This applicant proposes a program of research to prepare her for a career in academic medicine and basic science research studying male reproductive/urologic tract function, addressing the regulation of epididymal luminal acidification under physiological and pathophysiological conditions, during adulthood and postnatal development. The research will be conducted in the laboratory of Dr. Sylvie Breton at the Program in Membrane Biology and Renal Unit, Massachusetts General Hospital. The epididymis and vas deferens (VD) are the sites where spermatozoa mature and are stored in an immotile state. The luminal environment of these organs is tightly regulated and maintained at an acidic pH in order to keep spermatozoa from achieving motility. In particular, the applicant will study the mechanism by which an alkaline epididymal/VD luminal pH stimulates H+ATPase trafficking from subapical vesicles to the apical plasma membrane in clear cells. In addition, she will further investigate the role of the bicarbonate-activated """"""""soluble"""""""" adenylyl cyclase (sAC), cAMP, and downstream effectors (such as PKA and Epac) on this trafficking phenomenon. Other sources of cAMP, such as G protein-regulated transmembrane adenylyl cyclases, and other second messenger molecules (such as cGMP) in clear cells will also be investigated. These studies will also evaluate the effects of steroid hormone agonists and antagonists (DES, ethinyl estradiol, GnRH antagonist and flutamide) and toxic compounds (Cadmium and Lead) have on the epididymal cellular profile, protein expression, H+ATPase trafficking and function, and on the cellular profile of the epididymis/VD during adulthood. The work proposed in this application will be carried out on rat and/or mouse epididymal/VD epithelial cells during adulthood and postnatal development, in vivo, in situ, and in vitro, using a multidisciplinary approach to evaluate protein expression and vesicle recycling mechanisms. The techniques proposed include microspectrofluorescence, cell biological, molecular biological techniques, and the use of a proton-selective self-referencing electrode. Newly available transgenic mice expressing enhanced green fluorescence protein (EGFP) specifically in clear cells (B1-EGFP mice) will provide a unique model to examine the development and modulation of epithelial cell phenotypes in response to various manipulation.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08HD045524-04
Application #
7072186
Study Section
Pediatrics Subcommittee (CHHD)
Program Officer
Rankin, Tracy L
Project Start
2004-07-01
Project End
2009-06-30
Budget Start
2006-07-01
Budget End
2007-06-30
Support Year
4
Fiscal Year
2006
Total Cost
$129,600
Indirect Cost
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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Gong, Fan; Alzamora, Rodrigo; Smolak, Christy et al. (2010) Vacuolar H+-ATPase apical accumulation in kidney intercalated cells is regulated by PKA and AMP-activated protein kinase. Am J Physiol Renal Physiol 298:F1162-9
Banerjee, Sanjay K; McGaffin, Kenneth R; Pastor-Soler, Nuria M et al. (2009) SGLT1 is a novel cardiac glucose transporter that is perturbed in disease states. Cardiovasc Res 84:111-8
Hallows, Kenneth R; Wang, Huamin; Edinger, Robert S et al. (2009) Regulation of epithelial Na+ transport by soluble adenylyl cyclase in kidney collecting duct cells. J Biol Chem 284:5774-83
Hallows, Kenneth R; Alzamora, Rodrigo; Li, Hui et al. (2009) AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells. Am J Physiol Cell Physiol 296:C672-81
Pastor-Soler, Nuria M; Hallows, Kenneth R; Smolak, Christy et al. (2008) Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells. Am J Physiol Cell Physiol 294:C488-94
Estilo, Genevieve; Liu, Wen; Pastor-Soler, Nuria et al. (2008) Effect of aldosterone on BK channel expression in mammalian cortical collecting duct. Am J Physiol Renal Physiol 295:F780-8
Bouley, Richard; Pastor-Soler, Nuria; Cohen, Ori et al. (2005) Stimulation of AQP2 membrane insertion in renal epithelial cells in vitro and in vivo by the cGMP phosphodiesterase inhibitor sildenafil citrate (Viagra). Am J Physiol Renal Physiol 288:F1103-12
Pastor-Soler, Nuria; Pietrement, Christine; Breton, Sylvie (2005) Role of acid/base transporters in the male reproductive tract and potential consequences of their malfunction. Physiology (Bethesda) 20:417-28
Beaulieu, Valerie; Da Silva, Nicolas; Pastor-Soler, Nuria et al. (2005) Modulation of the actin cytoskeleton via gelsolin regulates vacuolar H+-ATPase recycling. J Biol Chem 280:8452-63