+CPT +TPA 
FIGURE 4. Desensitization of proenkephalin gene expression. SI analysis in 
which two parallel sets of plates of C6-D2 cells were treated and 
assayed. In both sets of plates, forskolin (10 pM) and I MX (0.5 
mM) were added at time 0. In the set of plates represented by 
the first 8 lanes (times Oto 6 hours from the left), cpt-cAMP (200 
pM) was added 1.5 hours after forskolin and I MX. The addition 
of cpt-cAMP has no effect on the normal time course of mRN A 
accumulation (compare with figure 2); thus, by the 6-hour time 
point (4.5 hours after addition of cpt-cAMP), no correctly initiated 
mRN A is observed. In the set of plates represented by the last 
3 lanes, TP A (50 nM) was added instead of cpt-cAMP 1.5 hours 
after forskolin and IMX. The time points shown are for 3, 4, and 
6 hours total (1.5, 2.5, and 4.5 hours after addition of TP A, 
respectively). Compared with the corresponding cpt-cAMP- 
treated plates, there is a new burst of transcription with maximal 
RNA accumulation 2.5 hours after the addition of TP A and 
appreciable mRN A remaining after 6 hours total (4.5 hours after 
the addition of TPA). The inset demonstrates the effectiveness of 
cpt-cAMP (200 pM) on induction of pENKAT-12 in C6-D2 cells not 
pretreated with forskolin or IMX after 1.5 and 2 hours. 
33 
