Inhibition of calcium-independent luminal uptake of L-dopa by calmodulin antagonists in immortalized rat capillary cerebral endothelial cells

The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca(2+)/calmodulin mediated pathways on the luminal uptake of L-DOPA through the L-type amino acid transporter in immortalized rat capillary cerebral endothelial (REB-4) cells. Non-linear analysis of the saturation curve for L-DOPA revealed a K(m)value (in microM) of 71+/-9 and a V(max)value of 17+/-1 (in nmol mg protein/6 min). L-DOPA uptake at the luminal cell border was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 m m), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC(50)=140 microM). The Ca(2+)/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC(50)s of 23 and 33 microM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutyrate, staurosporine and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 microM) concentration of L-DOPA. It is concluded that L-DOPA uptake in RBE-4 cells is promoted through the L-type amino acid transporter and appears to be under the control of calmodulin mediated pathways.     

Regulation of P-glycoprotein by orphan nuclear receptors in human brain microvessel endothelial cells

In mammalian systems, pregnane X receptor (PXR) and constitutive androstane receptor (CAR) have been recognized as xenobiotic-sensors which can up-regulate the functional expression of drug transporters, such as P-glycoprotein (P-gp). In the brain, an increase in P-gp expression can further limit drug permeability across the blood-brain barrier (BBB) and potentially reduce CNS pharmacotherapy efficacy. At present, the involvement of human PXR (hPXR) and CAR (hCAR) in the regulation of P-gp expression at the human BBB is unknown. In this study, we investigate the role of hPXR and hCAR in the regulation of P-gp expression using a human cerebral microvessel endothelial cell culture system. We demonstrate that activation of hPXR and hCAR by their respective ligands leads to P-gp induction at both mRNA and protein levels, while pharmacological inhibitors of hPXR and hCAR prevent ligand-mediated P-gp induction. Ligand-induced nuclear translocation of hPXR is observed, although such effect could not be demonstrated for hCAR. Furthermore, down-regulation of hPXR and hCAR proteins using small-interfering RNA decreased P-gp expression. Our findings provide first evidence for P-gp regulation by hPXR and hCAR at the human BBB and suggest insights on how to achieve selective P-gp regulation at this site.     

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.     

On the mechanism of oleate transport across human brain microvessel endothelial cells

The blood–brain barrier formed by the brain capillary endothelial cells provides a protective barrier between the systemic blood and the extracellular environment of the CNS. As most fatty acids in the brain enter from the blood, we examined the mechanism of oleate (C18:1) transport across primary human brain microvessel endothelial cells (HBMEC). The permeability of [1-¹⁴C]oleate was determined using confluent cells grown on Transwell® inserts in both the absence or presence of bovine serum albumin in the basolateral media, and following inhibition of various fatty acid transporters. The passage of [1-¹⁴C]oleate across confluent HBMEC monolayers was significantly enhanced when fatty acid free albumin was present in the basolateral media. The presence of the non-specific fatty acid uptake inhibitor phloretin significantly decreased [1-¹⁴C]oleate uptake by HBMEC and the subsequent release of [1-¹⁴C]oleate into the basolateral medium. Knockdown of fatty acid transport protein-1 or fatty acid translocase/CD36 significantly decreased [1-¹⁴C]oleate transport across the HBMEC monolayer from either apical as well as basolateral sides. The findings indicate that a fatty acid acceptor is a requirement for oleate transport across HBMEC monolayers. In addition, transport of oleate across HBMEC is, in part, a transcellular process mediated by fatty acid transport proteins.     

Modulation of uptake of organic cationic drugs in cultured human colon adenocarcinoma Caco-2 cells by an ecto-alkaline phosphatase activity

Alkaline phosphatase (ALP) refers to a group of nonspecific phosphomonoesterases located primarily in cell plasma membrane. It has been described in different cell lines that ecto-ALP is directly or indirectly involved in the modulation of organic cation transport. We aimed to investigate, in Caco-2 cells, a putative modulation of 1-methyl-4-phenylpyridinium (MPP(+)) apical uptake by an ecto-ALP activity. Ecto-ALP activity and (3)H-MPP(+) uptake were evaluated in intact Caco-2 cells (human colon adenocarcinoma cell line), in the absence and presence of a series of drugs. The activity of membrane-bound ecto-ALP expressed on the apical surface of Caco-2 cells was studied at physiological pH using p-nitrophenylphosphate as substrate. The results showed that Caco-2 cells express ALP activity, characterized by an ecto-oriented active site functional at physiological pH. Genistein (250 micro M), 3-isobutyl-1-methylxanthine (1 mM), verapamil (100 micro M), and ascorbic acid (1 mM) significantly increased ecto-ALP activity and decreased (3)H-MPP(+) apical transport in this cell line. Orthovanadate (100 micro M) showed no effect on (3)H-MPP(+) transport and on ecto-ALP activity. On the other hand, okadaic acid (310 nM) and all trans-retinoic acid (1 micro M) significantly increased (3)H-MPP(+) uptake and inhibited ecto-ALP activity. There is a negative correlation between the effect of drugs upon ecto-ALP activity and (3)H-MPP(+) apical transport (r = -0.9; P = 0.0014). We suggest that apical uptake of organic cations in Caco-2 cells is affected by phosphorylation/dephosphorylation mechanisms, and that ecto-ALP activity may be involved in this process.     

Up-regulation of P-glycoprotein expression by glutathione depletion-induced oxidative stress in rat brain microvessel endothelial cells

Glutathione (GSH) depletion has been implicated in the pathogenesis of neurological diseases. During GSH depletion, cells of the blood-brain barrier (BBB) are subjected to chronic oxidative stress. In this study, we investigated the effect of such stress, produced with the GSH synthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO), on expression of P-glycoprotein (Pgp) in primary cultured rat brain microvessel endothelial cells that comprise the blood-brain barrier (BBB). Application of BSO to cell monolayers at concentrations up to 800 microm caused increases in expression of Pgp. Concentrations >or= 400 microm BSO decreased cell viability. Application of 200 microm BSO caused a significant increase in Pgp function activity, as assessed by rhodamine 123 (Rh123) accumulation experiments. At this concentration, BSO produced time-dependent decreases in levels of intracellular GSH and increases in levels of intracellular reactive oxygen species (iROS). The increases were also observed within 48 h following BSO treatment in mdr1a and mdr1b mRNA. Exposure of cells to BSO for 24 h produced maximal effects in the accumulation of iROS, and in expression and function of Pgp. The ROS scavenger N-acetylcysteine prevented ROS generation and attenuated the changes of both expression and activity of Pgp induced by BSO. Therefore, the transport of Pgp substrates may be affected by changing Pgp expression under conditions of chronic oxidative stress induced by GSH depletion.     

Glutamate up-regulates P-glycoprotein expression in rat brain microvessel endothelial cells by an NMDA receptor-mediated mechanism

The accumulation of glutamate in the extracellular space in the central nervous system (CNS) plays a major part in ischemic and anoxic damage. In this study, we examined the effect of glutamate on the expression and activity of P-glycoprotein (P-gp) in rat brain microvessel endothelial cells (RBMECs) making up the blood-brain barrier (BBB). The level of P-gp expression significantly increased in RBMECs after the treatment of 100 microM glutamate. At this concentration, glutamate also enhanced rat mdr1a and mdr1b mRNA levels determined by RT-PCR analysis. Flow cytometry was used to study P-gp activity by analysis of intracellular rhodamine123 (Rh123) accumulation. Overexpression of P-gp resulted in a decreased intracellular accumulation of Rh123 in RBMECs. Glutamate-induced increase of intracellular reactive oxygen species (ROS) was observed by using the 2',7'-dichlorofluorescein (2',7'-DCF) assay. MK-801, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, and ROS scavenger N-acetylcysteine obviously blocked ROS generation and attenuated the changes of both expression and activity of P-gp induced by glutamate in RBMECs. These data suggested that glutamate up-regulated P-gp expression in RBMECs by an NMDA receptor-mediated mechanism and that glutamate-induced generation of ROS was linked to the regulation of P-gp expression. Therefore, transport of P-gp substrates in BBB appears to be affected during ischemic and anoxic injury.     

Brain capillary endothelial cells mediate iron transport into the brain by segregating iron from transferrin without the involvement of divalent metal transporter 1

Rats were studied for [(59)Fe-(125)I]transferrin uptake in total brain, and fractions containing brain capillary endothelial cells (BCECs) or neurons and glia. (59)Fe was transported through BCECs, whereas evidence of similar transport of transferrin was questionable. Intravenously injected transferrin localized to BCECs and failed to accumulate within neurons, except near the ventricles. No significant difference in [(125)I]transferrin distribution was observed between Belgrade b/b rats with a mutation in divalent metal transporter I (DMT1), and Belgrade +/b rats with regard to accumulation in vascular and postvascular compartments. (59)Fe occurred in significantly lower amounts in the postvascular compartment in Belgrade b/b rats, indicating impaired iron uptake by transferrin receptor and DMT1-expressing neurons. Immunoprecipitation with transferrin antibodies on brains from Belgrade rats revealed lower uptake of transferrin-bound (59)Fe. In postnatal (P)0 rats, less (59)Fe was transported into the postvascular compartment than at later ages, suggesting that BCECs accumulate iron at P0. Supporting this notion, an in situ perfusion technique revealed that BCECs accumulated ferrous and ferric iron only at P0. However, BCECs at P0 together with those of older age lacked DMT1. In conclusion, BCECs probably mediate iron transport into the brain by segregating iron from transferrin without involvement of DMT1.     

Restricted transport of anti-transferrin receptor antibody (OX26) through the blood-brain barrier in the rat

Anti-transferrin receptor IgG2a (OX26) transport into the brain was studied in rats. Uptake of OX26 in brain capillary endothelial cells (BCECs) was > 10-fold higher than isotypic, non-immune IgG2a (Ni-IgG2a) when expressed as % ID/g. Accumulation of OX26 in the brain was higher in 15 postnatal (P)-day-old rats than in P0 and adult (P70) rats. Iron-deficiency did not increase OX26 uptake in P15 rats. Three attempts were made to investigate transport from BCECs further into the brain. (i) Using a brain capillary depletion technique, 6-9% of OX26 was identified in the post-capillary compartment consisting of brain parenchyma minus BCECs. (ii) In cisternal CSF, the volume of distribution of OX26 was higher than for Ni-IgG2a when corrected for plasma concentration. (iii) Immunohistochemical mapping revealed the presence of OX26 almost exclusively in BCECs; extravascular staining was observed only in neurons situated periventricularly. The data support the hypothesis of facilitated uptake of OX26 due to the presence of transferrin receptors at the blood-brain barrier (BBB). However, OX26 accumulation in the post-capillary compartment was too small to justify a conclusion of receptor-mediated transcytosis of OX26 occurring in BCECs. Accumulation of OX26 in the post-capillary component may result from a diphasic transport that involves high-affinity accumulation of OX26 by the BCECs, clearly exceeding that of Ni-IgG2a, followed by a second transport mechanism that releases OX26 non-specifically further into the brain. The periventricular localization suggests that OX26 probably also derives from transport across the blood-CSF barrier.     

Growth of brain microvessel endothelial cells on collagen gels: Applications to the study of blood-brain barrier physiology and CNS inflammation

Summary Brain microvessel endothelial cells (BMEC) exhibit the tendency to migrate through 3.0-vm pore semipermeable inserts and establish monolayers on both apical and basal filter surfaces. This can potentially lead to complications in accurately assessing a wide variety of physiologic parameters uniquely associated with these cells. To avoid this problem, we have explored growing BMEC on Transwell filters coated with hydrated collagen gels. BMEC seeded on such gels grow as a monolayer until confluency, but do not invade the subendothelial collagen matrix or the underlying support filter. Furthermore, BMEC grown in this manner exhibit biochemical, morphologic, and electrophysiologic properties reflective of the endothelial cells that comprise the blood-brain barrier in vivo. Although the collagen gel acts as an impenetrable barrier to BMEC, and thus ensures the growth of only a single layer of cells, it nevertheless can be infiltrated by monocytes that have been stimulated by a chemotaxin to undergo diapedesis. Thus, growing BMEC on collagen gel-coated Transwells has broad applications for the in vitro study of both blood-brain barrier physiology as well as the mechanisms underlying central nervous system inflammation.     

Transcellular transport of CCL2 across brain microvascular endothelial cells

The means by which the chemokine CCL2 produced in the brain parenchyma can recruit leukocytes lying behind the highly impervious endothelium of the blood–brain barrier (BBB) has remained a paradox. As other chemokines have been evidenced to stimulate their own synthesis and release by peripheral microvascular endothelial cells, and/or undergo transcytosis in the abluminal-to-luminal direction, we determined whether CCL2 experiences similar fates across brain microvascular endothelial cells (BMEC). Using cultured BMEC as a paradigm of the BBB, it was observed that exogenous unlabeled CCL2 actually depressed the release of endogenous CCL2, and further caused diminished CCL2 mRNA levels in these cells. On the other hand, exogenous ¹²⁵I-labeled CCL2 exhibited transport across BMEC in a manner that was sensitive to temperature, competition by excess unlabeled CCL2 but not unlabeled CCL3, knockdown of caveolin-1/caveolae, and elimination of the cognate CCL2 receptor CCR2. These results implied a facet of CCL2 transport by a transcellular mechanism partly involving binding of CCL2 to CCR2, and subsequent transfer to caveolae vesicles for transcytosis. This notion was supported by double-label immuno-electronmicroscopy, which revealed co-localization of caveolin-1 with exogenous CCL2, during this chemokine's transit across BMEC. Collectively, these findings provide a rationale by which CCL2, deposited on the abluminal side of the brain microvasculature during inflammatory episodes, can be relayed across the BBB to foster leukocyte recruitment.     

The blood-brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells

Antibodies against receptors that undergo transcytosis across the blood-brain barrier (BBB) have been used as vectors to target drugs or therapeutic peptides into the brain. We have recently discovered a novel single domain antibody, FC5, which transmigrates across human cerebral endothelial cells in vitro and the BBB in vivo. The purpose of this study was to characterize mechanisms of FC5 endocytosis and transcytosis across the BBB and its putative receptor on human brain endothelial cells. The transport of FC5 across human brain endothelial cells was polarized, charge independent and temperature dependent, suggesting a receptor-mediated process. FC5 taken up by human brain endothelial cells co-localized with clathrin but not with caveolin-1 by immunochemistry and was detected in clathrin-enriched subcellular fractions by western blot. The transendothelial migration of FC5 was reduced by inhibitors of clathrin-mediated endocytosis, K+ depletion and chlorpromazine, but was insensitive to caveolae inhibitors, filipin, nystatin or methyl-beta-cyclodextrin. Following internalization, FC5 was targeted to early endosomes, bypassed late endosomes/lysosomes and remained intact after transcytosis. The transcytosis process was inhibited by agents that affect actin cytoskeleton or intracellular signaling through PI3-kinase. Pretreatment of human brain endothelial cells with wheatgerm agglutinin, sialic acid, alpha(2,3)-neuraminidase or Maackia amurensis agglutinin that recognizes alpha(2,3)-, but not with Sambucus nigra agglutinin that recognizes alpha(2,6) sialylgalactosyl residues, significantly reduced FC5 transcytosis. FC5 failed to recognize brain endothelial cells-derived lipids, suggesting that it binds luminal alpha(2,3)-sialoglycoprotein receptor which triggers clathrin-mediated endocytosis. This putative receptor may be a new target for developing brain-targeting drug delivery vectors.     

Quantitative detection of blood-brain barrier-associated enzymes in cultured endothelial cells of procine brain microvessels

Summary The present study deals with a rapid and convenient assay for blood-brain barrier (BBB)-associated enzymes, γ-glutamyl transpeptidase (γ-GTP) and alkaline phosphatase (ALP), in cultured endothelial cells and other cells. These enzyme activities in cultured cells could be efficiently measured by direct incubation of each substrate in the culture plates without pretreatment of the cells. This new direct in situ-in plate assay was more rapid and convenient than conventional ex-plate assays, and these assays gave similar values for specific enzyme activities. γ-GTP and ALP activities could be detected by this in situ method in primary-cultured endothelial cells of porcine brain microvessels, but their levels were lower than those before culture. The degree of loss due to culture differed, between γ-GTP and ALP; a relatively large amount of ALP remained but the γ-GTP level decreased greatly In this direct in situ-in plate assay, cultured porcine aortic endothelial cells exhibited negligibly small activities for both enzymes, whereas cultured astroglial cells of neonatal porcine brain showed moderate γ-GTP activity and a trace of ALP activity. This direct in situ-in plate assay can be used for microculture and automatic measurement and offers a convenient means for studying the possible regulatory mechanisms of the expression of the BBB-associated enzymes.     

Bradykinin receptor‐1 activation induces inflammation and increases the permeability of human brain microvascular endothelial cells

Neuroinflammatory disorders such as Alzheimer's and Parkinson's diseases are characterised by chronic inflammation and loss of vascular integrity. Bradykinin 1 receptor (B1R) activation has been implicated in many neuroinflammatory diseases, but the contribution of B1R to inflammation and vascular breakdown is yet to be determined. As a result, the present study evaluated the effect of B1R stimulation using Des‐Arg‐9‐BK on the cytokine profile and junctional properties of human cerebral microvascular endothelial cells (hCMVECs). Results showed that stimulation of B1R receptors increased secretion of pro‐inflammatory cytokines, interleukin‐6 (IL‐6), IL‐8, intracellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1) and monocyte chemoattractant protein‐1 (MCP‐1), but decreased the expression of vascular endothelial growth factor (VEGF), a cytokine and growth factor required for maintenance of the vasculature. B1R stimulation also resulted in the loss of occludin expression at tight junctions with no change in VE‐cadherin expression. There was also a significant increase in permeability to Evans blue albumin, suggesting an increase of vascular permeability. Taken together, these results suggest that B1R activation that occurs in neuroinflammatory diseases may contribute to both the inflammation and loss of blood‐brain barrier integrity that is characteristic of these diseases.     

Modulation of P-glycoprotein function by sphingosine kinase-1 in brain endothelial cells

P-glycoprotein (P-gp), an ABC-transporter highly expressed in brain capillaries, protects the brain by extruding xenobiotics. However, its overexpression has also been associated with the multidrug resistance phenotype in tumors. Here, we have investigated the regulation of P-gp transport activity by sphingosine kinase 1 (SphK-1) in brain endothelial cells. We first demonstrated that SphK-1 is overexpressed in endothelial cells (EC) isolated from rat brain tumors compared with EC from normal brain. We also provide evidence that the overexpression of SphK-1 in the cerebral EC line RBE4 leads to the up-regulation of P-gp, both at the gene and protein levels, and that this modulation depends on the catalytic activity of SphK-1. Moreover, we determined the effect of sphingosine-1-phosphate (S1P), the product of SphK-1, on P-gp function. S1P strongly stimulates P-gp transport activity, without modulating its expression. Finally, we found that the S1P-mediated stimulation of P-gp activity is mediated by S1P-1 and S1P-3 receptors at the RBE4 cell surface. Altogether, these results indicate that SphK-1 and its product S1P are involved in the control of P-gp activity in RBE4 cells. Since SphK-1 is overexpressed in EC from brain tumors, these data also suggest that this kinase and its product could contribute to the acquisition and the maintenance of the multidrug resistance phenotype in brain tumor-derived endothelial cells.     

Functional expression of the serotonin transporter in immortalized rat brain microvessel endothelial cells

There is evidence from recent studies that the brain endothelium (of capillaries and/or larger vessels) may serve as a specific target for serotonin [5-hydroxytryptamine (5-HT)]. This neurotransmitter is expected to be involved in the regulation of the blood-brain barrier (BBB) permeability and/or of the cerebral blood flow via receptor-mediated mechanisms. Effective control of these processes depends on a speedy uptake and metabolism of released 5-HT molecules. To realize this, a similar mechanism of 5-HT uptake as in brain may exist at the BBB. In this study, we have demonstrated using RT-PCR that 5-HT transporter mRNA is present in the brain endothelium and that a saturable transport system for 5-HT is functionally expressed in immortalized rat brain endothelial cells (RBE4 cells). These cells take up [3H]5-HT by an active saturable process with a Km value of 397 +/- 64 nmol/L and a transport capacity of 51.7 +/- 3.5 pmol x g(-1) x min(-1). The 5-HT uptake depends on Na+, as indicated by the replacement of NaCl by LiCl. The 5-HT uptake was sensitive to specific 5-HT transport inhibitors such as paroxetine, clomipramine, fluoxetine, and citalopram but not to inhibitors of the vesicular amine transporter such as reserpine or tetrabenazine. Our results demonstrate that cerebral endothelial cells are able to participate actively in the removal and metabolism of the released 5-HT, which supports the concept of direct serotoninergic regulation of the BBB function.     

Biochemical characteristics of primary and passaged cultures of primate brain microvessel endothelial cells

Rhesus macaque monkey brain microvessel endothelial cells (BMECs) were isolated and grown in culture in an effort to establish an appropriate primate in vitro model of the endothelial component of the blood-brain barrier. The presence of Factor VIII antigen, alkaline phosphatase, -glutamyl transpeptidase, lactate dehydrogenanse, total protein, and the passive permeability properties was documented for both primary and passaged cultures. Primate BMECs were shown to exhibit similar morphological and biochemical properties described for other BMEC culture systems derived from other species. In addition, the passaged primate BMECs were particularly notable for the changes in enzyme activities and total protein that parallel age-dependent changes in brain capillary endothelia. This study provides further support for the possible application of BMEC culture systems in investigations of blood-brain barrier functions under normal, aging, and diseased conditions.To whom to address reprint requests. Phone (913)864-3609; FAX (913)864-3578.