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Carnitine is known to accumulate in brain, therefore transport of carnitine through the blood–brain barrier was studied in anin vitrosystem using bovine brain capillary endothelial cells (BBCEC) grown on filter inserts in a co-culture system with glial cells. Long-term exposure of BBCEC to carnitine resulted in a high accumulation of long-chain acyl carnitines, which decreased dramatically upon removal of carnitine. Kinetic analysis of carnitine accumulation indicated a possibility of functioning of more than one transporter. BBCEC were incubated in the presence of substrates and inhibitors of known carnitine transporters added from either apical or basolateral side. Inhibition by replacement of sodium and expression of OCTN2 (RT-PCR) were in agreement withearlier reports on the functioning of OCTN2 in apical membrane. For the first time, functioning of OCTN2 was demonstrated in the basolateral membrane, as well as functioning in both membranes of a low affinity carnitine transporter B0,+. Expression of B0,+ in BBCEC was confirmed by RT-PCR. These results suggest that OCTN2 and B0,+ could be involved in carnitine transport in both the apical and basolateral membrane. [ABSTRACT FROM AUTHOR]

Glucosyl-1,5-benzodiazepin-2,4-diones were synthesized in order to study the influence of the glucidic moiety on the amphiphilic behaviour. The glucosyl groups include 6-deoxy-D-glucopyranos-6-yl and 6-deoxy-3-O-R-D-glucopyranos-6-yl(R = n − CnH2n+1; n = 1, 8, 10 and 12). Variation in the length of the hydrocarbon chain allowed comparison of such amphiphilic data as water solubility(Sw) and surface tension(γ) values. At 25°C, the glucopyranosyl benzodiazepines with R = H and CH3 show a higher water solubility than the starting 1,5-benzodiazepin-2,4-diones. Some other glucidic benzodiazepine derivatives with an appropriate alkyl chain at C-3 carbon of the D-glucopyranose present a variable hydrosolubility and surface tensionγ values close to 43 to 46 mN · m−1 at the corresponding saturation. Moreover, according to preliminary tests, these compounds seem to show a better affinity for the blood brain barrier. [ABSTRACT FROM AUTHOR]

The binding profile of [3H]BHDP ([3H]N-benzyl-N′-(2-hydroxy-3,4-dimethoxybenzyl)-piperazine) was evaluated. [3H]BHDP labelled a single class of binding sites with high affinity (Kd=2–3 nM) in rat liver mitochondria and synaptic membranes. The pharmacological characterization of these sites using σ reference compounds revealed that these sites are σ receptors and, more particularly, σ1 receptors. Indeed, BHDP inhibited [3H]pentazocine binding, a marker for σ1 receptors, with high affinity in a competitive manner. BHDP is selective for σ1 receptors since it did not show any relevant affinity for most of the other receptors, ion channels or transporters tested. Moreover, in an in vitro model of cellular hypoxia, BHDP prevented the fall in adenosine triphosphate (ATP) levels caused by 24 h hypoxia in cultured astrocytes. Taken together, these results demonstrate that [3H]BHDP is a potent and selective ligand for σ1 receptors showing cytoprotective effects in astrocytes. [Copyright &y& Elsevier]

Abstract: The passage of either unesterified docosahexaenoic acid(DHA) or lysophosphatidylcholine-containing DHA (lysoPC-DHA) through an invitro model of the blood-brain barrier was investigated. The model wasconstituted by a brain capillary endothelial cell monolayer set over themedium of an astrocyte culture. Cells were incubated for 4 h with a mediumdevoid of serum, then the endothelial cell medium was replaced by the samemedium containing labeled DHA or lysoPC-DHA and incubations were performed for2 h. DHA uptake by cells and its transfer to the lower medium (astrocytemedium when they were present) were measured. When the lower medium frompreincubation and astrocytes were maintained during incubation, the passage oflysoPC-DHA was higher than that of unesterified DHA. The passage of both formsdecreased when astrocytes were removed. The preference for lysoPC-DHA was notseen when the lower medium from preincubation was replaced by fresh medium,and was reversed when albumin was added to the lower medium. A preferentiallysoPC-DHA passage also occurred after 2 h with brain endothelial cellscultured without astrocytes but not with aortic endothelial cells cultured andincubated under the same conditions. Altogether, these results suggest thatthe blood-brain barrier cells released components favoring the DHA transferand exhibit a preference for lysoPC-DHA. [ABSTRACT FROM AUTHOR]

Metabolic labelling of mouse splenocytes with radioactive mannose indicates that the glycosylation process is accompanied by the release of soluble oligomannoside material. Chase experiments with an excess of unlabelled mannose indicate that the radioactivity is mainly chased from oligosaccharide-PP-Dol (PP-Dol = diphosphodolichol): 10% is recovered as (Man)9(GlcNAc)2-P, (Man)9(GlcNAc)2, (Man)9GlcNAc and (Man)5GlcNAc, and 90% is rapidly degraded further. Tunicamycin inhibits both oligosaccharide-PP-Dol synthesis and the formation of the oligosaccharide material to the same extent. The results thus indicate that these soluble oligomannoside structures represent the main steps of the oligosaccharide-PP-Dol catabolic pathway, starting with the cleavage of the diphosphate bond. However, it cannot be excluded that part of this material is released from newly formed glycoproteins. The soluble oligomannoside material does not contain glucose residues despite the fact that part of the oligosacharide-PP-Dol is glucosylated and it was shown, by the use of glucosidase I inhibitors (castanospermine, deoxynojirimycin) that, after cleavage, the glycan moiety of glucosylated oligosaccharide-PP-Dol is first rapidly deglucosylated. These experiments provide a physiological basis to our previous results obtained in vitro and allow the definition of further steps in the catabolic pathway of oligosaccharide-PP-Dol. [ABSTRACT FROM AUTHOR]

Purpose. The objective of this work was to assess, in vitro, the passage of P-glycoprotein dependent drugs across brain capillary endothelial cells, when these drugs are associated with a reversing agent. Methods. An in vitro model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes was used. Results. We demonstrate that P-glycoprotein expression is upregulated by the presence of astrocytes. Uptake in the cells and transport across endothelial cell monolayers of vincristine, cyclosporin A and doxorubicin were studied. Using S9788 or verapamil as reversing agents, we found an increase in vincristine transport across the endothelial cell monolayers. On the other hand, the association of S9788 or verapamil with cyclosporin A failed to increase the transport of this drug. An increase in the transport of doxorubicin from luminal to abluminal compartment was also observed, due to endothelial cell monolayer breakdown. Conclusions. Using this model, it is possible to predict the passage of a P-glycoprotein dependent drug to the brain or its sequestration in brain capillary endothelial cells when this drug is associated with a reversing agent, or its toxicity on the blood-brain barrier integrity. [ABSTRACT FROM AUTHOR]

Acylated proteins play a crucial role in cellphysiology because of their increased interaction withmembranes. Their isolation is difficult as aconsequence of their low cellular concentration andtheir chemical preparation is problematic due tosolubility problems. Through the use of reversedmicelles, we produced tens of milligrams of acylatedribonucleases A, chosen as a model, purified them bysemi-preparative high performance liquidchromatography (HPLC) and characterized them by analyticalHPLC, capillary electrophoresis, mass spectrometry, peptide mapping, Edman degradation and enzyme activity. We nextscrutinized the interaction with an in vitro blood-brainbarrier model and demonstrated that palmitoylated andstearoylated ribonucleases A are transported from onecompartment to the other across the cellular monolayer,in contrast to the native enzyme. [ABSTRACT FROM AUTHOR]