Mechanisms of Calcium Entry in the Intact Human Lens

Abstract

Purpose: : Calcium (Ca²⁺) has been shown to have an important role in cataract. How Ca²⁺ enters the lens, however, is less well understood. We have, therefore, investigated Ca²⁺ influx in the intact human lens.

Methods: : Ca²⁺ changes in equatorial (E) and central anterior (CA) epithelial cells of freshly isolated intact human lenses were monitored using a Ca²⁺-indicator (Fluo4) and confocal microscopy. Gene expression and protein levels were investigated by QRT PCR and Western blotting.

Results: : Un-stimulated Ca²⁺-influx was greater in E than CA cells. ATP (10 µM) induced Ca²⁺ responses were smaller in CA compared to E cells, indicating differences in store capacity and/or Ca²⁺-influx. Ca²⁺ store depletion by application of either ATP (100µM) or thapsigargin (TG; 1µM) in Ca²⁺-free conditions, revealed greater relative store capacity and Ca²⁺-influx in E than CA cells. However, major differences in the characteristics of Ca²⁺-influx were found between the 2 regions, that were dependent on the method used to deplete the Ca²⁺-stores. Ca²⁺-influx, induced following store depletion by ATP, was blocked by La³⁺ (0.5 µM) applied acutely to the Ca²⁺-influx, in both regions and was inhibited by 2APB (50 µM) only in E cells. Ca²⁺-influx in E cells, induced following store depletion by TG, however, was insensitive to La³⁺ (10uM) applied acutely to the Ca²⁺-influx and was stimulated by 2APB. In CA cells Ca²⁺-influx induced by TG was blocked by La³⁺ (0.5 µM) and was insensitive to 2APB. Interestingly, adding La³⁺ (0.5 µM) before Ca²⁺ was readmitted to the perusate greatly potentiated Ca²⁺-influx, induced by TG, in E but not in CA cells. RNA and protein for Orai1 and STIM1 were detected in CA and E epithelial samples. Expression of TRPC3 was restricted to E cells while there was greater expression of TRPC1 in CA cells.

Conclusions: : Greater Ca²⁺ store capacity and Ca²⁺-influx in E compared to CA cells reflect underlying differences in proliferation and differentiation between the regions. Relatively small resting Ca²⁺-influx in CA epithelium suggests that store operated Ca²⁺-entry (SOCE) is the main route of Ca²⁺-influx in these cells. Major differences in the characteristics of Ca²⁺-influx between the regions of the epithelium indicate a complicated interplay between SOCE and TRPC channels. In vivo, receptor activation will modulate Ca²⁺-influx into the lens and inappropriate activity may be a contributory factor in cortical cataract.

Keywords: ion channels • receptors: pharmacology/physiology • second messengers: pharmacology/physiology