cGMP Metabolism in Retinal Pigment Epithelial Cells

Abstract

Abstract: : Purpose: Retinal detachment was found to be associated with a decrease in cGMP levels in vitreous fluid as compared to controls (La Heij et al, Br J Ophthalmology, 2003). To investigate whether this could be due to an altered cGMP metabolism, we studied cGMP pathways in RPE cells following stimulation of guanylyl cyclases and simultaneous inhibition of phosphodiesterases. Methods: At both six hours and twenty–four hours after plating (D407) RPE cells, the cells were incubated in Krebs buffer containing 1mM isobutylmethylxantine (IBMX), a non–selective phosphodiesterase inhibitor. The cells were then stimulated with 0.1 mM sodium nitroprusside (SNP), a nitric oxide (NO) donor or 100 nM atrial natriuretic peptide (ANP). After fixation with paraformaldehyde the cells were immunostained for cGMP using anti–formaldehyde–fixed cGMP antiserum. RPE layers were cut from pig eyes into oblong pieces of 1 cm and incubated in Krebs containing 1mM IBMX. They were stimulated with SNP or ANP. After fixation the pieces were processed for immunocytochemistry. A cGMP radioimmunoassay (RIA) was used to confirm the immunocytochemical data. Results: The soluble cyclase in the human RPE cells could be stimulated with SNP and the particulate cyclase could be activated with ANP to increase the cGMP production. In non–confluent cells a large number of cells were cGMP positive. When the cells became more confluent the amount of SNP or ANP responsive cells decreased. These findings are consistent with the results of the experiments using the pig RPE layers, whereby only few cells responded to cyclase stimulation. In addition all cells stained immunopositive for NOS as well as for the ß1–subunit of the soluble guanylyl cyclase. Conclusions: These results indicate that human RPE cells are capable of producing cGMP after stimulation with SNP or ANP. The amount of cGMP produced is dependent on the differentiation state of the cells. Based on these findings it is unlikely that cGMP metabolism of RPE cells is responsible for the observed decrease in cGMP concentration in vitreous fluid after retinal detachment.

Keywords: retinal pigment epithelium • metabolism • nitric oxide