Brain Microvessel Endothelial Cell Monolayers Research Articles

Bile acids permeabilize the blood brain barrier after bile duct ligation in rats via Rac1-dependent mechanisms

The blood brain barrier tightly regulates the passage of molecules into the brain and becomes leaky following obstructive cholestasis. The aim of this study was to determine if increased serum bile acids observed during cholestasis permeabilize the blood brain barrier. Rats underwent bile duct ligation or deoxycholic or chenodeoxycholic acid injections and blood brain barrier permeability assessed. In vitro, the permeability of rat brain microvessel endothelial cell monolayers, the expression and phosphorylation of occludin, ZO-1 and ZO-2 as well as the activity of Rac1 was assessed after treatment with plasma from cholestatic rats, or bile acid treatment, in the presence of a Rac1 inhibitor. Blood brain barrier permeability was increased in vivo and in vitro following bile duct ligation or treatment with bile acids. Associated with the bile acid-stimulated increase in endothelial cell monolayer permeability was elevated Rac1 activity and increased phosphorylation of occludin. Pretreatment of endothelial cell monolayers with a Rac1 inhibitor prevented the effects of bile acid treatment on occludin phosphorylation and monolayer permeability. These data suggest that increased circulating serum bile acids may contribute to the increased permeability of the blood brain barrier seen during obstructive cholestasis via disruption of tight junctions.

The permeation of dynorphin A 1–6 across the blood brain barrier and its effect on bovine brain microvessel endothelial cell monolayer permeability

Dynorphin A 1–17 (Dyn A 1–17) is an endogenous neuropeptide known to act at the kappa opioid receptor; it has been implicated in a number of neurological disorders, including neuropathic pain, stress, depression, and Alzheimer's and Parkinson's diseases. The investigation of Dyn A 1–17 metabolism at the blood–brain barrier (BBB) is important since the metabolites exhibit unique biological functions compared to the parent compound. In this work, Dyn A 1–6 is identified as a metabolite of Dyn A 1–17 in the presence of bovine brain microvessel endhothelial cells (BBMECs), using LC–MS/MS. The transport of Dyn A 1–6 at the BBB was examined using this in vitro cell culture model of the BBB. Furthermore, the permeation of the BBB by the low molecular weight permeability marker fluorescein was characterized in the presence and absences of Dyn A 1–6.

Brain Pharmacokinetics of Two Prolyl Oligopeptidase Inhibitors, JTP-4819 and KYP-2047, in the Rat

Prolyl oligopeptidase (PREP) inhibitors are potential drug candidates for the treatment of neurological disorders, but little is known about their ability to cross the blood-brain barrier and to reach the target site. This study characterizes brain pharmacokinetics of two potent PREP inhibitors, JTP-4819 and KYP-2047. Firstly, the in vitro permeability (P(app) ) of JTP-4819 and KYP-2047 through a bovine brain microvessel endothelial cell monolayer was assessed. Then, the in vivo brain/blood ratio was determined for the total brain and plasma concentrations and also for the unbound extracellular drug concentrations after a single dose (50 μmol/kg i.p.). KYP-2047 had a significantly higher P(app) than JTP-4819. In vivo, KYP-2047 had higher total and unbound brain/blood ratios. KYP-2047 was equally distributed between the cortex, hippocampus and striatum. In the case of JTP-4819, the unbound brain extracellular concentrations could not be readily predicted from the unbound blood levels, probably because of its poor membrane penetration properties. KYP-2047 displayed a better ability to reach the intracellularly located brain PREP, and it inhibited this enzyme more effectively than JTP-4819 after an equimolar single dose. In conclusion, KYP-2047 showed better brain penetration characteristics than JTP-4819 both in vitro and in vivo. KYP-2047 is a brain-penetrating, potent and long-acting PREP inhibitor; thus, it represents a convenient pharmacological tool for assessing the potential of PREP as a drug target.

In-vitro characterization of blood-brain barrier permeability to delta sleep-inducing peptide.

The diffusion of delta sleep-inducing peptide (DSIP) across the blood-brain barrier (BBB) has been investigated with an in-vitro model comprised of primary cultures of brain microvessel endothelial cell (BMEC) monolayers. The BMEC monolayers were mounted in a side-by-side diffusion apparatus and the transendothelial flux of DSIP analysed by HPLC with UV detection at 280 nm. The transendothelial flux of the peptide was linear with time and increasing concentrations of DSIP (non-saturable), but was not altered by reduced temperature. The apparent permeability coefficient for DSIP penetration of BMEC monolayers was in a range similar to water-soluble substances (e.g. fluorescein, fluorescein isothiocyanate dextrans) that penetrate the blood-brain barrier to a limited degree based on molecular weight. DSIP flux across the BMEC monolayers was also found to be bidirectional, insensitive to metabolic inhibitors, and not altered by high concentrations of tryptophan. Little degradation (apparent t1/2 about 10 h) of DSIP to major metabolites, tryptophan (trp) and des-trp DSIP, occurred over the time of the diffusion experiments. The results of these studies support and confirm observations in-vivo indicating that intact DSIP crosses the BBB by simple transmembrane diffusion.

Dendritic Cell Transmigration through Brain Microvessel Endothelium Is Regulated by MIP-1α Chemokine and Matrix Metalloproteinases

Dendritic cells (DCs) accumulate in the CNS during inflammatory diseases, but the exact mechanism regulating their traffic into the CNS remains to be defined. We now report that MIP-1alpha increases the transmigration of bone marrow-derived, GFP-labeled DCs across brain microvessel endothelial cell monolayers. Furthermore, occludin, an important element of endothelial tight junctions, is reorganized when DCs migrate across brain capillary endothelial cell monolayers without causing significant changes in the barrier integrity as measured by transendothelial electrical resistance. We show that DCs produce matrix metalloproteinases (MMP) -2 and -9 and GM6001, an MMP inhibitor, decreases both baseline and MIP-1alpha-induced DC transmigration. These observations suggest that DC transmigration across brain endothelial cell monolayers is partly MMP dependent. The migrated DCs express higher levels of CD40, CD80, and CD86 costimulatory molecules and induce T cell proliferation, indicating that the transmigration of DCs across brain endothelial cell monolayers contributes to the maintenance of DC Ag-presenting function. The MMP dependence of DC migration across brain endothelial cell monolayers raises the possibility that MMP blockers may decrease the initiation of T cell recruitment and neuroinflammation in the CNS.

Characterization of P-glycoprotein Inhibition by Major Cannabinoids from Marijuana

The ATP-dependent drug efflux transporter P-glycoprotein (P-gp) plays a significant role in the absorption and disposition of many compounds. The purpose of this study was to investigate the possible interaction of P-gp with each of four major marijuana constituents: Delta(9)-tetrahydrocannabinol (THC), 11-nor-Delta(9)-tetrahydrocannabinol-carboxylic acid (THC-COOH), cannabinol (CBN), and cannabidiol (CBD). The results of a P-gp ATPase activity screen showed that THC-COOH, CBN, THC, and CBD all stimulated P-gp ATPase activity with a Michaelis-Menten parameter (V(max)/K(m)) value of 1.3, 0.7, 0.1, and 0.05, respectively. Furthermore, CBD showed a concentration-dependent inhibitory effect on verapamil-stimulated ATPase activity with an IC(50) value of 39.6 microM, whereas all other tested cannabinoids did not display appreciable inhibitory effects. Thus, the inhibitory effects of CBD on P-gp transport were further studied. At concentrations ranging from 5 to 100 microM, CBD robustly enhanced the intracellular accumulation of known P-gp substrates rhodamine 123 and doxorubicin in a concentration-dependent manner in Caco-2 and LLC-PK1/MDR1 cells. An IC(50) value of 8.44 microM was obtained for inhibition of P-gp function in LLC-PK1/MDR1 cells as determined by flow cytometry using rhodamine 123 as a fluorescence probe. Following exposure to 30 microM CBD, the apparent permeability coefficient of rhodamine 123 across Caco-2 and rat brain microvessel endothelial cell monolayers was increased to 2.2- and 2.6-fold in the apical-to-basolateral direction but decreased to 0.69- and 0.47-fold in the basolateral-to-apical direction, respectively. These findings indicate that CBD significantly inhibits P-gp-mediated drug transport, suggesting CBD could potentially influence the absorption and disposition of other coadministered compounds that are P-gp substrates.

Polypeptide Point Modifications with Fatty Acid and Amphiphilic Block Copolymers for Enhanced Brain Delivery

There is a tremendous need to enhance delivery of therapeutic polypeptides to the brain to treat disorders of the central nervous system (CNS). The brain delivery of many polypeptides is severely restricted by the blood-brain barrier (BBB). The present study demonstrates that point modifications of a BBB-impermeable polypeptide, horseradish peroxidase (HRP), with lipophilic (stearoyl) or amphiphilic (Pluronic block copolymer) moieties considerably enhance the transport of this polypeptide across the BBB and accumulation of the polypeptide in the brain in vitro and in vivo. The enzymatic activity of the HRP was preserved after the transport. The modifications of the HRP with amphiphilic block copolymer moieties through degradable disulfide links resulted in the most effective transport of the HRP across in vitro brain microvessel endothelial cell monolayers and efficient delivery of HRP to the brain. Stearoyl modification of HRP improved its penetration by about 60% but also increased the clearance from blood. Pluronic modification using increased penetration of the BBB and had no significant effect on clearance so that uptake by brain was almost doubled. These results show that point modification can improve delivery of even highly impermeable polypeptides to the brain.

Human immunodeficiency virus type 1 transport across the in vitro mouse brain endothelial cell monolayer

Human immunodeficiency virus type 1 (HIV-1) is associated with a neuroinflammatory dementia. Cognitive impairment remains a common complication of late-stage HIV-1 infection. Previous studies have shown that entry of HIV-1 into the central nervous system (CNS) occurs soon after infection. For these reasons, it is important to understand how HIV-1 crosses the BBB. We used primary mouse brain microvessel endothelial cell (MBEC) monolayer models to study interactions between brain endothelial cells and radioactively labeled HIV-1 CL4 ( 131I-HIV-1), which had been rendered noninfectious with aldithiol, and compared to radioactively labeled bovine serum albumin ( 131I-BSA or 125I-BSA) and detected HIV-1 on MBEC monolayer with electron microscopic analysis. The permeability of the monolayers to HIV-1 was measured by determining the percent material transported (PMT). Luminal to abluminal PMT of 131I-HIV-1 was 4.65 times greater than that of the much smaller 131I-BSA, showing that the MBEC monolayer is more permeable to HIV-1 than to BSA. Electron microscopy showed that HIV-1 was transported through a trans-cellular pathway from luminal side to basolateral space with some virus associated with the nucleus. Unlabeled HIV-1 did not affect the transport of 131I-HIV-1 or break down the MBEC monolayer. Wheatgerm agglutinin (WGA) increased 131I-HIV-1 penetration across the MBEC monolayer, consistent with absorptive endocytosis as the mechanism for HIV-1 penetration. The enhanced transport of HIV-1 was unidirectional, as the abluminal to luminal PMT of 131I-HIV-1 was not different from that of BSA nor enhanced by WGA. Characterization of the radioactivity transported from the luminal to abluminal chamber on Sepharose 4B-200 columns showed the transported radioactivity represented intact virus. MBEC monolayers preloaded from the luminal surface with 131I-HIV-1 showed most of the virus was retained by the endothelial cells, while the remainder was effluxed mainly to the luminal surface. MBEC monolayers preloaded from the abluminal surface with 131I-HIV-1 retained little virus and most of the virus was effluxed mainly to the abluminal surface. In conclusion, cell-free, intact 131I-HIV-1 crossed brain endothelial cell monolayers unidirectionally in the luminal to abluminal direction through an adsorptive endocytotic pathway. HIV-1 taken up from luminal side by monolayers of brain endothelial cells was mainly released to the luminal side. HIV-1 efflux mechanisms are different from influx mechanisms.

A Fluorometric Screening Assay for Drug Efflux Transporter Activity in the Blood-Brain Barrier

To examine the capability of a fluorometric assay to identify and characterize the drug efflux interactions of a broad spectrum of drug agents in an in vitro model of the blood-brain barrier (BBB). Various concentrations of drug agent (1, 10, and 100 microM) were evaluated for their effect on the cellular accumulation of the P-glycoprotein (P-gp) probe R123 (3.2 microM), and the mixed P-gp and multidrug resistance-associated protein (MRP) probe, BCECF (1 microM), in bovine brain microvessel endothelial cell (BBMEC) monolayers. Drugs demonstrating a significant effect were further quantitated using an expanded concentration range and a nonlinear regression curve fit to determine the potency (IC50) and efficacy (Imax) of the drug for P-gp and/or MRP. Several of the 36 therapeutic agents examined showed drug efflux transporter interactions in BBMEC monlayers. Melphalan and risperidone significantly enhanced the accumulation of R123 over control (1.47- and 1.82-fold, respectively) with resulting IC50s of 1.4 and 14.6 microM, respectively. Chlorambucil and valproic acid significantly enhanced the accumulation of BCECF compared to control monolayers (2.02- and 4.01-fold, respectively) with resulting IC50s of 146.1 and 768.5 microM, respectively. The current study demonstrates the feasibility of a fluorometric assay consisting of R123 and BCECF in assessing the drug efflux interactions of a variety of drugs in the BBB.

Effect of Heat Preconditioning on the Uptake and Permeability of R123 in Brain Microvessel Endothelial Cells during Mild Heat Treatment

The purpose of this study was to assess the effect of mild heat and heat preconditioning on the uptake and permeability of a P‐glycoprotein (P‐gp) substrate, rhodamine 123 (R123), in a cell culture model of the blood–brain barrier (BBB). An immediate goal was to determine whether prior mild heat treatment could render brain microvessel endothelial cells more resistant to future heat stress and affect BBB drug permeation by future ultrasound‐induced mild heat (USMH) treatment. To address this issue, the expression level of two proteins, P‐gp and heat shock protein 70 (Hsp70), and their effects on uptake of R123 and permeability of R123 and [14C]‐sucrose in combination with mild heat and P‐gp modulator PSC833 during and after mild heat treatment in heat‐preconditioned and heat‐unconditioned bovine brain microvessel endothelial cell (BBMEC) monolayers were studied. Mild heat caused a significant increase in BBB permeability of R123 and [14C]‐sucrose when compared with control and PSC833. Exposure of BBMECs to heat preconditioning caused a slight but insignificant decrease in cellular uptake of R123 both during and immediately after mild heat treatment. Heat preconditioning also caused a slight but insignificant decrease in permeability of R123 and [14C]‐sucrose in BBMEC monolayers during mild heat treatment. Because exposure of BBMEC monolayers to mild heat did not affect P‐gp expression but slightly affected Hsp70 expression, a heat preconditioning that results in a reinforcement of the BBB other than increased expression of P‐gp is suggested. However, heat preconditioning is not sufficient to override the permeation‐enhancing effects of mild heat because mild heat caused a significant increase in R123 uptake and permeability of R123 and [14C]‐sucrose in both heat‐preconditioned and heat‐unconditioned cells. Because Hsp70 is known to play a major role in cellular repair and protective mechanisms, our results would imply a relative benign nature of mild heat treatment. Because heating produced by ultrasonic waves can be controlled and localized to a small volume within the tissue, the present results also suggest that USMH could play a pivotal role in the treatment of brain tumors and other brain‐related diseases.

Computation of log BB values for compounds transported through carrier-mediated mechanisms using in vitro permeability data from brain microvessel endothelial cell (BMEC) monolayers.

To explore the possibility of determining in vivo log BB values (the logarithm value of brain to plasma concentration ratio) from in vitro permeability data measured in brain microvessel endothelial cell (BMEC) monolayers. An equilibrium mathematical model was developed: log BB = log(Ca/Cb) + log Kbr:pl, where Cb and Ca are the drug concentrations at equilibrium in the basolateral (B) and apical (A) sides of BMECs in an A-to-B directional diffusion system and Kbr:pl is the brain-plasma partition coefficient. With this model, murine log BB values were calculated for 24 pharmaceutical compounds, mostly Pgp substrates. Calculated log BB values correlated well to experimental values (r2 = 0.854, slope = 0.907 +/- 0.080), demonstrating that the model could reasonably predict brain penetration for compounds that are involved in carrier-mediated transport mechanisms. For a second data set that included volatile organic compounds (log BB = log Kbr:pl), log Kbr:pl values were also shown to correlate well with their respective experimental log BB values (r2 = 0.876, slope = 0.973 +/- 0.082), demonstrating that log Kbr:pl is an excellent descriptor for log BB when a compound penetrates the blood-brain barrier by passive diffusion only. The equilibrium model demonstrated a reasonable ability to compute in vivo log BB values, regardless of the involvement or mechanisms of carrier-mediated transport.

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.

Enhancement of Transport of D-Melphalan Analogue by Conjugation with L-Glutamate across Bovine Brain Microvessel Endothelial Cell Monolayers

In this paper, the L-glutamate (L-Glu) transport system was targeted to improve the delivery of a model compound, p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD), through the blood-brain barrier (BBB) in vitro cell culture model. D-MOD is an analogue of an antitumor agent D-melphalan. To target the L-Glu transport system, D-MOD was conjugated to L-Glu to give D-MOD-L-Glu conjugate. D-MOD and D-MOD-L-Glu transport properties were evaluated using the bovine brain microvessel endothelial cell (BBMEC) monolayers. The results suggest that D-MOD-L-Glu conjugate permeates through the BBMEC monolayers more readily than the parent D-MOD. The improvement of transport may be due to the recognition of D-MOD-L-Glu by the L-Glu transport system. The transport mechanism was evaluated using several different experiments including: (a) concentration-dependent studies; (b) temperature-dependent studies; (c) substrate inhibition studies; and (d) metabolic inhibitor studies. The D-MOD-L-Glu transport was inhibited by the change of temperature from 37°C to 4°C. At higher concentrations, the transport of D-MOD-L-Glu reached plateau due to saturation. Furthermore, some amino acids (i.e., L-Glu, L-Asp, D-Asp, and L-Gln) inhibited the transport of D-MOD-L-Glu; presumably the conjugate was competing with these amino acids for the same transport system. Metabolic inhibitors (i.e., 2,4-dinitrophenol and sodium azide) suppressed the transport of the conjugate. However, the conjugate was not transported by monocarboxylic acid, dipeptide and neutral amino acid transporters. In conclusion, the L-Glu transport system can be utilized to facilitate a non-permeable drug across the BBB by conjugating the drug with L-Glu amino acid.

Determination of angiotensin II in blood–brain barrier permeability studies using microbore LC with p-nitrophenyl-2,5-dihydroxyphenylacetate bis-tetrahydropyranyl ether as a pre-separation electrochemical labeling reagent

Angiotensin II has been determined using microbore LC after pre-column derivatization with the novel derivatization reagent, p-nitrophenyl 2,5-dihydroxyphenylacetate, bis-tetrahydropyranyl ether (NDTE). Derivatization of trace concentrations of angiotensin II, angiotensin III and angiotensin II 3–8 was complete after 4 h at 25°C and resulted in the formation of an electrochemically active derivative possessing a hydroquinone. After suitable sample preparation, samples were analyzed using gradient elution microbore LC-EC with a radial-flow electrochemical thin-layer detection cell set at a potential of +250 mV vs. Ag/AgCl, [Cl −]=15 mM. The concentration limit of detection for angiotensin II was found to be 62.5 nM (39 fmol injected). Samples obtained during in vitro blood–brain barrier transport studies were subjected to an appropriate preparation protocol subsequently and analyzed for angiotensin II using the described method. Semi-linear permeation of angiotensin II across a cultured, bovine brain microvessel endothelial cell monolayer was monitored for 2 h at concentrations ranging from 80 to 495 nM (84–518 ng ml −1).

Increased permeability of primary cultured brain microvessel endothelial cell monolayers following TNF-α exposure

TNF-α is a cytokine that produces increased permeability in the peripheral vasculature; however, little is known about the effects of TNF-α on the bloodbrain barrier (BBB). Using primary cultured bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB, this study shows that TNF-α produces a reversible increase in the permeability of the brain microvessel endothelial cells. The BBMEC monolayers were pre-treated with 100 ng ml of TNF-α for periods ranging from 2 to 12 hours. Permeability was assessed using three molecular weight markers, fluorescein (376 MW), fluorescein-dextran (FDX-4400; 4400 MW), and FDX-70000 (MW 70000). The permeability of BBMEC monolayers to all three fluorescent markers was increased two-fold or greater in the TNF-α treatment group compared to control monolayers receiving no TNF-α. Significant changes in permeability were also observed with TNF-α concentrations as low as 1 ng ml . These results suggest that TNF-α acts directly on the brain microvessel endothelial cells in a dynamic manner to produce a reversible increase in permeability. Exposure of either the lumenal or ablumenal side of BBMEC monolayers to TNF-α resulted in similar increases in permeability to small macromolecules, e.g. fluorescein. However, when a higher molecular weight marker was used (e.g. FDX-3000), there was a greater response following lumenal exposure to TNF-α. Together, these studies demonstrate a reversible and time dependent increase in brain microvessel endothelial cell permeability following exposure to TNF-α. Such results appear to be due to TNF's direct interaction with the brain microvessel endothelial cell.

Expression of Multidrug Resistance-Associated Protein (MRP) in Brain Microvessel Endothelial Cells

Multidrug resistance-associated protein (MRP) is a recently identified drug efflux transport system that actively transports organic acids and selected glucuronide or glutathione conjugates out of the cell. The current study presents, for the first time, both functional and biochemical data demonstrating the presence of MRP in the brain microvessel endothelial cells that form the blood-brain barrier (BBB). Using known MRP inhibitors, such as indomethacin and probenecid, fluorescein accumulation in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers was significantly enhanced compared to control. The specificity of the MRP inhibitors on cellular fluorescein accumulation was confirmed using both MRP positive (Panc-1) and MRP negative (KBv) cell lines. Furthermore, western blot analysis using a specific antibody for MRP (MRPm6) and RT-PCR studies using a complementary sequence probe for human MRP demonstrate the expression of MRP in BBMEC. Previous studies have demonstrated the significance of the P-glycoprotein drug efflux transporter in the BBB. Given its function as a drug efflux transport system, it is anticipated that MRP in the BBB will also have an important role in limiting the exposure of the brain to many endogenous and exogenous compounds, including both toxic and therapeutic agents.

Effect of tumor necrosis factor-alpha on the permeability of bovine brain microvessel endothelial cell monolayers

The administration of chemotherapy to patients with tumors of the central nervous system is often blocked by the blood-brain barrier. Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that promotes vascular permeability in addition to its pro-inflammatory effects. However. no direct evidence exists as to whether TNF-alpha may increase permeability of the BBB. We evaluated the effect of TNF-alpha on the transport of cisplatin (COOP) or high molecular weight dextran labeled with fluorescein isothiocyanate (FITC-dextran) across bovine brain microvessel endothelial cell (BMEC) monolayers that was conducted on side-by-side diffusion chambers in vitro. The permeability coefficient for the transport of COOP across the untreated monolayer was 3.80 x 10-5 cm sec-1 at 30 minutes. After treating the BMEC monolayer with TNF-alpha (50 U ml-1 and 500 U ml-1) for 36 hours, the PC of COOP increased significantly to 8.94 x 10-5/ and 14.43 x 10-5 cm sec-1 respectively (p< 0.01). TNF-alpha had no effect on the transport of FITC-dextran across the BMEC monolayers. Electron microscopy showed that the tight junctions between the BMECs persisted even after treatment with TNF-alpha, whereas they had been partially disrupted following exposure to mannitol, 1600 mOsm kg-1• TNF-alpha selectively promoted the in vitro permeability of the blood-brain barrier to COOP without disrupting tight junctions. This system could be used as a model for experimental studies of chemotherapy. Findings suggested that the combined administration of TNF-alpha and COOP may be clinically useful. [Neural Res 1997; 19: 369-376]

Use of rhodamine 123 to examine the functional activity of P-glycoprotein in primary cultured brain microvessel endothelial cell monolayers

The fluorescent dye, rhodamine 123, was used to evaluate the functional activity of the P-glycoprotein (P-gp) efflux transport system in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers. Rhodamine 123 accumulation was increased significantly in BBMEC monolayers treated with the P-gp modifying agent, cyclosporin A (CSA). Rhodamine 123 accumulation was also increased by other P-gp modifying agents. The rank effectiveness of these agents in increasing rhodamine 123 accumulation in BBMEC monolayers was CSA = dipyridamoleverapamil = quinidine. The maximal increase in rhodamine 123 accumulation in CSA treated BBMEC monolayers was approximately 3 fold greater than in control monolayers and was qualitatively similar to that observed with 3H-vincristine. Comparison of functional activity with the biochemical expression of P-gp in BBMEC monolayers and in an established tumor cell line that over-expresses P-gp indicate that functional activity may be a more descriptive measure of the importance of this drug efflux system than protein expression. Furthermore, these studies suggest that accumulation of rhodamine 123 in BBMEC monolayers can be used to quantitatively examine P-gp activity in the blood-brain barrier.

Signal transduction and glial cell modulation of cultured brain microvessel endothelial cell tight junctions.

Noncontact coculture of postconfluent bovine brain microvessel endothelial cell (BMEC) monolayers with rat C6 glioma cells results in markedly decreased transmonolayer permeability measured by transendothelial electrical resistance (TER) and solute flux. An elevation in adenosine 3',5'-cyclic monophosphate (cAMP) in response to forskolin correlates with an increase in TER through a threshold event; however, unlike forskolin, the severalfold increase in TER induced by C6 cells is cAMP independent. Activation of protein kinase C (PKC) enhances the C6 cell-induced increase in TER, and PKC inhibition blocks C6 cell induction. Treatment of control or C6 cell-induced BMEC monolayers with pertussis toxin immediately and irreversibly obliterates TER, without an apparent change in guanosine 3',5'-cyclic monophosphate levels or viability, indicating the importance of a G protein-mediated event. A similar effect is observed with transforming growth factor-beta 1, and both responses are polarized, since the loss in TER is significantly faster in BMEC monolayers exposed basolaterally. These results suggest that astroglia modulate the blood-brain barrier through a cAMP-independent PKC-dependent pathway maintained by a G protein-coupled mechanism.

21-aminosteroid and 2-(aminomethyl)chromans inhibition of arachidonic acid-induced lipid peroxidation and permeability enhancement in bovine brain microvessel endothelial cell monolayers

Selected 21-aminosteroids (U74500A, U74006F, and U74389G) and a 2-(aminomethyl)chromans (U78517F) were tested for their efficacy in preventing arachidonate-induced lipid peroxidation and permeability alterations in brain microvessel endothelial cells (BMECs). The 21-aminosteroids and 2-(aminomethyl)chromans were effective in varying degrees in inhibiting (U74500A = U78517F > U74006F = U74389G) concentration- and time-dependent arachidonate-induced thiobarbituric acid reactive substances (TBARS) production by BMECs. Arachidonate produced a corresponding concentration-dependent increase in BMEC monolayer permeability to the membrane impermeant marker, sucrose. Pretreatment of BMEC monolayers with either the 21-aminosteroids or the 2-(aminomethyl)chromans completely blocked the arachidonate-induced increase in permeability to sucrose. Our results demonstrated that these membrane-associating antioxidants were particularly effective in preventing both arachidonic acid-induced lipid peroxidation and permeability changes in BMEC monolayers. However, concentrations of some antioxidants that only partially inhibited TBARS production, completely inhibited the arachidonic acid-induced enhancement in BMEC monolayer permeability. Therefore, arachidonic acid-induced effects on BMEC permeability were likely due in part to both lipid peroxidation and direct or indirect effects of the fatty acid on membrane integrity. This study provides further support for the application of primary cultures of BMECs as a useful in vitro system to evalulate mechanisms through which mediators of disease or injury states compromise blood-brain barrier integrity.