Role of Gs Protein in Adrenomedullin Stimulated hRPE Cell Proliferation

Abstract

Abstract: : Purpose: Adrenomedullin (ADM) is a mitogen for human retinal pigment epithelial cells (hRPE) and stimulates cAMP in rat2 fibroblasts. Since G proteins play a role in cAMP mediated signaling pathways, we investigated the role of Gs protein in ADM induced proliferation of hRPE cells and have identified the presence of ADM receptors on hRPE cells. Methods: Primary hRPE cell cultures were established from human eyes obtained from the Michigan Eye Bank. Cells were allowed to grow to confluence and treated with ADM and elevated glucose. Cell proliferation was monitored by 3H-thymidine (3H-thy) incorporation. 125I-ADM was used to demonstrate receptor binding and bound material was also analyzed by SDS-PAGE analysis. The cAMP acetylation assay kit was used to measure intracellular cAMP. The role of Gs protein in cell proliferation was assessed by preincubating hRPE cells with cholera toxin (CT)(200 ng/well for 2 hrs) which is known to activate adenylyl cyclase by catalyzing the ADP ribosylation of Gs protein. Proliferation was assessed by measuring 3H-thy incorporation. Data were analyzed by Student 't' test. p<0.05 was considered to be significantly different. Results: ADM stimulated 3H-thy incorporation into hPRE cells in a dose dependent manner. Increasing concentrations of ADM inhibited binding of 125I-ADM to hRPE cells. SDS-PAGE analysis showed a single band that was eliminated by excess cold ADM (10-7 M). ADM (10-7 M) significantly stimulated intracellar cAMP formation (13.4±2.9 vs.10.7±2,mean±SEM, p<0.05,N=3) in hRPEcells. Further, ADM did not stimulate 3H-thy incorporation in hRPE cells preincubated with CT (200 ng/well for 2 hrs). Conclusion: ADM stimulates hRPE cell proliferation by interacting with its own receptor and stimulating Gs protein mediated cAMP pathways. ADM may play a role in the pathogenesis of proliferative eye disease.

Keywords: retinal pigment epithelium • second messengers • growth factors/growth factor receptors