Cyclic AMP-dependent regulation of tyrosine hydroxylase mRNA and immunofluorescence levels in rat retinal precursor cells.
Stimulation of tyrosine hydroxylase (TH) gene transcription by cyclic AMP (cAMP) has been clearly established in adrenal medula cells and neural-crest-derived cell lines but information on this mechanism is still lacking in dopaminergic neurons. Because they are easily amenable to in vitro experiments, dopaminergic amacrine cells of the retina might constitute a valuable model system to study this mechanism. We have used real-time reverse transcription with the polymerase chain reaction to quantify TH mRNA levels in the rat retina during post-natal development and in retinal precursor cells obtained from neonatal rats and cultured for 3 days in serum-free medium. Whereas the TH mRNA concentration remains consistantly low in control cultures, treatment with cAMP-increasing agents (forskolin, membrane depolarization, phosphodiesterase inhibitors) is sufficient to raise it to the level observed in adult retina (15-fold increase). Treatment of the cultured cells can be delayed by up to 2 days with identical results at the TH mRNA level, thus ruling out a survival-promoting effect of cAMP. TH immunofluorescence has confirmed cAMP-dependent regulation of TH expression at the protein level and indicates that the frequency of TH-positive cells in the cultures is similar to that observed in the adult retina. Selective phosphodiesterase inhibitors suggest that PDE4 is the major subtype involved in the dopaminergic amacrine cell response. Our data clearly establish the cAMP-dependent regulation of TH mRNA and immunofluorescence levels in retinal precursor cells. The possible role of this regulation mechanism in the developmental activation of TH gene expression is discussed.
Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, CNRS, Poitiers, France. pierre.voisin@univ-poitiers.fr
Pierre
P
Voisin
Marianne
M
Bernard
D000818:Animals; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase