This grant proposes to grow identified pigmented and non-pigmented epithelial cells from ciliary body in tissue culture and to study their physiology and pharmacology using patch-clamp techniques and fluorescence spectrophotometry. We shall attempt to develop improved tissue culture methods for reproducibly producing groups of ciliary body cells and eventually monolayers from rabbit and from human eyes. We shall isolate markers for both pigmented and non-pigmented cells using monoclonal antibodies which have been developed against purified populations of pigmented or non- pigmented cells.
The aim of these experiments is to develop methods which will allow us, for example, to add antibody to a dish of cultured cells to identify the non-pigmented cells prior to making patch-clamp recordings from them. We then proposed to use both excised-patch and whole-cell methods, in order to characterize both single membrane channels and voltage-dependent currents from cultured epithelial cells. The purpose of these experiments is to provide a complete characterization of epithelial cell ion permeabilities. Whole-cell methods will then be used to voltage- clamp these cells, in order to study changes in conductance produced by substances known to have effects on the intraocular pressure (beta-adrenergic agonists, cyclic AMP, dopamine). Finally, fluorescent dye methods will be used to measure pH and free Ca2+ concentration, both from cells in tissue culture and in suspensions, as well as from the non-pigmented cells in situ. Cells will be loaded with the pH-sensitive dye BCECF or the Ca2+- sensitive dye Fura 2. A dual excitation spectro-fluorometer together with image analysis will be used to investigate the mechanisms of pH regulation in these cells and the possible role of Ca2+ as a second messenger.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY007568-05
Application #
3264585
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1988-07-01
Project End
1994-06-30
Budget Start
1992-07-01
Budget End
1994-06-30
Support Year
5
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Cilluffo, M C; Esqueda, E; Farahbakhsh, N A (2000) Multiple receptor activation elicits synergistic IP formation in nonpigmented ciliary body epithelial cells. Am J Physiol Cell Physiol 279:C734-43
Cilluffo, M C; Xia, S L; Farahbakhsh, N A et al. (1998) Synergistic receptor-activated calcium increases in single nonpigmented epithelial cells. Invest Ophthalmol Vis Sci 39:1429-35
Cilluffo, M C; Farahbakhsh, N A; Fain, G L (1997) Functional and morphological differentiation of nonpigmented ciliary body epithelial cells grown on collagen rafts. In Vitro Cell Dev Biol Anim 33:546-52
Fain, G L; Farahbakhsh, N A (1989) Voltage-activated currents recorded from rabbit pigmented ciliary body epithelial cells in culture. J Physiol 418:83-103