**5.2. Ca2+-activated potassium channels**

vasculature [42]. The constitutive model of VSMC-specific MR inactivation reported a similar basal BP decrease in 5-month-old MR-KO mice [22]. The BP phenotype in both inactivated

role for VSMC-MR in BP regulation. Interestingly, tamoxifen-inducible VSMC-MR inactivation prevented the in vivo increase in BP induced by Ang II infusion but not by aldosteronesalt challenge [22, 42]. Inactivation of VSMC-MR was also shown to decrease the contractile response to KCl and extracellular Ca2+ [62]. The role of the vascular MR could also depend on the vascular bed that is considered. In the future, the use of transgenic models will allow us to decipher the contribution of endothelial MR and VSMC-MR in the different vascular beds

**5. Calcium handling proteins are targets of vascular MR receptors**

1.2α1C, Ca<sup>v</sup>

Recognized as a universal second messenger in various cellular processes and cell types, Ca2+ signal plays a critical role in many cellular processes, including, but not limited to, gene transcription and excitation-contraction (EC) coupling [91, 92]. Although almost all biological responses are mediated by Ca2+-dependent and Ca2+-controlled processes, Ca2+ signals need to be finely coordinated and precisely regulated. Ubiquitously expressed in the whole body,

1.2 is the main route of Ca2+ entry in VSMCs, essential for vascular EC coupling and control of myogenic tone [93]. As a heteromultimeric channel, L-type Ca2+ channel (LTTC) is formed

1.2β, Ca<sup>v</sup>

hypertensive properties [94–96], although its effectiveness has been achieved only in a subset

tissue-specific transcripts of *Cacna1c* driven by two alternative promoters P1 and P2, encoding,

N-terminal region [98]. In VSMCs, LTTC is activated in response to the membrane depolarization, allowing a small fraction of Ca2+ influx, which is sufficient to trigger VSMC contraction. Thus, sustained voltage-dependent Ca2+ influx through the LTCCs maintains a tonic level of vasoconstriction and provides an excitatory template upon which endogenous vasoactive

Although previous studies have demonstrated that aldosterone modulates VSMC Ca2+ currents [99–101], the mechanisms remain to be determined. A landmark study showed that VSMC-specific MR knockout mice (VSMC-MR-KO) are protected against the age-associated rise of BP [42]. Importantly, aged VSMC-MR-KO mice showed decreased myogenic tone and attenuated vascular contraction in mesenteric arteries in response to a LTCC opener.

sion. However, this phenomenon seems indeed to be an age-dependent effect, since a latter

from aged VSMC-MR-KO mice, suggesting that MR may regulate VSMC Ca<sup>v</sup>

study did not validate, at protein level, the downregulation of Ca<sup>v</sup>

1.2α2δ, and Ca<sup>v</sup>

1.2-LNT) and for a short "vascular/brain" (Ca<sup>v</sup>

1.2α1C subunit was dramatically downregulated in aortas

1.2α1C region, has also been the target of drugs with anti-

1.2α1C channels are expressed as two distinct

1.2γ. Undebatable the

1.2-SNT)

1.2 expres-

1.2 in aortas from young

intake and renal MR function supporting a

VSMC-MR model mice is independent of Na<sup>+</sup>

and the possible implication in BP regulation [90].

**5.1. L-Type Ca2+ channel**

74 Calcium and Signal Transduction

by four associated subunits: Ca<sup>v</sup>

main subunit, the pore-forming Cav

respectively, for a long "cardiac" (Ca<sup>v</sup>

Moreover, mRNA level of Ca<sup>v</sup>

of hypertensive patients [97]. Importantly Ca<sup>v</sup>

substances may act to modulate arterial diameter and BP.

Cav

Ca2+-activated potassium channels (KCa), mainly the large conductance KCa channels (BKCa), have been recognized as another important target of MR in blood vessels [105]. BKCa plays a critical role in limiting arterial contraction by producing VSMC hyperpolarization through transient outward K<sup>+</sup> current in response to increased intracellular Ca2+ concentration [106]. However, three subtypes of KCa have been identified in blood vessels and categorized according to their conductance: small (SKCa), intermediate, and BKCa. Small- and intermediate-conductance channels are mainly expressed in the ECs, while BKCa channels are predominately expressed in VSMCs.

Previous studies have shown that increased plasma aldosterone concentration enhances vascular KCa function [105]. Oppositely, it was demonstrated that mice lacking the poreforming BKCaα subunit led to an elevation of BP resulting from hyperaldosteronism, which was accompanied by decreased serum K<sup>+</sup> levels, as well as increased vascular tone in small arteries [107]. Accordingly, impaired acetylcholine-mediated relaxation in isolated coronary arteries has been shown in mice model with cardiac-specific overexpression of aldosterone synthase (MAS mice) [30]. These findings correlate with decreased mRNA and protein expression of BKCa α and β1 subunits in the heart and coronary artery of MAS mice. Moreover, in vitro treatment of rat aortic VSMCs with increasing concentrations of aldosterone led to a reduced BKCa subunit expression in a concentration-dependent manner. Thus, these findings suggest that augmented local aldosterone production likely acts in a paracrine fashion way suppressing BKCa expression in the surrounding coronary VSMC, thereby contributing to the impaired endothelium-dependent VSMC relaxation. Intriguingly, despite aged VSMC-MR-KO mice displaying lower BP than age-matched WT mice, no significant changes were observed in aortic mRNA expression and function of BKCa in mesenteric VSMC [42]. Furthermore, aldosterone-treated aorta for 24 h with 10−8 M of aldosterone did not modify mRNA expression of BKCa α and β1 subunits [19]; thus, further studies are needed to clarify the effect of MR activation in the expression and activity of BKCa channels.

As mentioned above, SKCa is predominantly expressed in ECs, where it contributes to endothelium-derived hyperpolarization (EDH) of VSMC resulting in vasorelaxation of resistance arteries [108]. In a previous study, circulating aldosterone level was significantly higher in mice fed with high-fat diet (HFD) compared to lean mice; however, despite the restoration of endothelium-dependent vasodilation, eplerenone treatment further increased plasma aldosterone levels of HFD-fed obese mice [109]. Recently, using similar obese model, plasmatic aldosterone concentration was also augmented in male and female mice, whereas no change was found in endothelial cell-specific MR knockout mice (EC-MR-KO) subjected to HFD [110]. In males, obesity impaired NO-dependent vasodilation of resistance arteries, which was compensated by enhancement of EDH of VSMC along with an increase in mesenteric protein expression of SKCa3, while any change was observed in EC-MR-KO. On the other hand, in females, EDH component of VSMC relaxation was impaired, whereas the expression of SKCa3 remained unchanged in control and EC-MR-KO underwent to HFD [110]. Altogether, these results uncover distinct sex-specific mechanisms driving vascular dysfunction, suggesting personalized therapies to prevent vascular disorders.

serum to preserve the contractile phenotype, displayed higher coronary contractility in both endothelium-denuded and endothelium-intact rings, while co-treatment with spironolactone prevented this effect [115]. Recently, we demonstrated that rat aorta treated with aldosterone (10 nM for 24 h) did not reveal changes in the expression of TRPC1, C3, C4, C5, and C6 [19]. Altogether, these studies suggest a concentration-dependent increase of TRPC channels, since we have previously demonstrated an upregulation of TRPC1, C4, and C5 in cardiomyocytes upon aldosterone concentrations higher than 100 nM [43]. Moreover, one of the features of metabolic syndrome is the elevated plasma aldosterone level [116], which has been associated with increased TRPC1 and TRPC6 expression in coronary arteries compared

Mineralocorticoid Receptor in Calcium Handling of Vascular Smooth Muscle Cells

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

77

Another critical component of SOCE is the protein Orai (comprising Orai1, Orai2, and Orai3), which forms a family of highly Ca2+-selective channels that are regulated by stromal-interacting molecules (STIM1 and STIM2) [111, 112]. In neonatal cardiomyocytes, Orai1 was significantly increased by 100 nM and 1 μM aldosterone treatment, whereas lower concentrations (1 and 10 nM) had no effect [43]. However, Stim1 expression remained unchanged even at the highest concentration tested (1 μM) [43]. Similarly, we recently observed, in blood vessels, that treatment with 10 nM of aldosterone for 24 h does not affect the expression of either Orai1 or

Hypertension is a substantial public health problem, affecting 25% of the adult population in industrialized societies. This disorder is a major risk factor for many common causes of morbidity and mortality including stroke, myocardial infarction, congestive heart failure, and end-stage renal disease. Thus, substantial effort has been devoted to defining the pathogenesis of BP variation. Aldo, through the activation of MR in tubular epithelial cells, has a wellknown function on water balance and BP homeostasis. The renal hemodynamic consequences

and water retention and K<sup>+</sup>

hypertension. However, the kidney is no longer regarded as the primary site for mineralocorticoid modulation of BP. MR is consistently expressed in both ECs and VSMCs of blood vessels, and its activation by Aldo at pathological concentrations (10 nM) is associated with several types of vascular dysfunction, including atherosclerosis and hypertension. However, despite the recent understanding about the mechanisms involved in the activation of MR mainly in pathological conditions, further research is still required to determine the physi-

The authors thank the program Conacyt-Anuies ECOS-Nord (Evaluation-Orientation de la COopération Scientifique, M13S01), PRODEP-SEP program, and Fundación Miguel Alemán,

secretion—ultimately lead to

to lean pigs [32].

Stim1 [19].

**6. Perspectives**

of excess mineralocorticoids—Na<sup>+</sup>

**Acknowledgements**

ological role of MR-VSMCs in blood vessels.

A.C., for providing funding to this work.

#### **5.3. Transient receptor potential channels**

In VSMC, Ca2+ entry from the extracellular space involves a variety of plasmalemmal Ca2+ channels, which also involve the superfamily of transient receptor potential (TRP) channels, such as TRPC (canonical), TRPM (melastatin), TRPV (vanilloid), and TRPP (polycystin) [111]. Widely expressed in visceral and vascular SMC, changes in the expression and activity of these channels are implicated in a variety of physiological and pathophysiological consequences [112]. Although TRPM subfamily contains eight isoforms (TRPM1–8), which exhibit a variety of cation permeability, only TRPM6 and M7 seems to be Ca2+ and Mg2+ permeable. Interesting, aldosterone (100 nM) transiently upregulates mRNA TRPM7 expression in rat VSMC from 2 to 6 h after the onset of treatment, restoring to control level after 24 h of treatment [113]. However, up to date, no studies have been done to evaluate whether aldosterone modulates the expression of TRPV and TRPP channels.

Originally thought to contribute solely to restoring Ca2+ concentration under store depletion, creating a capacitative Ca2+ entry, commonly termed as store-operated Ca2+ entry (SOCE), the role of SOCE is much more diverse than just refilling Ca2+ stores [114] but also contributing to vascular contractility, VSMC proliferation, and differentiation [112]. In addition, Ca2+ entry from the extracellular space may also occur through Ca2+-permeable store-independent channels, named as receptor-operated channels (ROC), which their activity depends on second messengers produced by downstream effectors from a vast array of G protein-coupled receptors [114]. TRPC subfamily comprises seven members (TRPC1–TRPC7), with the TRPC2 gene being a pseudogene in humans [114].

Although TRPC1 seems to be the most abundant isoform expressed in rat mesenteric arteries, only the expression of TRPC6 was increased in deoxycorticosterone acetate (DOCA)-salt hypertensive rats [31]. Moreover, A7r5 cells treated with aldosterone (1 μM for 24 h) also displayed increased mRNA and protein levels of TRPC6 [31]. Accordingly, it was shown that coronary rings cultured for 7 days with aldosterone (100 nM), without fetal bovine serum to preserve the contractile phenotype, displayed higher coronary contractility in both endothelium-denuded and endothelium-intact rings, while co-treatment with spironolactone prevented this effect [115]. Recently, we demonstrated that rat aorta treated with aldosterone (10 nM for 24 h) did not reveal changes in the expression of TRPC1, C3, C4, C5, and C6 [19]. Altogether, these studies suggest a concentration-dependent increase of TRPC channels, since we have previously demonstrated an upregulation of TRPC1, C4, and C5 in cardiomyocytes upon aldosterone concentrations higher than 100 nM [43]. Moreover, one of the features of metabolic syndrome is the elevated plasma aldosterone level [116], which has been associated with increased TRPC1 and TRPC6 expression in coronary arteries compared to lean pigs [32].

Another critical component of SOCE is the protein Orai (comprising Orai1, Orai2, and Orai3), which forms a family of highly Ca2+-selective channels that are regulated by stromal-interacting molecules (STIM1 and STIM2) [111, 112]. In neonatal cardiomyocytes, Orai1 was significantly increased by 100 nM and 1 μM aldosterone treatment, whereas lower concentrations (1 and 10 nM) had no effect [43]. However, Stim1 expression remained unchanged even at the highest concentration tested (1 μM) [43]. Similarly, we recently observed, in blood vessels, that treatment with 10 nM of aldosterone for 24 h does not affect the expression of either Orai1 or Stim1 [19].
