Connexins (Cxs) form connexons within the plasma membrane to create domains referred as gap junctions. The intercellular channels established by the pairing of two connexons allow for the diffusion of ions and molecules between adjacent cells. Vascular Cxs play a role in the contractility of the vessel wall by allowing the exchange of signaling molecules to coordinate the activity of the smooth muscle cells (SMCs) and the endothelial cells (ECs). In the vascular wall, four Cxs are mostly expressed: Connexin37 (Cx37) and Cx40 in ECs and Cx43 and Cx45 in SMCs. To investigate the effects of chronic hypertension on vascular Cxs in the aorta, Wild-type (WT) mice and mice lacking Cx40 (Cx40-/-), a genetic model of renin-dependent hypertension were subjected to either a two-kidney, one-clip (2K1C) procedure associated with elevated angiontensinII levels (AngII) or to N-nitro-l-arginine-methyl-ester (L-NAME) treatment, which induce renin-dependent and -independent hypertension, respectively. All hypertensive mice featured a thickened aortic wall, increased levels of Cx37 and Cx45 in SMCs, and of Cx40 in ECs (except in Cx40-/- mice). Cx43 was up-regulated only in the SMCs of renin-dependent models of hypertension. Blockade of the renin-angiotensin system of Cx40-/- mice normalized blood pressure and prevented both aortic thickening and Cx alterations. Ex vivo exposure of WT aortas, carotids, and mesenteric arteries to physiologically relevant levels of AngII increased the levels of Cx43, but not of other Cxs. In the aortic SMCs line A7r5, AngII activated kinase-dependent pathways and induced binding of the nuclear factor-kappa B (NF-kappaB) to the Cx43 gene promoter, increasing Cx43 expression. These data suggest that Cx43 is selectively increased in renin-dependent hypertension via an AngII activation of the extracellular signal-regulated kinase and NF-kappaB pathways. Upon agonist stimulation, ECs trigger smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). We investigated the link between the expression of endothelial Cxs (Cx40 and Cx37) and endothelial nitric oxide synthase (eNOS) expression and function in the mouse aorta. We demonstrated that acetylcholine (ACh)- and ATP-induced endothelium-dependent relaxations solely depend on NO release in both WT and Cx40-/- mice, but are markedly weaker in Cx40-/- mice. Consistently, both basal and ACh- or ATP-induced NO production were decreased in the aorta of Cx40-/- mice. Altered relaxations and NO release from aorta of Cx40-/- mice were associated with lower expression levels of eNOS in the aortic endothelium of Cx40-/- mice. We further demonstrate that eNOS, Cx40, and Cx37 tightly interact with each other at intercellular junctions in the aortic endothelium of WT mice, suggesting that the absence of Cx40 in association with a marked decrease of Cx37 levels in ECs from Cx40-/- mice participate to the decreased levels of eNOS. To investigate changes in vascular Cxs and eNOS during hypertension in mice aorta, WT mice and Cx40-/- were subjected to either one-kidney, one-clip (1K1C; volume overload model) or 2K1C surgery procedure. Mean blood pressure was 10% higher in the 1K1C Cx40-/- mice compared to the other hypertensive models (Cx40-/-, 2K1C Cx40-/-, 2K1C WT and 1K1C WT), due to the association of a renin-dependent and -independent hypertension. eNOS phosphorylation was specifically increased in the aortic endothelium of 1K1C Cx40-/- mice showing that the increased mechanical forces impinging on the aortic wall were elicited by the association of renin-dependent and independent hypertension, further suggesting a compensatory mechanism associated with decreased eNOS levels in the Cx40-/- mice. Finally, to investigate the role of Cx40 in the propagation of Ca2+ waves in the endothelial cells of the mouse aorta, we performed Ca2+ imaging on intact aortic endothelium from both WT and Cx40-/- mice. ACh induced Ca2+ signaling in a fraction of ECs expressing the M3 muscarinic receptors. Inhibition of intercellular communication using carbenoxolone or octanol fully blocked the propagation of ACh-induced Ca2+ transients toward adjacent cells in WT and Cx40-/- mice. As compared to WT, Cx40-/- mice displayed a reduced propagation of ACh-induced Ca2+ waves, indicating that Cx40 contributes to the spreading of Ca2+ signals. These findings provide evidence that 1) different types of hypertension are associated with a differential regulation of Cxs expression in SMC and ECs layers; 2) ECs Cxs play a role in the regulation of eNOS expression and function in response to hypertension; 3) Cx40 and Cx37 contribute to the propagation and amplification of the Ca2+ signaling triggered by ACh in ECs expressing the M3 muscarinic receptors.