3.3 The in vitro NO production induced by BK-(1-7) or BK-(1-5) is not mediated by activation of the kinin receptors
To evaluate whether BK-(1-7) or BK-(1-5) were agonists at the kinin receptors in vitro, we pre-incubated the cells with a selective antagonist for B1 receptors, Lys-des-Arg9-BK-(1-9) or a selective antagonist for B2 receptors, HOE-140. In this manuscript we will also refer to them as B1RB or B2RB, respectively, standing for B1 or B2 receptor blockage. In human glioblastoma cell line (U-87 MG), NO production induced by BK-(1-9) was only abolished by the selective B2 receptor antagonist (Figure 4A). However, the NO production induced by BK-(1-7) (Figure 4B) or BK-(1-5) (Figure 4C) were not affected by neither B1nor B2 receptor antagonists. We observed the same response profile in neonatal rat cardiomyocytes, where the response to BK-(1-9) (Figure 4D) was abolished by the selective antagonist of B2 receptors. No alteration of NO production induced by BK-(1-7) (Figure 4E) or BK-(1-5) (Figure 4F) was observed when the cells were incubated with antagonists of B1 or B2 receptors. These results suggest that the two fragments of BK-(1-9) does not act upon the activation of B1 and/or B2 receptors to induce NO production in vitro.
Male mice with knockout for either B1 (B1RKO) or B2 receptors (B2RKO) were used to further study our initial results. We found that BK-(1-9) induced NO production only in ventricular cardiomyocytes isolated from B1RKO mice (Figure 4G). On the other hand, BK-(1-7) (Figure 4H) and BK-(1-5) (Figure 4I) induced NO production in ventricular cardiomyocytes isolated from either B1RKO or B2RKO mice.
We employed qPCR to confirm that the cells used in this study expressed the mRNA for B1 and B2 receptors. As shown in Figure 4J, the mRNA for both receptors was highly expressed in U-87 MG cells, although the expression level of B1receptor mRNA was at least twice higher than the B2receptor mRNA. The mRNA for B2 receptors, but not that for B1 receptors, was detected in neonatal male rat cardiomyocytes (Figure 4K) and in male adult mouse ventricular myocytes (Figure 4L). Taken together, these results indicate that BK-(1-7) and BK-(1-5) do not act via B1 or B2 receptors to stimulate NO production in vitro.