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Flavin-Containing Monooxygenase (FMO) Protein Expression and Its Activity in Rat Brain Microvascular Endothelial Cells

DOI: 10.4236/pp.2013.41001    5,282 Downloads   8,547 Views   Citations

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.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

E. Sakurai, Y. Ueda, Y. Mori, Y. Shinmyouzu and E. Sakurai, "Flavin-Containing Monooxygenase (FMO) Protein Expression and Its Activity in Rat Brain Microvascular Endothelial Cells," Pharmacology & Pharmacy, Vol. 4 No. 1, 2013, pp. 1-6. doi: 10.4236/pp.2013.41001.

References

[1] J. R. Cashman, J. R. Celestial and A. R. Leach, “Enantioselective N-Oxygenation of Chlorpheniramine by the Flavin-Containing Monooxygenase from Hog Liver,” Xenobiotica, Vol. 22, No. 4, 1992, pp. 459-469. doi:10.3109/00498259209046658
[2] J. F. Ghersi-Egea, A. Minn and G. Siest, “A New Aspect of the Protective Functions of the Blood-Brain Barrier: Activities of Four Drug-Metabolizing Enzymes in Isolated Brain Microvessels,” Life Sciences, Vol. 42, No. 24, 1988, pp. 2515-2523. doi:10.1016/0024-3205(88)90351-7
[3] T. Hannsson, N. Tindberg, M. Inglman-Sundberg and C. Kohler, “Regional Distribution of Ethanol-Inducible Cytochrome P-450 IIE1 in the Rat Central Nervous System,” Neuroscience, Vol. 34, No. 2, 1990, pp. 451-453. doi:10.1016/0306-4522(90)90154-V
[4] J. R. Cashman, “Structural and Catalytic Properties of the Mammalian Flavin-Containing Monooxygenase,” Chemical Research in Toxicology, Vol. 8, No. 2, 1995, pp. 165- 181. doi:10.1021/tx00044a001
[5] J. C. Craig and K. K. Purushothaman, “An Improved Preparation of Tertiary Amine N-Oxides,” Journal of Organic Chemistry, Vol. 35, No. 5, 1970, pp. 1721-1722. doi:10.1021/jo00830a121
[6] N. Ichikawa, K. Naora, H. Hirano, M. Hashimoto, S. Masumura and K. J. Iwamoto, “Isolation and Primary Culture of Rat Cerebral Microvascular Endothelial Cells for Studying Drug Transport in Vitro,” Journal of Pharmacological and Toxicological Methods, Vol. 36, No. 1, 1996, pp. 45-52. doi:10.1016/1056-8719(96)00072-X
[7] J. Yamakami, E. Sakurai, T. Sakurada, K. Maeda and N. Hikichi, “Stereoselective Blood-Brain Transport of Histidine in Rats,” Brain Research, Vol. 812, No. 1-2, 1998, pp. 105-112. doi:10.1016/S0006-8993(98)00958-5
[8] M. A. K. Markwell, S. M. Haas, L. L. Bieder and N. E. Tolberg, “A Modification of the Lowry Procedure to Simplify Protein Determination in Membrane and Lipoprotein Samples,” Analytical Biochemistry, Vol. 87, No. 1, 1978, pp. 206-210. doi:10.1016/0003-2697(78)90586-9
[9] F. M. Farin, T. H. Pohlman and C. J. Omiecinski, “Expression of Cytochromes P450s and Microsomal Epoxide Hydrolase in Primary Cultures of Human Umbilical Vein Endothelial Cells,” Toxicology Applied Pharmacology, Vol. 124, No. 1, 1994, pp. 1-9. doi:10.1006/taap.1994.1001
[10] W. F. Graier, S. Simecek and M. Sturek, “Cytochrome P450 Mono-Oxygenase-Regulated Signaling of Ca+ Entry in Human and Bovine Endothelial Cells,” Journal of Physiology, Vol. 482, No. 2, 1995, pp. 259-274.
[11] J. J. Stegemann, M. E. Hahn, R. Weisbrod, B. R. Woodin, J. S. Joy, S. Najibi and R. A. Cohen, “Induction of Cytochrome P450 1A1 by Aryl Hydrocarbon Receptor Agonists in Porcine Aorta Endothelial Cells in Culture and Cytochrome P450 1A1 Activity in Intact Cells,” Molecular Pharmacology, Vol. 47, No. 2, 1995, pp. 296-306.
[12] A. S. Adeagbo, “Endothelium-Derived Hyperprolarizing Factor: Characterization as a Cytochrome P450 1A-Linked Metabolite of Arachidonic Acid in Perfused Rat Mesenteric Prearteriolar Bed,” American Journal of Hypertension, Vol. 10, No. 7, 1997, pp. 763-771. doi:10.1016/S0895-7061(97)00057-5
[13] D. M. Ziegler, “Flavin-Containing Monooxygenases: Catalytic Mechanism and Substrate Specificities,” Drug Metabolism Reviews, Vol. 19, No. 1, 1988, pp. 1-32. doi:10.3109/03602538809049617
[14] V. L. Burnett, M. P. Lawton and R. M. Philpot, “Cloning and Sequencing of Flavin-Containing Monooxygenases FMO3 and FMO4 from Rabbit and Characterization of FMO3,” Journal of Biological Chemistry, Vol. 269, No. 19, 1994, pp. 14314-14322.
[15] R. N. Hines, J. R. Cashman, R. M. Philpot, D. E. Williams and D. M. Ziegler, “The Mammalian Flavin-Containing Monooxygenases: Molecular Characterization and Regulation of Expression,” Toxicology Applied Pharmacology, Vol. 125, No. 1, 1994, pp. 1-6. doi:10.1006/taap.1994.1042
[16] D. M. Ziegler, “An Overview of the Mechanism, Substrate Specificities, and Structure of FMOs,” Drug Metabolism Reviews, Vol. 34, No. 3, 2002, pp. 503-511. doi:10.1081/DMR-120005650
[17] V. Lattard, C. Longin-Sauvageon, S. K. Krueger and D. E. Williams, “The FMO2 Gene of Laboratory Rats, as in Most Humans, Encodes a Truncated Protein,” Biochemical Biophysical Research Communications, Vol. 292, No. 2, 2002, pp. 558-563. doi:10.1006/bbrc.2002.6656
[18] F. Kasuya, K. Igarashi and M. Fukui, “Metabolism of Chlorpheniramine in Rat and Human by Use of Stable Isotopes,” Xenobiotica, Vol. 21, No. 1, 1991, pp. 97-109. doi:10.3109/00498259109039454
[19] R. J. Krause, S. L. Ripp, P. J. Sausen, L. H. Overby, R. M. Philpot and A. A. Elfarra, “Characterization of the Methionine S-Oxidase Activity of Rat Liver and Kidney Microsomes: Immunochemical and Kinetic Evidence for FMO3 Being the Major Catalyst,” Archives Biochemistry Biophysics, Vol. 333, No. 1, 1996, pp. 109-116. doi:10.1006/abbi.1996.0370

  
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