Endothelin-Induced Changes of Secondary Messengers in Cultured Corneal Endothelial Cells

Abstract

The effect of endothelins on corneal endothelial cells is not well understood. We have investigated the effects of endothelin-1 (ET-1), endothelin-2 (ET-2) and endothelin-3 (ET-3) on bovine corneal endothelial cellular proliferation and the secondary messenger changes in cells in the presence of ET-1. It was found that the ³H-thymidine uptake was enhanced by ET-1 significantly, whereas ET-2 and ET-3 had no effect. ET-1 remarkably affects the increase of corneal endothelial cells on ³H-thymidine, ³H-leucine, and ³H-uridine uptakes in a dose-dependent manner. The 50% effective concentrations (EC₅₀) for ET-1, as measured by ³H-thymidine uptake, ³H-uridine uptake, and ³H-leucine uptake were 10^-8.78 M, 10^-8.53 M and 10^-8.04 M, respectively. It was found that endothelin-1 increased intracellular calcium concentration by using the method of preloading with Fura-2-AM and assaying with spectrophotometry. The cellular IP_l, IP₂, and IP₃ were also stimulated in the presence of ET-1. Moreover, ET-1 enhanced the basal cellular cAMP and cGMP concentrations in corneal endothelial cells in a dose-dependent manner. Immunofluorescent staining revealed that ET-1 increased the fibronectin protein concentration and changed protein distribution in corneal endothelial cells. These findings indicate that endothelin-1 increases in cell proliferation and biological changes may be involved in changing intracellular calcium mobility, increasing intracellular phosphoinositides, enhancing intracellular cGMP and cAMP accumulation, and fibronectin protein synthesis.