 |
 Previous Article | Next Article 
The Journal of Neuroscience, August 1, 2000, 20(15):5874-5879
 -Opioid Receptor Agonists Modulate Visceral Nociception at a
Novel, Peripheral Site of Action
S. K.
Joshi1,
Xin
Su1,
Frank
Porreca2, and
G. F.
Gebhart1
1 Department of Pharmacology, College of Medicine, The
University of Iowa, Iowa City, Iowa 52242, and 2 Department
of Pharmacology, University of Arizona, Tucson, Arizona 85721
 |
ABSTRACT |
 -opioid receptor agonists (-ORAs) have been shown to modulate
visceral nociception through an interaction with a peripheral, possibly
novel, -opioid-like receptor. We used in the present experiments an
antisense strategy to further explore the hypothesis that -ORA
effects in the colon are produced at a site different from the cloned
-opioid receptor (KOR). An antisense oligodeoxynucleotide (ODN) to the cloned rat KOR was administered intrathecally (12.5 µg, twice daily for 4 d) to specifically knock-down the cloned KOR. Efficacy of the KOR antisense ODN treatment was behaviorally evaluated by assessing the antinociceptive effects of peripherally administered - (EMD 61,753 and U 69,593), µ- (DAMGO) and  -
(deltorphin) ORAs in the formalin test. Intrathecal antisense, but not
mismatch ODN blocked the actions of EMD 61,753 and U 69,593 without
affecting the actions of DAMGO or deltorphin; a complete recovery of
antinociceptive actions of the -ORA EMD 61,753 was observed 10 d after the termination of antisense ODN treatment. In contrast, the
ability of EMD 61,753 to dose-dependently attenuate responses of pelvic
nerve afferent fibers to noxious colonic distension was unaffected in
the same rats in which the antisense ODN effectively knocked-down the
KOR as assessed in the formalin test. Additionally, Western blot
analysis demonstrated a significant downregulation of KOR protein in
the L4-S1 dorsal root ganglia of antisense, but not mismatch
ODN-treated rats. The present results support the existence of a
non--opioid receptor site of action localized in the colon.
Key words:
peripheral opioids; nociception; colorectal distension; formalin test; antisense; visceral pain
| |
INTRODUCTION |
Effects of opioid receptor agonists
(ORAs) are mediated by one of the three opioid receptors: µ, , or
. Because of undesirable effects like respiratory depression,
tolerance, constipation, and abuse potential associated with µ-ORAs,
there is considerable interest in developing therapeutically useful
agonists at other opioid receptors. -ORAs exert potent
visceral antinociceptive effects by acting at peripheral sites.
-ORAs, but not µ- or -ORAs, dose-dependently attenuate
responses of decentralized pelvic nerve afferent fibers to noxious
colonic as well as urinary bladder distension, providing evidence for a
peripheral site of action for these agonists (Sengupta et al., 1996 ; Su
et al., 1997a,b). In addition, -ORAs are effective when injected
directly into a peripheral site (e.g., tail; Kolesnikov et al., 1996)
or intracolonically (Su et al., 2000).
Although only one -opioid receptor (KOR 1) has been cloned and
characterized from rat CNS (Minami et al., 1993), binding studies
suggest the existence of at least four different KOR subtypes (Pasternak, 1993). The existence of multiple subtypes of KOR is further
supported by data obtained in assays of cutaneous nociception (Zukin et
al., 1988; Clark et al., 1989). We have previously reported that
-ORAs inhibit the response of pelvic nerve afferent fibers to
noxious colonic or bladder distension (Sengupta et al., 1996; Su et
al., 1997a,b) as well as the pressor and visceromotor responses to
colonic distension in awake, unrestrained rats (Burton and Gebhart,
1998). In all these experiments, naloxone partially antagonized the
actions of the -ORAs tested, but two -receptor selective antagonists, nor-binaltorphimine (nor-BNI) and
2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-3- isothiocyanatophenyl)-2-(1-pyrrolidinyl)-ethyl]acetamide
(DIPPA), were ineffective. Furthermore, the mean effective doses
for the receptor-selective -ORAs examined in the
electrophysiological studies were virtually the same, ranging between 2 and 10 mg/kg, which is in contrast to 100-fold differences reported in
the literature for these same -ORAs in other models (Chang et al.,
1984; Devlin and Shoemaker, 1990; Nock et al., 1990; Paul et al.,
1990). These findings led us to speculate that the -ORAs tested
modulate visceral nociception through an interaction with a peripheral,
novel, site of action that is different from the cloned KOR.
Antisense oligodeoxynucleotides (ODNs) have proven to be a valuable
tool to study the pharmacology of opioid receptors (Pasternak and
Standifer, 1995). There is evidence that intrathecal administration of
antisense ODNs can also cause a "knock-down" of peripheral opioid
receptors, presumably by inhibiting the synthesis of receptors in the
dorsal root ganglion (Bilsky et al., 1996; Khasar et al., 1996). In the
present study, we have used antisense strategy to both assess the
peripheral visceral antinociceptive role, if any, of the cloned KOR and
further explore the hypothesis that the -ORA effects in the colon
are produced at a site different from the cloned KOR.
Parts of this paper have been published previously in abstract
form (Joshi et al., 1999).
| |
MATERIALS AND METHODS |
Animals. Male Sprague Dawley rats (Harlan,
Indianapolis, IN) were housed one or two per cage with ad
libitum access to food and water and were maintained on a 12 hr
light/dark cycle (lights on 6:00 A.M. to 6:00 P.M.) in the Association
For Assessment and Accreditation of Laboratory Animal
Care-approved animal care facility. All experimental procedures
were approved by the Institutional Animal Care and Use Committee, The
University of Iowa.
Experimental objectives. The goal of the present experiments
was to evaluate the role of the cloned KOR in peripheral somatic (hindpaw) and visceral (colon) tissues using antisense ODN-mediated receptor knock-down. Rats received antisense or mismatch ODNs targeting
the cloned KOR or saline intrathecally for 4 d. The antinociceptive effects of -, µ-, and -ORAs, administered
peripherally, were tested in the formalin test. After testing the
peripheral actions of -ORAs in antisense and mismatch ODN-treated
rats in the formalin test, electrophysiological evaluation of a -ORA in visceral nociception was tested in the same rats. To verify whether
the ODNs caused a decrease in KOR expression in the dorsal root
ganglia, Western blot analysis was performed. In a separate group of
animals, the recovery of antinociceptive actions of -ORAs 6 and
10 d after the termination of antisense ODN treatment was also
assessed in the formalin test. The experimental strategy is diagrammed
in Figure 1.
View larger version (39K):
[in this window]
[in a new window]
|
Figure 1.
Diagram illustrating the experimental strategy to
study the effects of KOR knock-down using antisense ODNs. Antisense ODN
targeting the cloned rat KOR was administered intrathecally into the
lumbosacral area twice daily (dosing interval 10-12 hr) for 4 consecutive days, each dose containing 12.5 µg of ODN in a 5 µl
volume followed by a 10 µl saline flush. The efficacy of the
antisense ODN treatment to cause a knock-down of the peripheral KOR was
evaluated using the formalin test. Rats showing a block of the
peripheral analgesic actions of the -ORAs in the formalin test were
subsequently examined electrophysiologically to test -ORA modulation
of visceral nociception.
|
|
Synthesis and administration of ODNs. Antisense and mismatch
ODNs having a phosphodiester backbone, derived from the 5' end of the
coding sequence of the cloned rat KOR (nucleotides 6-25), were
synthesized by Integrated DNA Technologies (Iowa City, IA) and
reconstituted in sterile deionized water before use. Phosphodiester ODNs have been shown to be stable in CSF and devoid of many
nonspecific effects associated with phosphothiorate ODNs (Wahlestedt,
1994) and hence are preferred for intrathecal administration. The
sequences of the two ODNs used in these experiments were as follows:
5'-GGAAAATCTG GATGGGGGAC-3'(antisense) and 5'-GGAAATACTG
GTAGGGGAGC-3'(mismatch). The sequences were selected to ensure that
their cross-hybridization with - and µ-opioid receptors was minimal.
Rats were deeply anesthetized with an intraperitoneal injection of
sodium pentobarbital (45 mg/kg, Nembutal; Abbott Laboratories, Abbott
Park, IL) and a catheter (8.5 cm; PE-10) was passed to the lumbosacral
intrathecal space through an incision in the dura over the
atlantooccipital joint. Animals were allowed at least 3 d to
recover from surgery before testing. Antisense or mismatch ODNs were
injected intrathecally twice daily (dosing interval 10-12 hr) for 4 consecutive days, each dose of ODN containing 12.5 µg in a 5 µl
volume followed by a 10 µl saline flush.
Behavioral study. The formalin test was performed on
antisense and mismatch ODN-treated and saline-treated rats on the
morning of the fifth day, ~12 hr after the last intrathecal injection of ODN or saline. Different groups of rats (n = 5-10)
received 100 nmol in 5 µl of selective - (EMD 61,753 and U
69,593), µ- (DAMGO), or - (deltorphin) opioid agonists or saline,
injected subcutaneously into the dorsum of the right hindpaw, 10 min
before formalin injection (50 µl, 2.5%, s.c.). Rats were then placed in observation chambers and observed for paw-flinching behavior. Flinching of the injected paw is a consistent component of
formalin-induced behavior and has been advocated as a more robust
parameter, when compared to paw licking, and less contaminated by other
non-nociceptive behavioral changes (Tjolsen et al., 1992). Flinches
were counted in bins of 5 min each, starting with the formalin
injection and continuing for 50 min. The flinch response was divided
into first and second phases by summing the total number of flinches
occurring between 0 and 15 and between 15 and 50 min, respectively. In
some experiments, the selective -opioid receptor antagonist nor-BNI (50 nmol in 5 µl) was injected into the hindpaw simultaneously with
EMD 61,753. Recovery of antinociceptive actions of EMD 61,753 was also
assessed in a separate group of animals 6 and 10 d after the
termination of antisense ODN treatment.
Electrophysiological study. The experimental procedures for
recording responses of pelvic nerve afferent fibers to noxious colorectal distension have been described in detail (Sengupta et al.,
1996; Su et al., 1997b) and are only briefly summarized here. Rats
treated with either antisense or mismatch ODN and previously tested
with formalin were anesthetized with sodium pentobarbital (Nembutal)
and mechanically ventilated with room air. A femoral artery and vein
were catheterized for measurement of arterial pressure and
administration of pentobarbital, respectively. The left carotid artery
was catheterized for subsequent drug administration. Core body
temperature was maintained at 37°C. The lower abdomen was exposed by
a 3- to 4-cm-long incision laterally at the left flank. A flaccid,
flexible latex balloon 6- to 7-cm-long and 2-3 cm in diameter was
inserted intra-anally into the descending colon and rectum. The balloon
catheter was connected to a distension control device via a low-volume
pressure transducer, and the colon was distended with air. The left
pelvic nerve was isolated from the surrounding fatty tissues, and a
pair of Teflon-coated stainless steel wires that were stripped at the
tips were wrapped around the pelvic nerve and sealed with a nonreactive
silicon gel (Wacker Silicone Corporation, Adrian, MI). The hypogastric,
pudendal, and femoral nerves were isolated and transected. The sciatic
nerve was approached through the ischiatic notch and transected.
The lumbosacral spinal cord was exposed by laminectomy (T13-S1), and
the rat was suspended from thoracic vertebral and ischia clamps. The
dorsal skin was reflected laterally and tied to make a pool for mineral
oil. The dura membrane was carefully removed, and the spinal cord was
covered with warm (37°C) mineral oil. The S1 dorsal root was
decentralized close to its entry to the spinal cord. Recordings of
single units (from a fine filament of the dorsal root) were made from
the distal cut end of the central process of the S1 dorsal root, ~3
hr after the conclusion of formalin testing. Action potentials,
monitored continuously by analog delay, were processed through a window
discriminator, and the frequency of impulses was counted using the
Spike2/CED 1401 program.
Pelvic nerve input to the S1 dorsal root was identified first by
electrical stimulation of the pelvic nerve (one 0.5 msec square wave
pulse at 3-8 V). If a fiber responded to distension, the effect of
-ORA EMD 61,753 given intra-arterially was tested on responses to
noxious distension (80 mmHg, 30 sec) of the colon. Each dose of the
drug was administered 2 min before the onset of distension.
Dose-response relationships in antisense and mismatch ODN-treated rats
were obtained by giving cumulative doses of EMD 61,753 (0.5, 1, 2, 4, 8, 16, and 32 mg/kg; doses administered at 4.5 min intervals).
Drugs. EMD 61,753 (MW: 469.1, a gift from Dr. Andrew Barber;
E. Merck, Darmstadt, Germany) was dissolved in 10% DMSO. DAMGO (MW:
523.7; Sigma, St. Louis, MO) and nor-BNI dihydrochloride (MW: 734.7;
Research Biochemicals, Natick, MA) were dissolved in saline. U 69,593 (MW: 521.5; Sigma) was dissolved in ethanol and
[D-Ala2] deltorphin (MW:
782.89; Research Biochemicals) was dissolved in 100% DMSO. The lack of
effects of DMSO, ethanol, and saline were determined in preliminary experiments.
Western blot analysis. In a separate group of saline,
antisense, or mismatch ODN-treated animals, Western blot analysis was performed to assess KOR expression levels in L4, L5, L6, and S1 dorsal
root ganglia (DRG). Tissue homogenates were prepared in a phosphate
lysis buffer (10 mM Tris, 1% Triton X-100, 5 mM Na2EDTA, 50 mM NaCl,
30 mM sodium pyrophosphate, 50 mM sodium
orthovanadate, 0.5% sodium deoxycholate, and 50 µg/ml PMSF) from DRG
of rats 12 hr after the last bolus injection of either ODNs or saline. Soluble extracts were resolved by SDS-PAGE and transferred to polyvinylidene fluoride (Millipore, Bedford, MA). Immunoblots were
blocked overnight with 5% milk in Tris-buffered saline (TBS) at 4°C
and then incubated overnight at 4°C with an affinity-purified antibody raised against an internal region of the cloned rat KOR (PharMingen KA8) diluted 1:1000 in 5% milk and TBS. Immunoblots were
then incubated for 2 hr at room temperature with anti-rabbit IgG-HRP
secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA; SC-2004)
diluted 1:5000 in 5% milk and TBS. Immunoreactive proteins were
visualized with enhanced chemiluminescence (Amersham, Arlington
Heights, IL; NA934).
Data analysis. All experimental groups in the behavioral
study consisted of at least five animals, and data are presented as
mean ± SEM. The data were analyzed by one-way ANOVA with Tukey's test for post hoc comparisons. A value of p < 0.05 was considered statistically significant in all tests.
| |
RESULTS |
Effects of selective ORAs on formalin-produced flinching in KOR
antisense ODN-treated rats
Injection of formalin into the dorsal surface of the hindpaw
produced characteristic biphasic flinching behavior with clear first
(0-15 min) and second (15-50 min) phases (Figs.
2A,
3A, 4A,
5A). On average, rats that
received pretreatment with saline (10 min before formalin) flinched a
total of 345 ± 20 times between 15 and 50 min after formalin.
Pretreatment with 100 nmoles of selective - (EMD 61,753 and U
69,593), µ- (DAMGO), or - (deltorphin) ORAs significantly
decreased the number of second phase flinches after their
administration, 10 min before formalin at the same site (Figs.
2B, 3B, 4B). The first
phase of the response to formalin was not significantly altered by
pretreatment with the ORAs tested. The second phase antinociceptive
actions of the two -ORAs tested were significantly blocked by
intrathecal treatment with KOR antisense, but not mismatch ODN
treatment (Figs. 2B, 3B). In contrast,
neither KOR antisense nor mismatch ODN treatment had any effect on the antinociceptive actions of either DAMGO or deltorphin (Fig. 4). Additionally, administration of KOR antisense ODN alone had no effect
on formalin-induced flinching behavior (Figs. 2B,
3B, 4B). The antinociceptive action of EMD
61,753 was also blocked by the -opioid receptor-selective antagonist
nor-BNI (50 nmoles, injected simultaneously with EMD 61,753), thus
demonstrating that peripheral agonist effects were selective for KOR
(Fig. 2B).
View larger version (33K):
[in this window]
[in a new window]
|
Figure 2.
Effect of -ODN treatment on peripheral EMD
61,753 analgesia in the formalin flinch test. Formalin (50 µl, 2.5%)
was injected into the dorsal surface of the rat hindpaw 10 min after
the injection of drugs or saline at the same site. A,
Time course of formalin-induced flinching behavior for the different
treatment groups exhibits a characteristic biphasic response. Data are
summarized in B. One hundred nanomoles of the -ORA
EMD 61,753 (EMD) significantly blocked the
formalin-induced second phase flinching response (an
asterisk indicates a p value of <0.05).
The antinociceptive effects of EMD were blocked by KOR antisense, but
not mismatch ODN treatment. Fifty nanomoles of the -opioid receptor
antagonist nor-BNI injected simultaneously with EMD antagonized the
antinociceptive actions of EMD. Administration of -ODN alone did not
affect the formalin response.
|
|
View larger version (29K):
[in this window]
[in a new window]
|
Figure 3.
Effect of -ODN treatment on peripheral U 69,593 analgesia in the formalin flinch test. Formalin (50 µl, 2.5%) was
injected into the dorsal surface of the rat hindpaw 10 min after the
injection of drug or saline at the same site. A, Time
course of formalin-induced flinching behavior for the different
treatment groups exhibits a characteristic biphasic response. Data are
summarized in B. One hundred nanomoles of the -ORA U
69,593 (U69) significantly blocked the formalin-induced
second phase flinching response. The antinociceptive effects of U69
were blocked by KOR antisense, but not mismatch ODN treatment.
Administration of -ODN alone did not affect the formalin response.
*p < 0.05.
|
|
View larger version (27K):
[in this window]
[in a new window]
|
Figure 4.
Effects of -ODN treatment on peripheral
antinociceptive actions of µ-ORA (DAMGO) and -ORA (deltorphin) in
the formalin flinch test. One hundred nanomoles of drug were
administered 10 min before formalin in the dorsal surface of the rat
hindpaw. A illustrates the time course of the
formalin-induced flinching response; the data are summarized in
B. Both DAMGO and deltorphin significantly attenuated
the second phase flinching response (p < 0.05). Administration of KOR antisense ODN had no effect on the
antinociceptive actions of either of these two drugs.
|
|
View larger version (30K):
[in this window]
[in a new window]
|
Figure 5.
Recovery of antinociceptive actions of -ORAs 6 and 10 d after the termination of KOR antisense ODN treatment. One
hundred nanomoles of the -ORA EMD 61,753 (EMD) was
administered 10 min before formalin in the dorsal surface of the rat
hindpaw in all cases. A, Time course of formalin-induced
flinching behavior; the data are summarized in B.
Administration of -ODN significantly blocked the antinociceptive
actions of EMD (p < 0.05). A partial
recovery of antinociceptive actions of EMD was seen 6 d after the
termination of -ODN treatment, and a complete recovery was observed
after 10 d.
|
|
To verify that the blockade of antinociceptive actions of -ORAs is
not attributable to a generalized neurotoxicity brought about by
antisense ODN administration, effects of EMD 61,753 were assessed in
the formalin test 6 and 10 d after the termination of antisense
ODN treatment. A partial recovery of the antinociceptive actions of EMD
61,753 was seen 6 d after the last antisense ODN injection, and a
complete recovery was observed after 10 d (Fig. 5).
Effects of EMD 61,753 on responses of pelvic nerve afferent fibers
to noxious colonic distension
Electrophysiological evaluation of -ORA modulation of pelvic
nerve afferent fiber responses to colonic distension was performed in
mismatch and antisense ODN-treated rats after the formalin test.
Recordings were made from a total of 14 pelvic nerve sensory fibers in
the decentralized S1 dorsal root. In contrast to the observations in
the formalin test, in which the antinociceptive actions of the -ORA
EMD 61,753 were blocked by KOR antisense ODN treatment, EMD 61,753 dose-dependently attenuated the responses of pelvic nerve afferent
fibers to noxious colonic distension (80 mmHg) in both KOR antisense
and mismatch ODN-treated rats (Fig. 6).
The estimated ED50 values (dose-attenuating
response magnitude to 50% of control) and 95% confidence intervals
for EMD 61,753 in antisense (7 mg/kg; 1.7-12.4 mg/kg) and mismatch (6 mg/kg; 0.6-11.37 mg/kg) ODN-treated rats did not differ from each
other or from the ED50 of EMD 61,753 determined
in other experiments (7.9 mg/kg; Sengupta et al., 1999).
View larger version (24K):
[in this window]
[in a new window]
|
Figure 6.
Effect of KOR antisense and mismatch ODN treatment
on EMD 61,753 modulation of visceral nociception. ODN-treated rats,
previously tested with formalin, were deeply anesthetized and were
examined electrophysiologically. EMD 61,753 dose-dependently inhibited
the responses of the pelvic nerve afferent fibers to noxious colonic
distension (80 mmHg, 30 sec). There were no differences in effects of
the drug in antisense or mismatch ODN-treated rats.
|
|
Effects of ODN treatment on KOR protein levels in rat DRG
Western blot analysis was performed to compare KOR protein content
in L4, L5, L6, and S1 DRGs of rats treated with saline, antisense ODN,
or mismatch ODN. Immunoblots of tissue extracts showed immunoreactive
proteins at molecular masses of ~43 and 70 kDa in the DRG of
saline-treated control animals (Fig. 7,
lanes labeled C). The KOR 1 cDNA clone predicts a
protein with a molecular mass of ~43 kDa (Arvidsson et al., 1995).
The protein detected at 70 kDa most likely corresponds to a
posttranslationally modified form of the KOR. A clear downregulation of
KOR protein was observed in antisense ODN-treated (Fig. 7,
lanes labeled AS) but not in mismatch ODN-treated
animals (Fig. 7, lanes labeled MM).
View larger version (43K):
[in this window]
[in a new window]
|
Figure 7.
Immunoblot showing the effects of ODN treatment on
KOR protein levels in rat DRG. Tissue extracts were prepared from L4,
L5, L6, and S1 DRG of saline (C), antisense
ODN-treated (AS), and mismatch ODN-treated
(MM) rats. Samples were normalized for protein
content using BCA quantification. Each lane was loaded with ~100 µg
of protein, and immunoreactive bands were revealed at 43 and 70 kDa
with an antibody directed against an internal region of the cloned rat
KOR. Antisense ODN, but not mismatch ODN treatment, caused a clear
downregulation of KOR protein at all the DRGs examined. Similar results
were obtained from duplicate experiments.
|
|
| |
DISCUSSION |
The present experiments support the hypothesis that there exists
in the colon a novel site at which -ORAs significantly attenuate visceral nociception. Intrathecal administration of antisense, but not
mismatch ODNs to the cloned KOR blocked the antinociceptive effects of
-ORAs injected into the paw without affecting the actions of DAMGO
(µ-ORA) or deltorphin (-ORA). Significantly, the ability of the
-ORA EMD 61,753 to dose-dependently attenuate responses of pelvic
nerve afferent fibers to colonic distension was unaffected by the same
ODN treatment. We used Western blot analysis to verify that intrathecal
ODN administration causes a KOR protein knock-down in the L4-S1 DRG.
Antisense strategies in opioid pharmacology
Antisense ODNs have been extensively used to correlate the
pharmacology and molecular biology of opioid receptors (Pasternak and
Standifer, 1995; Hutcheson et al., 1999). The turnover rate of opioid
receptors is ~4 d in vivo (Pasternak, 1993), which
necessitated administration of the KOR antisense ODN for 4 d so
that not only new protein synthesis was blocked, but also the
pre-existing protein was cycled out.
Antisense ODNs targeting the KOR have been shown to selectively inhibit
-ORA antinociception in rats (Adams et al., 1994) and, more
recently, to cause hypertension after injection into the hippocampus
(Wright et al., 1999). Intrathecal administration of antisense ODNs has
also been demonstrated to cause a knock-down of peripheral proteins.
For example, intrathecal administration of antisense ODN targeting the
µ-opioid receptor significantly decreased peripheral
DAMGO-produced inhibition of prostaglandin E2 hyperalgesia and also
DAMGO-induced inhibition of voltage-gated Ca2+ currents in cultured rat DRG neurons
(Khasar et al., 1996). Intrathecal administration of -opioid
receptor (DOR) ODN selectively blocks the antinociceptive effects of
peripherally administered -ORAs (Bilsky et al., 1996). Radioligand
binding experiments in the same study indicated an ~50% decrease in
-opioid receptors in the lumbar spinal cord. A large proportion of
-opioid receptors in rat spinal cord are located on primary afferent
nerve terminals (Dado et al., 1993), suggesting that one of the sites
of action of intrathecally administered DOR antisense ODN is the DRG,
where the opioid receptors are synthesized and subsequently transported to central and peripheral terminals. Likewise, we verified using Western blot analysis that lumbosacral intrathecal administration of
antisense ODNs directed toward the cloned KOR causes a KOR knock-down
in the L4-S1 DRG. Although the KOR downregulation we observed was
almost complete, it is possible that the antibody could not detect some
KOR protein that persisted in DRG after the antisense ODN treatment. We
have also behaviorally demonstrated the efficacy of the antisense ODN,
which selectively blocked the antinociceptive actions of peripherally
administered -ORAs in the formalin test, an effect not produced by
administration of mismatch ODN. Moreover, the antisense ODN was
selective in its effect; the efficacy of the µ- and -ORAs in the
formalin test was not affected.
Role of -ORAs in modulating visceral nociception
Whereas -ORAs are well documented to be antinociceptive in
cutaneous models of pain (Leighton et al., 1988; Herraro and Headley, 1993), several recent studies point toward a significant role in
modulating visceral pain. The role of the KOR gene product in the
perception of visceral chemosensitivity has been demonstrated in
KOR-deficient mice, which have a decreased threshold to a noxious visceral chemical stimulus when compared to their wild-type littermates (Simonin et al., 1998). A peripheral site of action of -ORAs in the
viscera has been recently documented in reports in which -ORAs, but
not µ- or -ORAs, dose-dependently inhibited responses of
mechanosensitive pelvic nerve afferent fibers innervating the urinary
bladder or colon (Sengupta et al., 1996; Su et al.,
1997a,b). Interestingly, naloxone at non-receptor-selective doses was
able to partially antagonize the effects of the -ORAs tested, but the KOR-selective antagonists nor-BNI and DIPPA were without effect (Su
et al., 1997a). Additionally, the ED50 values for
the receptor-selective -ORAs examined were virtually identical,
ranging between 2 and 10 mg/kg (Su et al., 1997b), which is in contrast
to the broad range of antinociceptive doses reported in the literature
for these same -ORAs (Chang et al., 1984; Devlin and
Shoemaker, 1990; Nock et al., 1990; Paul et al., 1990). This led us to
hypothesize that -ORAs mediate their antinociceptive actions in the
viscera by acting at a -opioid-like receptor unlike the KOR cloned
and characterized from the CNS or any opioid receptor subtype
documented in the literature. Consistent with this hypothesis, the
-ORA EMD 61,753 in the present study was observed to
dose-dependently inhibit responses of pelvic nerve afferent fibers to
noxious colonic distension in antisense-ODN treated rats in which a
knock-down of the cloned KOR is demonstrated.
Visceral -opioid agonist site of action
The present results, combined with our previous
electrophysiological data, suggest that -ORAs attenuate responses to
noxious colonic distension by acting at a receptor that is distinct
from the KOR cloned in the CNS. The present experiments neither
document nor localize such a receptor. An immunohistochemical study
using antibodies raised to the cloned KOR demonstrated that -opioid receptors, although not present in the smooth muscle cells in the rat
colon, were present on myenteric and submucosal plexus neurons as well
as on interstitial cells of Cajal (Bagnol et al., 1997). At present, there is no evidence for the existence of any opioid receptor associated with the peripheral endings of pelvic nerve
sensory fibers. Accordingly, it is possible that -ORA effects on
pelvic nerve sensory fibers arise indirectly through action at the
-opioid receptor associated with neurons of the intrinsic nervous
system of the gut. Intrinsic and extrinsic primary afferent nerve
terminals, however, are not anatomically organized in a manner that
would make such an interaction likely. That is, intrinsic primary
afferent nerve terminals are not "presynaptic" to terminals of
extrinsic neurons. Intrinsic primary afferent neurons have been shown
to interact with each other and with second order neurons (interneurons
and motor neurons) of the enteric nervous system, but not with
extrinsic primary afferent neurons such as studied here (Furness et
al., 1998).
The existence of KOR 1 splice variants has been suggested in a recent
report (Pasternak et al., 1999). In this study, the analgesic actions
of  -neoendorphin and dynorphin B in mice were antagonized by nor-BNI
but not blocked by antisense ODN targeting exon 1 of the KOR. Although
we find nor-BNI to be without effect in our electrophysiology studies,
we cannot rule out the possibility that -ORA effects in the viscera
are mediated by a splice variant encoded by the KOR 1 gene. Such a
splice variant would neither be affected by the present antisense ODN
treatment nor be recognized by the antibody used in the Western blot
analysis. Further studies using antisense ODN sequences targeting other
regions of the cloned KOR mRNA would therefore be informative, although
an antibody that can be used to assess the differential knock-down of
various KOR 1 splice variants is not currently available. Alternately, the receptor in the viscera may be heterodimeric. A recent report (Jordan and Devi, 1999) demonstrated the ability of - and -opioid receptors to form a heterodimeric receptor with altered ligand binding
and functional properties. However, the inability of -ORAs to
modulate the responses of mechanosensitive pelvic nerve afferent fibers
innervating the colon (Sengupta et al., 1996) and the comparable efficacy of subtype-selective -ORAs (Su et al., 1997b) makes us
believe that the presently investigated visceral receptor does not
represent such a - heterodimer. We have previously eliminated the
possibility that this novel receptor may be an orphan receptor like ORL
1 at which the endogenous orphanin FQ/nociceptin peptide acts (Meunier
et al., 1995; Reinscheid et al., 1995) because
nociceptin had no effect on pelvic nerve afferent fibers to colorectal
distension (V. Julia and G. F. Gebhart, unpublished
observations). It remains to be tested whether this novel receptor
maybe an opioid-somatostatin-like receptor. Two novel, related genes,
named GPR7 and GPR8, have been described which encode receptors with
structural features in common with both opioid and somatostatin
receptors and also bind several opioid drugs (O'Dowd et al., 1995).
Finally, several -ORAs, in addition to their opioid effects, have
been demonstrated to possess Na+ channel
blocking properties (Wong et al., 1990; Pugsley et al., 1993). Although
-ORAs have no effect on conduction velocity or amplitude of action
potentials of pelvic nerve afferent fibers (Sengupta et al., 1996; Su
et al., 1997a,b), and -ORA effects are partially reversed by
naloxone, we cannot completely rule out the possibility that -ORA
actions are mediated by Na+ channel blockade.
In summary, the present experiments provide further evidence for the
existence of a novel, peripheral -opioid-like receptor localized in
the colon. Agonists directed toward this receptor are attractive
targets as analgesics, potentially free of the undesirable side effects
associated with activation of central -opioid receptors and could
provide relief for visceral pain states like inflammatory bowel
disease, for which satisfactory treatment is not currently available.
| |
FOOTNOTES |
Received Jan. 4, 2000; revised May 9, 2000; accepted May 11, 2000.
This work was supported by National Institutes of Health Grant
NS 19912. We thank Mike Burcham for preparation of the figures and Alex
Eapen for help with the Western blot technique.
Correspondence should be addressed to S. K. Joshi, Department of
Pharmacology, BSB, The University of Iowa, Iowa City, IA 52242. E-mail: shailen-joshi{at}uiowa.edu.
| |
REFERENCES |
-
Adams JU,
Chen X,
DeRiel JK,
Adler MW,
Liu-Chen LY
(1994)
Intracerebroventricular treatment with an antisense oligonucleotide to a kappa opioid receptor, inhibited kappa-agonist induced analgesia in rats.
Brain Res
667:129-132[Medline].
-
Arvidsson U,
Riedl M,
Chakrabarti S,
Vulchanova L,
Lee JH,
Nakano AH,
Lin X,
Loh HH,
Law PY,
Wessendorf MW,
Elde R
(1995)
The -opioid receptor is primarily postsynaptic: combined immunohistochemical localization of the receptor and endogenous opioids.
Proc Natl Acad Sci USA
92:5062-5066[Abstract/Free Full Text].
-
Bagnol D,
Mansour A,
Akil H,
Watson SJ
(1997)
Cellular localization and distribution of the cloned mu and kappa opioid receptors in the gastrointestinal tract.
Neuroscience
81:579-591[Web of Science][Medline].
-
Bilsky EJ,
Wang T,
Lai J,
Porreca F
(1996)
Selective blockade of peripheral delta opioid agonist induced antinociception by intrathecal administration of delta receptor antisense oligodeoxynucleotide.
Neurosci Lett
220:155-158[Web of Science][Medline].
-
Burton MB,
Gebhart GF
(1998)
Effects of kappa-opioid receptor agonists on responses to colorectal distension in rats with and without acute colonic inflammation.
J Pharmacol Exp Ther
285:707-715[Abstract/Free Full Text].
-
Chang KJ,
Blanchard SG,
Cuatrecasas P
(1984)
Benzomorphan sites are ligand recognition sites of putatative epsilon-receptors.
Mol Pharmacol
26:484-488[Abstract].
-
Clark JA,
Liu L,
Price M,
Hersh B,
Edelson M,
Pasternak GW
(1989)
Kappa opiate receptor multiplicity: evidence for two U50-488-sensitive 1 subtypes and novel 3 subtype.
J Pharmacol Exp Ther
251:461-468[Abstract/Free Full Text].
-
Dado RJ,
Law PY,
Loh HH,
Elde R
(1993)
Immunofluorescent identification of a delta (delta)-opioid receptor on primary afferent nerve terminals.
NeuroReport
5:341-344[Web of Science][Medline].
-
Devlin T,
Shoemaker WJ
(1990)
Characterization of kappa opioid binding using dynorphin A1-13 and U69,593 in the rat brain.
J Pharmacol Exp Ther
253:749-759[Abstract/Free Full Text].
-
Furness JB,
Kunze WAA,
Bertrand PP,
Clerc N,
Bornstein JC
(1998)
Intrinsic primary afferent neurons of the intestine.
Prog Neurobiol
54:1-18[Web of Science][Medline].
-
Herraro JF,
Headley PM
(1993)
Functional evidence for multiple receptor activation by -ligands in the inhibition of spinal nociceptive reflexes in the rat.
Br J Pharmacol
110:303-309[Web of Science][Medline].
-
Hutcheson DM,
Sanchez-Blazques P,
Rodriquez-Diaz M,
Garzon J,
Schmidhammer H,
Borsodi A,
Roques BP,
Maldonado R
(1999)
Use of selective antagonists and antisense oligonucleotides to evaluate the mechanisms of BUBU antinociception.
Eur J Pharmacol
383:29-37[Medline].
-
Jordan BA,
Devi LA
(1999)
G-protein-coupled receptor heterodimerization modulates receptor function.
Nature
399:697-700[Medline].
-
Joshi SK,
Su X,
Gebhart GF
(1999)
Further evidence for the existence of a novel, peripheral kappa-opioid-like receptor localized in the colon.
Soc Neurosci Abstr
25:925.
-
Khasar SG,
Gold MS,
Dastmalchi S,
Levine JD
(1996)
Selective attenuation of mu-opioid receptor-mediated effects in rat sensory neurons by intrathecal administration of antisense oligodeoxynucleotides.
Neurosci Lett
218:17-20[Web of Science][Medline].
-
Kolesnikov Y,
Jain S,
Wilson R,
Pasternak GW
(1996)
Peripheral 1-opioid receptor-mediated analgesia in mice.
Eur J Pharmacol
310:141-143[Medline].
-
Leighton GE,
Rodriguez RE,
Hill RG,
Hughes J
(1988)
-Opioid agonists produce antinociception after i.v. and i.c.v. but not intrathecal administration in the rat.
Br J Pharmacol
93:553-560[Web of Science][Medline].
-
Meunier J-C,
Mollereau C,
Toll L,
Suaudeau C,
Moisand C,
Alvinerie P,
Butour JL,
Guillemot JC,
Ferrara P,
Monsarrat B,
Mazarguil H,
Vassart G,
Parmentier M,
Costentin J
(1995)
Isolation and structure of the endogenous agonist of opioid receptor-like ORL 1 receptor.
Nature
337:532-535.
-
Minami M,
Toya T,
Katao Y,
Maekawa K,
Nakamura S,
Onogi T,
Kaneko S,
Satoh M
(1993)
Cloning and expression of a cDNA for the rat kappa-opioid receptor.
FEBS Lett
329:291-295[Web of Science][Medline].
-
Nock B,
Giordano AL,
Cicero TJ,
O'Connor LH
(1990)
Affinity of drugs and peptides for U69,593-sensitive and -insensitive kappa opiate binding sites: the U69,593-insensitive site appears to be the beta endorphin-specific epsilon receptor.
J Pharmacol Exp Ther
254:412-419[Abstract/Free Full Text].
-
O'Dowd BF,
Scheideler MA,
Nguyen T,
Cheng R,
Rasmussen JS,
Marchese A,
Zastawny R,
Heng HQ,
Tsui L-C,
Shi X,
Asa S,
Puy L,
George SR
(1995)
The cloning and chromosomal mapping of two novel human opioid somatostatin like receptor genes, GPR7 and GPR8, expressed in discrete areas of the brain.
Genomics
28:84-91[Web of Science][Medline].
-
Pasternak GW
(1993)
Pharmacological mechanisms of opioid analgesics.
Clin Neuropharmacol
16:1-18[Web of Science][Medline].
-
Pasternak GW,
Standifer KM
(1995)
Mapping of opioid receptors using antisense oligodeoxynucleotides: correlating their molecular biology and pharmacology.
Trends Pharmacol Sci
16:344-350[Medline].
-
Pasternak KR,
Rossi GC,
Zuckerman A,
Pasternak GW
(1999)
Antisense mapping KOR-1: evidence for multiple kappa analgesic mechanisms.
Brain Res
826:289-292[Web of Science][Medline].
-
Paul D,
Levison JA,
Howard DH,
Pick CG,
Hahn EF,
Pasternak GW
(1990)
Naloxone benzoylhydrazone (NalBzoH) analgesia.
J Pharmacol Exp Ther
255:769-774[Abstract/Free Full Text].
-
Pugsley MK,
Saint DA,
Penz WP,
Walker MJ
(1993)
Electrophysiological basis for the antiarrhythmic actions of the agonist PD129290 and its RR(+)-enantiomer PD129289.
Br J Pharmacol
110:1579-1585[Web of Science][Medline].
-
Reinscheid RK,
Nothacker HP,
Bourson A,
Ardati A,
Henningsen RA,
Bunzow JR,
Grandy DK,
Lengen H,
Monsma Jr FJ,
Civelli O
(1995)
Orphanin FQ: a neuropeptide that activates an opioid like G protein-coupled receptor.
Science
270:792-794[Abstract/Free Full Text].
-
Sengupta JN,
Su X,
Gebhart GF
(1996)
Kappa, but not mu or delta opioids attenuate responses to distension of pelvic nerve afferents innervating the colon of the rat.
Gastroenterology
111:968-980[Web of Science][Medline].
-
Sengupta JN,
Snider A,
Su X,
Gebhart GF
(1999)
Effects of kappa opioids in the inflamed rat colon.
Pain
79:175-185[Web of Science][Medline].
-
Simonin F,
Valverde O,
Smadja C,
Slowe S,
Kitchen I,
Dierich A,
Meur ML,
Roques BP,
Maldonado R,
Kieffer BL
(1998)
Disruption of the -opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective -agonist U-50,488H and attenuates morphine withdrawal.
EMBO J
17:886-897[Web of Science][Medline].
-
Su X,
Sengupta JN,
Gebhart GF
(1997a)
Effects of opioids on mechanosensitive pelvic nerve afferent fibers innervation the urinary bladder of the rat.
J Neurophysiol
77:1566-1580[Abstract/Free Full Text].
-
Su X,
Sengupta JN,
Gebhart GF
(1997b)
Effects of kappa opioid receptor-selective agonists on responses of pelvic nerve afferents to noxious colorectal distension.
J Neurophysiol
78:1003-1012[Abstract/Free Full Text].
-
Su X,
Julia V,
Gebhart GF
(2000)
Effects of intracolonic opioid receptor agonists on polymodal pelvic nerve afferent fibers in the rat.
J Neurophysiol
83:963-970[Abstract/Free Full Text].
-
Tjolsen A,
Berge O-C,
Hunskaar S,
Rosland JH,
Hole K
(1992)
The formalin test: an evaluation of the method.
Pain
51:5-17[Web of Science][Medline].
-
Wahlestedt C
(1994)
Antisense oligonucleotide strategies in neuropharmacology.
Trends Pharmacol Sci
15:42-46[Medline].
-
Wong TM,
Lee AY,
Tai KK
(1990)
Effect of drugs interacting with opioid receptors during normal perfusion or ischaemia and reperfusion in the isolated heart-an attempt to identify cardiac opioid receptor subtypes involved in arrhythmogenesis.
J Mol Cell Cardiol
22:1167-1175[Web of Science][Medline].
-
Wright RC,
McConnaughey MM,
Phan TA,
Ingenito AJ
(1999)
-opioid receptor antisense oligonucleotide injected into rat hippocampus causes hypertension.
Eur J Pharmacol
377:57-61[Medline].
-
Zukin RS,
Eghbali M,
Olive D,
Unterwald EM,
Tempel A
(1988)
Characterization and visualization of rat and guinea pig brain opioid receptors: evidence for 1 and 2 opioid receptors.
Proc Natl Acad Sci USA
85:4061-4065[Abstract/Free Full Text].
Copyright © 2000 Society for Neuroscience 0270-6474/00/20155874-06$05.00/0
This article has been cited by other articles:
|
|
|
|
 
T. Kitamura, M. Ogawa, and Y. Yamada
The Individual and Combined Effects of U50,488, and Flurbiprofen Axetil on Visceral Pain in Conscious Rats
Anesth. Analg.,
June 1, 2009;
108(6):
1964 - 1966.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. U. De Schepper, B. Y. De Winter, L. Van Nassauw, J.-P. Timmermans, A. G. Herman, P. A. Pelckmans, and J. G. De Man
TRPV1 receptors on unmyelinated C-fibres mediate colitis-induced sensitization of pelvic afferent nerve fibres in rats
J. Physiol.,
November 1, 2008;
586(21):
5247 - 5258.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Jimenez, M. M. Puig, and O. Pol
Antiexudative Effects of Opioids and Expression of {kappa}- and {delta}- Opioid Receptors during Intestinal Inflammation in Mice: Involvement of Nitric Oxide
J. Pharmacol. Exp. Ther.,
January 1, 2006;
316(1):
261 - 270.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. K. Joshi, K. Lamb, K. Bielefeldt, and G. F. Gebhart
Arylacetamide {kappa}-Opioid Receptor Agonists Produce a Tonic- and Use-Dependent Block of Tetrodotoxin-Sensitive and -Resistant Sodium Currents in Colon Sensory Neurons
J. Pharmacol. Exp. Ther.,
October 1, 2003;
307(1):
367 - 372.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. Pol, J. R. Palacio, and M. M. Puig
The Expression of {delta}- and {kappa}-Opioid Receptor Is Enhanced during Intestinal Inflammation in Mice
J. Pharmacol. Exp. Ther.,
August 1, 2003;
306(2):
455 - 462.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. K. Joshi and G. F. Gebhart
Nonopioid Actions of U50,488 Enantiomers Contribute to Their Peripheral Cutaneous Antinociceptive Effects
J. Pharmacol. Exp. Ther.,
June 1, 2003;
305(3):
919 - 924.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. N. Sengupta, B. K. Medda, and R. Shaker
Effect of GABAB receptor agonist on distension-sensitive pelvic nerve afferent fibers innervating rat colon
Am J Physiol Gastrointest Liver Physiol,
December 1, 2002;
283(6):
G1343 - G1351.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Su, S. K. Joshi, S. Kardos, and G. F. Gebhart
Sodium Channel Blocking Actions of the kappa -Opioid Receptor Agonist U50,488 Contribute to Its Visceral Antinociceptive Effects
J Neurophysiol,
March 1, 2002;
87(3):
1271 - 1279.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Ji and R. J. Traub
Spinal NMDA Receptors Contribute to Neuronal Processing of Acute Noxious and Nonnoxious Colorectal Stimulation in the Rat
J Neurophysiol,
October 1, 2001;
86(4):
1783 - 1791.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. G. Hill
Book Review: Molecular Basis for the Perception of Pain
Neuroscientist,
August 1, 2001;
7(4):
282 - 292.
[Abstract]
[PDF]
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
