinity chromatography. The yield of poly(A)+ mRNA was—15-19 #g/g of brain or choroid plexus, 14-17 μg per batch of isolated capillaries in a single bovine forebrain (190 g), and 6-12 μg/107 ECL cells. Northern blot analysis showed characteristic and undegraded 2.1-and 1.7-kb actin transcripts in brain capillaries and a 2.1-kb actin mRNA in brain and ECL cells. Northern analysis was also used to quantify the glucose transporter type I transcript, which is very rare in basal ECL cells, and this mRNA was shown to be up-regulated by glucose deprivation. This method represents a significant improvement in the mRNA yield for brain capillaries or cultured endothelial cells compared with the conventional two-step method.

Notes: Abstract: Tryptophan is the only amino acid in the circulation that is bound by albumin, and previous studies have suggested that the brain tryptophan supply is a function of either the free or the albumin-bound pool of tryptophan in blood. Since the albumin molecule per se does not cross the brain capillary wall, i.e., the blood-brain barrier (BBB), the transport of tryptophan from the circulating albumin-bound pool may involve enhanced dissociation of tryptophan from the albumin binding sites within the cerebral microcirculation. This hypothesis was confirmed in the present studies wherein the dissociation constant (KaD) of albumin binding of tryptophan in the rat or rabbit brain microcirculation was measured in vivo. Brain extraction data for [14C]tryptophan determined with the carotid artery injection technique were fit to the Kety-Renkin-Crone equation modified for protein-bound solute. The KaD of albumin binding in the rat or rabbit brain microcirculation under pentobarbital anesthesia was 1.7 ± 0.1 and 3.9 ± 1.0 mM, respectively, as compared to the KD value measured in vitro with equilibrium dialysis, 0.13 ± 0.03 mM. In contrast, the KaD value of albumin binding of tryptophan in vivo in the rabbit brain microcirculation was reduced by ether anesthesia to value of 2.1 ± 0.4 mM. This reduction in the KaD under ether anesthesia was associated with a 2.5-fold increase in cerebral blood flow. In addition, dialyzed rabbit serum caused a statistically significant inhibition in [14C]tryptophan influx during ether, but not pentobarbital, anesthesia. The BBB permeability-surface area product (PS) was estimated along with the KaD parameter, and this PS value in the rabbit brain, 0.13 ml/min/g, was identical to the quantity [(Vmax/Km) +KD] measured from estimates of the constants of saturable (Km, Vmax) and nonsaturable (KD) transport of tryptophan through the BBB. In conclusion, the present studies demonstrate that tryptophan is transported into brain from the circulating albumin-bound pool via an enhanced dissociation mechanism and that this process varies among species and with changing anesthesia and cerebral hemodynamics.

Notes: Abstract: 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-ßlood 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, 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.

Notes: Abstract: The expression of the blood-brain barrier GLUT1 glucose transporter is down-regulated in brain capillary endothelial cells in tissue culture. Consequently, the study of the regulation of this low-abundance transcript requires the isolation of poly(A)+ mRNA from relatively large numbers of brain endothelial cells in culture (˜107). Therefore, in order to facilitate studies with smaller amounts of cells, we describe here a quantitative polymerase chain reaction (PCR) assay to measure the mRNA of GLUT1 and the mRNA of the housekeeping gene, actin, which is used as standard control. Bovine brain endothelial cells were grown as either a primary culture (EP cells) or as a brain endothelial cell line (ECL cells) in 25-mm 6-well cluster dishes, and total or poly(A)+ RNA was isolated. Following synthesis of cDNA with AMV reverse transcriptase and oligo(dT)18 primer, PCR was performed with sense and antisense primers for bovine GLUT1 and 7-actin, respectively. Reactions were performed in the presence of 2.5 μCi of [α-32P]dCTP, and products were resolved in agarose gels and quantified by scanning densitometry of autoradiograms. A direct relationship between RNA-cDNA and PCR products was observed for GLUT1 after 30 cycles, and for actin after 15 PCR cycles. The method was reproducible within specified ranges of starting RNA-derived cDNA, and the intraassay coefficient of variation averaged 7.2 β 1.8%. The GLUT1/actin mRNA ratio was as follows: brain capillaries β EP 〉 ECL. In addition, it is demonstrated that tumor necrosis factor-α induced a three-to fourfold increase in the GLUT1/actin mRNA ratio in ECL cells. This method provides a 100-200-fold increase in the sensitivity of detection of blood-brain barrier GLUT1 transcript in bovine brain capillary endothelial cells in tissue culture compared with the conventional northern blotting technique using poly(A)+ mRNA.

Notes: Abstract: Capillaries in vertebrate brain have unique permeability properties that make up the blood–brain barrier (BBB). Although it is known that capillaries are innervated by nerve endings of intracerebral origin and that brain capillary function is likely acutely regulated by neuronal inputs, the possible mechanisms of neuronal regulation of capillary function are at present unknown. One possible mode of regulation is via the phosphorylation of brain capillary proteins. The present studies characterize, for the first time, the major phosphoproteins in the bovine brain capillary using both intact bovine brain capillaries and plasma membrane fractions from bovine brain capillaries. The patterns of endogenous phosphorylation of capillary proteins are compared to similar patterns obtained with synaptosomal (P2) fractions from bovine brain. The major findings of this study are: (a) The activity of protein phosphorylation in brain capillaries is localized almost exclusively to the capillary plasma membrane, and is nearly comparable to the activity of protein phosphorylation in synaptosomal membranes. (b) A major phosphoprotein doublet in the capillary fraction comigrates on a sodium dodecyl sulfate gel with a major phosphoprotein doublet of approximate molecular weight of 80K in the synaptosomal fraction, and the latter is presumed to be synapsin I; in dephosphorylation assays the synaptosomal 80K phosphoprotein doublet is not subject to measurable dephosphorylation, whereas the capillary 80K doublet is subject to rapid dephosphorylation, and is essentially completely dephosphorylated within 5 s at 0°C. (c) A prominent triplet of phosphoproteins with molecular weight of 50–55K is present in the capillary fraction, and is not present in the synaptosomal fraction; thus, this 50—55K triplet of phosphoproteins appears specific for brain capillaries. In summary, these studies provide the basis for future investigations of a protein phosphorylation paradigm in regard to the rapid control of brain capillary function by brain.

Notes: Abstract: Recent studies have indicated that the blood-brain barrier GLUT1 glucose transporter is under post-transcriptional regulation. To begin functional mapping of the GLUT1 transcript, in the present investigation we studied the translational efficiency of capped full-length synthetic GLUT1 mRNA, and both 5′- and 3′-untranslated regions (UTRs) deleted GLUT1 mRNAs. Deletion of 5′- and 5′-/3′-UTRs markedly reduced the translation efficiency of the human (h) GLUT1 transcript in the rabbit reticulocyte lysate (RRL), and this effect was not modified by addition of microsomes to the translation system. The putative role of these hGLUT1 5′-UTR cis-acting elements was studied using the luciferase expression vector pGL2. DNA corresponding to the hGLUT1 5′-UTR generated by PCR was subcloned at the HindIII site of the pGL2 located upstream of the luciferase 5′-UTR. Transfection of brain endothelial cultured cells with pGL2 containing most of the hGLUT1 5′-UTR (nucleotides 1–171) markedly increased the expression of luciferase, and disruption of luciferase-leading sequence with an unrelated 171-nucleotide fragment decreased its expression. Insertion of nucleotides 1–96 of the hGLUT1 5′-UTR retained most of the stimulatory effect, and nucleotides 123–171 produced 64% of maximal induction. On the contrary, clones containing nucleotides 79–171 and 154–171 of bGLUT1 5′-UTR had marginal effects on luciferase expression. The present data provide evidence suggesting that the 5′-UTR of the GLUT1 mRNA contains cis-acting elements involved in the translational control of the GLUT1 gene in mammalian cells.

Notes: Abstract: The posttranscriptional regulation of glucose transporter GLUT1 gene expression may be mediated by specific interactions of cytosolic proteins and regulatory cis-elements within the untranslated regions (UTRs) of the GLUT1 mRNA. These putative cis/trans interactions were examined in the present studies with RNase T1 protection assays using 32P-labeled GLUT1 3′-UTR prepared from transcription plasmids and cytosolic proteins from C6 rat glioma cells. RNase T1 mapping studies localized a cis-element to nucleotides 2,170–2,207 on the bovine GLUT1 mRNA 3′-UTR. Ultraviolet cross-linking of RNA/protein complexes identified two complexes having molecular masses of 88 and 44 kDa. Competition studies with synthetic RNA and oligodeoxynucleotides showed the 88-kDa complex reacted with nucleotides 2,180–2,197 and that the 44-kDa complex reacted with sequences within nucleotides 1,717–2,132 of the bovine GLUT1 mRNA. The GLUT1 3′-UTR between nucleotides 2,100 and 2,300 was generated by polymerase chain reaction and subcloned at a unique Pf/MI site within the 3′-UTR of a luciferase gene within the mammalian expression vector pGL2. Transfection of C6 rat glioma cells with the luciferase expression vector containing this portion of the GLUT1 3′-UTR resulted in a sixfold increase in luciferase gene expression in C6 cells. The identification of these cis/trans mechanisms provides support for the hypothesis that the posttranscriptional regulation of GLUT1 gene expression may be mediated by the interaction of specific cytosolic proteins with the GLUT1 mRNA 3′-UTR.