Puromycin selectively increases mdr1a expression in immortalized rat brain endothelial cell lines

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from blood to brain extracellular fluid. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain endothelium and contributes to the formation of a functional barrier to lipid-soluble drugs such as anticancer agents. The mdr1a P-gp-encoding gene is exclusively expressed in the rodent BBB. Primary cultures of rat brain endothelial cells and GP8.3 cells showed a dramatic decrease in mdr1a mRNA level and some expression of mdr1b mRNA. GPNT cells, derived from GP8.3 cells after transfection with a puromycin resistance gene, were chronically treated with 5 microg/mL puromycin, a P-gp substrate. Compared with rat brain endothelial cells and GP8.3 cells, GPNT cells exhibited a very high level of expression of mdr1a mRNA together with a moderate level of mdr1b mRNA expression. Accordingly, P-gp expression and activity were strongly increased. When GP8.3 and puromycin-starved GPNT cells were treated with puromycin, mdr1a expression was selectively increased. High expression of mdr1a mRNA in GPNT cells may thus be related to the chronic treatment with puromycin. We conclude that GPNT cells may be used as a valuable rat in vitro model for studying the regulation of mdr1a expression at the BBB level.     

Functional expression of the P-glycoproteinmdr in primary cultures of bovine cerebral capillary endothelial cells

The P-glycoproteinmdr is expressed not only in tumoral cells, but also in nontransformed cells, including the specialized endothelial cells of brain capillaries which build up the blood-brain barrier. Since all previously identified blood-brain barrier markers are rapidly lost when cerebral capillary endothelial cells are maintained in primary culture, we have investigated whether P-glycoprotein (P-gp) would follow the same rule, in order to address the influence of the cerebral environment on the specific P-gp expression in the brain endothelium. As compared to freshly isolated purified cerebral capillaries, P-glycoprotein was detected by immunochemistry at a high level in 5–7 day primary cultures. In our culture conditions, P-glycoprotein was immunodetected at a lower molecular weight than that found in freshly isolated capillaries. Enzymatic deglycosylation led to the same 130 kDa protein for both fresh and cultured samples, suggesting that P-gp post-translational modifications were altered in primary cultures. However, studies on the uptake and efflux of the P-gp substrate [³H]vinblastine, and on the effect of variousmdr reversing agents on the uptake and efflux, clearly indicated that the efflux pump function of the P-glycoprotein was maintained in primary cultures of bovine cerebral capillary endothelial cells. P-Glycoprotein may thus represent the first blood-brain barrier marker which is maintained in cerebral endothelial cells cultured in the absence of factors originating from the brain parenchyma.Abbreviations BBB blood-brain barrier - BCEC brain capillary endothelial cells - -GT -glutamyltranspeptidase - HBSS Hank's balanced salt solution - Mab monoclonal antibody - mdr multidrug resistance - P-gp P-glycoprotein     

Multidrug Resistance-Related Transport Proteins in Isolated Human Brain Microvessels and in Cells Cultured from These Isolates

Abstract: The multidrug transporter, P-glycoprotein (Pgp), at the blood-brain barrier is thought to be important for limiting access of toxic agents to the brain, but controversy surrounds its cellular location, whether on endothelium or on adjacent astrocyte foot processes. In the present study, the distribution of protein and mRNA for Pgp and for another transporter, multidrug resistance-associated protein (MRP), is compared with that for the endothelial marker, platelet-endothelial cell adhesion molecule-1 (PECAM-1) and for the astrocyte-derived glial fibrillary acidic protein (GFAP) in microvessels isolated from human brain and in cells grown from these microvessels. Activities of the multidrug transporters are assessed in the cultured cells from the effects of transport inhibitors on intracellular [³H]vincristine accumulation. The isolated microvessels show strong immunocytochemical staining for Pgp and PECAM-1 and little or no staining for GFAP and MRP, and they contain mRNAs detectable by RT-PCR encoding only Pgp and PECAM-1, but not GFAP or MRP. Thus, Pgp may well be synthesised and expressed on cells within the microvessels rather than on adherent astrocyte foot processes. In cells grown from the microvessels, although PECAM-1 remains, Pgp expression decreases and MRP appears. Evidence suggests these multidrug transporters are functionally active in the cultured cells.     

Effect of chronic exposure to morphine on the rat blood-brain barrier: focus on the P-glycoprotein

Morphine may affect the properties of the blood-brain barrier (BBB) by modifying the expression of certain BBB markers. We have determined the effect of chronic morphine treatment on the expression and function of some BBB markers in the rat. The mRNAs of 19 selected genes encoding caveolins, endothelial transporters, receptors and tight junctions proteins in the total RNA of isolated cortex microvessels were assayed by quantitative RT-PCR (qRT-PCR). The expression of genes Mdr1a, Mrp1, Bcrp, Glut-1 and Occludin, was slightly increased, while that of Flk-1 was decreased in microvessels from morphine-treated rats. The expression of the Mrd1a and Mdr1b genes encoding the P-glycoprotein (P-gp) also increased in the whole hippocampus and cortex of morphine-treated rats. The Mdr1a gene induction (1.38-fold) observed by qRT-PCR was also confirmed using in situ hybridization technique (1.40-fold). Immunoblotting revealed an increase in P-gp expression in the hippocampus (1.8-fold) and cortex (1.36-fold) of morphine-treated rats, but no effect in isolated microvessels. In contrast, morphine treatment increased by 1.48-fold the expression of P-gp in a large vessel-enriched fraction. The integrity of the BBB, measured by in situ brain perfusion of [(14)C]-sucrose, and the activity of P-gp at the BBB, measured with the P-gp substrate [(3)H]-colchicine, were not modified by morphine. Immunohistofluorescence experiments revealed that P-gp expression is restricted to large vessels and microvessels in control rats and that morphine treatment did not induce the expression of P-gp in the brain parenchyma (astrocytes or neurons). Taken together, our results showed that chronic morphine treatment does not significantly alter BBB integrity or P-gp activity. The impact of morphine-mediated P-gp induction observed in large vessels remains to be determined in terms of brain disposition of drugs that are P-gp substrates.     

Dexamethasone Regulation of P-Glycoprotein Activity in an Immortalized Rat Brain Endothelial Cell Line, GPNT

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain capillary endothelial cells, thus forming a functional barrier to lipid-soluble drugs, notably, antitumor agents. It is of interest to develop an in vitro BBB model that stably expresses P-gp to investigate the mechanisms of regulation in expression and activity. The rat brain endothelial cell line, GPNT, was derived from a previously characterized rat brain endothelial cell line. A strong expression of P-gp was found in GPNT monocultures, whereas the multidrug resistance-associated pump Mrp1 was not expressed. The transendothelial permeability coefficient of the P-gp substrate vincristine across GPNT monolayers was close to the permeability coefficient of bovine brain endothelial cells cocultured with astrocytes, a previously documented in vitro BBB model. Furthermore, the P-gp blocker cyclosporin A induced a large increase in apical to basal permeability of vincristine. Thus, P-gp is highly functional in GPNT cells. A 1-h treatment of GPNT cells with dexamethasone resulted in decreased uptake of vincristine without any increase in P-gp expression. This effect could be mimicked by protein kinase C (PKC) activation and prevented by PKC inhibition, strongly suggesting that activation of P-gp function may involve a PKC-dependent pathway. These results document the GPNT cell line as a valuable in vitro model for studying drug transport and P-gp function at the BBB and suggest that activation of P-gp activity at the BBB might be considered in chemotherapeutic treatment of cancer patients.     

Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells

Rat brain microvessel endothelial cells were immortalized by transfection with a plasmid containing the E1A adenovirus gene. One clone, called RBE4, was further characterized. These cells display a nontransformed phenotype and express typical endothelial markers, Factor VIII-related antigen and Bandeiraea simplicifolia binding sites. When RBE4 cells were grown in the presence of bFGF and on collagen-coated dishes, confluent cultures developed sprouts that extend above the monolayer and organized into three-dimensional structures. The activity of the blood-brain barrier-associated enzyme, gamma-glutamyl transpeptidase (γGTP), was expressed in these structures, not in the surrounding monolayer. Similar results were obtained with the microvessel-related enzyme alkaline phosphatase (ALP). Addition of agents that elevate intracellular cAMP reduced the formation of three-dimensional structures, but every cell inside the aggregates still expressed γCTP and ALP activities. Such structures, associated with high levels of γCTP and ALP activities, were also induced by astroglial factors, including (1) plasma membranes from newborn rat primary astrocytes or rat glioma C6 cells, (2) C6 conditioned media, or (3) diffusible factors produced by primary astrocytes grown in the presence of, but not in contact with RBE4 cells. RBE4 cells thus remain sensitive to angiogenic and astroglial factors for the expression of the blood-brain barrier-related γCTP activity, as well as for ALP activity, and could constitute the basis of a valuable in vitro model of the blood-brain barrier. © 1994 wiley-Liss, Inc.     

Modulation of insulin transport in rat brain microvessel endothelial cells by an ecto-phosphatase activity.

The physiological function of alkaline phosphatase (ALP) remains controversial. It was recently suggested that this membrane-bound enzyme has a role in the modulation of transmembranar transport systems into hepatocytes and Caco-2 cells. ALP activity expressed on the apical surface of blood-brain barrier cells, and its relationship with (125)I-insulin internalization were investigated under physiological conditions using p-nitrophenylphosphate (p-NPP) as substrate. For this, an immortalized cell line of rat capillary cerebral endothelial cells (RBE4 cells) was used. ALP activity and (125)I-insulin internalization were evaluated in these cells. The results showed that RBE4 cells expressed ALP, characterized by an ecto-oriented active site which was functional at physiological pH. Orthovanadate (100 microM), an inhibitor of phosphatase activities, decreased both RBE4-ALP activity and (125)I-insulin internalization. In the presence of L-arginine (1 mM) or adenosine (100 microM) RBE4-ALP activity and (125)I-insulin, internalization were significantly reduced. However, D-arginine (1 mM) had no significant effect. Additionally, RBE4-ALP activity and (125)I-insulin internalization significantly increased in the presence of the bioflavonoid kaempferol (100 microM), of the phorbol ester PMA (80 nM), IBMX (1 mM), progesterone (200 microM and 100 microM), beta-estradiol (100 microM), iron (100 microM) or in the presence of all-trans retinoic acid (RA) (10 microM). The ALP inhibitor levamisole (500 microM) was able to reduce (125)I-insulin internalization to 69.1 +/- 7.1% of control. Our data showed a positive correlation between ecto-ALP activity and (125)I-insulin incorporation (r = 0.82; P < 0.0001) in cultured rat brain endothelial cells, suggesting that insulin entry into the blood-brain barrier may be modulated through ALP.     

Effects of LPS stimulation on the expression of prostaglandin carriers in the cells of the blood-brain and blood-cerebrospinal fluid barriers.

Prostaglandins produced in cerebral endothelial cells (CECs) are the final signal transduction mediators from the periphery to the brain during fever response. However, prostaglandins are organic anions at physiological pH, and they enter cells poorly using simple diffusion. Several transporters have been described that specifically transport prostaglandins across cell membranes. We examined the expression of the two principal prostaglandin carriers, prostaglandin transporter (PGT), and multidrug resistance-associated protein 4 (MRP4) in cells of the blood-brain barrier and in choroid epithelial cells in vitro as well as in vivo in rat brain in control conditions and after lipopolysaccharide (LPS) challenge. We detected PGT in primary cultures of rat CECs, astrocytes, pericytes, and choroid epithelial cells. LPS stimulation had no effect on the expression level of PGT in these cells; however, after LPS stimulation the polarized, dominantly luminal, expression pattern of PGT significantly changed. MRP4 is also expressed in CECs, and its level was not influenced by LPS treatment. In rat brain, PGT was highly expressed in the supraoptic and paraventricular nuclei of the hypothalamus, in the ependymal cell layer of the third ventricle, and in the choroid plexus. LPS treatment increased the expression of PGT in the supraoptic and paraventricular nuclei. Our results suggest that PGT and MRP4 likely play a role in transporting prostaglandins through the blood-brain and blood-cerebrospinal fluid barriers and may be involved in the maintenance of prostaglandin homeostasis in the brain and in the initiation of fever response.     

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.     

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.     

Regulation of brain endothelial cells migration and angiogenesis by P-glycoprotein/caveolin-1 interaction

We have investigated the involvement of P-glycoprotein (P-gp)/caveolin-1 interaction in the regulation of brain endothelial cells (EC) migration and tubulogenesis. P-gp overexpression in MDCK-MDR cells was correlated with enhanced cell migration whereas treatment with P-gp inhibitors CsA or PSC833 reduced it. Transfection of RBE4 rat brain endothelial cells with mutated versions of MDR1, in the caveolin-1 interaction motif, decreased the interaction between P-gp and caveolin-1, enhanced P-gp transport activity and cell migration. Moreover, down-regulation of caveolin-1 in RBE4 cells by siRNA against caveolin-1 stimulated cell migration. Interestingly, the inhibition of P-gp/caveolin-1 interaction increased also EC tubulogenesis. Furthermore, decrease of P-gp expression by siRNA inhibited EC tubulogenesis. These data indicate that the level of P-gp/caveolin-1 interaction can modulate brain endothelial angiogenesis and P-gp dependent cell migration.     

Expression of P-glycoprotein in human cerebral cortex microvessels.

P-Glycoprotein (P-gp) is an ATP-dependent efflux transporter that extrudes non-polar molecules, including cytotoxic substances and drugs, from the cells. It was initially found in cancer cells and then was shown to be a normal component of complex transport systems working at the blood-brain barrier (BBB). Previous studies have demonstrated that, in the brain, P-gp is localized on the luminal plasmalemma of BBB endothelial cells and that it may interact with the caveolar compartment of these cells. The aim of this study was to identify the site of cellular expression of P-gp in human brain in situ and to morphologically determine whether an association may exist between P-gp and caveolin-1, a structural and functional protein of the caveolar frame. The study was carried out on human cerebral cortex by immunoconfocal microscopy with antibodies to both P-gp and caveolin-1. The results show that P-gp marks the microvessels of the cortex and that the transporter is localized in the luminal endothelial compartment, where it co-localizes with caveolin-1. The demonstration of this co-localization of P-gp with caveolin-1 contributes a morphological backing to biochemical studies on P-gp/caveolin-1 relationships and leads us to suggest that interactions between these molecules may occur at the BBB endothelia.     

Effects of selected vasoactive substances on adenylate cyclase activity in brain,isolated brain microvessels,and primary cultures of brain microvessel endothelial cells

The specific activity of adenylate cyclase was assayed in homogenates of gray matter, freshly isolated and primary cultured microvessel endothelial cells from bovine cerebral cortex. Specific activities for the tissues were 14.6±2.1, 15.6±2.7, and 8.4±1.5 pmol cAMP/mg protein/min±SD for gray matter, cultured microvessels, and freshly isolated microvessels, respectively. Adenylate cyclase associated with gray matter and cultured microvessels was sensitive to histamine and selected catecholamines. Perhaps due to metabolic deficiencies, adenylate cyclase of freshly isolated microvessels exhibited little or no response to either the catecholamines or histamine. Angiotensin II stimulated adenylate cyclase of both freshly isolated and cultured microvessels but had no effect on gray matter. Bradykinin did not stimulate cAMP generation in any of the tissues. Overall results support the role of cAMP in regulating brain microvessel functions and suggest that primary cultures of brain microvessels may be useful in examining cAMP-mediated biochemical pathways at the blood-brain barrier.     

TNF activates P-glycoprotein in cerebral microvascular endothelial cells.

BACKGROUND/AIMS: Multidrug resistance proteins (MDRs, including P-glycoproteins) are efflux pumps that serve important biological functions but hinder successful drug delivery to the CNS. Many chemotherapeutic agents, anti-epileptics, anti-HIV drugs, and opiates are substrates for MDRs. Therefore, understanding the regulation of MDRs in the endothelial cells composing the blood-brain barrier has therapeutic implications. METHODS: We used microarray, real time RT-PCR, Western blotting, and uptake of vinblastine by RBE4 cerebral endothelial cells to test the effects of tumor necrosis factor alpha (TNF) on the expression and functions of P-glycoprotein (MDR1). RESULTS: The proinflammatory cytokine TNF specifically induced the expression and enhanced the function of MDR1 in RBE4 cells. The persistent upregulation of MDR1 mRNA was shown by cDNA microarray at 6, 12, and 24 h after TNF treatment. This was confirmed by real-time RT-PCR between 2 and 24 h. MDR1 protein expression was increased 6 to 24 h after TNF treatment and resulted in a significant reduction in the cellular uptake of (3)H-vinblastine. CONCLUSION: The drug efflux transporter in cerebral endothelial cells can be upregulated by TNF. This suggests that adjunctive anti-TNF treatment has novel therapeutic potential in conditions such as brain cancer, epilepsy, neuroAIDS, and chronic pain.