Department of Pharmacology, College of Medicine, The University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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Su, X.,
V. Julia, and
G. F. Gebhart.
Effects of Intracolonic Opioid Receptor Agonists on Polymodal
Pelvic Nerve Afferent Fibers in the Rat.
J. Neurophysiol. 83: 963-970, 2000.
We studied the effects of
intracolonic administration of opioid receptor agonists (ORAs) on
responses of pelvic nerve afferent fibers to colorectal distension
(CRD) and heat. Single-fiber recordings were made from the
decentralized S1 dorsal rootlet in the rat. An ~7-cm length of
descending colon was isolated in situ to permit intracolonic perfusion
with Krebs solution, which, when the outflow was clamped, was used to
distend the colon. Responses to noxious CRD (40 mmHg, 30 s) were
tested after intracolonic instillation of µ-, - or
-ORAs.
Intracolonic administration of the
-ORAs EMD 61,753 (n = 5/12) and U62,066 (n = 8/11), but not either the µ-ORA fentanyl or the
-ORA SNC-80,
concentration-dependently inhibited responses of afferent fibers. For
fibers unaffected by intracolonic administration of EMD 61,753 or
U62,066, intra-arterial administration of
-ORAs was effective.
Forty-one of 54 mechanosensitive fibers also responded to intracolonic
instillation of heated Krebs solution (50°C). Intra-arterial
injection of fentanyl or SNC-80 did not attenuate responses to heat.
Either intracolonic or intra-arterial administration of EMD 61,753 or
U62,066, however, inhibited afferent fiber responses to heat. These
results document that mechanical and thermal sensitivity of polymodal
pelvic nerve afferent fibers innervating the rat colon can be inhibited
peripherally by intracolonic instillation of
-ORAs.
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INTRODUCTION |
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Studies in humans and nonhuman animals have
established that both nonpainful and painful sensations from the distal
gut and urinary bladder are conveyed by the pelvic nerve to the spinal cord (e.g., Head 1893; White 1943
; see
Ness and Gebhart 1990
for review). We previously
reported that
-, but not either µ- or
-opioid receptor agonists
(ORAs) dose-dependently inhibit responses of mechanosensitive pelvic
nerve afferent fibers to noxious colorectal or urinary bladder
distension in the rat (Sengupta et al. 1996
; Su
et al. 1997a
,b
). Considerable efforts have been made to develop
-ORAs with limited access to the CNS, where they produce
unacceptable sedation and dysphoria (Barber et al. 1994
;
Rogers et al. 1992
; Shaw et al.
1989
). One such compound, EMD 61,753, is a peripherally restricted
-ORA (Barber et al. 1994
) that inhibits
responses of pelvic nerve afferent fibers to colorectal distension
(CRD) (Sengupta et al. 1999
). Because these recordings
were made in experiments in which pelvic nerve input was decentralized
at the dorsal root, the site of action of the
-ORAs was limited to
the periphery, either at a peripheral receptor in the tissue, axon, and/or cell body in the dorsal root ganglion (DRG). Regarding the
latter, we studied the effects of
-, µ-, and
-ORAs on
high-voltage-activated calcium channels in colon sensory neurons,
concluding that only part of the dose-dependent attenuation by
-ORAs
of responses to noxious CRD can be accounted for by an action at the
level of the DRG (Su et al. 1998b
). In the present
study, we examined ORA effects on responses to noxious CRD, limiting
effects of drugs to the periphery by intracolonic administration.
We have to date only examined -ORA effects on mechanical stimulation
of the colon and bladder. It has been suggested that
-ORAs are more
effective against noxious mechanical stimuli than noxious heat
stimulation (Abbott et al. 1986
; Millan
1989
, 1990
; Tyers 1980
). A recent
report (Gschossmann et al. 1997
) suggested that
fedotozine, a drug with agonist efficacy at the
-opioid receptor,
inhibited a mechanically stimulated, but not capsaicin-stimulated Ca2+ increase in DRG cells, again suggesting modality
selective actions of
-ORAs. Other work, however, suggests that the
reported relative selectivity of
-ORAs against mechanical input is
more related to stimulus intensity than to stimulus modality (e.g.,
Millan 1989
; Parsons and Headley
1989a
,b
).
Because mechanosensitive pelvic nerve afferent fibers innervating the
colon of the rat are activated by noxious chemical and/or thermal
stimuli (i.e., are polymodal) (Su and Gebhart 1998b), the objectives of this study were twofold: 1) to examine
the effects of intracolonic administration of ORAs on responses of
mechanosensitive pelvic nerve afferent fibers to noxious CRD and
2) to examine the effects of ORAs on the responses of
mechanosensitive pelvic nerve afferent fibers to noxious heat
stimulation. Some of these data have been reported in abstract form
(Su et al. 1998a
).
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METHODS |
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General procedures
Male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing
410-530 g were used. Food, but not water, was withheld for 24 h
before an experiment. Rats were anesthetized initially with 40-45
mg/kg ip pentobarbital sodium (Nembutal, Abbott Laboratories, North
Chicago, IL); anesthesia was maintained by infusion of pentobarbital (5-10 mg · kg1 · h
1 iv). A femoral artery and vein were
catheterized for measurement of arterial pressure and administration of
drugs, respectively; the trachea was also cannulated to provide
artificial ventilation. Rats were paralyzed with pancuronium bromide
(initial 0.3 mg/kg iv; supplemental 0.2-0.3 mg · kg
1 · h
1 iv).
Core body temperature was maintained at 36°C by a
hot-water-circulating heating pad placed under the rat and an overhead
feedback-controlled heat lamp (thermoprobe inserted into the thoracic
esophagus). At the end of experiments, rats were killed by an overdose
of intravenous pentobarbital. The experimental protocol was approved by
the Institutional Animal Care and Use Committee of The University of Iowa.
Surgical procedures
The surgical procedures have been described in detail (Su
and Gebhart 1998a,b
) and are only briefly summarized here. The
lower abdomen was exposed by a 4- to 5-cm-long incision laterally at the left flank. An ~7 cm length of descending colon was exposed and
isolated in situ. The blood supply and nerves innervating the colon
remained intact. Both the proximal end and anus end of the descending
colon were catheterized to permit intracolonic perfusion of the colon
with Krebs-Henseleit (Krebs) solution. The pelvic nerve was isolated
from the surrounding fatty tissues, and a pair of Teflon-insulated
stainless steel wires were wrapped around the pelvic nerve and sealed
with nonreactive Reprosil (hydrophilic vinyl polysiloxane, type
I; The L. D. Caulk Division, Dentsply International, Millord, DE).
The abdomen was closed with silk sutures. The lumbosacral spinal cord
was exposed by laminectomy (T13-S2), the dura
was carefully removed, and the spinal cord was covered with warm
(37°C) mineral oil.
The isolated colon was connected to a pressurized fluid reservoir
through the proximal catheter, and intracolonic pressure was measured
through a fine catheter (polyethylene tubing, PE-60) placed in the
colon from the proximal end. The pressure reservoir was connected to a
distension control device via a low-volume pressure transducer (see
Gebhart and Sengupta 1995). At rest, 37°C Krebs
solution (0 mmHg) remained in the colon. For phasic, constant pressure
distension (5-60 mmHg, 30 s), 37°C Krebs solution was
introduced via the proximal catheter, and the distal catheter was clamped.
Thermal stimulation of the colon was produced by changing the
temperature of the Krebs solution with which the isolated colon was
perfused. In these experiments, the colon was continuously perfused
with heated Krebs solution, and intracolonic pressure was 5 mmHg
(i.e., perfusion pressure was 5 mmHg with the distal outflow open). To
monitor the temperature of the perfusate, a thermoprobe (Physitemp,
type IT-1E; Physitemp Instruments, Clifton, NJ) was introduced into the
colon via the anal catheter. Thermal stimulation of the colon was
produced by ramp increases in temperature (37-50°C, ~480 s)
without changing intracolonic pressure while outflow was open.
Recording of afferent nerve action potentials
The S1 dorsal root was decentralized close to its entry to the spinal cord. Recordings were made from the distal cut end of the central processes of primary afferent fibers. The dorsal rootlet was split into thin bundles, and a fine filament was isolated to obtain a single unit. Electrical activity of the single unit was recorded by placing the teased fiber over one arm of a bipolar silver-silver chloride electrode; a fine strand of connective tissue was placed across the other pole of the electrode. Action potentials were monitored continuously by analogue delay and displayed on a storage oscilloscope after initial amplification through a low-noise AC differential amplifier. The action potentials were processed through a window discriminator and counted using the spike2/CED 1401 data acquisition program. Peristimulus time histograms (1-s binwidth), intracolonic pressure, intracolonic temperature, colonic distending pressures, and blood pressure were displayed on-line continuously.
Experimental protocol
CHARACTERIZATION OF AFFERENT FIBERS.
Mechanosensitive pelvic nerve input to the S1 dorsal root was
identified by electrical stimulation of the pelvic nerve (single pulse
0.5-ms square wave at 4-10 V) and CRD (40 mmHg, 3-5 s). Fibers with
conduction velocities (CV) <2.5 m/s were considered unmyelinated
C-fibers and those with CV >2.5 m/s were considered thinly myelinated
A-fibers.
EFFECTS OF INTRACOLONIC ADMINISTRATION OF ORAS ON
MECHANONOCICEPTION.
The effects of ORAs were tested on responses of afferent fibers to 40 mmHg CRD. The volume of Krebs solution that produced 40 mmHg distension
was 5.2-5.4 ml. The desired concentrations of ORAs were obtained by
addition to the Krebs solution in the reservoir before distension of
the colon. Drug remained in the colon for 12 min during which three
40-mmHg distensions at 4-min intervals were given, followed by
intracolonic instillation of the next, higher concentration of ORA.
Concentration-response relationships for fentanyl and SNC-80 were
obtained by intracolonic perfusion, sequentially, of
105, 5 × 10
5,
10
4 M or 10
4, 5 × 10
4 and 10
3 M drug
concentration; EMD 61,753 and U62,066 were given in concentrations of
10
4, 5 × 10
4 and
10
3 M. At the greatest concentration of
-ORA
tested (10
3M), ~2.5 mg of EMD 61,753 or 1.9 mg of U62,066 were given intracolonically in the volume required to
distend the colon to 40 mmHg. Intra-arterial cumulative doses of EMD
61,753 and U62,066 were 0.5, 0.5, 1, 2, 4, 8, and 16 mg/kg (total
cumulative dose, 32 mg/kg).
EFFECTS OF ORAS ON NOXIOUS HEAT STIMULATION.
The effects of fentanyl and SNC-80, each at a dosage of 300 µg/kg
intra-arterially, were tested on responses to intracolonic perfusion of
heated Krebs solution. The effects of -ORAs on responses to heat
were tested on 15 fibers, four after intracolonic administration of
10
3 M EMD 61,753 (n = 3) or
U62,066 (n = 1), and 11 after intra-arterial administration of a total 16 mg/kg of EMD 61,753 (n = 3) or U62,066 (n = 8).
Data analysis
The resting activity of a fiber was counted for 60 s before CRD, and the response was determined as the increase in discharge during stimulation above its resting activity. SRFs to graded CRD were plotted for each individual fiber, and a least-squares regression line was obtained from the linear part of the SRF. The regression line was then extrapolated to the ordinate (representing distension pressure) to estimate the distension response threshold.
To estimate the response threshold to thermal stimulation, the mean and standard deviation (SD) of the resting activity was determined. Threshold was defined as the temperature at which unit activity increased >2 SD above resting activity. For fibers with no or low background activity, the response threshold was considered that temperature at which the fiber began and continued to discharge. Unit activity during thermal stimulation was counted in 10-s bins, and the maximum response during thermal stimulation was defined as that bin with the greatest number of counts.
All data are expressed as means ± SE. Results were analyzed using
Student's t-test or an ANOVA for repeated measures. The inhibitory dose 50 (ID50; dose to produce 50%
inhibition of the response to distension) and 95% confidence intervals
were calculated from the 20-80% component of the cumulative
dose-response curve (Tallarida and Murray 1991). A value
of P < 0.05 was considered statistically significant.
Drugs
Krebs solution of the following composition (in mM) 118.0 NaCl, 0.7 KCl, 24.0 NaHCO3, 1.2 MgSO3, 2.5 CaCl2, 1.1 KH2PO4, and 10.0 glucose, pH 7.3-7.4, was prepared from chemicals purchased from Sigma Chemical (St. Louis, MO). Naloxone hydrochloride (MW: 363.8; Sigma), and fentanyl citrate (MW: 528, Abbott Laboratories, North Chicago, IL) were dissolved in saline. U62,066 (MW: 356.5, Research Biochemicals, Natick, MA) was dissolved in 10% methanol. SNC-80 (MW: 449.6, Tocris Cookson, St. Louis, MO) and EMD 61,753 (MW: 469.1, a gift from Dr. Andrew Barber, E. Merck, Darmstadt, Germany) was dissolved in 10% DMSO.
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RESULTS |
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Fiber sample
A total of 70 mechanosensitive afferent fibers in the S1 dorsal
root were studied; 24 (34%) were myelinated A-fibers (mean conduction velocity: 4.9 ± 0.6 m/s, mean ± SE), and 46 (66%) were unmyelinated C-fibers (mean conduction velocity: 1.7 ± 0.1 m/s). Forty-one of 54 (76%) mechanosensitive fibers tested also
responded to noxious heat; 27 were C-fibers and 14 were myelinated
A
-fibers. Sixty-three of the total 70 fibers had an ongoing
discharge (mean: 0.8 ± 0.2 imp/s; range: 0.01-6.9 imp/s); 7 fibers had no resting activity. There was no significant difference
(P > 0.05) between the resting activities of C- (mean:
0.8 ± 0.2 imp/s; n = 46) and A
- (mean:
0.5 ± 0.2 imp/s; n = 24) fibers. All fibers gave
monotonic increases in response to increasing pressures of CRD.
Extrapolation of the linear portion of SRFs of these fibers revealed
two populations of fibers: a large group of fibers had low thresholds
(LT) for response (mean: 2.2 ± 0.4 mmHg; n = 67),
and a smaller group of fibers had high thresholds (HT) for response
(mean: 28.4 ± 1.3 mmHg; n = 3). SRFs of
individual fibers are plotted in Fig. 1; the insets illustrate the mean SRFs of each group of fibers.
The characteristics of this sample of S1 pelvic nerve afferent fibers are similar to what we found in an earlier study (Su and Gebhart 1998b
).
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Effects of intracolonic ORAs on responses to noxious CRD
We previously reported that responses to repetitive CRD at 40 mmHg
were reproducible (Su and Gebhart 1998b) and in the
present study tested responses to repetitive CRD after intracolonic
administration of drug vehicle (methanol or DMSO). None of the fibers
exhibited any change in response magnitude or pattern to repeated
distension at a 4-min interval between distensions. Figure
2 shows the response of one fiber to 10 successive colonic distensions and the responses of each of 4 fibers to
repeated CRD (see also Fig. 4 in which responses of 21 fibers do not
change with repeated distension).
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µ- AND - ORAS.
The effects of intracolonic administration of fentanyl, a high efficacy
µ-opioid receptor-selective agonist, and SNC-80, a
-opioid
receptor-selective agonist, were tested on the responses to noxious CRD
(40 mmHg, 30 s). Fentanyl, but not SNC-80, increased the resting
activity of 7/11 fibers (see Fig. 3 for
example) from a mean 0.9 ± 0.4 imp/s to 2.0 ± 0.5 imp/s
(10
5 M), 2.6 ± 0.5 imp/s (5 × 10
5 M) and 2.3 ± 0.5 imp/s
(10
4 M), respectively (P < 0.05). Neither fentanyl (n = 11) nor SNC-80 (n = 10), however, altered responses to noxious CRD.
Figure 3 illustrates the absence of effects of these drugs on responses of different fibers. The data are summarized in Fig.
4. Responses to CRD of five fibers (3 after SNC-80 and 2 after fentanyl) were subsequently examined and
inhibited after intracolonic administration of EMD 61,753 (see
-ORAS).
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-ORAS.
Intracolonic administration of EMD 61,753 did not change resting
activity, but concentration- and time-dependently inhibited responses
to noxious CRD in 5/12 afferent fibers tested; EMD 61,753 was
ineffective in 7/12 fibers. Six of 7 fibers unaffected by intracolonic
administration of EMD 61,753 were then tested after intra-arterial
injection of EMD 61,753; responses to CRD were dose-dependently
inhibited by EMD 61,753 in all six fibers. Examples are given in Fig.
5A; data are summarized in
Fig. 6. The mean maximum inhibition by
EMD 61,753 given by the intracolonic route was to 49.3 ± 5.9%
(n = 5) of control. The mean dose that inhibited responses to CRD to 50% of control (ID50) by the
intra-arterial route of administration was 6.5 ± 0.002 mg/kg.
Compared with a previous study of the effects of EMD 61,753 given
systemically (Sengupta et al. 1999
), there was no
difference between the slopes of the EMD 61,753 dose-response functions
(
3.1 ± 0.6 vs.
2.1 ± 0.4), despite the difference in
the method (fluid vs. air) and intensity (40 and 80 mmHg) of colonic
distension.
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Effects of -ORAs on intracolonic pressure
The two -ORAs tested here did not significantly alter colonic
compliance. Intracolonic pressures during 40 mmHg CRD were changed a
mean +1.2 ± 0.8,
0.3 ± 1.4 and
1.3 ± 1.4 mmHg
after intracolonic administration of 10
4,
5 × 10
4 and 10
3 M
of EMD 61,753, respectively (n = 3). Similarly, in five
rats, mean intracolonic pressures changed after U62,066
(10
4, 5 × 10
4 and
10
3 M) a mean
1.1 ± 0.6,
3 ± 1.7 and
2.8 ± 1.8 mmHg, respectively.
Effects of ORAs on responses to noxious heat
We established in previous experiments (Su and Gebhart
1998b) that two successive heat stimuli as used here (10-min
interval) did not alter mean response threshold (45.0 ± 1.4°C
vs. 45.4 ± 1.3°C), mean resting activity (1.4 ± 0.5 imp/s
vs. 2.5 ± 0.8 imp/s), or mean response magnitude (maximum
response magnitude was 106.9 ± 18.5% of 1st heat trial).
Intra-arterial administration of the µ-ORA fentanyl (300 µg/kg) did
not change the response threshold (44.6 ± 0.8 vs. 40.8 ± 0.7°C), resting activity (0.2 ± 0.1 vs. 2.8 ± 1.3), or
the maximum response to heat (138.7 ± 21.6% of 1st trial) in six
of seven heat-sensitive fibers (Fig.
7A). One fiber did not respond
to heat after administration of fentanyl, but exhibited an
afterdischarge when intracolonic temperature returned to control
(37°C). In seven other heat-sensitive fibers, the -ORA SNC-80 (300 µg/kg) did not change either response threshold (44.8 ± 0.4 vs.
42.0 ± 1.1°C), resting activity (0.2 ± 0.1 vs. 0.8 ± 0.2), or the maximum response to heat (139.7 ± 33.5% of 1st
trial).
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In contrast, the -ORA EMD 61,753 (n = 3, intra-arterial 16 mg/kg; n = 3, intracolonic,
10
3 M) totally blocked responses to heat in
four fibers tested and attenuated response in the other two fibers (1 intra-arterial and 1 intracolonic) to 73 and 9% of control. Response
thresholds of these two fibers were not affected by EMD 61,753 (45 and
46.5°C before and 47.5 and 46°C after EMD 61,753, respectively).
Similarly, U62,066 (n = 8, intra-arterial 16 mg/kg;
n = 1, intracolonic 10
3 M)
completely blocked responses to heat in eight fibers, having no effect
in one fiber after intra-arterial administration. Respective examples
are shown in Fig. 7, which also illustrates that responses to heat can
recover 20 min after U62,066 administration.
In seven of these fibers, the effects of EMD 61,753 or U62,066 were tested on responses of pelvic nerve afferents to both mechanical and heat stimuli (intracolonic administration of EMD 61,753, n = 3; intra-arterial administration of EMD 61,753, n = 3; intra-arterial administration of U62,066, n = 1). EMD 61,753 or U62,066 attenuated the responses of pelvic nerve afferent fibers to both mechanical and heat stimuli in all seven fibers tested.
The effects of intra-arterial administration of U62,066 on responses of three mechanosensitive afferent fibers to noxious heat were tested before and 10 min after intra-arterial administration of naloxone (2 mg/kg). U62,066 (16 mg/kg) inhibited responses to heat in all three fibers. After naloxone treatment, the response to heat by U62,066 was 106.2 ± 23.5% of control; an example is shown in Fig. 8.
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DISCUSSION |
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We previously documented that intra-arterial administration of
-, but neither µ- nor
-ORAs dose-dependently attenuated
response of mechanosensitive pelvic nerve afferent fibers to noxious
CRD (Sengupta et al. 1996
, 1999
;
Su et al. 1997a
). Similarly, in the present study,
intracolonic administration of
-, but neither µ- nor
-ORAs
concentration-dependently attenuated response of mechanosensitive
pelvic nerve afferent fibers to noxious CRD. In addition, we previously
reported that mechanosensitive pelvic nerve afferent fibers also
responded to noxious heat stimulation (Su and Gebhart
1998b
). The present study confirms the polymodal character of
mechanosensitive pelvic nerve afferent fibers innervating the rat
colon; 78% of mechanosensitive fibers tested were also heat sensitive.
Like responses to CRD, responses to noxious heat were attenuated by
-, but neither µ- nor
-ORAs. Drug administration in these
experiments was principally by the intra-arterial route, but
intracolonic administration of EMD 61,753 similarly attenuated responses to intracolonic perfusion of heated Krebs solution. Naloxone
antagonized the effects of the
-ORA U62,066 on both noxious
mechanical and heat stimuli, providing evidence that the effects
observed were produced at an opioid-like receptor, consistent with our
previous reports (Sengupta et al. 1996
,
1999
; Su et al. 1997a
,b
). In those
experiments where naloxone was administered intracolonically before
U62,066, we noted no change in responses to noxious CRD, suggesting
that no opioid tone was present peripherally or contributed to outcomes.
Intracolonic -ORA effects on mechanonociception
These results extend our previous findings and establish a
peripheral (colonic) site of action for -ORAs (in addition to or
distinct from the cell soma in the DRG). Systemic administration of
-ORAs in previous work and in the present study uniformly inhibited
responses to CRD (Sengupta et al. 1996
,
1999
; Su et al. 1997a
). Intracolonic
administration of
-ORAs attenuated responses to fluid CRD of only 13 of the 23 fibers tested. This may reflect the location of some fiber
terminals relatively more removed from the mucosal surface of the
colon, where drugs may not have penetrated. For nine fibers unaffected
by intracolonic administration of
-ORAs, intra-arterial
administration of
-ORAs dose-dependently attenuated responses to
noxious CRD. The magnitude of inhibition of responses to CRD differed
between the different routes of drug administration, supporting the
notion that there are two (or more) sites of action. In this and
previous studies (Sengupta et al. 1996
; Su et al. 1997a
), intra-arterial administration of
-ORAs typically
inhibited responses to
10% of control (e.g., see Fig.
5B); the doses of EMD 61,753 and U62,066 in the present
study that produced 50% attenuation of the response to CRD were 6.5 and 3.5 mg/kg, respectively. Intracolonic administration of
-ORAs
attenuated responses to CRD to ~40-50% of control, which is not
inconsistent with the maximum systemic concentration of drug estimated
to result from intracolonic instillation of 10
3
M EMD 61,753 or U62,066 if all drug entered the circulation (i.e., 5.06 and 3.86 mg/kg, respectively).
Although some drug administered into the colon likely entered the
systemic circulation, only if we assume that all or most of the drug
did so (see above), and did so over a relatively short period of time
(effects of -ORAs were apparent when tested 6 min after intracolonic
administration of the 1st concentration tested,
10
4 M; see Fig. 5A), can the present
results be interpreted to involve other than a local, colonic site of
action. This interpretation is supported by the antagonistic efficacy
of naloxone when both naloxone and U62,066 were given intracolonically
and the failure of intracolonic naloxone to antagonize intra-arterial
U62,066. That responses of fibers unaffected by intracolonic
administration of
-ORAs were significantly attenuated when the same
drugs were given intra-arterially (suggesting that drug given
intracolonically does not escape in sufficient concentration into the
systemic circulation) also supports a peripheral site of action of
-ORAs.
The inhibitory effects of the -ORAs produced after intracolonic
administration also are not due to a change in compliance of the colon;
intracolonic pressure was not changed by the maximum concentration of
-ORAs administered into the colon. This is consistent with a
previous finding that another
-ORA, U50,488, did not produce a
significant change in tension of either longitudinal or circular muscle
of the colon (Su et al. 1997a
). We also failed to
observe significant effects of
-ORAs on the tone or contractility of urinary bladder smooth muscle (Su et al. 1997b
). In
complementary studies,
-ORAs have been reported to not affect
gastrointestinal transit in rats (La Regina et al. 1988
;
Tavani et al. 1984
).
µ-, -, and
-opioid receptors or their messenger RNAs have been
documented to be present in spinal DRG cells in the rat, preferentially
small DRG cells (Ji et al. 1995
; Minami et al. 1995
; Schafer et al. 1994
). Since they were
first discovered, it was appreciated that opioid receptors were present
in the intestine (Pert and Snyder 1973
). Pharmacological
and electrophysiological investigations provided early evidence for
opioid effects localized to the gastrointestinal tract, and binding
studies subsequently localized opioid receptors to nerves and smooth
muscle in the gastrointestinal tract (Daniel and Fox-Threlkeld
1989
; Kuemmerle et al. 1992
; Miller and
Kirning 1989
). Recent studies using antibodies raised to the
cloned µ- and
-receptors found that smooth muscle cells in the rat
colon contained neither µ- nor
-receptors (Bagnol et al.
1997
; Fickel et al. 1997
). Both receptor types,
however, were present on myenteric and submucosal plexus neurons as
well as on interstitial cells, suggesting possible roles
related to absorption, secretion, motility, and visceral sensation.
Resolution of which opioid receptors associated with different cells
relate to which of the many effects of opioids in the colon requires further investigation. We believe that the inhibitory effects of
-opioid receptor agonists reported here occur in consequence of
activation of opioid-like receptors associated with the receptive endings of pelvic nerve afferent fibers and/or neurons of the enteric
nervous system. Finally, a recent study (Simonin et al. 1998
) in
-opioid receptor-deficient mice documented a
significantly enhanced sensitivity to intraperitoneal injection of
acetic acid, a model of chemical visceral nociception. In the
aggregate, the present and other results demonstrate that the
antinociceptive effect of
-ORAs occurs at a peripheral receptor
likely associated with afferent nerves innervating the colon. In
support, Diop et al. (1996)
reported in an abstract that
a colonic intramural injection of the
-ORA U50,488 (100 µg)
significantly attenuated the stretching and contractions produced by a
colonic intramural injection of Formalin (5%, 50 µl) in the rat.
-ORA effects on colonic thermonociception
With the use of cutaneous models of nociception, it was initially
reported that -ORAs were more effective against mechanical (pressure) stimuli than noxious thermal stimulation (Millan
1989
, 1990
; Tyers 1980
). When
mechanical and thermal stimulus intensities are matched to produce
equivalent response magnitudes, however, Parsons and Headley
(1989a
,b
) found no difference in the ability of
-ORAs to
attenuate responses to these different modalities of stimulation. In
the present study, only
-ORAs and not either a µ- or
-ORA
attenuated responses to noxious visceral mechanical and thermal
stimuli. Because thermal stimulation of the colon repeated more than
twice, even at a 10- to 20-min interstimulus interval, sensitizes
subsequent responses of pelvic nerve afferent fibers (Su and Gebhart,
unpublished observations), we could not perform a quantitative,
dose-dependent study of
-ORA effects on colonic thermonociception.
Comparing responses to thermal stimulation after drug with its predrug
control response, we nevertheless obtained clear evidence for
significant
-ORA attenuation in response magnitude, whether given
intra-arterially or intracolonically.
Like joints (Schmidt 1996), skeletal muscle
(Kumazawa and Mizumura 1976
; Mense 1996
),
dura (Bove and Moskowitz 1997
), cornea (Gallar et
al. 1993
), splanchnic nerve afferent fibers innervating the
mesentery (Adelson et al. 1996
, 1997
),
and superior spermatic nerve innervation of the testis and/or
epididymus (Kumazawa and Mizumura 1980a
,b
;
Kumazawa et al. 1987
), mechanosensitive colonic afferent
fibers also transduce noxious thermal energy (Su and Gebhart
1998b
; and this study). In support, in whole cell patch-clamp recordings from adult colon sensory neurons, we (Su et al.
1999
) found that nearly one-half of the DRG cells studied were
capsaicin sensitive, consistent with their polymodal character (see
Bevan and Szolcsányi 1990
) and with documentation
that the recently cloned vanilloid receptor at which capsaicin acts is
a heat-transducing channel (Caterina et al. 1997
).
Opioids are fairly selective in their ability to attenuate noxious
inputs and have no or modest effects on other sensory modalities. Detailed information about the association of opioid receptors with
polymodal nociceptors, however, is still lacking. Polymodal nociceptor
axons have been described as terminating in a "chain of beads" or
as having multiple sensor sites; some of these are able to be activated
by mechanical stimuli, and others are depolarized by noxious heat or
chemical stimuli (Szolcsányi 1993). Functionally,
-ORAs inhibit responses of visceral polymodal afferent fibers to
mechanical and thermal stimuli, and the pharmacology of modulation of
heat and pressure stimuli by
-ORAs was indistinguishable in the
present study.
Implications
Intracolonic administration of -ORAs could provide a useful
route of administration for reduction of visceral pain, reducing drug
access to the CNS where
-ORAs produce undesirable effects. In
acutely irritated (HAc-, xylenes- or mustard oil-treated colon or
bladder) (Sengupta et. 1996
; Su et al.
1997a
,b
) or chronically inflamed colon (Sengupta et al.
1999
), neither µ- nor
-ORAs attenuated responses to
distension, whereas the inhibitory effects of
-ORAs were
significantly greater in inflamed colon (Langlois et al. 1994
; Sengupta et al. 1999
). This outcome is
consistent with other reports that opioids are more effective at
peripheral opioid receptors in the presence of inflammation
(Ferreira and Nakamura 1979
; Stein et al.
1988
, 1989
). Accordingly, intracolonic
administration of
-ORAs may be an effective analgesic in visceral
pain states such as irritable bowel disease.
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ACKNOWLEDGMENTS |
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The authors thank M. Burcham for producing the graphics and S. Birely for preparing the manuscript.
This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-19912.
Present address of V. Julia: Institut de Recherche Jouveinal/Parke-Davis, 3-9 rue de la Loge, BP 1000, 94265 Fresnes Cedex, France.
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FOOTNOTES |
---|
Address for reprint requests: X. Su, Dept. of Pharmacology, Bowen Science Bldg., The University of Iowa, Iowa City, IA 52242.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 July 1999; accepted in final form 13 October 1999.
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REFERENCES |
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