Opioid-induced regulation of gene expression in PC12 cells stably transfected with mu-opioid receptor.
It has been postulated that opiates induce addictive behaviour via changes in gene expression. PC12 cells were stably transfected with the recombinant human mu-opioid receptor (MOR) to study opioid-induced gene expression. Expression was verified by binding assay, immunocytochemistry, and immunblotting experiments. Forskolin-induced cAMP formation was inhibited by [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO 1 microM), a specific MOR agonist. This effect was completely antagonized by naloxone. By using cDNA arrays, including approximately 1,200 well-defined genes normally expressed in neural tissue, we monitored semi-quantitative changes in gene expression after 3 h short-term exposure to DAMGO. Incubation with DAMGO increased mRNA levels for 13 genes and down-regulated 13 other genes. Annexin V, RGS4 and CREB genes showed pronounced increase in expression after stimulation with DAMGO. Quantitative RT-PCR confirmed that DAMGO increased mRNA levels of Annexin V, an apoptosis-induced gene. We suggest that the PC12 cell transfected with the recombinant human MOR is a useful tool for identification of opioid-induced genes that may provide information on opiate effects of relevance for dependence.
Zarnegar
Parisa
P
Department of Clinical Neuroscience, Center for Molecular Medicine, Karolinska Institutet, SE-171 76 Stockholm, Sweden. parisa.zarnegar@cns.ki.se
Persson
Anders I
AI
Ming
Yu
Y
Terenius
Lars
L
D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D017304:Annexin A5 / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D009270:Naloxone / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D009294:Narcotics / Q000494:pharmacology*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016716:PC12 Cells; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D011869:Radioligand Assay / Q000379:methods; D051381:Rats; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014162:Transfection / Q000379:methods
D000701:Analgesics, Opioid; D017304:Annexin A5; D009292:Narcotic Antagonists; D009294:Narcotics; D012333:RNA, Messenger; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D005576:Colforsin; D009270:Naloxone; D000242:Cyclic AMP