. The uroepithelium, which lines the inner surface of the bladder, forms an impermeable barrier and also functions as an integral part of a ?sensory web.? Through uroepithelial-associated channels and receptors, the uroepithelium receives sensory ?input? such as changes in hydrostatic pressure and binding of mediators such as ATP. These input signals stimulate membrane turnover in the outermost umbrella cell layer and release of sensory ?output? from the uroepithelium in the form of mediators that communicate changes in the uroepithelial milieu to the underlying tissues, altering the function of the bladder. Adenosine is a universally produced nucleoside that participates in the normal function of all organ systems, and preliminary data indicates that the uroepithelium is a site of adenosine biosynthesis and expresses all four adenosine receptors (A1, A2a, A2b, and A3). Adenosine binding to these receptors stimulates exocytosis in the umbrella cell layer. The goal of this proposal is to test the hypothesis that adenosine, acting through uroepithelial-associated adenosine receptors, functions as a sensory input molecule that stimulates membrane turnover in the umbrella cell layer and alters bladder function.
The first aim of this proposal will define the mechanism and function of adenosine biosynthesis and turnover by the uroepithelium. Selective inhibitors of enzymes/transporters as well as knockout mice lacking expression of adenosine kinase and adenosine deaminase will be used to define the mechanisms of adenosine production and turnover by the uroepithelium. Adenosine and its metabolites will be measured with a novel, highly sensitive and specific 3D-ion trap HPLC-mass spectrometric method.
The second aim will define if uroepithelial-associated adenosine receptors modulate bladder function. The Cre/loxP system will be used to generate mice lacking uroepithelial expression of A1 and A2a receptors. Bladder function will be studied in these mice as well as in knockout mice globally lacking A1, A2a, A2b, and A3 receptor expression in all tissues.
The third aim will define the role of adenosine and adenosine receptors in modulating membrane turnover in umbrella cells. Selective agonists and knockout mice lacking uroepithelial or global expression of A1 and A2a receptors will be used to define if these receptors are important for regulating endocytosis/exocytosis in the uroepithelium.
The fourth aim will explore which signaling pathways act downstream of adenosine receptors to modulate membrane turnover in the uroepithelium. A combined biochemical/pharmacological approach and permeabilized cell systems will be used to define which G proteins and second messenger pathways are involved in regulating membrane traffic in the umbrella cell layer. The results of these studies will increase our understanding of the role of adenosine and its receptors in the uroepithelial-associated sensory web, and will ultimately allow us to understand how perturbations in uroepithelial-associated receptor expression and signal output can contribute to bladder diseases such as interstitial cystitis and detrusor overactivity.

Public Health Relevance

. Adenosine is a natural product of cells that regulates the function of the cardiovascular, neuronal, and renal organ systems. This grant will explore if adenosine regulates the delivery and recovery of membrane at the free surface of the cells lining the bladder, and whether adenosine affects bladder filling and voiding. Understanding adenosine function is important because it will help us to understand how the cells lining the bladder can aid in regulating the function of the bladder and how to treat diseases like interstitial cystitis and bladder overactivity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK077777-05
Application #
8197700
Study Section
Urologic and Kidney Development and Genitourinary Diseases Study Section (UKGD)
Program Officer
Mullins, Christopher V
Project Start
2008-02-15
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2013-11-30
Support Year
5
Fiscal Year
2012
Total Cost
$408,034
Indirect Cost
$136,987
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Hanna-Mitchell, Ann T; Ruiz, Giovanni W; Daneshgari, Firouz et al. (2013) Impact of diabetes mellitus on bladder uroepithelial cells. Am J Physiol Regul Integr Comp Physiol 304:R84-93
Khandelwal, Puneet; Prakasam, H Sandeep; Clayton, Dennis R et al. (2013) A Rab11a-Rab8a-Myo5B network promotes stretch-regulated exocytosis in bladder umbrella cells. Mol Biol Cell 24:1007-19
Carattino, Marcelo D; Prakasam, H Sandeep; Ruiz, Wily G et al. (2013) Bladder filling and voiding affect umbrella cell tight junction organization and function. Am J Physiol Renal Physiol 305:F1158-68
Butterworth, Michael B; Edinger, Robert S; Silvis, Mark R et al. (2012) Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC). Am J Physiol Renal Physiol 302:F581-90
Prakasam, H Sandeep; Herrington, Heather; Roppolo, James R et al. (2012) Modulation of bladder function by luminal adenosine turnover and A1 receptor activation. Am J Physiol Renal Physiol 303:F279-92
Yu, Weiqun; Hill, Warren G; Apodaca, Gerard et al. (2011) Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium. Am J Physiol Renal Physiol 300:F49-59
Khandelwal, Puneet; Ruiz, Wily G; Apodaca, Gerard (2010) Compensatory endocytosis in bladder umbrella cells occurs through an integrin-regulated and RhoA- and dynamin-dependent pathway. EMBO J 29:1961-75
Jackson, Edwin K; Ren, Jin; Mi, Zaichuan (2009) Extracellular 2',3'-cAMP is a source of adenosine. J Biol Chem 284:33097-106
Khandelwal, Puneet; Abraham, Soman N; Apodaca, Gerard (2009) Cell biology and physiology of the uroepithelium. Am J Physiol Renal Physiol 297:F1477-501