Cataract and posterior capsule opacification (PCO) are major drains on health budgets throughout the world and it is a long-term objective of this project to develop strategies to prevent both of these conditions. Calcium is known to play a critical role in cortical cataract, and activation of the protease calpain is implicated. More recently it has been revealed that calcium also has an important role in normal human lens cell signaling through calcium mobilization from endoplasmic reticulum stores, via G-protein coupled receptors. Agonists for these receptors include ATP and acetylcholine and the latter has been implicated in cataract formation.
The specific aims are firstly to elucidate the sites of agonist-induced calcium release from intracellular stores in the intact human lens. Secondly to localize the store-operated calcium entry pathways and study their activation and inactivation characteristics. Thirdly to localize where a calcium increase leads to calpain activation and loss of transparency, and to test calpain antagonists.
The final aim will be to develop and test hydrophobic intraocular lens coatings to prevent PCO, based on knowledge of calcium cell signaling antagonists such as thapsigargin (Tg). Since there are considerable interspecies differences in lens behaviour a significant aspect of project design has been the development of techniques, including long-term culture, employing only human lens and capsular bag preparations. Techniques include confocal fluorescent (Fluo-3) imaging for cell calcium dynamics and electrophysiological and radiotracer methods to characterize the calcium entry pathways. Calpain activation will be measured with a fluorescent probe, lens opacification localized and quantified by digital imaging, and proteolysis studied by immunocytochemistry and gel electrophoresis. Calpain antagonists will be tested to determine those which best preserve lens transparency. The capsular bag culture system will be invaluable for testing the growth-inhibiting (and hence PCO inhibiting) properties of intraocular lenses (IOLs) coated with thapsigargin. 3H-labeled Tg will be used to optimize the coating technology. The ability of Tg-coated IOLs to abolish agonist-induced calcium signaling in capsular bag preparations will serve as an accurate assessment of coating efficacy. This investigation aims therefore not only to elucidate the role of calcium in the normal human lens, but also to identify strategies for preventing both cortical cataract and PCO based on that knowledge.
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