**2. Mineralocorticoid receptor structure and expression in vascular tissues**

The MR is a ligand-activated transcription factor that belongs to the nuclear receptor superfamily [3]. MR was originated by a process of gene duplication from a common ancestor that diverged into the glucocorticoid receptor (GR) and the MR [4]. In 1987 the group of Arriza et al. cloned the MR from human placenta [5]. The human MR (hMR) is coded by a unique gene (*Nr3C2*, due to its belonging to the subfamily 3 of nuclear receptors, group C, member 2) located in chromosome 4, locus q31.1, and with a length of about 75Kb. The gene encodes a polypeptide chain of 984 aa (~107 KDa) [5]. The orthologous gene in rat encodes for a protein of 981 aa (**Figure 1**) and share an identity of 90.1% with the hMR. *Nr3C2* contains 10 exons; the first 2 of them (1α and 1β) comprise the 5′-noncoding sequences, whereas the following exons (2–9) are harboring the functional domains of the protein. It has been reported that at

**Figure 1.** Mineralocorticoid receptor structure. Linear representation of rat MR sequence with respective protein domains. The MR contains an N-terminal transactivation domain of variable lengths (A/B) and a DNA binding domain (DBD) with two zinc fingers involved in the recognition of specific DNA sequences within the promoters of target genes and named hormone response elements (HRE); a flexible hinge is connecting the DBD to the ligand-binding domain (LBD) in the C-terminal region. The residues N767, Q773, R814, and T942 are part of the ligand-binding pocket. The MR also contains three nuclear localization signals (NL0, NL1, and NL2) and multiple phosphorylation sites, and between them is Ser843 that is a target of calcium/calmodulin-dependent protein kinase type II (CamKII).

least three variants of MR mRNA (α, β, and γ) are encoded in a tissue-specific manner under the control of different gene promoters [6].

The MR receptor has the same protein structure as other members of the nuclear receptor superfamily. The MR is composed of an A/B domain (1–604 aa) with a transactivation function and several serine and threonine phosphorylation sites [7] and a DNA binding domain (DBD, 604–699 aa) with two zinc finger motifs that recognize DNA-specific sequences named hormone response elements (HRE), normally found in the promoters of target genes [8]. After the DBD, the hinge D region (670–733 aa) is found and, finally, the C-terminal region (734–981 aa) that is harboring the ligand-binding domain (LBD) with a pocket (which comprises Asp767, Gln773, Arg814, and Thr942) involved in the recognition of agonist and antagonist [9] (**Figure 1**).

#### **2.1. Mineralocorticoid receptor expression in different tissues**

ion channels that participate in Ca2+ handling of vascular smooth muscle cells and the thera-

In 1972, Crabbé demonstrated that Aldo was interacting with cytoplasmic receptors and that the steroid-protein complex acted as activator triggering the synthesis of mRNA and proteins [1]; thus, Aldo was the first identified mammalian steroid hormone that exerted transcriptional actions via some kind of cytoplasmic/nuclear receptors [2]. Several classes of mineralocorticoid receptors were identified in both epithelial and surprisingly in non-epithelial tissues such as cardiomyocytes, endothelial cells (ECs), and vascular smooth muscle cells (VSMCs)

peutic implications for hypertension and cardiovascular diseases.

[2], auguring the future actions of Aldo in the cardiovascular system.

**tissues**

66 Calcium and Signal Transduction

**2. Mineralocorticoid receptor structure and expression in vascular** 

The MR is a ligand-activated transcription factor that belongs to the nuclear receptor superfamily [3]. MR was originated by a process of gene duplication from a common ancestor that diverged into the glucocorticoid receptor (GR) and the MR [4]. In 1987 the group of Arriza et al. cloned the MR from human placenta [5]. The human MR (hMR) is coded by a unique gene (*Nr3C2*, due to its belonging to the subfamily 3 of nuclear receptors, group C, member 2) located in chromosome 4, locus q31.1, and with a length of about 75Kb. The gene encodes a polypeptide chain of 984 aa (~107 KDa) [5]. The orthologous gene in rat encodes for a protein of 981 aa (**Figure 1**) and share an identity of 90.1% with the hMR. *Nr3C2* contains 10 exons; the first 2 of them (1α and 1β) comprise the 5′-noncoding sequences, whereas the following exons (2–9) are harboring the functional domains of the protein. It has been reported that at

**Figure 1.** Mineralocorticoid receptor structure. Linear representation of rat MR sequence with respective protein domains. The MR contains an N-terminal transactivation domain of variable lengths (A/B) and a DNA binding domain (DBD) with two zinc fingers involved in the recognition of specific DNA sequences within the promoters of target genes and named hormone response elements (HRE); a flexible hinge is connecting the DBD to the ligand-binding domain (LBD) in the C-terminal region. The residues N767, Q773, R814, and T942 are part of the ligand-binding pocket. The MR also contains three nuclear localization signals (NL0, NL1, and NL2) and multiple phosphorylation sites, and between

them is Ser843 that is a target of calcium/calmodulin-dependent protein kinase type II (CamKII).

The direct and specific actions of Aldo require MR expression in target tissues. For a long time, it was thought that MR was expressed exclusively in kidney epithelial cells and that Aldo was secreted only by the adrenal gland. However, a cumulative evidence has showed the presence of MR in non-epithelial tissues, such as the colon, salivary glands, trachea, heart [10], adipocytes [11], brain [5], skeletal muscle [12], leucocytes, macrophages [13], and vessels [14–19] (**Table 1**).



Im, immunohistochemistry; NB, Northern blot; PCR, polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; WB, Western blot technique.

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

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 [3 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. Interestingly, neither immunostaining nor [<sup>3</sup> H]Aldo binding allowed the detection of MR in 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 cardiovascular system supporting its direct role in vascular pathophysiology.

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

**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

Mineralocorticoid Receptor in Calcium Handling of Vascular Smooth Muscle Cells

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

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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

higher transcriptional efficiency than the MR-cortisol complex [9].

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

1; *Orai1*, ORAI calcium release-activated calcium modulator 1.
