The corneal epithelium provides an essential barrier function which protects the cornea against pathogenic invasion and loss of its optical transparency. Following corneal epithelial wounding, the cytokine endothelial accelerates healing. This hastening of wound closure may include increases in migration, proliferation and differentiation. Even though there is endothelin receptor subtype heterogeneity in the corneal epithelium, little is known about the cell signaling mechanisms which link receptor stimulation to the mediation of these responses. The focus of this proposal is to determine if there is any difference between the cell signaling mechanisms of the endothelin receptors in the proliferating basal and differentiating suprabasal layers of the corneal epithelium. To evaluate this question, we will determine if endothelin has different effects on cell volume regulatory responses in primary subconfluent proliferating cultures of the bovine corneal epithelium and in differentiating suprabasal cells of the intact bovine corneal epithelium. Their effects on cell volume regulation are evaluated because this response is a reflection of individual ion fluxes which account for osmotically driven water flow. The measurements of volume changes in the cultured cells are made simultaneous to those of changes in intracellular [Ca2+] and pH. To monitor these changes, the technique uses microfluorometric measurements of changes in the concentration of intracellularly trapped dyes to report relative volume. By using pH or Ca2+ sensitive dyes and measuring the fluorescence resulting from excitation at the ion sensitive and isosbestic wavelengths with an imaging system, cell volume changes and intracellular ion activities are concomitantly measured. Preliminary results show that the technique is appropriate for characterizing volume regulatory responses which occur subsequent to dilution of the bathing solution by 55%. These regulatory volume decreases (RVD) reflect increases in net ion transport which restore the match between intracellular and extracellular osmolality. Simultaneous to this response there are large transient changes in intracellular (Ca2+] whereas in other nonregulating cells these transients are not seen. To characterize cell volume regulation in the intact suprabasal epithelial layers of the cornea, cell height is used as an index of volume and it is measured with a video microscopy technique. In another preliminary study, it is apparent that these cells perform RVD and can also restore themselves to their control volume during exposure to a hypertonic stress (i.e. regulatory volume increase, RVI). To ascertain if membrane ion transport changes are part of the linkage involved in the control of proliferation by endothelin receptors, ionic transport will be perturbed. If such an alteration affects this control, membrane ion transport changes contribute to endothelin mediated cell signaling and stimulation of proliferation in cultured cells. Taken together, these results could impact on the development of drugs which can selectively stimulate cell proliferation.
Showing the most recent 10 out of 73 publications
