Correspondence:
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Corresponding author: Pinar Ulker, Department of Physiology, Akdeniz University, Medical Faculty, Kampus, 07070, Antalya, Turkey. Tel.: +90 2422496960; Fax: +90 2422274483; E-mail: [email protected].
Abstract: BACKGROUND:Rho-kinase, an effector of the small GTPase RhoA, is known to be a novel inhibitory regulator of eNOS in endothelial cells under basal conditions and disease states. However, although RBC possesses active RhoA/Rho-kinase pathway, Rho-kinase mediated eNOS regulation has not been investigated in RBC, so far. OBJECTIVE:The aim of the present study is to investigate whether eNOS activity is regulated by Rho-kinase under basal conditions and to evaluate whether inhibition of this enzyme causes eNOS activation and intracellular NO production in RBC. METHODS:RBC packeds were isolated from healthy volunteers and resuspended in Hepes solution at a hematocrit of 0.01 l/l. Intracellular NO and Ca+2 levels and eNOS activation measured by flow cytometry in response to Rho-kinase inhibitors, fasudil and Y-27632, in the absence and presence of NOS, and PI3K inhibitors. RESULTS:Rho-kinase inhibitors fasudil and Y-27632 found to increase intracellular NO concentrations. These inhibitors also cause enhancement of intracellular Ca+2 and serine 1177 phosphorylated eNOS levels. Besides, although these responses have shown to be suppressed by NOS enzyme, PI3K inhibition had no effect on this mechanism. CONCLUSIONS:The results of the present study demonstrated that RBC eNOS enzyme activity is regulated by inhibitory Rho-kinase pathway under basal conditions and inhibition of this pathway enhances the activity of eNOS in RBC. This activation is mediated by both intracellular Ca+2 and Serine 1177 phosphorylated eNOS increment, with no contribution of AKT activation, in RBC. The mechanism we described here gives first evidences about Rho-kinase mediated eNOS regulation in RBC under basal conditions. This pathway could also be more important under disease states.
Keywords: Red blood cells, Rho-kinase, nitric oxide, nitric oxide synthase
