Acute exposure of cerebellar granule neurons to ethanol suppresses stress-activated protein kinase-1 and concomitantly induces AP-1

The current studies were designed to examine the mechanisms of acute effect s of ethanol on cerebellar granule neurons (CGNs) during neurodevelopment, with specific reference to activator protein-1 (AP-1). CGNs, isolated from 3-day-old Sprague-Dawley rats and cultured for 3 days, were exposed to 0, 2 2.5, and 100 mM ethanol for 1 h. Gel shift assays performed on the nuclear protein extracts showed increased AP-1 and heat shock factor-1 (HSF-1) tran scriptional activation in response to ethanol. Western blots and RT-PCR sho wed increased c-JUN and phosphorylated c-JUN (serine 73) protein, as well a s c-jun mRNA. Ethanol paradoxically decreased the activity of stress-activa ted protein kinase-1 (SAPK-1) while increasing p44 and p42 mitogen-activate d protein kinase (MAPK) activity. The protein synthesis-inhibiting and SAPK -1 activity- inducing antibiotic, anisomycin (30 and 500 muM) decreased AP- 1 transcriptional activation to 47 and 23% of control values, respectively. The anisomycin effect was enhanced in the presence of 100 mM ethanol. Simi larly, cycloheximide decreased ethanol-induced AP-1 transcriptional activat ion. Pretreatment with the MAPK kinase (MEK) pathway inhibitor PD98059 resu lted in decreases in both ethanol-induced and control AP-1 DNA binding. Thu s this acute ethanol-induced increased AP-1 transcriptional activation requ ires protein synthesis and involves MEK-independent increased MAPK phosphor ylation, on the one hand, and decreased SAPK-1 activity on the other. The e thanol effect is thus ascribed to the activities of alternate kinase pathwa ys and/or the inhibition of (a) protein phosphatase(s). Exposure of CGNs to ethanol for 24 h resulted in decreased AP-1 DNA binding, an observation th at could have consequences for overall neuronal function under chronic expo sure conditions. (C) 2001 Academic Press.