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G. Duncan, J. D. Rhodes; Imaging Calcium Dynamics of Epithelial and Fibre Cells in the Intact Human Lens. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4428.
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Changes in intracellular calcium have a role in normal lens homeostasis and also in cortical cataract and so calcium regulatory mechanisms were investigated in epithelial and fibre cells in the intact human lens.
Cytoplasmic Ca2+ levels were monitored in anterior and equatorial epithelial cells in the intact perifused human lens by wide-field microscopy using the fluorescent dye Fura-2 and by confocal microscopy using Fluo-4. In both cases the lenses were loaded with esterified dye for 1.5 hours at room temperature. For confocal fibre investigations de-esterified Fluo-4 was injected directly into the cells. Lenses were then placed anterior face down in a plastic chamber and perifused with physiological saline (AAH) which could be modified by adding agonists and antagonists or by altering calcium or sodium levels.
Wide-Field: The Ca2+ level in anterior cells was unaffected by exposure to Ca2+-free (1mM EGTA) or Na+-free (Na+ replaced by n-methyl glucamine+) solutions, while equatorial cell calcium decreased on exposure to Ca2+-free and increased on exposure to Na+-free solutions. After exposure to acetylcholine (10µM), Ca2+ levels in anterior cells were sensitive to external Ca2+, revealing a capacitative Ca2+ entry (CCE) pathway and ATP (10µM) activated a similar pathway in equatorial cells. The kinetics of the CCE pathways were investigated by readmitting Ca2+ after emptying the store by the same agents in Ca2+ -free solution. The calcium increases observed were proportionately greater at the equator than at the anterior and both were increased on exposure to Na+-free solutions. All readmission influxes were significantly reduced by La3+ (10µM) and 2-APB (10µM). Confocal Microscopy: Single cell measurements at the equator revealed a prolonged, homogeneous response to EGF, accompanied by activation of the CCE, while similar measurements at the anterior revealed brief, oscillating responses in a small proportion of cells. The same anterior population gave homogeneous and sustained responses to acetylcholine. The calcium control properties of the fibre cells resembled those of the overlying equatorial cells.
Compared to anterior cells, equatorial cells have an increased sensitivity to growth factors such as EGF, a greater resting Ca2+ influx and a proportionately greater CCE pathway. The larger calcium load on these cells and on the fibres helps explain why cortical cataract is initiated in this region.
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