REGULATION OF CREB PHOSPHORYLATION BY DOPAMINE AND CYCLIC AMP IN THE DEVELOPING AVIAN RETINA: ANALYSIS OF DIFFERENT SIGNALING PATHWAYS INVOLVING CALCIUM AND SOLUBLE ADENYLYL CYCLASE

Chemical messengers and signaling pathways regulate neuronal survival and differentiation during central nervous system development. Cyclic AMP plays a key role in these processes, largely through activating the transcription factor CREB. Dopamine controls cyclic AMP levels since early stages of chicken retina development. This study examined the effects of dopamine and forskolin-induced adenylyl cyclase activation on CREB phosphorylation throughout retinal development. Chicken embryo retinas from different developmental stages were dissected, preincubated in Hank’s saline with or without antagonists or inhibitors, then treated with stimulatory agents. Retinal lysates were analyzed by western blot. Dopamine or the D1 agonist SKF38393 increases CREB phosphorylation in E10, but not in E16 retinas, and these effects are inhibited by the D1 antagonist SCH23390 or adenylyl cyclase inhibitor SQ22536. Forskolin increases phospho-CREB levels in E10 in a concentration- and time-dependent manner; however, its effect is notably reduced in E16. Forskolin's effect in E10 is unaffected by the PKA inhibitor KT5720, or the EPAC inhibitors ESI-05 or CE3F4. Unlike dopamine, forskolin’s effect is not blocked by SQ22536 but is reduced by soluble adenylyl cyclase antagonists (LRE1, biothionol, TDI11861), the calcium chelator BAPTA-AM, and the CAMK inhibitor KN93. Furthermore, forskolin promotes CREB phosphorylation and raises intracellular calcium levels in retinal cultures. Immunocytochemistry shows that glial cells and neurons display strong staining for soluble adenylyl cyclase in these cultures. Our findings indicate that dopamine and forskolin activate distinct pathways leading to CREB phosphorylation and underscore the important role of soluble adenylyl cyclase in early retinal development.