Role of protein kinase C in estrogen protection against apoptotic cerebellar cell death in ethanol-withdrawn rats

Results of studies from our laboratory have shown that administration of 17beta-estradiol (E-2) reduces cerebellar neuronal damage during ethanol withdrawal (EW). In the current study, we examined mechanisms underlying E-2 protection against EW-associated cerebellar damage by assessing apoptotic indicators: DNA fragmentation, caspase-3 activity, and protein kinase C (PKC) activity. Ovariectomized rats, implanted with E-2 or oil pellets, received ethanol [7.5% weight/volume (wt./vol.)] (EW/E-2 group and EW/Oil group, respectively) chronically (for 5 weeks) or control dextrin diet (Dextrin/Oil group). At day 14 of EW, cerebelli were collected for the terminal deoxynucleotidyltransferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL) assay to detect DNA fragmentation and for inummohistochemistry to detect caspase-3 activation. A separate group of rat cerebelli was prepared to assess for total PKC activity, as well as for activity of a specific PKC isozyme, epsilon (PKCkappa), by using an in vitro [gamma-P-32]ATP phosphorylation assay at days I and 14 of EW. Results indicated that rats in the EW/Oil group had more DNA fragments and caspase-3-positive neuronal cells than observed for control rats, and these effects were inhibited by E-2 treatment. For total PKC activity at day I of EW, rats in the EW/E-2 group had a lower cytosolic PKC activity than observed for either rats in the EW/Oil group or control rats. At day 14 of EW, both EW groups had a lower total PKC activity than observed for control rats. For PKCepsilon activity, rats in the EW/E-2 group had a lower cytosolic PKCe activity than observed for rats in the EW/Oil group or for control rats at day 1, and they had a lower membrane PKCepsilon activity at day 14 of EW than observed for control rats. These findings support the suggestion that E2 protects against cerebellar neuronal damage in ethanol-withdrawn rats by inhibition of DNA fragmentation and caspase-3 activation, and that reduced PKC activity may be involved in the protection. (C) 2003 Elsevier Inc. All rights reserved.