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

Abstract

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-1␤-bound microbeads in vivo. Two weeks after the operation, phorbol ester caused coronary spasm in vivo and coronary hypercontractions in vitro at the IL-1␤-treated segment; both were significantly inhibited by hydroxyfasudil, a specific Rho-kinase inhibitor. Guanosine 5Ј-[␥-thio]triphosphate (GTP␥S), which activates Rho with a resultant activation of Rho-kinase, enhanced Ca 2ϩ sensitization of permeabilized vascular smooth muscle cells, which were resistant to the blockade of PKC by calphostin C. The GTP␥S-induced Ca 2ϩ sensitization was greater in the spastic segment than in the control segment. Western blot analysis revealed that only PKC␦ 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 PKC␦ may be involved. Thus, the strategy to inhibit Rho-kinase rather than PKC may be a more specific and useful treatment for coronary spasm. Key Words: arteriosclerosis Ⅲ coronary disease Ⅲ smooth muscle Ⅲ signal transduction C oronary artery spasm plays an important role in the pathogenesis of a variety of ischemic heart diseases, including not only variant angina but also other forms of angina, myocardial infarction, ventricular arrhythmias, and sudden death. 1,2 However, the intracellular mechanism for the spasm remains to be elucidated.We previously developed a porcine model of coronary spasm in which the spasm was repeatedly induced by autacoids, such as serotonin and histamine, at atherosclerotic lesions made by a combination of balloon endothelial removal and high-cholesterol feeding. [3][4][5] We subsequently developed a porcine model in which long-term adventitial treatment with interleukin-1␤ (IL-1␤), one of the major inflammatory cytokines, induces arteriosclerotic changes and vasospastic responses of the coronary artery. 6 -15 Since the histological changes and vasospastic responses in our porcine models are similar to those observed in humans, our models may be useful to elucidate the mechanisms of the spasm in humans.We recently demonstrated that in our porcine models, coronary artery spasm induced by serotonin is mediated by protein kinase C (PKC) 5,7 and Rho-kinase. 9,11-15 Rho-kinase, one of the major target proteins of small Rho GTPase, 16,17 was upregulated in spastic coronary segment, leading to an increased phosphorylation of myosin-binding subunit of myosin phosphatase (MLCPh) and the resultant suppression of MLCPh. 11-15 Indeed, we recently demonstrated that Rho-kinase is substantially involved in hypercontractions of isolated arteriosclerotic human arteries in v...

Evidence for Rho-Kinase Activation in Patients With Pulmonary Arterial Hypertension

“…These findings are consistent with our previous studies with MCT-induced PH in rats and hypoxia-induced PH in mice, 10,11 and previous clinical studies of patients with PAH. [23][24][25][26][27][28][29][30][31][32][33][34][35] Furthermore, we were able to demonstrate that inhibition of Rho-kinase abolished VSMC hypercontraction of pulmonary arteries from IPAH patients. This finding also is consistent with our previous clinical study that found acute inhibition of Rho-kinase improves pulmonary hemodynamics in PAH patients.…”

“…The present findings support our previous findings in animal models of PAH, and during right-heart cardiac catheterization in patients with PAH. 9,[25][26][27][28][29][30][31][32] To the best of our knowledge, this is the first study to provide direct evidence for Rho-kinase activation in patients with PAH.…”

“…The present finding in patients with PAH is consistent with our previous finding in a porcine model of coronary spasm that PKC (especially PKCδ) and Rho-kinase coexist in the intracellular signaling pathway, with PKC located upstream of RhoA/Rho-kinase, suggesting that a strategy to inhibit Rho-kinase rather than PKC would be more specific and useful for the treatment of the vasospastic disorders. 28 …”

Newborn lamb coronary artery reactivity is programmed by early gestation dexamethasone before the onset of systemic hypertension

“…Abnormal activation of the Rho/Rho-kinase pathway has been shown to play a role in a variety of diseases, including hypertension and coronary vasospasm (7,22). In both groups of lambs presently evaluated, the Rho-kinase inhibitor Y-27632 significantly decreased the responses to ET-1 (by 65-67%) but did not eliminate the dexamethasoneinduced hypercontractile state.…”

The Role of Rho Kinase and Extracellular Regulated Kinase-Mitogen-Activated Protein Kinase in α2-Adrenoceptor-Mediated Vasoconstriction in the Porcine Palmar Lateral Vein

“…PKC␦ has been identified in porcine coronary arteries (Kandabashi et al, 2003), although attempts to determine whether PKC␦ is present in the porcine palmar lateral vein by Western blotting, using the same antibodies, proved inconclusive (data not shown). On the other hand, like PRK, PKC causes inhibition of myosin phosphatase through phosphorylation of CPI-17 rather than phosphorylation of MYPT.…”