Increased basal phosphorylation of detrusor smooth muscle myosin in alloxan-induced diabetic rabbit is mediated by upregulation of Rho-kinase beta and CPI-17.
Urinary bladder dysfunction caused by the alteration of detrusor smooth muscle (DSM) is one of the complications of diabetes. It is well established that smooth muscle contractility is regulated by an elevation of cytosolic Ca(2+) via myosin light chain (MLC) phosphorylation. However, recent studies have shown the modulation of MLC phosphorylation without a rise in Ca(2+) in smooth muscle and that two key molecules (Rho-kinase and CPI-17) are involved in the regulation of calcium sensitization. This study investigates the effect of diabetes on DSM calcium sensitization. Diabetes was induced by alloxan in New Zealand White rabbits, and age-matched rabbits given 5% sucrose in the drinking water served as control for diuresis. Two-dimensional gel electrophoresis showed that basal MLC phosphorylation level was significantly higher in diabetic animals than normal or diuretic controls, and Rho-kinase-specific inhibitor, Y-27632, decreased MLC phosphorylation level. Adding Y-27632 to bethanechol-precontracted DSM strips can induce muscle relaxation, but it occurred much more slowly in diabetic samples compared with controls. RT-PCR, Western blot analysis, and immunohistochemistry revealed the overexpression of Rho-kinase beta and CPI-17 at both mRNA and protein levels in response to diabetes. In conclusion, our results demonstrate that Rho-kinase contributes to DSM MLC phosphorylation and there is a higher basal MLC phosphorylation level in diabetic DSM. Our results also suggest that this high basal MLC phosphorylation may be due to the upregulation of Rho-kinase and CPI-17. Thus Rho-kinase- and CPI-17-mediated Ca(2+) sensitization might play a role in diabetes-induced alteration of the detrusor contractility and bladder dysfunction.
Chang
Shaohua
S
Division of Urology, Department of Pathobiology, University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, USA.
Hypolite
Joseph A
JA
DiSanto
Michael E
ME
Changolkar
Arun
A
Wein
Alan J
AJ
Chacko
Samuel
S
D000577:Amides / Q000494:pharmacology; D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D003928:Diabetic Nephropathies / Q000097:blood; D004231:Diuresis; D004232:Diuretics / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D011817:Rabbits; D001743:Urinary Bladder / Q000503:physiopathology; D054460:rho-Associated Kinases
D000577:Amides; D001786:Blood Glucose; C491521:CPI-17 protein, Oryctolagus cuniculus; D004232:Diuretics; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; D011725:Pyridines; C108830:Y 27632; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D010749:Phosphoprotein Phosphatases; D009218:Myosins