RBC-NOS-dependent S-nitrosylation of cytoskeletal proteins improves RBC deformability

Abstract

Background: Red blood cells (RBC) possess a nitric oxide synthase (RBC-NOS) whose activation depends on the PI3-kinase/Akt kinase pathway. RBC-NOS-produced NO exhibits important biological functions like maintaining RBC deformability. Until now, the cellular target structure for NO, to exert its influence on RBC deformability, remains unknown. In the present study we analyzed the modification of RBC-NOS activity by pharmacological treatments, the resulting influence on RBC deformability and provide first evidence for possible target proteins of RBC-NOS-produced NO in the RBC cytoskeletal scaffold.

Methods/findings: Blood from fifteen male subjects was incubated with the NOS substrate L-arginine to directly stimulate enzyme activity. Direct inhibition of enzyme activity was induced by L-N5-(1-Iminoethyl)-ornithin (L-NIO). Indirect stimulation and inhibition of RBC-NOS were achieved by applying insulin and wortmannin, respectively, substances known to affect PI3-kinase/Akt kinase pathway. The NO donor sodium nitroprusside (SNP) and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) were additionally applied as NO positive and negative controls, respectively. Immunohistochemical staining was used to determine phosphorylation and thus activation of RBC-NOS. As a marker for NO synthesis nitrite was measured in plasma and RBCs using chemiluminescence detection. S-nitrosylation of erythrocyte proteins was determined by biotin switch assay and modified proteins were identified using LC-MS. RBC deformability was determined by ektacytometry. The data reveal that activated RBC-NOS leads to increased NO production, S-nitrosylation of RBC proteins and RBC deformability, whereas RBC-NOS inhibition resulted in contrary effects.

Conclusion/significance: This study first-time provides strong evidence that RBC-NOS-produced NO modifies RBC deformability through direct S-nitrosylation of cytoskeleton proteins, most likely α- and β-spectrins. Our data, therefore, gain novel insights into biological functions of RBC-NOS by connecting impaired RBC deformability abilities to specific posttranslational modifications of RBC proteins. By identifying likely NO-target proteins in RBC, our results will stimulate new therapeutic approaches for patients with microvascular disorders.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Changes of RBC-NOSSer1177 levels after RBC incubation with RBC-NOS stimulants, inhibitors and NO controls.

(A) Bars show statistical analysis of gray values [au] after a 60 min incubation of RBCs with insulin and wortmannin, respectively. Application of RBC-NOS stimulant insulin led to a significant increase in enzyme phosphorylation compared to control (P<0.01). Wortmannin (P<0.001) application decreased phosphorylation, respectively. (B) Pictures show representative RBC-NOSSer1177 staining after 60 min incubation, respectively. Magnification for all images was 400-fold. Data in (A) are presented as mean±S.E.M., n = 6.

Figure 2. Changes in nitrite concentration after modification of RBC-NOS activity, and plasma ADMA content after L-arginine application.

Nitrite was measured as representative of NO synthesis after 60 min incubation of RBCs with RBC-NOS stimulants, inhibitors and NO controls. (A) RBC nitrite level significantly increased after L-arginine (P<0.01), insulin (P<0.01) and SNP (P<0.01) application, while L-NIO (P<0.001), wortmannin (P<0.01) and cPTIO (P<0.05) significantly decreased RBC nitrite content compared to control samples. (B) Plasma ADMA concentration was additionally measured after L-arginine incubation to present an explanation for the L-arginine paradox. Bars show that ADMA concentration in the plasma fraction significantly increased upon substrate addition (P<0.001). (C) Plasma nitrite concentration was not altered by the applied substances, except SNP. Data in (A–C) are presented as mean±S.E.M., n = 15.

Figure 3. S-nitrosylation of RBC proteins after modification of RBC-NOS-dependent NO production.

(A) Representative western blot bands after 60 min incubation of RBCs with RBC-NOS stimulants, inhibitors and NO controls are demonstrated. The image shows two distinctive bands with varying intensity depending on RBC-NOS modulation. LC-MS/MS analysis identified 240 kDa band as possible α- spectrin and 220 kDa band as possible β-spectrin. (B) Calculation of relative intensity in relation to the control sample revealed that S-nitrosylation of both, α- and β-spectrin, significantly increased upon L-arginine, insulin and SNP incubation and significantly decreased upon L-NIO, wortmannin and cPTIO incubation, respectively. Data in (B) are presented as mean±S.E.M., n = 6.

Figure 4. Maximum deformability (EI max) after RBC incubation with RBC-NOS stimulants, inhibitors and NO controls and correlation of EI max with S-nitrosylation of α- and β-spectrin.

(A) Bars show that EI max significantly increased after 60 min incubation with L-arginine (P