The overall organization of urinary acidification has been defined by a variety of studies of the intact kidney and individual nephrons in vivo, yet relatively little is known about the transport processes across the individual cell membranes--their biochemical nature, their rate-limiting factors, and their organization within the epithelial cell layer. The proposed research extends our work on the control of the rate of urinary acidification in the turtle urinary bladder and focuses on the nature of the transport processes across the cell membranes, i.e. the proton pump at the luminal membrane and the efflux of bicarbonate across the serosal membrane. The proton pump characteristics will be analyzed according to a kinetic model consisting of two components: a catalytic unit responsible for active H+ translocation and a membrane channel with a finite resistance to H+ flow. This model simulates the observed relationship between transport rate (JH) and Delta MuH in the linear region and will be tested in the nonlinear regions at large and small Delta MuH. In addition to kinetic factors, JH is regulated by the number of H+ pumps present in the luminal membrane of the carbonic anhydrase(CA) containing cell population. The role of endocytosis and exocytosis will be examined by morphometric techniques during CO2 stimulation of JH with and without agents inhibiting cytoskeletal function. In a combined biochemical and morphologic approach to the isolation of the H+ pumps, we will test the hypothesis that a distinctive rod-shaped intramembrane particle that occurs in abundance in the luminal and vesicular membranes of the CA cells contains components of the H+-translocating ATPase. Freeze fracture studies of membrane fractions will be correlated with the activity of oligomycin- and ouabain-resistant ATPase. In related efforts, we will examine two HC03- transport systems which depend on Cl- and occur in the same epithelium, one in series with the H+ pump and one parallel to the H+ pump. The possibility will be explored that the parallel HCO3- secretory system occurs in a subpopulation of CA cells and is composed of the same transport elements in a different arrangement across the two cell membranes. These studies should advance the understanding of the cellular mechanisms of urinary acidification and provide new insights into epithelial acid-base transport in general.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK030693-13
Application #
3483578
Study Section
Special Emphasis Panel (NSS)
Project Start
1981-07-01
Project End
1995-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
13
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Connecticut
Department
Type
Schools of Medicine
DUNS #
City
Farmington
State
CT
Country
United States
Zip Code
06030
Kohn, O F; Hand, A R; Mitchell, P P et al. (1997) Intra- and submembrane particle densities during CO2 stimulation of H+ secretion in turtle bladder. Am J Physiol 272:F491-7
Steinmetz, P R; Kohn, O F; Hand, A R (1996) Scales of urine acidification: apical membrane-associated particles in turtle bladder. Kidney Int 49:1655-9
Kohn, O F; Mitchell, P P; Steinmetz, P R (1993) Sch-28080 inhibits bafilomycin-sensitive H+ secretion in turtle bladder independently of luminal [K+] Am J Physiol 265:F174-9
Kohn, O F; Mitchell, P P; Steinmetz, P R (1990) Characteristics of apical Cl-HCO3 exchanger of bicarbonate-secreting cells in turtle bladder. Am J Physiol 258:F9-14
Palmisano, J; Mitchell, P P; Steinmetz, P R (1989) NBD-taurine uptake by alpha-type carbonic anhydrase cells of turtle bladder. Am J Physiol 257:F1015-20
Silveira, J E; Perez, S E; Cirne, B R et al. (1989) Characteristics of toad bladder urinary acidification. Braz J Med Biol Res 22:1163-70
Steinmetz, P R (1988) Electrogenic proton transport by intercalated cells of tight urinary epithelia. Ciba Found Symp 139:122-38