The regulation of prodynorphin gene expression in cultured spinal cord cells: involvement of second messengers.
The regulation of prodynorphin (proDYN) mRNA levels by cAMP and protein kinase C (PKC) pathways was studied in cultured rat spinal cord cells. Spinal cord cells were cultured from 14 day (E 14) embryos of Sprague-Dawley rats. After 7 days in vitro, the spinal cord cells were incubated with either forskolin (5 microM) or phorbol-13-myristate acetate (PMA; 2.5 microM) for 1, 3, 6, 9, 12, or 24 h and the total RNA was isolated for Northern blot analyses. The proDYN mRNA level began to increase 1 h, then reached and remained at a peak 3-6 h after stimulation by forskolin or PMA. proDYN mRNA levels in forskolin treated cells decreased slightly from their peak after 9 h of treatment, whereas the level of proDYN mRNA returned to the basal level in PMA-treated cells. Pretreatment of cells with cycloheximide (a protein synthesis inhibitor; 10 microM) did not affect the forskolin- or PMA-induced increase in proDYN mRNA, but pretreatment with nimodipine (a L-type Ca2+ channel blocker; 2 microM), omega-conotoxin (a N-type Ca2+ channel blocker; 1 microM), or KN-62 (a Ca2+/calmodulin-dependent protein kinase II inhibitor; 5 microM) inhibited induction of proDYN mRNA both by forskolin and PMA. Additionally, dexamethasone did not affect the expression of proDYN mRNA level induced by forskolin. Our results suggest that proDYN mRNA levels in spinal cord cells is regulated by both cAMP and PKC pathways. Calcium influx through both L- and N-type calcium channels and Ca2+/calmodulin-dependent protein kinase II appear to be involved in the increase of proDYN mRNA levels induced by either forskolin or PMA. Furthermore, ongoing protein synthesis is not required for forskolin- or PMA-induced responses.
Ha
T S
TS
Department of Pharmacology, College of Medicine, Hallym University, Chunchon, Kangwon-Do, Korea.
Kim
Y H
YH
Song
D K
DK
Wie
M B
MB
Suh
H W
HW
D000818:Animals; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004622:Embryo, Mammalian; D004745:Enkephalins / Q000096:biosynthesis*; D007700:Kinetics; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011498:Protein Precursors / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015290:Second Messenger Systems; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D004745:Enkephalins; D011498:Protein Precursors; D012333:RNA, Messenger; D005576:Colforsin; C033660:preproenkephalin; D000242:Cyclic AMP; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate