Abstract 861: Acute Activation of Inducible Nitric Oxide Synthase (iNOS) by B1 Receptor Signaling Generates “Super-High Output” NO in Human Endothelial Cells
NO mediates normal vascular functions at low concentrations generated by receptor-mediated activation of eNOS. Inflammatory conditions upregulate iNOS expression, which is thought to generate unregulated, “high output” NO that can cause tissue damage. We studied inflammatory endothelial NO production by pre-treating human lung microvascular endothelial cells (HLMVEC) with cytokines (20 ng/ml IL-1β + 200 U/ml interferon-γ) for 16 h, which increased iNOS expression by 7-fold. Cells were then incubated in Arg-free medium for 2 h and after addition of 1 mM Arg, NO output was measured in real time with a porphyrinic electrode. Arg generated a prolonged peak of “high output” NO (maximum = 295 ± 22 nM NO at 40 – 60 min) that lasted 80 –90 min and was due to iNOS as 4 μM 1400W blocked it. Cytokine-treated HLMVEC also expressed B1 kinin receptors (B1R). A B1 R agonist, 100 nM des-Arg10-kallidin (DAKD), produced “super-high output” NO (maximum = 832 ± 37 nM) with a total output over 80 min almost 3-fold higher than Arg alone. Surprisingly, this was due to activation of iNOS as 4 μM 1400W blocked it. Angiotensin converting enzyme inhibitors can also directly activate B1R and 100 nM enalaprilat stimulated a similar iNOS response. B1R-dependent activation of iNOS was mediated by Gαi as pertussis toxin inhibited it. Furthermore, cytokine-treated HLMVEC transfected with a constitutively active Gαi QL mutant (to bypass B1R) generated super-high output NO (450 ± 40 nM) in response to 1 mM Arg (non-transfected cells = 200 ± 25 nM NO) but did not generate additional NO after B1R agonist. βγ signaling was also involved; transfection with the C-terminus of the β-adrenergic receptor kinase to scavenge βγ inhibited the response whereas the βγ activating peptide mSIRK stimulated NO production, but inhibited further B1R-mediated NO. Downstream mediators of the response include Src, Raf, and Erk1/2 as B1R mediated NO production was inhibited by the Src inhibitor PP2, Raf kinase inhibitor I, and the MEK inhibitor PD 98059. B1R agonist also activated Erk1/2 in cytokine-treated HLMVEC, with a time course similar to that of NO production. Thus, a novel B1R signaling pathway leads to acute activation of iNOS and “super-high output” NO that could affect the endothelial barrier under inflammatory conditions.