FIGURE 2. SI analysis showing the time course of accumulation of correctly 
initated mRNA from pENKAT-12 in stably transfected C6 glioma 
cells (C6-D2). The protected band is 70 bp as expected for this 
probe. The numbers represent time in hours after replacing the 
growth media with low serum media (0. 1 percent fetal bovine 
serum) containing 10 pM forskolin and 0.5 mM I MX. Rapid 
transcriptional changes are more easily seen with pENKAT-12 
than with the endogenous proenkephalin gene because the CAT 
mRNA is relatively unstable; therefore, basal levels of mRNA are 
low, leaving changes unmasked. 
et al. 1983; Tang et al. 1983; Romano et al. 1987; Le Moine et al. 1990). 
Because haloperidol has also been shown to induce expression of c -fos, zif/ 
268, and other lEGs in striatum (Dragunow et al. 1990; Miller 1990; Robertson 
and Fibiger 1992; Nguyen et al., in press), a role for lEGs in the regulation of 
proenkephalin gene expression is under investigation. However, in such 
chronic stimulation paradigms an additional difficulty must be taken into 
account: I EG expression is transient, and over a time course of minutes to 
hours, the composition of AP-1 complexes is altered (Sonnenberg et al. 1989b). 
Indeed, not only does c -fos expression rapidly return to basal levels, it also 
becomes refractory to further activation (desensitization). The same is true of 
cAMP-inducible proenkephalin gene expression in C6 glioma cells. As shown 
in figure 2, forskolin and IMX-stimulated levels of pENKAT-12 mRNA decline to 
undetectable levels by 6 hours after the onset of stimulation. To determine 
whether expression had been desensitized, either the stable cAMP analog cpt- 
31 
