**3.2. MR posttranslational modifications**

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

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

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 actinomycin D (a transcriptional inhibitor) supporting an MR-dependent effect of Aldo in vessels [16, 19, 30, 41]. Furthermore, an increasing body of evidence has underlined the ability of MR to modulate the expression of ion channels in several vascular beds, unveiling the role of MR in vascular physiology and pathology [19, 30, 42, 43].
