Abstract 426: Complete Deletion of All Nitric Oxide Synthase Genes Causes Activation of the Renin-Angiotensin-Aldosterone Axis in Mice in Vivo
Background: We have recently developed mice lacking all three nitric oxide synthase (NOS) genes: nNOS, iNOS, and eNOS (PNAS 2005). The triply n/i/eNOS−/− mice spontaneously developed coronary arteriosclerosis and acute myocardial infarction (AMI) with a shorter survival. To elucidate its molecular mechanism(s), in this study, we tested our hypothesis that the renin-angiotensin-aldosterone (RAA) axis is activated in those mice.
Methods and Results: Experiments were performed in 3-month-old male wild-type (WT), singly nNOS−/−, iNOS−/−, eNOS−/−, and triply n/i/eNOS−/− mice. Plasma renin activity (ng/ml/hr, RIA) and plasma aldosterone levels (pg/mL, ELISA) were both significantly elevated only in the triply n/i/eNOS−/− mice (39±5 and 561±14), but not in any singly nNOS−/− (22±8 and 341±34), iNOS−/− (22±3 and 372±14), or eNOS−/− mice (29±2 and 366±40), as compared with the WT mice (22±3 and 315±17) (both P<0.05, n=5–7). Furthermore, cardiac angiotensin-converting enzyme activity (unit/g protein, Kasahara method) and cardiac angiotensin II type 1 receptor (AT1) expression (/actin, Western blotting) were also both significantly enhanced only in the triply n/i/eNOS−/− mice (140±16 and 38±6) compared with the WT mice (140±16 and 38±6) (both P<0.05, n=5– 6). In addition, plasma angiotensin II levels (nmol/L, RIA) were highest in the triply n/i/eNOS−/− mice, while all the singly nNOS−/− (86±25), iNOS−/− (85±7), eNOS−/− (78±20), and the triply n/i/eNOS−/− (136±21) mice showed significantly higher plasma angiotensin II levels as compared with the WT mice (37±7) (all P<0.05, n=5– 8). Importantly, long-term oral treatment with olmesartan (AT1 blocker, 5 mg/kg/day) for 10 months significantly inhibited coronary arteriosclerosis (intima/media ratio, 1.58±0.04 to 0.01±0.01), reduced the incidence of AMI (100% to 42%), and improved the prognosis of those mice (15% to 61%) (all P<0.05, n=6–12).
Conclusions: These results demonstrate that complete deficiency of all NOSs results in activation of the RAA axis in mice in vivo. Our findings also indicate that this mechanism could play a role in the pathogenesis of AMI in the presence of defective NOS system, suggesting the therapeutic importance of the RAA axis inhibitors to prevent AMI in humans.