0022-3565/98/2842-0611$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
JPET 284:611–617, 1998
Vol. 284, No. 2
Printed in U.S.A.
Tyr-W-MIF-1 Attenuates Down-Regulation of Opiate Receptors
in SH-SY5Y Human Neuroblastoma Cells1
LAURA M. HARRISON, ABBA J. KASTIN and JAMES E. ZADINA
Tulane University School of Medicine and Veterans Affairs Medical Center, New Orleans, Louisiana
Accepted for publication October 27, 1997
This paper is available online at http://www.jpet.org
Differences between in vivo and in vitro preparations in
studies of receptor down-regulation may help explain some
mechanisms of opiate tolerance. Down-regulation has been
easier to demonstrate in vitro than in vivo (Zadina et al.,
1995). For example, delta agonists down-regulate receptors
in cell lines containing only delta receptors (Chang et al.,
1982; Law et al., 1982, 1983), and morphine and mu agonists
down-regulate receptors in cell lines containing mu receptors
(Werling et al., 1989). Down-regulation of both mu and delta
receptors has been demonstrated in the human neuroblastoma cell lines SH-SY5Y and SK-N-SH in response to chronic
morphine and selective agonists (Carter and Medzhiradsky,
1993; Baumhaker et al., 1993; Zadina et al., 1990, 1993a,
1994a).
Early in vivo studies showed no change in receptor number
after exposure to chronic agonists (Klee and Streaty, 1974;
Hollt et al., 1975). More recent studies have shown downregulation in response to chronic etorphine (Tao et al., 1987),
PL017 (Tao et al., 1990) and DPDPE (Tao et al., 1991). A
Received for publication July 21, 1997.
1
This work was supported by the VA and National Institute on Drug Abuse
Predoctoral Training Grant DA05645 to L.M.H. Preliminary reports of these
results were presented at the International Narcotics Research Conference in
Long Beach, CA in July 1996, and at the Society for Neuroscience Meeting,
Washington, DC, November 1996.
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ABSTRACT
Down-regulation of opiate receptors is demonstrated more
easily in vitro than in vivo. The possible role of endogenous
opiate-modulating peptides in preventing such down-regulation was investigated by addition of Tyr-W-MIF-1 to an in vitro
preparation, the human neuroblastoma cell line SH-SY5Y, in
which down-regulation of opiate receptors has been demonstrated previously. Although both morphine and Met-enkephalin down-regulated mu and delta receptors after chronic (24 h)
exposure in serum-free medium, Tyr-W-MIF-1, at doses of up
to 100 mM, did not affect receptor number when administered
alone. This lack of effect could not be attributed to degradation
of the peptide during chronic treatment because high-performance liquid chromatography showed that 79% of the peptide
remained intact after a 24-h incubation. When coadministered
with 3 mM morphine, Tyr-W-MIF-1 dose-dependently attenuated morphine-induced down-regulation of both mu and delta
receptors. Down-regulation of mu receptors by the selective
agonist PL017 was also attenuated by Tyr-W-MIF-1, but downregulation of delta receptors by the selective agonist DPDPE
was not. These studies indicate that endogenous opiate modulators may play a role in opiate tolerance at the level of
receptor down-regulation.
major difference exists, however, between in vitro and in vivo
studies, because down-regulation in response to morphine
has been observed only rarely in vivo (Bhargava and Gulati,
1990). Indeed, under the same conditions in which etorphine
down-regulates receptors, morphine increases receptor number (Tao et al., 1987). The only preparation in which downregulation in response to morphine is observed consistently
in vivo is the prenatal or early neonatal rat (Tempel et al.,
1988).
These studies highlight two of the important features of
opiate receptor down-regulation: a) in vitro preparations,
which exhibit down-regulation to many different agonists,
may lack one or more components of the adaptation response
to chronic drug exposure that is present in mature animals;
b) the manner in which cells adapt to chronic morphine
seems to depend on the characteristics of the specific cells
studied. Different brain regions and cell lines, for example,
may have different complements of G-proteins or efficiencies
of coupling to intracellular signaling pathways.
One possibility contributing to the tolerance response and
to the lack of receptor down-regulation in mature animals is
the presence of an endogenous “opiate-modulating system,”
such as the Tyr-MIF-1 family of peptides. Tyr-MIF-1 (TyrPro-Leu-Gly-NH2) and Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2)
have been isolated from human and bovine brain (Hackler et
ABBREVIATIONS: DAMGO, Tyr-D-Ala-Gly-N-MePhe-Gly-ol; CTAP, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2; ICI 174,864, N,N-diallyl-Tyr-AibAib-Phe-Leu-OH([N,N-diallyl-Tyr1,Aib2,3]Leu-enkephalin); DPDPE, Tyr-D-Pen-Gly-Phe-Pen ([D-Pen2,D-Pen5]-enkephalin); PL017, Tyr-Pro-NMePhe-D-Pro-NH2; EDTA, ethylenediaminetetraacetic acid; MS, morphine sulfate; RA, retinoic acid; HPLC, high-performance liquid chromatography; EF, Eagle’s Minimum Essential Medium 1 F12; EFN, EF 1 N2 supplement; EFF, EF 1 fetal bovine serum.
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Vol. 284
Methods
Materials. All trans-retinoic acid was obtained from Sigma
Chemical Co. (St. Louis, MO), [3H]DAMGO (60 Ci/mmol) from Amersham (Arlington Heights, IL), [3H]pCl-DPDPE (40 Ci/mmol) from
Dupont New England Nuclear (Wilmington, DE) and fetal bovine
serum from Intergen (Purchase, NY). Tyr-W-MIF-1 was synthesized
in our laboratory by solution-phase methods. PL017 and DPDPE
were obtained from Multiple Peptide Systems through the National
Institute on Drug Abuse.
Cell culture conditions. SH-SY5Y human neuroblastoma cells
were cultured, harvested and prepared for binding assays as described previously (Zadina et al., 1993a, 1994a). Cells of passage
24 –31 were cultured in a medium (EFF) containing a 1:1 ratio of
Eagle’s Minimum Essential Medium and F12 (Gibco no. 41500 – 034
and no. 21700 – 075; Grand Island, NY) with 10% fetal bovine serum,
100 mg/ml streptomycin and 100 IU/ml penicillin (Gibco). They were
grown at 37°C in a humidified atmosphere containing 5% CO2. At
about 90% confluence, cells were differentiated into a neuronal phenotype with RA (10 mM). RA was administered, with the media
change, every 2 days for the last 6 days of culture, for a total of three
treatments. To avoid metabolism of administered peptides, cells
were switched to a serum-free medium (EFN), with the medium
supplement N2 [containing insulin (bovine, 5 mg/ml), transferrin
(human, 100 mg/ml), progesterone (20 nM), putrescine (100 mM), and
Na selenite (30 nM)], with the last RA treatment. Drugs were administered for the last 24 h of culture.
Plasma membrane preparation. Cells were rinsed three times
with serum-free medium (EF) and then dissociated with Ca11/
Mg11-free phosphate-buffered saline containing 0.04% EDTA. After
centrifugation (183 3 g for 7 min at 4°C) for removal of the phosphate-buffered saline and EDTA, cells were reconstituted in 50 mM
TRIS, vortexed and homogenized with a polytron at setting 6 (23,000
rpm) for 20 sec. The homogenate was centrifuged at 30,000 3 g for 20
min, and the pellet was incubated for 1 h at room temperature in 50
mM TRIS/100 mM NaCl to remove endogenous ligands. After another centrifugation at 30,000 3 g for 20 min, the pellet was reconstituted in STEM (0.25 M sucrose, 5 mM TRIS, 0.5 mM EDTA and 1
mM MgSO4), and protein samples were taken. The sample was
divided into equal aliquots and, after a final centrifugation, resuspended in 2 ml STEM for freezing. Protein was measured by the
method of Lowry et al. (1951) with bovine serum albumin as
standard.
Opiate receptor assays. Saturation binding assays were performed in 50 mM TRIS with 0.1% bovine serum albumin at a final
volume of 600 ml. For mu receptor assays, aliquots (200 ml, containing 100 mg protein) of membranes were incubated for 90 min at 23°C
with varying concentrations (0.08 –5 nM) of [3H]DAMGO. Nonspecific binding was measured in the presence of 1 mM DAMGO. For
delta receptor assays, membranes were incubated for 4 h at 23°C in
the presence of varying concentrations (0.08 –5 nM) of [3H]pClDPDPE, with nonspecific binding measured in the presence of 10 mM
naltrexone. Bound and free ligands were separated by filtration.
Specific binding at low concentrations of isotope were 70 to 85% for
[3H]DAMGO and 50 to 65% for [3H]pCl-DPDPE.
HPLC. Tyr-W-MIF-1 was iodinated by the chloramine T method,
and the monoiodinated fraction was isolated by HPLC. The specific
activity (2100 Ci/mmol) was diluted with nonradioactive Tyr-WMIF-1 to a final concentration of 200,000 cpm/30 mM Tyr-W-MIF-1 in
7.5 ml EFN/10 mM RA. SH-SY5Y cells were incubated at 37°C in this
medium in T-25 flasks for 1 min or 2, 5 or 24 h. At the end of the
incubation, 3.5 ml of medium was transferred to 3.5 ml ice-cold
ethanol for 30 min, centrifuged at 3500 rpm for 30 min and the
supernatant dried down (Speed-Vac, Savant Instruments, Hicksville, NY). The extract was reconstituted in 400 ml of 0.1% trifluoroacetic acid/water (HPLC buffer A) and fractionated by HPLC on a
Vydac C18 no. 201HS54 (46 3 250 mm) column. The gradient for
buffer B (methanol in 0.1% trifluoroacetic acid) increased from 20 to
45% during 20 min, then remained at 45% for 30 min, increased to
65% in 0.5 min, remained at 65% for 20 min, then increased to 80%.
Retention times of synthetic standards for Tyr-W-MIF-1 and its
fragments were determined with the same column and gradient.
Statistical analyses. Values (mean 6 S.E.M.) are represented as
percent of control to normalize differences in Bmax from one assay to
the next. Percent attenuation was calculated as follows: [(% decrease
in Bmax induced by 3 mM MS) 2 (% decrease in Bmax induced by 3 mM
MS 1 Tyr-W-MIF-1)]/% decrease in Bmax induced by 3 mM MS.
Multiple experiments were analyzed by analysis of variance followed
by Duncan’s range test.
Results
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al., 1993; Erchegyi et al., 1992; Horvath and Kastin, 1989,
1990), and they both bind selectively to the mu receptor (Ki 5
1 mM for Tyr-MIF-1 and Ki 5 70 nM for Tyr-W-MIF-1)
(Zadina et al., 1994c). They exhibit opiate agonist and antagonist properties in the guinea pig ileum, with antagonism
most easily observed in tolerant tissue (Erchegyi et al., 1992,
1993; Zadina et al., 1992). Tyr-MIF-1 has a significantly
longer half-life in neonatal versus adult plasma, a finding
that suggests the possible postnatal appearance of its degradative enzymes (Kastin et al., 1994).
In this study, we used an in vitro preparation, the human
neuroblastoma cell line SH-SY5Y, in which opiate receptor
down-regulation by morphine and selective agonists has already been demonstrated (Zadina et al., 1990, 1993a, 1994a),
to test whether Tyr-W-MIF-1 can block this agonist-induced
decrease in receptor number. We chose Tyr-W-MIF-1 instead
of Tyr-MIF-1 because of its higher affinity for the mu receptor. Serum-free medium was used to avoid degradation of the
peptide, and therefore we characterized the suitability of this
culture condition for the study of receptor regulation in SHSY5Y cells.
Suitability of serum-free culture conditions for
study of receptor regulation. To study the role of a peptide in receptor regulation, it was necessary to culture the
cells in a serum-free medium to avoid degradation of the
peptide. However, because the presence of serum was required for initial seeding and growth of the cells, they were
cultured in a serum-containing medium (EFF) until 48 h
before harvest. At this point, they were transferred to a
medium (EFN) containing the supplement N2 and were allowed to adjust to these new conditions for 24 h before drug
treatment. The switch to serum-free conditions caused a
significant change in the mu:delta ratio from 1.9:1 in EFF to
1.3:1 in EFN [F(1,12) 5 7.90, P , .05]. The serum may
contain opiate-like substances, as demonstrated in both binding and guinea pig ileum assays (Zadina et al., 1994a). Therefore, the removal of the serum, rather than the addition of
N2, would appear to be the more likely explanation for the
change in receptors.
The stability of Tyr-W-MIF-1 in the serum-free medium
was tested directly by incubation of a radiolabeled form of the
peptide with the cultured cells for various time points; samples of the medium were tested by HPLC. The percent of
intact Tyr-W-MIF-1 remaining after 2, 5 and 24 h of incubation was 95, 92.1 and 79.2, which indicates that the peptide
was quite stable in the EFN medium for the duration of the
experiments described in this study. Figure 1 shows the
1998
HPLC profile from the 24-h sample (solid line) compared
with that exposed for only 1 min (dashed lines). There was a
small peak at the position of free Tyr, which indicates some
cleavage of this N-terminal residue.
Because Tyr-W-MIF-1 remained mostly intact in our culture conditions, we next tested whether the peptide affected
mu or delta receptor number when administered alone for
24 h. The peptide had no effect on mu or delta receptor
number at a dose of 30 mM (data not shown) or 100 mM (fig.
2). This lack of effect could not be attributed to degradation of
the peptide or to an inability of agonists to down-regulate
receptors in serum-free medium. As shown in figure 2, under
the same experimental conditions, the peptide Met-enkephalin at a dose of 10 mM down-regulated both mu (67 6 1% of
control) and delta (31 6 5% of control) receptors. Thus,
chronic Tyr-W-MIF-1 does not down-regulate receptors under conditions in which a known peptide agonist is capable of
down-regulating receptors.
Fig. 2. Mu receptor and delta receptor number expressed as percent of
control Bmax after 24 h incubation with Tyr-W-MIF-1 or Met-enkephalin
in serum-free medium. Neither mu nor delta receptor number was decreased by Tyr-W-MIF-1 under the same conditions in which Met-enkephalin (Met-enk) reduced mu receptors to 67 6 1% of control and delta
receptors to 31 6 5% of control (n 5 2).
613
In addition to Met-enkephalin, chronic morphine also
down-regulates receptors under conditions in which Tyr-WMIF-1 does not change receptor number. Down-regulation of
mu and delta receptors was dose-dependent, with 1, 3 and 10
mM doses of morphine reducing the Bmax of mu receptors to
73 6 7, 57 6 11 and 52 6 6% of control and the Bmax of delta
receptors to 99 6 2, 60 6 4 and 46 6 12% of control (data not
shown). This agrees with our earlier finding of dose-responsive down-regulation of opiate receptors in EFF medium
(Zadina et al., 1993a).
The ability of an antagonist to up-regulate receptors in
serum-free medium was also tested. Naloxone previously has
been shown to up-regulate mu and delta receptors after
chronic treatment in SH-SY5Y cells (Zadina et al., 1993a,
1994a). Here we compared the ability of naloxone to upregulate receptors in EFF versus EFN. Figure 3 shows that
naloxone retains its ability to up-regulate receptors in serumfree medium. Mu and delta receptors were up-regulated
about equally in the two culture conditions (24 6 5% in EFF
vs. 28 6 11% in EFN for mu and 31 6 9% in EFF vs. 43 6 19%
in EFN for delta). This finding confirms our earlier preliminary results (Zadina et al., 1994a) with a supplement (B27,
Gibco) that is more complex than the N2 supplement used
here. Together, these results argue that mechanisms other
than, or in addition to, displacement of opiates in the serum
account for the ability of naloxone to up-regulate receptors.
Attenuation of morphine-induced down-regulation
of both mu and delta receptors by Tyr-W-MIF-1. Of the
doses of morphine tested for down-regulation of opiate receptors in EFN (1, 3 and 10 mM), the intermediate dose was
chosen for the interaction studies because it afforded enough
down-regulation to detect enhanced or attenuated down-regulation but avoided the possibility that a supramaximal dose
of morphine could prevent detection of attenuation. The dose
of Tyr-W-MIF-1 was varied (10, 30, 100 mM) so that it represented 3 times, 10 times or 33 times the dose of morphine,
and the two drugs were administered to the cells simultaneously for 24 h. Down-regulation of mu receptors by morphine was attenuated by Tyr-W-MIF-1 in a dose-dependent
manner. Figure 4 shows the percent attenuation of mor-
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Fig. 1. HPLC chromatogram of 125I-Tyr-W-MIF-1 after 1 min (dashed
line) and 24 h (solid line). At 24 h, 79% of the cpm eluted at the position
of the intact peptide.
Tyr-W-MIF-1 and Down-regulation
Fig. 3. Up-regulation of mu and delta receptors in serum-containing
medium (EFF) and serum-free medium (EFN), expressed as percent
increase in Bmax, after 24 h incubation with 10 mM naloxone (Nlx). Values
are means 6 S.E.M. for three to five experiments.
614
Harrison et al.
phine-induced down-regulation of mu receptors by increasing
doses of Tyr-W-MIF-1. The 10 mM dose of Tyr-W-MIF-1 had
no effect, but down-regulation was significantly attenuated
by 30 mM (28 6 9%, P , .05) and 100 mM (49 6 8%, P , .01)
doses of Tyr-W-MIF-1.
In addition to its effect on mu receptor down-regulation,
Tyr-W-MIF-1 also attenuated morphine-induced down-regulation of delta receptors, as shown in figure 5. The attenuation (54 6 9%) of morphine-induced down-regulation by 30
mM Tyr-W-MIF-1 was significant (P , .05), but the higher
dose (100 mM) was less effective (39 6 8%, P . .1).
Effects of Tyr-W-MIF-1 on selective agonist-induced
down-regulation of opiate receptors. In addition to morphine, selective agonists also have been shown to down-
Fig. 5. Attenuation of delta receptor down-regulation by Tyr-W-MIF-1.
The main figure illustrates percent attenuation with increasing doses of
Tyr-W-MIF-1 (*P , .05 compared with control cells given morphine but
no Tyr-W-MIF-1). Inset shows the results expressed as percent of control
Bmax. Coincubation with 30 mM and 100 mM (1 W30 and 1 W100) but not
10 mM (1 W10) Tyr-W-MIF-1 resulted in smaller decreases from control
Bmax. Values are mean 6 S.E.M. for two to three experiments.
regulate opiate receptors in SH-SY5Y cells (Zadina et al.,
1994a). We therefore tested the ability of Tyr-W-MIF-1 to
attenuate this down-regulation by selective agonists. Figure
6 shows the dose-responsive attenuation by Tyr-W-MIF-1 of
mu receptor down-regulation induced by PL017, a mu selective agonist. PL017 alone (1 mM) decreased mu receptor Bmax
by 52%, in agreement with our earlier work (Zadina et al.,
1994a). Tyr-W-MIF-1 attenuated this effect at 30 mM (29 6
1%) and 100 mM (68 6 2%) doses. This pattern parallels that
seen with morphine-induced down-regulation of mu receptors.
The delta selective agonist DPDPE (5 nM) caused a large
(80%) reduction in delta receptors, in agreement with our
previous findings (Zadina et al., 1994a). However, Tyr-WMIF-1 caused only a small attenuation of the down-regulation, and this effect was not dose-responsive (18, 20 and 17%
attenuation with 10, 30 and 100 mM Tyr-W-MIF-1, respectively; data not shown). Thus, the effect of Tyr-W-MIF-1 on
selective agonist-induced down-regulation of delta receptors
was different from its effect on morphine-induced down-regulation of delta receptors.
Discussion
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Fig. 4. Attenuation of mu receptor down-regulation by Tyr-W-MIF-1.
The main figure illustrates increasing percent attenuation with increasing doses of Tyr-W-MIF-1 (*P , .05; **P , .01 compared with control cells
given morphine but no Tyr-W-MIF-1). Inset shows the results expressed
as percent of control Bmax. Coincubation with increasing doses of Tyr-WMIF-1 (1 WI0, 1 W30 and 1 W100) resulted in smaller decreases from
control Bmax. Values are mean 6 S.E.M. for two to three experiments.
Vol. 284
In this study, we established the suitability of serum-free
culture conditions for the study of opiate receptor regulation
and used this preparation to examine the effects of the opiate-modulating peptide Tyr-W-MIF-1 on agonist-induced decreases in receptor number. Tyr-W-MIF-1 remained largely
intact during 24 h of culture in serum-free medium, but did
not affect receptor number when administered alone. However, Tyr-W-MIF-1 dose-dependently attenuated morphineinduced down-regulation of both mu and delta receptors. The
effect of Tyr-W-MIF-1 on selective agonist-induced downregulation revealed differences between mu and delta receptors; the PL017-induced down-regulation of mu receptors
was attenuated by Tyr-W-MIF-1, but the DPDPE-induced
down-regulation of delta receptors was not. These studies
represent the first demonstration of attenuation by an endogenous ligand of morphine-induced changes in receptor
Fig. 6. Attenuation by Tyr-W-MIF-1 of PL017-induced down-regulation
of mu receptors. Results are expressed as percent attenuation (mean 6
S.E.M. for two separate experiments) with increasing doses of Tyr-WMIF-1.
1998
615
model of tolerance and dependence, such peptides counteract
the actions of administered opiates. However, the mechanism
of this counteraction is unknown and has not been studied at
the cellular level. The use of a preparation in which morphine-induced down-regulation has been demonstrated (SHSY5Y cells) and of a peptide (Tyr-W-MIF-1) which binds to
the mu opiate receptor affords a unique opportunity to study
this endogenous peptide’s ability to counteract the actions of
morphine at the cellular and receptor level during chronic
administration.
Tyr-W-MIF-1 attenuated morphine-induced down-regulation of the mu receptor in a dose-dependent manner, but it
did not affect receptor number when administered alone.
These results suggest that it may be working as a partial
agonist. Although it binds to the mu receptor, the affinity of
Tyr-W-MIF-1 (70 nM) is far less than the affinity of morphine
for this receptor (Zadina et al., 1994c). However, at the two
higher doses used here, when it is present in a 10-fold or
33-fold excess over morphine, it may be able to compete with
morphine for binding to the receptor. If Tyr-W-MIF-1 has
lower efficacy at this receptor, it could prevent morphine’s
agonist activity without having appreciable activity of its
own. Thus, by acting as a partial agonist, it could attenuate
morphine’s actions at the mu receptor and the subsequent
down-regulation of this site. This would be in agreement with
studies involving the guinea pig ileum, in which Tyr-MIF-1
and Tyr-W-MIF-1 increase their antagonist activity when the
receptor reserve is low, such as in tolerant tissue (Erchegyi et
al., 1992,1993; Zadina et al., 1992). Met-enkephalin has been
shown to negatively modulate morphine analgesia, but this
action is blocked by the delta antagonist ICI 174864 and thus
does not result from partial agonism at the mu receptor
(Jiang et al., 1990). The partial agonism by an endogenous
ligand suggested by the present studies therefore is an unusual phenomenon at the opiate receptor.
The attenuation of morphine-induced down-regulation of
the delta receptor was unexpected and suggests that the
mechanism of action of Tyr-W-MIF-1 at this receptor may not
be simple partial agonism. The affinity of Tyr-W-MIF-1 for
the delta receptor (15 mM) is 200-fold lower than its affinity
for the mu receptor (Zadina et al., 1994c). Even in a 10- and
33-fold excess over morphine, it seems unlikely that the
peptide could compete for binding at the delta receptor. It has
been suggested that down-regulation of the delta receptor by
morphine in SK-N-SH cells occurs through the mu receptor
(Baumhaker et al., 1993), which might account for the ability
of a mu-acting peptide to attenuate delta down-regulation.
However, we have shown previously that in SH-SY5Y cells
morphine down-regulates delta receptors even when the mu
receptor is blocked by the selective antagonist CTAP (Zadina
et al., 1994a). Thus, attenuation of delta receptor down-regulation in this preparation seems unlikely to occur through
the mu receptor.
Alternatively, Tyr-W-MIF-1 could be acting through a nonopiate, high-affinity binding site which it shares with TyrMIF-1 and which is present in SH-SY5Y cells (Zadina et al.,
1990, 1993a). However, Tyr-MIF-1 inhibits cAMP in SHSY5Y cells in a naloxone-reversible manner, which suggests
activity at opiate receptors rather than at this nonopiate site
(Zadina et al., 1991). Tests of other signaling pathways and
development of antagonists for this site will be necessary to
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number and suggest a mechanism whereby endogenous antiopiates can counteract the effects of chronic morphine.
Several peptides have been proposed to play a role in
opiate tolerance and dependence by acting as “antiopiates.”
According to the proposed model, administration of exogenous opiates, such as morphine, causes the release of endogenous antiopiate peptides. These peptides counteract the effects of morphine, thereby causing a need for increased doses
of the drug to achieve a given effect (tolerance). When the
drug is removed, the continued presence of the antiopiate
peptide in the absence of opiate drug causes the symptoms of
withdrawal (Rothman, 1992; Zadina et al., 1994b). When we
introduced the antiopiate concept with MIF-1 (Pro-Leu-GlyNH2) in 1979, we predicted that other types of antiopiates
would be found (Kastin et al., 1979). Among the proposed
antiopiates is neuropeptide FF, whose actions in opiate tolerance and dependence have been relatively well characterized. The level of this peptide in the cerebrospinal fluid is
increased after chronic morphine (Malin et al., 1990b), and it
can attenuate morphine antinociception (Yang et al., 1985)
and precipitate withdrawal (Malin et al., 1990a). Antibodies
to this peptide can attenuate naloxone-induced withdrawal
(Malin et al., 1990b) and reverse morphine tolerance (Lake et
al., 1991). It does not, however, bind to opiate receptors and
therefore must exert its antiopiate actions through a homeostatic process (Allard et al., 1989; Raffa et al., 1994).
The Tyr-MIF-1 peptides display antiopiate activity and can
be described as “opiate modulating,” because the peptides
exhibit both agonist and antagonist properties, depending on
the preparation. As an example of an opiate agonist property,
Tyr-W-MIF-1 induces naloxone-reversible analgesia after either intracerebroventricular or intrathecal routes of administration (Gergen et al., 1996a; Zadina et al., 1993b). Also, in
SH-SY5Y cells, Tyr-MIF-1 inhibits cAMP in a naloxone-reversible manner (Zadina et al., 1991). Examples of antagonist
properties are the ability of Tyr-MIF-1 to precipitate withdrawal (Malin et al., 1993) and to antagonize stress-induced
(Galina and Kastin, 1987) as well as morphine-induced analgesia (Kastin et al., 1984).
The opiate modulating nature of the peptides is best demonstrated within a single preparation in which the peptides
can exhibit either agonism or antagonism, depending on the
conditions of testing. This was first demonstrated with the
guinea pig ileum. In ilea from naive animals, the peptides
inhibit electrically induced contractions, but in ilea from
animals made tolerant to morphine or with “receptor reserve” reduced by alkylating agents, they antagonize opiateinduced contractions (Erchegyi et al., 1992, 1993; Zadina et
al., 1992). Tyr-W-MIF-1 can bind to both mu1 and mu2 receptors (Zadina et al., 1996) and can induce analgesia in the
mouse through a mu2 mechanism but antagonize mu1 analgesia induced by morphine and DAMGO (Gergen et al.,
1996b).
A key characteristic of Tyr-MIF-1 peptides is their ability
to bind to opiate receptors. For an antiopiate to account for
the high degree of opiate tolerance that can be attained, it
must be able to bind to the opiate receptor (Smith et al.,
1988). Tyr-MIF-1 peptides are the only proposed antiopiates
(excluding forms of opioids themselves) that have the ability
to bind to opiate receptors. Both Tyr-MIF-1 and Tyr-WMIF-1 bind selectively to mu over delta and kappa receptors
(Zadina et al., 1994c). According to the antiopiate peptide
Tyr-W-MIF-1 and Down-regulation
616
Harrison et al.
Acknowledgments
The authors thank Dr. Laszlo Hackler for synthesis of Tyr-WMIF-1 and NIDA for the PL017 and DPDPE.
References
Akiyama K, Gee KW, Mosberg HI, Hruby VJ and Yamamura HI (1985) Characterization of [3H][2-D-penicillamine, 5-D-penicillamine]-enkephalin binding to delta
opiate receptors in the rat brain and neuroblastoma-glioma hybrid cell line (NG
108 –15). Proc Natl Acad Sci USA 82:2543–2547.
Allard S, Geoffre S, Legendre P, Vincent JD and Simonnet G (1989) Characterization
of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study. Brain Res 500:169 –176.
Arden JR, Segredo V, Wang Z, Lameh J and Sadee W (1995) Phosphorylation and
agonist-specific intracellular trafficking of an epitope-tagged mu-opioid receptor
expressed in HEK 293 cells. J Neurochem 65:1636 –1645.
Baumhaker Y, Gafni M, Keren O and Sarne Y (1993) Selective and interactive
down-regulation of mu- and delta-opioid receptors in human neuroblastoma SKN-SH cells. Mol Pharmacol 44:461– 467.
Bhargava H and Gulati A (1990) Down-regulation of brain and spinal cord mu-opiate
receptors in morphine tolerant-dependent rats. Eur J Pharmacol 190:305–311.
Carter BD and Medzhiradsky F (1993) Receptor mechanisms of opioid tolerance in
SH-SY5Y human neural cells. Mol Pharmacol 43:465– 473.
Chang K-J, Eckel RW and Blanchard SG (1982) Opioid peptides induce reduction of
enkephalin receptors in cultured neuroblastoma cells. Nature 296:446 – 448.
Erchegyi J, Kastin AJ and Zadina JE (1992) Isolation of a novel tetrapeptide with
opiate and antiopiate activity from human cortex: Tyr-Pro-Trp-Gly-NH2 (Tyr-WMIF-1). Peptides 13:623– 631.
Erchegyi J, Zadina JE, Qui X-D, Kersh D, Ge L-J, Brown MM and Kastin AJ (1993)
Structure-activity relationships of analogs of the endogenous brain peptides TyrMIF-1 and Tyr-W-MIF-1. Pept Res 6:31–37.
Galina ZH and Kastin AJ (1987) Tyr-MIF-1 attenuates antinociceptive responses
induced by three models of stress-analgesia. Br J Pharmacol 90:669 – 674.
Gergen KA, Zadina JE, Kastin AJ and Paul D (1996a) Intrathecal Tyr-W-MIF-1
produces potent, naloxone-reversible analgesia modulated by alpha2-adrenoceptors. Eur J Pharmacol 298:235–239.
Gergen KA, Zadina JE and Paul D (1996b) Analgesic effects of Tyr-W-MIF-1: a mixed
mu2-opioid receptor agonist/mu1-opioid receptor antagonist. Eur J Pharmacol
316:33–38.
Hackler L, Kastin AJ, Erchegyi J and Zadina JE (1993) Isolation of Tyr-W-MIF-1
from bovine hypothalami. Neuropeptides 24:159 –164.
Heyman JS, Vaught JL, Mosberg HI, Haaseth RC and Porreca F (1989) Modulation
of mu-mediated antinociception by delta agonists in the mouse: selective potentiation of morphine and normorphine by [D-Pen2,D-Pen5]enkephalin. Eur J Pharmacol 165:1–10.
Hollt V, Dum J, Blasig J, Schubert P and Herz A (1975) Comparison of in vivo and
in vitro parameters of receptor binding in naive and tolerant/dependent rats. Life
Sci 16:1823–1828.
Horvath A and Kastin AJ (1989) Isolation of tyrosine-melanocyte-stimulating hormone release-inhibiting factor-1 from bovine brain tissue. J Biol Chem 264:2175–
2179.
Horvath A and Kastin AJ (1990) Evidence for the presence of Tyr-MIF-1 (Tyr-ProLeu-Gly-NH2) in human brain cortex. Int J Pept Protein Res 36:281–284.
Jiang Q, Mosberg HI and Porreca F (1990) Modulation of the potency and efficacy of
mu-mediated antinociception by delta agonists in the mouse. J Pharmacol Exp
Ther 254:683– 689.
Kastin AJ, Olson RD, Ehrensing RH, Berzas MC, Schally AV and Coy DH (1979)
MIF-1’s differential actions as an opiate antagonist. Pharmacol Biochem Behav
11:721–723.
Kastin AJ, Stephens E, Ehrensing RH and Fischman AJ (1984) Tyr-MIF-1 acts as an
opiate antagonist in the tail-flick test. Pharmacol Biochem Behav 21:937–941.
Kastin AJ, Hahn K, Banks WA and Zadina J (1994) Delayed degradation of TyrMIF-1 in neonatal rat plasma. Peptides 8:1561–1563.
Keith DE, Murray SR, Zaki PA, Chu PC, Lissin DV, Kang L, Evans CJ and Von
Zastrow M (1996) Morphine activates opioid receptors without causing their rapid
internalization. J Biol Chem 271:19021–19024.
Klee WA and Streaty RA (1974) Narcotic receptor sites in morphine-dependent rats.
Nature 248:61– 63.
Lake JR, Hammond MV, Shaddox RC, Hunsicker LM, Yang HY-T and Malin D
(1991) IgG from neuropeptide FF antiserum reverses morphine tolerance in the
rat. Neurosci Lett 132:29 –32.
Law PY, Hom DS and Loh HH (1982) Loss of receptor activity in neuroblastoma x
glioma NG108 –15 hybrid cells after chronic opiate treatment: a multiple step
process. Mol Pharmacol 22:1– 4.
Law PY, Hom DS and Loh HH (1983) Opiate receptor down-regulation and desensitization in neuroblastoma x glioma NG108 –15 hybrid cells are two separate
cellular adaptation processes. Mol Pharmacol 24:413– 424.
Lowry OH, Rosebrough NJ, Farr AL and Randall RJ (1951) Protein measurement
with the Folin phenol reagent. J Biol Chem 193:265–275.
Malin DH, Lake JR, Fowler DE, Hammond MV, Brown SL, Leyva JE, Prasco PE and
Dougherty TM (1990a) FMRF-NH2-like mammalian peptide precipitates opiatewithdrawal syndrome in the rat. Peptides 11:277–280.
Malin DH, Lake RJ, Hammond MV, Fowler DE, Rogillio RB, Brown SL, Sims JL,
Leecraft BM and Yang HY-T (1990b) FMRF-NH2-like mammalian octapeptide:
Possible role in opiate dependence and abstinence. Peptides 11:969 –972.
Malin DR, Zadina JE, Lake JR, Rogillio RB, Leyva JE, Benson TM, Corriere LS,
Handunge BP and Kastin AJ (1993) Tyr-MIF-1 precipitates abstinence syndrome
in morphine-dependent rats. Brain Res 610:169 –171.
Raffa RB, Kim A, Rice KC, Decosta BR, Codd EE and Rothman RB (1994) Low
affinity of FMRFamide and four FaRPs (FMRFamide-related peptides), including
the mammalian-derived FaRPs F-8-Famide (NPFF) and A-18-Famide, for opioid
mu, delta, kappa1, kappa2a, or kappa2b receptors. Peptides 15:401– 404.
Rothman RB (1992) A review of the role of anti-opioid peptides in morphine tolerance
and dependence. Synapse 12:129 –138.
Smith AP, Law PY and Loh HH (1988) Role of opioid receptors in narcotic dependence/tolerance, in The Opiate Receptors (Pasternak G, ed) pp 441– 485, Humana
Press, Clifton, NJ.
Tao P-L, Law P-Y and Loh HH (1987) Decrease in delta and mu opioid receptor
binding capacity in rat brain after chronic etorphine treatment. J Pharmacol Exp
Ther 240:809 – 816.
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define its role, if any, in the modulation of down-regulation
observed in the present studies.
The effects of Tyr-W-MIF-1 on selective agonist-induced
down-regulation reveal differences between mu and delta
receptors, as well as between morphine and other ligands.
The PL017-induced down-regulation of mu receptors was
attenuated by Tyr-W-MIF-1, but the DPDPE-induced downregulation of delta receptors was not. The lack of attenuation
at delta receptors could be caused by the high affinity of
DPDPE for these receptors (Akiyama et al., 1985) or by its
high efficacy in down-regulating delta receptors. We have
shown that in SH-SY5Y cells DPDPE down-regulates delta
receptors with an IC50 of 0.5 nM and a maximal effect of
about 80% reduction of Bmax, compared with an IC50 of 179
nM and maximal effect of 62% reduction for PL017 (Zadina et
al., 1994a). It could be argued that the inability of Tyr-WMIF-1 to attenuate DPDPE-induced down-regulation of delta
receptors is caused by the high degree of down-regulation
achieved by the dose (5 nM) used here. However, this dose is
just enough to produce maximal down-regulation, whereas
the dose of PL017 chosen (1 mM) is well above that required
to achieve maximal down-regulation of mu receptors (Zadina
et al., 1994a).
The ability to modulate actions of morphine but not of
other ligands, including peptides, has been demonstrated
previously for the enkephalin analog DPDPE (Heyman et al.,
1989). Such findings, as well as those presented here, suggest
differences in the ways cells respond to morphine as compared with other opiate ligands. As mentioned, in vivo studies have demonstrated down-regulation of opiate receptors in
response to many ligands, but rarely to morphine. More
recently, it has been demonstrated that cloned mu and delta
receptors expressed in HEK 293 cells internalize in response
to enkephalins and etorphine but not in response to morphine under the same conditions (Keith et al., 1996; Arden et
al., 1995). Thus, morphine may induce unique changes in a
cell’s responsiveness, and the type of cell and presence of
modulators can affect the responsiveness.
In summary, we have demonstrated that under appropriate culture conditions an endogenous peptide can attenuate
morphine-induced down-regulation. In addition, these findings demonstrate another example of different responses of
cells to morphine and other opiate ligands, illustrated by the
failure of Tyr-W-MIF-1 to attenuate DPDPE-induced downregulation even though it can attenuate morphine-induced
down-regulation of delta receptors. The in vitro condition,
with the addition of this endogenous peptide, may help clarify the in vivo condition, in which significant down-regulation
in response to morphine is not easily demonstrated. These
findings, therefore, indicate a role for endogenous opiate
modulators in the mechanism of tolerance and dependence
and suggest a specific cellular mechanism, down-regulation
of receptors, that is altered by these peptides.
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1998
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regulation by morphine and up-regulation by naloxone in SH-SY5Y human neuroblastoma cells. J Pharmacol Exp Ther 265:254 –262.
Zadina JE, Kastin AJ, Kenigs V, Bruno C and Hackler L (1993b) Prolonged analgesia
after intracerebroventricular Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2). Neurosci Lett
155:220 –222.
Zadina JE, Harrison LM, Ge L-J, Kastin AJ and Chang SL (1994a) Differential
regulation of mu and delta opiate receptors by morphine, selective agonists and
antagonists and differentiating agents in SH-SY5Y human neuroblastoma cells.
J Pharmacol Exp Ther 270:1086 –1096.
Zadina JE, Kastin AJ, Ge L-J and Chang SL (1994b) Novel peptides and mechanisms
of opiate tolerance. Regul Pept Suppl 1:S195–S196.
Zadina JE, Kastin AJ, Ge L-J and Hackler L (1994c) Mu, delta and kappa opiate
receptor binding of Tyr-MIF-1 and of Tyr-W-MIF-1, its active fragments and two
potent analogs. Life Sci 55:PL461–PL466.
Zadina JE, Kastin AJ, Harrison LM, Ge L-J and Chang SL (1995) Opiate receptor
changes after chronic exposure to agonists and antagonists. Ann NY Acad Sci
257:353–361.
Zadina JE, Paul D, Gergen KA, Ge L-J, Hackler L and Kastin AJ (1996) Binding of
Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) and related peptides to mu1 and mu2 opiate
receptors. Neurosci Lett 215:65– 69.
Send reprint requests to: James E. Zadina, Ph.D., Research Service (151),
VA Medical Center, 1601 Perdido Street, New Orleans, LA 70146.
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2016
Tao P-L, Lee H-Y, Chang L-R and Loh HH (1990) Decease in mu-opioid receptor
binding capacity in rat brain after chronic PL017 treatment. Brain Res 526:270 –
275.
Tao P-L, Tsai C-L, Chang L-R and Loh HH (1991) Chronic effect of [D-Pen2,DPen5]enkephalin on rat brain opioid receptors. Eur J Pharmacol 201:209 –214.
Tempel A, Habas J, Paredes W and Barr GA (1988) Morphine-induced downregulation of mu-opioid receptors in neonatal rat brain. Dev Brain Res 41:129 –133.
Werling L, McMahon PN and Cox BM (1989) Selective changes in mu opioid receptor
properties induced by chronic morphine exposure. Proc Natl Acad Sci USA 86:
6393– 6397.
Yang HY-T, Fratta W, Majane EA and Costa E (1985) Isolation, sequencing, synthesis and pharmacological characterization of two brain neuropeptides that modulate the actions of morphine. Proc Natl Acad Sci USA 82:7757–7761.
Zadina JE, Chang SL, Ge L-J, Kastin AJ and Harlan RE (1990) Down-regulation of
mu opiate receptors by morphine and presence of Tyr-MIF-1 binding sites in
SH-SY5Y human neuroblastoma cells, in New Leads in Opioid Research (van Ree
JM, Mulder AH, Wiegant VM and van Wimersma Greidanus TB eds) pp 151–153,
Excerpta Medica, New York.
Zadina JE, Chang SL, Ge L-J and Kastin AJ (1991) Naloxone-reversible inhibition of
cyclic AMP production by Tyr-MIF-1 in SH-SY5Y human neuroblastoma cells. Soc
Neurosci Abstr 17:811.
Zadina JE, Kastin AJ, Kersh D and Wyatt A (1992) Tyr-MIF-1 and hemorphin can
act as opiate agonists as well as antagonists in the guinea pig ileum. Life Sci
51:869 – 885.
Zadina JE, Chang S-L, Ge L-J and Kastin AJ (1993a) Mu opiate receptor down-
Tyr-W-MIF-1 and Down-regulation