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. 2007 Apr;35(4):682-8.
doi: 10.1124/dmd.106.012823. Epub 2007 Jan 12.

Detection of human CYP2C8, CYP2C9, and CYP2J2 in cardiovascular tissues

Tracy C Delozier  1 , Grace E Kissling, Sherry J Coulter, Diana Dai, Julie F Foley, J Alyce Bradbury, Elizabeth Murphy, Charles Steenbergen, Darryl C Zeldin, Joyce A Goldstein
Affiliations

Detection of human CYP2C8, CYP2C9, and CYP2J2 in cardiovascular tissues

Tracy C Delozier et al. Drug Metab Dispos. 2007 Apr.

Abstract

The cytochrome P450 (P450) enzymes CYP2C8, CYP2C9, and CYP2J2 metabolize arachidonic acid to epoxyeicosatrienoic acids, which are known to be vital in regulation of vascular tone and cardiovascular homeostasis. Because there is limited information regarding the relative expression of these P450 enzymes in cardiovascular tissues, this study examined the expression of CYP2C8, CYP2C9, and CYP2J2 mRNA and protein in human heart, aorta, and coronary artery samples by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. CYP2J2 and CYP2C9 mRNA levels were highly variable in human hearts, whereas CYP2C8 mRNA was present in lower abundance. CYP2J2 mRNA was approximately 10(3) times higher than CYP2C9 or CYP2C8 in human heart. However, CYP2C9 mRNA was more abundant than CYP2J2 or CYP2C8 in one ischemic heart. In human aorta, mean CYP2C9 mRNA levels were approximately 50 times higher than that of CYP2J2 and 5-fold higher than that of CYP2C8. In human coronary artery, mean values for CYP2C9 mRNA were approximately 2-fold higher than that of CYP2J2 mRNA and 6-fold higher than that of CYP2C8 mRNA. Immunoblotting results show relatively high levels of CYP2J2 and CYP2C8 protein in human hearts, which was confirmed by immunohistochemistry. CYP2C9 protein was also detected at high levels in one ischemic heart by immunoblotting. CYP2C9 was present at higher levels than CYPJ2 in aorta and coronary artery, whereas CYP2C8 protein was below the limits of detection. The expression of CYP2J2 and CYP2C8 in human heart, and CYPC9 and CYP2J2 in aorta and coronary artery is consistent with a physiological role for these enzymes in these tissues.

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Figures

Figure 1
Figure 1
(A) Expression of CYP2C8, CYP2C9 and CYP2J2 in human heart by real–time PCR. GAPDH was used as an internal control to normalize all target values. Derivations of triplicate samples are shown and experimental data are mean ± SE. (B) Expression of CYP2C8, CYP2C9 and CYP2J2 in human aorta by real–time PCR. (C) Expression of CYP2C8, CYP2C9 and CYP2J2 human coronary artery tissues by real–time PCR. aCYP2J2 greater than CYP2C8, p<0.05; bCYP2J2 greater than CYP2C9 p<0.05, cCYP2C9 greater than CYP2J2 p<0.05; dCYP2C9 greater than CYP2C8, p<0.05 (pairwise comparisons were performed using Mann-Whitney one-sided p-values).
Figure 1
Figure 1
(A) Expression of CYP2C8, CYP2C9 and CYP2J2 in human heart by real–time PCR. GAPDH was used as an internal control to normalize all target values. Derivations of triplicate samples are shown and experimental data are mean ± SE. (B) Expression of CYP2C8, CYP2C9 and CYP2J2 in human aorta by real–time PCR. (C) Expression of CYP2C8, CYP2C9 and CYP2J2 human coronary artery tissues by real–time PCR. aCYP2J2 greater than CYP2C8, p<0.05; bCYP2J2 greater than CYP2C9 p<0.05, cCYP2C9 greater than CYP2J2 p<0.05; dCYP2C9 greater than CYP2C8, p<0.05 (pairwise comparisons were performed using Mann-Whitney one-sided p-values).
Figure 1
Figure 1
(A) Expression of CYP2C8, CYP2C9 and CYP2J2 in human heart by real–time PCR. GAPDH was used as an internal control to normalize all target values. Derivations of triplicate samples are shown and experimental data are mean ± SE. (B) Expression of CYP2C8, CYP2C9 and CYP2J2 in human aorta by real–time PCR. (C) Expression of CYP2C8, CYP2C9 and CYP2J2 human coronary artery tissues by real–time PCR. aCYP2J2 greater than CYP2C8, p<0.05; bCYP2J2 greater than CYP2C9 p<0.05, cCYP2C9 greater than CYP2J2 p<0.05; dCYP2C9 greater than CYP2C8, p<0.05 (pairwise comparisons were performed using Mann-Whitney one-sided p-values).
Figure 2
Figure 2
Immunoblotting of human heart microsomes and aorta and coronary artery lysates utilizing CYP2C8, CYP2C9 and CYP2J2 antibodies (A)- human heart microsomes, (B)- human aorta tissue lysates, (C)- human coronary artery tissue lysates.
Figure 2
Figure 2
Immunoblotting of human heart microsomes and aorta and coronary artery lysates utilizing CYP2C8, CYP2C9 and CYP2J2 antibodies (A)- human heart microsomes, (B)- human aorta tissue lysates, (C)- human coronary artery tissue lysates.
Figure 2
Figure 2
Immunoblotting of human heart microsomes and aorta and coronary artery lysates utilizing CYP2C8, CYP2C9 and CYP2J2 antibodies (A)- human heart microsomes, (B)- human aorta tissue lysates, (C)- human coronary artery tissue lysates.
Figure 3
Figure 3
(A) Hematoxylin and Eosin staining of heart G. (B) IHC staining for CYP2J2 in heart (20x). Endothelial cells are stained (arrow) while staining is absent in the negative control, normal immune serum, (inset). (C) IHC staining for CYP2C8 in heart (20x). Patchy cytoplasmic staining is observed in the myocytes. Endothelial cells are also stained (arrow). Staining is absent in the negative control, normal immune serum, (inset).
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