Striatal proenkephalin gene induction: coordinated regulation by cyclic AMP and calcium pathways

Abstract

Enkephalin modulates striatal function, thereby affecting motor performance and addictive behaviors. The proenkephalin gene is also used as a model to study cyclic AMP-mediated gene expression in striatal neurons. The second messenger pathway leading to proenkephalin expression demonstrates how cyclic AMP pathways are synchronized with depolarization. We show that cyclic AMP-mediated regulation of the proenkephalin gene is dependent on the activity of L-type Ca2+ channels. Inhibition of L-type Ca2+ channels blocks forskolin-mediated induction of proenkephalin. The Ca2+-activated kinase, Ca2+/calmodulin kinase, as well as the cyclic AMP-activated kinase, protein kinase A (PKA), are both necessary for the induction of the proenkephalin promoter. Similarly, both kinases are needed for the L-type Ca2+ channel-mediated induction of proenkephalin. This synchronization of second messenger pathways provides a coincidence mechanism that gates proenkephalin synthesis in striatal neurons, ensuring that levels are increased only in the presence of activated PKA and depolarization.

Fig. 1

The L-type Ca²⁺ channel agonist FPL 64176 induces proenkephalin synthesis in primary striatal culture. (A) Proenkephalin gene levels are upregulated by the L-type Ca²⁺ channel agonist FPL 64176 (20 μM). This upregulation is blocked by the L-type Ca²⁺ channel antagonist nifedipine (20 μM). (B) The proenkephalin construct pENKAT12, transfected into primary striatal neurons, is induced by FPL 64176 (20 μM). The induction is blocked by nifedipine (20 μM). The construct with a shorter promoter, Δ80, is not induced by FPL 64176. Average fold induction of n=4–7±S.E.M. is shown. *P<0.001.

Fig. 2

Proenkephalin induction by forskolin is inhibited by the L-type Ca²⁺ channel antagonist nifedipine, by the CaMK inhibitor KN62, and by the PKA inhibitor H89. (A) Forskolin (10 μM)-induced expression of the proenkephalin gene is blocked by nifedipine (20 μM). (B) The proenkephalin construct pENKAT12, transfected into primary striatal neurons, is induced by forskolin (10 μM). The induction is blocked by nifedipine (20 μM). The Δ80 construct is not induced by forskolin. (C) pENKAT12-induction by forskolin (10 μM) is blocked by KN62 (30 μM), and by H89 (20 μM), (D). Average fold induction of n=7–10±S.E.M. is shown. * P<0.001.

Fig. 3

Proenkephalin induction by FPL 64176 is blocked by the CaMK inhibitor KN62 and by the PKA inhibitor H89. (A) FPL 64176 (20 μM)-mediated induction of the proenkephalin gene is blocked by KN62 (30 μM). (B) FPL 64176 (20 μM)-mediated induction of the proenkephalin construct pENKAT12 is blocked by KN62 (30 μM). (C) FPL 64176 (20 μM)-mediated induction of the proenkephalin gene is blocked by H89 (20 μM). (D) pENKAT12-induction by FPL 64176 (20 μM) is blocked by H89 (20 μM). Average fold induction of n=4–7±S.E.M. is shown. * P<0.001.