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-1beta-bound microbeads in vivo. Two weeks after the operation, phorbol ester caused coronary spasm in vivo and coronary hypercontractions in vitro at the IL-1beta-treated segment; both were significantly inhibited by hydroxyfasudil, a specific Rho-kinase inhibitor. Guanosine 5'-[gamma-thio] triphosphate (GTPgammaS), 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 GTPgammaS-induced Ca2+ sensitization was greater in the spastic segment than in the control segment. Western blot analysis revealed that only PKCdelta 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 PKCdelta may be involved. Thus, the strategy to inhibit Rho-kinase rather than PKC may be a more specific and useful treatment for coronary spasm.