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NEUROPHARMACOLOGY
Laboratory of Molecular Neuropharmacology, Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, New York (C.L.N., A.J.J., E.B., G.W.P.); and the Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, Montreal, Canada (I.B., T.M.-D.N., P.W.S.)
Received January 30, 2003; accepted March 25, 2003.
 |
Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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,
-imido)triphosphate
together reduced specific [3H][Dmt1]DALDA binding levels
by almost 75%. Competition studies confirmed the µ-selectivity of the
binding, with Ki values that were not appreciably
different from those seen against [3H]DAMGO. In guanosine
5'-O-(3-[35S]thio)-triphosphate
([35S]GTPS) binding assays in brain and spinal cord
membranes, [Dmt1]DALDA was more potent than DAMGO, but showed
plateaus suggestive of a partial agonist. [Dmt1]DALDA bound to
µ-opioid receptor clone 1 (MOR-1) and its splice variants with high
affinity. Unlike [3H]DAMGO, [3H][Dmt1]DALDA
seemed to label both agonist and antagonist conformations of MOR-1 expressed
in Chinese hamster ovary cells. In [35S]GTPS assays
[Dmt1]DALDA showed high efficacy with all the MOR-1 variants, but
its potency (EC50) varied markedly among some of the splice
variants despite similar affinities in receptor binding assays. Although
[3H][Dmt1]DALDA is a very potent µ-selective
analgesic, its binding characteristics and its ability to stimulate
GTPS binding differed from that of the classical µ-opioid peptide
DAMGO.
;
Payne and Pasternak, 1992;
Rossi et al., 1995;
Pasternak, 2001).
µ-Opioids, such as heroin, morphine-6-glucuronide (M6G),
6-acetylmorphine, and fentanyl retain their analgesic potencies in CXBK mice,
a strain that is insensitive to morphine
(Rossi et al., 1996), and they
display limited cross-tolerance to morphine in animal studies
(Lange et al., 1980;
Rossi et al., 1996) and
clinically (Cherny et al.,
2001). The antagonist 3-methylnaltrexone selectively blocks the
actions of heroin, 6-acetylmorphine, and M6G more potently than morphine
(Brown et al., 1997a;
Walker et al., 1999).
Antisense mapping studies on the cloned µ-opioid receptor clone 1 (MOR-1)
find different sensitivity profiles for morphine compared with heroin and M6G
(Rossi et al., 1995,
1996). In addition, M6G,
heroin and 6-acetylmorphine retained their analgesic activity in a knockout
mouse with disruption of exon 1 of the MOR-1 receptor that was totally
insensitive to morphine (Schuller et al.,
1999). Although exon 1 has been deleted, there is still expression
of MOR-1 splice variants in this knockout mouse, as demonstrated by both
reverse transcriptase-polymerase chain reaction and immunohistochemistry
(Schuller et al., 1999;
Pan et al., 2001; C. Abbadie,
J. Pintar, and G. W. Pasternak, unpublished observations).
The dermorphin analog [Dmt1]DALDA
(H-Dmt-D-Arg-Phe-Lys-NH2; Dmt,
2',6'-dimethyltyrosine)
(Schiller et al., 2000)
displays a very unique pharmacology that differs from that of morphine, as
evidenced by its insensitivity to antisense probes that reduce morphine (exons
1, 4, 5, 6, 7, 8, 9, and 10) or M6G (exon 2) analgesia, its incomplete
cross-tolerance to morphine (Neilan et
al., 2001; Riba et al.,
2002), and persistent analgesia in a MOR-1 knockout mouse
(Neilan et al., 2003). This
peptide also is an effective analgesic in morphine-insensitive CXBK mice
(Neilan et al., 2001).
[Dmt1]DALDA has a long duration of action and is metabolically
stable with limited penetration of the placental barrier
(Szeto et al., 2001).
Radioligand binding studies have proved to be a valuable tool in investigating
the mechanisms of action of µ-opioids. Detailed binding experiments
reported over twenty years ago first proposed multiple classes of µ-binding
sites (Wolozin and Pasternak,
1981). More recent studies using [3H]M6G also support
the concept of µ-receptor heterogeneity
(Brown et al., 1997b). In an
attempt to further investigate the more unusual aspects of its pharmacology,
we now have characterized the receptor binding profile of
[3H][Dmt1]DALDA.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). The
peptide was purified by preparative reversed-phase chromatography, and its
structure was confirmed by fast atom bombardment mass spectrometry. Catalytic
tritiation of this precursor peptide was performed at the Institute of
Isotopes (Budapest, Hungary), resulting in the tritiated peptide
[3H][Dmt1]DALDA with a specific activity of 47 Ci/mmol.
[3H]DAMGO, specific activity 50 Ci/mmol, and
[3H]naloxone, specific activity 60 Ci/mmol, were purchased from
PerkinElmer Life Sciences (Boston, MA). Unlabeled [Dmt1]DALDA was
synthesized in the laboratory of P. W. Schiller. All other ligands were
obtained from the Research Technology Branch of the National Institute on Drug
Abuse (Rockville, MD).
Receptor Binding.Membranes were prepared as reported previously
(Clark et al., 1988,
1989). Tissue or cells were
homogenized in 50 volumes of 50 mM Tris buffer, pH 7.7, containing 10 µM
phenylmethylsulfonyl fluoride, 100 mM NaCl, and 1 mM K+ EDTA. The
homogenate was incubated for 15 min at 25°C, centrifuged at
39,000g for 45 min, resuspended in 0.32 M sucrose, and frozen at
–80°C. Binding was performed in potassium phosphate buffer (50 mM;
pH 7.4), containing MgSO4 (either 1 or 5 mM, respectively) for
assays with [3H][Dmt1]DALDA or [3H]DAMGO.
Reactions were incubated at 25°C for 60 min, with the exception of
dissociation and association binding which used the indicated time points.
Assay volumes were 3 ml for calf thalamic and frontal cortex membranes, 2 ml
for guinea pig cerebellum membranes and calf striatal membranes, and 1 ml for
spinal cord, whole mouse brain, and CHO cell membranes. Protein concentration
was 3 mg/ml wet weight of tissue for all calf brain and guinea pig cerebellar
membranes and 150 µg for binding assays using CHO cell membranes. Reactions
were terminated by rapid filtration over glass fiber filters that then were
subjected to liquid scintillation counting. For assays using
[3H][Dmt1]DALDA, filters were soaked in 0.5% (w/v)
polyethylimine solution for 5 min before using. Nonspecific binding, which
typically represented 15% of total binding, was determined using levallorphan
(1 µM) or [Dmt1]DALDA (1 µM) for assays using
[3H]DAMGO and [3H][Dmt1]DALDA, respectively,
with no appreciable difference in levels of nonspecific binding. Only specific
binding is reported. Saturation and competition studies were analyzed using
nonlinear regression analysis with the Prism program (GraphPad Software Inc.,
San Diego, CA).
[35S]GTPS assays were performed in Tris buffer (50 mM; pH
7.4) containing EGTA (0.2 mM), NaCl (100 mM), and MgCl2 (3 mM).
Membranes (25–50 µg) were incubated for 1 h at 30°C with GDP (30
µM), [35S]GTPS (0.05 nM), and unlabeled ligand at varying
concentrations. Reactions then were terminated by rapid filtration and subject
to liquid scintillation counting as described above. All values were
normalized as a percentage of the stimulation by DAMGO at 10 µM.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Association experiments using calf striatal membranes showed that [3H][Dmt1]DALDA binding (0.1 nM) reached maximum levels within 60 min (Fig. 2A). The observed half-life of dissociation (t1/2) was 16.1 ± 3.3 min. A semilog plot of the [3H][Dmt1]DALDA dissociation (Fig. 2B) revealed a linear relationship consistent with the labeling of a single population of receptors. Saturation binding in striatal membranes under equilibrium conditions showed that the peptide labeled a single population of receptors, affording a KD value of 0.22 ± 0.02 nM and a Bmax value of 255 ± 14 fmol/mg protein (Fig. 3).
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Sensitivity toward Ions and Nucleotides. µ-Opioid receptor
binding is modulated by a variety of ions and nucleotides, with the binding of
agonists and antagonists generally affected in opposite ways
(Pert et al., 1973;
Pasternak et al., 1975;
Childers and Snyder, 1980). In
general divalent cations enhance µ-opioid agonist binding, leaving
antagonist binding unaffected. Conversely, sodium ions and guanine nucleotides
markedly reduce agonist, but not antagonist, binding.
Compared with [3H]DAMGO binding,
[3H][Dmt1]DALDA binding was relatively insensitive to
any of the divalent cations (Fig.
4), although MgCl2 (1 mM) did modestly enhance binding
by approximately 15% (Fig. 3;
P < 0.01). We observed no difference between MgCl2 and
MgSO4 and therefore routinely included MgSO4 (1 mM) in
all [3H][Dmt1]DALDA binding assays. Manganese ions
potentiate the binding of the µ-opioid agonist
[3H]dihydromorphine (Pasternak
et al., 1975). Similarly, MnCl2 (1 mM) increased
[3H]DAMGO binding. However, [3H][Dmt1]DALDA
binding was unaffected. Similarly, [3H][Dmt1]DALDA
binding was not influenced by CaCl2, despite the modest lowering of
[3H]DAMGO binding.
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Sodium ions diminish the binding of [3H]dihydromorphine while
enhancing the binding of the antagonist [3H]naloxone
(Pert et al., 1973). Guanine
nucleotides also selectively diminish agonist binding. In our studies, we
observed a similar decline in [3H]DAMGO binding
(Fig. 5). Yet, neither
Na+ ions alone nor the GTP analog Gpp(NH)p alone affected
[3H][Dmt1]DALDA binding to an appreciable degree,
although the combination of NaCl (50 mM) and Gpp(NH)p (0.1 mM) reduced binding
by almost 75% (P < 0.01). The sensitivity of
[3H][Dmt1]DALDA binding to the combination is consistent
with the established agonist nature of the drug
(Childers and Snyder, 1980;
Selley et al., 2000). However,
its insensitivity to sodium ions or the GTP analog alone is somewhat unusual
and sets it apart from other µ-opioid agonists.
|
Binding Selectivity Profile and Regional Distribution of
[3H][Dmt1]DALDA and [3H]DAMGO Binding. We
next compared the binding selectivity profile of
[3H][Dmt1]DALDA to that of [3H]DAMGO. Full
competition curves were generated, and Ki values were
determined for both radioligands (Table
1). All compounds show similar potencies against both
radioligands. [3H][Dmt1]DALDA binding showed a
selectivity typical for µ-radioligands, with the traditional µ-opioids
DAMGO, morphine, methadone, and fentanyl all potently lowering binding as
effectively as against [3H]DAMGO. The 
-selective peptide
[D-Pen2,D-Pen5]-enkephalin and the
-selective drug U50,488H displayed low affinities, consistent with the
µ-selectivity of the radioligands. None of the Hill slopes for any of the
ligands tested differed significantly from unity.
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Functional Assessment of [Dmt1]DALDA and DAMGO in
[35S]GTPS Binding Assays. The efficacy and
potency of [Dmt1]DALDA was tested in vitro in an attempt to further
understand the highly potent nature of the compound in vivo.
[35S]GTPS binding was carried out in calf striatal
membranes, whole mouse brain, and mouse spinal cord membranes after
stimulation with either [Dmt1]DALDA or DAMGO
(Fig. 6).
[Dmt1]DALDA (EC50 = 12.2 ± 2.7 nM) was 26-fold
more potent than DAMGO (EC50 = 322 ± 21 nM) in calf striatal
membranes, 34-fold more potent in mouse spinal cord (EC50 = 10.1
± 1.3 nM compared with EC50 = 343 ± 49 nM for DAMGO),
and 80-fold more potent in whole mouse brain (EC50 = 2.9 ±
0.2 nM compared with EC50 = 228 ± 32 nM for DAMGO). Despite
its greater potency, [Dmt1]DALDA had a ceiling effect in striatal
membranes, implying that it was a partial agonist. This was more pronounced in
the whole brain membrane assays, where the maximal value was only
approximately 75% that of DAMGO stimulation at 10 µM. Only the spinal cord
assay revealed agonist activity for [Dmt1]DALDA that was similar to
that of DAMGO.
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[Dmt1]DALDA Interactions with the Cloned MOR-1 Splice
Variants. A number of MOR-1 splice variants have recently been isolated
and cloned (Bare et al., 1994;
Zimprich et al., 1995; Pan et
al., 1999,
2000,
2001). First, we determined
the affinity of [3H][Dmt1]DALDA for a series of murine
MOR-1 variants expressed in CHO cells
(Table 2). With the MOR-1
clone, [3H][Dmt1]DALDA was about 3-fold more potent than
[3H]DAMGO in receptor binding assays. Yet, with the MOR-1C and
MOR-1D clones, the two radioligands were equipotent. Their equivalent potency
in the MOR-1C and MOR-D clones also contrasted with their relative potencies
in calf striatal membranes, where [Dmt1]DALDA was 5- to 10-fold
more potent in the competition studies
(Table 1).
|
In saturation studies using membranes from cells transfected with the MOR-1 clone, [3H][Dmt1]DALDA labeled approximately twice as many receptors as [3H]DAMGO (P < 0.05) (Table 3). Interestingly, [3H][Dmt1]DALDA labeled the same number of receptors in these membranes as the opioid antagonist [3H]naloxone, suggesting that [3H][Dmt1]DALDA may label both agonist and antagonist conformations of the receptor.
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We next compared [Dmt1]DALDA and DAMGO in GTPS binding
assays (Table 2). All
[Dmt1]DALDA values were normalized to DAMGO at 10 µM. The
potency of DAMGO for the variants was similar, ranging from about 50 to 70 nM.
In contrast, [Dmt1]DALDA values ranged over 10-fold among the
clones. This was quite unexpected in view of the similar receptor binding
affinities of [Dmt1]DALDA for the clones and the small structural
differences among the variant receptors. The relative potency of
[Dmt1]DALDA to DAMGO for each variant, defined as the ratio of
their EC50 values, also varied markedly. The MOR-1 cells displayed
the greatest difference, where [Dmt1]DALDA was almost 100-fold more
potent than DAMGO (P < 0.05). However, this may be an
overrepresentation of the difference in efficacy between them because
[Dmt1]DALDA also has a higher binding affinity. To compensate for
this, we also calculated the relative potency of stimulation of the drugs in
which the EC50 ratio is divided by the ratio of the
KD values. Although only an estimate, the value takes into
consideration differences between the binding affinities of the drugs for the
receptors in assessing their relative potencies in the stimulation of
GTPS binding. [Dmt1]DALDA still was almost 30-fold more
effective in stimulating GTPS binding in MOR-1 than DAMGO after taking
into consideration the differences in binding affinities. We also observed a
similar enhanced relative potency for the other clones, but they were more
modest, ranging from only 3.5-fold for MOR-1E to 10.4-fold for MOR-1D
(Table 2). In all cases,
[Dmt1]DALDA activated GTPS binding far more potently than
DAMGO. Together, these observations illustrate differences between the two
drugs in their activation of these receptors.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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S binding assays that
distinguish it from DAMGO.
µ-Opioid receptor binding has been extensively characterized over the
past 30 years. A variety of ions and treatments have documented the ability to
distinguish between agonist and antagonist binding, findings that were then
generalized to other G protein-coupled receptors. For example, the ability of
sodium ions to selectively enhance antagonist and inhibit agonist binding was
first described with µ-opioid receptors
(Pert et al., 1973), as were
the ability of a number of divalent cations to selectively enhance agonist
binding (Pasternak et al.,
1975). Guanine nucleotides also distinguish between agonist and
antagonist binding, lowering the binding of agonists but not antagonists
(Childers and Snyder,
1980).
Our current studies confirmed these effects with [3H]DAMGO.
However, [3H][Dmt1]DALDA binding was different. All of
the previous studies with [Dmt1]DALDA clearly show that the peptide
is a µ-opioid agonist whose analgesic responses in vivo are inhibited by
the µ-selective antagonist -funaltrexamine. Although a reduction in
specific [3H][Dmt1]DALDA binding was observed after
addition of both NaCl and Gpp(NH)p to the binding buffer, its insensitivity to
sodium ions or Gpp(NH)p alone clearly distinguished it from most other
µ-opioid agonists. Nor was it increased to the same degree as
[3H]DAMGO by divalent cations. Although these differences are
subtle, they imply differences in the mode of binding/activation of
µ-opioid receptors by [Dmt1]DALDA.
Differences between [Dmt1]DALDA and DAMGO also were observed
functionally in the GTPS binding studies. [Dmt1]DALDA is far
more potent in vivo than DAMGO (Neilan et
al., 2001; Shimoyama et al.,
2001; Riba et al.,
2002). Thus, it was not surprising that it remained more potent in
the GTPS binding studies. However, the presence of ceiling effects
compared with DAMGO in both mouse brain and calf striatum imply that it is a
partial agonist in these tissues with an efficacy below that of DAMGO, whereas
it retained full agonist activity in the spinal cord. Although its maximal
stimulation varied among the tissues, its EC50 value remained the
same.
The increased spinal cord efficacy observed for [Dmt1]DALDA is
consistent with its extraordinary intrathecal analgesic potency in vivo.
However, other factors may also be important. Synergistic interactions between
µ-opioid and 
2-agonists at the level of the spinal cord
have been widely reported (Ossipov et al.,
1989,
1990;
Fairbanks et al., 2002), and
in a recent study the 2-adrenergic antagonist yohimbine
significantly attenuated [Dmt1]DALDA-mediated intrathecal analgesia
in rats (Shimoyama et al.,
2001). Therefore, it is interesting to speculate whether the
increased efficacy of [Dmt1]DALDA in spinal cord membranes is due
in part to an interaction with other receptors, such as the
2-receptor. However, in preliminary studies yohimbine at
concentrations as high as 100 nM failed to compete
[3H][Dmt1]DALDA binding, leaving open how these
receptors may be interacting.
The Oprm gene, which encodes MOR-1 and its variants, is large
(>250 kb) and has a complex pattern of alternative splicing
(Bare et al., 1994;
Zimprich et al., 1995; Pan et
al., 1999,
2000,
2001). In vivo and in vitro
studies using brain and/or spinal cord membranes, the overall actions of
[Dmt1]DALDA presumably reflect the summation of its interactions
with a variety of splice variants of the µ-receptor. Therefore, we also
examined the effects of [Dmt1]DALDA on a series of variants
individually expressed in CHO cells. These variants differ from MOR-1 itself
and each other only at the tip of the intracellular COOH tail of the receptor.
All these variants selectively bind µ-opioids with high affinity, but show
subtle differences in affinity for certain compounds across the variants,
particularly the endogenous ligands -endorphin and dynorphin A (Pan et
al., 1999,
2000,
2001). These variants also
differ functionally among themselves. Internalization has been well described
for MOR-1 (Burford et al.,
1998; Keith et al.,
1998). In these studies, DAMGO, but not morphine, internalizes
MOR-1. When expressed in HEK293 cells, however, MOR-1D and MOR-1E internalize
in response to either DAMGO or morphine treatment, whereas MOR-1 and MOR-1C
only internalize in response to DAMGO treatment and not with morphine
(Koch et al., 2001). In vivo,
MOR-1 internalizes in response to DAMGO, but not morphine, whereas MOR-1C
internalizes with both (Abbadie and
Pasternak, 2001). The ability of morphine to internalize MOR-1C in
neurons in the brain and not HEK293 cells may be due to a variety of issues,
such as the overexpression of the protein in the HEK293 cells, the different
environment of the receptor, and the repertoire of associated proteins.
In binding studies with the splice variants
[3H][Dmt1]DALDA displayed modest differences from DAMGO.
Looking at the ratios of KD values for the two
radioligands, MOR-1C and MOR-1D showed virtually no difference in affinity,
whereas [3H][Dmt1]DALDA was almost 4-fold more potent
than [3H]DAMGO against MOR-1 itself. The most intriguing difference
between the binding of the two radioligands involved the number of sites each
labeled. G protein-coupled receptors are thought to have both agonist and
antagonist conformations, with agonists labeling only the agonist conformation
and antagonists labeling both. Thus, it was not surprising to see
[3H]naloxone labeling about twice as many sites as
[3H]DAMGO in membranes from MOR-1-expressing CHO cells. We did not
anticipate seeing [3H][Dmt1]DALDA label the same number
of receptors as the antagonist [3H]naloxone. This suggests that
[3H][Dmt1]DALDA was labeling both agonist and antagonist
states of the receptor. The ability of [Dmt1]DALDA to label the
antagonist conformation of the receptor may help explain the relative
insensitivity of [3H][Dmt1]DALDA binding to either
sodium or divalent cations and to GTPS and is consistent with its
apparent partial agonist actions in the brain GTPS binding studies.
The functional actions of [Dmt1]DALDA with the various MOR-1
variants further illustrated differences among them. In the transfected cell
lines, [Dmt1]DALDA seemed to have full agonist activity, giving
maximal responses equivalent to those of DAMGO. The difference in structure
among the variants is limited to the terminal amino acids of the intracellular
COOH tail, far away from the binding pocket formed by the transmembrane
domains. Although prior work from our laboratory has shown subtle differences
in affinity of several endogenous opioid peptides among the variants, most
µ-drugs show little difference (Pan et al.,
1999,
2000,
2001). Therefore, we
anticipated similar affinities of the variants for
[3H][Dmt1]DALDA in receptor binding assays, but we did
not expect the major differences in its stimulation of
[35S]GTPS binding. [Dmt1]DALDA stimulated
GTPS binding about 10- fold more effectively in the MOR-1-expressing
cell membranes than in those expressing either MOR-1C or MOR-1E. Although
these studies help with our understanding of [Dmt1]DALDA actions,
they also provide important insights into the variants themselves. As noted
above, the structural differences among them are quite small, being restricted
to the terminal amino acids in the intracellular COOH tail. This variability
in response to [Dmt1]DALDA would suggest that the tip of the COOH
tail is important in modulating the efficacy of µ-opioids, possibly by
influencing the association of the receptor with other proteins.
The relative ability of [Dmt1]DALDA to stimulate
[35S]GTPS binding compared with DAMGO also was interesting.
In all cases, [Dmt1]DALDA was more potent. The greatest difference
was seen with MOR-1 itself where the EC50 of [Dmt1]DALDA
was almost 100-fold lower than that of DAMGO. Direct comparisons can be
somewhat misleading because [Dmt1]DALDA has a higher binding
affinity than DAMGO for MOR-1 sites. Even after taking the relative affinity
of the drugs to take into consideration, we estimated that
[Dmt1]DALDA was still almost 30-fold more effective than DAMGO in
stimulating [35S]GTPS binding. Although
[Dmt1]DALDA also was more active with the other variants, the
difference for MOR-1E was only 3.5-fold. Although these are only rough
estimates of the relative potencies of the two drugs, they do raise the
possibility of activation differences for the two drugs and among the various
variants.
The pharmacology of [Dmt1]DALDA is complex and not easily explained. Binding studies imply that it is a partial agonist and capable of labeling both agonist and antagonist receptor conformations. Yet, [Dmt1]DALDA activates G proteins far more potently than DAMGO, even when taking their relative receptor binding affinities into consideration. Further studies into the pharmacology of [Dmt1]DALDA may provide valuable insights into the action of µ-analgesics.
|
Footnotes |
|---|
ABBREVIATIONS: M6G, morphine-6-glucuronide; MOR-1, µ-opioid
receptor clone-1; [Dmt1]DALDA,
H-Dmt-D-Arg-Phe-Lys-NH2; Dmt,
2',6'-dimethyltyrosine; DAMGO,
[D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin;
CHO, Chinese hamster ovary; GTPS, guanosine
5'-O-(3-thio)triphosphate; Gpp(NH)p, guanosine
5'-(,-imido)triphosphate; HEK, human embryonic kidney;
U50,488H,
(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide
methane-sulfonate hydrate; CTAP,
D-Phe-c[Cys-Tyr-D-Trp-Arg-Thr-Pen]-Thr-NH2.
Address correspondence to: Dr. Gavril Pasternak, Department of Neurology, 1275 York Ave., New York, NY 10021. E-mail: pasterng{at}mskcc.org
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