Maintenance of cell volume is a homeostatic process expressed in all animal cells. The necessary water movement across the plasma membrane is coupled to solute transport mechanisms that are also involved in other cellular activities such as the accumulation of glucose, pHi regulation or vectorial fluid transport. The corneal endothelium is endowed with an active ion pump mechanism responsible for vectorial fluid transport and hence the maintenance of corneal hydration, a determinant of corneal transparency. Since this fluid transport and cell volume regulation must be intertwined, it is imperative to establish the mechanisms of volume maintenance and regulation to obtain a complete understanding of the fluid transport. In addition, it is of clinical significance to understand how fluid transport is affected during times of osmotic stress such as during contact lens wear and diabetes. The focus of this study is to establish the principles of volume regulation upon acute and sustained disturbances leading to short and long term volume regulations, respectively/ Short term volume regulation will be studied by following changes in cell volume and intracellular ionic activities to identify the mechanisms and second messengers involved. Experiments will employ anisosmotic perturbations, acidosis, hypoxia, ion substitutions and transport-specific inhibitors. Long term volume regulation will be examined in the context of adaptation to sustained anisosmotic loads by adjusting the levels of organic osmolytes (such as amino acids, myo-inositol and sorbitol). Isolated in vitro rabbit corneas and cultured bovine endothelial cells that are confluent and polarized on either glass coverslips or permeable supports will be used. The dynamics of cell volume will be measured using athe sensitive light scattering technique and by following the concentration of a fluorescent dye used as a volume marker. The long term goal of this study is to understand the interaction of the fluid transport, pHi regulation and volume regulation activities of the corneal endothelium under normal conditions and during metabolic stress, hyperglycemia and anisosmotic loads.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
1R01EY011107-01
Application #
2165380
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1995-09-30
Project End
1998-09-29
Budget Start
1995-09-30
Budget End
1996-09-29
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Other Health Professions
Type
Schools of Optometry/Ophthalmol
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Gomes, Priya; Srinivas, Sangly P; Vereecke, Johan et al. (2005) ATP-dependent paracrine intercellular communication in cultured bovine corneal endothelial cells. Invest Ophthalmol Vis Sci 46:104-13
Gomes, Priya; Srinivas, Sangly P; Van Driessche, Willy et al. (2005) ATP release through connexin hemichannels in corneal endothelial cells. Invest Ophthalmol Vis Sci 46:1208-18
Srinivas, S P; Satpathy, M; Gallagher, P et al. (2004) Adenosine induces dephosphorylation of myosin II regulatory light chain in cultured bovine corneal endothelial cells. Exp Eye Res 79:543-51
Jans, Danny; Srinivas, S P; Waelkens, Etienne et al. (2002) Hypotonic treatment evokes biphasic ATP release across the basolateral membrane of cultured renal epithelia (A6). J Physiol 545:543-55
Srinivas, Sangly P; Ong, Angeline; Goon, Leanne et al. (2002) Lysosomal Ca(2+) stores in bovine corneal endothelium. Invest Ophthalmol Vis Sci 43:2341-50
Jans, Danny; De Weer, Paul; Srinivas, S P et al. (2002) Mg(2+)-sensitive non-capacitative basolateral Ca(2+) entry secondary to cell swelling in the polarized renal A6 epithelium. J Physiol 541:91-101
Srinivas, S P; Mutharasan, R; Fleiszig, S (2002) Shear-induced ATP release by cultured rabbit corneal epithelial cells. Adv Exp Med Biol 506:677-85
Srinivas, Sangly P; Ong, Angeline; Zhai, Chang-bin et al. (2002) Inhibition of carbonic anhydrase activity in cultured bovine corneal endothelial cells by dorzolamide. Invest Ophthalmol Vis Sci 43:3273-8
Srinivas, S P; Guan, Y; Bonanno, J A (1999) Swelling activated chloride channels in cultured bovine corneal endothelial cells. Exp Eye Res 68:165-77
Srinivas, S P; Bonanno, J A; Hughes, B A (1998) Assessment of swelling-activated Cl- channels using the halide-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl)quinolinium. Biophys J 75:115-23

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