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.
Xie
Zhongwen
Z
Departments of Physiology, University of Kentucky, 509 Wethington Building, 900 South Limestone, Lexington, KY 40536, USA.
Su
Wen
W
Guo
Zhenheng
Z
Pang
Huan
H
Post
Steven R
SR
Gong
Ming C
MC
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D015151:Immunoblotting; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D008638:Mesenteric Arteries; D051379:Mice; D008810:Mice, Inbred C57BL; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000503:physiopathology; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015854:Up-Regulation; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
C459092:2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine; D000577:Amides; D004791:Enzyme Inhibitors; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins; D008301:Maleimides; D009124:Muscle Proteins; D010750:Phosphoproteins; C491520:Ppp1r14a protein, mouse; D011725:Pyridines; C108830:Y 27632; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D043263:Myosin-Light-Chain Phosphatase; D020742:rhoA GTP-Binding Protein; D005947:Glucose; C070515:bisindolylmaleimide I