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Increased basal phosphorylation of detrusor smooth muscle myosin in alloxan-induced diabetic rabbit is mediated by upregulation of rho-kinase β and CPI-17

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Increased basal phosphorylation of detrusor smooth muscle myosin in alloxan-induced diabetic rabbit is mediated by upregulation of rho-kinase β and CPI-17
  See discussions, stats, and author profiles for this publication at: Increased basal phosphorylation of detrusorsmooth muscle myosin in Alloxan induceddiabetic rabbit is...  Article   in  American journal of physiology. Renal physiology · April 2006 DOI: 10.1152/ajprenal.00235.2005 · Source: PubMed CITATIONS 45 READS 20 6 authors , including:Joseph A HypoliteUniversity of Pennsylvania 61   PUBLICATIONS   1,102   CITATIONS   SEE PROFILE Michael E DisantoAlbert Einstein College of Medicine 72   PUBLICATIONS   1,359   CITATIONS   SEE PROFILE Alan J WeinUniversity of Pennsylvania 776   PUBLICATIONS   23,167   CITATIONS   SEE PROFILE Samuel ChackoUniversity of Pennsylvania 147   PUBLICATIONS   2,896   CITATIONS   SEE PROFILE All content following this page was uploaded by Samuel Chacko on 19 December 2013. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  1 Increased basal phosphorylation of detrusor smooth muscle myosin in Alloxan-induced diabetic rabbit is mediated by up-regulation of Rho-kinase and CPI-17 Shaohua Chang, Joseph A. Hypolite, Michael E. DiSanto 1 ,Arun Changolkar, Alan J. Wein and Samuel Chacko* Divison of Urology and Department of Pathobiology, University of Pennsylvania Running title: Diabetes up-regulates Rho-kinase & CPI-17 in detrusor *Corresponding Author: 3010 Ravdin-Courtyard, HUP University of Pennsylvania 3400 Spruce Street Philadelphia, PA 19104 USA Phone (215) 662-6870 Fax (215) 349-5026 This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants P50 DK52620 and DK55042. 1 Dr. DiSanto is presently at Albert Einstein College of Medicine, Bronx, NY Articles in PresS. Am J Physiol Renal Physiol (October 4, 2005). doi:10.1152/ajprenal.00235.2005 Copyright © 2005 by the American Physiological Society.  2 Abstract 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 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-match rabbits given 5% sucrose in the drinking water served as control for diuresis. 2D 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 bethenachol-precontracted DSM strips can induce muscle relaxation, but it occurred much more slowly in diabetic samples compared with controls. RT-PCR, Western-blot and immuno-histochemistry revealed the over-expression of Rho-kinase  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 up-regulation 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. Key Words: Diabetes, Detrusor, Rho-Kinase, CPI-17  3 INTRODUCTION Urinary bladder dysfunction is a common complication of diabetes mellitus. The effect of diabetes on the bladder is a delay in the first sensation to void during filling and increased bladder capacity, interpreted as sensory impairment. This altered urodynamic response then progresses with the development of poor contractility, and leads to impaired bladder emptying and residual urine (7). These common diabetic complications of the urinary bladder function are thought to be due to the downstream effects of hyperglycemia on the nerve and muscle. However, the underlying mechanisms of diabetes-induced alteration in DSM contractility and bladder dysfunction are not well understood. The induction of experimental diabetes in the rat and rabbit with STZ and alloxan provide useful models to study various functional, biochemical and  pharmacological alterations that occur in the bladder (10; 18). There are several studies indicating that these diabetic complications may be due to neuronal changes (13-16; 28), downstream effects of oxidative stress (1; 5; 23) and bladder hypertrophy (17; 22).  Normal regulation of the smooth muscle contractility is crucial for normal bladder function. Many investigators have shown diabetes-induced alteration in detrusor contractility, either decreased force generation (5; 10; 18; 25) or increased force generation (13; 27; 29) depending on agonists used and experimental conditions. These different contractile responses may be due to diabetes-induced alteration of certain specific receptors, which in turn alters the contractile characteristics. The information regarding the direct effects of diabetes on general smooth muscle contractility or the regulation of smooth muscle contractility is very limited. A recent study shows decreased  bladder smooth muscle contraction in diabetes with higher MLC phosphorylation levels  4 (25). Phosphorylation and dephosphorylation of MLC is the major regulatory mechanism of smooth muscle contractility, which is modulated by MLC kinase and MLC  phosphatase (MP). MLC kinase is a Ca 2+ /camodulin dependent kinase and its regulation is already well known. Recent studies have focused on the regulation of MP and discovered two important proteins that regulate MP activity, Rho-kinase and CPI-17. Rho-kinase is activated by the small GTPase RhoA and inhibits MP through  phosphorylating its myosin binding subunit (12). CPI-17 is a protein phosphatase1 inhibitor and its phosphorylated form has been shown to inhibit MP (6; 8). The inhibition of MP by Rho-kinase and CPI-17 decreases the MP activity and raises the level of MLC  phosphorylation without an increase in calcium concentration. This phenomenon is known as calcium sensitization of smooth muscle (3; 24). In this study, we investigate the effect of diabetes on the regulation of DSM calcium sensitization, especially on MLC  phosphorylation, expression of Rho-kinase and CPI-17 in DSM.
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