**3. Mineralocorticoid receptor: mechanism of activation and regulation in vascular tissues**

In basal conditions, the MR is located at the cytoplasm forming a complex with heat-shock proteins, Hsp90 and Hsp70, that stabilize its structure in a conformation where the ligandbinding site is ready to interact with the hormone [24, 25]. Once the ligand is bound, the MR is subjected to a series of conformational changes (**Figure 2**). The interaction between N-terminal and C-terminal domains of MR induces the dissociation of heat-shock proteins and corepressors, allowing MR translocation to the nucleus. However, it has been reported also that Hsp90 follows MR into the nucleus [26]. Inside the nucleus the MR dimerizes, binds to HRE, and recruits a co-regulator complex to induce the transcription of target genes [27, 28]. Interestingly, in some animal models of kidney and cardiac injury that showed normal plasma Aldo concentrations, the MR is activated through a ligand-independent pathway, involving a direct interaction between a small GTPase, Ras-related C3 botulinum toxin substrate 1 (Rac1) and the MR [29], though evidence of this pathway is still lacking in vessels.

Mineralocorticoid Receptor in Calcium Handling of Vascular Smooth Muscle Cells http://dx.doi.org/10.5772/intechopen.79556 69

**Figure 2.** Schematic representation of the MR activation mechanism by Aldo in vascular smooth muscle cells. Once MR binds Aldo in the cytoplasm, it is subjected to conformational changes that allow the dissociation of heat-shock proteins (Hsp70/90), the unmasking of nuclear localization signal, and finally the MR translocation to the nucleus where the MR dimerizes (homodimer complex) and binds to hormone response elements (HRE). The MR dimer recruits a co-regulator complex for regulating the transcription of target genes. Both Aldo and cortisol bind to MR with similar affinity. The mechanism that confers MR selectivity for Aldo depends on the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11 βHSD2), which converts cortisol to cortisone, the latter has a low affinity for MR. Some of the MR target genes in SMC from different vascular beds are listed [19, 30–32]. Abbreviations: *Cacna1a*, VDCC subunit alpha1 A; *Kcnma1*, BKCa channel (alpha subunit); *Kcnmb1*, BKCa channel (beta subunit 1); *Trpc1*, transient receptor potential cation channel subfamily c member 1; *Trpc6*, transient receptor potential cation channel subfamily c member 6; *Stim1*, stromal interaction molecule 1; *Orai1*, ORAI calcium release-activated calcium modulator 1.

The high homology between the LBD structure of MR and GR receptors helps to explain why the MR binds cortisol and corticosterone and glucocorticoids with similar affinity with Aldo [33]. Cortisol and Aldo bind to hMR with similar affinity [5, 9]. Because the serum levels of cortisol are higher (from 100 to 1000 times more) than Aldo, then it is expected that the occupancy of MR by cortisol predominates. However, a mechanism that confers MR selectivity for Aldo depends on the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2), which converts cortisol to cortisone, the latter has a low affinity for MR [34]. Several papers have demonstrated the co-expression of MR with a functional 11βHSD2 in different vascular beds including mesenteric [15] and coronary arteries [16, 35], though with an indirect determination in CA [36]. These studies support that vascular tissues are Aldo-specific targets and that the action of 11βHSD2 is the physiological mechanism that excludes the activation of MR by cortisol. Furthermore, these data support that the MR-Ado complex has greater stability and higher transcriptional efficiency than the MR-cortisol complex [9].

#### **3.1. Importation of MR into the nucleus**

Specifically in blood vessels, the group of Marc Lombѐs showed for the first time the expression of MR in ECs and SMCs of the aorta and pulmonary artery by immunostainings and

Im, immunohistochemistry; NB, Northern blot; PCR, polymerase chain reaction; RT-PCR, reverse transcription

**Tissue or cell type Detection method Reference** *Umbilical vein* [23] Adipocytes Im [11] Macrophages PCR, Im [13] Lymphocytes PCR [13]

H]Aldo binding [10]. Although with a low level of immunostaining signal, the MR was found in small arteries such as the carotid, humeral, mesenteric, coronary, and renal arteries.

the vena cava and portal vein [10]. Hatakeyama et al. detected MR mRNA in primary cultures of ECs and SMCs from human pulmonary arteries [14]. Later, Takeda et al. demonstrated the presence of MR mRNA in mesenteric arteries of stroke-prone spontaneously hypertensive rats (SHRSP) [15]. Using RT-PCR the MR mRNA was detected in a human aorta [21]. Jaffe and Mendelsohn also demonstrated that MR was expressed in VSMCs from aorta and heart vasculature, specifically in coronary arteries [16]. In the case of cerebral arteries (CA), MR expression has been showed by Western blots, where MR levels were higher in CA from females than males [18]. All of these data established the foundation for understanding MR action in vascular tissues. Nowadays we know that MR is indeed expressed in the cardiovas-

In basal conditions, the MR is located at the cytoplasm forming a complex with heat-shock proteins, Hsp90 and Hsp70, that stabilize its structure in a conformation where the ligandbinding site is ready to interact with the hormone [24, 25]. Once the ligand is bound, the MR is subjected to a series of conformational changes (**Figure 2**). The interaction between N-terminal and C-terminal domains of MR induces the dissociation of heat-shock proteins and corepressors, allowing MR translocation to the nucleus. However, it has been reported also that Hsp90 follows MR into the nucleus [26]. Inside the nucleus the MR dimerizes, binds to HRE, and recruits a co-regulator complex to induce the transcription of target genes [27, 28]. Interestingly, in some animal models of kidney and cardiac injury that showed normal plasma Aldo concentrations, the MR is activated through a ligand-independent pathway, involving a direct interaction between a small GTPase, Ras-related C3 botulinum toxin substrate 1 (Rac1)

H]Aldo binding allowed the detection of MR in

[3

Interestingly, neither immunostaining nor [<sup>3</sup>

polymerase chain reaction; WB, Western blot technique.

**Table 1.** Cell types and tissues expressing MR.

68 Calcium and Signal Transduction

**regulation in vascular tissues**

cular system supporting its direct role in vascular pathophysiology.

**3. Mineralocorticoid receptor: mechanism of activation and** 

and the MR [29], though evidence of this pathway is still lacking in vessels.

Immunostainings of MR in SMC from coronary arteries have showed the presence of MR in both cytoplasm and nucleus, even in the absence of a ligand. After exposure to Aldo, MR 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 mechanism of its transcriptional activity [37].

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

Mineralocorticoid Receptor in Calcium Handling of Vascular Smooth Muscle Cells

http://dx.doi.org/10.5772/intechopen.79556

cannot fully explain the development of hypertension and associated cardiovascular mortality; but these actions are still poorly understood. In fact, there are multiple clinical studies in which mineralocorticoid receptor antagonists (MRA) reduce the incidence of heart attacks

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

Vessel injury induces a pathological response termed vascular remodeling which contributes to human ischemic vascular disease. Adverse vascular remodeling limits vessel lumen diameter and increases vascular stiffness associated with fibrosis, thereby contributing to organ ischemia and hypertension. MR activation contributes to vascular remodeling by acting synergistically with endothelial damage, angiotensin II (Ang II), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) [52–55]. These processes involve both genomic (upregulation of genes involved in cell migration, proliferation, and matrix modulation) and non-genomic mechanisms (via MAPK and the c-Src/Rho) [56]. In the pulmonary artery, MR activation induced the proliferation of VSMC, an effect prevented by spironolactone [57]. Moreover, in a VSMC-MR knockout mouse model, carotid injury-induced and

handling alone

71

**4. Pathological role of vascular mineralocorticoid receptor in blood** 

It has been confirmed that MR presents extrarenal actions [42, 44, 45] as Na<sup>+</sup>

consequently increasing vasoconstriction and blood pressure (BP) [51].

of MR in vascular physiology and pathology [19, 30, 42, 43].

and cardiovascular mortality [46, 47].

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

**4.2. Role of MR in vascular remodeling**

**vessels**
