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

Erik Elebring,Mathias Hallberg,Anna Casselbrant,Mats Larhed,Rebecka Isaksson,Lars Fändriks

doi:10.1016/j.ejphar.2019.172855

Erik Elebring, Mathias Hallberg + Show 4 more

Open Access

https://doi.org/10.1016/j.ejphar.2019.172855

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Abstract

The angiotensin II type 2 receptor (AT2) 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 AT2 receptor ligands on nitric oxide production in murine macrophages as a potential assay to determine the functional activity of an AT2 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 AT2 receptor agonist, compound C21, we conclude that activation of AT2 receptor reduces nitric oxide production in M1 macrophages. Furthermore, the AT2 receptor selective ligand compound C38, a regioisomer of C21, reported as a selective AT2 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 AT2 receptor ligands, foreseen to be valuable in future drug discovery programs.

Highlights

The octa-peptide angiotensin II (AngII) is the major effector of the renin-angiotensin-aldosterone system (RAAS)
RAAS is well known for its role in blood pressure regulation and fluid-electrolyte balance, an effect exerted by the AngII type 1 receptor (AT1) (Raizada et al, 1993)
Mouse macrophage cell line J774.2 and RAW264.7 was purchased from SigmaAldrich (Stockholm, Sweden; cat. no. 85011428 and 91062702) and the cells were cultured in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum, 1% non-essential amino acids, and 100 IU/ml penicillin-streptomycin

Summary

Introduction

The octa-peptide angiotensin II (AngII) is the major effector of the renin-angiotensin-aldosterone system (RAAS). RAAS is well known for its role in blood pressure regulation and fluid-electrolyte balance, an effect exerted by the AngII type 1 receptor (AT1) (Raizada et al, 1993). During tissue damage the AT2 receptor is upregulated (Altarche-Xifro et al, 2009; Busche et al, 2000; Gallinat et al, 1998; Li et al, 2005; Nakajima et al, 1995; Nio et al, 1995), and it has been shown to inhibit cell proliferation, cause vasodilation, exhibit neuronal protective and regenerative properties, be involved in apoptosis, and modulate inflammation (Lu et al, 2004; Ruiz-Ortega et al, 2000; Steckelings et al, 2010; Sumners et al, 2015; Suzuki et al, 2003; Uhal et al, 1998; Unger et al, 2015; Wang et al, 1998; Yamada et al, 1996). The AT2 receptor has emerged as a promising new drug target (Foulquier et al, 2013; Padia and Carey, 2013; Unger et al, 2015)

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