**2. Endothelin-1**

Endothelins are peptide hormones, composed of 21 amino acid residues, capable of performing autocrine and paracrine functions. There are three distinct subtypes of endothelin, endothelin-1 (ET-1), endothelin-2 (ET-2), and endothelin-3 (ET-3). Endothelin-1 and endothelin-2 bind to endothelin receptor A (ETA) and endothelin receptor B (ETB), which are G-protein-coupled receptors, whereas endothelin-3 has a lower affinity for the ETA subtype. Endothelin-1 is considered a potent sub-nanomolar vasoconstrictor in the human cardiovascular system [5].

The vasoconstrictor mechanism of action was obtained from a culture medium of bovine aortic endothelial cells, described and characterized for the first time in 1985 by Hickey et al. It was from a potent contraction derived from an endogenous vasoactive peptide of the vascular endothelium [6]. A few years later, Yanagisawa and his group, in 1998, isolated endothelin to investigate its potential as a vasoconstrictor. Concluding with their research, endothelin, as well as neurotoxins, act directly on membrane ion channels, suggesting that the action of endothelin is closely associated with the influx of Ca2+, through Ca2+ channels dependent on dihydropyridine, a calcium channel blocker that acts on smooth muscle cell [7].

Endothelins are synthesized into preprohormones and transformed posttranslationally into active peptides (**Figure 1**). Endothelin-1, which has been extensively studied, is synthesized with 212 amino acid residues (preproET-1), which are cleaved by an endopeptidase into Big-Et-1 (proET1), with 39 amino acid residues. This proET1 is in turn cleaved by endothelin converting enzyme (ECE), resulting in the active peptide hormone with 21 amino acid residues that play an important role in physiology [8].

Some works associate protein disulfide isomerase (PDI), based on evidence that shows the presence of PDI in the membrane of human erythrocytes, with the activity of the Gardos channel. Furthermore, a study by Prado and colleagues in 2013 showed that in the presence of endothelin-1, PDI activity increased, through a mechanism that includes casein kinase II. The Gardos channel is a K+ channel activated by Ca2+, in erythrocytes, being this channel related to homeostasis, so when this channel is activated, the cell dehydrates, leading to cell disorder and cell death. Prado suggests, as part of his research, the use of endothelin-1 receptor antagonists as a therapeutic target for sickle cell disease [9].

The ETB receptor has also been described in murine erythrocytes, and its presence has already been suggested in human erythrocytes. Foller and Rivera carried out studies showing the effect of endothelin-1 on erythrocytes regulation of Gardos channel activity, and this effect is due to the ET-1 binding to the ETB receptor. This effect interferes with the dehydration of sickled erythrocytes with a protective effect on the programmed death of murine erythrocytes (**Figure 2**) [10, 11].

*Hormones Action on Erythrocytes and Signaling Pathways DOI: http://dx.doi.org/10.5772/intechopen.110096*

#### **Figure 1.**

*Biosynthesis and amino acid sequence and structure of endothelin-1, endothelin-2, and endothelin-3 and related sarafotoxins. ET-2 and ET-3 differ from ET-1 by two and five amino acids, respectively, while sarafotoxin differs by seven amino acids [8].*

A study carried out by George et al. in 2013 demonstrated the presence of the erythrocyte ETB receptor on the membrane of healthy and sickle cell anemia patients, associating the increased production of reactive oxygen species (ROS) to the presence of endothelin-1, concluding that these ROS were generated from erythrocyte NADPH oxidase (**Figure 3**). They demonstrated the oxidation of HbS, induced damage to the structure of red blood cells, increasing lysis, deformation, and vaso-occlusive process in a patient with sickle cell anemia. The inflammatory process that is increasing due to these processes alters plasma proteins, leukocytes, and endothelial cells, impairing the inflammatory condition. In addition, extracellular signaling molecules associated with this entire process end up acting back on erythrocytes *via* cell surface receptors, activating signaling pathways, such as the PKC and Rac pathways [12].

Prado in 2013 also showed a relationship between increased protein disulfide isomerase (PDI) activity and ET-1. Researchers performed experiments using BERK mice, which are mice mutated for sickle cell anemia, where they showed that reducing PDI activity improved hematological parameters and it was also possible to notice that there was modulation in the effects of ET-1 against the Gardos channel, further suggesting that this modulation occurred *via* the ETB receptor, which is present on the erythrocyte membrane [9]. These findings corroborate the study by Rivera in 2002, which showed that ET-1 induced changes in red blood cell volume and increased K+

#### **Figure 2.**

*Effects of ET-1 and its receptor antagonists on dehydration of oxygenated and non-oxygenated sickle cells [10].*

#### **Figure 3.**

*Representation of nitric oxide synthase, ROS metabolism, and the presence of ROS enzyme inhibitors [12].*

flux *in vivo.* Activation of endothelin receptors on healthy erythrocytes regulates the site of Ca2+ affinity or an undefined Ca2+-dependent regulatory protein related to the Gardos channel. In the same study, they investigated whether this modulation would occur *via* the ETB receptor, using the receptor's antagonist, BQ-788, and showed a decrease in ET-1-induced activation of Gardos channel, both in healthy erythrocytes and in sickle erythrocytes [13].

Although the literature demonstrated the presence of ETB in erythrocytes, it is not well documented whether ETA is present and whether this receptor also modulates cell physiology. It is also important to compare the expression levels of these receptors in disease conditions compared to healthy counterparts to position the importance of endothelins in the disease process. Furthermore, it can be concluded that this endothelin-erythrocyte relationship is a powerful target therapeutic option in cases of anemia and also chronic pain, as reported by Smith et al. [14].

## **3. Thyroid-stimulating hormone (TSH)**

Thyroid-stimulating hormone activity in the pituitary gland was first reported in the 1920s. However, it was not until the 1980s that the hormone thyrotropin (THS)

*Hormones Action on Erythrocytes and Signaling Pathways DOI: http://dx.doi.org/10.5772/intechopen.110096*

had a detailed description of its structure [15, 16]. TSH is a glycoprotein produced by the thyrotrophs of the anterior pituitary gland. Thyrotropin-releasing hormone (TRH) is responsible for stimulating the synthesis and secretion of TSH, and inhibition is done through negative feedback by thyroid hormones (triiodothyronine and thyroxine—T3 and T4) [15, 17].

TSH, acting through the thyroid-stimulating hormone receptor (thyrotropin receptor—TSHR), is a G-protein-coupled receptor with seven transmembrane domains, and this protein can be Gq or Gs [18, 19]. Gs activities are mainly mediated by increased adenylate cyclase (AC) activity, which generates an increase in intracellular cAMP. This increase leads to direct activation of protein kinase A (PKA), activating CREB, or PKA activating the family pathway Ras. The Gq pathway mediates the activation of phospholipase C (PLC) and the Gβγ subunit, which will participate in the activation of second messengers, activating the Ras/Ras/Mek/Erk or PI3K/Akt pathways (**Figure 4**) [18–20].

In 2007, Balzan et al. identified TSHR in human erythrocyte membranes by Western blot (**Figure 5**). After the identification of TSHR, new research began on which pathways this hormone would act on the erythrocyte. In 2009, Balzan et al., demonstrated that TSH binds to TSHR in erythrocytes and modulates Na+ /K+ -ATPase, suggesting a new signaling pathway [21]. In 2020, Mendonça-Reis [22] showed that TSH at different concentrations (1–5 mIU/L) improved the resistance of red blood cells to hemolysis and this effect was caused by inhibition of the AMPK-dependent pathway and concomitant activation of the signaling pathway PI3K/Akt (**Figure 6**).

#### **Figure 4.**

*Simplified illustrative diagrammatic image of the main signaling pathways involved with the G protein. Five pathways can be seen from left to right: cAMP/PKA/ERK or PKA/CREB, PI3/Akt/mTOR, PKC/NFB, PKC/craf/ERK/p90RSK, and Ras/c-Raf/ERK [18].*

**Figure 5.** *Identification of TSH receptor on human erythrocyte membranes by Western blot [21].*

#### **Figure 6.**

*Illustrative image demonstrating that TSH improves erythrocyte resistance to hemolysis in a situation of inducedosmotic stress, TSH binds to its receptor (TSHr) on the erythrocyte membrane, inhibiting the AMPK-dependent pathway (stimulated by AICAR) and concurrently activating the PI3K (inhibited by wortmannin)/Akt (inhibited by Akt 1/2 inhibitor) signaling pathway [23].*

These new studies also opened a new range of research involving these receptors and diseases. For example, in the study by Ref. [24], it was suggested that erythrocyte Na+ /K+ -ATPase would be a good biochemical marker for subclinical hypothyroidism, as it is sensitive to subtle changes in thyroid function in this situation [24].

Patients with sickle cell disease (SCD) have also been found to have clinical hypothyroidism and high concentrations of TSH (6.4 mIU/L) [25]. And in ElAlfy et al. [26] observed impaired thyroid microcirculation and decreased thyroid volume among patients with SCD, and these factors were related to disease duration, but the results were not related to thyroid function, suggesting that these disorders can happen independently of the accumulation of iron [26].

On this basis, what can be observed from the identification of a functional TSH receptor in erythrocytes and some clarified pathways is that this hormone can modulate cell behavior and fate. All of these studies demonstrate in several ways the importance of continuing to elucidate the functions of TSH and its receptor on erythrocytes and how they may be involved in the pathophysiology of several diseases and serve as indicators of physiological changes.
