Morphine and Endomorphins Differentially Regulate μ-Opioid Receptor mRNA in SHSY-5Y Human Neuroblastoma Cells

Abstract

A sensitive quantitative-competitive reverse transcriptase-polymerase chain reaction method was developed to measure -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 M) 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.Opioid receptors belong to the G protein-coupled receptor superfamily. In the nervous system, opioid agonists stimulate heterotrimeric proteins of the G i/o family and control neuronal function through several receptor-effector mechanisms, including activation of receptor-operated potassium channels, inhibition of voltage-gated calcium channels and the inhibition of adenylyl cyclase (Ueda et al

Expression and regulation of the mu opioid peptide receptor in TPA-differentiated HL-60 promyelocytic leukemia cells

“…A more sensitive method, quantitative competitive reverse-transcriptase polymerase chain reaction (QC-RT-PCR), has been shown to detect MOPR mRNA in the brain as well as on SHSY-5Y neuroblastoma cells [12]. The primers for the recombinant RNA (rcRNA) internal standard were constructed by synthesizing two oligomers of approximately 70 bp as described previously [12,13], and contained sequences for the T7 promoter, MOPR mRNA, a spacer gene, β-actin, and a poly (dT) tail in the forward primer, and sequences for the spacer gene, a target gene hybridization probe, the MOPR mRNA reverse primer, and poly (dT)18 in the reverse primer. PCR was performed essentially as described previously [12].…”

“…This concentration of TPA has been shown to be sufficient to induce differentiation of various cells [12]. The cell culture medium containing TPA was changed every 48 h until the completion of the differentiation period.…”

“…A concentration of 10 μM morphine was previously shown to be effective for these experiments using TPA-differentiated cells [12]. Morphine (10 μM) with and without 100 μM naloxone was added to the media 24 h after the final treatment with TPA, and the cells were prepared 24 h later for RNA isolation for real-time RT-PCR analysis.…”

“…QC-RT-PCR was carried out as previously described [12], except that the rcRNA was used as a competitive template instead of genomic DNA. For each sample, 8 aliquots of total RNA (100 ng) were prepared, and a factorial dilution series of the rcRNA internal standard was spiked into these aliquots.…”

“…The primers for the recombinant RNA (rcRNA) internal standard were constructed by synthesizing two oligomers of approximately 70 bp as described previously [12,13], and contained sequences for the T7 promoter, MOPR mRNA, a spacer gene, β-actin, and a poly (dT) tail in the forward primer, and sequences for the spacer gene, a target gene hybridization probe, the MOPR mRNA reverse primer, and poly (dT)18 in the reverse primer. PCR was performed essentially as described previously [12]. The PCR products were diluted 1:100 in DNAse free water, and 2 μl of the diluted products were re-amplified.…”