Abstract
The aim of this study was to examine whether flavin-containing monooxygenase (FMO) protein was expressed in cultured rat brain microvascular endothelial cells (BMECs), which constitute the blood-brain barrier (BBB), and whether N-oxide from the tertiary amine, d-chlorpheniramine, was formed by FMO in rat BMECs. BMECs were isolated and cultured from the brains of three-week-old male Wistar rats. The expression of FMO1, FMO2 and FMO5 proteins was confirmed in rat BMECs by western blotting analysis using polyclonal anti-FMO antibodies, but FMO3 and FMO4 proteins were not found in the rat BBB. Moreover, N-oxide of d-chlorpheniramine was formed in rat BMECs. The intrinsic clearance value for N-oxidation at pH 8.4 was higher than that at pH 7.4. Inhibition of N-oxide formation by methimazole was found to be the best model of competitive inhibition yielding an apparent Ki value of 0.53 μmol/L, suggesting that N-oxidation was catalyzed by FMOs in rat BMECs. Although FMO activity in rat BMECs was lower than that in SD rat normal hepatocytes (rtNHeps), we suggest that rat BMECs enzymes can convert substrates of exogenous origin for detoxification, indicating that BMECs are an important barrier for metabolic products besides hepatic cells.
Highlights
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
The aim of this study was to examine whether flavin-containing monooxygenase (FMO) protein was expressed in cultured rat brain microvascular endothelial cells (BMECs), which constitute the blood-brain barrier (BBB), and whether N-oxide from the tertiary amine, d-chlorpheniramine, was formed by FMO in rat BMECs
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
Summary
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. 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]. They are able to protect the brain from chemical insult. 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. Little is known about the presence of FMOs localized in the BBB
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