Angiotensin II causes nitric oxide synthase 3 uncoupling in the thick ascending limb

Abstract

In the thick ascending limb (TAL) luminal flow increases NO production which inhibits flow‐mediated O2− production via a cGMP/PKG dependent pathway. Angiotensin II (Ang II) acutely stimulates TAL NO production by activating the AT2 receptor and stimulates O2− production via the AT1 receptor. We hypothesized that Ang II‐stimulated NO inhibits Ang II‐stimulated O2− via a cGMP‐dependent process. We measured NO production by fluorescence microscopy in isolated TAL. NO production was increased by 0.025 ± 0.004, 0.023 ± 0.014, 0.028 ± 0.007 arbitrary units (AU)/sec by 1, 10 and 100 pM Ang II, respesctively. Scavenging O2− with tempol did not affect Ang II‐stimulated NO production. We then measured O2− production in isolated TAL using a lucigenin‐based assay. Ang II (1 nM) increased O2− production by 1.77 ± 0.26 to 2.62 ± 0.36 AU (p<0.01). Adding L‐arginine (L‐Arg), the substrate for NO production, had no effect on angiotensin II‐induced O2−. In the absence of L‐Arg the NOS inhibitor, L‐NAME, reduced Ang II‐stimulated O2− by 0.82 ± 0.31 AU (p<0.05). In the presence of L‐Arg L‐NAME reduced O2− production by 0.71 ± 0.24 AU, however L‐NAME had no effect on basal O2− production. We conclude that: 1) Ang II increases both O2− production and NO production; 2) unlike flow‐induced NO, Ang II‐induced NO production does not inhibit Ang II‐induced O2− production; and 3) Ang II causes NOS uncoupling. Supported by NIH 5P01HL028982.