Endothelial dysfunction is a major risk factor for vascular complications associated with diabetes. The purpose of this study was to evaluate the possible link between oxidative stress and alteration in calcium homeostasis in dysfunctional endothelial cells from db/db diabetic mice, 20 or 45 week old. Impaired flow‐induced dilation in mesenteric arteries and acetylcholine‐induced relaxation in aorta due to a decreased nitric oxide (NO) bioavailability were already shown in this model. For this study, mice aortic endothelial cells (MAEC) were isolated from vessel explants. Oxidative stress and Ca2+ homeostasis in MAEC were simultaneously recorded with DHE and Fura‐2 probes using an inverted epi‐fluorescence microscope. Compared to control mice, oxidative stress in MAEC from db/db mice increased with age leading to a progressive alteration of Ca2+ homeostasis, characterized by a rise in basal cytosolic calcium and a decrease in amplitude of acetylcholine‐induced calcium peak. Interestingly, a NO donor reduced oxidative stress and improved calcium signaling in endothelial cells from db/db mice. These results suggest that reduced NO bioavailability contributes to oxidative stress and abnormal calcium homeostasis in endothelial cells during progression of diabetes.
NO controls the activity of canonical transient receptor potential channel (TRPC) in endothelial cells. Our group already provided evidences that reduced NO bioavailability was directly linked to increased basal cytosolic Ca2+ in a cellular model of endothelial dysfunction. The aim of this study was to evaluate the role of TRPC3 channels in Ca2+ homeostasis of aortic porcine endothelial cells (PAEC). In PAEC at P1, producing NO, specific inhibition of TRPC3 by Pyr3 induced a concentration‐dependent decrease in Ca2+ entry as shown by reduced basal [Ca2+]i and decreased Ca2+ peak amplitude in response to bradykinin. Similar modifications were observed in the presence of Orai1 inhibitor or in a Ca2+‐free buffer while OAG, a specific TRPC3 activator, didn't modify Ca2+ homeostasis. In dysfunctional PAEC at P4, model of endothelial dysfunction associated with basal increased of [Ca2+]i, Pyr3 normalized the basal [Ca2+]i in a concentration‐dependent manner while the activation of TRPC3 by OAG over increased it. These results indicate that calcium entry by TRPC3 and Orai1 channels is involved in regulation of cytosolic calcium in functional endothelial cells. In dysfunctional endothelial cells with limited NO production, an increased expression or activity of TRPC3 may participate to the abnormal Ca2+ homeostasis
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