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Amritlal Mandal, Mohammad Shahidullah, Nicholas A. Delamere; Hyposmotic Stress Causes a Cytoplasmic Calcium Increase in Cultured Lens Epithelium by a Mechanism Involving P2-purinoceptors and Src Family Tyrosine Kinase. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1529.
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Many cells respond to osmotic stress initially by releasing ATP which has been proposed to act as an extracellular signal for purinergic receptors. In previous studies by ourselves and others, receptor-triggered increases of calcium and activation of Src family tyrosine kinases (SFKs) have been observed in lens epithelium. Here we examined the effect of hyposmotic stress on cytoplasmic calcium. Studies were conducted to test whether the calcium response involves purinergic receptors and SFKs.
Porcine lens epithelium was established in primary culture. Cytoplasmic calcium was measured in Fura 2-loaded cells exposed to hypotonic solution (200 mOsm). SFK activation was examined by Western blot analysis. ATP release into the medium was measured by luciferin-luciferase bioluminescence assay.
Exposure of lens epithelial cells to hyposmotic solution caused an immediate increase in cytoplasmic calcium concentration. The selective SFK inhibitor PP2 (10µM) suppressed the calcium increase. In parallel experiments, hyposmotic medium was found to cause SFK activation as evidenced by an increase in phosphoY416 band density. PP2 inhibited SFK activation. The hypotonicity-induced cytoplasmic calcium increase also was largely prevented by suramin (50µM). The ability of suramin to prevent the calcium response points to the involvement of P2-purinoceptors. Consistent with this notion, hypotonicity was found to cause release of ATP into the bathing medium.
The results suggest that the hyposmotic stress-induced cytoplasmic calcium increase is the result of an interaction between purinergic P2 receptors and Src family tyrosine kinase. The signaling mechanism may be involved in the lens response to osmotic stress.
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