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.

[1]  M. Ellenberg.  Development of urinary bladder dysfunction in diabetes mellitus. , 1980, Annals of internal medicine.

[2]  G. Burnstock,et al.  The smooth muscle of rat bladder in the early stages of streptozotocin-induced diabetes. , 1984, British journal of urology.

[3]  J. Belis,et al.  Abnormalities of rat bladder contractility in streptozotocin-induced diabetes mellitus. , 1986, The Journal of pharmacology and experimental therapeutics.

[4]  R. Weiss,et al.  Differential Regulation of Bladder β-Adrenergic and Muscarinic Cholinergic Receptors in Experimental Diabetes , 1991, Diabetes.

[5]  T. Tammela,et al.  Factors underlying the increased sensitivity to field stimulation of urinary bladder strips from streptozotocin‐induced diabetic rats , 1994, British journal of pharmacology.

[6]  R. Cohen,et al.  Altered contractility of urinary bladder in diabetic rabbits: relationship to reduced Na+ pump activity. , 1996, The American journal of physiology.

[7]  S. Bursell,et al.  Amelioration of Vascular Dysfunctions in Diabetic Rats by an Oral PKC β Inhibitor , 1996, Science.

[8]  Juei-Tang Cheng,et al.  Alterations in urinary bladder synaptosomal neurotransmitter concentrations in two-week streptozotocin-induced diabetic rats , 1996, Neuroscience Letters.

[9]  Kozo Kaibuchi,et al.  Regulation of Myosin Phosphatase by Rho and Rho-Associated Kinase (Rho-Kinase) , 1996, Science.

[10]  D. Birenbaum,et al.  Exogenous NO inhibits basal NO release from vascular endothelium in vitro and in vivo. , 1996, The American journal of physiology.

[11]  M. Eto,et al.  Molecular cloning of a novel phosphorylation‐dependent inhibitory protein of protein phosphatase‐1 (CPI17) in smooth muscle: its specific localization in smooth muscle 1 , 1997 .

[12]  M. Eto,et al.  Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: its specific localization in smooth muscle. , 1997, FEBS letters.

[13]  I. Wendt,et al.  Effects of streptozotocin-induced diabetes mellitus on intracellular calcium and contraction of longitudinal smooth muscle from rat urinary bladder. , 2000, The Journal of urology.

[14]  M. Kodama,et al.  Influence of 5‐hydroxytryptamine and the effect of a new serotonin receptor antagonist (sarpogrelate) on detrusor smooth muscle of streptozotocin‐induced diabetes mellitus in the rat * , 2000, International journal of urology : official journal of the Japanese Urological Association.

[15]  J. Calvert,et al.  Upregulation of small GTPase RhoA in the basilar artery from diabetic (mellitus) rats. , 2002, Life sciences.

[16]  T. Ma,et al.  Time-dependent urinary bladder remodeling in the streptozotocin-induces diabetic rat model , 2002, Acta Diabetologica.

[17]  T. Haystead,et al.  Smooth Muscle Myosin Phosphatase-associated Kinase Induces Ca2+ Sensitization via Myosin Phosphatase Inhibition* , 2002, The Journal of Biological Chemistry.

[18]  A. Somlyo,et al.  Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. , 2003, Physiological reviews.

[19]  A. Wein,et al.  Increased contractility of diabetic rabbit corpora smooth muscle in response to endothelin is mediated via Rho-kinase β , 2003, International Journal of Impotence Research.

[20]  S. Yuan,et al.  Upregulation of PKC genes and isozymes in cardiovascular tissues during early stages of experimental diabetes. , 2003, Physiological genomics.

[21]  John H. Zhang,et al.  Increased RhoA translocation in renal cortex of diabetic rats. , 2003, Life sciences.

[22]  S. Howell,et al.  Novel in vitro and in vivo phosphorylation sites on protein phosphatase 1 inhibitor CPI-17. , 2003, Biochemical and biophysical research communications.

[23]  K. Sakamoto,et al.  Augmentation of rat urinary bladder relaxation mediated by beta1-adrenoceptors in experimental diabetes. , 2003, European journal of pharmacology.

[24]  S. Chacko,et al.  Diabetes decreases rabbit bladder smooth muscle contraction while increasing levels of myosin light chain phosphorylation. , 2004, American journal of physiology. Renal physiology.

[25]  G. Burnstock,et al.  Rat bladder in the early stages of streptozotocin-induced diabetes: adrenergic and cholinergic innervation , 2004, Diabetologia.

[26]  S. Carrier,et al.  Oxidative stress plays a role in diabetes-induced bladder dysfunction in a rat model. , 2004, Urology.

[27]  S. Naito,et al.  Ca2+ sensitization in contraction of human bladder smooth muscle. , 2004, The Journal of urology.

[28]  J. Bauer,et al.  Oxidant driven signaling pathways during diabetes: role of Rac1 and modulation of protein kinase activity in mouse urinary bladder. , 2004, Biochimie.

[29]  A. Wein,et al.  Over expression of smooth muscle thin filament associated proteins in the bladder wall of diabetics. , 2005, The Journal of urology.

[30]  A. Wein,et al.  Diabetes induced decrease in detrusor smooth muscle force is associated with oxidative stress and overactivity of aldose reductase. , 2005, The Journal of urology.

[31]  A. Seftel.  RhoA/Rho-kinase suppresses endothelial nitric oxide synthase in the penis: a mechanism for diabetes-associated erectile dysfunction. , 2005, The Journal of urology.