Fonction, expression et localisation cellulaire du récepteur B1 des kinines chez le rat diabétique
Kinins are vasoactive and pro-inflammatory peptides involved in a number of biological and inflammatory processes. They act on two types of receptors coupled to G proteins, GPCR, called B1 and B2 receptors. Although B2R plays a protective role in the cardiovascular system through the activation of endothelial nitric oxide synthase (eNOS), B1R is associated to insulin resistance, an early event in type 2 diabetes. This is mediated through the regulation and activation of the inducible nitric oxide synthase (iNOS) and increased production of superoxide anion (O2●‒) involving NADPH oxidase in the vascular system of diabetic rats. In two studies conducted in parallel, we considered the hypothesis that the inhibition of: a) iNOS (article 1), or b) kininase 1 (carboxypeptidase M, CPM), the key enzyme involved in the synthesis of endogenous B1R agonists (article 2), exerts the same beneficial effects as the B1R antagonist (SSR240612) in insulin resistance. Male Sprague-Dawley rats were made insulin resistant with a drinking solution containing 10% D-glucose for a period of 9 weeks. Control rats received tap water. We therefore evaluated the effects of a one-week treatment with 1400W, a selective inhibitor of iNOS, or with Mergetpa, a non-selective inhibitor of CPM. Rats fed a high glucose diet displayed hyperglycemia, hyperinsulinemia, insulin resistance, body weight gain and increased expression of inflammatory markers (B1R, CPM, iNOS and IL-1β) in the renal cortex, the aorta and the liver. Markers of oxidative stress (superoxide anion and nitrotyrosine) also increased in various tissues including the vascular system. Both treatments with 1400W and Mergetpa reversed and normalized most of these alterations. These data showed that the pharmacological blockade of iNOS or kininase1 (CPM) has similar beneficial effects to a 1-week treatment with a B1R antagonist (SSR240612). In the model of insulin resistant rats, overexpression of B1R increases O2●‒ production by activation of NADPH oxidase, whereas antagonism of B1R prevents this production in the aorta (Dias et al., 2010; Dias et al., 2012 b). This last observation led us to hypothesize that B1R is colocalised with NADPH oxidase, which was the subject of a third study (article 3) in which we demonstrated that B1R is colocalised with NADPH oxidase and its activation increases the production of O2●‒ via protein kinase C. Male rats were made diabetic with a single dose of streptozotocin (STZ), a model of type 1 diabetes. Two weeks later, O2●‒ production was measured in the aorta rings in response to the B1R agonist, by the lucigenin chemiluminescence method. Many inhibitors have been added to block total PKC (Ro-31-8220), PKCβ1/2 (LY333531) or NADPH oxidase (diphenyleneiodonium). The cellular localization of B1R with NADPH oxidase was studied in the aorta, popliteal artery, glomerulus and renal arteries by immunofluorescence and confocal microscopy with markers of endothelial cells, macrophages and vascular smooth muscle cells. Although B1R has been widely found in all these cell types, it has rarely been found in the endothelium of the aorta. The maximal response to the B1R agonist to enhance O2●‒ was inhibited by all inhibitors. Finally, the immunofluorescent intensity of labelling of B1R, NOX1 and NOX2 (isoforms of NADPH oxidase) was significantly increased in endothelial cells, vascular smooth muscle cells, and macrophages in the diabetic aorta where they displayed cellular colocalization. . The results of this last study show that B1R colocalizes with NADPH oxidase on the same cell type and increases the production of O2●‒ via PKC β1/2 (isoform of PKC). This suggests that the detrimental role of B1R manifest itself in part by an increase in oxidative stress from invasive immune cells in diabetes. Therefore, B1R represents a potential therapeutic target in the treatment of diabetes.
GPCR diabetes type 1 and 2 kinin receptors insulin resistance iNOS CPM NADPH oxidase PKC iNOS CPM PKC