Abstract
Purpose::
To identify potential signal molecules secreted by retinal pigment epithelium (RPE) in response to visual manipulation of retinal activity that could also serve as growth modulators. It is generally accepted that ocular growth is under local retinal control. It also has been demonstrated that apomorphine, a dopamine receptor agonist, inhibits myopic changes in experimental chick models, and that cultured embryonic chick RPE cells secrete TGF-beta, which has been linked to scleral remodeling. The effect of apomorphine on TGF-beta secretion by RPE was examined here.
Methods::
RPE cells were harvested from embryonic White Leghorn chicks (Gallus gallus) (E10-12, 95% purity under dissection microscope) and cultured in 96-wells cell culture plates in DMEM medium with 20% FBS for 5 days. Cells then were starved for 18 hr by replacing the DMEM medium with serum free medium before being treated with apomorphine. Half of the wells were treated with 2µM of apomorphine for 6 hours, while the other half were given only serum free medium as a control (n=6 in each case). An ELISA immunoassay kit (R&D) was used to determine the expression levels of TGFß2 in the supernatant of the culture. To check for the presence of mRNA for TGFß2, rt-PCR was performed on total RNA extracted from both cultured RPE cells and cells isolated from the eyes of adult chicken (40 days) using an RNeasy kit.
Results::
The ELISA assay demonstrated that RPE cells secrete TGFß2, with the apomorphine treatment increasing secretion by 1.4 fold (treated vs. control, p<0.05). rt-PCR confirmed the presence of TGFß2 and in both cultured embryonic and isolated adult RPE cells.
Conclusions::
TGFß2 mRNA is present in chick RPE cells and its protein expression is up-regulated by apomorphine, a dopamine receptor agonist. This result provides a possible signal pathway for the anti-myopia effect of this drug.
Keywords: retinal pigment epithelium • growth factors/growth factor receptors • dopamine