Spatial variations in the membrane properties of differentiating fibre cells isolated from the rat lens

Abstract

The ocular lens is a dense avascular structure composed of a bulk of terminally differentiated elongated fibre cells. During differentiation, lens fibre cells lose their nuclei and organelles and are laid down in successive layers to create a pseudocrystalline cellular architecture, where extracellular space dimensions are kept below the wavelength of visible light. Disruption of this precise structural arrangement leads to the development of cataract and eventual blindness. Differences in membrane transport within different regions of the lens, connected by gap junctions, are thought to generate a unique microcirculatory system which is essential for lens transparency - regulating tissue volume and ionic homeostasis and supplying internalised fibre cells with metabolic and antioxidant support. While the spatial differences in membrane transport thought to drive the lens internal circulation system have been inferred from intact-lens measurements, they have not yet been confirmed at the cellular level. Problems isolating and working with lens fibre cells has lead to a historical lack of information about the role of these cells in lens physiology. This thesis describes the development and characterisation of a viable isolated fibre cell preparation, representing the first investigation of physiological membrane behaviour in single fibre cells isolated from the rat lens. It was found that by blocking non-selective cation channels activated by fibre cell dissociation with ionic gadolinium, isolated fibre cells of a range of lengths could be maintained viable for several hours in the presence of physiological [Ca2+]o. Furthermore, fibre cell length in vitro was similar to that measured in the intact lens, allowing the spatial localisation of measured membrane properties within the outer lens cortex. The measured membrane properties of isolated fibre cells indicate that membrane conductances are altered during fibre cell differentiation, thus creating spatial differences in membrane transport across the lens radius. The measured transition from K+-selectivity towards Na+/Cl- selectivity during fibre cell elongation confirms inferred properties derived from intact-lens studies. These alterations in the membrane properties of differentiating fibre cells are arranged spatially in a manner appropriate for the generation of the circulating ion fluxes thought to form the lens internal circulation system. To investigate the role of fibre cell membrane properties in both physiological and pathological circumstances, modulation of fibre cell membrane properties was demonstrated by osmotic and pharmacological means. Data obtained indicates that fibre cells respond to osmotic or metabolic insult by altering their membrane behaviour. The physiological and pathological alteration in fibre cell membrane properties described directly answers significant outstanding questions in lens physiology, and has the potential to generate new therapeutic interventions in the development of cataract