Abstract
Abstract: :
Purpose:Adrenomedullin (ADM) is a vasorelaxant peptide originally identified from human pheochromocytoma. Subsequent study revealed that ADM are expressed in various human tissues and cells and has a variety of biologic actions.In eyes, ADM has a relaxant effect on iris sphincter muscle and the concentrations of ADM inceased in the vitreous fluid of patients with proliferative vitreoretinopathy . Recently, it has been shown that ADM is secreted from cultured human retinal pigment epithelial (RPE) cells and hypoxia increases the expression. But the physiological role(s) and mechanism of pharmacological effects of ADM on RPE are still unclear. Here we investigated whether the actions of ADM on RPE through two independent signal transduction pathways, cAMP accumulation and Ca2+ mobilization. Methods:RPE cells were isolated and cultured from eye bank donor eyes. To measure [Ca2+], we incubated RPE with the fluorescent Ca2+indicator fluo 3AM. Fluorescence was visualized using a laser scanning confocal microscope after cells stimulated with ADM (10-7 to 10 -12M).Cellular cAMP and cGMP were measured with commercial enzyme immunoassay kits using the manufacturer’s non-acetylation. Results:Adrenomedullin induced a [Ca2+]decrease in RPE in a dose-dependent manner(10-7 to 10-12M). The minimal response was observed at 10-12M and the maximal response was observed at 0.1 µM. Pretreatment of cells with N -nitro-L-arginine methyl ester, an NO synthase inhibitor, vanished the [Ca2+] decrease by ADM, suggesting that the [Ca2+] decrease was mediated by NO. Addition of ADM for 15 min dose-dependently (10-7 to 10-10M) increased intracellular cAMP levels of RPE. An effective response was observed at 10-10M and the maximal effect was observed at 10-7M. ADM (10-7 to 10-10M) significantly decrease the cGMP level in a dose-dependent manner. Pretreatment of RPE with N -nitro-L-arginine methyl ester, further the decrease of cGMP initiated by ADM. Conclusion: The results suggest that the physiological role(s) and mechanism of pharmacological effects of ADM on RPE are through at least two mechanisms, a direct action on RPE to increase intracellular cAMP and decrease [Ca2+] mobilization.
Keywords: 567 retinal pigment epithelium • 334 calcium • 581 signal transduction: pharmacology/physiology