+ Site Statistics
+ Search Articles
+ Subscribe to Site Feeds
EurekaMag Most Shared ContentMost Shared
EurekaMag PDF Full Text ContentPDF Full Text
+ PDF Full Text
Request PDF Full TextRequest PDF Full Text
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter
Follow on LinkedInFollow on LinkedIn

+ Translate

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

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

Plos One 8(2): E56759-E56759

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. 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. 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.

(PDF emailed within 0-6 h: $19.90)

Accession: 055333606

Download citation: RISBibTeXText

PMID: 23424675

DOI: 10.1371/journal.pone.0056759

Related references

Decreased cyclin-dependent kinase 5 (cdk5) activity is accompanied by redistribution of cdk5 and cytoskeletal proteins and increased cytoskeletal protein phosphorylation in p35 null mice. Journal of Neuroscience 23(33): 10633-10644, 2003

Respiratory substrates regulate S-nitrosylation of mitochondrial proteins through a thiol-dependent pathway. Chemical Research in Toxicology 27(5): 794-804, 2015

Effects of superoxide anions on rbc deformability and membrane proteins impaired rbc deformability due to degradation of membrane proteins. Biorheology 29(1): 37, 1992

Cellular deformability as an indicator of cytoskeletal viscoelasticity. Biorheology 27(1): 106, 1990

Hydrolysis of cytoskeletal proteins by the Ca2+-dependent protease during platelet activation. Advances in Experimental Medicine and Biology 192: 201-213, 1985

Activation of mitogen-activated protein kinase cascade and phosphorylation of cytoskeletal proteins after neurone-specific activation of p21ras. II. Cytoskeletal proteins and dendritic morphology. Neuroscience 105(4): 1041-1054, 22 August, 2001

In vitro and in situ proteolysis of some cytoskeletal proteins by the Ca-2+-dependent protease in Allomyces. Experientia (Basel) 50(ABSTR ): A88, 1994

Calcium-calmodulin-dependent phosphorylation of cytoskeletal proteins from adrenal cells. Molecular and Cellular Endocrinology 74(2): 109-123, 1990

Modulation of the voltage-dependent inactivation of cardiac L-type Ca channels by cytoskeletal proteins. Japanese Journal of Pharmacology 79(SUPPL 1): 39P, 1999

Time-dependent alteration of cytoskeletal proteins in cerebral cortex of rat during 2,5-hexanedione-induced neuropathy. Neurochemical Research 32(8): 1407-1414, 2007

Analysis of calcium-dependent proteinase and cytoskeletal proteins in muscle from patients with inflammatory myopathy. Annals of Neurology 54(Suppl 7): S44-S45, 2003

Is cytoskeletal tension a major determinant of cell deformability in adherent endothelial cells?. American Journal of Physiology 274(5 Pt 1): C1283-9, 1998

PH and phospholipid-dependent properties of profilin Mechanistic implications for cytoskeletal regulatory proteins at the plasma membrane. Molecular Biology of the Cell 11(Supplement): 551a, 2000