Localization and Interaction between Kinin B1 Receptor and NADPH Oxidase in the Vascular System of Diabetic Rats.

Youssef Haddad,Réjean Couture

doi:10.3389/fphys.2017.00861

Abstract

Kinin B1 receptor (B1R) enhanced superoxide anion () production in the vasculature of diabetic rats. This study investigates the induction and distribution of B1R in diabetic blood vessels and addresses the hypothesis that B1R is co-localized with NADPH oxidase (NOX1 and NOX2) and produces its activation via protein kinase C (PKC). Diabetes was induced in rats with streptozotocin (STZ 65 mg.kg−1, i.p.). Two weeks later, the production of was measured in aorta rings in response to the B1R agonist (Sar[D-Phe8]-des-Arg9-BK, 20 μM) by the method of lucigenin-enhanced chemiluminescence. Various inhibitors were added (10 μM) to block PKCtotal (Ro-31-8220), PKCβ1/2 (LY333531), or NADPH oxidase (Diphenyleneiodonium). The cellular localization of B1R was studied in the aorta, popliteal artery, and renal glomerulus/arteries by immunofluorescence and confocal microscopy with markers of endothelial cells (anti-RECA-1), macrophages (anti-CD11), vascular smooth muscle cells (anti-SMA), and NADPH oxidase (anti-NOX1 and NOX2). Although B1R was largely distributed in resistant vessels, it was sparsely expressed in the aorta's endothelium. The greater basal production of in STZ-diabetic aorta was significantly enhanced by the B1R agonist (15–45 min). The peak response to the agonist (30 min) was inhibited by all inhibitors. Immunofluorescent staining for B1R, NOX1, and NOX2 was significantly increased in endothelial cells, vascular smooth muscle cells, and macrophages of STZ-diabetic aorta on which they were found co-localized. Data showed that B1R enhanced by activating vascular NADPH oxidase through PKCβ1/2. This was substantiated by the cellular co-localization of B1R with NOX1 and NOX2 and opens the possibility that B1R-enhanced oxidative stress is derived from vascular and infiltrating immune cells in diabetes.

Highlights

Sustained hyperglycemia-induced oxidative stress exposes individuals to chronic, low-grade inflammation, and contributes to diabetes and its complications
To assess the activity of nicotnamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase in the production of O2− evoked by the B1 receptor (B1R) agonist, its enzyme substrate NADPH was added to the vials
Oxidase, diphenyleneiodonium (DPI, 10 μM), an inhibitor of NADPH oxidase (Massart et al, 2013; Song et al, 2015) was added to the vials 30 min prior to the addition of the B1R agonist. Data show that both the basal production of O2− at time 0 min and that induced by 30 min stimulation with the B1R agonist were significantly blocked by DPI, confirming that O2− derived from NADPH oxidase (Figure 2B)

Summary

Introduction

Sustained hyperglycemia-induced oxidative stress exposes individuals to chronic, low-grade inflammation, and contributes to diabetes and its complications. A recent concept involving B1R in the propagation of inflammation has been proposed in human vascular disease, which consists in the transfer to target organ recipient cells of a massive release of circulating leukocyte-derived microvesicles bearing functional B1R (Kahn et al, 2017)

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Discussion

Conclusion