Evidence for Protein Kinase C-Mediated Activation of Rho-Kinase in a Porcine Model of Coronary Artery Spasm

Objective-We have recently demonstrated that protein kinase C (PKC) and Rho-kinase play important roles in coronary vasospasm in a porcine model. However, it remains to be examined whether there is an interaction between the two molecules to cause the spasm. Methods and Results-A segment of left porcine coronary artery was chronically treated with IL-β-bound microbeads in vivo. Two weeks after the operation, phorbol ester caused coronary spasm in vivo and coronary hypercontractions in vitro at the IL-β-treated segment; both were significantly inhibited by hydroxyfasudil, a specific Rho-kinase inhibitor. Guanosine 5′-[γ-thio] triphosphate (GTPγS), which activates Rho with a resultant activation of Rho-kinase, enhanced Ca²⁺ sensitization of permeabilized vascular smooth muscle cells, which were resistant to the blockade of PKC by calphostin C. The GTPγS-induced Ca²⁺ sensitization was greater in the spastic segment than in the control segment. Western blot analysis revealed that only PKCδ isoform was activated during the hypercontraction. Conclusions-These results demonstrate that PKC and Rho-kinase coexist on the same intracellular signaling pathway, with PKC located upstream on Rho-kinase, and that among the PKC isoforms, only PKCδ may be involved. Thus, the strategy to inhibit Rho-kinase rather than PKC may be a more specific and useful treatment for coronary spasm.