Reduced Intercellular Communication and Altered Morphology of Bovine Corneal Endothelial Cells with Prolonged Time in Cell Culture

Purpose: Mechanical stimulation induces intercellular Ca2+ waves in the corneal endothelium. The extent of the wave propagation is dependent on the activity of gap junctions, hemichannels, and ectonucleotidases. To further establish the use of a cell culture model to investigate intercellular communication, in this study, we have characterized the changes in the Ca2+ wave propagation in bovine corneal endothelial cells with prolonged time in culture. Materials and Methods: Freshly isolated BCEC were cultured for a short term (8 to 14 days; referred to as "short term") and a long term (21 to 30 days; referred to as "long term"). Cell surface area and size were measured by confocal microscopy and flow cytometry, respectively. Calcium wave propagation was assayed by imaging spread of the Ca2+ waves elicited by mechanical stimulation. ATP release was assayed using Luciferin-Luciferase bioluminescence technique. Results: Cells cultured for a long term showed larger surface area and size compared to those cultured for a short term, but a reduced spread of the Ca2+ wave. Exposure to exogenous apyrases, which can rapidly hydrolyze extracellular ATP, reduced the spread of the Ca2+ wave in both groups. The fractional decrease, however, was smaller in cells cultured for a long term. Exposure to ARL-67156 to inhibit the ectonucleotidases led to a larger enhancement of the active area in cells cultured for a long term. However, the active areas of the two groups were not significantly different in the presence of the drug. Furthermore, ATP release in response to mechanical stimulation was lower in cells cultured for a long term in the absence of ARL-67156 but not in its presence. Conclusions: BCEC cultured for a long term show an increase in cell surface area and cell size similar to the effect of aging in human corneas. Moreover, the cells cultured for a long term showed a reduced ATP-dependent paracrine intercellular communication, largely due to an increase in the activity of the ectonucleotidases.