An ex vivo study of nitric oxide efflux from human erythrocytes in both genders

1 Introduction

Nitric oxide (NO) is a gas signalling molecule that modulates neurological activity, blood flow and endothelial function [2, 16]. Once synthesized in endothelial cells by endothelial nitric oxide synthase (eNOS), NO can diffuse to the adjacent smooth muscle cells or to the vascular lumen [3]. In the vascular lumen NO can be captured by red blood cells (RBCs) that store and release this molecule, regulating erythrocytes’ hemorheological properties and their ability to transit through the capillaries [11]. When scavenged by the erythrocyte, NO enters through the protein of membrane band 3 protein and binds to haemoglobin (Hb) [4, 6].

Another protein located on the outer surface of the erythrocyte membrane is acetylcholinesterase (AChE), known as a marker of membrane integrity [1].

An in vitro study showed that erythrocyte AChE activity values in female were significantly higher than in male [11]. Once the active complex AChE-ACh triggers a signal transduction mechanism that leads to the release of NO from erythrocyte [6, 14], we hypothesised that differences between genders verified in AChE enzymatic activity could also represent differences of NO efflux from the RBC.

Thus, the main objective of this ex vivo study was to compare the amount of NO efflux obtained from erythrocytes of healthy donors in males and females.

2 Materials and methods

3 Results

Results showing values of NO efflux from erythrocytes and membrane AChE enzyme activity obtained from the 66 healthy volunteers divided according the gender, with age between 40 and 60 years old, can be observed in Table 1.

Regarding NO efflux from erythrocytes no significant differences between men and women was observed (Table 1). That is also the case of enzymatic activity of AChE in erythrocytes membranes, where no difference between genders was verified (Table 1).

It was necessary to perform an interquatile range as a strong measure of the spread values obtained in both variables.

Comparing the values of NO efflux from the erythrocytes to the quartiles of AChE enzyme activity values, there were no significant differences between men and women Fig. 1.

At variance the values of AChE enzyme activity in the third quartile of NO were significantly higher (p < 0.05) in women in comparison to men Fig. 2.

4 Discussion

This study shows that there are no differences between the values of erythrocyte NO efflux obtained from men and women in the same range of age (40–60 years old).

The principle of the amperometric method for NO measurements of the release of NO from the RBC suspensions is based in the pathway initiated by binding of ACh to membrane AChE. The active complex ACh-AChE nearing band-3 protein associates with protein Gi protein resulting in decrease of cAMP levels and increase in protein C kinase enzyme activity which activate protein tyrosine kinase (PTK) that phosphorylated protein band-3, and promotes NO release [7].

In the present ex vivo study, there are not significant differences in the values of erythrocyte AChE enzyme activity between men or women, being the concentration of NO efflux from erythrocytes no significantly different in both genders.

Nevertheless, in Sandra Hilário’s work the values of erythrocyte AChE enzyme activity were significantly higher in women than in men [11]. It is important to note the range of age of the participants. In Hilário’s study, the average age was of 24±2 years, while we recruited healthy donors between 40 and 60 years old. Another difference that must be highlighted refers to the blood collection, which was done in fast humans in Hilario‘s study at variance with the present study where blood samples were collected after the breakfast.

In healthy premenopausal women the production of NO is greater, in comparison to men, due to ovarian hormones (endothelium-derived NO-dependent vasodilatation is enhanced by oestrogen involved in up regulating NO synthase) [12], which may contribute to a low risk of cardiovascular events in women of reproductive age [9].

Although our ex vivo study illustrates slightly higher NO efflux values in women in comparison to men, that is not statistically significant for all the quartiles values of AChE enzyme activity.

However, in the 3^rd quartile of NO efflux the values of AChE enzyme activity are higher in women than in man. This result is in accordance with the positive relation between the two variables, verified in in vitro studies [13]. It was also evidenced that when band 3 protein is phosphorylated, AChE showed higher enzyme activity and consequently favoured the efflux of NO from erythrocytes [4].

Since AChE is a marker of hypertension [15], we can assume that the majority of individuals, situated in a normal range of AChE enzymatic activity (200–300 U/min/mg Hb), are not hypertensive, which is in accordance with the protocol of recruitment of healthy donors. Thus, as erythrocyte membrane is one of the factors responsible for the maintenance of normal blood rheology and tissue oxygenation [18], healthy blood donors evidenced an erythrocyte membrane integrity able to furnish nitric oxide. Despite all literature about erythrocytes, only a little is known about its properties and functions differences between genders. However, animal and human studies demonstrate that gender of subjects affect some vascular responses, which may denote important implications in the severity and incidence of diseases (such as heart disease, obesity and rheumatoid arthritis [17]), and medication applied to different genders.

5 Conclusion

The present work suggests that NO efflux from healthy humans’ erythrocytes do not change with gender.

More studies are needed to understand molecular differences between male and female in order to determine how gender influences susceptibility to disease and functioning of organ systems in order to improve suitable therapies according the gender.