Porcine Brain Capillary Endothelial Cells Research Articles

Interaction of the mitotic kinesin Eg5 inhibitor monastrol with P-glycoprotein

Monastrol is the first characterised small molecule inhibitor of the motor protein Eg5 involved in bipolar mitotic spindle assembly. Eg5 localises to microtubules in mitosis, but not to interphase microtubules, suggesting that Eg5 inhibitors may be useful to specifically target proliferating tumour tissue, thereby avoiding dose-limiting neuropathy observed with other antimicrotubule agents like taxanes or vinca alkaloids. Because other antimicrotubule agents fail in multidrug resistance associated with P-glycoprotein (Pgp) over-expression, we investigated the interaction of monastrol with Pgp in vitro. By means of the calcein assay (with P388/dx cells and primary porcine brain capillary endothelial cells) and confocal laser-scanning microscopy (with L-MDR1 cells) we demonstrated that monastrol is a weak inhibitor of Pgp in vitro, with f2 values being about two orders of magnitude greater than those of the well-known inhibitors verapamil and quinidine. Monastrol also induces Pgp in vitro as measured by mRNA expression in LS180 cells after incubation with monastrol. However, its effect is weak compared to rifampicin. Whilst it reveals weak inhibitory and inductive characteristics, monastrol appears to be not transported by Pgp, as indicated by the lack of difference in the antiproliferative effect of this compound in cell lines with and without over-expression of Pgp. The observed interaction profile of monastrol with Pgp is promising for the development of other more potent Eg5 inhibitors.

Influence of Hydrocortisone on the Mechanical Properties of the Cerebral Endothelium In Vitro

Cerebral endothelial cells accomplish the barrier functions between blood and brain interstitium. Structural features are the tight junctions between adjacent endothelial cells and the formation of marginal folds at the cell-cell contacts. The glucocorticoid hydrocortisone (HC) has been reported to enforce the blood-brain-barrier in vitro measurable by an increase of the transendothelial electrical resistance. This study shows the impact of HC on the mechanical and morphological properties of confluent cell layers of brain microvascular endothelial cells. HC induces an increase in height of these marginal folds and a reduction of the intercellular contact surface. These morphological changes are accompanied by changes in cell elasticity. Staining of fibrous actin indicates that HC induces a reorganization of the actin cortex. The quantitative determination of the local elastic properties of cells reveals for the first time an HC-induced increase of the representative Young’s modulus according to cytoskeletal rearrangements. For this study, cells of two different species, porcine brain capillary endothelial cells and murine brain capillary endothelial cells, were used yielding similar results, which clearly demonstrates that the HC effect on the cell elasticity is species independent.

Evaluation of the immortalized human brain capillary endothelial cell line BB19 as a human cell culture model for the blood–brain barrier

In vitro models of the blood–brain barrier (BBB) play a major role in the study of BBB permeability of drug candidates. However, most established in vitro models use cells of non-human origin, which is not optimal for the prediction of brain permeability in humans. The aim of this study was to assess the human brain capillary endothelial cell line BB19 for its usefulness as an in vitro model of the human BBB. Restrictive tight junctions are a prerequisite for drug transport studies. Sucrose permeability of BB19 cells on different filters was compared to porcine brain capillary endothelial cells (BCEC). Tightness of BB19 cell monolayers still needs further optimization. Hardly any discrimination between Sucrose and Propranolol ( P app = 1.30 × 10 −5 vs. 2.18 × 10 −5 cm/s) was seen. Cells showed an improvement towards a more primary BCEC morphology with C6 conditioned medium, dexamethasone, and 1,25-dihydroxyvitamin D 3. The presence of P-glycoprotein (P-gp), MRP4 and BCRP, ABC-transporters located in the BBB, has been shown on mRNA level, by immunostaining, and western blot. MRP1, MRP2, MRP5, OAT3, and OAT4 were also detected by RT-PCR. Functional properties of the BBB were shown with uptake of propranolol, morphine, and sucrose. Uptake studies with daunomycin and the P-gp inhibitor verapamil showed functional activity of P-gp. We conclude that BB19 cells might be feasible as a human in vitro model of the BBB for drug uptake studies. However, for the assessment of transport studies, further improvements of this model are necessary.

Inhibition of P-Glycoprotein Function by Several Antidepressants may not Contribute to Clinical Efficacy

In many depressive patients the negative feedback mechanism of the HPA (hypothalamic-pituitary-adrenocortical) axis is impaired. It has been suggested that antidepressants inhibit membrane glucocorticoid transporters like P-Glycoprotein (Pgp) and hence enhance the intracellular glucocorticoid concentration, leading to an increased glucocorticoid-receptor mediated gene transcription and therefore to normalization of the function of the HPA axis. The aim of this study is to investigate inhibition of Pgp by several different antidepressants. We characterized the inhibitory potencies of the antidepressants in two in vitro assays by using calcein-AM as Pgp substrate. The two different cell-systems expressing Pgp were: 1. PBCEC (porcine brain capillary endothelial cells) as model for the blood-brain-barrier, and 2. A human lymphocytic leukaemia cell line CEM and the multi-drug-resistant (MDR) cell line VLB-100, expressing Pgp as model for the human protein. All of the antidepressants tested inhibit the transport of calcein-AM by Pgp in the micromolecular range. Because this inhibition is only seen at concentrations above therapeutically relevant plasma levels, their effect my not play a role for the mechanism of action of the antidepressants tested.

In vitro study of malaria parasite induced disruption of blood–brain barrier

The mechanism of blood–brain barrier breakdown in the complex pathogenesis of cerebral malaria is not well understood. In this study, primary cultures of porcine brain capillary endothelial cells (PBCEC) were used as in vitro model. Membrane-associated malaria antigens obtained from lysed Plasmodium falciparum schizont-infected erythrocytes stimulated human peripheral blood mononuclear cells (PBMC) to secrete tumor necrosis factor alpha. In co-cultivation with the brain endothelial cell model, the malaria-activated PBMC stimulated the expression of E-selectin and ICAM-1 on the PBCEC. Using electric cell–substrate impedance sensing, we detected a significant decrease of endothelial barrier function within 4 h of incubation with the malaria-activated PBMC. Correspondingly, immunocytochemical studies showed the disruption of tight junctional complexes. Combination of biochemical and biophysical techniques provides a promising tool to study changes in the blood–brain barrier function associated with cerebral malaria. Moreover, it is shown that the porcine endothelial model is able to respond to human inflammatory cells.

Regulated expression of endothelial lipase by porcine brain capillary endothelial cells constituting the blood–brain barrier

Normal neurological function depends on a constant supply of polyunsaturated fatty acids to the brain. A considerable proportion of essential fatty acids originates from lipoprotein-associated lipids that undergo uptake and/or catabolism at the blood-brain barrier (BBB). This study aimed at identifying expression and regulation of endothelial lipase (EL) in brain capillary endothelial cells (BCEC), major constituents of the BBB. Our results revealed that BCEC are capable of EL synthesis and secretion. Overexpression of EL resulted in enhanced hydrolysis of extracellular high-density lipoprotein (HDL)-associated sn-2-labeled [(14)C]20 : 4 phosphatidylcholine. [(14)C]20 : 4 was recovered in cellular lipids, indicating re-uptake and intracellular re-esterification. To investigate local regulation of EL in the cerebrovasculature, BCEC were cultured in the presence of peroxisome-proliferator activated receptor (PPAR)- and liver X receptor (LXR)-agonists, known to regulate HDL levels. These experiments revealed that 24(S)OH-cholesterol (a LXR agonist), bezafibrate (a PPARalpha agonist), or pioglitazone (a PPARgamma agonist) resulted in down-regulation of EL mRNA and protein levels. Our findings implicate that EL could generate fatty acids at the BBB for transport to deeper regions of the brain as building blocks for membrane phospholipids. In addition PPAR and LXR agonists appear to contribute to HDL homeostasis at the BBB by regulating EL expression.

Rat brain endothelial cell lines for the study of blood-brain barrier permeability and transport functions.

(1) In vitro models of the BBB have been developed from cocultures between bovine, porcine, rodent or human brain capillary endothelial cells with rodent or human astrocytes. Since most in vivo BBB studies have been performed with small laboratory animals, especially rats, it is important to establish a rat brain endothelial (RBE) cell culture system that will allow correlations between in vitro and in vivo results. The present review will constitute a brief description of the best characterized RBE cell lines (RBE4, GP8/3.9, GPNT, RBEC1, TR-BBBs and rBCEC4 cell lines) and will summarize their recent and important contribution to our current knowledge of the BBB transport functions and permeability to blood-borne solutes, drugs, and cells. (2) In most cases, primary cultures of RBE cells were transduced with an immortalizing gene (SV40 or polyoma virus large T-antigen or adenovirus E1A), either by transfection of plasmid DNA or by infection using retroviral vectors. In one case however, the conditionally immortalized TR-BBB cell line was derived from primary cultures of brain endothelial cells of SV40-T-expressing transgenic rats. (3) All cell lines appear to have an endothelial morphology. The absence of foci formation would mean that the cells are not transformed. The endothelial origin is shown by the expression of Factor VIII-related antigen. Immortalized RBE cells express all the enzymes and transporters that are considered as specific for the blood-brain barrier endothelium, with similar characteristics to those expected from in vivo analyses, but at a significantly lower level. Some RBE cell lines are responsive to astroglial factors, such as RBE4 cells, rBEC4, and TR-BBB cells. None of the immortalized RBE cell lines appear to generate the necessary restrictive paracellular barrier properties that would allow to use them in transendothelial permeability screening. (4) RBE cell lines have been used to demonstrate that transporters such as organic cation transporter/carnitine transporter, serotonin transporter, and the ATA2 system A isoform are expressed in rat brain endothelium. When the transporter is shown to be expressed with the same properties in the immortalized RBE cells as in vivo, regulation studies may be initiated even if the transporter is down-regulated. Pharmacological applications have been proposed with well-characterized transporters such as monocarboxylic acid transporter-1, large neutral amino acid tansporter-1, nucleoside carrier systems, and P-glycoprotein. RBE cell monolayers have also been used to investigate the mechanism of the transendothelial transport of large molecules, such as immunoliposomes or nanoparticles, potentially useful as drug delivery vectors to the brain. (5) RBE4 and GP8 cell lines have been extensively used to demonstrate that intercellular adhesion molecule-1 (ICAM-1) engagement in brain endothelial cells triggers multiple signal transduction pathways. Using functional assays, it was established that ICAM-1 signaling is intimately and actively involved in facilitating lymphocyte infiltration. (6) Several RBE cell lines have been described, which constitute tentative in vitro models of the rat BBB. The major limitation of these models generally appears to be due to their relatively high paracellular permeability to small molecules, thus limiting their use for permeability studies. The strategies developed for the production of these RBE cell lines will enable the characterization of still more efficient permeability models, as well as the immortalization of human brain endothelial cells.

Modulation of P-Glycoprotein Function by St John’s Wort Extract and its Major Constituents

Recent data suggest some relevant drug interactions caused by St John's wort extract, which can be explained by interactions with the Cytochrome P450 system or P-Glycoprotein (Pgp). Interaction with Pgp, including activation, inhibition and induction, can lead to altered plasma or brain levels of Pgp substrates. The aim of the present study was to investigate the possible interactions of St John's wort extract and most relevant constituents with the transport activity of Pgp. We characterized the modulatory potencies in two in vitro assays using calcein-AM, first in VLB cells (a human lymphocytic leukemia cell line expressing Pgp) and second in PBCEC cells (porcine brain capillary endothelial cells). The extract, as well as some of the tested constituents modulate the transport by Pgp in the micromolecular range. Quercetin and hyperforin seem to be most potent. These findings suggest the possibility of drug interactions at the level of the gastro-intestinal absorption of drugs. Plasma levels of the constituents of St John's wort are very likely too low to interfere with Pgp at the blood-brain-barrier with the possible exception of quercetin.

529 Nonlinear pharmacokinetic modeling of XAA296 administered to patients with advanced solid tumors once every 3 weeks (q3w) intravenously (IV) in a phase I clinical trial

Thai medicine has a long tradition of tonifying medicinal plants. In the present investigation, we studied the flower extracts of Jasminum sambac, Mammea siamensis, Mesua ferrea, Michelia alba, Mimusops elengi, and Nelumbo nucifera and speculated that these plants might influence metabolism and substance flow in the body.Isolation of porcine brain capillary endothelial cells (PBCECs) as well as multidrug-resistance CEM/ADR5000 leukemia cells, MDA-M;B-231 breast cancer, U-251 brain tumor, and HCT-116 colon cancer cells were used. The calcein-acetoxymethylester (AM) assay was used to measure inhibition of P-glycoprotein transport. XTT and resazurin assays served for measuring cytotoxicity.The extracts revealed cytotoxicity towards CCRF-CEM leukemia cells to a different extent. The strongest growth inhibition was found for the n-hexane extracts of Mammea siamensis and Mesua ferrea, and the dichloromethane extracts of Mesua ferrea and Michelia alba. The flower extracts also inhibited P-glycoprotein function in porcine brain capillary endothelial cells and CEM/ADR5000 leukemia cells, indicating modulation of the blood–brain barrier and multidrug resistance of tumors. Bioactivity-guided isolation of coumarins from Mammea siamensis flowers revealed considerable cytotoxicity of mammea A/AA, deacetylmammea E/BA and deacetylmammea E/BB towards human MDA-MB-231 breast cancer, U-251 brain tumor, HCT-116 colon cancer, and CCRF-CEM leukemia cells.The plants analyzed may be valuable in developing novel treatment strategies to overcome the blood–brain barrier and multidrug-resistance in tumor cells mediated by P-glycoprotein.

Scavenger receptor class B, type I mediates uptake of lipoprotein-associated phosphatidylcholine by primary porcine cerebrovascular endothelial cells

We previously reported that scavenger receptor class B, type I (SR-BI) mediates uptake of lipoprotein-associated cholesteryl ester and Vitamin E by porcine brain capillary endothelial cells (pBCECs). In the present study we investigated whether SR-BI is capable of mediating phosphatidylcholine (PC) uptake by pBCECs from low- and high density lipoproteins. SR-BI-overexpressing CHO cells and pBCECs showed significantly enhanced uptake rates of PC from both lipoprotein classes. In addition, preincubation of pBCECs in the presence of both lipoprotein species resulted in a significant increase of the cellular fatty acid content, particularly linoleic and arachidonic acid. Our results suggest that uptake of lipoprotein-associated PC by the cerebrovasculature via SR-BI could generate a pool of lipids containing polyunsaturated fatty acids available for transport into deeper regions of the brain.

Uptake and transport of high-density lipoprotein (HDL) and HDL-associated alpha-tocopherol by an in vitro blood-brain barrier model.

The present study aimed to investigate pathways that contribute to uptake and transcytosis of high-density lipoproteins (HDLs) and HDL-associated alpha-tocopherol (alpha TocH) across an in vitro model of the blood-brain barrier (BBB). In primary porcine brain capillary endothelial cells HDL-associated alpha TocH was taken up in 10-fold excess of HDL holoparticles, indicating efficient selective uptake, a pathway mediated by scavenger receptor class B, type I (SR-BI). SR-BI was present in caveolae of brain capillary endothelial cells and expressed almost exclusively at the apical membrane. Disruption of caveolae with methyl-beta-cyclodextrin (CDX) resulted in (mis)sorting of SR-BI to the basolateral membrane. Immunohistochemistry of porcine brain cryosections revealed SR-BI expression on brain capillary endothelial cells and presumably astrocytic endfeet. HDL-associated [(14)C]alpha TocH taken up by brain capillary endothelial cells was recovered in sucrose gradient fractions containing the majority of cellular caveolin-1, the major caveolae-associated protein. During mass transfer studies using alpha TocH-enriched HDL, approximately 50% of cellular alpha TocH was recovered with the bulk of cellular caveolin-1 and SR-BI. Efflux experiments revealed that a substantial amount of cell-associated [(14)C]alpha TocH could be mobilized into the culture medium. In addition, apical-to-basolateral transport of HDL holoparticles and HDL-associated alpha TocH was saturable. Results from the present study suggest that part of cerebral apolipoprotein A-I and alpha TocH originates from plasma HDL transcytosed across the BBB and that caveolae-located SR-BI facilitates selective uptake of HDL-associated alpha TocH at the BBB.

Tyrosine phosphatase inhibition induces loss of blood–brain barrier integrity by matrix metalloproteinase-dependent and -independent pathways

Tight junctions between endothelial cells of brain capillaries form the structural basis of the blood–brain barrier (BBB), which controls the exchange of molecules between blood and CNS. Regulation of cellular barrier permeability is a vital and complex process involving intracellular signalling and rearrangement of tight junction proteins. We have analysed the impact of tyrosine phosphatase inhibition on tight junction proteins and endothelial barrier integrity in a primary cell culture model based on porcine brain capillary endothelial cells (PBCEC) that closely mimics the BBB in vitro. The tyrosine phosphatase inhibitor phenylarsine oxide (PAO) induced increased matrix metalloproteinase (MMP) activity, which was paralleled by severe disruption of cell–cell contacts and proteolysis of the tight junction protein occludin. ZO-1 and claudin-5 were not affected. Under these conditions, the transendothelial electrical resistance (TEER) was markedly reduced. PAO-induced occludin proteolysis could be prevented by different MMP inhibitors. Pervanadate (PV) reduced the TEER similar to PAO, but did not increase MMP activity. Cell–cell contacts of PV-treated cells appeared unaffected, and occludin proteolysis did not occur. Our results suggest that tyrosine phosphatase inhibition can influence barrier properties independent of, but also correlated to MMPs. Evidence is given for a role of MMPs in endothelial tight junction regulation at the BBB in particular and probably at tight junctions (TJs) in general.

A porcine astrocyte/endothelial cell co-culture model of the blood–brain barrier

A method for the isolation of porcine atrocytes as a simple extension of a previously described procedure for isolation of brain capillary endothelial cells from adolescent pigs [Methods Cell Sci. 17 (1995) 2] is described. The obtained astroglial culture purified through two passages and by the method of the selective detachment was validated by a phase contrast microscopy and through an immunofluorescent assay for the glial fibrillary acidic protein (GFAP). Porcine astrocytes were co-cultivated with porcine brain capillary endothelial cells (PBCEC) for the development of an in vitro blood–brain barrier (BBB) model. The model was visualized by an electron microscopy and showed elevated transendothellial electrical resistance and reduced inulin permeability. To our knowledge, this is the first report for the establishment of a porcine astrocyte/endothelial cell co-culture BBB model, which avoids interspecies and age differences between the two cell types, usually encountered in the other reported co-culture BBB models. Considering the availability of the porcine brain tissue and the close physiological and anatomical relation between the human and pig brain, the porcine astrocyte/endothelial cell co-culture system can serve as a reliable and easily reproducible model for different in vitro BBB studies.

Modulation of transendothelial permeability and expression of ATP-binding cassette transporters in cultured brain capillary endothelial cells by astrocytic factors and cell-culture conditions.

Confluent cell monolayers of brain capillary endothelial cells (BCEC) are used widely as an in vitro cell culture model of the blood-brain barrier. The present study describes the influence of cell-culture conditions on tight junctions, filamentous-actin cytoskeleton, and expression of ATP-binding cassette (ABC) transporters in primary cell cultures of porcine BCEC. Astrocyte as well as C6 glioma-conditioned cell culture medium was used in combination with retinoic acid, dexamethasone, cyclic adenosine monophosphate (cAMP) analogs, or 1,25-dihydroxyvitamin D3. It was shown that C6-conditioned medium led to a reorganization of filamentous actin and to an improved staining of zonula occludens-associated protein-1 (ZO-1). Further optimization of these culture conditions was achieved with cAMP analogs and dexamethasone. Retinoic acid, as well as 1,25-dihydroxyvitamin D3, did not improve cellular tight junctions as judged by filamentous actin, ZO-1 rearrangement, and transcellular electrical resistance (TER) measurements. However, these morphological changes did not influence the paracellular permeability of the extracellular marker sucrose. Expression of ABC transporters such as P-glycoprotein, multidrug resistance-associated protein-1(MRP1), and MRP2 were compared by measuring messenger RNA (mRNA) levels in whole-brain tissue, isolated brain capillaries, and cultured cells. In freshly isolated BCEC, mRNA levels of MRP2 and P-glycoprotein dropped by two- to sevenfold, respectively, whereas MRP1 mRNA levels were slightly increased. During cell culture, mRNA levels of MRP1 and MRP2 decreased by up to fivefold, while P-glycoprotein levels remained constant. These results were unaltered by different cell-culture conditions. In conclusion, the present study suggests that paracellular permeability, as well as mRNA expression of the studied ABC transporters in primary cultures, of porcine BCEC are insensitive toward changes in cell-culture conditions.

Interaction of antiepileptic drugs with human P-glycoprotein in vitro.

About one-third of epilepsy patients are resistant to treatment with antiepileptic drugs (AEDs). This refractoriness is not fully understood, but is thought to be attributed to overexpression of multidrug transporters at the blood-brain barrier, particularly P-glycoprotein (Pgp). In certain cases pharmacoresistance can be overcome by add-on therapy, raising the question of whether the coadministered drugs act as inhibitors of Pgp. Indeed, several AEDs are substrates and to some extent also inducers of Pgp. To date nothing is known about possible Pgp inhibitory activities of AEDs. Therefore, we investigated whether AEDs commonly used in mono or add-on therapy inhibit Pgp using a calcein acetoxymethylester uptake assay with L-MDR1 cells and primary porcine brain capillary endothelial cells, as well as confocal laser-scanning microscopy with L-MDR1 cells and bodipy-verapamil as Pgp substrate. In the calcein assay only carbamazepine inhibited Pgp, which was confirmed in confocal laser-scanning microscopy. In this assay, also phenytoin, lamotrigine, and valproate revealed Pgp inhibitory potency. Because Pgp inhibition by AEDs appeared at significantly higher concentrations than that of well known inhibitors and because the effective concentrations exceeded therapeutic AED plasma concentrations, Pgp inhibition appears not to be of clinical relevance, at least in antiepileptic monotherapy. However, it is possible that this inhibition may contribute to the effectiveness of certain add-on therapies.

Serum-derived factors weaken the barrier properties of cultured porcine brain capillary endothelial cells in vitro

Cultured cerebral capillary endothelial cells are often used as a functional in vitro model of the blood–brain barrier (BBB) to determine drug uptake or to study barrier properties. Usually serum is supplemented to these cultures for cell proliferation. Here, we demonstrate the effect of serum and the serum-derived factors lysophosphatidic acid (LPA) and vascular endothelial growth factor (VEGF) on the barrier properties of cultured porcine brain capillary endothelial cells (PBCEC). Serum prevents tight junction formation of confluent PBCEC monolayers and moreover, opens already established tight junctions shown by decreasing transendothelial electrical resistances (TER). These effects are highly polarised with serum almost exclusively acting from the basolateral side of the cell culture. Immunocytochemistry of PBCEC revealed a delocalisation of the cell border lining tight junction proteins ZO-1, occludin and claudin-5 when serum was added. A serum fraction of 67 kDa was isolated by size-exclusion chromatography, identified as albumin and found to cause a serum-like decrease of the TER. However, fatty acid-free serum albumin does not develop this barrier weakening effect, indicating that small protein-bound factors might be responsible. For instance, serum-bound LPA demonstrated a TER-decreasing effect as well, but in contrast to serum mainly when added to the apical side of PBCEC. Addition of VEGF caused a serum-like decrease of the TER with the same polar effect; however, VEGF will be denatured by heat and could thus not be the heat-sensitive factor. Thus, we hypothesise that serum contains a variety of factors which weaken the tightness of a PBCEC monolayer from the apical side as expected but also from the basolateral side. Although the structure of the 67 kDa factor could not be analysed, this finding is of importance for in vitro models not only of the blood–brain barrier mostly using serum-containing media.

Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood–brain barrier

The blood–brain barrier (BBB) plays the predominant role in controlling the passage of endogenous and xenobiotic substances between the circulating blood and the extracellular fluid environment of the brain. The transfer of compounds is strictly regulated by brain capillary endothelial cells (BCEC), which are interconnected to each other by well developed tight junctions, without fenestrations. Although hydrophobic molecules such as nicotine and ethanol readily cross the BBB by diffusion, the brain microvasculature shows a highly restrictive permeability to hydrophobic antitumor agents. So far, this multidrug resistance has been almost exclusively attributed to the most prominent member of the ATP-binding cassette (ABC) transporter family, P-glycoprotein located in the luminal membrane of brain capillary endothelial cells and to a minor extent to the multidrug resistance-associated proteins (MRPs). The brain multidrug resistance protein (BMDP) has recently been discovered at the porcine BBB and was shown to be highly homologous to the human breast cancer resistance protein (BCRP/ABCG2). Here, we demonstrate by northern blot and RT-PCR analysis that BMDP mRNA is more highly expressed in the capillary endothelial cells compared to other cell types of the brain. Immunocytochemistry of porcine BCEC showed a clear plasma membrane localisation of BMDP. Analysis of the total mRNA pool revealed that BMDP is more strongly expressed than P-glycoprotein and MRP1. Consistently, first transport studies indicate that active exclusion of the chemotherapeutic drug daunorubicin from the central nervous system is mediated mainly by this new transporter compared to P-glycoprotein or MRP1. Thus, we hypothesise that BMDP might play an important role in the exclusion of xenobiotics from the porcine brain.

Polyunsaturated fatty acids induce tight junctions to form in brain capillary endothelial cells

Tight junctions create a rate-limiting barrier to the diffusion of solutes between vertebrate epithelial cells and endothelial cells. They are also controlled within individual cells by a variety of physiologically relevant signals. We investigated the effects of polyunsaturated fatty acids on the formation of tight junctions in brain capillary endothelial cells, monitoring the transepithelial electrical resistance, and analyzed the expression of occludin messenger RNA. Brain-capillary endothelial cells were grown to confluence on filters and exposed to eicosapentaenoic acids, gamma linolenic acid and linoleic acid. Transepithelial electrical resistance was determined with voltage-measuring electrodes. The messenger RNA expression of occludin was quantitated by real-time quantitative reverse transcriptase–polymerase chain reaction. The basal resistance across monolayers of porcine brain capillary endothelial cells was 83±8.1 Ω cm 2. Cells cultured in eicosapentaenoic acids and gamma linolenic acid, but not linolenic acid, displayed a 2.7-fold increase in transepithelial electrical resistance at 10 μM in brain capillary endothelial cells. The expression level of occludin messenger RNA increased markedly immediately after the exposure to eicosapentaenoic acids or gamma linolenic acid. Following an 8 h exposure to exogenous eicosapentaenoic acids or gamma linolenic acid, occludin messenger RNA levels were significantly increased. In addition, the rise in transepithelial electrical resistance induced by eicosapentaenoic acids and gamma linolenic acid was markedly inhibited by the tyrosine kinase inhibitors genistein and PP2 and protein kinase C inhibitor, calphostin C. In contrast, the rise in transepithelial electrical resistance induced by eicosapentaenoic acids and gamma linolenic acid was not inhibited by the PI 3-kinase inhibitor, LY294002. We conclude that eicosapentaenoic acids and gamma linolenic acid increased the transepithelial electrical resistance and the expression of occludin messenger RNA in brain capillary endothelial cells. This gamma linolenic acid and eicosapentaenoic acid induced assembly of tight junction is likely to be regulated by protein kinase C and tyrosine kinase activity.

Inhibition of P-Glycoprotein by Newer Antidepressants

Pharmacokinetic drug-drug interactions often occur at the level of P-glycoprotein (Pgp). To study possible interactions caused by the newer antidepressants we investigated citalopram, fluoxetine, fluvoxamine, paroxetine, reboxetine, sertraline, and venlafaxine and their major metabolites desmethylcitalopram, norfluoxetine, paroxetine-metabolite (paroxetine-M), desmethylsertraline, N-desmethylvenlafaxine, and O-desmethylvenlafaxine for their ability to inhibit Pgp. Pgp inhibition was studied by a fluorometric assay using calcein-acetoxymethylester as Pgp substrate and two different cell systems: L-MDR1 cells (model for human Pgp) and primary porcine brain capillary endothelial cells (pBCECs, model for the blood-brain barrier). Both cell systems proved to be suitable for the evaluation of Pgp inhibitory potency of drugs. All antidepressants tested except O-desmethylvenlafaxine showed Pgp inhibitory activity with sertraline, desmethylsertraline, and paroxetine being the most potent, comparable with the well known Pgp inhibitor quinidine. In L-MDR1 cells fluoxetine, norfluoxetine, fluvoxamine, reboxetine, and paroxetine-M revealed intermediate Pgp inhibition and citalopram, desmethylcitalopram, venlafaxine, and N-desmethylvenlafaxine were only weak inhibitors. The ranking order was similar in pBCECs. The fact that some of the compounds tested exert Pgp inhibitor effects at similar concentrations as quinidine suggests that pharmacokinetic drug-drug interactions between the newer antidepressants and Pgp substrates should now be thoroughly studied in vivo.

Compound profiling for P-glycoprotein at the blood-brain barrier using a microplate screening system.

The purpose of this study was to establish a fluorescent dye (calcein-acetoxymethylester; calcein-AM)-based assay to rapidly screen compounds for interactions with p-glycoprotein (p-gp) at the blood-brain barrier and to determine whether such an assay can be useful for kinetic analysis. Porcine brain capillary endothelial cells (PBCECs) were isolated and cultured in 96-well plates. Cells were incubated with calcein-AM in the absence and presence of substrates and inhibitors of ABC transporters and the extent of intracellularly appearing fluorescence was monitored with a fluorescence plate reader in a time-and a concentration-dependent manner. PBCECs showed stable expression of p-gp and as a result calcein-AM was extruded by the cells. In the presence of p-gp substrates and inhibitors a significant increase of intracellular fluorescence was observed (decreased calcein-AM efflux), the increase being well correlated with the p-gp affinity of the compounds used. Inhibitors of Mrpl and Mrp2 did not influence fluorescence intensity. Time-dependent readouts and Michaelis-Menten kinetic analysis separated inhibitors into those showing competitive, mixed and non-competitive inhibition of p-glycoprotein-mediated transport. The calcein-AM-assay based on PBCECs can be used as a rapid microplate screening system for interactions of drugs with p-glycoprotein at the blood-brain barrier and represents therefore a useful tool in the profiling of drugs. In addition, convenient kinetic assays can provide information about the mode of interaction.