Abstract
A sensitive quantitative-competitive reverse transcriptase-polymerase chain reaction method was developed to measure μ-opioid receptor (MOR) mRNA expression in SHSY-5Y neuroblastoma cells. Differentiation of SHSY-5Y cells with either retinoic acid (RA) or 12-o-tetradecanoyl-phorbol-13-acetate (TPA) significantly increased MOR mRNA levels. Morphine treatment (10 μM) for 24 h decreased MOR mRNA levels in control, as well as RA- and TPA-differentiated cells. In contrast, chronic exposure to the opioid peptides endomorphin-1 or endomorphin-2 significantly increased MOR mRNA levels in undifferentiated and RA-differentiated cells. An opioid antagonist, naloxone, reversed the morphine and endomorphin-1 and -2 effects on MOR mRNA levels in undifferentiated SHSY-5Y cells, but naloxone had differential reversing effects on the agonists' regulation of MOR mRNA in RA- or TPA-differentiated cells. To investigate whether the changes in MOR mRNA expression paralleled changes in MOR receptor function, intracellular cAMP accumulation in SHSY-5Y cells was measured. After chronic treatment with morphine, forskolin-induced cAMP levels in SHSY-5Y cells were significantly higher than those of untreated control cells. In contrast, forskolin-induced cAMP accumulation levels were lower in cells treated with endomorphin-1 or -2 than in untreated control cells. Together, our studies indicate that the opioid alkaloid morphine and the opioid peptides endomorphin-1 and -2 differentially regulate MOR mRNA expression and MOR function in SHSY-5Y cells.
Footnotes
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This study was partially supported by Public Health Service National Institutes of Health R01 DA-07058 and K02 DA-016149 (to S.L.C.).
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DOI: 10.1124/jpet.103.048694.
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ABBREVIATIONS: MOR, μ-opioid receptor; RA, retinoic acid; TPA, 12-o-tetradecanoyl-phorbol-13-acetate; QC-RT-PCR, quantitative-competitive reverse transcriptase-polymerase chain reaction; bp, base pair; rcRNA, reconstructed RNA from cDNA; IS, internal standard; ANVOA, analysis of variance; EtOH, ethanol.
- Received January 2, 2003.
- Accepted April 21, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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