Skip to main content
SpringerLink
Log in

Determination of the Dominant Arachidonic Acid Cytochrome P450 Monooxygenases in Rat Heart, Lung, Kidney, and Liver: Protein Expression and Metabolite Kinetics

  • Research Article
  • Published:
The AAPS Journal Aims and scope Submit manuscript

Abstract

Cytochrome P450 (P450)-derived arachidonic acid (AA) metabolites serve pivotal physiological roles. Therefore, it is important to determine the dominant P450 AA monooxygenases in different organs. We investigated the P450 AA monooxygenases protein expression as well as regioselectivity, immunoinhibition, and kinetic profile of AA epoxygenation and hydroxylation in rat heart, lung, kidney, and liver. Thereafter, the predominant P450 epoxygenases and P450 hydroxylases in these organs were characterized. Microsomes from heart, lung, kidney, and liver were incubated with AA. The protein expression of CYP2B1/2, CYP2C11, CYP2C23, CYP2J3, CYP4A1/2/3, and CYP4Fs in the heart, lung, kidney, and liver were determined by Western blot analysis. The levels of AA metabolites were determined by liquid chromatography–electrospray ionization mass spectroscopy. This was followed by determination of regioselectivity, immunoinhibition effect, and the kinetic profile of AA metabolism. AA was metabolized to epoxyeicosatrienoic acids and 19- and 20-hydroxyeicosatetraenoic acid in the heart, lung, kidney, and liver but with varying metabolic activities and regioselectivity. Anti-P450 antibodies were found to differentially inhibit AA epoxygenation and hydroxylation in these organs. Our data suggest that the predominant epoxygenases are CYP2C11, CYP2B1, CYP2C23, and CYP2C11/CYP2C23 for the heart, lung, kidney, and liver, respectively. On the other hand, CYP4A1 is the major ω-hydroxylase in the heart and kidney; whereas CYP4A2 and/or CYP4F1/4 are probably the major hydroxlases in the lung and liver. These results provide important insights into the activities of P450 epoxygenases and P450 hydroxylases-mediated AA metabolism in different organs and their associated P450 protein levels.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

REFERENCES

  1. Panigrahy D, Kaipainen A, Greene ER, Huang S. Cytochrome P450-derived eicosanoids: the neglected pathway in cancer. Cancer Metastasis Rev. 2010;29(4):723–35.

    Article  PubMed  CAS  Google Scholar 

  2. Roman RJ. P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev. 2002;82(1):131–85.

    PubMed  CAS  Google Scholar 

  3. Sudhahar V, Shaw S, Imig JD. Epoxyeicosatrienoic acid analogs and vascular function. Curr Med Chem. 2010;17(12):1181–90.

    Article  PubMed  CAS  Google Scholar 

  4. Keseru B, Barbosa-Sicard E, Popp R, Fisslthaler B, Dietrich A, Gudermann T, et al. Epoxyeicosatrienoic acids and the soluble epoxide hydrolase are determinants of pulmonary artery pressure and the acute hypoxic pulmonary vasoconstrictor response. FASEB J. 2008;22(12):4306–15.

    Article  PubMed  Google Scholar 

  5. Pokreisz P, Fleming I, Kiss L, Barbosa-Sicard E, Fisslthaler B, Falck JR, et al. Cytochrome P450 epoxygenase gene function in hypoxic pulmonary vasoconstriction and pulmonary vascular remodeling. Hypertension. 2006;47(4):762–70.

    Article  PubMed  CAS  Google Scholar 

  6. Salvail D, Cloutier M, Rousseau E. Functional reconstitution of an eicosanoid-modulated Cl− channel from bovine tracheal smooth muscle. Am J Physiol Cell Physiol. 2002;282(3):C567–77.

    PubMed  CAS  Google Scholar 

  7. Roman RJ, Maier KG, Sun CW, Harder DR, Alonso-Galicia M. Renal and cardiovascular actions of 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids. Clin Exp Pharmacol Physiol. 2000;27(11):855–65.

    Article  PubMed  CAS  Google Scholar 

  8. Sacerdoti D, Gatta A, McGiff JC. Role of cytochrome P450-dependent arachidonic acid metabolites in liver physiology and pathophysiology. Prostaglandins Other Lipid Mediat. 2003;72(1–2):51–71.

    Article  PubMed  CAS  Google Scholar 

  9. Yoshida S, Hirai A, Tamura Y. Possible involvement of arachidonic acid metabolites of cytochrome P450 monooxygenase pathway in vasopressin-stimulated glycogenolysis in isolated rat hepatocytes. Arch Biochem Biophys. 1990;280(2):346–51.

    Article  PubMed  CAS  Google Scholar 

  10. Zou AP, Fleming JT, Falck JR, Jacobs ER, Gebremedhin D, Harder DR, et al. 20-HETE is an endogenous inhibitor of the large-conductance Ca(2+)-activated K+ channel in renal arterioles. Am J Physiol. 1996;270(1 Pt 2):R228–37.

    PubMed  CAS  Google Scholar 

  11. Aboutabl ME, Zordoky BN, El-Kadi AO. 3-methylcholanthrene and benzo(a)pyrene modulate cardiac cytochrome P450 gene expression and arachidonic acid metabolism in male Sprague Dawley rats. Br J Pharmacol. 2009;158(7):1808–19.

    Article  PubMed  CAS  Google Scholar 

  12. Yousif MH, Benter IF, Roman RJ. Cytochrome P450 metabolites of arachidonic acid play a role in the enhanced cardiac dysfunction in diabetic rats following ischaemic reperfusion injury. Auton Autacoid Pharmacol. 2009;29(1–2):33–41.

    Article  PubMed  CAS  Google Scholar 

  13. Lv X, Wan J, Yang J, Cheng H, Li Y, Ao Y, et al. Cytochrome P450 omega-hydroxylase inhibition reduces cardiomyocyte apoptosis via activation of ERK1/2 signaling in rat myocardial ischemia-reperfusion. Eur J Pharmacol. 2008;596(1–3):118–26.

    Article  PubMed  CAS  Google Scholar 

  14. Kroetz DL, Huse LM, Thuresson A, Grillo MP. Developmentally regulated expression of the CYP4A genes in the spontaneously hypertensive rat kidney. Mol Pharmacol. 1997;52(3):362–72.

    PubMed  CAS  Google Scholar 

  15. Certikova Chabova V, Kramer HJ, Vaneckova I, Thumova M, Skaroupkova P, Tesar V, et al. The roles of intrarenal 20-hydroxyeicosatetraenoic and epoxyeicosatrienoic acids in the regulation of renal function in hypertensive Ren-2 transgenic rats. Kidney Blood Press Res. 2007;30(5):335–46.

    Article  PubMed  CAS  Google Scholar 

  16. Jacobs ER, Effros RM, Falck JR, Reddy KM, Campbell WB, Zhu D. Airway synthesis of 20-hydroxyeicosatetraenoic acid: metabolism by cyclooxygenase to a bronchodilator. Am J Physiol. 1999;276(2 Pt 1):L280–8.

    PubMed  CAS  Google Scholar 

  17. Quigley R, Baum M, Reddy KM, Griener JC, Falck JR. Effects of 20-HETE and 19(S)-HETE on rabbit proximal straight tubule volume transport. Am J Physiol Ren Physiol. 2000;278(6):F949–53.

    CAS  Google Scholar 

  18. Zordoky BN, Aboutabl ME, El-Kadi AO. Modulation of cytochrome P450 gene expression and arachidonic acid metabolism during isoproterenol-induced cardiac hypertrophy in rats. Drug Metab Dispos. 2008;36(11):2277–86.

    Article  PubMed  CAS  Google Scholar 

  19. Imaoka S, Hashizume T, Funae Y. Localization of rat cytochrome P450 in various tissues and comparison of arachidonic acid metabolism by rat P450 with that by human P450 orthologs. Drug Metab Pharmacokinet. 2005;20(6):478–84.

    Article  PubMed  CAS  Google Scholar 

  20. Thum T, Borlak J. Gene expression in distinct regions of the heart. Lancet. 2000;355(9208):979–83.

    Article  PubMed  CAS  Google Scholar 

  21. Minamiyama Y, Takemura S, Akiyama T, Imaoka S, Inoue M, Funae Y, et al. Isoforms of cytochrome P450 on organic nitrate-derived nitric oxide release in human heart vessels. FEBS Lett. 1999;452(3):165–9.

    Article  PubMed  CAS  Google Scholar 

  22. Delozier TC, Kissling GE, Coulter SJ, Dai D, Foley JF, Bradbury JA, et al. Detection of human CYP2C8, CYP2C9, and CYP2J2 in cardiovascular tissues. Drug Metab Dispos. 2007;35(4):682–8.

    Article  PubMed  CAS  Google Scholar 

  23. Marji JS, Wang MH, Laniado-Schwartzman M. Cytochrome P-450 4A isoform expression and 20-HETE synthesis in renal preglomerular arteries. Am J Physiol Ren Physiol. 2002;283(1):F60–7.

    CAS  Google Scholar 

  24. Barakat MM, El-Kadi AO, du Souich P. L-NAME prevents in vivo the inactivation but not the down-regulation of hepatic cytochrome P450 caused by an acute inflammatory reaction. Life Sci. 2001;69(13):1559–71.

    Article  PubMed  CAS  Google Scholar 

  25. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193(1):265–75.

    PubMed  CAS  Google Scholar 

  26. Gharavi N, El-Kadi AO. tert-Butylhydroquinone is a novel aryl hydrocarbon receptor ligand. Drug Metab Dispos. 2005;33(3):365–72.

    Article  PubMed  CAS  Google Scholar 

  27. Nithipatikom K, Grall AJ, Holmes BB, Harder DR, Falck JR, Campbell WB. Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome P450 metabolites of arachidonic acid. Anal Biochem. 2001;298(2):327–36.

    Article  PubMed  CAS  Google Scholar 

  28. Powell PK, Wolf I, Jin R, Lasker JM. Metabolism of arachidonic acid to 20-hydroxy-5,8,11,14-eicosatetraenoic acid by P450 enzymes in human liver: involvement of CYP4F2 and CYP4A11. J Pharmacol Exp Ther. 1998;285(3):1327–36.

    PubMed  CAS  Google Scholar 

  29. Xu F, Falck JR, Ortiz de Montellano PR, Kroetz DL. Catalytic activity and isoform-specific inhibition of rat cytochrome p450 4F enzymes. J Pharmacol Exp Ther. 2004;308(3):887–95.

    Article  PubMed  CAS  Google Scholar 

  30. Brash AR. Arachidonic acid as a bioactive molecule. J Clin Invest. 2001;107(11):1339–45.

    Article  PubMed  CAS  Google Scholar 

  31. Wu S, Moomaw CR, Tomer KB, Falck JR, Zeldin DC. Molecular cloning and expression of CYP2J2, a human cytochrome P450 arachidonic acid epoxygenase highly expressed in heart. J Biol Chem. 1996;271(7):3460–8.

    Article  PubMed  CAS  Google Scholar 

  32. Capdevila JH, Falck JR, Harris RC. Cytochrome P450 and arachidonic acid bioactivation. Molecular and functional properties of the arachidonate monooxygenase. J Lipid Res. 2000;41(2):163–81.

    PubMed  CAS  Google Scholar 

  33. Choudhary D, Jansson I, Stoilov I, Sarfarazi M, Schenkman JB. Metabolism of retinoids and arachidonic acid by human and mouse cytochrome P450 1b1. Drug Metab Dispos. 2004;32(8):840–7.

    Article  PubMed  CAS  Google Scholar 

  34. Poloyac SM, Tortorici MA, Przychodzin DI, Reynolds RB, Xie W, Frye RF, et al. The effect of isoniazid on CYP2E1- and CYP4A-mediated hydroxylation of arachidonic acid in the rat liver and kidney. Drug Metab Dispos. 2004;32(7):727–33.

    Article  PubMed  CAS  Google Scholar 

  35. Laethem RM, Balazy M, Falck JR, Laethem CL, Koop DR. Formation of 19(S)-, 19(R)-, and 18(R)-hydroxyeicosatetraenoic acids by alcohol-inducible cytochrome P450 2E1. J Biol Chem. 1993;268(17):12912–8.

    PubMed  CAS  Google Scholar 

  36. Theken KN, Deng Y, Kannon MA, Miller TM, Poloyac SM, Lee CR. Activation of the acute inflammatory response alters cytochrome P450 expression and eicosanoid metabolism. Drug Metab Dispos. 2011;39(1):22–9 [Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov’t].

    Article  PubMed  CAS  Google Scholar 

  37. Nguyen X, Wang MH, Reddy KM, Falck JR, Schwartzman ML. Kinetic profile of the rat CYP4A isoforms: arachidonic acid metabolism and isoform-specific inhibitors. Am J Physiol. 1999;276(6 Pt 2):R1691–700.

    PubMed  CAS  Google Scholar 

  38. Carroll MA, Balazy M, Huang DD, Rybalova S, Falck JR, McGiff JC. Cytochrome P450-derived renal HETEs: storage and release. Kidney Int. 1997;51(6):1696–702.

    Article  PubMed  CAS  Google Scholar 

  39. Wu S, Chen W, Murphy E, Gabel S, Tomer KB, Foley J, et al. Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes. J Biol Chem. 1997;272(19):12551–9.

    Article  PubMed  CAS  Google Scholar 

  40. Holla VR, Makita K, Zaphiropoulos PG, Capdevila JH. The kidney cytochrome P-450 2C23 arachidonic acid epoxygenase is upregulated during dietary salt loading. J Clin Invest. 1999;104(6):751–60.

    Article  PubMed  CAS  Google Scholar 

  41. Imig JD. Epoxides and soluble epoxide hydrolase in cardiovascular physiology. Physiol Rev. 2012;92(1):101–30.

    Article  PubMed  CAS  Google Scholar 

  42. Capdevila JH, Karara A, Waxman DJ, Martin MV, Falck JR, Guenguerich FP. Cytochrome P-450 enzyme-specific control of the regio- and enantiofacial selectivity of the microsomal arachidonic acid epoxygenase. J Biol Chem. 1990;265(19):10865–71.

    PubMed  CAS  Google Scholar 

  43. Imig JD, Navar LG, Roman RJ, Reddy KK, Falck JR. Actions of epoxygenase metabolites on the preglomerular vasculature. J Am Soc Nephrol. 1996;7(11):2364–70.

    PubMed  CAS  Google Scholar 

  44. Ito O, Nakamura Y, Tan L, Ishizuka T, Sasaki Y, Minami N, et al. Expression of cytochrome P-450 4 enzymes in the kidney and liver: regulation by PPAR and species-difference between rat and human. Mol Cell Biochem. 2006;284(1–2):141–8.

    Article  PubMed  CAS  Google Scholar 

  45. Helvig C, Dishman E, Capdevila JH. Molecular, enzymatic, and regulatory characterization of rat kidney cytochromes P450 4A2 and 4A3. Biochemistry. 1998;37(36):12546–58.

    Article  PubMed  CAS  Google Scholar 

  46. Wang MH, Guan H, Nguyen X, Zand BA, Nasjletti A, Laniado-Schwartzman M. Contribution of cytochrome P-450 4A1 and 4A2 to vascular 20-hydroxyeicosatetraenoic acid synthesis in rat kidneys. Am J Physiol. 1999;276(2 Pt 2):F246–53.

    PubMed  CAS  Google Scholar 

  47. Imig JD. Epoxygenase metabolites. Epithelial and vascular actions. Mol Biotechnol. 2000;16(3):233–51.

    Article  PubMed  CAS  Google Scholar 

Download references

ACKNOWLEDGMENTS

The authors wish to thank Dr. Dion Brocks for his valuable comments on this manuscript. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR) MOP 106665 to AOSE. AAE is the recipients of Egyptian Government Scholarship. BNMZ is the recipient of Alberta Innovates-Health Solutions Studentship. AA-M is the recipient of Alberta Ingenuity Graduate Scholarship and Izaak Walton Killam Memorial Graduate Scholarship.

Conflict of Interest

The authors declare no conflict of interest.

Author information

Authors and Affiliations

  1. Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E1

    Ahmed A. El-Sherbeni, Beshay N. M. Zordoky, Anwar Anwar-Mohamed & Ayman O. S. El-Kadi

  2. Medicinal and Pharmaceutical Chemistry Department (Pharmacology Group), Pharmaceutical and Drug Industries Research Division, National Research Centre, 12311 Dokki, Cairo, Egypt

    Mona E. Aboutabl

Authors
  1. Ahmed A. El-Sherbeni
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mona E. Aboutabl
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Beshay N. M. Zordoky
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anwar Anwar-Mohamed
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ayman O. S. El-Kadi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ayman O. S. El-Kadi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

El-Sherbeni, A.A., Aboutabl, M.E., Zordoky, B.N.M. et al. Determination of the Dominant Arachidonic Acid Cytochrome P450 Monooxygenases in Rat Heart, Lung, Kidney, and Liver: Protein Expression and Metabolite Kinetics. AAPS J 15, 112–122 (2013). https://doi.org/10.1208/s12248-012-9425-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1208/s12248-012-9425-7

Key words

Search

Navigation