Contribution of cytochrome P450 metabolites to bradykinin-induced vasodilation in endothelial NO synthase deficient mouse hearts

Abstract

We have characterized the contribution of endothelial nitric oxide synthase (eNOS), cyclo-oxygenase (COX) and cytochrome P450 (CYP450) to the bradykinin (BK)- induced vasodilation in isolated hearts from wildtype (WT) and eNOS deficient mice (eNOS-/-). The endothelium-dependent vasodilation by bradykinin (BK, 1 microM) was significantly lower in eNOS-/- hearts than that in WT hearts (+247% and +325% of basal flow, respectively), while there was no difference in the endothelium-independent vasodilation by adenosine. In WT hearts, the BK-induced vasodilation was markedly attenuated following inhibition of NOS with ETU (10 microM) but not after COX inhibition with diclofenac (3 microM) (P<0.01). In line with this finding, Bk did not increase the cardiac prostacyclin release as measured by ELISA for 6-keto-PGF1alpha in the coronary venous effluent. In eNOS-/- hearts, the flow response to BK was insensitive to both NOS and COX inhibition. The NOS/COX-independent vasodilatory factor which remained under L-NMMA+DF application was almost completely eliminated by either clotrimazole (3 microM), miconazole (0.5 microM) or 17-ODYA (1 microM), suggesting that it was a metabolite of CPY450 enzymes. Sulfaphenazole (10 microM), a CYP450 2C inhibitor, exerted only a minimal inhibitory effect. In eNOS-/- hearts the effect of CYP450 inhibitors on the BK response was significantly more pronounced than in WT hearts, indicating an enhanced contribution of CYP450 enzymes. These findings suggest that in isolated mouse hearts the BK-induced vasodilation is mediated by NO and CYP450 metabolites but not by prostaglandins. The CYP450 dependent vasodilator was was functionally up-regulated in eNOS-/- hearts and thus likely to compensate for the loss of eNOS in the coronary circulation.

Figure 1

Coronary vasodilation induced by BK (1 μM; light grey) and adenosine (1 μM dark grey). When applied repeatedly with time intervals of 28 – 35 min, both vasodilators elicited similar vasodilatory responses.

Figure 2

Effects of NOS (A) and COX (B) inhibition on BK-induced vasodilation. (A) acute NOS inhibition by ETU (10 μM) significantly reduced basal and the BK-induced flow response in WT hearts (n=6) but had no effects in eNOS−/− hearts (n=5). (B) COX inhibition by diclofenac (3 μM) did not alter the BK-induced flow response both in WT (n=6) and eNOS−/− hearts (n=5). ^**P<0.01 vs control; #P<0.05 vs WT.

Figure 3

Effects of CYP450 inhibitors on BK-induced vasodilation. The L-NMMA+DF insensitive vasodilation induced by BK was completely eliminated by each of the three CYP450 inhibitors (clotrimazole, n=4; miconazole, n=5 or 17-ODYA, n=5), while the endothelium-independent vasodilation to adenosine was not affected. The horizontal lines indicate the average of basal coronary flow. ^**P<0.01 vs control.

Figure 4

Effects of CYP450 inhibitors on BK-induced vasodilation in WT and eNOS−/− hearts. Clotrimazole (3 μM, A), Miconazole (0.5 μM, B), 17-ODYA (1 μM, C) and sulphaphenazole (10 μM) were tested in WT and in eNOS−/− hearts (n=5 – 9). ##P<0.01 vs control; #P<0.05 vs control; ^**P<0.01 and ^*P<0.05 vs flow reduction in wildtype, two-way ANOVA.