Copyright © 2005, European Society of Cardiology
Up-regulation of CPI-17 phosphorylation in diabetic vasculature and high glucose cultured vascular smooth muscle cells
aDepartments of Physiology, University of Kentucky, 509 Wethington Building, 900 South Limestone, Lexington, KY 40536, United States
bMolecular and Biomedical Pharmacology, University of Kentucky, Lexington, KY 40536, United States
cGraduate Center for Nutritional Sciences, University of Kentucky, Lexington, KY 40536, United States
* Corresponding author. Department of Physiology, University of Kentucky, 509 Wethington Building, 900 South Limestone, Lexington, KY 40536, United States. Tel.: +859 323 4933x81361; fax: +859 257 3646. Email address: mcgong2{at}uky.edu
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.
KEYWORDS Diabetes; Vascular smooth muscle contraction; Protein phosphorylation; Signal transduction; G-protein
1 These authors contributed equally to this work.