Insulin - mediated stimulation of C U T and cJun in the developing retina neurons involves

Neuronal differentiation is considered to be sensitive to signals from the environment somounding the differentiating cells [I]. Such signals can be contact mediated or modulated by diffusible factors such as growth factors [2]. Our previous studies have shown that the expression of acetylcholine synthesizing enzyme, choline acetyl transferase (ChAT) is highly dependent on the cell to cell interactions and the presence of insulin (1). In the embryonic chick retina, ChAT starts appearing at about 4-5 days and increases bimodally through hatching at day 21 with subsequent increases later in CNS maturation [I]. We have recently shown that insulin-mediated stimulation of ChAT involves activation of c-jun as antisense oligonucleotide to c-jun inhibited the expression of ChAT [3]. This study was conducted to examine the signalling mechanisms involved in the insulin mediated increase in the expression of c-jun and thus ChAT in the embryonic chick retina neurons. For this purpose, retina tissue was prepared from 8-1 1 day chick embryos as described previously [3,4]. Intact retinas (4-6) were placed into sterile 25 ml Erlenmeyer flasks and rotation incubated at 370C in sterile Tyrode's salt solution. For experiments with insulin, it was added to a final concentration of 100 ng/ml. Exposure was stopped after 5 min by the addition of hormone free Tyrode's salt soltuion. Retinas were collected immediately, or at different time intervals after treatment. If overnight incubation was necessary, Dulbecco's modified Eagle's medium (DMEM) was used instead of Tyrode's salt solution. Primary cultures for antisense experiments were prepared from 8-11 day chick embryo retinas. Retinas from embryos in ovo were dissected free of other tissues, treated with trypsin and dispersed into single cells as described previously [3,4]. These were plated on polyomithine coated dishes in serumfree DMEM with pencillin (50 U h l ) and streptomycin (50 pg/ml). The cultures were maintained at 38 "C with 5% C& over for 24 h before they were used for antisense experiments. For Western blotting, retina tissue was sonicated in lysis buffer (0.1 M NaCI, 0.01 M Tris-CI, pH 7.6, 0.001 M EDTA, 1 pg/ml PMSF) for 20 sec, supernatant collected and used for polyacrylamide gel electrophoresis for subsequent immunoblotting of ChAT, c-jun or phosphatidyl-inositoI3-kinase (PI 3-kinase). The activity of PI 3-kinase in retina tissue was determined as described [5 ] . Exposure of retina tissue to insulin caused transient increase in the c-jun protein. Treatment of retina tissue with c-jun antisense oligonucleotides inhibited the expression of ChAT protein indicating the involvement of c-jun in this cascade [5]. Since insulin is known to activate PI 3-kinase [6], we investigated the role of this protein in the insulin-mediated stimulation of c-jun and ChAT. Results in Figure 1 show that activity of PI 3-kinase rises rapidly more than 5-fold within 3 minutes of exposure to insulin. Pretreatment of retina tissue with fungal metabolite, wortmannin (10 nM), a specific inhibitor of PI 3-kinase [7], led to inhibition of increase in c-jun indicating that the c-jun activation occurs distal to that of PI 3-kinase. Since insulin is also well known to stimulate S6 kinase activity (8), one likely possibility of this increase may be the consequence of PI 3-kinase activation. To examine that we used rapamycin, an inhibitor of S6-kinase (8). We found that pretreatment with rapamycin (10 ng/ml) blocked the rise in active c-jun as determined by immunoblotting. 6 i i 6 i time (min)