Interactive Role of Protein Kinase C-δ with Rho-Kinase in the Development of Cerebral Vasospasm in a Canine Two-Hemorrhage Model

Abstract

Background: We previously reported that protein kinase C (PKC)-δ was initially translocated from the cytosol to the membrane fraction (on day 4), followed by PKC-α, with the progression of cerebral vasospasm after subarachnoid hemorrhage (SAH) on day 7. Rho/Rho-kinase pathways have also been proposed to be involved in the vasospasm. Thus we investigated the interactive role of Rho-kinase and PKC in the development of cerebral vasospasm after SAH. Methods: The cerebral vasospasm was produced using a ‘two-hemorrhage’ canine model. The animals were treated with Y-27632, a Rho-kinase inhibitor, and rottlerin, a PKC-δ inhibitor, both injected into the cisterna magna. Results: Y-27632 inhibited the vasospasm, 20-kDa myosin light chain (MLC₂₀) phosphorylation, and PKC-δ translocation after the second injection of autologous blood on day 4. In contrast, Y-27632 did not affect the vasospasm on day 7. Rottlerin also inhibited the vasospasm on day 4, but had no effect on MLC₂₀ phosphorylation and RhoA translocation. The vasospasm was accompanied with the phosphorylation of caldesmon (CaD), an actin-linked regulatory protein, which was strongly attenuated by Y-27632 and rottlerin. The application of PKC-δ to skinned strips of isolated canine basilar arteries caused a contraction and an increase in CaD phosphorylation. Conclusion: The development of cerebral vasospasm after SAH (on day 4) is caused by at least two mechanisms: one involves MLC₂₀ phosphorylation mediated by the inhibition of MLC₂₀ phosphatase by Rho-kinase, and the other CaD phosphorylation mediated by the activation of PKC-δ by Rho-kinase, which results in the alleviation of the inhibition by CaD of myosin Mg²⁺-ATPase activity.