Determination of the dominant arachidonic acid cytochrome p450 monooxygenases in rat heart, lung, kidney, and liver: protein expression and metabolite kinetics

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.

Fig. 1

P450 protein expression in heart, lung, kidney, and liver. Microsomal protein was isolated from the heart, lung, kidney, and liver and separated on a 10% SDS-PAGE. CYP2B1/2, CYP2C11, CYP2C23, CYP2J3, CYP4A1/2/3, CYP4Fs, and actin proteins were detected by the enhance chemiluminescence method. The graph represents the amount of protein normalized to the loading control (mean ± SEM, n = 3), and the results are expressed as a percentage of the liver protein expression value (*p < 0.05) compared with the liver

Fig. 2

Kinetic profile of arachidonic acid metabolism by P450 epoxygenases in the heart, lung, kidney, and liver microsomal incubates. In a 200-μL total volume, 100 (heart and lung) and 200 μg (kidney and liver) of microsomal protein pooled from five rats were incubated with arachidonic acid for 30 min for heart and lung and 15 min for kidney and liver. The experimental values for arachidonic acid metabolism were expressed as mean ± SEM. Each point was measured in triplicate

Fig. 3

Kinetic profile of arachidonic acid metabolism by P450 hydroxylases in the heart, lung, kidney, and liver microsomal incubates. In a 200-μL total volume, 100 (heart and lung) and 200 μg (kidney and liver) of microsomal protein pooled from five rats were incubated with arachidonic acid for 30 min for heart and lung and 15 min for kidney and liver. The experimental values for arachidonic acid metabolism were expressed as mean ± SEM. Each point was measured in triplicate

Fig. 4

Regioselectivity of arachidonic acid epoxygenation and hydroxylation by the heart, lung, kidney, and liver microsomal fraction. The regioselectivity was determined using different AA concentrations ranging between 16 and 81 μM. Data were expressed as mean ± SEM. The y-axis indicates the percentage of metabolite formation to total investigated P450-derived arachidonic acid metabolites formation