Direct stimulation of angiotensin II type 2 receptor reduces nitric oxide production in lipopolysaccharide treated mouse macrophages.

The angiotensin II type 2 receptor (AT) is upregulated after tissue damage and mediates protective functions in the renin-angiotensin-aldosterone system (RAAS). One of these is to inhibit inducible nitric oxide synthase (iNOS) in activated macrophages. In the present study, we assessed the effect of AT receptor ligands on nitric oxide production in murine macrophages as a potential assay to determine the functional activity of an AT receptor ligand. Mouse macrophage J744.2 and RAW264.7 were cultivated in lipopolysaccharide (LPS) to induce M1 differentiation and increase iNOS expression. Using Griess reagent and spectrophotometric analysis, the nitric oxide levels were determined, while employing Western blot and immunocytochemistry to determine basal protein expression. Using the first reported selective non-peptide AT receptor agonist, compound C21, we conclude that activation of AT receptor reduces nitric oxide production in M1 macrophages. Furthermore, the AT receptor selective ligand compound C38, a regioisomer of C21, reported as a selective AT receptor antagonist exhibits a similar effect on nitric oxide production. Thus, we propose C38 acts as a partial agonist in the macrophage system. Monitoring nitric oxide attenuation in M1 J744.1 and RAW264.7 macrophages provides a new method for characterizing functional activity of AT receptor ligands, foreseen to be valuable in future drug discovery programs.