An early and transient period of protein synthesis is required for induction of neuropeptide Y-mRNA by phorbol ester and forskolin in aggregate cultures of fetal brain cells.
We have previously shown that both forskolin (F) and phorbol ester (P) induce neuropeptide Y (NPY) production by aggregate cultures formed from dissociated fetal rat brains. In this study, we addressed the question: Do F/P induce NPY-mRNA in aggregate cultures and if so, does induction require active protein kinases and on-going protein synthesis? On Northern blots, the NPY cDNA hybridized to a single species of about 0.8 kb. F and P each induced a time-dependent increase in NPY-mRNA relative abundance, and this was inhibited by staurosporine, an inhibitor of both protein kinase A and C. Cycloheximide (CHX) inhibited F/P induction of mRNA in a time-dependent manner. When aggregates were incubated with F+P for a total 12 h period, CHX added along with F+P (0 h) completely inhibited, CHX added 1.5 h after F+P partially inhibited, and CHX added 6 h after F+P did not inhibit the increase in NPY-mRNA. To rule out the possibility that this inhibitory profile reflects toxicity of CHX, blots were re-hybridized with a SRIF cRNA probe and, as expected for SRIF gene, a 12 h exposure to CHX did not inhibit F+P induction of SRIF-mRNA. Close inspection of the blots (derived from 1.5% agarose gels) suggested the presence in F/P-treated aggregates of 2 species NPY-mRNA [approximately 0.75 and approximately 0.85 kb, most likely differing in the length of poly(A) tail (Jamal et al., 1991)]; size-fractionation on a higher resolution gel (3% agarose) resolved the F/P induced mRNA into 2 distinct bands, while mRNA from untreated cultures migrated as a single band--the 0.75 kb.(ABSTRACT TRUNCATED AT 250 WORDS)
Magni
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Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas 75235-9032.
Barnea
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D000818:Animals; D015152:Blotting, Northern; D001921:Brain / Q000166:cytology* / Q000196:embryology* / Q000378:metabolism; D001923:Brain Chemistry; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology*; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000032:analysis / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005260:Female; D005333:Fetus / Q000166:cytology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D017781:Genes, Immediate-Early / Q000235:genetics; D009478:Neuropeptide Y / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology*; D011494:Protein Kinases / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013004:Somatostatin / Q000235:genetics; D013997:Time Factors; D014158:Transcription, Genetic / Q000235:genetics*
D009478:Neuropeptide Y; D010703:Phorbol Esters; D012333:RNA, Messenger; D005576:Colforsin; D013004:Somatostatin; D004247:DNA; D003513:Cycloheximide; D011494:Protein Kinases