Dept. of Pathophysiology, Jagiellonian University Medical College, Cracow, Poland
Dept. of Integrative Biology and Pharmacology, University of Texas Medical School,
Houston, USA
Changes of intestinal motility and transit produced by tolerance to and dependence upon
morphine have been partly attributed to peripheral mechanisms. We evaluated the effect
of chronic peripheral morphine administration and peripheral µ-receptor blockade on
vagal afferent activity (VAA) and c-Kit positive intramuscular cells of Cajal (ICCs). Ten
rats were subjected to chronic subcutaneous morphine infusion for 72 h with subsequent
VAA recording. Potential frequency was evaluated within recordings before and after µ
receptor blockade by D-Phe -Cys -Tyr -D-Trp -Orn -Thr -Phe -Thr (CTOP) i.p. injections.
Afterwards the rats were sacrificed and intramuscular c-Kit antigen expression was
assessed by image analysis within removed fragments of duodenum and ascending
colon. An equal group of rats served as a control for VAA and c-Kit expression.
Analysis of VAA revealed similar frequencies of potentials in morphine tolerant /
dependent rats before CTOP and in the controls. CTOP increased potential frequency in
the morphine group which effect was visible mostly within the first 20 minutes (p=0.01).
The morphine infused animals presented also higher c-Kit expression in both the
duodenum (p<0.001) and the ascending colon (p<0.001) in comparison to the control
group. Results of our study may indicate the involment of both the intestinal wall and
the long vago-vagal reflexes in tolerance to and dependence upon opioids.
K e y w o r d s : c-Kit antigen, morphine dependence, morphine tolerance, vagal afferent
Opioids are the principal agents in treatment of severe acute and chronic pain.
Their antinociceptive effect is produced by activation of the µ receptors located
not only in the central nervous system (CNS) but also in the peripheral tissues (1).
Unfortunately gastrointestinal GI dysmotility and constipation are one of main
side effects of the opioid therapy. Several studies have supported the hypothesis
of involvement of both the CNS and the enteric nervous system (ENS) receptors
in the attenuation of gastrointestinal (GI) myoelectrical activity, motility and
transit by morphine (2, 3, 4, 5). Therefore the central and peripheral receptors
have been also suspected in development of the intestinal tolerance and
dependence. Both phenomena have been frequently reported as the effects of
chronic administration of morphine. GI tolerance has been defined as the decrease
overtime of the motility response to the chronically administered drug whereas
dependence was assessed by the generation of characteristic withdrawal
syndrome due to interruption of the medication. In the experimental studies use
of suitable µ receptor antagonists develops the clinical syndrome of withdrawal
with increase of motility and intense diarrhoeas. Previous studies on morphine
revealed mutual dependence of both phenomena based on the mechanisms of
intracellular adaptation to the administered drug including the receptor
quantitative and structural changes, second messengers changes and alterations of
neuronal membrane potential (6).
In the current study we have been interested in the involvement of peripheral
ENS µ2 receptors in mechanisms of the intestinal tolerance to and dependence
upon morphine. We have hypothesised that if the peripheral receptors are
involved the development of tolerance / dependence would produce changes in
both the intestinal wall and the long vago-vagal reflex activity. Our purpose was
the evaluation of the influence of chronic, peripheral administration of morphine
and peripheral injections of µ-receptor antagonist D-Phe -Cys -Tyr -D-Trp -Orn Thr -Phe -Thr (CTOP) on tyrosine kinase receptor (c-Kit antigen) and vagal
afferent activity (VAA). According to the previous studies we have presumed that
intramuscular c-Kit expression could reflect activity of the interstitial cells of
Cajal (ICCs), which generate the intestinal base electrical rhythm (BER) (7, 8).
Peripheral administration of CTOP, the agent that doesn't cross the blood-brain
barrier revealed the peripheral mechanisms of the induced morphine withdrawal.
Twenty male Wistars rats, weighting 200 g were used in our experiments. During the study
animals were housed in single cages in the temperature and humidity controlled room with 12-hour
light-dark cycle (light on at 7 a.m.). Food and water were available ad libitum. The rats were divided
into 2 equal group. One group was continuously infused with morphine for 72 h, for the
development of tolerance and dependence, whereas the other served as controls. All animals
underwent the recordings of VAA in left vagus nerve and the assessment of c-Kit antigen expression
in duodenum and ascending colon. All experimental protocols were approved by Local Bioethical
Committee of the Polish Board of Scientific Investigations in Cracow.
Surgery procedures
Placement of osmotic minipumps
For the procedure the animals were anaesthetised by intraperitoneal (i.p.) injections of ketamine
- Ketamina 10% (Biowet, Poland), 100 mg/kg and xylasine - Sedazin (Biowet, Poland), 2 mg/kg.
No information about the prolonged influence of the drugs applied on the GI motility has been
available so far. The osmotic minipumps (Alzet, model 2001, Durect Corporation, USA) were filled
with 200 µl of morphine solution and then placed subcutaneously (s.c.) in the lumbar region of the
animal's back. The procedure was performed to produce 72 h of the continuous s.c. administration
of morphine, 1 mg/kg/h The dose was chosen to establish blood concentration of morphine that
resulted in decrease of the intestinal transit by 50% (9).
Placement of electrodes on left vagus nerve
The procedure was performed in the rats under the ketamine / xylasine anaesthesia after 72 h of
continuous morphine administration. During the surgery and the subsequent recordings a
thermostatically controlled heating pad maintained rectal temperature at 37 ± 1 °C. The left vagus
nerve was uncovered on the animal neck, isolated from the cervical artery and cut possibly
proximally for the access to the distal nerve trunk of 1.5 cm length. The cuff electrode consisted of
the silver wire of diameter 75 µm (A-M Systems, Carlsborg, USA) and the elastic tube 1,5 mm of
diameter and 1,5 cm of length was installed in the distal end of the nerve trunk accordingly to the
data previously published (10). In order to avoid postoperative damage response the latency period
of 15 min before the commencement of the VAA recording was applied.
Experimental procedures
VAA evaluation
VAA in the left vagus nerve was recorded under the ketamine / xylasine anaesthesia
subsequently to the placement of the cuff electrode. Both groups of animals the morphine and the
control were subjected to the 60-min VAA recordings. In the morphine group initial 30 min of
fasting recording was followed by i.p. CTOP (Sigma, USA) injection (1 mg/kg) and another 30 min
of recording. Use of CTOP, specific µ receptor antagonist, allowed evoking the morphine
withdrawal syndrome (11). The potentials were amplified by the BIO Amp (ADInstruments,
Australia) amplifier and analysed spectrally using the Spike Histogram (ADInstruments) software.
The recordings obtained were divided into 10-min intervals. Four subsequent 10-min intervals, one
before and three after the CTOP injection were subjected to the analysis, which allowed for better
exploration of vagal response to CTOP. Frequencies of the potentials were compared between the
controls and morphine animals before CTOP injections for evaluation of the tolerance. Dependence
upon morphine was evaluated by comparison of VAA frequency before and after CTOP. After the
procedure animals were killed by overdosage of the anaesthetics. Fragments of the duodenum and
the ascending colon were removed for intramuscular c-Kit assessment.
Intramuscular c-Kit assessment
The intestinal fragments removed were fixed with alcohol and immersed with paraffin. The 45 µm thick slices prepared, containing longitudinal sections of the bowel wall, were deparaffinated
and marked using the rabbit monoclonal antibodies anty-CD117 (c-Kit Antibody C-19, Santa Cruz
Biotechnology, USA) and the dye set En Vision (DAKO Corporation, USA) for presentation of the
c-Kit antibodies. The entire surface of c-Kit antigen expression [mm2] per 1 mm of the section was
evaluated using the optical microscope Axiophot (Zeiss, Germany), co-operating with the software
applied for the morphometrical measurement.
Statistical analysis
The results were performed as mean values ± standard deviations (SD). The results obtained
from the same group of the animals were subjected to the ANOVA analysis of variance with the
Rat TB M14
nerve X (mV)
Fig.1. Vagal afferent activity (VAA) recorded (Spike Histogram, ADInstruments) in left vagus nerve
of the control (A) and the morphine tolerant and dependent rat (B). [1] - i.p. injection of CTOP.
"post hoc" t-Student test for multiple comparisons. The t-Student test for two populations of images
was applied for the analysis of the results obtained from two compared groups. For each test p<0.05
was considered as statistically significant.
Effect of chronic morphine administration on VAA
Frequency of VAA recorded in the rats influenced by morphine for 72 h was
similar to those obtained from the control animals (0.45±0.2 vs. 0.58±0.3 Hz,
p>0.05). Analysis of the left vagus nerve afferent activity in the morphine infused
animals revealed short lasting, significant response to i.p. injection of µ-receptor
antagonist. After CTOP VAA frequency increased, which was visible within the
first (0.78±0.3, p=0.02) and second (0.7±0.3, p=0.01) 10-min. interval. Frequency
of VAA recorded within the last period, over 20 min after CTOP administration,
was similar to those obtained prior to the m-receptor blockade (Fig. 1, 2).
Effect of chronic morphine administration on c-Kit
Period dominant freguency [Hz
In the control group comparison of c-Kit expression areas in the duodenum
and the ascending colon revealed the prevalence of their expression in the large
bowel (105 ± 15 vs. 78 ± 16 10-4 mm2, p<0.001). Significant differences of cKit expression were also visible between the compared groups. The animals
tolerant to and dependent upon morphine presented bigger than the controls
areas of c-Kit expression in the examined bowel fragments. Such differences
were found in both duodenum (97 ± 15 vs. 78 ± 16 10-4 mm2, p<0.01) and
ascending colon (160 ± 65 vs. 105 ± 15 10-4 mm2, p<0.01) and confirmed by the
applied analysis of variance (Fig. 3, 4).
after 1 h
Fig.2. Quantification
of VAA period dominant
frequency in the controls
and the morphine tolerant
/ dependent rats, before and
after i.p. injections of
CTOP. Values plotted as
means ± standard
Fig.3. Areas of c-Kit antigen expression in longitudinal sections of duodenum (A) and ascending
colon (B) of morphine tolerant and dependent rat (magnification - 360 x).
Chronic administration of morphine inhibits GI motility and transit, which
effect induces constipation. Previous observations suggest that intestinal
dysmotility may result from both central and peripheral action of morphine (4,
12). Simultaneously tolerance and dependence develop due to chronic morphine
administration. Both phenomena are clinically independent however they possess
common mechanisms based on receptor dysfunction and changes of neuronal
Area of c-Kit expression [1 x 10 -4 mm2 x mm-1]
Fig.4. Quantification of
c-Kit antigen expression in
duodenum and ascending
colon of the controls and
the morphine tolerant
/ dependent rats. Values
plotted as means ± standard
membrane potential (6). Suggestion of the central mechanisms of tolerance to and
dependence upon morphine is based on the well-described opioid action on CNS
(13). However regarding the intestinal tolerance / dependence the involvement of
the peripheral mechanisms located within the ENS or the intestinal wall can not
be excluded. Our purpose in the current study was the evaluation of the long
vago-vagal reflexes and the intramuscular c-Kit expression in the small and large
bowel in rats tolerant to and dependent upon morphine. We hypothesised that the
coexistence of changes in both evaluated parameters might indicate the
involvement of peripheral mechanisms in intestinal response to chronic morphine
administration. Evaluation of the long vago-vagal reflexes was based on VAA
measurement. Vagal afferent fibres serve as the afferent branch of the reflex and
their activity depends on stimulation of receptors located in the intestinal wall
(14,15). During the study we applied chronic peripheral infusions of morphine
from the subcutaneously implanted osmotic pumps. Our previous results showed
that the time of tolerance development depends on the opioid dose (16). In our
study we applied the standard D50 dose of morphine that results in 50% inhibition
of the transit. For the experimental development of withdrawal syndrome we used
the i.p. injections of CTOP, a µ receptor antagonist that doesn't cross the bloodbrain barrier (6). The dose of CTOP was matched with dose of morphine based
on our previous observations. According to the data available intensity of the
dependence symptoms may depend on the dose of antagonist which interacts
competitively with the agonist and the receptor (17).
The results obtained by Williams et al. (9) who evaluated the influence of
morphine on the colon contractility and transit suggested that the intestinal
tolerance could be visible on the first day of the chronic drug infusion. Physical
dependence and intestinal dysmotility in reaction to the applied naloxone were
observed after 72 h of the morphine infusion. Therefore in our study we haven't
expected changes of the fasting VAA after 72 h of morphine administration.
Decrease of the fasting VAA after 72 h would have meant that tolerance still
hadn't developed. However we expected the response of vagal activity to the
peripheral µ receptors blockade by CTOP. Our expectations were based on the
other results that confirmed the coexistence between GI dysmotility and changes
of VAA. Blackshaw (18) observed excitation of the vagal afferent fibres during
gastric or oesophageal distension in the anaesthetised ferrets. Similarly Thor et al.
(19) revealed increased vagal afferent response to gastric distension (GD) in rats,
whereas Ozaki et al. (20) didn't observe decreased VAA reaction to GD after
morphine. Those results may suggest that morphine doesn't affect the vagal
afferent fibres directly. On the contrary Kaczyñska et al. (21) suggested that
opioid - receptor activity on vagal afferents maight have been involved in apnoea
an hypotension due to morphine administration.
Regarding these expectations in our study we haven't observed changes of
VAA frequency due to the development of tolerance to morphine. No significant
differences were visible between the recordings obtained in the controls and the
morphine rats before CTOP. Intraperitoneal injection of CTOP produced strong
increase of the VAA frequency in the rats suspected of dependence upon
morphine. Peripheral action of the injected µ-receptor antagonist suggested that
the observed withdrawal response was located inside the intestinal (ENS) µ2
receptors because their blockade revealed transient hyperactivity of the long
vago-vagal reflex.
The observed changes of VAA were compared to the expression of the
intramuscular c-Kit antigen in the duodenum and colon. We presumed that the
intestinal c-Kit expression might reflect the ICCs activity and the intestinal ability
to generate BER (7). Changes in c-Kit expression seem to play the fundamental
role in the intestinal dysmotility related to chronic morphine administration. It has
been previously evidenced that c-Kit antigen blockade decreases the capability of
BER generation by ICCs and facilitates their transformation to typical muscular
cells (22). In our study we evaluated expression of the intramuscular c-Kit,
situated on the border of the longitudinal and circular musculature of the
duodenal and the colon wall. Our study showed significantly bigger areas of c-Kit
expression in both small and large intestine, in the rats chronically infused with
morphine. Such increase of c-Kit expression may be responsible not only for the
development of tolerance to the administered opioid but also for the excessive
myoelectrical and motility withdrawal response due to rebound effect after
peripheral µ2-receptor blockade.
In summary in the examined animals we observed the involvement of the long
vago-vagal reflexes in tolerance to and dependence upon chronically
administered morphine. Changes of the vago-vagal reflex activity coexisted with
increased expression of c-Kit antigen in both the duodenum and the ascending
colon. Our results suggest the involvement of peripheral µ2 receptors located in
ENS in development of tolerance to and dependence upon morphine.
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R e c e i v e d : October 4, 2005
A c c e p t e d : February 3, 2006
Author's address: Tomasz Banach M.D., Ph.D., Dept. of Pathophysiology Jagiellonian
University Medical College, ul. Czysta 18, 31-543 Kraków, Poland