application) Diabetes in humans is associated with a spectrum of voiding dysfunctions characterized by impaired bladder sensation, increased postvoidal residual volume and decreased detrusor contractility that may progress in detrusor areflexia and diminished urinary flow. Studies on animal models, particularly the streptotzotozin (STZ) induced diabetic rats and Brattleboro (BB) rats with hereditary diabetic insipidus (di/di), show changes similar to those in human diabetes, e.g., a greater bladder capacity with impaired voiding function. Data from the proposed studies will provide the basic information regard the effect of diabetes on detrusor contractility and bladder function, and test the underlying hypothesis that diabetes has an effect on the molecular mechanisms that regulate force generation and maintenance that are required to empty the urinary bladder. To address this hypothesis, we propose to use detrusor smooth muscle from diabetic (STZ induced diabetes BB rats), insulin treated, and control rats (non diabetic osmotic diuresis, and normal) and to determine how the regulatory mechanisms controlled (through protein protein interaction or enzymatically) by these proteins are altered. Specifically. we will determine: (1) whether the lack of intravesical pressure and over distension of the diabetic bladder is due to a low level of myosin light chain (MLC) phosphorylation or a change in the site of phosphorylation of the MLC in the detrusor smooth muscle at the resting tone or during force development; (2) whether the activities and expression of MLC kinases and/or the protein C kinase (PCK) isoforms, implicated in signaling pathways, are altered in the detrusor in diabetes; (3) whether changes in the expression of smooth muscle specific caldesmon (h-caldesmon), the thin filament component that regulates actin-myosin interactions, in the detrusor in diabetes is responsible for the inability for the inability of diabetic smooth muscle to maintain force; (4) whether thre is a change in the expression of smooth muscle myosin isoforms in response to diabetes; and (5) whether the actin/activated ATPase activity of myosin in the detrusor is altered in diabetic bladders. We proposed to use muscles from bladders and urethras of diabetic, non diabetic osmotic diuretic and normal rats. The expression of myosin will be studied using reverse transcribed polymerase chain reaction (PCR), quantitative comparative PCR, and Northern and Western blot analyses. The function of myosin isoforms will be analyzed by measuring the action-myosin ATPase activity and the movement of actin filaments over myosin heads in the in vitro motility assays. Detrusor contractility will be analyzed by force measurements of intact muscle strips. Alterations in the contractile apparatus and regulation of contraction, independent of the membranes and sarcoplasmic reticulum, will be determined using chemically """"""""skinned"""""""" muscle strips. An understanding of the molecular events that lead to contractile dysfunctions in diabetes is crucial in order to target the molecular steps for the development for pharmacological gents to treat diabetic cystopathy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK055042-02
Application #
2906345
Study Section
Special Emphasis Panel (ZDK1-GRB-4 (O1))
Program Officer
Mullins, Christopher V
Project Start
1998-09-30
Project End
2003-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Pathology
Type
Schools of Veterinary Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Changolkar, Arun K; Hypolite, Joseph A; Disanto, Michael et al. (2005) Diabetes induced decrease in detrusor smooth muscle force is associated with oxidative stress and overactivity of aldose reductase. J Urol 173:309-13
Chang, Shaohua; Hypolite, Joseph A; Velez, Marielena et al. (2004) Downregulation of cGMP-dependent protein kinase-1 activity in the corpus cavernosum smooth muscle of diabetic rabbits. Am J Physiol Regul Integr Comp Physiol 287:R950-60
Su, Xiaoling; Changolkar, Arun; Chacko, Samuel et al. (2004) Diabetes decreases rabbit bladder smooth muscle contraction while increasing levels of myosin light chain phosphorylation. Am J Physiol Renal Physiol 287:F690-9
Chang, S; Hypolite, J A; Changolkar, A et al. (2003) Increased contractility of diabetic rabbit corpora smooth muscle in response to endothelin is mediated via Rho-kinase beta. Int J Impot Res 15:53-62
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Arafat, H A; Kim, G S; DiSanto, M E et al. (2001) Heterogeneity of bladder myocytes in vitro: modulation of myosin isoform expression. Tissue Cell 33:219-32