Up-regulation of CPI-17 phosphorylation in diabetic vasculature and high glucose cultured vascular smooth muscle cells.
OBJECTIVE: Contractile responses are significantly increased in vascular smooth muscle tissues isolated from type 2 diabetic db/db mice (hyperreactivity). However, the molecular mechanisms underlying this hyperreactivity are largely unknown. The current study investigates the roles of RhoA, ROCK (rho kinase), PKC (protein kinase C), and CPI-17 (protein kinase C-potentiated phosphatase inhibitor of 17 kDa), molecules shown to play pivotal physiological roles regulating smooth muscle contraction, in diabetes-associated vascular smooth muscle hyperreactivity. METHODS: Experiments utilized db/db mouse mesenteric arteries and aortas and primary rat aortic smooth muscle cells (VSMCs) cultured in high or normal glucose. RhoA, ROCK, and CPI-17 protein expression and activity were determined by immunoblotting for total or phosphorylated proteins. RhoA activity was determined by subcellular fractionation and pull-down assays. Isometric contractions were determined using isolated mesenteric artery strips. RESULTS: Active phosphorylated CPI-17 and total and active membrane- bound RhoA were significantly increased in db/db mouse mesenteric arteries and aortas. High glucose time-dependently activated RhoA, ROCK, and CPI-17 in VSMCs. Moreover, inhibiting either RhoA with C3 exoenzyme or ROCK with Y-27632 or H-1152 for 30 min diminished high glucose- induced CPI-17 phosphorylation. Inhibiting protein kinase C (PKC) with GF109203X for 30 min did not inhibit high glucose-induced CPI-17 phosphorylation. Interestingly, when added at the same time as high glucose for a total of 48 h, GF109203X diminished high glucose-induced RhoA and ROCK activation as well as CPI-17 phosphorylation, suggesting PKC is required for high glucose-induced RhoA/ROCK activation and consequently CPI-17 phosphorylation. Importantly, in isolated db/db mouse mesenteric arteries, inhibiting ROCK with Y-27632 or H-1152 significantly alleviated the contractile hyperreactivity in response to phenylephrine or high potassium. CONCLUSIONS: Diabetes and high glucose activate RhoA, ROCK, and CPI-17, which in turn contribute to diabetic vascular smooth muscle hyperreactivity.[1]References
- Up-regulation of CPI-17 phosphorylation in diabetic vasculature and high glucose cultured vascular smooth muscle cells. Xie, Z., Su, W., Guo, Z., Pang, H., Post, S.R., Gong, M.C. Cardiovasc. Res. (2006) [Pubmed]
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