Effects of NO on the expression and function of bradykinin type 2 receptors. Role for angioedema

Purpose: The pathophysiology of angioedema induced by angiotensin-converting enzyme inhibitors (ACEi) is not well understood. While activation of bradykinin (BK) type 2 receptors (B2) has been established, the triggering events remain obscure. In this study we investigated effect of NO as one product of B2 stimulation. Methods: Brain (bEND3), human umbilical vein (HUVEC) and porcine aortic endothelial cells (PAEC) were incubated with the NO-donors DEA/NO (10μM), DETA/NO (100μM) and SNAP (1-100μM) for 3, 6 and 12 h. B2 expression was assessed by RT-PCR and westernblot using monoclonal B2 antibodies staining either glycosylated or unglycosylated B2. Function of B2 was determined by monitoring iCa2+ upon activation with BK. Likewise, B2 protein levels were evaluated in aorta, heart and lung of (1) two different eNOS-/- strains, (2) mice with endothelial-specific overexpression of eNOS (eNOS-tg), (3) in C57Bl/6 mice treated with NOS inhibitor L-nitroarginine (L-NA). Here, B2 function was evaluated in aortic rings. Results: After 3h incubation with DEA/NO of bEND3, B2-mRNA was unchanged (160±32%, n=6, P>0.05). Likewise, B2 protein levels remained stable (98±13.6%, n=8, P>0.05). Similar results were obtained in PAECs and HUVECs and after extended incubation time and increased concentration. There was also a similar rise of iCa2+ in response to 0.01-100 μM BK in bEND3 treated with DEA/NO or vehicle as demonstrated by almost identical flourescence signals. These results obtained in cell lines and primary cultured PAEC were confirmed using transgenic mice with different levels of vascular NO-bioavailability. In aorta, neither the complete lack of endothelial NO in eNOS-/- (93±26%, n=5, P>0.05), nor overexpression in eNOS-tg (112±13.7%, n=10, P>0.05) changed B2 protein expression and a similar observation was made in C56Bl/6 treated with L-NA (110±19.4%, n=5, P>0.05). Aortic constrictor responses to 10 μM BK (related to maximal constriction to KCl) were identical in C57Bl/6 (24.3±2.7%, n=7) and eNOS-tg (30±11.8%, n=4, P>0.05). However, B2 signaling involved generation of NO as constrictor responses were significantly increased following L-NA in both C57BL/6 (64±18.3%, n=4) and eNOS-tg (67±3.6%, n=4) and a similar constriction was observed in eNOS-/- without (61±10.9%, n=7) and with L-NA (61±14.6%, n=6). None of these responses differed significantly. Conclusion: These data demonstrate that NO does not regulate vascular B2 gene expression and function suggesting that changes of B2 expression by this product of B2 activation are unlikely involved in triggering ACEi-induced angioedema.