Flavin-Containing Monooxygenase (FMO) Protein Expression and Its Activity in Rat Brain Microvascular Endothelial Cells ()
1. Introduction
Brain microvascular endothelial cells (BMECs) are a major structural and functional component of the bloodbrain barrier (BBB) that maintains homeostasis of the central nervous system. BMECs have unique and efficient protective systems controlling the passage of materials from the blood into the brain. The first is a permeability barrier and the second seems to be a metabolic barrier, formed by enzymes in the endoplasmic reticulum of endothelial cells which metabolize some permeable molecules recognized as substrates. Previous data indicate that isolated brain capillaries contain activities of enzymes involved in drug metabolism such as cytochrome P450s (CYPs) [1], NADPH-cytochrome P450 reductase, 1-naphthol glucuronyltransferase, and epoxide hydrolase [2]. Therefore, they are able to protect the brain from chemical insult. The possible implication of CYP 2E1 in rat vascular metabolism of arachidonic acid to vasoactive compounds has been also suggested, resulting in a functional role for this isoform [3]. On the other hand, like CYP, flavin-containing monooxygenases (FMOs) are microsomal enzymes that require NADPH and O2, and catalyze the oxidation of nucleophilic tertiary amines to N-oxides and secondary amines to hydroxylamines and oximes. Moreover, humans and other mammals express five different FMOs (FMO1, FMO2, FMO3, FMO4, and FMO5) in a speciesand tissue-specific manner [4]. However, little is known about the presence of FMOs localized in the BBB.
In the present study, we examined whether cultured rat microvascular endothelial cells (BMECs) have the ability to form N-oxide from the tertiary amine, d-chlorpheniramine, and whether FMOs proteins are expressed in rat BMECs.
2. Experimental
2.1. Materials
d-Chlorpheniramine N-oxide was synthesized, as described by Craig and Purushothaman [5], and separated by HPLC as described below. d-chlorpheniramine maleate, glucose 6-phosphate dehydrogenase, NADP, glucose 6-phosphate were obtained from Wako Pure Chemicals (Osaka, Japan). Gentamicin sulfate and amphotericin B were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (D-MEM/F-12), heparin, piperazine-N’-(2-ethane-sulfonic acid) (HEPES), dispase, epidermal growth factor (EGF), fetal bovine serum (FBS), and donor horse serum (HS) were obtained from Gibco BRL, Life Technologies (Rockville, MD). Dextran T-70 and Percoll were obtained from Pharmacia (Uppsala, Sweden). Collagenase/- dispase was purchased from Boehringer Mannheim (Mannheim, Germany). Acetylated low density lipoprotein labeled with a fluorescent probe, 1,1’-dioctadecyl- 3,3,3’,3’tetramethyl-indocarbocyamine perchlorate (DiIAc-LDL), was obtained from Biomedical Technologies (Stoghton, MA, USA). SD rat normal hepatocytes (rtNHeps) were purchased from Takara Bio Products (Ootu, Japan). All other chemicals were of reagent grade and commercially available.
2.2. Isolation and Culture of Rat BMECs
Three-week-old male Wistar rats purchased from Japan SLC (Hamamatsu, Japan) were housed at a constant temperature (23˚C ± 1˚C) and constant humidity (55% ± 5%), with automatically controlled lighting (07:00-19:00). Rat BMECs were isolated using a technique described previously [6,7]. Twenty rats were killed by decapitation. After removal of the choroid plexuses and diencephalon, cerebral cortical tissues were cleaned of meninges and superficial blood vessels, minced into 1 to 2 mm cubes, and incubated in M199 containing 0.2% dispase at 37˚C for 120 min. The dispase suspension was spun at 1000 × g for 10 min and then the pellet was resuspended in M199 containing 13% dextran. After centrifugation at 5800 × g and 4˚C for 10 min, the fat pad and dextran solution were collected into a new tube and spun again. Two pellets were collected and resuspended in the new dextran solution and spun. The resulting tissue pellet was resuspended in M199 and filtered through a 300-μm mesh. The microvessel suspension was dissociated using 0.1% collagenase/dispase in M199 at 37˚C for 60 min. After enzyme treatment, microvessels were collected by centrifugation at 1000 × g for 10 min, and resuspended in M199. The suspension was layered on a Percoll gradient formed by centrifugation of 50% Percoll at 26,000 × g at 4˚C for 60 min and then centrifuged at 600 × g for 10 min. Three layers were observed after Percoll gradient centrifugation. Endothelial cell aggregates formed a band around the middle third of the gradient, and the entire middle layer was collected from gradients. Cells were resuspended in M199 and collected by centrifugation at 600 × g for 10 min. Cell suspensions were seeded onto collagen-coated 75 cm2 tissue culture flasks (Iwaki GlassFunabashi, Japan). Cells were allowed to attach and grow to monolayers at 37˚C in a humidified atmosphere of 5% CO2/95% air. The culture medium (D-MEM/F-12 containing 14 mmol/L of sodium bicarbonate, 20 μg/mL of EGF, 50 μg/mL of gentamycin-amphotericin B, 10 U/mL of heparin, 5% FBS, and 5% HS) was changed every 3 days. A subculture was performed when cells reached confluence, after approximately 10 - 12 days. Cells were trypsinized at a ratio of 1:3 after reaching confluence using 0.025% trypsin in HBSS containing 0.02% EDTA. Secondary subcultured cells (5 × 105 cells∙cm−2) were grown on collagen-coated 225 cm2 tissue culture flasks. All metabolism experiments were performed on only second passaged BMECs after cells reached confluence, in approximately 10 - 12 days.
2.3. Western Blot Analysis
BMECs were washed with ice-cold phosphate buffered saline (PBS (-)). BMECs were then harvested and the pellet was lysed in RIPA buffer (Nacalai tesque, Kyoto, Japan). Lysates were incubated for 15 min on ice prior to sonication and then centrifuged at 10,000 × g for 20 min at 4˚C. Protein content was determined by a BCA protein kit (Thermo Scientific, Rockford, USA). Equivalent protein aliquots (5.0 μg) were separated by 10% SDS-PAGE and transferred electrophoretically to an Immobilon-P transfer membrane (Millipore Corporation, Billerica, MA, USA). The membrane was blocked with Blocking One (Nacalai tesque, Kyoto, Japan) and then incubated with primary antibodies against FMO1 (Rabbit polyclonal IgG at dilution of 1:1500, Santa Cruz), FMO2 (Rabbit polyclonal IgG at dilution of 1:1000, Protein Tech Group Inc., Chicago, USA), FMO3 (Goat polyclonal IgG at dilution of 1:1000, Santa Cruz, California, USA), FMO4 (Rabbit polyclonal IgG at dilution of 1:1000, Protein Tech Group Inc., Chicago, USA), or FMO5 (Rabbit polyclonal IgG at dilution of 1:1000, Protein Tech Group Inc., Chicago, USA). These primary antibodies used in this experiment have specificity reaction in human, rat and mouse, respectively. Blots were incubated with HRP-conjugated anti-rabbit antibody (GE Healthcare UK Ltd., Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England) or anti-goat antibody (Santa Cruz, Chicago, USA), and detected with ECL Plus Western blotting detection reagents (GE Healthcare UK Ltd., Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England). The antibody of each FMO used shows no cross-reactivity with the other FMO isoforms.
2.4. Enzyme Assay
For enzyme kinetic studies, cultured rat BMECs were homogenized in 0.1 mol/L phosphate buffer (pH 7.4 and 8.4, respectively). Incubation vessels contained rat BMECs (0.25 mg protein/mL), MgCl2 (25 mmol/L), glucose 6- phosphate (6.7 mmol/L), nicotine amide (2.5 mmol/L), and glucose 6-phosphate dehydrogenase (1 unit/mL) in a total volume of 100 μL. d-Chlorpheniramine-dissolved 0.1 mol/L phosphate buffer (pH 7.4 and 8.4, respectively) was the substrate at a final concentration in the range of 0.08 to 1.04 mmol/L. After addition of NADP (0.5 mmol/L in 0.1 mol/L phosphate buffer), the mixtures were incubated for 2 min at 37˚C in a shaking water bath. At the end of the incubation, 600 μL of acetonitrile was added as a reaction stopper. After vortex mixing, the deproteinized sample was centrifuged at 9000 × g for 15 min. The supernatant was evaporated for 5 min, and 50 μL of the residues was injected onto a HPLC column. Protein concentrations were assayed by the method of Markwell et al. [8].
Moreover, cultured rat BMECs and rtNHeps were homogenized in 0.1 mol/L phosphate buffer (pH 7.4), respectively. The enzyme was incubated with 0.16 mmol/L d-chlorpheniramine for 2 min at 37˚C in a shaking water bath.
2.5. Inhibition Study
Methimazole was used for the inhibition study of FMO. Metabolism of d-chlorpheniramine (0.16 mmol/L) was examined with or without methimazole (10−8 - 10−3 mol/L) by the method described above. Inhibition kinetics for methimazole were determined in BMECs homogenates using the standard assay procedure with eight to nine different d-chlorpheniramine concentrations and methimazole (1 μmol/L). Data were analyzed by nonlinear regression analysis to allow determination of the type of inhibition and inhibitory constants.
2.6. HPLC Condition
HPLC was performed with a Shimazu (Kyoto, Japan) LC-6A apparatus equipped with a UV detector (Shimazu SPD-10AVP, Kyoto, Japan) and LiChrospher Si60 column (250 × 4.6 mm i.d., 5 μm particle size, Kanto Chemical Co., INC, Tokyo, Japan). Material was eluted with 73:25:2, v/v, acetonitrile-methano l% - 28% ammonia water at a flow rate of 1 mL/min at 30˚C, and the absorption at 228 nm was measured. The peak of synthesized d-chlorpheniramine N-oxide was symmetrical and clearly separated from other peaks. The calibration curve for metabolites was linear over the concentration range 50 μg/mL and the lower limit for quantitation was 0.1 μg/mL.
2.7. Data Analysis
The formation of metabolites from d-chlorpheniramine was calculated as nmol formed/min/mg protein. Kinetic data were fit to the Michaelis-Menten equation for a oneenzyme or two-enzyme system using the non-linear leastsquares regression analysis program MULTI, and apparent Km and Vmax values were estimated. Values are presented as mean ± s.e.m. of means for n experiments. Comparisons of data among groups were carried out using an analysis of variance and Dunnett’s post hoc multiple-comparisons test. Differences were considered significant at P < 0.05 (two-tailed).
3. Results
3.1. The Formation Rate of d-Chlorpheniramine N-Oxide in Rat BMECs
Figure 1 shows respective Michaelis-Menten plots for the formation of d-chlorpheniramine N-oxide from dchlorpheniramine incubated with cultured rat BMECs and the concentration of d-chlorpheniramine (0.08 - 1.04 mmol/L). The apparent Km and Vmax values for N-oxidation at pH 7.4 and 8.4 are listed in Table 1. Consequently, the intrinsic clearance value (Vmax/Km) for N-oxidation at pH 8.4 was higher than that at pH 7.4. As shown in an Eadie-Hofstee plot, the formation of N-oxide was analyzed to be monophasic at pH 7.4 and 8.4, respectively.