Rho-kinase is a negative regulator of red blood cell eNOS under basal conditions

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.

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.

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.

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.

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.