expression in response to cAMP and Ca ++ in a fashion that mimics physiologic 
regulation in many cell types (Kobierski et al. 1991). 
THE PROENKEPHALIN GENE CAN BE REGULATED AS AN IEG IN ITS 
OWN RIGHT 
The cAMP- and Ca ++ -inducible ENKCRE-2 site within the proenkephalin gene 
is quite similar to the cAMP- and Ca ++ -inducible calcium-response element 
that is involved with the rapid induction of c -fos in response to depolarization 
(Sheng et al. 1988). Indeed, in transformed cell types, such as C6 glioma cells 
and PCI 2 cells, the induction of proenkephalin gene expression by second 
messengers is quite similar to the induction of c -fos: It is rapid, transient, and 
independent of new protein synthesis. In fact, in such cell types the regulation 
of proenkephalin gene expression in response to second messengers is 
essentially indistinguishable from that of c-fos and other lEGs. 
Activation of the proenkephalin promoter has been studied in C6 glioma cell 
lines, which also express the endogenous rat proenkephalin gene (Yoshikawa 
and Sabol 1986), by stably transfecting the plasmid pENKAT-12, which 
contains human proenkephalin gene regulatory sequences (Comb et al. 1986). 
In pENKAT-12, human proenkephalin gene sequences from nucleotides -193 to 
+70 (with respect to the mRNA cap site), containing the proenkephalin second 
messenger-inducible enhancer, are fused to the bacterial chloramphenicol 
acetyltransferase (CAT) transcription unit. The best characterized of the C6 
glioma stably transfected lines is the C6-D2 line (Nguyen et al. 1990). When 
C6-D2 cells are treated with forskolin (10 ^M) and the phosphodiesterase 
inhibitor 3-isobutyl-1-methylxanthine (IMX) (0.5 mM), CAT mRNA correctly 
initiated from the proenkephalin promoter is detectable by SI analysis within 
15 minutes (figure 2); mRNA levels peak by 90 to 120 minutes and then 
rapidly decline. By 6 hours after addition of forskolin, levels of correctly initiated 
mRNA have returned to basal levels. Moreover, the protein synthesis inhibitors 
cycloheximide (100 ^M) or anisomycin (10 |iM) added to the cells just prior to 
addition of forskolin and IMX have no effect on onset of mRNA appearance, 
consistent with activation being caused by a rapid posttranslational modification 
of preexisting proteins (figure 3). This pattern of activation is consistent with 
second messenger-dependent posttranslational modification (phosphorylation) 
of a preexisting transcription factor such as jun - D (Kobierski et al. 1991). 
IEG-TARGET INTERACTIONS IN CHRONIC STIMULATION PARADIGMS 
MUST TAKE DESENSITIZATION INTO ACCOUNT 
Proenkephalin mRNA and peptides have also been shown to be induced by 
antipsychotic drugs in rat striatum following 2 to 3 weeks of treatment (Sabol 
30 
