Endothelin-1 modulates hemoglobin-mediated signaling in cerebrovascular smooth muscle via RhoA/Rho kinase and protein kinase C

Endothelin- 1 ( ET- 1) and oxyhemoglobin ( OxyHb) have been implicated in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage. However, the contribution of ET- 1 to this condition has not been definitely established. In this study, we investigated whether threshold concentration of ET- 1 enhances cerebrovascular smooth muscle ( CVSM) contraction to OxyHb by activating the RhoA/ Rho kinase and protein kinase C ( PKC) pathways. CVSM contraction was measured in endothelium- denuded rabbit basilar arteries. Cytosolic and particulate fractions of CVSM cells were examined for RhoA and PKC reactivity with specific antibodies using immunoblotting procedures. ET- 1 ( 0.1 nM) alone did not produce any significant contraction, but it markedly potentiated the magnitude ( 223% of control) and rate ( 149% of control) of contraction in response to OxyHb, which was attenuated by the inhibitors of Rho kinase Y- 27632 and HA- 1077. ET- 1- mediated potentiation of the contraction was also inhibited by inhibitors of PKC, Ro- 32- 0432, and GF- 109203X. BQ- 123 prevented potentiation of vasoconstriction mediated by ET- 1, indicating that the action of ET- 1 was mediated by the endothelin type A receptor. Pretreatment with ET- 1 significantly enhanced OxyHb- mediated RhoA translocation in CVSM cells and intact basilar arteries. ET- 1 also caused potentiation of PKC-epsilon expression in membranes of CVSM cells exposed to OxyHb for 10 and 60 min but did not markedly change the distribution of PKC-alpha. Thus, in CVSM, threshold concentration of ET- 1 potentiates contraction induced by OxyHb via RhoA/ Rho kinase- and PKC-epsilon- dependent mechanisms. This process may contribute to the pathological contraction of cerebral arteries observed after subarachnoid hemorrhage.