Serum deprivation can suppress receptor-mediated calcium signaling in pterygial-derived fibroblasts.

PURPOSE: Pterygium is characterized as invasive, proliferative fibrovascular altered conjunctival tissue. The extensive vascular network is likely to significantly contribute to the progression of the disease. In the present study, we investigated the effects of reduced serum (to mimic a suppressed blood supply) on cell signaling events and the functional role of the calcium store in cultured, pterygial-derived fibroblasts.

METHODS

Pure fibroblast cultures were established from cell outgrowths of pterygial tissue. Growth and migration of pterygial-derived fibroblast was evaluated using a patch growth assay, MTS assay, and a scratch wound assay. Intracellular calcium levels were determined using Fura-2 detection in response to ligand stimulation using a 96-well plate format.

RESULTS

A progressive increase in serum concentration resulted in promotion of pterygial cell growth detected using the MTS assay. A significant increase in intracellular calcium level was observed in response to histamine (10 and 100 μM), ATP (10 and 100 μM), acetylcholine (10 and 100 μM) and epidermal growth factor (10 ng/mL) in serum-maintained cells. However, no significant changes were observed when cells were maintained in serum-free medium. Thapsigargin (1 μM), a Ca-ATPase inhibitor, induced a significantly greater increase in intracellular calcium level in the serum-maintained group relative to serum-starved cells. In both cases, elevation of intracellular calcium was reduced when calcium-free bathing medium was used. Preincubation of cells with 1 μM thapsigargin ablated ligand-induced calcium responses. Disruption of calcium signaling through thapsigargin treatment significantly perturbed cell growth and migration.

CONCLUSIONS

Receptor-induced calcium signaling activity is suppressed in pterygial-derived fibroblasts in response to serum deprivation. This correlates with reduced growth rates and a depleted endoplasmic reticulum calcium store. The store plays a key role in cell growth and migration of pterygial-derived fibroblasts. Therefore, the strategic reduction of the vascular network in pterygium will affect the calcium store level and in turn affect functional responses associated with pterygia.