Acetylcholinesterase of erythrocytes

Erythrocytes or red blood cells (RBC) are more than sacks of oxyhemoglobin or deoxyhemoglobin during the semi-life of 120 days in blood circulation [1]. Erythrocytes comport different signaling pathways which includes the final stage of apoptosis, also called eryptosis [2,3]. Exovesicules enriched with acetylcholinesterase (AChE) originated from membranes of aged erythrocytes appear in plasma [4]. Kinetic changes of the AChE enzyme have been observed in old erythrocytes [5]. Previously, AChE in erythrocytes was evidenced as a biomarker of membrane integrity [6]. Later on, increased impairment values of AChE enzyme activities were observed in several diseases as will be described below.

Human erythrocyte acetylcholinesterase (AChE) discovered by Alles and Haves in 1940 was later, in 1961, classified as EC.3.1-1.7 by the Enzyme Commission [20,21]. Only in 1975 the appropriate process of extraction and purification of the erythrocyte membrane AChE confirmed it as a glycoprotein [22]. Later in 1985 it was shown that this enzyme, located in the external leaflet of the erythrocyte membrane, is a dimeric protein [23]. The catalytic efficiency of the dimeric form of AChE depends on the amphipathic medium of extraction and purification [24,25]. AChE belongs to the glycosylphosphatidylinositol (GPI)-anchored protein family and bears the Yta blood group antigen [26,27].

Implications of Erythrocyte Membrane Acetylcholinesterase Enzyme Activity in Disease

In 1973 erythrocyte AChE enzyme activity was reported as a marker of membrane integrity [38].

Changes in RBC AChE enzyme activity were evidenced in health and disease states. Reduced erythrocyte AChE activity in aged humans and in neonates related to adult man was evidenced [5,38]. Sub-fractions of RBCs of different ages, prepared in vitro, showed that the oldest RBCs present an AChE enzyme activity that is lower than the young RBC subpopulations, erythrocyte AChE being considered a biomarker of aging [39]. Increased RBC AChE enzyme activity has been evidenced in blood samples taken from healthy females [40].

In healthy females, adrenaline decreases AChE activity when α- and β-adrenergic receptors are blocked and an inverse significant correlation between erythrocyte membrane rigidity and AChE activity has also been registered. In erythrocytes from healthy males, adrenaline increases AChE activity when no adrenergic receptors are blocked [5]. An opposite profile of erythrocyte membrane fluidity under an adrenaline effect was observed in relation to that obtained in AChE activity in both genders [5]. Peripheral blood from males and females has shown echinocytes when adrenaline is present [5].

The discovery of sex-related differences in erythrocyte AChE activity and in the membrane hydrophobic region fluidity under the adrenaline influence can contribute to understanding different responses, attitudes, and behaviors with respect to stress situations, usually verified in both genders. The existence of certain gender characteristics, at the cellular level, has important implications in disease and medication responses.

Lower RBC AChE enzyme activity among farmers exposed to pesticides has been reported [41]. A recent evaluation of longitudinal changes of AChE and paraoxonase-1 enzyme activities in greenhouse workers, over a crop season, reveals that the decrease in RBC AChE results from an indirect effect of pesticides, generating oxidant molecules, inducing lipid peroxidation, and consequently interfering with the erythrocyte membrane’s integrity [41,42].

Patients with paroxysmal nocturnal hemoglobinuria and others with hemolytic anemia have been characterized by lower levels of erythrocyte AChE [5].

In non-insulin diabetes mellitus patients undergoing routine angiography, an impairment in RBC AChE enzyme activity and a lower fluidity in the hydrophobic erythrocyte membrane domain after fluorescein injection was verified [43]. The less active state of AChE probably results from conformational molecular changes occurring in AChE due to its tail insertion nearby or on high rigidity membrane domains [43].

Ex vivo studies using blood samples obtained from patients suffering different diseases, namely Parkinson, essential hypertension, glaucoma, retinal vasculitis, amyotrophic lateral sclerosis (ALS), and Hirschsprung’s disease, have evidenced augmented levels of RBC AChE enzyme activity [44,45,46,47,48,49]. Erythrocyte AChE is considered a biomarker of essential hypertension, glaucoma, ALS, neurotoxicity, and pesticide poisoning and a diagnostic marker in Hirschsprung’s disease [45,46,48,49,50,51,52]. Higher AChE enzyme activity in RBCs were verified in glaucoma, essential hypertension, and ALS, which are inflammatory vascular diseases characterized by a presence in the blood of high inflammatory molecule concentrations, reactive oxygen species, and reactive nitrogen species [45,46,49,50,53,54]. Consequently, erythrocyte AChE is considered a marker of inflammation [54,55,56]. The modulation of AChE enzyme activity by its natural substrate acetylcholine or by a strong inhibitor, such as velnacrine, showed ACh with an anti-inflammatory effect characterized by its protective action before inflammation development [56]. These anti-inflammatory characteristics of ACh (acetylcholine) were observed in vivo, by intravital microscopy, in an experimental animal model, by the quantification of pro-inflammatory cytokines production and by the visualization and quantification of leukocyte recruitment which includes the number of rolling and adherent leukocytes and their rolling velocities [55].

The erythrocyte membrane AChE’s enzyme activity values

The erythrocyte membrane AChE’s enzyme activity values are implicated as a biomarker of membrane integrity (normal), aging (lower), gender (higher in females than in males), inflammation (higher), neurotoxicity (higher), and pesticide poisoning (higher). The erythrocyte AChE is used as diagnostic marker in Hirschsprung’s disease. The active state of AChE is modulated by the membrane band 3 protein phosphorylation, meaning that this enzyme activity can be manipulated from inside the erythrocytes.

Erythrocyte AChE is a biomarker of inflammation and is involved in the white blood cells approaches to the endothelial vessel wall and in the production of pro-inflammatory cytokines.

At microcirculation, blood flow through small vessels favors gas exchanges, such as the exchange between oxygen and nitric oxide with carbon dioxide, delivers nutrients metabolites, and removes waste products. Erythrocytes deliver NO (nitric oxide) in tissues with lower oxygen partial pressure (PaO₂) and scavenge it at high PaO₂ through the band 3 protein [80]. The ability of erythrocytes to deliver or retain NO depends of the membrane integrity, of the AChE activation state and of its molecular protein conformations.

The signal transduction pathways associated with NO mobilization in erythrocytes were described under the influence of the endogenous plasma compounds, namely, ACh and fibrinogen, whose levels increase in inflammation. There are specific key points in those pathways where activators or inhibitor molecules of AChE, PTK (protein tyrosine kinase), PTP (protein tyrosine phosphatase), AC (adenylyl cyclase), PDE3(phosphorylates phosphodiesterase-3) and PKC (protein kinase C ) change the NO (nitric oxide) efflux from erythrocytes, which, in the future, might be considered as therapeutic targets in vascular inflammatory diseases.

It is mandatory to highlight the new function for erythrocyte enzyme membrane AChE acting as receptor for hydrophilic blood circulating molecules in the NO signal transduction pathway.

PS: Novichok is a group of organophosphate chemicals that act as nerve agents and were designed for use as weapons of chemical warfare.

Mechanism of action and toxicity

Novichok agents are anticholinesterases (also known as acetylcholinesterase inhibitors). They bind to the enzyme acetylcholinesterase and thereby prevent the destruction of the neurotransmitter acetylcholine within the nervous system. The buildup of acetylcholine at its sites of action results in the continuous stimulation of the parasympathetic nervous system, causing decreases in heart rate and blood pressure and increases in the secretion of body fluids. The agents are also suspected of targeting neurons more generally in the central and peripheral nervous systems.

Novichok agents are severely toxic. Poisoning can occur by inhalation, ingestion, or skin contact. Absorbed in small quantities, the agents induce convulsions, muscle weakness, nausea, paralysis, vomiting, and difficulty breathing due to the secretion of fluids into the airways. In larger doses, the respiratory muscles become paralyzed and death occurs, typically by asphyxiation. Those who survive poisoning are at risk of experiencing long-lasting or permanent neurological damage.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6151671/#:~:text=Erythrcito%20AChE%20is%20a%20biomarcador,producción%20of%20pro%2Dinflamatoria%20citocinas .

https://www.nature.com/articles/pr197347.pdf

https://www.britannica.com/technology/Novichok