FIGURE 3. Independence from new protein synthesis. SI analysis showing 
the time course of accumulation of correctly initiated mRNA from 
pENKAT-12 in C6-D2 cells in response to forskolin (10 pM) and 
I MX (0.5 mM). Anisomycin (10 pM) was added just before the 
forskolin and IMX. Anisomycin causes superinduction of 
proenkephalin mRNA levels , consistent with inhibition of synthesis 
of a cAMP-inducible transcriptional repressor or inhibition of 
synthesis of a labile mRNAse. 
cAMP (200 |iM) or the phorbol ester 12-0-tetradecanoyl-phorbol-14 acetate 
(TPA) (50 nM) was added 90 minutes after the addition of forskolin and IMX, 
by which time transcriptional activation appears to have peaked (figure 4). 
Cpt-cAMP, which is a potent activator of proenkephalin gene expression (figure 
4 inset), had no effect on proenkephalin expression when cells were pretreated 
with forskolin and IMX for 90 minutes (i.e., the addition of cpt-cAMP did not 
produce a new peak of mRNA synthesis or in any way prolong the time course 
of mRNA accumulation). On the other hand, TPA, which activates protein 
kinase C rather than the cAMP-dependent protein kinase, did produce a new 
32 
