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Vol. 293, Issue 2, 662-669, May 2000
Department of Molecular Pharmacology and Neuroscience, Nagasaki University School of Pharmaceutical Sciences, Nagasaki, Japan
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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We studied the acute tolerance liability of peripheral
opioid analgesia in mice. The analgesia was assessed by the inhibition of bradykinin (BK)-induced nociceptive action by using a newly developed flexor reflex paradigm. Morphine [intraplantarly
(i.pl.)] given ipsilaterally to BK showed a dose-dependent
reduction of the BK (2 pmol) responses, whereas the administration of
10 nmol of morphine into the contralateral side failed to show any
significant analgesic effects. Furthermore, DAMGO
([D-Ala2,MePhe4,Gly-ol5]-enkephalin),
a µ-opioid receptor (MOR) agonist, and U-69593, a 
-opioid receptor
(KOR) agonist, but not DSLET
([D-Ser2]Leu-enkephalin-Thr6), a
-opioid receptor agonist, showed similar analgesia on the BK
responses. The morphine- or U-69593
[(5
,7,8
)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8yl] benzeneacetamide]-induced analgesia was markedly attenuated by the intrathecal injection of each antisense oligodeoxynucleotide for
the MOR or KOR, respectively, suggesting that these peripheral analgesia are mediated through MORs and KORs located on nociceptor endings, respectively. As BK response was completely recovered to the control level 4 h after morphine (3 nmol i.pl.) or U-69593 (10 nmol i.pl.) administration, these compounds were challenged again
to see the inhibition of BK responses. Although morphine analgesia by
the second challenge was markedly attenuated, U-69593 analgesia was
not. The attenuated morphine analgesia was completely reversed by the
pretreatment of calphostin C, Go6976, or HBDDE, a protein kinase
C inhibitor, but not by KT-5720, a protein kinase A inhibitor. These
results suggest that selective acute tolerance of peripheral morphine
analgesia, but not U-69593 analgesia, through MORs and KORs located on
polymodal nociceptors, respectively, in the bradykinin-nociception test
in mice was mediated through protein kinase C activation.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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It
is accepted that opioid analgesia is particularly related to the
activation of opioid receptors within the central nervous system, such
as periaqueductal gray matter (Hosobuchi et al., 1977
), nucleus
reticularis para-gigantocellularis (Takagi, 1980), nucleus raphe
magnus (Dickenson et al., 1979), and dorsal horn of spinal cord (Yeung
and Rudy, 1980). However, there are also accumulating reports
that opioids exert potent peripheral analgesia (Khasar et al., 1996;
Kolesnikov et al., 1996a,b; Zhou et al., 1998). Indeed, opioid
receptors were found on peripheral nerve endings (nociceptor endings)
of thinly myelinated (A-fiber) and unmyelinated (C-fiber) sensory
neurons in animals (Hassan et al., 1993; Stein, 1995), and
significant amounts of opioid receptor mRNA were also found in the
dorsal root ganglia (Schafer et al., 1995).
In many nociception tests to evaluate analgesic actions, various
mechanical and thermal stimulations have been used. Although these
stimulations are supposed to directly drive mechanothermal A- and
polymodal C-fiber nociceptors, they are also expected to drive indirect
mechanisms, including a release of pain-producing substances such as
bradykinin (BK), histamine, serotonin, ATP, and potassium ion (for a
review, see Ueda, 1999). Therefore, it is difficult to identify or
discuss the molecular basis of mechanism in pain production and
analgesia. Recently, we developed a simple and sensitive peripheral
nociception test in mice (Inoue et al., 1998a; Ueda, 1999), where we
assessed the flexor responses in mice after the local administration of
a pain-producing substance, such as BK or substance P. Because BK is
known to stimulate C-fiber nociceptors, the analgesia evaluated as an
inhibition of BK responses would be more likely attributed to the
action on polymodal nociceptors. The fact that the cell body of sensory
neurons is located in the distance from nociceptor endings gives some
advantages to this paradigm of nociception test, because the selective
reduction of expression of specific molecules in nociceptors can be
performed by intrathecal injection with antisense
oligodeoxynucleotide (AS-ODN; Ueda, 1999) and the signaling mechanisms
in nociceptor endings without effects on the cell body can be discussed.
Prolonged and repeated exposure to opioid agonists reduces the
responsiveness of opioid receptors to its agonist over time. This loss
of receptor function was hypothesized to contribute to the opiate
tolerance, dependence, and addiction in humans (Nestler, 1992).
Substantial experimental evidence has divided this loss of function
into separate but related receptor events: 1) down-regulation, 2)
desensitization, and 3) internalization. The mechanisms of opioid
tolerance observed in cell culture studies have been well discussed,
but little is known of such mechanisms in in vivo studies of opioid
analgesia. Here, we demonstrate that some types of opioid receptors may
be involved in the peripheral analgesia through an inhibition of BK
stimulation of polymodal nociceptors in mice and the selective acute
tolerance to the peripheral analgesia of morphine (a µ-opioid
agonist), but not U-69593-induced (a -opioid agonist) one through a
protein kinase C (PKC)-mediated mechanisms.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals.
Male ddY-strain mice weighing from 20 to 22 g
were kept in a room maintained at 21 ± 2°C with free access to
a standard laboratory diet (MF; Oriental Yeast, Tokyo, Japan) and tap
water. Procedures were approved by the Nagasaki University Animal Care
Committee and amplified with the recommendations of the International
Association for the Study of Pain (Zimmermann, 1983).
Drugs.
The drugs used were morphine (obtained from Takeda
Chemical Industries, Osaka, Japan); DAMGO, DSLET, U-69593,
naloxone,
D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-amide (CTOP), nor-binaltorphimine (nor-BNI), and BK (all obtained from Sigma, St. Louis, MO); calphostin C and KT-5720 (obtained from Kyowa
Medics, Tokyo, Japan); and Go6976, HBDDE, and Rottlerin (obtained from Calbiochem, La Jolla, CA). All drugs except for U-69593,
calphostin C, KT5720, Go6976, HBDDE, and Rottlerin were dissolved in
physiological saline. U-69593 was dissolved in 1.5% ethanol.
Calphostin C, KT-5720, Go6976, HBDDE, and Rottlerin were dissolved in
30% dimethyl sulfoxide. In many experiments, drugs except for
calphostin C and KT-5720 were given by intraplantar (i.pl.)
injection in a volume of 2 µl. Calphostin C, KT-5720, Go6976, HBDDE,
and Rottlerin were given by i.pl. injection in a volume of 5 µl. In
some experiments, drugs were given in a large volume (0.1 ml/10 g
b.wt.) to the back (s.c.). The AS-ODN (5'-GCC GGC GCT GCT GTC CAT-3')
and its missense oligodeoxynucleotide (MS-ODN; 5'-GCC GGC GGT GCT GCT
CAT-3') for µ-opioid receptor (MOR; Min et al., 1994) and the AS-ODN
(5'-GGT GCC TCC AAG GAC TAT CGC-3') and its MS-ODN (5'-GGG TCC CTC AAG
GCA TAC TGC-3') for -opioid receptor (KOR; Chien et al., 1994), were
synthesized, freshly dissolved in physiological saline, and used for
intrathecal (i.t.) injection according to the protocol of Hylden and
Wilcox (1980) in a volume of 2 µl on the 1st, 3rd, and 5th days. On
the 6th day, mice were used for assessing the opioid analgesia. The MOR1 antiserum (Schulz et al., 1998) for Western blot experiments was a
gift from Dr. V. Hollt (Otto-von-Guericke University, Magdeburg, Germany).
Evaluation of Analgesia on Nociceptive Flexor Responses.
All
experiments were performed in compliance with the relevant laws and
institutional guidelines. Experiments were performed, as described
earlier (Inoue et al., 1998a,b; Ueda, 1999). Briefly, mice were lightly
anesthetized with ether and held in a cloth sling with their four limbs
hanging free through holes. The sling was suspended on a metal bar. All
limbs were tied with strings; three were fixed to the floor, and the
other one was connected to an isotonic transducer and recorder.
Mice were lightly anesthetized with ether and a small incision was made
in the surface of the right hind-limb planta. Two polyethylene cannulae
(0.61-mm outer diameter) filled with drug solution were connected to
separate microsyringes, respectively. One cannula was filled with BK or saline, and the other with test drugs. Because we used light and soft
polyethylene cannulae, they did not fall off the paw during the
experiments. All experiments were started after the complete recovery
of mouse from the light ether anesthesia (20-30 min) and the
confirmation that the i.pl. injection of saline did not show any
significant flexor responses. BK was given i.pl. at 10 and 5 min before
and 5, 10, 20, and 30 min after opioid or vehicle injection. In most
experiments, the results were expressed as percent analgesia, using the
following equation: (1 
BK response (millimeters) after test
drug administration/the average of twice control BK responses) × 100 (%)). In some experiments, analgesia was also evaluated by the
area under the analgesic curve (AUC) obtained by plotting
analgesia (%) on the ordinate, and time after morphine (i.pl.)
administration (minutes) on the abscissa. In this case, morphine
analgesia was assessed by percentage of the maximal AUC, which
represents the analgesia when BK response is completely inhibited
during periods from 5 to 30 min after drug injection. Thus, the maximal
AUC was calculated to be 2500 (% · minutes). The median analgesic
dose (AD50) was calculated from the linear
regression curve of the percentage of maximal against log dose of
opioid. To get S.E.M., we carried out five separate experiments,
in which three different doses of morphine were tested.
Western Blot Analysis.
SDS-polyacrylamide gel
electrophoresis by using 10% polyacrylamide gel and immunoblot
analysis were performed as described (Yoshida and Ueda, 1999).
Visualization of immunoreactive bands was performed by using an
enhanced chemiluminescent substrate for detection of horseradish
peroxidase, Super Signaling Substrate (Pierce Chemical Co., Rockford,
IL). The intensities of immunoreactive bands were analyzed by NIH Image
after scanning exposed films.
Statistical Analysis. The data were analyzed using Student's t test after multiple comparisons of the ANOVA. The criterion of significance was set at P < .05. All results are expressed as the mean ± S.E.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Dose-Dependent Analgesia by Local Application of Morphine.
The
local application of BK at 2 pmol into the planta of hind limb (i.pl.)
produced a nociceptive flexor response, and there were stable responses
in amplitude on repeated applications every 5 min (Inoue et al., 1997).
The mean ± S.E. of BK responses (2 pmol) corresponds to the force
of 6.86 ± 0.25 g (n = 50). BK in ranges of
0.02 to 20 pmol (i.pl.) showed such responses in a dose-dependent manner and the average of nociceptive dose (±S.E.M.) showing 50% of
maximal reflex was 0.71 ± 0.09 pmol (n = 5). The
BK (2 pmol)-induced responses were completely abolished by the i.pl.
injection of B2-type BK receptor antagonist
(Inoue et al., 1997).
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Peripheral Morphine Analgesia.
When a higher amount of
morphine at 10 nmol, which corresponds to 0.18 mg/kg, was injected
locally into the contralateral side of hind limb to the BK injection
side or systemically into the s.c. space of the back, there was no
significant suppression of BK responses for 30 min (Fig.
2A). On the other hand, the morphine analgesia by i.pl. injection was abolished by the ipsilateral injection
of naloxone (1 nmol), but not by the contralateral one (Fig. 2B).
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Opioid Receptor Type-Specific Peripheral Analgesia.
DAMGO, a
specific MOR agonist, at 1 nmol (i.pl.) showed a potent analgesia by
suppressing BK responses for 30 min (Fig.
3A). The analgesia by DAMGO lasts for 120 min. The AD50 was 33 ± 4 pmol
(n = 5) for DAMGO (Table
1), and this value was 8 times lower than
that for morphine (274 pmol). On the other hand, 3 nmol of U-69593, a
specific KOR agonist, showed analgesic effects equivalent to those of
morphine at 1 nmol (Fig. 3A), and were also completely recovered after
180 min (data not shown). The AD50 of
U-69593 was 1045 ± 102 pmol (n = 5, Table 1).
However, DSLET, a specific -opioid receptor (DOR) agonist, at a dose
up to 30 nmol showed no significant effects (Fig. 3A).
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-type), were carried out. The
analgesic effects of 1 nmol of DAMGO or 3 nmol of U-69593 were markedly
blocked by equimoles of antagonists, respectively (Fig. 3B).
Blockade of Opioid Analgesia by Intrathecal Injection of
AS-ODNs for Opioid Receptors.
To clarify the opioid receptor type
specificity and the site of action, the pretreatment of AS-ODN for each
type of opioid receptor was carried out. In the control experiments,
there was no significant difference in the morphine (i.pl.) analgesia
between untreated mice (Fig. 1B) and the ones pretreated with MS-ODN
for MOR (µ-MS) as shown in Fig. 4A.
When the AS-ODN for MOR (µ-AS) was given, the morphine analgesia was
markedly attenuated and there was a significant difference from the
µ-MS data (Fig. 4A). The µ-AS did not affect the peripheral
analgesia by U-69593, a KOR agonist (Fig. 4B). Similarly, the
pretreatments with KOR-AS-ODN (-AS) significantly attenuated the
U-69593 (i.pl.)-induced analgesia (Fig. 4C), but not the morphine
(i.pl.) analgesia (Fig. 4D). When dorsal root ganglions (DRGs)
treated with ODNs for MOR were analyzed by Western blot analysis, it
was revealed that immunoreactive signals of MOR were significantly
reduced by AS-ODNs, but not by MS-ODNs, as shown in Fig.
5. On the other hand, we could not detect
any signal for KOR from the DRG and brain preparations in the Western
blot analysis using commercially available antibody against KOR.
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Development of Acute Tolerance on Morphine-Peripheral
Analgesia.
To evaluate the development of peripheral acute
tolerance by morphine pretreatment, mice were given 3 nmol of
morphine (i.pl.), a dose 3 times higher than the maximal dose (1 nmol)
for peripheral analgesia. When the nociceptive response by BK (2 pmol)
was assessed at different times after the first morphine treatment, the
time-dependent recovery was observed between 1 and 4 h (Fig.
6A). Because the BK response at 4 h
after morphine treatment was equivalent to that with vehicle treatment,
we decided to use this morphine pretreatment and assess the analgesic
action by the second morphine given 4 h after the first challenge
for additional experiments. As shown in Fig. 6B, the morphine (3 nmol
i.pl.) injection to mice pretreated with vehicle showed a time course
of analgesia similar to that of 1 nmol of morphine in naive mice, as
seen in Fig. 1B. However, there was a marked decrease in the analgesia
by 3-nmol morphine in morphine (3 nmol)-pretreated mice (Fig. 6B). When
various doses of morphine were given to assess the analgesic activity
in morphine-pretreated mice, the dose-dependent analgesia was observed
while the dose range was shifted to a higher one than that in naive
mice (Fig. 6C). The AD50 of morphine in
the pretreated mice became 10 nmol, a 30-times-higher dose than that
(0.3 nmol) in naive mice. There was no change in U-69593 analgesia in
morphine-treated mice (Fig. 6D); therefore, we suppose that the acute
tolerance to morphine is due to the functional change at the receptor
level.
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PKC Involvement in the Acute Tolerance of Morphine-Induced
Peripheral Analgesia.
To characterize the mechanism of acute
tolerance of peripheral analgesia, we assessed the effects of protein
kinase inhibitors on it. When mice were pretreated with calphostin C, a
PKC inhibitor or KT-5720, a cyclic AMP-dependent protein kinase (PKA)
inhibitor, there was no significant change in the BK-induced
nociceptive activity 4 h after such inhibitor injection, compared
with vehicle-treated mice (Fig. 7A),
although the BK responses were partially inhibited 30 min after the
injection of 10 nmol of calphostin C (41 ± 8% inhibition,
n = 5). When mice were pretreated with calphostin C
together with the first challenge of morphine, the acute tolerance was
completely abolished, whereas there was no significant change with
KT-5720 (Fig. 7B). There was no significant change in morphine analgesia with the submaximal dose (0.3 nmol) by pretreatment with
calphostin C alone (AUC in pretreatment with vehicle, 1157 ± 333% · min; calphostin C, 1091 ± 200% · min).
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and 
isoforms
(Wenzel-Seifert et al., 1994) completely reversed the development of
morphine tolerance in a dose-dependent manner. Similarly, HBDDE, known
as a specific inhibitor of the PKC and isoforms (Kashiwada et
al., 1994) also completely reversed it in a dose-dependent manner (Fig.
8B). However, less evident reversal of morphine tolerance development
was observed with Rottlerin, which is known as a specific inhibitor of
the PKC isoform (Lu et al., 1997), as shown in Fig. 8C.
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Lack of Acute Tolerance of U-69593-Induced Peripheral
Analgesia.
As in the case of morphine, mice were pretreated with
U-69593 at 10 nmol (i.pl.), a dose that is 3 times higher than the
maximal dose. The complete recovery of BK responses was also observed 4 h after this pretreatment, as seen in the case with morphine (Fig. 9A). The analgesia by the second
challenge with U-69593 (10 nmol) in vehicle-pretreated mice (Fig. 9B)
was slightly more potent than in the case with 3 nmol of this compound
in naive mice (Fig. 3A). Unlike in the case with morphine, however,
there was no significant difference in the U-69593 (10 nmol
i.pl.)-induced analgesia between vehicle- and U-69593-pretreated mice
(Fig. 9B).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The peripheral nociception test used in this study has been
developed for the purpose of analyzing in vivo signaling mechanisms at
the level of nociceptor endings (Inoue et al., 1998a; Ueda, 1999). This
test has several advantages over many other nociception tests. First,
it is sensitive enough to assess very weak and short-acting nociceptive
responses induced by a local application of small amounts of
pain-producing substances (Inoue et al., 1997, 1998a,b). Second, the
nociceptive responses in this test are attributed to relatively simple
molecular and neuronal mechanisms because a single species of molecule
is used for the stimulation of specified receptors on nociceptor
endings. Third, because peripheral nociceptors are distant from the
dorsal root ganglion containing cell bodies of sensory neurons, the
targeting of specific protein expression in nociceptive neurons by
AS-ODN (i.t.) techniques is available without effects in peripheral cells.
Here we observed the peripheral morphine analgesia against such BK responses. The local application of morphine into the ipsilateral side of hind paw to the BK-injection side caused a potent inhibition of BK responses (we call it analgesia). However, there was no significant inhibition even when morphine at a 10-times-higher dose was given into the contralateral side or systemically into the back. In addition, the morphine analgesia was blocked by naloxone given into the ipsilateral, but not the contralateral, side. Thus, it is evident that the analgesia by morphine given i.pl. is due to mechanisms on peripheral sites. From the evidence that µ-AS, but not µ-MS pretreatment abolished the morphine-induced peripheral analgesia, it is concluded that the site of morphine action is located on nociceptor endings, as mentioned above.
The type specificity has been often discussed in the opioid
analgesia. As well reported with the central opioid analgesia, the MOR agonists, such as morphine and DAMGO, show more potent analgesia than the DOR or KOR agonists (Hong and Abbott, 1995; Coggeshall et al., 1997). Indeed, U-69593, an agonist with high affinity and specificity to KOR showed 5 or 50 times less potent action
in the AD50 values than morphine or DAMGO,
respectively. DSLET, a representative DOR agonist, however, showed no
significant analgesia (Fig. 3, Table 1). This was supported by the
report that DOR function was not observed in the electrophysiological studies with DRG (Abdulla and Smith, 1998), although the gene expression of three types of opioid receptors is observed in the DRG
(Hassan et al., 1993; Maekawa et al., 1994; Coggeshall et al., 1997).
The lack of DOR function in the peripheral system may be explained by
the finding that DORs in the DRG are more located in the Golgi
apparatus or in vesicular membranes than in the plasma membranes (Zhang
et al., 1998a).
Here we demonstrated that the peripheral morphine analgesia developed acute tolerance in this unique paradigm of experiments. In this study, we took more care in the recovery from the analgesia by the first morphine challenge. As the BK responses when evaluated as percentage of maximal reflex were stable from one mouse to another (see data in Figs. 6-9), the recovery of the BK responses from morphine analgesia could be examined without assessing the control BK response of each mouse before morphine injection. Finally, it was determined that 4 h is required for complete recovery from morphine (or U-69593) analgesia. Under this condition, there was a marked reduction in the morphine analgesia on the second challenge. This finding strongly suggests that the acute tolerance was developed to peripheral morphine analgesia. As higher doses (10 and 30 nmol i.pl.) of morphine still have potent analgesic potencies, the tolerance may be attributed to the desensitization to morphine, but not to the down-regulation, including MOR degradation.
Previously, we reported that DOR is desensitized to repeated challenges
of the agonist at a supramaximal concentration in the
Xenopus oocytes expressing DOR clone (Ueda et al., 1995). As
this desensitization was recovered after the 60-min absence of agonist
challenges, it may not be attributed to the down-regulation. Instead,
this reduced response was recovered by the treatment with calphostin C,
a PKC inhibitor, but not by inhibitors of other protein kinases, such
as PKA and calcium-calmodulin kinase II. In that report, the DOR
agonist-induced desensitization was not affected by the stimulation of
M2-muscarinic receptor, which shares common
signaling including activation of Gi1 and
phospholipase C (PLC) with DOR. Thus, PKC activation might be a
downstream mechanism of DOR stimulation, and might desensitize its
receptor. Similar desensitizations were also observed with MOR
and KOR in Xenopus oocytes (Ueda et al., 1996). These
findings were also confirmed by other reports using in vivo or culture
preparations (Cai et al., 1997; Narita et al., 1997; Kramer and
Simon, 1999). Furthermore, such a PKC-mediated desensitization
mechanism has been also confirmed by the in vivo experiments in which
the analgesic action of DAMGO given i.c.v. was reduced by the i.c.v.
pretreatment of this opioid peptide; calphostin C attenuated this
desensitization (Narita et al., 1997).
The calphostin C-induced reversal of the development of morphine
tolerance was confirmed by this study using the peripheral nociception
test. One of the advantages of this study is that PKC mechanisms in the
acute tolerance could be discussed at the level of well defined
nociceptor endings, but not at the level of the central nervous system,
consisting of complicated neuronal networks. The involvement of PKC was
also confirmed by Go6976 and HBDDE. However, less evident reversal of
morphine tolerance development was observed with Rottlerin. Therefore,
the PKC isoforms involved in the development of morphine tolerance are
characterized to be members of conventional PKC (cPKC) isoforms, which
include , , and isoforms, and possess binding sites for
phorbol ester, Ca2+, and ATP (Nishizuka, 1992).
It is unlikely that this mechanism uses PKC isoforms, a
member of novel PKC (nPKC) isoform including , 
, 
, and 
isoforms, and possessing binding sites for phorbol ester and ATP, but
not Ca2+. However, the involvement of atypical
PKC (aPKC) isoform remains to be examined because membrane-permeable
inhibitors of such isoforms are not available.
Recently, the PKC isoform has been reported to be involved in
the BK-induced potentiation of neuronal response to painful heat
(Cesare et al., 1999). More recently, there has been a report that the
PKC isoform may be involved in the mechanical, thermal, and chemical
hyperalgesia in experiments using PKC knockout mice (Khasar et al.,
1999). These findings may be consistent with the present results in
which BK responses were partially inhibited by calphostin C at 30 min
after injection. However, it is unlikely that the inhibitory
actions of PKC inhibitors on BK responses affect the BK sensitivity for
assessment of morphine analgesia 4 h after calphostin C injection
because BK responses themselves were completely recovered at that time
(Fig. 7A). As the membrane-permeable inhibitors for the PKC isoform
are not available, the involvement of this isoform in the development
of morphine tolerance remains to be determined. It is curious to know
whether Rottlerin has an inhibitory action on the PKC isoform, which
is classified into the nPKC family, as well as the PKC isoform.
Regarding the molecular mechanisms of opioid tolerance cAMP hypothesis
proposed by Sharma et al. (1975), this hypothesis is now modified by
the view that opioid mechanism are attenuated by the up-regulation of
cAMP and its downstream-signaling molecules during chronic opioid
treatments (Nestler et al., 1994). However, in this study, KT-5720 did not affect the morphine tolerance. These findings suggest
that PKC mechanisms are involved in the development of acute morphine
tolerance, whereas cAMP or its downstream mechanisms, including PKA
mechanisms, are not.
In conclusion, we demonstrated that the peripheral MOR- and
KOR-mediated analgesia was observed in a newly developed BK nociception test. We also demonstrated that the acute tolerance is selectively observed with morphine analgesia through MOR, and PKC is likely involved in the acute tolerance. As KOR was reported to be easily internalized by KOR agonist treatment whereas MOR is resistant to the
internalization by morphine treatment (Sternini et al., 1996; Koch et
al., 1998; Zhang et al., 1998b; Li et al., 1999), the present finding
showing the selective and acute tolerance of morphine analgesia may be
explained by the resistance of MOR to internalization. Histochemical or
immune electron microscopical demonstration of opioid receptor
internalization corresponding to the acute opioid analgesic tolerance
should be an important subject in the future. The present paradigm of
peripheral analgesic tests in mice would be useful for the study of in
vivo signaling of opioid tolerance.
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Acknowledgments |
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We thank F. Fujiwara, T. Yamada, and H. Nakayamada for technical help and Dr. Volker Hollt for the gift of µ-opioid receptor antiserum.
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Footnotes |
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Accepted for publication January 25, 2000.
Received for publication August 31, 1999.
1 Parts of this study were supported by Grants-in-Aid from the Ministry of Education, Science, Culture and Sports of Japan, and a grant from The Suzuken Memorial Foundation. M.I. is a Research Fellow of the Japan Society for the Promotion of Science.
Send reprint requests to: Hiroshi Ueda, Ph.D., Dept. of Molecular Pharmacology and Neuroscience, Nagasaki University School of Pharmaceutical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. E-mail: ueda{at}net.nagasaki-u.ac.jp
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Abbreviations |
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BK, bradykinin;
nor-BNI, nor-binaltorphimine;
AUC, area under the analgesic curve;
CTOP, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-amide;
DAMGO, [D-Ala2,MePhe4,Gly-ol5]-enkephalin;
DSLET, [D-Ser2]Leu-enkephalin-Thr6;
DRGs, dorsal root ganglions;
HBDDE, 2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanol dimethyl
ether;
U-69593, (5,7,8)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8yl]
benzeneacetamide;
AS-ODN, antisense oligodeoxynucleotide;
MS-ODN, missense oligodeoxynucleotide;
i.pl., intraplantar(ly);
MOR, µ-opioid
receptor;
KOR, -opioid receptor;
i.t., intrathecal;
AD50, median analgesic dose;
DOR, -opioid receptor;
PKC, protein kinase C;
PKA, cyclic AMP-dependent protein kinase.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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mutant mice.
Neuron
24:
253-260[Medline]. opioid receptor coupled to Gi1-phospholipase C activation in Xenopus oocytes.
J Neurosci
15:
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, 1.0 nmol; 
,
0.3 nmol; 
, 0.1 nmol) or vehicle (
). C, dose dependence of
morphine-induced analgesia. The results were expressed as percentage of
maximal AUC, as described in the text. The maximal AUC was represented
the analgesia when BK response is completely inhibited during periods
from 5 to 30 min. Data represent the mean ± S.E. from five
separate experiments. *P < .05, compared with
value of vehicle-treated group. There was no significant difference in
the control BK responses among different groups.
, saline) or naloxone (
