**2. Estradiol effects on the fluid intake**

Thirst and sodium appetite and the water and sodium intake resulting comprise motivated behaviors involved in the regulation of the hydromineral and cardiovascular homeostasis [3]. A number of epidemiological, clinical, and genetic studies in humans and animals have been showing a link between chronically high salt consumption and the development of hypertension (for review see [24]). Although the involved mechanisms are not fully understood, it is known that occurs increased in the sympathetic and RAS activities, besides be associated with a reduced ability of the kidney to excrete large amounts of salt [23, 24]. Nevertheless, women during their reproductive period have a lower incidence of hypertension than age-matched men [39, 40]. On the other hand, women and females tend to have salt sensitivity and increased blood pressure at postmenopausal or reproductive senescence, suggesting sex differences in body fluid balance and blood pressure regulation [3, 24]. In fact, E2 is known to mediate hydromineral homeostasis and blood pressure mainly by attenuating RAS activity (**Figure 1**). E2 replacement therapy decreases ANGII-induced water and sodium intake [3, 9, 41]; blood pressure increased induced by ANGII and development of hypertension in spontaneously hypertensive rats (for review see [40, 42]); angiotensin-converting enzyme (ACE) and renin activities [43–45]; AT1 mRNA expression, ANGII-AT1 binding, and ANGII-induced Fos immunoreactivity in the OVLT and SFO [28, 46–48]. Moreover, women during reproductive period have lower (pro)renin and renin plasma levels than men [49]. In rats, females at proestrus and estrus respond less to RAS activation and ANGII administration than at other stages of the cycle [3, 50].

*Non-Reproductive Effects of Estradiol: Hydromineral Homeostasis Control DOI: http://dx.doi.org/10.5772/intechopen.95348*

#### **Figure 1.**

*Schematic summary displays the cascade leading to peripheral ANGII formation and subsequent AT1 receptor activation and the E2 influence on RAS components. Importantly, all these RAS components are also expressed within the brain, leading to central ANGII formation [19].*

Although E2 and progesterone have complementary actions in reproductive function, regarding RAS, studies have shown that progesterone has an opposite effect of E2 [43, 51] and is not involved in water regulation in response to ANGII [52].

#### **2.1 Interaction between E2 and ANGII signaling on the fluid intake**

Interestingly, in ovariectomized (OVX) rats and during their period of most active, i.e., at night, both water and sodium intakes induced by ANGII require p38 MAPK, JNK, and PKC signaling pathways. ANGII-induced sodium intake also requires ERK1/2 signaling pathway PKC-independent in female [9, 41]. These observations corroborate with Coble et al. [53], which also showed that PKC is involved in both ANGII-induced water and sodium intake in the SFO in male mice. However, Daniels et al. [8] showed that PKC signaling is exclusively involved in ANGII-induced water intake in male rats. Thus, these divergent results suggest a

sexually dimorphic aspect to the AT1 signaling pathway involved in fluid intake induced by ANGII. Indeed, several studies report that the RAS is differentially regulated in males and females [3, 39, 40, 42].

In the brain, ERα is extensively distributed in the neurons of the nuclei of the basal forebrain, such as lamina terminalis. SFO neurons have been shown to express both AT1 and ERα [5, 33]. Evidence from our group showed that E2 impairs ANGII signaling [9, 41, 54] besides decreased AT1 mRNA expression in the SFO [28]. For example, E2 attenuates JNK phosphorylation as well as prevents p38 MAPK phosphorylation induced by ANGII in the lamina terminalis. Moreover, E2 attenuates ANGII-induced ERK1/2 phosphorylation within the SFO. These mechanisms can explain, at least in part, the E2 inhibitory effect on the fluid intake induced by ANGII.

An important feature of GPCRs is that they are rapidly phosphorylated by specific GPCR kinases (GRKs) in their serine and threonine residues within the intracellular loop and carboxyl-terminal tail domains. GRK family members selectively phosphorylate agonist activated GPCRs, promoting the binding with cytosolic cofactor proteins termed arrestins, which uncouple GPCRs from G proteins, interrupting the signaling pathway. This process is referred to as desensitization, which occurs within seconds to minutes (for review see [55, 56]). Nevertheless, it is currently known that arrestins can also act as scaffolds to recruit signaling molecules, such as ERK1/2 and JNK, to increase the repertoire of receptor responses. When β-arrestin binds to the AT1 receptor that is phosphorylated by GRK5 or GRK6, it functions as an intracellular signaling adapter leading to robust ERK1/2 activation [57, 58]. In this context, evidence from our group showed that E2 reduced the expression of GRK5 in the lamina terminalis [54]. This observation suggests that E2 may also impair the ANGII signaling pathway by decreasing the activation of ERK1/2 via negative regulation of the GRK5, which can be relevant to the inhibitory effect of E2 on sodium intake induced by ANGII.

Furthermore, Almeida-Pereira et al. [9] showed that the inhibitory effect of E2 on ANGII-induced water and sodium intake requires the ERK1/2 and JNK signaling pathways. Because these inhibitory effects of E2 were quickly reversed by the central inhibition of ERK1/2 and JNK activities, suggesting that there is the involvement of a non-genomic effect of ER agonism. E2 replacement therapy also induces ERK1/2 and JNK phosphorylation in the lamina terminalis [9], and these proteins are involved in the AT1 receptor desensitization process [59, 60]. These observations point to the idea that ERK1/2 and JNK activation from E2 signaling may contribute to the AT1 receptor desensitization process in the lamina terminalis.

Regarding PKC signaling, central PKC inhibition maintains the inhibitory effect of E2 on ANGII-induced fluid intake, and when analyzing PKC activation, E2 induced an increase in PKC (specifically alpha isoform) translocation to the plasmatic membrane in the lamina terminalis structures. Thus, perhaps PKC is not involved in the inhibitory effect of E2 on fluid intake induced by ANGII or E2 may change other protein activation from the sequence of PKC signaling cascade. Indeed, E2 prevents p38 MAPK phosphorylation induced by ANGII and does not activate p38 MAPK in the lamina terminalis [41]. In addition, as already described, E2 attenuates ERK1/2 and JNK phosphorylation in response to ANGII. Lastly, besides GRK, it is known that PKC also phosphorylates the AT1 receptor, exposed or not to agonists, inducing its desensitization [56]. Taken together, these findings lead to the hypothesis that E2 can modulate AT1 desensitization by PKC activation in the lamina terminals. A summary of all these signaling interactions is provided in **Figure 2**.

*Non-Reproductive Effects of Estradiol: Hydromineral Homeostasis Control DOI: http://dx.doi.org/10.5772/intechopen.95348*

#### **Figure 2.**

*Schematic summary of the proposed interaction between E2 and ANGII signaling within lamina terminalis structures involved in water and sodium intake. E2 impairs MAPKs phosphorylation in response to ANGII by inducing AT1 desensitization, reduced GRK5 expression, and (or) phosphatase activation (not identified), which leads to ANGII-induced fluid intake reduction. A possible explanation for the E2-induced AT1 desensitization is through ER-mediated ERK1/2 and (or) JNK signaling. Another is through PKC activation mediated by ER inducing AT1 phosphorylation and, consequently, its desensitization. Legend: continuous arrow indicates stimulation and dashed arrow indicates inhibition.*

### **3. Estradiol effects on the neurohypophysial hormone release**

It is widely known that OT plays a pivotal role in parturition and lactation by inducing contractions of the uterus and myoepithelial cells of the breast alveoli. In addition, OT participates in the hydromineral balance as a regulator of blood volume by eliciting natriuresis and sodium appetite inhibition [15–17]; and participates in the blood pressure control through its vascular and cardiac relaxation effects [61, 62]. In the central nervous system, OT acts as a neurotransmitter involved in sex and maternal behavior (for review see [63]).

The plasma hyperosmolality is the major stimulus for AVP secretion following by hypotension or decreased blood volume [64]. Thus, through its main antidiuretic effect, AVP plays a fundamental role in hydromineral homeostasis as a regulator of plasma osmolality [11]. Regarding blood pressure control, as described previously, AVP increases sympathetic outflow and peripheral vascular tone by eliciting vasoconstriction and increase consequently the blood pressure [11, 21].

In situ hybridization studies reported a wide distribution of mRNA expression for ERβ in the brain of rats, including in the neurons of SON and the parvo and magnocellular divisions of PVN [65, 66]. Hrabovszky et al. [67] showed that OT and AVP neurons from PVN and SON co-express mRNA for ERβ. These findings provide a neuroendocrine basis for E2 influence on the hypothalamo-neurohypophysial system by acting directly on the PVN and SON neurons. Importantly, E2 can also regulate neurohypophysial hormone release through ERα activation present in the lamina terminal structures and via connections with PVN and SON [33]. In this context, E2 is known to modulate OT release positively, besides increases mRNA expression for OT in the PVN and SON [68–70]. Conversely, the influence of E2 on AVP secretion is complex with controversial data in the literature. Nevertheless, in general, studies point out that there is a positive correlation between AVP secretion and E2 plasma levels associated with body water retention. Moreover, E2 decreases the osmotic threshold for AVP stimulation during dehydration without affect renal

free water clearance, suggesting that E2 may alter renal sensitivity to AVP or even interfere with AVP action in the kidney [71–73].

#### **3.1 Interaction between E2 and ANGII signaling on the OT and AVP secretion**

A decrease in blood volume and increased renal sympathetic outflow stimulate renin release from the kidney, which results in increased circulating levels of ANGII [3, 11]. Furthermore, during hypovolemia and hypotension, AVP secretion is stimulated mainly by neural (from cardiac baroreceptors and afferent inputs from the brainstem that project to the SON and PVN) and humoral (i.e., ANGII) signals. In dehydration and hyperosmolality conditions, both AVP and OT secretion are stimulated by an osmoregulatory circuit comprising osmoreceptors activation and axonal projections from the basal forebrain to the PVN and SON besides the intrinsic osmosensitivity of magnocellular neurons of the PVN and SON [11, 64]. Thus, hypovolemia (from hemorrhage or dehydration) and hyperosmolality (from dehydration) stimulate RAS activation as well as AVP and OT secretion.

Evidence from our group provides interesting insights that neurohypophysial secretion in response to ANGII involves distinct signal transduction pathways in OVX rats. We reported for the first time that PKC/p38 MAPK signaling is involved in ANGII-induced OT release while AVP release requires ERK1/2 and p38 MAPK signaling PKC-independent [9, 41]. However, Felgendreger et al. [20] showed that the ERK1/2 activation induced by endogenous ANGII is not involved in AVP secretion in male rats. These divergent results suggest a sexually dimorphic aspect to the AT1 signaling pathway involved in ANGII-induced AVP secretion. As already described, several studies reported that the RAS is differentially regulated in males and females, which can be attributed to differences in gonadal and steroid profiles [3, 39, 40, 42].

Concerning E2 modulation on ANGII-induced AVP and OT release, it was observed that E2 inhibits AVP and OT secretion in response to ANGII by impairing ERK1/2 and p38 MAPK phosphorylation, respectively, in the PVN and SON [9, 41]. MAPK proteins are inactivated by phosphatases, such as mitogen-activated protein kinase phosphatases (MKPs). MPKs are dual-specificity protein phosphatases (also known as DUSPs) that dephosphorylate both tyrosine and threonine residues on MAPK members [74]. MAPK phosphatase 1 (MKP-1) was the first of the MKPs to be characterized and is known for dephosphorylating all three major classes of MAPK (ERK, p38 MAPK, and JNK) being expressed in the brain besides many others tissues [75]. MKP-1 is under the positive regulation of E2 in the PVN and SON and thereby can participate in the inhibitory effect of E2 by eliciting ERK1/2 and p38 MAPK dephosphorylation [9]. In addition, it was hypothesized that E2 inhibits ANGII-induced AVP release via PKC-mediated MKP-1 induction and consequent ERK1/2 dephosphorylation, involving E2 non-genomic signaling [54]. A summary is provided in **Figure 3**.

Importantly, ANGII can also stimulate AVP and OT release by acting in the lamina terminalis structures which send angiotensinergic projections to magnocellular neurons of the PVN and SON [5, 13]. Thus, E2 can indirectly modulate AVP and OT release induced by ANGII via afferent inputs from the basal forebrain that project to the SON and PVN. Nevertheless, as E2 increases MKP-1 specifically in the PVN and SON and not in lamina terminalis structures, it is suggested that E2 plays an important direct modulation on AVP and OT release induced by ANGII in hypothalamic nuclei [9, 41]. In the same sense, Vilhena-Franco et al. [76] showed that E2 modulates AVP secretion in response to water deprivation via a direct mechanism mediated by ERβ expressed in the SON and PVN. Additionally, others studies have

*Non-Reproductive Effects of Estradiol: Hydromineral Homeostasis Control DOI: http://dx.doi.org/10.5772/intechopen.95348*

#### **Figure 3.**

*Schematic summary of the regulatory mechanisms of E2 proposed on ANGII-induced OT and AVP release. Top: E2 can indirectly modulate AVP and OT release induced by ANGII via angiotensinergic afferent inputs from the lamina terminalis structures that project to the SON and PVN. E2 impairs ANGII signaling in neurons of the lamina terminalis leading thereby to the OT and AVP neurons decreased activity in the SON and PVN. Bottom: E2 directly acts in OT and AVP neurons in the PVN and SON, preventing MAPKs phosphorylation through PKC/MKP-1 signaling pathway. Legend: continuous arrow indicates stimulation and dashed arrow indicates inhibition.*

demonstrated that E2 modulates OT and AVP release directly via its ERβ or mER (mainly AVP release) in magnocellular neurons of the PVN and SON [77–79].

#### **3.2 Interaction between ovarian hormones, phoenixin and AVP release**

A novel peptide, conserved across species, was recently described and named phoenixin (PNX). PNX is produced in the brain and heart binding selectively in the pituitary gland, ovary, and brain. The hypothalamus was identified to produce the most PNX of all tissues examined as well as presented the highest binding of labeled PNX [80]. PNX has been implicated to play an important role in the hypothalamic–pituitary-gonadal axis control by increasing the gonadal release hormone (GnRH) expression and its receptor in the hypothalamus besides increasing luteinizing hormone (LH) release [80, 81]. Using a deductive ligand-receptor matching strategy (U.S. Patent #9, 146, 240, B2), the orphaned G protein-coupled receptor (GPR)173 was identified to be a candidate PNX receptor [82].

The novel reproductive neuropeptide PNX and its receptor, GPR173, were also identified in magnocellular neurons of the PVN and SON, suggesting the participation of PNX on the hypothalamo-neurohypophysial system control and hence of the hydromineral homeostasis. [80, 82]. In fact, Gasparini et al. [83] demonstrated that PNX induces AVP release through its candidate receptor, GPR173, besides depolarizes magnocellular neurons of the PVN. Interestingly, PNX does not modulate OT release. Despite there are no differences between males and females on PNX-induced AVP release, a potential estrogen response element (ERE) upstream of PNX was identified, suggesting that ovarian hormones, especially E2, can modulate PNX [80, 83]. Consistent with this idea, ovarian failure induced by OVX induces downregulation of PNX compared with intact females in the hypothalamus [84].

Circulating levels of ovarian steroids progesterone, and, mainly 17β-estradiol, increase throughout the pregnancy, reaching maximum values at the end of pregnancy in women [85, 86]. On the other hand, in rats, peripheral plasma levels of estradiol increase across pregnancy with a concomitant decrease in progesterone [87]. However, in both humans and animals, it is observed changes in hydromineral homeostasis over the pregnancy, such as blood volume expansion and low osmolality associated with a reduced threshold for hyperosmotic stimulation of AVP secretion. Thus, AVP secretion is paradoxically elevated during this hypervolemic and hyponatremic state of the pregnancy [32, 88] although is important to maintain water homeostasis offsetting AVP that is metabolized by vasopressinase in humans [89]. A dysfunction in the metabolism of vasopressinase and AVP can predispose women to develop cardiovascular diseases associated with hydromineral imbalance, as pregnancy-induced hypertension, that occurs after the second trimester of pregnancy [89, 90].

In this context, our group demonstrated upregulation of GPR173 during late post-puberty in the PVN, and importantly, upregulation during the last third of pregnancy in the hypothalamus. Moreover, it was observed an increase in the hypothalamic levels of PNX and AVP across pregnancy compared with levels present during diestrus with a positive correlation between both peptides [84]. Thus, these results suggest an important role of PNX on AVP release during late pregnancy, which can help to provide potential pharmacological targets for preventing the development of cardiovascular diseases across pregnancy.
