Intensive exercise induces changes of endothelial nitric oxide synthase pattern in human erythrocytes
Introduction
Nitric oxide (NO) is known to be synthesized in variety of different organs or tissues playing a multiplicity of physiological roles. NO characterizes a highly reactive, short-lived and gaseous diffusible molecule that interacts with different biological agents either by covalent or redox reactions. NO functions as a potent regulator of vascular tone, cytotoxic agent, neurotransmitter, and as an antioxidant by its reaction with superoxide to form peroxynitrite. Additionally, NO may serve as a modulator of overall microvascular integrity, function and oxygen transport. NO is synthesized from l-arginine pathway in a variety of cell types, including neutrophils, macrophages, platelets, endothelial, smooth muscle as well as parenchymal cells by a family of isoenzymes, so-called nitric oxide synthases (NOSs) [1]. The three NOS isoforms have been categorized as type I (neuronal NOS, nNOS), characterized by an inducible expressed and calcium-dependent NOS isoform. Type II (inducible NOS, iNOS) describes a calcium-independent NOS isoform. The third NOS isoform (endothelial NOS, eNOS) is characterized by its constitutive expression and its calcium dependence. But it is now apparent that all three isoforms can be induced, although different stimuli are responsible for the distinct isoforms [2]. Different authors demonstrated that under normal conditions, endothelial cells produce a small amount by eNOS in response to either receptor-mediated stimuli or to shear stress-mediated vasodilatory stimuli [3]. Because of its gaseous properties, NO diffuses down its concentration gradient from endothelial cells to smooth muscle cells to induce vasodilation. A further important regulatory role of NO may be due to its ability to regulate erythrocyte deformability [4]. It is well established that NO is involved in key physiological functions, such as anti-apoptotic activity [5] and mitogen-activated protein (MAP) kinase signaling [6]. Therefore, NO is an important factor in maintaining the integrity of blood by regulating resistance vessel diameter, blood rheology and blood vessels.
NO is typically produced by endothelial cells in the vascular system through the conversion of the l-arginine to l-citrulline using molecular oxygen. In the vascular bed, NO synthesis has been attributed exclusively to the endothelial NOS isoform (eNOS). The endothelial NOS characterizes a constitutive, double acetylated membrane protein that can be localized within the plasma membrane as well as in the Golgi region of endothelial cells [7]. Initially this enzyme was thought to be a solely calmodulin-regulated molecule, it now becomes clear that eNOS has evolved to be tightly controlled by several co-factors and post-translational modifications, phosphorylation on multiple residues, and regulated protein–protein interactions [8], [9]. Michel and Feron demonstrated that the eNOS activation is translocation-dependent. The authors showed that an increased intracellular calcium concentration leads to the dissociation of eNOS from its inhibitory plasmalemmal binding partner, the caveolin-1. This dissociation facilitates a binding of eNOS to calmodulin to induce NO synthesis [10]. Beside the intracellular calcium concentration, different phosphorylation states are key regulators of eNOS activity. Three major phosphorylation states have been described. These are the phosphorylations at the residues serine116 (phospho-eNOSSer116), serine1177 (phospho-eNOSSer1177), and threonine495 (phospho-eNOSThr495) of the eNOS isoform. The most potent triggers of eNOS phosphorylation are bloodstream-mediated shear stress and the encroachment of the vascular endothelium. Shear stress induces an activation of phosphoidylinositol-3 kinase (PI-3 kinase) and subsequently an activation of protein kinase B (Akt) is mediated. The Akt signaling pathway is important because it phosphorylates the eNOS at its residue phospho-eNOSSer1177. This phosphorylation enhances the NO production [11], [12]. Additionally, the NO production will be reduced through the phosphorylation of phospho-eNOSSer116, because of lower electron transfers [13]. A phosphorylation of the eNOS enzyme at its residue phospho-eNOSThr495 results in a decreased release of NO because of the inhibitory effect of this phosphorylation state on eNOS activity. This event goes along with an up-regulation of protein kinase C (PKC) [11].
Different authors have described that human blood and, in particular hemoglobin-carrying red blood cells (RBCs), also called erythrocytes, display a prominent pool for NO [14]. This is due to the fact that in the human vasculature, RBCs display the major scavenger of NO, because of the high hemoglobin (Hb) content in RBCs. In RBCs NO is converted to nitrate by HbFe(II)O2. On the other hand HbFe(II) binds to NO that subsequently forms HbFe(II)NO [15]. Different authors showed that the NO bioavailability is strongly regulated by RBCs and by the potential of RBCs to consume NO. Han et al. [16] demonstrated that hypoxic RBCs consume large amounts of NO. This event is subjected to the oxygen release from HbFe(II)O2 under hypoxic conditions. When hypoxic RBCs were subsequently exposed to NO, HbFe(II)NO will be formed and the NO consumption rates by RBCs in the vasculature will increase. These results might be interesting for NO regulation under intensive exercise conditions. It is well-established that intensive exercise can cause local hypoxic situations [17] that might trigger the described NO metabolism within RBCs. Former studies revealed that a NOS isoform might be resident in RBCs [18]. There remains an ongoing debate as to whether RBCs possess a NOS isoform and where this possible isoform might be located. However, very recently we were able to demonstrate that erythrocytes comprise a functional NO synthase located within the plasma membrane and the cytoplasm. The NO synthesis is dependent on the amount of l-arginine, the local calcium concentration as well as the phosphorylation state of PI-3 kinase and influences deformability of erythrocytes as well as platelet aggregation [19].
Acute and/or chronic exercise, and especially the resulting shear stress, are important stimuli for NO production in endothelial cells [1]. Endothelial cell generated NO interacts with RBCs via binding to the heme portion of hemoglobin to form S-nitrosohemoglobin [20] and inducing the formation of methemoglobin [21], because hemoglobin presents both nitrite oxidase and reductase activity, depending on its oxygenation state [5]. Until today, it is still unknown whether exercise influences the regulation of erythrocyte eNOS content and activity. On the one hand it seems to be obvious that exercise affects the plasma membrane of RBCs and therefore impairs their deformability. This effect might result in a reduced oxygen supply in peripheral capillaries [22], [23]. On the other hand exercise induces NO release in erythrocytes and thus improves the deformability of RBCs [4]. Sureda and co-workers recently described a possible NO production in blood cells in general after exercise. But the data does not specifically deal with RBCs [24]. Therefore, the present study aims to investigate the possible influence of a two-day long highly intensive training camp on the eNOS isoform in human RBCs by describing effects of intensive exercise on eNOS content and the phosphorylation states at Ser116, Ser1177, and Thr495 of the eNOS in human erythrocytes. Additionally, in the present study nitrotyrosine was focused. By producing large amounts of NO it seems to be well established that NO reacts with the superoxide anion (O2−). This reaction results in the formation of peroxynitrite and subsequently nitrotyrosine will be generated. Therefore, nitrotyrosine may function as an indirect marker for the determination of NO production in the circulation [25].
Section snippets
Selection of subjects
Twelve male international-class field hockey players participated in this study. Characteristics of basic anthropometric and physical parameters of the subjects are summarized in Table 1. In addition, all subjects abstained from alcohol consumption for 24 h prior to and during the training intervention and were non-smokers. All subjects gave written informed consent to contribute to the study. The protocols used in the study have been granted a license from the institutional office to conduct
Exercise and eNOS content in erythrocytes
Statistical analysis of the gray values of athletes’ erythrocytes against eNOS revealed significant differences between the pre-training camp conditions compared to the post-training camp conditions. After the conducted training camp eNOS was decreased (pre: 14.29 ± 0.80 vs. post: 10.99 ± 0.79, p = 0.000045). The data are presented in Fig. 2A. The offered pictures in Fig. 2B and C of stained erythrocytes show well-defined results as eNOS in erythrocytes of the athletes is higher before the training
Discussion
The present study aimed to investigate the influence of a two-day long intensive training camp on different NOS and phosphorylation states of eNOS in erythrocytes of international-class field hockey players. It was demonstrated that intensive exercise induces significant down-regulations of eNOS, activated eNOS, phosphorylated eNOSSer1177, eNOS, and nitrotyrosine. Phosphorylated eNOSSer116 and phosphorylated eNOSThr495 were not affected by exercise input. The severity of the two-day long
Conclusion
In conclusion the present study provides evidence that human erythrocytes contain eNOS in different phosphorylation and activation stages. This is a new observation supporting the recent findings of Kleinbongard and colleagues [19] who described a functional eNOS located in human erythrocytes. Therefore, the current results may underline the hypothesis that NO synthesis within the cardiovascular system is not strictly connected to endothelial cells, but also that erythrocytes possess the
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