Brain Microvessel Endothelial Cells Research Articles

Generic fast gradient liquid chromatography/tandem mass spectrometry techniques for the assessment of the in vitro permeability across the blood-brain barrier in drug discovery.

Rapid, generic gradient liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays, designed to accelerate sample analyses, have been developed to keep pace with the productivity of advanced synthetic procedures. In this study, LC/MS/MS was combined with an in vitro, cell-based, blood-brain barrier (BBB) model to evaluate the potential of new chemical entities (NCEs) to cross the BBB. This in vitro assay provides the permeability of discovery compounds across a monolayer of a primary culture of bovine brain microvessel endothelial cells in a fraction of the time that is required for in vivo studies (brain/plasma concentrations), using only 2 mg of the compound. The results are consistent with in vivo brain/plasma concentration ratio data.

Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation.

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJ) that are critical for maintaining brain homeostasis and low permeability. Both integral (claudin-1 and occludin) and membrane-associated zonula occluden-1 and -2 (ZO-1 and ZO-2) proteins combine to form these TJ complexes that are anchored to the cytoskeletal architecture (actin). Disruptions of the BBB have been attributed to hypoxic conditions that occur with ischemic stroke, pathologies of decreased perfusion, and high-altitude exposure. The effects of hypoxia and posthypoxic reoxygenation in cerebral microvasculature and corresponding cellular mechanisms involved in disrupting the BBB remain unclear. This study examined hypoxia and posthypoxic reoxygenation effects on paracellular permeability and changes in actin and TJ proteins using primary bovine brain microvessel endothelial cells (BBMEC). Hypoxia induced a 2.6-fold increase in [(14)C]sucrose, a marker of paracellular permeability. This effect was significantly reduced (~58%) with posthypoxic reoxygenation. After hypoxia and posthypoxic reoxygenation, actin expression was increased (1.4- and 2.3-fold, respectively). Whereas little change was observed in TJ protein expression immediately after hypoxia, a twofold increase in expression was seen with posthypoxic reoxygenation. Furthermore, immunofluorescence studies showed alterations in occludin, ZO-1, and ZO-2 protein localization during hypoxia and posthypoxic reoxygenation that correlate with the observed changes in BBMEC permeability. The results of this study show hypoxia-induced changes in paracellular permeability may be due to perturbation of TJ complexes and that posthypoxic reoxygenation reverses these effects.

Regulation of [(3)H]MPP(+) transport by phosphorylation/dephosphorylation pathways in RBE4 cells: role of ecto-alkaline phosphatase.

The aim of this study was to investigate the role of phosphorylation/dephosphorylation mechanisms at the blood-brain barrier (BBB) in the uptake of organic cations. The experiments were performed using RBE4 cells, an immortalized, rat brain microvessel endothelial cell line, an in vitro model of the BBB. The modulation of the uptake of 1-methyl-4-phenylpyridinium (MPP(+)), a model organic cation, at the apical membrane of RBE4 cells was studied. Agents that stimulate protein kinase A, but not protein kinase C, produced a moderate inhibition (approximately 18% reduction) of uptake of [(3)H]MPP(+) by RBE4 cells. Okadaic acid, an inhibitor of protein serine/threonine phosphatase, did not affect uptake of (3)H-MPP(+), but the alkaline phosphatase (ALP) inhibitor levamisole markedly reduced (3)H-MPP(+) uptake. The activity of membrane-bound ALP expressed on the apical surface of RBE4 cells was studied at pH 7.4 using p-nitrophenylphosphate as substrate. Kaempferol, progesterone, 3-isobutyl-1-methylxanthine, all- trans-retinoic acid and iron stimulated ecto-ALP activity and uptake of [(3)H]MPP(+) in RBE4. Orthovanadate (a protein tyrosine phosphatase inhibitor) markedly inhibited both ecto-ALP activity and uptake of [(3)H]MPP(+) by RBE4 cells. In conclusion, these results suggest that apical transporter(s) of MPP(+) in RBE4 cells may be under the control of phosphorylation/dephosphorylation mechanisms, being active in the dephosphorylated state. A physiological role for ALP in the modulation of organic cation transport in the BBB is suggested.

Comparison of in vitro BBMEC permeability and in vivo CNS uptake by microdialysis sampling

The studies presented in this report were designed to assess the correlation of the bovine brain microvessel endothelial cell (BBMEC) apparent permeability coefficient ( P app) and in vivo BBB penetration using microdialysis sampling. A mathematical model was developed to describe the relationship of brain extracellular fluid (ECF) concentration to free drug in plasma. The compounds studied have a broad range of physico-chemical characteristics and have widely varying in vitro and in vivo permeability across the blood–brain barrier (BBB). BBMEC permeability coefficients vary in magnitude from a low of 0.9×10 −5 cm/s to a high value of 7.5×10 −5 cm/s. Corresponding in vivo measurements of BBB permeability are represented by clearance (CL in) into the brain ECF and range from a low of 0.023 μl/min/g to a high of 12.9 μl/min/g. While it is apparent that in vitro data from the BBMEC model can be predictive of the in vivo permeability of a compound across the BBB, there are numerous factors both prior to and following entry into the brain which impact the ultimate uptake of a compound. Even in the presence of high BBB permeability, factors such as high plasma protein binding, active efflux across the BBB, and metabolism within the CNS can greatly limit the ultimate concentrations achieved. In addition, concentrations in the intracellular space may not be the same as concentrations in the extracellular space. While these data show that the BBMEC permeability is predictive of the in vivo BBB permeability, the complexity of the living system makes prediction of brain concentrations difficult, based solely on the in vitro measurement.

Ultrasound-induced mild hyperthermia as a novel approach to increase drug uptake in brain microvessel endothelial cells.

Drug delivery to the central nervous system (CNS) is limited by the blood-brain barrier (BBB). Thus, a noninvasive and reversible method to enhance BBB permeation of drugs is highly desirable. In the present work, we studied if ultrasound-induced mild hyperthermia (USHT, 0.4 watts (W)/cm2 at 41 degrees C) can enhance drug absorption in BBB endothelial cells, and we elucidated the mechanism of USHT on cellular accumulation. To accomplish these aims, we studied the effects of hyperthermia (41 degrees C), USHT, P-glycoprotein (P-gp) modulator (PSC 833), and combination of USHT and PSC 833 on accumulation of P-gp substrate (R123) and non-P-gp substrates (sucrose, 2-deoxyglucose, and antipyrine) in monolayers of primary bovine brain microvessel endothelial cells (BBMEC). USHT, through its thermal effect, produces a significant (relative to controls; no USHT) and comparable increase in R123 accumulation with PSC 833. We also demonstrate that USHT increases permeability of hydrophobic (R123 and [14C]-antipyrine) and not hydrophilic molecules ([14C]-sucrose and 2-[3H]-deoxy-D-glucose). The enhanced permeability is reversible and size dependent, as USHT produces a much larger effect on cellular accumulation of [14C]-antitpyrine (molecular weight of 188 D) than that of R123 (molecular weight of 380.8 D). Although USHT increases membrane permeability, it did not affect P-gp activity or the activity of glucose transporters. Our results point to the potential use of USHT as a reversible and noninvasive approach to increase BBB permeation of hydrophobic drugs, including P-gp-recognized substrates.

Vascular endothelial cells express isoforms of protein kinase A inhibitor.

The expression and function of the endogenous inhibitor of cAMP-dependent protein kinase (PKI) in endothelial cells are unknown. In this study, overexpression of rabbit muscle PKI gene into endothelial cells inhibited the cAMP-mediated increase and exacerbated thrombin-induced decrease in endothelial barrier function. We investigated PKI expression in human pulmonary artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs). RT-PCR using specific primers for human PKI alpha, human PKI gamma, and mouse PKI beta sequences detected PKI alpha and PKI gamma mRNA in all three cell types. Sequencing and BLAST analysis indicated that forward and reverse DNA strands for PKI alpha and PKI gamma were of >96% identity with database sequences. RNase protection assays showed protection of the 542 nucleotides in HBMEC and HPAEC PKI alpha mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKI gamma mRNA. Western blot analysis indicated that PKI gamma protein was detected in all three cell types, whereas PKI alpha was found in HBMECs. In summary, endothelial cells from three different vascular beds express PKI alpha and PKI gamma, which may be physiologically important in endothelial barrier function.

Investigation of substance P transport across the blood-brain barrier

The transport of substance P (SP) was investigated using the bovine brain microvessel endothelial cell culture model of the blood-brain barrier (BBB). The samples were derivatized precolumn with naphthalene dialdehyde, then analyzed by cyclodextrin-modified micellar electrokinetic chromatography with laser-induced fluorescence detection. SP crossed the BBB in both the apical-to-basolateral and basolateral-to-apical directions through an active transport mechanism. The transport of SP from the apical side was demonstrated to be via transcytosis. The N-terminal (SP 1–4) and C-terminal (SP 3–11) fragments were also found to permeate the BBB from the apical side.

Hypoxia-Induced Hyperpermeability in Brain Microvessel Endothelial Cells Involves VEGF-Mediated Changes in the Expression of Zonula Occludens-1

In vivo, hypoxia is known to damage the blood–brain barrier (BBB) leading to the development of vasogenic brain edema. Primary cultures of porcine brain derived microvascular endothelial cells were used as an in vitro BBB model to evaluate the mechanisms by which hypoxia regulates paracellular permeability. Paracellular passage across endothelial cell monolayers is regulated by specialized intercellular structures like the tight junctions (TJ). Zonula occludens-1 (ZO-1), a protein of the TJ, lines the cytoplasmic face of intact TJ. The continuity of the ZO-1 expression was disrupted during 24 h of hypoxia which correlated with a decrease of the protein level to 32 ± 8% and with a twofold increase in the phosphorylation of ZO-1 in comparison to values determined at the start of the experiment. The localization and expression level of ZO-1 were maintained during hypoxia in the presence of a polyclonal antibody to vascular endothelial growth factor (VEGF) demonstrating that hypoxia-induced changes of the ZO-1 expression are mediated by VEGF. The effect of hypoxia on the ZO-1 distribution probably is not tissue- or cell-specific because similar changes of ZO-1 distribution were observed when the rat brain endothelial cell line RBE4 or the murine epithelial cell line CSG was used. Furthermore, ZO-1 changes correlated with small changes in actin distribution. These results suggest that hypoxia increases the paracellular flux across the cell monolayer via the release of VEGF, which in turn leads to the dislocalization, decreased expression, and enhanced phosphorylation of ZO-1.

Uptake of Cationized Albumin Coupled Liposomes by Cultured Porcine Brain Microvessel Endothelial Cells and Intact Brain Capillaries

The suitability of protein-coupled liposomes as drug carriers for brain specific targeting was investigated using albumin (BSA) and cationized albumin (CBSA), respectively, as model proteins. Liposomes coated with polyethylene glycol (sterically stabilized, PEG-liposomes) were prepared from phosphatidylcholine, cholesterol, and a PEG-derivatized phospholipid and covalently coupled to thiolated BSA or CBSA. Liposomes were loaded with carboxy-fluorescein and rhodamine-labeled dipalmitoyl-phosphatidylethanolamine as hydrophilic and lipophilic marker compounds, respectively. The interaction of these constructs with monolayers of porcine brain capillary endothelial cells (BCEC) and freshly isolated porcine brain capillaries was studied by means of fluorescence assays and confocal laser scanning fluorescence microscopy (CLSFM). In contrast to BSA, CBSA was rapidly taken up by cultured BCECs. BSA-coupled liposomes did not interact with endothelial cells, whereas CBSA-coupled liposomes bound to cellular surfaces and exhibited time dependently a high intracellular accumulation. CBSA-conjugated liposomes were also taken up by intact brain capillaries. Cellular uptake could be inhibited by free cationized albumin, phenylarsineoxide, nocodazole, and filipin, but not by dansylcadaverine, suggesting a caveolae-mediated incorporation process. Immunostaining demonstrated a high expression of caveolin in the capillary endothelium. In conclusion, liposomes coupled to CBSA are taken up into brain endothelium via an endocytotic pathway and may therefore be a suitable carrier for drug delivery to the brain.

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.

Nanosized cationic hydrogels for drug delivery: preparation, properties and interactions with cells

A new family of nanoscale materials on the basis of dispersed networks of cross-linked ionic and nonionic hydrophilic polymers is being developed. One example is the nanosized cationic network of cross-linked poly(ethylene oxide) (PEO) and polyethyleneimine (PEI), PEO-cl-PEI nanogel. Interaction of anionic amphiphilic molecules or oligonucleotides with PEO-cl-PEI results in formation of nanocomposite materials in which the hydrophobic regions from polyion-complexes are joined by the hydrophilic PEO chains. Formation of polyion-complexes leads to the collapse of the dispersed gel particles. However, the complexes form stable aqueous dispersions due to the stabilizing effect of the PEO chain. These systems allow for immobilization of negatively charged biologically active compounds such as retinoic acid, indomethacin and oligonucleotides (bound to polycation chains) or hydrophobic molecules (incorporated into nonpolar regions of polyion–surfactant complexes). The nanogel particles carrying biological active compounds have been modified with polypeptide ligands to enhance receptor-mediated delivery. Efficient cellular uptake and intracellular release of oligonucleotides immobilized in PEO-cl-PEI nanogel have been demonstrated. Antisense activity of an oligonucleotide in a cell model was elevated as a result of formulation of oligonucleotide with the nanogel. This delivery system has a potential of enhancing oral and brain bioavailability of oligonucleotides as demonstrated using polarized epithelial and brain microvessel endothelial cell monolayers.

The Kinetics, Function, and Regulation of P-Selectin Expressed by Human Brain Microvessel Endothelial Cells in Primary Culture

P-selectin is an endothelial cell adhesion glycoprotein expressed on the cell surface early in inflammation where it binds to blood leukocytes. This study examines the expression, function, and regulation of P-selectin in primary cultures of human brain microvessel endothelial cells (HBMEC). Surface expression of P-selectin was minimal in unstimulated HBMEC; however, it was significantly augmented upon stimulation with histamine (10−7–10−3 M) and thrombin (0.01–1 U/ml). Expression increased rapidly at 10 min and remained elevated at 60 min. Immunogold electron microscopy showed that histamine (10−7 M) increased surface expression preferentially on the apical surface of subconfluent monolayers. A cell binding assay showed that the adhesion of polymorphonuclear leukocytes (PMNs) to confluent monolayers was augmented after histamine treatment. Histamine-induced surface expression of P-selectin was blocked by the histamine H2 receptor antagonist cimetidine. The H1 receptor antagonist mepyramine had no effect. Expression was reduced by the extracellular calcium chelator EDTA and blocked by the cyclic AMP phosphodiesterase inhibitor rolipram. Thus histamine and thrombin both increase P-selectin expression in HBMEC. Histamine mediates expression through the H2, but not the H1, receptor and calcium, whereas expression is reduced by cyclic AMP. The histamine-induced expression increases PMN binding to the HBMEC. These data suggest that P-selectin plays a role in the recruitment of acute inflammatory cells to the CNS.

Occludin expression in microvessels of neoplastic and non-neoplastic human brain.

The tight junction protein occludin 'glues' normal, adjacent brain microvessel endothelial cells together. Malignant brain tumours cause cerebral oedema because they have leaky endothelial tight junctions, which allow plasma fluid to enter the brain from the microvessel lumen. In order to identify molecular abnormalities in tumour endothelial tight junctions, we investigated occludin expression in microvessels from adult human non-neoplastic brain tissue using immunohistochemistry and immunoblotting. The proportions of microvessels immunolabelling for occludin were >2/3 in 5/5 non-neoplastic brain tissue samples, >1/3 in 5/5 low grade (Daumas-Duport I or II) astrocytomas and <1/3 in 5/5 high grade (III or IV) astrocytomas and 6/6 metastatic adenocarcinomas. Six non-neoplastic brain tissue immunoblots gave a 55-kDa occludin band, three low-grade astrocytomas gave 55-kDa and 60-kDa bands, 13 high-grade astrocytomas gave 60-kDa or no band and four adenocarcinomas did not give an occludin band. Expression of 55-kDa occludin inversely correlated with the presence of contrast enhancement on computed tomograms (P < 0.001). Electron microscopy showed open endothelial tight junctions in 0/2 non-neoplastic human brain specimens and 2/2 high-grade astrocytomas. We suggest that loss of 55-kDa occludin expression in human brain tumours may contribute to endothelial tight junction opening. Characterizing the molecular pathology of brain endothelial tight junctions may facilitate the design of novel drugs against cerebral oedema.

Influence of Multidrug Resistance (MDR) Proteins at the Blood–Brain Barrier on the Transport and Brain Distribution of Enaminone Anticonvulsants

Previous in vitro studies evaluating the permeability of enaminones suggested that their blood-brain barrier (BBB) transport might be influenced by the presence of an efflux mechanism. Therefore, transport mechanisms responsible for these anticonvulsants across the BBB were examined. The transport of enaminones (1 x 10(-4) M) were evaluated over 120 min with verapamil (50 microM) and probenecid (100 microM) using bovine brain microvessel endothelial cells (BBMECs) to assess the role of multidrug resistant (MDR) transport proteins [i.e., P-glycoprotein (Pgp) and MDR protein 1 (MRP1)] on efflux, respectively. Uptake studies in the presence and absence of rhodamine 123 (R123; 3.2 and 5.0 microM) were also performed in a Pgp overexpressing cell line, MCF-7/Adr. Select enaminone esters (12.5 mg/kg) were administered intravenously to mdr 1 a/b (+/+), mdr 1 a/b (-/-) knockout and probenecid pretreated mice (20 +/- 5g). Enaminones and R123 were assayed with validated ultraviolet and fluorescence high-performance liquid chromatography methods, respectively. Verapamil and probenecid significantly ( p>0.05) inhibited the transport of select enaminone esters across BBMECs. Two enaminones caused a statistically significant increase in the uptake of R123 in MCF-7/Adr cells. Concentrations of select enaminones in mdr 1 a/b (-/-) mice brains were significantly higher ( p<0.05) compared with those in mdr 1 a/b (+/+) mice brains; however, no differences were observed in probenecid pretreated animals. Taken together, these results strongly suggest that Pgp may influence enaminone transport at the BBB and hence affect epilepsy treatment with these agents.

Investigation of the metabolism of substance P at the blood-brain barrier using capillary electrophoresis with laser-induced fluorescence detection.

Substance P (SP) metabolism was investigated upon exposure to a monolayer of bovine brain microvessel endothelial cells (BBMECs), a cell culture model of the blood-brain barrier. SP was incubated with the BBMECs and its metabolism was followed as a function of time over a 5-h period. The resulting samples were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA)/cyanide, separated, and detected using cyclodextrin-modified electrokinetic chromatography with laser-induced fluorescence detection (CDMEKC-LIF). Upon exposure to the BBMEC monolayer, SP rapidly degraded to produce the N-terminal (1-9), (1-4) and (1-7) and C-terminal (2-11) and (3-11) fragments. These results were compared with those in an earlier report from our laboratory, where SP metabolism was investigated in vivo by microdialysis sampling in rat striatum.

Functional feature of a novel model of blood brain barrier: studies on permeation of test compounds

Drug delivery to the central nervous system (CNS) is subject to the permeability limitations imposed by the blood–brain barrier (BBB). Several systems in vitro have been described to reproduce the physical and biochemical behavior of intact BBB, most of which lack the feature of the in vivo barrier. We developed a fully formed monolayer of RBE4.B immortalized rat brain microvessel endothelial cells (ECs), grown on top of polycarbonate filter inserts with cortical neuronal cells grown on the outside. Neurons induce ECs to synthesize and sort occludin to the cell periphery. Occludin localization is regulated by both compositions of the substratum and soluble signals released by cortical co-cultured neurons. The observed effects do not require strict physical contact among cells and neurons. To assess the physiological function of the barrier we examined the transendothelial transfer of three test compounds: dopamine, l-tryptophan and l-DOPA. Polycarbonate filter inserts, where ECs were co-cultured with neurons, were assumed as open two compartments vertical dynamic models. Permeation studies demonstrated that the ECs/neurons co-cultures possess permeability characteristics approaching those of a functional BBB: the system behaved as a selective interface that excludes dopamine permeation, yet permits l-tryptophan and l-DOPA to cross. The movement of test compounds from the donor to the acceptor compartment was observed at a distinct time from the start of co-culture. Transfer was determined using standard kinetic equations. Different performance was observed after 5 and 7 days of co-culture. After 5 days dopamine, l-tryptophan and l-DOPA passively permeate through the membrane as indicated by fittings with a first-order kinetic process equation. After 7 days of co-culture, occludin localizes at ECs periphery, dopamine does not cross the barrier to any further extent, while the transfer of l-tryptophan and l-DOPA fits well with a saturable Michaelis–Menten kinetic process, thus indicating the involvement of a specific carrier-mediated transport mechanism. Permeation studies confirmed that culture of ECs in the presence of neurons induces the characteristic permeability limitations of a functional BBB.

Rhesus macaque brain microvessel endothelial cells behave in a manner phenotypically distinct from umbilical vein endothelial cells

Activation of endothelium is a critical step in leukocyte recruitment to the CNS and in development of neurological diseases, such as HIV-associated dementia. Due to limited availability of early disease course data, it is important to develop in vitro models of the blood–brain barrier (BBB) that can be used to address these early events. No such model of the BBB has been established for the macaque. Here, we characterize rhesus microvascular brain endothelial cells (MBEC), comparing them with rhesus umbilical vein endothelial cells (RUVEC), and discuss their suitability for future use in developing in vitro models of simian immunodeficiency virus (SIV) neuropathogenesis. We conclude that MBEC are distinct from RUVEC with respect to growth characteristics, culture requirements, morphology and expression of surface molecules important for leukocyte adhesion and immune activation.

Correlation between bradykinin-induced blood–tumor barrier permeability and B2 receptor expression in experimental brain tumors

Localization of B2 receptors in brain tumor cells and microvessel endothelial cells of the brain tumors was investigated to study the differential sensitivity of brain tumors to bradykinin. The present study shows that B2 receptor expression levels vary in cultured RG2, C6 and 9L glioma cells as well as in the intracerebral tumors established with these cell lines in rats. The double immunohistochemical data indicate that B2 receptors are localized to tumor cells and not to the tumor capillaries. Immunostaining and Western blot analysis for B2 receptor showed that the B2 receptor expression was in the order C6>RG2>9L. The permeability studies on RG2, C6 and 9L tumors in rats showed that C6 tumor had the highest increase (178%) in Ki (unidirectional transport across blood–brain barrier (BBB)/blood–tumor barrier (BTB)), while 9L tumor had the least increase of Ki (35%) over the control group, following intracarotid infusion of bradykinin. We found a positive correlation (r = 0.965, p<0.001) between B2 receptor levels and bradykinin-induced increase in BTB permeability. We conclude that B2 receptors are localized to tumor cells and not to normal or tumor capillary endothelial cells. C6 tumor with highest B2 receptor expression was most responsive to bradykinin, while RG2 and 9L tumors with lower B2 receptor expression level were less sensitive to bradykinin with regard to BTB permeability.

Transport of choline and its relationship to the expression of the organic cation transporters in a rat brain microvessel endothelial cell line (RBE4)

The present study was undertaken to elucidate the functional characteristics of choline uptake and deduce the relationship between choline uptake and the expression of organic cation transporters in the rat brain microvessel endothelial cell line RBE4. Confluent RBE4 cells were found to express a high affinity choline uptake system. The system is Na +-independent and shows a Michaelis–Menten constant of approx. 20 μM for choline. The choline analogue hemicholinium-3 inhibits choline uptake in these cells with an inhibition constant of approx. 50 μM. The uptake system is also susceptible for inhibition by various organic cations, including 1-methyl-4-phenylpyridinium, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, clonidine, procainamide, and tetramethylammonium. The prototypical organic cation tetraethylammonium shows very little affinity for the choline uptake system in these cells. The inhibition of choline uptake by hemicholinium-3 is competitive. Northern analysis and RT-PCR show that these cells do not express the organic cation transporters OCT2 and OCT3. These cells do express, however, low levels of OCT1, but the functional characteristics of choline uptake in these cells are very different from the known properties of choline uptake via OCT1. The Na +-coupled high affinity choline transporter CHT1 is not expressed in these cells as evidenced by RT-PCR. This corroborates the Na +-independent nature of choline uptake in these cells. It is concluded that RBE4 cells express an organic cation transporter that is responsible for choline uptake in these cells and that this transporter is not identical to any of the organic cation transporters thus far identified at the molecular level in mammalian cells.

Role of angiotensin II type 1 receptor in the leucocytes and endothelial cells of brain microvessels in the pathogenesis of hypertensive cerebral injury

To elucidate the mechanisms of activation of polymorphonuclear neutrophils (PMNs) and their adhesion to endothelial cells in hypertensive cerebral injury, and to determine the effects of angiotensin II type 1 (AT1) receptor antagonism on PMNs and endothelial cells. We examined expression of AT1 receptor in PMNs in relation to that in endothelial cells of brain microvessels, using mature stroke-prone spontaneously hypertensive rats (SHRSP). To investigate the expression of AT1 receptor, we used 23-week-old male spSHRs and age-matched Wistar-Kyoto (WKY) rats. For the effects of AT1 receptor blockade, the AT1 receptor antagonist, TCV-116, was orally administered at a dosage of 0.5 mg/kg per day for 4 weeks in rats from age 19 weeks. A PMN-rich fraction was obtained by density gradient using Ficol-hypaque. AT1 receptor expression in PMNs was investigated by immunohistochemistry (avidin-biotinylated peroxidase complex method) and reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of macrophage-1 (Mac-1) in PMNs was examined by flow cytometry. Expression of intercellular adhesion molecule-1, glucose transporter-1 and fibrinogen in the cerebral cortex (occipital region) was investigated by immunohistochemistry. AT1 receptor was identified in PMNs by both immunohistochemistry and RT-PCR. It was also detected in the cerebral cortex. Expression in both types of cells was much more intense in spSHRs than in WKY rats. AT1 receptor antagonism ameliorated the enhanced expression of Mac-1 in PMNs. In addition, it was confirmed that enhanced expression of adhesion molecules and increased permeability of brain microvessels were decreased by AT1 receptor antagonism. The results indicate that both PMNs and brain microvessel endothelial cells possess AT1 receptor, that AT1 receptor antagonism ameliorates endothelial injury via inhibition of PMNs and endothelial cell adhesion, and that angiotensin II must be a key factor in hypertensive endothelial injury.