**4.1. Role of vascular MR in oxidative stress**

is located mainly in the nucleus [16]. Like other nuclear receptors, the MR is a protein of a considerable molecular mass (~107 KDa) that exceeds the calculated size for its passive diffusion through the nucleus; thus, it requires specific signals for its nuclear transport. Specifically the importation of MR into the nucleus is controlled through three nuclear localization signals (NLS): the first NLS (NL0) is a serine-/threonine-rich sequence located at the N-terminal region, the second is a NLS (NL1) located at the DBD, and the third is a NLS (NL2) within the LBD. The presence of several NLS in different regions of the MR structure suggests a redundant mechanism to assure its mobilization toward the nucleus as part of the essential

The MR is subjected to several posttranslational modifications (PTM) such as phosphorylation, ubiquitination, sumoylation, and acetylation that regulate its localization, activity, and stability. Phosphorylation is the most common PTM since the MR contains more than 30 (putative and experimentally assessed) phosphorylation sites that allow to consider it as a phosphoprotein (**Figure 2**). The multiple phosphorylation sites of the MR generate a double band in SDS-PAGE due to a shift of approximately 30 kDa in its apparent molecular mass. The physiological function of all these phosphorylation sites is still under study or completely unknown as in the case of vascular MR. A report has shown that the phosphorylation of Ser843 (in the LBD) prevents MR ligand binding and activation [38]. In contrast, Amazit et al. reported an increase in the MR phosphorylation after Aldo binding, suggesting a liganddependent process [39]. Walther et al. reported that phosphorylation of residues inside the NL0 might modulate the MR transport into the nucleus [37]. Finally, Faresse et al. found that the MR is monoubiquitinylated at its basal state and that the Aldo-stimulated MR phosphorylation induces its polyubiquitinylation, destabilization, and degradation [40]. The physiologi-

Aldo exerts its effects in vascular tissues via non-genomic (which are not subjects of this chapter) and genomic MR-dependent pathways. In the case of MR-dependent genomic actions in VSMCs, Jaffe and Mendelsohn investigated the MR-mediated gene transcription activity in SMC of human coronary arteries (HCSMCs) by microarray and quantitative RT-PCR assays. These researchers showed that Aldo modulated the expression of VSMC genes that contribute to vascular inflammation and fibrosis. Additionally, by using a MR response element (MRE) reporter driving the expression of the luciferase gene, they demonstrated that Aldo activated MR in HCSMCs. The Aldo transcriptional effects were regulated in a dose-dependent manner, starting at 1 nM, which is consistent with the Kd for Aldo-MR interaction of ~1–2 nM [16]. Similarly, Newfell et al. evaluated the gene expression profile in Aldo-treated aorta ex vivo, identifying 72 genes that were regulated by Aldo, some of them in a concentration-dependent fashion (1, 10, 30, and 100 nM). Between the Aldo-regulated genes, several of them are involved in oxidative stress, nitric oxide (NO) signaling, vascular proliferation, and fibrosis. Moreover, it has been showed that the MR transcriptional activity is blunted by MR antagonists, and

mechanism of its transcriptional activity [37].

cal role of MR-PTM has not been determined in VSMCs.

**3.3. Genomic effects of aldosterone in vessels**

**3.2. MR posttranslational modifications**

70 Calcium and Signal Transduction

In vitro and in vivo data suggest a vascular MR activation in stimulating oxidative stress, inhibiting vascular relaxation, and contributing to vessel inflammation, fibrosis, and remodeling. MR activation may promote vascular aging and atherosclerosis contributing to the pathophysiology of heart attack, stroke, and possibly hypertension [42]. The balance between damaging reactive oxygen species (ROS) and protective NO determinates the vascular oxidative stress. ROS interact with NO decreasing the NO bioavailability. In vivo experiments in rats support that activation of MR signaling contributes to the vascular dysfunction induced by βAR overstimulation associated with endothelial NO synthase uncoupling reducing NO production and bioavailability [48]. Meanwhile, in the presence of endothelial dysfunction, vascular injury, or high vascular oxidative stress (for instance, in patients with cardiovascular risk factors), ROS production increases via VSMC-MR-mediated activation of NADPH oxidase (a ROS generator) [23, 49, 50] promoting impaired EC-dependent vasorelaxation and consequently increasing vasoconstriction and blood pressure (BP) [51].
