Objective: To investigate the mechanism of retinal pigment epithelial (RPE) cell migration and proliferation at molecular level by measuring the concentrations of intracellular cyclic 3', 5'-adenosine monophosphate (cAMP) and calcium ion (Ca(2+)) level of frozen cultured RPE cells.
Methods: The culture of human RPE cells was frozen for 0s, 15s, 30s, 45s, 60s respectively at -70 degrees C. The concentrations of cAMP and Ca(2+) in cultured human cells were measured by Fura-2/AM fluorescent technique and endogenous protein combination method.
Results: After freeze for 15 s, the contents of cAMP and Ca(2+) in RPE cells were not changed significantly compared with the control (no freeze). After freeze for 30s, 45s, 60s respectively, the level of intracellular cAMP was significantly decreased, while the concentration of cytoplasmic free Ca(2+) was markedly increased, showing that the concentration of cAMP and Ca(2+) being antagonistic to each other. With the re-vitalization of RPE cells for 60 minutes, the intracellular free Ca(2+) gradually reduced.
Conclusions: The decreased concentration of cAMP and increased concentration of free Ca(2+) in RPE cells may be the effective mechanism of RPE cell migration and proliferation after cryotherapy, and it possibly plays an important role in the formation of proliferative vitreoretinopathy.