 |
 Previous Article | Next Article 
The Journal of Neuroscience, December 1, 2002, 22(23):10494-10500
Differential Mechanisms of Morphine Antinociceptive Tolerance
Revealed in  Arrestin-2 Knock-Out Mice
Laura M.
Bohn1,
Robert
J.
Lefkowitz2, and
Marc G.
Caron1
Howard Hughes Medical Institute Laboratories, Departments of
1 Cell Biology and 2 Biochemistry and Medicine,
Duke University Medical Center, Durham, North Carolina 27710
 |
ABSTRACT |
Morphine induces antinociception by activating µ opioid receptors
(µORs) in spinal and supraspinal regions of the CNS.  arrestin-2 (arr2), a G-protein-coupled receptor-regulating protein, regulates the µOR in vivo. We have shown previously that mice
lacking arr2 experience enhanced morphine-induced analgesia and do
not become tolerant to morphine as determined in the hot-plate test, a
paradigm that primarily assesses supraspinal pain responsiveness. To
determine the general applicability of the arr2-µOR interaction in
other neuronal systems, we have, in the present study, tested arr2 knock-out (arr2-KO) mice using the warm water tail-immersion paradigm, which primarily assesses spinal reflexes to painful thermal
stimuli. In this test, the arr2-KO mice have greater basal
nociceptive thresholds and markedly enhanced sensitivity to morphine.
Interestingly, however, after a delayed onset, they do ultimately
develop morphine tolerance, although to a lesser degree than the
wild-type (WT) controls. In the arr2-KO but not WT mice, morphine
tolerance can be completely reversed with a low dose of the classical
protein kinase C (PKC) inhibitor chelerythrine. These findings provide
in vivo evidence that the µOR is differentially regulated in diverse regions of the CNS. Furthermore, although arr2
appears to be the most prominent and proximal determinant of µOR
desensitization and morphine tolerance, in the absence of this
mechanism, the contributions of a PKC-dependent regulatory system
become readily apparent.
Key words:
morphine; µ opioid receptor; MOP; knock-out mice; arrestin; desensitization; G-protein-coupled receptors; tolerance; antinociception
| |
INTRODUCTION |
The regulation of G-protein-coupled
receptor (GPCR) signaling can determine the extent of drug effect. In
the continued presence of agonist, GPCR signaling is curtailed by a
process of desensitization that results in a disrupted signal to the
principal secondary messenger cascade. In the classic sense, GPCR
desensitization results after the GPCR is uncoupled from its G-protein.
Desensitization occurs when agonist-stimulated receptor is
phosphorylated by a GPCR kinase (GRK) and then attracts an arrestin
protein (Ferguson et al., 1998 ; Krupnick and Benovic, 1998; Lefkowitz,
1998). The arrestin binds to the phosphorylated receptor and thereby
acts as a damper to prevent further coupling of the receptor with the G-protein. Several reports have demonstrated the importance of GRKs and
arrestins in determining GPCR signaling in cell cultures, where it has
been shown that certain GRKs and arrestins can affect GPCR
desensitization. For example, the overexpression of GRK2 leads to
greater phosphorylation and desensitization of rat µ opioid receptors
(µORs), whereas an overexpression of arrestin (arr) can
increase the internalization of the µOR in transfected cell systems
(Whistler and von Zastrow, 1998; Zhang et al., 1998).
To gain a greater understanding of the contribution of arr2 to
opioid receptor signaling, we tested the effects of morphine in mice
lacking arr2. arr2-knock-out (arr2-KO) mice experience enhanced and prolonged antinociception in the 56°C hot-plate test after morphine treatment compared with their wild-type (WT) littermates (Bohn et al., 1999). Furthermore, these mice do not develop tolerance to morphine after acute or chronic treatment in this test of pain perception, yet they still develop morphine dependence (Bohn et al.,
2000b). These observations indicate that at least in this nociception
paradigm, functional desensitization of the µOR is required for
development of tolerance to morphine, whereas morphine dependence can
be dissociated from desensitization and tolerance.
The opioid regulation of pain perception involves µORs in both spinal
and supraspinal regions of the CNS (for review, see Cesselin et al.,
1999; Heinricher and Morgan, 1999). The spinal and supraspinal action
of opioids are both believed to contribute significantly to hot-plate
responses, although the supraspinal system is thought to be the more
prominent contributor. The spinal action of opioids is believed to
contribute to the spinal reflex demonstrated by rapid tail withdrawal
from a heat source (i.e., a tail flick). The spinal reflex associated
with pain perception is usually tested as the latency of time passed
before the tail is withdrawn. Mice that lack the µOR no longer
experience morphine antinociception in either of these tests,
indicating that the µOR is primarily responsible for mediating
morphine antinociception (Sora et al., 1997; Kieffer 1999). Regulation
of the µOR is therefore an attractive target for affecting the degree
of antinociception and tolerance induced by morphine. The overall
manifestation of morphine-induced antinociception and tolerance
undoubtedly involves complex neuronal interactions and many different
signaling components. As an example, there is much evidence that the
activation of PKC may play an integral part in the development of
morphine antinociceptive tolerance (Mayer and Price, 1976; Narita et
al., 1995; Li and Roerig, 1999; Granados-Soto et al., 2000; Inoue and
Ueda, 2000; Zeitz et al., 2001). Furthermore, the extent of µOR
desensitization in different brain regions of the rat was shown to
differ after chronic morphine treatment, suggesting that the receptor
could be differentially regulated (Noble and Cox, 1996). By using mice that lack an essential component of the desensitization process, arr2, we are able to assess the contribution of this and other mechanisms to the antinociceptive actions of morphine in various pain
perception paradigms. It is becoming increasingly apparent that the
regulation of a particular receptor may differ depending on its
cellular environment, be it different regions of the nervous system or
within different cell types. Using this model, we now demonstrate that
the spinal antinociceptive actions of morphine are regulated by
arr2- and PKC-dependent pathways, suggesting that, within the spinal
system, both of these regulatory mechanisms contribute to µOR regulation.
| |
MATERIALS AND METHODS |
Animals and drugs. The WT mice, heterozygotes, and
knock-out mice are generated as littermates from crossing heterozygous arr2 C57BL/6/129SvJ animals (over nine generations) as described previously (Bohn et al., 1999). Mice used in this study were
age-matched, 3- to 5-month-old male siblings weighing between 20 and 35 gm. In all experiments, WT littermates served as controls for the arr2+/ and arr2-KO mice, and all genotypes were evaluated
simultaneously. Groups of mice were only used in the individual
experiments indicated. To prevent damage to the tail, genotyping was
performed on DNA extracted for tissue punched from the ear of each
mouse. Experiments were conducted in accordance with National
Institutes of Health Guidelines for the Care and Use of
Laboratory Animals and with an approved animal protocol from the
Duke University Animal Care and Use Committee. Morphine sulfate and
naltrindole were purchased from Sigma (St. Louis, MO) and freshly
prepared in saline and water, respectively. Naltrexone (NTX) and
nor-binaltorphamine (nor-BNI) were purchased from Tocris Cookson
(Ballwin, MO) and freshly prepared in water. Chelerythrine chloride
(Calbiochem, San Diego, CA) was freshly prepared in DMSO and diluted
for a final injection volume in water at 2% DMSO. All compounds were injected at a volume of 10 µl/gm animal weight.
Warm water tail-immersion assay. Antinociception was
evaluated by measuring response latencies in the warm water
tail-immersion (tail-flick) assay (Janssen et al., 1963; Stone et al.,
1997). Response latencies were measured as the amount of time the
animal took to respond to the thermal stimuli. The warm water (43, 48, and 54°C) tail-flick test was performed by gently holding the mouse
in a terry cloth towel and immersing between 2 and 3 cm from the tip of
the tail into the water, and the response was defined as the removal of
the tail from the warm water as described previously (Bohn et al.,
2000c). Hot-plate (50, 53, and 56°C) experiments were performed as
described previously (Bohn et al., 1999). When exposed to the test
under the influence of morphine, mice were not permitted to exceed 30 sec of exposure to the thermal source to prevent prolonged painful
stimulation. The reported data account for this artificial ceiling as
well as for the basal responsiveness of each mouse to the test and is
presented as the percentage of maximum possible effect (%MPE), which
is calculated by the following formula: 100% × [(drug response
time basal response time)/(30 sec basal response
time)] = %MPE.
[35S]GTP S binding assays.
[35S]GTPS (1250 Ci/mmol; NEN, Boston,
MA) binding assays were performed on membranes from mouse spinal cords
as described previously (Bohn et al., 1999). Samples were placed on ice
and homogenized by polytron in membrane preparation buffer (50 mM Tris, pH 7.4, 1 mM EDTA,
and 3 mM MgCl2), and crude membranes were prepared by centrifugation at 20,000 × g for 30 min at 4°C. Membranes were resuspended in assay
buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 3 mM
MgCl2, and 0.2 mM EDTA)
containing 10 µM GDP. Reactions were terminated
by rapid filtration over GF/B filters (Brandel, Gaithersburg, MD) using
a Brandel cell harvester. Filters were washed three times with ice-cold
10 mM Tris-HCl, pH 7.4, and then counted in a
liquid scintillation counter. n represents membranes
prepared from striata from one mouse.
Statistical analysis. All statistical analyses were
calculated using GraphPad (San Diego, CA) Prism software.
| |
RESULTS |
Basal nociceptive thresholds
Basal response latencies were assessed using the warm water
tail-immersion test, and a difference between genotypes became readily
apparent. At 43, 48, and 54°C, the arr2-KO mice experience a
significantly greater delay in tail withdrawal than their WT counterparts (Fig. 1A).
Furthermore, this delay in tail withdrawal is also greater in the
arr2 heterozygotes, implicating the importance of the concentration
of arr2 in regulating µOR responsiveness (Zhang et al., 1998; Lowe
et al., 2002). The initial comparison of basal nociception to the
56°C hot-plate revealed that the WT and arr2-KO mice did not
differ in their response latencies (Bohn et al., 1999), and this
observation has been extended to hot-plate temperatures of 50 and
53°C as well (Fig. 1B) where again, no differences
are seen. The prolonged tail-flick latencies in the arr2-KO mice
come somewhat unexpectedly, considering that there are no differences
between genotypes observed in the hot-plate test. Furthermore, the
elevated tail-flick latencies were also present in arr2-KO mice that
had been back-crossed six generations to C57BL/6 mice (C57-WT, 1.8 ± 0.34 sec; C57-KO, 4.5 ± 0.3 sec), exemplifying the penetrance
and significance of the differences seen between the genotypes in the
tail-flick test.
View larger version (24K):
[in this window]
[in a new window]
|
Figure 1.
Basal tail-flick but not hot-plate response
latencies are prolonged in arr2-KO mice. A, Tail
flick. Mice of each genotype were assessed for their responsiveness in
the warm water tail-flick assay. Response latencies were assessed at
43, 48, and 54°C water temperatures; *p < 0.05, **p < 0.01, and ***p < 0.001 versus WT; #p < 0.05 versus
arr2+/; one-way ANOVA followed by Bonferroni's multiple
comparison test; n = 13-20 WT;
n = 6-8 arr2+/; n = 13-21 arr2-KO mice. B, Hot-plate. WT and arr2-KO
mice were assessed for paw-withdrawal latencies on the 50, 53, and
56°C hot plate. There were no differences in responses between the
littermates (n = 12-27).
|
|
[35S]GTPS binding in spinal
cord membranes
GPCR coupling to G-protein is an indicator of the potential of the
receptor to signal. Therefore, we assessed µOR coupling in membranes
prepared from mouse spinal cord. The µOR-selective agonist
D-Ala2-N-Me-Phe4-Glycol5]enkephalin
showed a robust stimulation of G-protein coupling in the WT mouse
membranes; however, there was a greater degree of agonist-dependent
coupling in the spinal cord membranes from the arr2-KO mice (Fig.
2). Radioligand binding assays in spinal cord membranes using [3H]naloxone (52 Ci/mmol; Amersham Biosciences, Piscataway, NJ) showed no difference
between genotypes in receptor binding (data not shown). It appears that
this enhanced coupling of the µOR in the arr2-KO mice correlates
with the enhanced basal antinociception seen in the tail-immersion
test. However, it should be noted that an enhanced coupling of the
µOR was also detected in periaqueductal gray and brainstem regions of
the mutant mice, and this was not reflected in differences in basal
pain perception as assessed in the hot-plate test (Bohn et al., 1999,
2000b). This apparent difference in the behavioral outcome could
reflect a more complex circuitry underlying central pain processing
systems.
View larger version (19K):
[in this window]
[in a new window]
|
Figure 2.
Enhanced µOR coupling to G-proteins in spinal
cord membranes from arr2-KO mice. Data were analyzed by nonlinear
regression using GraphPad Prism software and are presented as the
mean ± SEM of four experiments performed in triplicate, wherein
WT and arr2-KO spinal cord membranes were assayed simultaneously.
The percentage of agonist-stimulated binding over basal is expressed.
In the absence of agonist stimulation, basal
[35S]GTPS binding was as follows: WT, 1835 ± 251 cpm; arr2-KO, 1675 ± 223 cpm. The curves are
significantly different (p < 0.001)
compared with two-way ANOVA. DAMGO,
D-Ala2-N-Me-Phe4-Glycol5]enkephalin.
|
|
Antagonism of basal antinociception
To assess whether the increased tail-withdrawal latency was
mediated via the µOR, mice were treated with opioid receptor
antagonists. Naltrexone (2 mg/kg, i.p.) was used to reverse the
tail-flick latencies of the arr2-HT and -KO mice to the same levels
experienced by the WT mice (Fig.
3A). This effect was also seen
at water temperatures of 42 and 54°C (data not shown). Furthermore, a
fourfold lower dose of naltrexone reduced tail-flick latencies in the
arr2-KO mice (KO basal, 7.8 ± 1.2; KO plus NTX, 4.2 ± 0.2 sec; n = 7), whereas WT mice were not affected by
this dose (WT basal, 4.4 ± 0.8; WT plus NTX, 4.3 ± 0.3 sec;
n = 7). Naltrexone blocks µ,  , and  opioid
receptors; however, because there is not a highly selective µOR
antagonist that can be administered systemically, it was used in
parallel with antagonists selective for or opioid receptors
(Negus et al., 1993). Naltrindole ( selective; 2.5 mg/kg, s.c.) and
nor-binaltorphamine ( selective; 5 mg/kg, s.c.), which inhibit -
and -mediated antinociception (Matthes et al., 1998), did not affect
the basal tail-flick latencies in either genotype (Fig. 3B),
demonstrating that the enhanced basal antinociception is mediated
through the µOR.
View larger version (15K):
[in this window]
[in a new window]
|
Figure 3.
Antagonist reversal of enhanced
basal tail-flick latencies. Antagonists were injected 20 min before the
tail-flick test was performed. Water temperature was 48°C.
A, Naltrexone (2 mg/kg, i.p.) antagonizes tail-flick
latencies of arr2-KO and arr2+/ to that experienced by WT mice.
B, Naltrindole (2.5 mg/kg, s.c.) and nor-BNI (5 mg/kg,
s.c.) did not affect the enhanced latencies seen in the arr2-KO
mice; *p < 0.01 and **p < 0.001 versus WT basal; #p < 0.01 and
§p < 0.05 versus arr2+/ basal;
and ##p < 0.001 versus arr2-KO
basal; one-way ANOVA followed by Bonferroni's multiple comparison
test; n = 6-8 mice per genotype.
|
|
Morphine-induced antinociception
The arr2-KO mice display an increased sensitivity to morphine
in the hot-plate antinociception test (Bohn et al., 1999); therefore,
morphine-induced antinociception was assessed in the tail-immersion
test as well. Again, arr2-KO mice experience a more prolonged and
enhanced antinociceptive response compared with WT littermates after a
single morphine (10 mg/kg, s.c.) injection (Fig.
4). The data have been normalized to the
basal latency and the artificial ceiling of 30 sec to prevent
overexposure of the tail to the warm water. By normalizing to the basal
latency of each mouse, the effect of morphine can be considered over
the differences already seen in the basal latencies. Furthermore, the
tests with morphine were conducted at 54°C, a temperature at which
the smallest differences between WT and arr2-KO mice are seen.
View larger version (14K):
[in this window]
[in a new window]
|
Figure 4.
Morphine induces enhanced and prolonged tail-flick
latencies in arr2-KO mice. Morphine (10 mg/kg, s.c.) was injected,
and tail-withdrawal latencies were recorded 15, 30, 45, 60, 90, and 120 min later (54°C water). The genotypes are significantly different
(p < 0.0001) by two-way ANOVA;
n = 7 mice per genotype.
|
|
Morphine-induced antinociceptive tolerance
A disruption of µOR desensitization by elimination of arr2
results in a lack of morphine antinociceptive tolerance in the hot-plate test, as demonstrated previously in the arr2-KO mice (Bohn
et al., 2000b). This lack of antinociceptive tolerance was apparent in
three separate regimens of morphine treatment, which demonstrated that
the arr2-KO mice do not become tolerant after an acute challenge of
a large dose of morphine, nor do they develop tolerance after chronic
administration of the drug. The underlying circuitry regulating the
perception of pain in the warm water tail-immersion and hot-plate tests
is likely to differ (LeBars et al., 1976; Mayer and Price, 1976;
Mansour et al., 1988; Yaksh, 1997). Moreover, the contribution of
arr2 to each of these systems may also differ, as can be observed in
their basal pain response latencies in the two tests, wherein the
mutant mice only displayed an increased latency in the
tail-flick test. Given the potential for differences in the regulation
of these systems, the development of morphine tolerance was also
evaluated in the tail-immersion test.
Twenty-four hours after an acute challenge with either saline or a high
dose of morphine (100 mg/kg, s.c.), mice were assessed for their
responsiveness to the 54°C water tail immersion 30 min after morphine
(10 mg/kg, s.c.) treatment. As was observed previously in the hot-plate
test, the WT mice develop substantial tolerance to subsequent morphine
treatment, whereas the arr2-KO mice experience the full
antinociceptive effects of morphine in the tail-immersion test
regardless of whether they received saline or a large dose of morphine
the day before (Fig. 5). This absence of
the development of acute morphine tolerance suggests that, as in the
hot-plate test, arr2 plays an essential role in regulating µOR
responsiveness in the development of acute tolerance.
View larger version (16K):
[in this window]
[in a new window]
|
Figure 5.
arr2-KO mice do not develop acute morphine
tolerance. Mice were pretreated with either saline or morphine (100 mg/kg, s.c.), and 24 hr later, when they had returned to their basal
latencies, mice were administered morphine (10 mg/kg, s.c.).
Tail-withdrawal latencies (54°C water) were assessed 30 min after
this second injection. Although the WT mice experience tolerance 24 hr
after receiving the first injection of morphine
(p < 0.001 compared with saline
pretreatment), the arr2-KO mice do not. The arr2-KO mice
experience greater antinociception after morphine than WT mice
regardless of whether they were pretreated with morphine or saline
(p < 0.001). Analysis is by one-way ANOVA
followed by Bonferroni's multiple comparison test;
n = 6 mice per genotype.
|
|
The mechanisms underlying the development of acute and chronic morphine
tolerance have been considered to be the same (Fairbanks and Wilcox,
1997). However, when considering tolerance in direct relation to
desensitization of the receptor, the development of acute tolerance is
likely to be fundamentally different from chronic tolerance caused
by the desensitization requirements of a repeated low dose of
drug versus an immediate need to compensate for a single challenge with
an extremely high concentration of drug. The cellular adaptations
required to compensate for the challenge of a high concentration of
drug might differ at the receptor level from the cellular adaptations
required to compensate for repeated administrations of a lower dose of
drug. Therefore, we treated animals with morphine (10 mg/kg, s.c.) on a
daily basis and monitored their responsiveness in the tail-immersion
test. Although the onset of morphine tolerance is readily apparent in
WT mice after the third day of morphine treatment, the arr2-KO mice
still experience the full effect of morphine over this period.
Interestingly, after the fourth day of treatment, the arr2-KO mice
abruptly begin to lose their sensitivity to morphine (Fig.
6A). By the fifth day,
the arr2-KO mice are no different from the WT mice in their responsiveness to morphine, and both mice appear to be completely tolerant to morphine (10 mg/kg, s.c.) in the tail-immersion test throughout the seventh day. This was surprising, because the arr2-KO mice failed to develop morphine tolerance even after 9 d of
consecutive morphine administration in the hot-plate test paradigm. In
this experiment, the same mice that showed no responsiveness to
morphine in the tail-immersion test on the seventh day were also
subjected to the 56°C hot-plate test. Although the arr2-KO mice
experience tolerance in the tail-immersion test, they still experience
the antinociceptive effects of morphine in the hot-plate test (Fig. 6A, inset).
View larger version (23K):
[in this window]
[in a new window]
|
Figure 6.
Chronic morphine tolerance in WT and arr2-KO
mice. A, Mice received morphine (10 mg/kg, s.c.) each
day; 30 min later, tail-flick latencies were recorded (54°C water).
arr2-KO experience significantly enhanced morphine antinociception
compared with WT mice for the first 4 d (*p < 0.001; #p < 0.005), with a significant
decrease on days 4 and 5 (p < 0.02).
Student's t test; n = 7 mice per
genotype. Inset, On day 7, mice were placed on the hot
plate (56°C), and paw-withdrawal latencies were recorded.
B, A morphine dose-response curve was generated in
animals on day 1 and again after 7 d of daily morphine treatments.
In these studies, mice were not tested daily for antinociceptive
latencies. Some points at the lower doses are the averaged responses
after cumulative dosing of morphine as described previously (Bohn et
al., 2000a). These points were similar to results obtained with an
acute dose of the drug and were therefore averaged together.
ED50 values were calculated via nonlinear regression
analysis (GraphPad Prism), and 95% confidence intervals are as
follows: day 1 WT, 10.3 (7.6-13.7); arr2-KO, 4.6 (4.5-4.8); day 7 WT, 75.8 (51.3-112.1); arr2-KO, 34.5 (33.8-35.3) mg/kg.
|
|
To ascertain whether there is a difference in the degree of tolerance
developed between genotypes, a dose-response curve was generated on
days 1 and 7 of the chronic morphine tail-immersion regimen (Fig.
6B). On day 1, it is apparent that the arr2-KO mice are approximately twofold more sensitive to morphine than the WT
mice; this observation was also consistent with the hot-plate test
results (Bohn et al., 2000b). However, after chronic morphine treatment, the knock-out mice have developed morphine tolerance, as
have the WT mice. Although the arr2-KO mice, like the WT mice, experience tolerance to morphine, the concentration required to reach
half-maximal antinociception in the WT mice was considerably higher
than in the mutant mice. This suggests that although the arr2-KO
mice have become tolerant in the tail-flick assay, they have not
reached the same extent of tolerance as the WT mice.
By examining the antinociceptive properties of morphine using the
hot-plate and tail-immersion paradigms, we find that in both cases,
mice lacking arr2 are more sensitive to morphine. After chronic
morphine treatment, arr2-KO mice do not develop tolerance to the hot
plate; however, they do experience tolerance, albeit to a lesser
extent, in the tail-immersion test. These differences suggest that
perhaps in the spinal reflex test, there is another, arr2-independent regulatory mechanism that might contribute to the
modulation of morphine-induced antinociception. The concept that other
regulatory components could contribute to desensitizing the receptor
after chronic morphine is attractive and perhaps consistent with the
delayed onset of tolerance until the fourth day of treatment. Moreover,
mechanisms other than classical µOR desensitization have been
suggested previously for the development of morphine tolerance.
However, the arr2-KO mice do not develop the same degree of
tolerance as the WT mice, suggesting that the arr2-mediated
desensitization of the receptor may be the predominant mechanism, yet
other contributions to morphine tolerance clearly must be present.
Reversal of morphine tolerance in knock-out mice
One potential mechanism involves PKC activation, which has been
shown to contribute to µOR responsiveness. In several studies, PKC
inhibitors have been used to block the development of morphine tolerance in mice (Narita et al., 1995; Granados-Soto et al., 2000;
Inoue and Ueda, 2000). Because PKC inhibitors are not isoform selective, it has been difficult to attribute the actions of these inhibitors to a specific form of PKC. Recently, Zeitz et al. (2001) reported that mice lacking the PKC isoform experience slightly yet
significantly attenuated morphine tolerance in the tail-immersion test.
The fact that the PKC-KO mice still experience a significant degree
of tolerance could be attributable to the dominance of the still intact
arr2-mediated desensitization of the receptor. To test whether PKC
is contributing to the delayed and attenuated development of morphine
tolerance in the arr2-KO mice, we treated mice after 7 d of
chronic morphine (10 mg/kg, s.c., daily) with the PKC inhibitor
chelerythrine. When given 10 min before morphine, chelerythrine was
able to completely reverse morphine tolerance in the arr2-KO mice,
while having little to no effect on the tolerant WT mice (Fig.
7A).
View larger version (13K):
[in this window]
[in a new window]
|
Figure 7.
Chelerythrine reverses morphine
tolerance in arr2-KO mice. A, Mice that had been
treated for 7 d with morphine were then given one of the following
combinations: vehicle for 10 min plus morphine (10 mg/kg, s.c.) for 30 min, chelerythrine (5 mg/kg, i.p.) for 10 min plus vehicle for 30 min,
or chelerythrine (5 mg/kg, i.p.) for 10 min plus morphine (10 mg/kg,
s.c.) for 30 min. At the end of the 40 min total treatment time,
antinociceptive latencies were tested (54°C water).
**p < 0.001 versus all points; one-way ANOVA
followed by Bonferroni's multiples comparison test;
n = 6 (Morphine);
n = 3 (Chelerythrine);
n = 9 (Chelerythrine + Morphine).
B, Naive mice were treated with either vehicle for 10 min plus morphine (5 mg/kg, s.c.) for 30 min or chelerythrine (5 mg/kg, i.p.) for 10 min plus morphine (5 mg/kg, s.c.) for 30 min before
antinociceptive testing. **p < 0.001 versus WT;
n = 5-6, as assessed in A.
|
|
In previous studies in which PKC inhibitors have been reported to
reverse morphine tolerance, the drug was administered intrathecally (Granados-Soto et al., 2000; Inoue and Ueda, 2000) or
intracerebroventricularly (Smith et al., 1999). In the present study,
the dose of chelerythrine (5 mg/kg) was given intraperitoneally;
therefore, it is not surprising that the low concentration of the
inhibitor reaching the spinal sites is not sufficient to reverse
tolerance in the WT mice, wherein the arr2-mediated desensitization
remains intact. The effect of chelerythrine alone was also tested in
tolerant and naive animals. Chelerythrine did not affect
tail-withdrawal latencies in either genotype when given alone to
tolerant mice (Fig. 7A). To verify that chelerythrine does
not synergize with morphine to produce antinociception in the
arr2-KO mice, we tested chelerythrine with morphine (5 mg/kg, s.c.).
The lower dose of morphine was chosen to ensure that in the case of
synergism, the ceiling would not be reached; however, there is no
effect of chelerythrine on morphine-induced antinociception (Fig.
7B). Therefore, in the absence of the arr2-mediated
desensitization, the contributions of other systems, possibly
downstream of the immediate regulation of the µOR, become readily
apparent. Our data suggest that PKC activity contributes to the
desensitization of the µOR and hence to the delayed development of
morphine tolerance in the tail-flick assay in the arr2-KO mice.
| |
DISCUSSION |
The delayed tail-flick response latency displayed by the
arr2-KO mice represents the first phenotype reported in the mutant mice that occurs in the absence of agonist challenge. Although the
latency of the tail-flick response is prolonged in the mutant mice, the
response to the hot plate remains the same between the littermates
(Fig. 1). It should also be noted that there is a gene-dosage effect,
as can be seen in the delayed tail-withdrawal responses in the
heterozygotes. The enhanced tail-flick latency seen in the arr2-KO
mice correlates with increased µOR-G-protein coupling in membranes
prepared from spinal cord (Fig. 2), implying that the extended response
latencies in the warm water tail-immersion test may be caused by
increased activity at spinal µORs. This concept is supported by the
ability of naltrexone, but not nor-BNI or naltrindole, to inhibit this
effect in the knock-out animals (Fig. 3).
The arr2-KO mice represent an animal model in which the
morphine-induced desensitization of the µOR has been significantly impaired because of the loss of arr2. In addition to displaying an
enhanced and prolonged response to morphine in both hot-plate and
tail-immersion tests (Fig. 4) for pain perception, the arr2-KO mice
also do not develop acute tolerance after a single challenge of a large
dose of morphine in both tests (Fig. 5). However, after chronic daily
morphine administration, although the KO mice do not demonstrate
morphine tolerance in the hot-plate test, they do develop tolerance,
although to a lesser degree than WT mice, in the tail-immersion test
(Fig. 6). The enhanced and prolonged antinociception after a single
dose of morphine, the lack of acute tolerance in the arr2-KO mice,
and the fact that they show no sign of decreased morphine
responsiveness in the first 3 d of treatment suggest that arr2
is predominantly involved in the normal desensitization of the µOR
when drug is present. However, on the fourth day of morphine treatment,
the arr2-KO mice show the first signs of tolerance, and by the fifth
day, the mice no longer respond to 10 mg/kg morphine in the
tail-immersion test. This delayed onset of the development of tolerance
in the mutant mice implies the intervention of another mechanism.
There is good evidence supporting a role for PKC in morphine tolerance.
Several studies have shown that PKC levels and activity are increased
after chronic opiate treatment. Specifically, PKC and PKC
activity and expression were increased in rat dorsal horn membrane
preparations after chronic morphine; furthermore, the increased
activity could be blocked by chelerythrine after the chronic treatment
(Granados-Soto et al., 2000). Li and Roerig (1999) reported enhanced
PKC catalytic activity in mouse spinal cord after chronic morphine
treatment. In addition, studies in mice lacking PKC revealed a
modest attenuation of morphine tolerance in the tail-immersion test. In
these studies, because the degree of attenuation was small, these
authors suggested that other PKC isoforms, which are still present in
these mice, might be contributing to the induced tolerance as well. Our
data suggest that perhaps the remaining tolerance expressed by the
PKC-KO mice is not simply attributable to remaining PKC activity but
is much more likely the result of arr2-mediated desensitization of
the receptors, which is still intact in these mice.
It is not known whether PKC regulation of the µOR occurs via direct
phosphorylation of the receptor (Zhang et al., 1996) or whether the
kinase serves as an intermediate in another neuronal signaling system.
For example, increased PKC activity facilitates NMDA receptor
signaling. Glutamatergic activation of NMDA receptors is involved in
morphine tolerance, and the blockade of this system by NMDA receptor
antagonists, such as (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]
cyclohepten-5,10-imine maleate (MK-801), can attenuate the development of morphine tolerance (Trujillo and Akil, 1991; Gutstein and Trujillo, 1993; Allen and Dykstra, 1999). Fan et al. (1998) have
proposed that the activation of NMDA receptors enhances PKC activation
and the subsequent desensitization of opioid receptors in NG108-15
cells and in cultured neurons. PKC activation could also play a role in
the opioid receptor-mediated effects on tolerance (Vanderah et al.,
2001), because the receptor has been shown to signal through PKC
(Barg et al., 1993; Bohn et al., 2000a). Although we do not know at
what point PKC regulation of µOR signaling comes into play, the
importance of arr2-mediated receptor regulation in the development
of morphine tolerance is once again demonstrated. It is only in the
absence of arr2 that the role for chelerythrine-sensitive PKC
regulation of the µOR becomes readily apparent.
Mice lacking arr2 exhibit substantially enhanced morphine
responsiveness either in the hot-plate test or the tail-immersion antinociceptive test. Our previous study demonstrated that in the
absence of arr2, mice did not develop tolerance regardless of
whether morphine was given by acute, daily chronic, or constant chronic
administration (Bohn et al., 2000b). In this study, we have found that
µOR response to the antinociceptive drug morphine can be
alternatively regulated in different pain perception systems. It would
appear that the arr2-mediated mechanism of desensitization is the
major contributor to the development of tolerance in both supraspinal
and spinal systems. However, abrogation of this dominant regulatory
mechanism clearly reveals the contribution of a PKC-mediated mechanism
underlying the development of morphine tolerance in spinal systems.
| |
FOOTNOTES |
Received June 18, 2002; revised Sept. 19, 2002; accepted Sept. 20, 2002.
This work was supported by the National Institutes of Health, National
Institute on Drug Abuse Grants DA-14600 (L.M.B) and DA-13115 (M.G.C.).
M.G.C. and R.J.L are investigators of the Howard Hughes Medical
Institute. We thank S. Suter for care and genotyping of the mouse
colony and L. Dykstra and R. Gainetdinov for constructive reading of
this manuscript.
Correspondence should be addressed to Dr. Marc G. Caron, Duke
University Medical Center, Box 3287, Room 487 CARL, Research Drive,
Durham, NC 27710. E-mail: m.caron{at}cellbio.duke.edu.
| |
REFERENCES |
-
Allen RM,
Dykstra LA
(1999)
The competitive NMDA receptor antagonist LY235959 modulates the progression of morphine tolerance in rats.
Psychopharmacology (Berl)
142:209-214[Medline].
-
Barg J,
Belcheva MM,
Rowinski J,
Coscia CJ
(1993)
kappa-Opioid agonist modulation of [3H]thymidine incorporation into DNA: evidence for the involvement of pertussis toxin-sensitive G protein-coupled phosphoinositide turnover.
J Neurochem
60:1505-1511[Web of Science][Medline].
-
Bohn LM,
Lefkowitz RJ,
Gainetdinov RR,
Peppel K,
Caron MG,
Lin FT
(1999)
Enhanced morphine analgesia in mice lacking beta-arrestin 2.
Science
286:2495-2498[Abstract/Free Full Text].
-
Bohn LM,
Belcheva MM,
Coscia CJ
(2000a)
Mitogenic signaling via endogenous kappa-opioid receptors in C6 glioma cells: evidence for the involvement of protein kinase C and the mitogen-activated protein kinase signaling cascade.
J Neurochem
74:564-573[Web of Science][Medline].
-
Bohn LM,
Gainetdinov RR,
Lin FT,
Lefkowitz RJ,
Caron MG
(2000b)
Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence.
Nature
408:720-723[Medline].
-
Bohn LM,
Xu F,
Gainetdinov RR,
Caron MG
(2000c)
Potentiated opioid analgesia in norepinephrine transporter knock-out mice.
J Neurosci
20:9040-9045[Abstract/Free Full Text].
-
Cesselin F,
Benoliel J-J,
Bourgoin S,
Collin E,
Pohl M,
Hamon M
(1999)
Spinal mechanisms of opioid analgesia.
In: Opioids in pain control: basic and clinical aspects (Stein C,
ed), pp 70-95. New York: Cambridge UP.
-
Fairbanks CA,
Wilcox GL
(1997)
Acute tolerance to spinally administered morphine compares mechanistically with chronically induced morphine tolerance.
J Pharmacol Exp Ther
282:1408-1417[Abstract/Free Full Text].
-
Fan GH,
Zhao J,
Wu YL,
Lou LG,
Zhang Z,
Jing Q,
Ma L,
Pei G
(1998)
N-Methyl-D-aspartate attenuates opioid receptor-mediated G protein activation and this process involves protein kinase C.
Mol Pharmacol
53:684-690[Abstract/Free Full Text].
-
Ferguson SS,
Zhang J,
Barak LS,
Caron MG
(1998)
Molecular mechanisms of G protein-coupled receptor desensitization and resensitization.
Life Sci
62:1561-1565[Web of Science][Medline].
-
Granados-Soto V,
Kalcheva I,
Hua X,
Newton A,
Yaksh TL
(2000)
Spinal PKC activity and expression: role in tolerance produced by continuous spinal morphine infusion.
Pain
85:395-404[Web of Science][Medline].
-
Gutstein HB,
Trujillo KA
(1993)
MK-801 inhibits the development of morphine tolerance at spinal sites.
Brain Res
626:332-334[Web of Science][Medline].
-
Heinricher MM,
Morgan MM
(1999)
Supraspinal mechanisms of opioid analgesia.
In: Opioids in pain control: basic and clinical aspects (Stein C,
ed), pp 46-69. New York: Cambridge UP.
-
Inoue M,
Ueda H
(2000)
Protein kinase C-mediated acute tolerance to peripheral mu-opioid analgesia in the bradykinin-nociception test in mice.
J Pharmacol Exp Ther
293:662-669[Abstract/Free Full Text].
-
Janssen PA,
Niemegeers CJE,
Dony JGH
(1963)
The inhibitory effect of fentanyl and other morphine-like analgesics on the warm water induced tail withdrawal reflex in rats.
Arzneimittelforschung
13:502-507[Medline].
-
Kieffer BL
(1999)
Opioids: first lessons from knockout mice.
Trends Pharmacol Sci
20:19-26[Medline].
-
Krupnick JG,
Benovic JL
(1998)
The role of receptor kinases and arrestins in G protein-coupled receptor regulation.
Annu Rev Pharmacol Toxicol
38:289-319[Web of Science][Medline].
-
LeBars D,
Menetrey D,
Besson JM
(1976)
Effects of morphine upon the lamina V type cells activities in the dorsal horn of the decerebrate cat.
Brain Res
113:293-310[Medline].
-
Lefkowitz RJ
(1998)
G protein-coupled receptors. III. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization.
J Biol Chem
273:18677-18680[Free Full Text].
-
Li Y,
Roerig SC
(1999)
Alteration of spinal protein kinase C expression and kinetics in morphine, but not clonidine, tolerance.
Biochem Pharmacol
58:493-501[Medline].
-
Lowe JD,
Celver JP,
Gurevich VV,
Chavkin C
(2002)
Mu opioid receptors desensitize less rapidly than delta opioid receptors due to less efficient activation of arrestin.
J Biol Chem
277:15729-15735[Abstract/Free Full Text].
-
Mansour A,
Khachaturian H,
Lewis ME,
Akil H,
Watson SJ
(1988)
Anatomy of CNS opioid receptors.
Trends Neurosci
11:308-314[Web of Science][Medline].
-
Matthes HW,
Smadja C,
Valverde O,
Vonesch JL,
Foutz AS,
Boudinot E,
Denavit-Saubie M,
Severini C,
Negri L,
Roques BP,
Maldonado R,
Kieffer BL
(1998)
Activity of the delta-opioid receptor is partially reduced, whereas activity of the kappa-receptor is maintained in mice lacking the µ-receptor.
J Neurosci
18:7285-7295[Abstract/Free Full Text].
-
Mayer DJ,
Price DD
(1976)
Central nervous system mechanisms of analgesia.
Pain
2:379-404[Web of Science][Medline].
-
Narita M,
Mizoguchi H,
Tseng LF
(1995)
Inhibition of protein kinase C, but not of protein kinase A, blocks the development of acute antinociceptive tolerance to an intrathecally administered mu-opioid receptor agonist in the mouse.
Eur J Pharmacol
280:R1-R3[Web of Science][Medline].
-
Negus SS,
Henriksen SJ,
Mattox A,
Pasternak GW,
Portoghese PS,
Takemori AE,
Weinger MB,
Koob GF
(1993)
Effect of antagonists selective for mu, delta and kappa opioid receptors on the reinforcing effects of heroin in rats.
J Pharmacol Exp Ther
265:1245-1252[Abstract/Free Full Text].
-
Noble F,
Cox BM
(1996)
Differential desensitization of mu and delta opioid receptors in selected neural pathways following chronic morphine treatment.
Br J Pharmacol
117:161-169[Web of Science][Medline].
-
Smith FL,
Lohmann AB,
Dewey WL
(1999)
Involvement of phospholipid signal transduction pathways in morphine tolerance in mice.
Br J Pharmacol
128:220-226[Web of Science][Medline].
-
Sora I,
Takahashi N,
Funada M,
Ujike H,
Revay RS,
Donovan DM,
Miner LL,
Uhl GR
(1997)
Opiate receptor knockout mice define mu receptor roles in endogenous nociceptive responses and morphine-induced analgesia.
Proc Natl Acad Sci USA
94:1544-1549[Abstract/Free Full Text].
-
Stone LS,
MacMillan LB,
Kitto KF,
Limbird LE,
Wilcox GL
(1997)
The 2a adrenergic receptor subtype mediates spinal analgesia evoked by 2 agonists and is necessary for spinal adrenergic-opioid synergy.
J Neurosci
17:7157-7165[Abstract/Free Full Text].
-
Trujillo KA,
Akil H
(1991)
Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801.
Science
251:85-87[Abstract/Free Full Text].
-
Vanderah TW,
Suenaga NM,
Ossipov MH,
Malan Jr TP,
Lai J,
Porreca F
(2001)
Tonic descending facilitation from the rostral ventromedial medulla mediates opioid-induced abnormal pain and antinociceptive tolerance.
J Neurosci
21:279-286[Abstract/Free Full Text].
-
Whistler JL,
von Zastrow M
(1998)
Morphine-activated opioid receptors elude desensitization by beta-arrestin.
Proc Natl Acad Sci USA
95:9914-9919[Abstract/Free Full Text].
-
Yaksh TL
(1997)
Pharmacology and mechanisms of opioid analgesic activity.
Acta Anaesthesiol Scand
41:94-111[Web of Science][Medline].
-
Zeitz KP,
Malmberg AB,
Gilbert H,
Basbaum AI
(2001)
Reduced development of tolerance to the analgesic effects of morphine and clonidine in PKC gamma mutant mice.
Pain
94:245-253[Medline].
-
Zhang J,
Ferguson SS,
Barak LS,
Bodduluri SR,
Laporte SA,
Law PY,
Caron MG
(1998)
Role for G protein-coupled receptor kinase in agonist-specific regulation of mu-opioid receptor responsiveness.
Proc Natl Acad Sci USA
95:7157-7162[Abstract/Free Full Text].
-
Zhang L,
Yu Y,
Mackin S,
Weight FF,
Uhl GR,
Wang JB
(1996)
Differential mu opiate receptor phosphorylation and desensitization induced by agonists and phorbol esters.
J Biol Chem
271:11449-11454[Abstract/Free Full Text].
Copyright © 2002 Society for Neuroscience 0270-6474/02/222310494-07$05.00/0
This article has been cited by other articles:
|
|
|
|
 
V. C. Dang, I. A. Napier, and M. J. Christie
Two Distinct Mechanisms Mediate Acute {micro}-Opioid Receptor Desensitization in Native Neurons
J. Neurosci.,
March 11, 2009;
29(10):
3322 - 3327.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Labrie, S. Duffy, W. Wang, S. W. Barger, G. B. Baker, and J. C. Roder
Genetic inactivation of D-amino acid oxidase enhances extinction and reversal learning in mice
Learn. Mem.,
December 30, 2008;
16(1):
28 - 37.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. L. Mathews, A. V. Smrcka, and J. M. Bidlack
A Novel G{beta}{gamma}-Subunit Inhibitor Selectively Modulates {micro}-Opioid-Dependent Antinociception and Attenuates Acute Morphine-Induced Antinociceptive Tolerance and Dependence
J. Neurosci.,
November 19, 2008;
28(47):
12183 - 12189.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. L. Schmid, K. M. Raehal, and L. M. Bohn
From the Cover: Agonist-directed signaling of the serotonin 2A receptor depends on {beta}-arrestin-2 interactions in vivo
PNAS,
January 22, 2008;
105(3):
1079 - 1084.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Zheng, H. H. Loh, and P.-Y. Law
-Arrestin-Dependent {micro}-Opioid Receptor-Activated Extracellular Signal-Regulated Kinases (ERKs) Translocate to Nucleus in Contrast to G Protein-Dependent ERK Activation
Mol. Pharmacol.,
January 1, 2008;
73(1):
178 - 190.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z.-Q. Zhao, Y.-J. Gao, Y.-G. Sun, C.-S. Zhao, R. W. Gereau IV, and Z.-F. Chen
Central serotonergic neurons are differentially required for opioid analgesia but not for morphine tolerance or morphine reward
PNAS,
September 4, 2007;
104(36):
14519 - 14524.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Fernandez-Duenas, O. Pol, P. Garcia-Nogales, L. Hernandez, E. Planas, and M. M. Puig
Tolerance to the Antinociceptive and Antiexudative Effects of Morphine in a Murine Model of Peripheral Inflammation
J. Pharmacol. Exp. Ther.,
July 1, 2007;
322(1):
360 - 368.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. A. Berg, A. M. Patwardhan, T. A. Sanchez, Y. M. Silva, K. M. Hargreaves, and W. P. Clarke
Rapid Modulation of {micro}-Opioid Receptor Signaling in Primary Sensory Neurons
J. Pharmacol. Exp. Ther.,
June 1, 2007;
321(3):
839 - 847.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Walwyn, C. J. Evans, and T. G. Hales
{beta}-Arrestin2 and c-Src Regulate the Constitutive Activity and Recycling of {micro} Opioid Receptors in Dorsal Root Ganglion Neurons
J. Neurosci.,
May 9, 2007;
27(19):
5092 - 5104.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. E. Groer, K. Tidgewell, R. A. Moyer, W. W. Harding, R. B. Rothman, T. E. Prisinzano, and L. M. Bohn
An Opioid Agonist that Does Not Induce {micro}-Opioid Receptor--Arrestin Interactions or Receptor Internalization
Mol. Pharmacol.,
February 1, 2007;
71(2):
549 - 557.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Li, M. L. Lee, M. R. Bruchas, G. C. Chan, D. R. Storm, and C. Chavkin
Calmodulin-Stimulated Adenylyl Cyclase Gene Deletion Affects Morphine Responses
Mol. Pharmacol.,
November 1, 2006;
70(5):
1742 - 1749.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Puckerin, L. Liu, N. Permaul, P. Carman, J. Lee, and M. A. Diverse-Pierluissi
Arrestin Is Required for Agonist-induced Trafficking of Voltage-dependent Calcium Channels
J. Biol. Chem.,
October 13, 2006;
281(41):
31131 - 31141.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. M. Raehal, J. K. L. Walker, and L. M. Bohn
Morphine Side Effects in {beta}-Arrestin 2 Knockout Mice
J. Pharmacol. Exp. Ther.,
September 1, 2005;
314(3):
1195 - 1201.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Zuo
The Role of Opioid Receptor Internalization and {beta}-Arrestins in the Development of Opioid Tolerance
Anesth. Analg.,
September 1, 2005;
101(3):
728 - 734.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Clark and J. R. Traynor
Endogenous Regulator of G Protein Signaling Proteins Reduce {micro}-Opioid Receptor Desensitization and Down-Regulation and Adenylyl Cyclase Tolerance in C6 Cells
J. Pharmacol. Exp. Ther.,
February 1, 2005;
312(2):
809 - 815.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. P. Bailey, E. Kelly, and G. Henderson
Protein Kinase C Activation Enhances Morphine-Induced Rapid Desensitization of {micro}-Opioid Receptors in Mature Rat Locus Ceruleus Neurons
Mol. Pharmacol.,
December 1, 2004;
66(6):
1592 - 1598.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. C. Dang and J. T. Williams
Chronic Morphine Treatment Reduces Recovery from Opioid Desensitization
J. Neurosci.,
September 1, 2004;
24(35):
7699 - 7706.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Tegeder and G. Geisslinger
Opioids As Modulators of Cell Death and Survival--Unraveling Mechanisms and Revealing New Indications
Pharmacol. Rev.,
September 1, 2004;
56(3):
351 - 369.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. M. Bohn, L. A. Dykstra, R. J. Lefkowitz, M. G. Caron, and L. S. Barak
Relative Opioid Efficacy Is Determined by the Complements of the G Protein-Coupled Receptor Desensitization Machinery
Mol. Pharmacol.,
July 1, 2004;
66(1):
106 - 112.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. M. Cox and A. T. Crowder
Receptor Domains Regulating {micro} Opioid Receptor Uncoupling and Internalization: Relevance to Opioid Tolerance
Mol. Pharmacol.,
March 1, 2004;
65(3):
492 - 495.
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Celver, M. Xu, W. Jin, J. Lowe, and C. Chavkin
Distinct Domains of the {micro}-Opioid Receptor Control Uncoupling and Internalization
Mol. Pharmacol.,
March 1, 2004;
65(3):
528 - 537.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. B. Bolger, A. McCahill, E. Huston, Y.-F. Cheung, T. McSorley, G. S. Baillie, and M. D. Houslay
The Unique Amino-terminal Region of the PDE4D5 cAMP Phosphodiesterase Isoform Confers Preferential Interaction with {beta}-Arrestins
J. Biol. Chem.,
December 5, 2003;
278(49):
49230 - 49238.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. M. Bohn, R. R. Gainetdinov, T. D. Sotnikova, I. O. Medvedev, R. J. Lefkowitz, L. A. Dykstra, and M. G. Caron
Enhanced Rewarding Properties of Morphine, but not Cocaine, in {beta}arrestin-2 Knock-Out Mice
J. Neurosci.,
November 12, 2003;
23(32):
10265 - 10273.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. D. Mandyam, D. R. Thakker, and K. M. Standifer
{micro}-Opioid-Induced Desensitization of Opioid Receptor-Like 1 and {micro}-Opioid Receptors: Differential Intracellular Signaling Determines Receptor Sensitivity
J. Pharmacol. Exp. Ther.,
September 1, 2003;
306(3):
965 - 972.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. L. Borgland, M. Connor, P. B. Osborne, J. B. Furness, and M. J. Christie
Opioid Agonists Have Different Efficacy Profiles for G Protein Activation, Rapid Desensitization, and Endocytosis of Mu-opioid Receptors
J. Biol. Chem.,
May 23, 2003;
278(21):
18776 - 18784.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
