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CELLULAR AND MOLECULAR
Department of Biology, Seton Hall University, South Orange, New Jersey (X.Y., A.D.B., S.L.C.); and Department of Virology, Wuhan University, Wuhan, China (X.M., W.X.L.)
Received January 2, 2003; accepted April 21, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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). Pharmacological studies using highly selective ligands
have classified opioid receptors into three subtypes: µ, 
, and
based upon a characteristic pharmacology
(Loh and Smith, 1990).
Molecular cloning studies over the past 10 years have confirmed the
pharmacological classification of the opioid receptors
(Evans et al., 1992;
Kieffer et al., 1992; Chen et
al.,
1993a,b;
Meng et al., 1993;
Wang et al., 1994). The
µ-opioid receptor (MOR) is the principal target of the clinically
efficacious opioids, such as morphine, as well as opioid peptides, such as the
endomorphins, the only endogenous opiates known to bind selectively and with
high affinity to the MOR. The clinical usefulness of opiates in controlling
pain is compromised by the development of opioid tolerance and dependence,
which remain significant drawbacks to the use of opiate drugs, including
morphine, as pain relievers (Harrison et
al., 1998). Although intensively investigated, the molecular
mechanisms underlying opioid tolerance and dependence are poorly
understood.
SHSY-5Y cells are human neuroblastoma cells, a subclone of the SK-N-SH cell
line (Kohl et al., 1980;
Kuramoto et al., 1981).
SHSY-5Y cells are of sympathetic adrenergic ganglionic origin
(Scott et al., 1986) and
express both µ- and -opioid receptors
(Yu and Sadee, 1988) in a
ratio of 5:1 based on quantitative receptor binding studies. A series of
biological and morphological changes have been shown to occur in SHSY-5Y cells
during differentiation (Pahlman et al.,
1990,
1995). Differentiation of
SHSY-5Y cells with either retinoic acid (RA) or
12-o-tetradecanoyl-phorbol-13-acetate (TPA) is associated with
considerable reduction in the proliferation rate and with induction of
neuritic processes (Scott et al.,
1986; Pahlman et al.,
1990; Kohring and Zimmermann,
1998). Of particular interest is that opioid receptor-mediated
signal transduction pathways are reportedly associated with RA- and
TPA-induced differentiation (Kohring and
Zimmermann, 1998). Using receptor binding studies, the MOR binding
sites in SHSY-5Y cells was shown to be enhanced after differentiation with
either RA or TPA (Zadina et al.,
1993,
1994). SHSY-5Y cells,
therefore, are ideal for investigating how MOR regulation is altered during
neuronal differentiation.
Our present study evaluates MOR mRNA expression to delineate the molecular basis of MOR regulation in SHSY-5Y cells. A sensitive and precise method of quantitative competitive reverse transcriptase polymerase chain reaction (QC-RT-PCR) has been developed using primers derived from the human MOR sequence to quantitatively measure MOR mRNA. Using QC-RT-PCR, differentiation with both RA and TPA was shown to increase MOR mRNA levels in SHSY-5Y cells.
In 1997, endomorphins-1 and -2, two endogenous tetrapeptide opioids with
high selectivity and affinity for the MOR, were discovered and shown to be
more potent analgesics than morphine
(Zadina et al., 1997).
However, differences between the actions of morphine and those of the
endomorphins have been noted. For example, chronic morphine treatment
down-regulates MOR membrane density
(Zadina et al., 1993) without
internalization of the MOR (Keith et al.,
1996; Zhang et al.,
1998), whereas endomorphins-1 and -2 have been shown to cause
rapid endocytosis of the MOR (McConalogue
et al., 1999).
In our study, in both undifferentiated and differentiated SHSY-5Y cells, MOR mRNA expression was decreased after chronic morphine treatment, whereas treatment with either endomorphin-1 or -2 increased MOR mRNA levels. We also observed that morphine and endomorphins have opposite effects on forskolin-induced intracellular cAMP levels. After chronic treatment with morphine, forskolin-induced intracellular cAMP accumulation in SHSY-5Y cells is higher than that of the control cells. However, forskolin-induced cAMP accumulation is lower in cells treated with endomorphin-1 or -2 than in control cells. Differentiation of SHSY-5Y cells with either RA or TPA seemed to decrease the effects of naloxone, an opioid antagonist, which was shown to reverse morphine's influence on both MOR mMRA expression and forskolin-induced cAMP accumulation. Together, our data suggest that morphine and endomorphins-1 and -2 may have differential effects on MOR receptor regulation in the SHSY-5Y neuroblastoma cell line.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Cell Line and Culture Conditions. SHSY-5Y human neuroblastoma cells (passages 20–30) were cultured in 10% EFN medium containing a 1:1 ratio of Eagle's minimum essential medium and F-12 with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). The cells were grown at 37°C in a humidified atmosphere containing 5% CO2.
Differentiation and Drug Treatment. At about 65 to 75% confluence,
SHSY-5Y cells were differentiated into a neuronal phenotype with RA or TPA as
described previously (Zadina et al.,
1993). Either RA (10 µM in 0.1% ethanol) or TPA (16 nM in 0.1%
ethanol) was added to the media every other day. For total RNA isolation, the
cells were cultured in T25-cm2/T75-cm2 flasks. Cells
were cultured in 24-well plates for cAMP accumulation assays. Test compounds
were added to the media 24 h after the final treatment with the
differentiating agent, and the cells were prepared 24 h later for either RNA
isolation or cAMP assay. To avoid the metabolism of the administered peptides,
the cells were switched to serum-free EFN medium that contains a 1:1 ratio of
Eagle's minimum essential medium and F-12 supplemented with insulin (bovine, 5
µg/ml), transferrin (human, 100 µg/ml), progesterone (20 nM), putrescine
(100 µM), and Na selenite (30 nM). Either endomorphin-1 or -2 was added
with the last RA treatment for the last 24 h of culture.
Structure and Construction of Internal Standard and µ-Opioid Receptor
Primers. Construction of the internal standard was accomplished by
synthesizing two oligonucleotides of approximately 75 bp each containing
sequences for the T7 promoter, the MOR target gene (mRNA), a random spacer,
and a housekeeping gene (
-actin). The forward primer of the
reconstructed RNA from cDNA (rcRNA) contained the T7 promoter, MOR mRNA
forward primer, random sequence, and -actin forward primer. The rcRNA
reverse primer contained the -actin reverse primer, random sequence, MOR
mRNA reverse primer, and a poly(dT) tail. The MOR forward primer
(5'-TAC-CGT-GTG-CTA-TGG-ACT-GAT-3') was from position 962, and the
reverse primer (5'-ATG-ATG-ACG-TAA-ATG-TGA-ATG-3') was from
position 1103 of the genomic MOR gene
(Wang et al., 1994). The
-actin forward primer (5'-AGA-CCT-CTA-TGC-CAA-CAC-AGT-3')
was from position 2753 in exon 5, and the reverse primer
(5'-GAC-ACA-CCT-AAC-CAC-CGA-GAT-3') was from position 3017 in exon
6. There is a 124-bp intron E between exons 5 and 6
(Nudel et al., 1983).
Therefore, the -actin primers will generate products of 161 bp from mRNA
and 285 bp from genomic DNA. All primers were synthesized and purified by
Oligo (Wilsonville, OR).
Reactions were conducted in a final volume of 50 µl containing PCR buffer, 3 mM MgCl2, 0.2 mM of each dNTP, 20 pmol of upper and lower rcRNA primer, 200 ng of genomic DNA from the SHSY-5Ycells or cDNA, and 1.25 units of TaqDNA polymerase (AmpliTaq; PerkinElmer Instruments, Norwalk, CT). The reactions were heated to 94°C for 3 min and immediately cycled 30 times through a 10-s denaturing step at 94°C, a 30-s annealing step at 59°C, and a 45-s extension step at 72°C. After the final cycle, a 5-min extension step at 72°C was included. The PCR products were diluted 1:100 in water, and 2 µl was reamplified using the conditions stated above. The second amplification PCR products were pooled and purified using the Magic Prep DNA purification system (Promega, Madison, WI). The pooled PCR products were transcribed into RNA by the T7 promoter using the Riboprobe Gemini II in vitro transcription system (Promega). The DNA templates were removed by digestion with DNase I after the transcription reaction. The rcRNA was extracted according to the procedure of the Riboprobe Gemini II in vitro transcription system (Promega), and quantitated by absorbance at 260 nm. Figure 1 shows the general procedure of the construction of the internal standard and the diagram of QC-RT-PCR.
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Quantitative Competitive RT-PCR. Total RNA was extracted from
SHSY-5Y cells with TRIzol reagents (Invitrogen). Competitive RT-PCR was
carried out with the internal standard (IS) rcRNA as the competitor as
described by Vanden Heuvel et al.
(1993). For each sample, six
aliquots of SH-SY5Y total RNA (50 ng each) were prepared, and a series of 1:2
dilutions, from 0.625 to 20 pg, of the rcRNA internal standard (231 bp) were
added to these aliquots. Reverse transcription of RNA was performed in a final
volume of 20 µl. The samples were incubated at 45°C for 30 min, and the
reverse transcriptase was inactivated by heating to 99°C for 5 min. To
these cDNA samples, a PCR master mixture containing PCR buffer, 1 unit of
TaqDNA polymerase, and 10 pmol each of the MOR primers were added to
bring the final volume to 50 µl. The reactions were heated to 94°C for
3 min and immediately cycled 30 times through a 20-s denaturing step at
94°C, a 20-s annealing step at 55°C, and a 20-s extension step at
72°C. After the final cycle, a 5-min extension step at 72°C was
included. Aliquots (15 µl) of the PCR products were electrophoresed on a 3%
NuSieve/1% agarose gel (FMC Bioproducts, Rockland, ME), visualized by ethidium
bromide staining, and analyzed using the AlphaEase Stand Alone software.
Quantitation of the amount of MOR mRNA present was determined as described
previously (Gilliland et al.,
1990; Vanden Heuvel et al.,
1993). By titrating the unknown amount of MOR cDNA template
against a dilution series containing known amounts of corresponding internal
standard template, one should be able to quantitate the amount of MOR mRNA.
After electrophoresis, bands corresponding to internal standard and MOR cDNA
were excised and a ratio of internal standard and MOR cDNA was calculated. As
would be predicted of competitive templates, a plot of the ratio of Internal
Standard to MOR cDNA versus the known concentration of input internal standard
is linear when plotted on a log-log scale
(Fig. 2). At the point where
MOR and internal standard products are equivalent (i.e., ratio = 1.0), the
starting concentration of MOR before PCR is equal to the known starting
concentration of the internal standard. Furthermore, we can obtain the amount
of the MOR mRNA from the extracted cell samples based on the known
concentration of the internal standard rcRNA. The QC-RT-PCR for the
housekeeping gene, -actin, was also performed with the 285-bp internal
standard as shown in Fig.
2.
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cAMP Accumulation Assay. The SHSY-5Y cells were subcultured in 24-well culture plates. For endomorphin-1 and -2 pretreatment, the 10% EFN growth medium was replaced with medium containing 10 µM of either endomorphin-1 or endomorphin-2, and the cells were incubated for 24 h. After treatment, the medium was removed and replaced with 0.5 ml of EFN medium containing 0.5 mM isobutylmethylxanthine, a phosphodiesterase inhibitor, to block the breakdown of cAMP in SH-SY5Y cells, and the cells were incubated for 30 min at 37°C. The culture medium was then removed and replaced with 0.5 ml of fresh medium with or without 25 µM forskolin. The cells were transferred to 37°C for 10 min. The medium was then removed, and the cells were rinsed once with 1 ml of phosphate-buffered saline. One-half milliliter of 0.1 N HCl was then added to lyse the cells, and the monolayers were frozen at –20°C. For determination of cAMP content, the monolayers were thawed, and the intracellular cAMP level from the cell lysate in each well was measured by radioimmunoassay (Amersham Biosciences Inc., Piscataway, NJ).
Data Analysis. For MOR mRNA quantification, each treatment was
performed in triplicate, and duplicate gel electrophoreses were carried out.
The cAMP accumulation assays were performed in duplicate on triplicate
samples. MOR and -actin mRNA expression of each sample was measured by
QC-RT-PCR, and the MOR mRNA level in each sample was normalized to the
-actin level as follows: [mRNAMOR]normalized =
[mRNAMOR]/[mRNA-actin].
All statistical data are presented as mean ± S.D. unless other wise noted and was analyzed using one-way ANOVA with post test Tukey where appropriate. Statistical significance was considered p < 0.05 (the P value of each comparison is shown in the legends of each figure).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Pahlman
et al., 1990,
1995;
Kohring and Zimmermann, 1998).
To determine whether MOR mRNA expression is altered during differentiation,
SHSY-5Y cells were treated with either RA (10 µMin 0.1% ethanol) for 6 days
or TPA (16 nM in 0.1% ethanol) for 4 days to induce differentiation. MOR mRNA
levels were then examined and compared with undifferentiated cells cultured in
either 0.1% ethanol (EtOH) or vehicle (growth medium). The MOR mRNA level
increased 1.3-fold after RA-induced differentiation
(Fig. 3A) and 1.7-fold after
TPA-induced differentiation (Fig.
3B) compared with undifferentiated SHSY-5Y cells.
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Effects of Morphine with and without Naloxone Cotreatment on MOR mRNA
Expression in SHSY-5Y Cells. To determine morphine's effects on the
transcriptional regulation of the MOR, we examined MOR mRNA levels after
chronic exposure to morphine in both undifferentiated SHSY-5Y cells and in
SHSY-5Y cells induced to differentiate with RA or TPA. We observed that
chronic morphine treatment (10 µM) for 24 h significantly decreased MOR
mRNA levels in undifferentiated cells (0.1% EtOH + vehicle-treated) by 47%,
and that cotreatment with the opioid receptor antagonist naloxone completely
blocked morphine's effects (Fig.
4A). Chronic morphine treatment also significantly decreased MOR
mRNA expression in both RA- and TPA-differentiated SHSY-5Y cells by 11 and
70%, respectively, compared with vehicle
(Fig. 4, B and C). These
results are consistent with the binding assay data previously reported by
Zadina et al. (1993). However,
cotreatment with morphine and naloxone (10 µM) partially blocked morphine's
effects in TPA-differentiated SHSY-5Y cells
(Fig. 4C), but did not block
morphine's effects on MOR mRNA expression in RA-differentiated SHSY-5Y cells
(Fig. 4B).
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Effects of Endomorphin-1 and -2 on MOR mRNA Expression in SHSY-5Y
Cells. Because endomorphins-1 and -2, two endogenous tetrapeptide opioids,
have been shown to have high selectivity and affinity for the MOR
(Zadina et al., 1997), we next
investigated whether treatment with either endomorphin-1 or -2 would affect
MOR mRNA regulation. Endomorphin-1 and -2 increased MOR mRNA levels in
undifferentiated SHSY-5Y cells 3.7- and 2.5-fold, respectively, compared with
vehicle (Fig. 5A), and also
increased MOR mRNA levels in RA-differentiated SHSY-5Y cells (1.8- and
2.3-fold, respectively) compared with vehicle
(Fig. 5B). Cotreatment with
naloxone and either endomorphin-1 or -2 completely blocked endomorphin's
effects in undifferentiated SHSY-5Y cells
(Fig. 5A). Interestingly,
although naloxone completely blocked endomorphin-2's effects in
RA-differentiated SHSY-5Y cells (Fig.
5B), it only partially blocked the effects of endomorphin-1.
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Effects of Morphine, Endomorphin-1 and -2, and Naloxone on
Forskolin-Stimulated cAMP Accumulation in SHSY-5Y Cells. It has been
reported that chronic exposure to morphine or to endomorphin-1 or -2 can
affect adenylyl cyclase activity
(Avidor-Reiss et al., 1995;
Monory et al., 2000). To
investigate whether the changes in MOR mRNA expression observed after morphine
or endomorphin treatment were associated with a change in adenylyl cyclase
activity, we examined forskolin-stimulated cAMP levels in undifferentiated
SHSY-5Y cells chronically treated with morphine or with enodmorphin-1 or -2.
Chronic morphine treatment resulted in a 20.1-fold up-regulation of cAMP
production after forskolin stimulation compared with only a 9.5-fold
forskolin-induced cAMP increase after vehicle treatment
(Fig. 6A). Conversely, after
endomorphin-1 and -2 treatments, forskolin-induced cAMP levels were observed
to be about 4.7-fold, which is significantly lower than the 8.9-fold of
forskolin-induced cAMP levels in the SHSY-5Y cells given vehicle treatment.
Cotreatment with the opioid antagonist naloxone inhibited morphine's and
endomorphin-1 and -2's effects on forskolin-stimulated cAMP accumulation,
returning the stimulated cAMP levels to vehicle control levels. These results
suggest that although morphine pretreatment results in a compensatory
up-regulation of adenylyl cyclase activity, the endomorphins serve to suppress
adenylyl cyclase activity.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Pahlman et al., 1990;
Kohring and Zimmermann, 1998),
although some phenotypic differences are noted in the differentiated cells. In
addition, both have been shown to have similar effects on MOR expression in
SHSY-5Y cells. Previous studies using receptor radioligand binding assays have
shown that MOR expression is increased after differentiation with either RA or
TPA (Zadina et al., 1993,
1994). Using QC-RT-PCR, we
have now shown that the increase in MOR in SHSY-5Y cells with either RA- or
TPA-induced differentiation takes place at the transcriptional level. We found
a 1.3- and 1.7-fold increase in MOR mRNA levels after RA- and TPA-induced
differentiation, respectively, compared with undifferentiated SHSY-5Y
cells.
The actions of opiates, such as morphine, are mediated via the MOR
(Loh and Smith, 1990),
although the molecular mechanisms governing receptor-mediated transcriptional
regulation are still not defined. Previous studies reported that MOR membrane
density in SHSY-5Y cells is decreased after chronic morphine treatment (Zadina
et al., 1993,
1994), however, MOR
endocytosis is not induced by morphine. To investigate whether morphine's
down-regulation of the MOR occurs at the transcriptional level, we measured
MOR mRNA expression after exposure to morphine for 24 h in undifferentiated
SHSY-5Y cells and found that MOR mRNA levels are decreased by chronic morphine
treatment. Because MOR expression in SHSY-5Y cells was up-regulated after
differentiation by both RA and TPA, we then compared morphine's effects on MOR
expression in differentiated SHSY-5Y cells and found that MOR mRNA expression
was also decreased by chronic morphine treatment in RA- and TPA-differentiated
cells, indicating that transcriptional regulation may be one of the mechanisms
underlying the reduction of MOR expression after chronic morphine treatment.
In addition, in undifferentiated SHSY-5Y cells, naloxone, an opioid receptor
antagonist, completely blocked morphine's down-regulation of MOR mRNA,
suggesting that morphine's effects on undifferentiated SHSY-5Y cells are
receptor-mediated. However, naloxone did not block morphine's down-regulation
of MOR mRNA levels in RA-differentiated SHSY-5Y cells and only partially
blocked morphine's effects in TPA-differentiated cells, suggesting that
differentiation may cause a modification in naloxone's reversing effects on
morphine's actions and that MOR regulation may be mediated through alternate
pathways during differentiation, depending on the inducing stimulus.
We also investigated the effects of endomorphin-1 and -2 on MOR mRNA
expression in SHSY-5Y cells. These two opioid peptides have been shown to be
more potent analgesics than morphine and have been suggested as endogenous
ligands for the MOR (Zadina et al.,
1997). Additionally, endomorphin analgesia has been shown to be
effectively blocked by naloxone (Zadina et
al., 1997; Goldberg et al.,
1998; Fischer and Undem,
1999). However, differences between the actions of morphine and
those of the endomorphins have been noted. For example, endomorphins-1 and -2
bind the MOR at the cell surface and cause rapid internalization, similar to
DAMGO, a MOR-specific synthetic peptide agonist
(McConalogue et al., 1999),
whereas morphine treatment down-regulates MOR membrane density
(Zadina et al., 1993) without
rapid MOR internalization (Keith et al.,
1996; Zhang et al.,
1998). It has been suggested that endomorphin-induced MOR
endocytosis may be the mechanism for MOR desensitization and down-regulation
(Harrison et al., 2000). In
our study, both endomorphin-1 and -2 significantly increased MOR mRNA levels
in both undifferentiated and RA-differentiated SHSY-5Y cells, thus lending
credence to the argument that MOR endocytosis may regulate the cell's
responsiveness to ligand stimulation. Endomorphin-induced MOR endocytosis may
act as a feedback mechanism to activate MOR gene transcription and restore
normal MOR membrane density.
Activation of opioid receptors stimulates inhibitory G proteins
(Gi) that suppress the activity of adenylyl cyclase and decrease
cAMP levels. To further investigate and compare the signaling pathways
associated with morphine and endomorphin regulation of the MOR, we examined
forskolin-induced intracellular cAMP levels in undifferentiated SHSY-5Y cells
after chronic exposure to either morphine or to endomorphin-1 or -2. Morphine
increased forskolin-induced intracellular cAMP levels in undifferentiated
SHSY-5Y cells, which is consistent with our previous report showing that
chronic morphine treatment can cause an adaptive sensitization of adenylyl
cyclase, resulting in a compensatory up-regulation of cAMP production
(Blake et al., 1997).
Conversely, forskolin-induced cAMP levels were significantly reduced in
undifferentiated SHSY-5Y cells chronically treated with either endomorphin-1
or -2. After chronic treatment with different MOR agonists, forskolin-induced
activity of adenylate cyclase in undifferentiated SHSY-5Y cells seemed to be
inversely correlated with expression of MOR mRNA. Consistent with our MOR mRNA
data, treatment of undifferentiated SHSY-5Y cells with naloxone completely
blocked the cAMP up-regulation by morphine and the down-regulation of cAMP by
endomorphin-1 and -2. Together, these data indicate that the differential
effects of MOR agonists on MOR mRNA expression and forskolin-induction of cAMP
levels in undifferentiated SHSY-5Y cells are mediated via MOR.
The morphine supersensitization results presented in the manuscript reflect
the inherent property of morphine, but not endomorphins, to activate an
adaptive adenylyl cyclase response after chronic exposure. The cAMP overshoot
is widely used as a model of opioid withdrawal. This adaptive response seems
to be a function of the agonist used in the chronic treatment paradigm,
because it is only observed when cells are treated with morphine or
[D-Ala2,N-MePhe4,Gly5-ol]-enkephalin,
but not with other opioid agonists such as levorphanol, methadone, etorphine,
or buprenorphine (Blake et al.,
1997). However, different isoforms of adenylyl cyclase also seem
to be involved in the superactivation response
(Avidor-Reiss et al., 1997).
The endomorphins' inability to activate an adaptive or overshoot response in
the SHSY5Y cells suggests that the removal of these opioid peptides after
chronic treatment is less likely to trigger a withdrawal response.
The putative promoter DNA sequence of the human MOR contains various
consensus sequences, including cAMP response elements
(Min et al., 1994). Therefore,
the transcription of MOR is expected to be under the influence by the
intracellular levels of cAMP and the signal transduction pathways mediated by
cAMP. Enhancing the activity of cAMP-dependent protein kinase pathways has
been shown to blunt MOR responsiveness in the animal studies
(Maldonado et al., 1996; Shen
et al., 2000). Activation of the cAMP-dependent protein kinase with forskolin
in the presence of phosphodiesterase inhibitor has been shown to be associated
with a decrease in the levels of MOR mRNA in SHSY-5Y cells. This suggests that
cAMP has a functional role in regulating expression of MOR gene transcripts
(Wallington et al., 2002).
After chronic treatment with different MOR agonists, forskolin-induced
activity of adenylate cyclase seemed to be inversely correlated with
expression of MOR mRNA in the undifferentiated SHSY-5Y cells. Our results
confirm previous studies where intracellular cAMP levels are associated with
MOR gene regulation via camp-dependent signal transduction mechanisms.
In summary, our studies indicate that morphine and endomorphins differentially regulated MOR mRNA and function in SHSY-5Y cells. Alkaloid opiates, such as morphine, and peptide opioids, such as endomorphins, may mediate distinct molecular events that are involved in the regulation of the MOR. To delineate these events, further investigation is needed in differential activation of various isoforms of adenyl cyclases in SHSY-5Y cells, especially after chronic treatment with these agonists.
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Acknowledgements |
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Footnotes |
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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.
Address correspondence to: Dr. Sulie L. Chang, Department of Biology, Seton Hall University, 400 South Orange Ave., South Orange, NJ 07079. E-mail: changsul{at}shu.edu
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