Hormones Action on Erythrocytes and Signaling Pathways

Abstract

Erythrocytes are the most abundant cell type in the human body, although considered as merely hemoglobin carriers for a long time. Extensive studies on its biochemical pathways, metabolism, and structure-activity relationship with a consistent number of publications demonstrated the presence of autocrine, paracrine, and endocrine hormone receptors. In this chapter, some of these hormones will be discussed, bringing attention to those that regulate erythrocyte survival, disease connection, and functionality.

1. Introduction

In the human body, there are several hormones that regulate different functions, such as growth and development, metabolism, electrolyte balance, and reproduction [1]. These functions have already been widely studied, and most of them are very well elucidated; even so, there are still aspects that have not yet been well explored. For example, the relationship between erythrocytes and hormones. Erythrocytes were treated as merely hemoglobin carriers for a long time, with small or none of the complex functions as seen currently.

It has already been seen that in healthy erythrocytes, adenosine can increase 2,3-bisphosphoglycerate (2,3-BPG) through activation of the ADORA2B receptor (A2B), suggesting that increasing adenosine levels increases oxygen release, which is positive for preventing tissue damage from acute ischemia. However, in sickly erythrocytes, the increase in oxygen release through the induction of 2,3-BPG by adenosine can be harmful, as oxygen release of oxygen induces the sickling of these erythrocytes [2].

In 1999, a study by Tuvia et al. showed that exposure of erythrocytes to adrenaline led to a concentration-dependent increase in erythrocyte deformability, and consequently, increased oxygen delivery to tissues [3]. Another study reported that adrenaline and epinephrine dose-dependently increase the rate of erythrocyte agglutination through alpha-1 adrenergic receptor activation. Furthermore, this study suggested that the effect of adrenaline was caused by an increase in Ca²⁺ entry into the erythrocyte, with consequent activation of the erythrocyte calmodulin, cyclooxygenase, and phospholipase A2 and leading to the release of K⁺ from erythrocytes through Ca²⁺-dependent K⁺ channels, which is considered a manifestation of eryptosis. In summary, the potentiation of α1AR activation increases, while β2AR decreases the rate of eryptosis [4].

These studies present modulation of erythrocyte function by surface receptors and some of the signaling pathways that might be triggered inside the cell. In this chapter, the focus is to explore the actions of three well-studied hormones on erythrocyte function and some reports regarding intracellular pathways involved in these modulations.

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].

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].

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].

Figure 2.

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

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].

Figure 3.

Representation of nitric oxide synthase, ROS metabolism, and the presence of ROS enzyme inhibitors [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⁺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) 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, 19, 20].

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/c- raf/ERK/p90RSK, and Ras/c-Raf/ERK [18].

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 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 induced-osmotic 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.

4. Angiotensin II

The renin-angiotensin aldosterone system is an endocrine system responsible for regulating normal blood pressure in vivo, through the regulation of extracellular volume, vascular structure, and integrity, maintaining homeostasis [27, 28]. One of its main components is angiotensin II (Ang II), a vasoactive octapeptide responsible for several actions in tissues [29].

Two subtypes of Ang II receptors have been identified in humans, ATR1 and ATR2, described as receptors formed by seven transmembrane domains coupled to the G protein (Figure 7), with ATR1 being the dominant subtype and being widely distributed in the endocrine, renal, cardiac, and nervous systems [30]. While ATR2, after birth, has low expression, persisting only in some organs, such as the brain, kidney, and peripheral vasculature, mediating the physiological effects of Ang II, and may increase its expression in pathological conditions, such as hypertension, cardiac, and renal failure [28, 31].

Figure 7.

Angiotensin II synthesis. Angiotensinogen is converted into angiotensin I by renin that in turn is converted into angiotensin II by the angiotensin converting enzyme. Angiotensin II can bind to ATR1 and ATR2.

There are studies about signaling pathways and the action of angiotensin in different cell types, however, there is little information in the literature about the actions of Ang II receptors on erythrocytes and their signaling, especially considering human erythrocytes.

In vitro experiments, carried out in 1997, showed a stimulatory effect of Ang II on erythropoiesis when erythroid progenitors were cultured in the presence of erythropoietin, demonstrating the importance of erythropoietin in erythropoiesis. Erythropoietin is an essential hormone for the regulation of erythropoiesis [32]. They also observed that the use of losartan (ATR1 antagonist, used to prevent vasoconstriction and volume expansion induced by circulating Ang II) blocked this stimulatory effect, demonstrating that Ang II, via the AT1 receptor, is responsible for mediating this stimulation [33, 34].

Reinforcing previous results in 2005, it was demonstrated that persistent activation of the renin-angiotensin system increases erythropoiesis in mice, in vivo, and that the most important receptor subtype responsible for this erythropoiesis was the AT1 receptor [35]. In 2015, in addition to reinforcing the idea that erythropoiesis and blood pressure are negatively regulated by inhibiting the ATR1 receptor, it was possible to say that the signaling pathways involved are complex and distinct since erythropoiesis is more resistant to inhibition of the ATR1 receptor than blood pressure control [30].

Some studies on the Ang II signaling pathway in erythrocytes have been described in order to elucidate the mechanism of the parasitic invasion of Plasmodium falciparum in erythrocytes. Research published in 2011 demonstrated that human erythrocytes express different Ang II receptors, namely ATR1, ATR2, and MAS receptors [36].

The levels of bradykinin (BK) and Ang- (1–7) increase in the presence of captopril (ECA inhibitor) in the supernatant of infected erythrocytes, decreasing parasite invasion and reducing PKA activity, through the association of receptors B2/MAS [37]. Thus, inhibition of protein kinase A (PKA) by the MAS receptor appears to be favorable to the erythrocyte against parasitic invasion.

Experiments in mice and humans showed that the activation of the ADORA2B-AMPK cascade, in the presence of angiotensin II, increased BPG mutase and consequently 2,3BPG, being beneficial for renal hypoxia, injury, proteinuria, and reduction of chronic kidney disease (CKD) [38].

In 2021, Guimares-Nobre and collaborators demonstrated that AT1 receptors and AT2 receptors, expressed in human erythrocytes, are capable of responding to osmotic stress situations in the presence of Ang II and its antagonists, losartan (ATR1 antagonist) and PD123319 (ATR2 antagonist). The study was carried out using modulators of signaling pathways, already recognized as activated by Ang II, in other cell types. As a result, it was possible to observe that, in osmotic stress, Ang II binds to the ATR2 receptor and reduces hemolysis through the PI3K/AKT and P38 pathways. However, when binding to ATR1, this protection did not occur (Figure 8) [39].

Figure 8.

Representative scheme of angiotensin II signaling pathway in erythrocytes in osmotic stress [39].

Furthermore, ex vivo experiments displayed that sickle cell erythrocytes treated with Ang II present increased cell deformability, decreased phosphatidyl serine translocation to the outer layer, and decreased hemoglobin S polymerization. All these factors are crucial findings for sickle cell disease outcomes (Figure 9) [40]. However, despite knowing this important action of Ang II in sickle cell erythrocytes, the precise mechanism of actions has not yet been demonstrated.

Figure 9.

Representative scheme of angiotensin II effects on sickle cell disease erythrocytes [39].

There is very little information regarding the importance of angiotensin II receptors on erythrocytes and the signaling pathways are triggered by it; however, researchers have been demonstrating that these receptors are functional and interfere in the survival of erythrocytes. Therefore, more studies are needed to expand this knowledge.