Adrenomedullin affects two signal transduction pathways and the migration in retinal pigment epithelial cells.
OBJECTIVE
Adrenomedullin is a multifunctional regulatory peptide known to inhibit the migration of smooth muscle cells. In vitro studies were performed to identify whether adrenomedullin (ADM) also inhibits the migration of RPE cells. The aberrant behavior of these cells is an early event in proliferative vitreoretinopathy, and these studies were designed to determine how ADM acts on RPE cells at the second-messenger level.


METHODS
Migration of cultured human RPE cells was determined by the Boyden chamber method, using 10% fetal calf serum (FCS) as a chemotactic factor. The attachment assay was performed on fibronectin, laminin, or poly-D-lysine-coated 96-well plates. RPE cells were incubated in PBS buffer with or without ADM for 15 minutes. Intracellular cAMP and cGMP changes were then measured by enzyme immunoassay (EIA). To determine the cytoplasmic free Ca2+ concentration ([Ca2+]i) response to ADM, fluo-3 AM-loaded RPE cells were imaged with a laser scanning confocal microscope, after stimulation with ADM (10(-12)-10(-7) M).


RESULTS
ADM exhibited a concentration-dependent inhibition of FCS-stimulated RPE cell migration. The maximum inhibitory effect of ADM, observed at 10(-7) M, on basal and FCS-induced RPE cell migration was approximately 53.8% and 43.8% of the control, respectively. Exogenously added ADM (10(-9)-10(-7) M) had no significant effect on RPE cell attachment on all tested substrates. ADM increased intracellular cAMP and decreased intracellular cGMP levels dose dependently (10(-10)-10(-7) M) in RPE. The maximum effect was observed at 10(-7) M. ADM also induced a [Ca2+]i decrease in a dose-dependent manner (10(-12)-10(-7) M). The maximum effect was observed at 0.1 micro M, at which point the level declined to 42.9% of the control.


CONCLUSIONS
ADM inhibits the migration of RPE cells in vitro by a mechanism that involves the reciprocal upregulation of cAMP and downregulation of cGMP, in association with reductions in [Ca2+]i. ADM-mediated fluctuations in [Ca2+]i, which are well known to be involved in cell migration, appear to be regulated in part by mechanisms involving cAMP synthase. Thus, it appears that ADM acts as a constitutive regulatory system to control aberrant RPE cell behavior and specific migration in response to inflammatory mediators.
Huang
Wei
W
Institute of Ophthalmology, Xijing Hospital, The Fourth Military Medical University, Xi'an, China. wei_huang@meei.harvard.edu
Wang
Lin
L
Yuan
Ming
M
Ma
Jixian
J
Hui
Yannian
Y
D053607:Adrenomedullin; D000814:Aniline Compounds / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D006801:Humans; D007124:Immunoenzyme Techniques; D018613:Microscopy, Confocal; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology*; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism; D015290:Second Messenger Systems / Q000187:drug effects*; D015854:Up-Regulation; D014665:Vasodilator Agents / Q000494:pharmacology*; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; D010455:Peptides; D014665:Vasodilator Agents; D014966:Xanthenes; D053607:Adrenomedullin; D005576:Colforsin; C059715:Fluo-3; D000242:Cyclic AMP; D006152:Cyclic GMP; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester