Visceral sensations are an important component of many clinical pain states. It is apparent that intrathecal pain relief may be more effective if appropriate combinations of drugs rather than a single agent can be used. The purpose of this study was to examine the relative contribution of opioid receptor subtypes to visceral antinociception using colorectal distension as a visceral pain model.
The minimum colorectal distending pressure necessary to evoke a visceromotor response (contraction of abdominal musculature) was determined before and after the administration of opioid agonists for the mu (morphine), delta ([D-Pen2, D-Pen5] enkephalin [DPDPE]), and kappa (U50,488H) opioid receptors. In addition to the three drugs administered intrathecally, U50, 488H was also administered intravenously.
Morphine and DPDPE produced a reversible increase in threshold for activation of the visceromotor response (50% maximum possible effect [MPE] at intrathecal doses of 2.2 and 16.4 micrograms, respectively). The maximum intrathecal dose of U50,488H (100 micrograms) produced only a 20% MPE. Intravenous U50,488H produced a 50% MPE at a dose of 2.6 mg/kg.
The results suggest that spinal mu- and delta- but not kappa-opioid receptors have a significant role in the modulation of visceral nociception induced by colorectal distension. In addition, the results indicate that activation of nonspinal kappa receptors may mediate visceral antinociception.
Methods: The minimum colorectal distending pressure necessary to evoke a visceromotor response (contraction of abdominal musculature) was determined before and after the administration of opioid agonists for the micro (morphine), delta ([D-Pen2, D-Pen5] enkephalin [DPDPE]), and kappa (U50,488H) opioid receptors. In addition to the three drugs administered intrathecally, U50,488H was also administered intravenously.
Results: Morphine and DPDPE produced a reversible increase in threshold for activation of the visceromotor response (50% maximum possible effect [MPE] at intrathecal doses of 2.2 and 16.4 micro gram, respectively). The maximum intrathecal dose of U50,488H (100 micro gram) produced only a 20% MPE. Intravenous U50,488H produced a 50% MPE at a dose of 2.6 mg/kg.
Conclusions: The results suggest that spinal micro- and delta- but not kappa-opioid receptors have a significant role in the modulation of visceral nociception induced by colorectal distension. In addition, the results indicate that activation of nonspinal kappa receptors may mediate visceral antinociception.
Key words: Analgesics, opioid: [D-Pen2, D-Pen5] enkephalin; morphine; U50,488H. Opioid receptor antagonists: naloxone; naltrindole. Pain, visceral: antinociception; colorectal distension.
DEEP pain associated with the viscera is different from somatic pain and is of clinical importance. Visceral pain has been less well studied than somatic pain, and we therefore need a better understanding of the pharmacologic control of visceral pain. With use of colorectal distension (CRD) as a reliable model of visceral pain, investigators have begun to focus on visceral antinociception and the mechanisms of visceral pain produced by mechanical distension of a hollow viscus. [2-6].
Opioids are of obvious importance in the control of pain. In experimental studies using somatic stimuli, it has been well established that micro-opioid receptor agonists such as morphine inhibit both nociceptive behavioral reflexes and neuronal activity at the spinal cord level. [7-9]Using the CRD test in the rat, Ness and Gebhart [2,3]and Maves and Gebhart demonstrated that intrathecally or intravenously administered morphine powerfully inhibited nociceptive cardiovascular and behavioral (visceromotor) reflexes and dorsal horn neuronal activity. To date, at least three opioid receptor subtypes (micro, delta, and kappa) have been shown to be involved in mediating the processing of a variety of nociceptive information, [10-13]yet the antinociceptive effects of the three opioid subtype agonists on visceral nociception have not been fully appreciated.
Using relatively highly selective micro-opioid, delta-opioid, and highly selective kappa-opioid agonists (morphine, DADLE, and U50,488H), respectively, Schmauss and Yaksh reported that spinal micro- and kappa- but not delta-opioid receptors were involved in modulating the writhing response of a rat to a chemically induced visceral stimulus. In contrast, Porreca et al., studying three agonists (DAGO, [D-Pen2, D-Pen5] enkephalin [DPDPE], and U50,488H) with the highest degree of selectivity for micro-, delta-, and kappa-opioid receptors, respectively, reported that spinal micro and delta receptors had a more important role than kappa receptors in inhibiting the writhing response in the mouse. Therefore, an important question is what role the spinal delta- and kappa-opioid agonists play in visceral pain modulation. .
Visceral pain and perhaps more importantly the adequate stimuli for the production of visceral pain are not well understood. Although the chemically induced writhing test is related to visceral pain, the reproducibility, reliability, and specificity of this test are questionable. On the other hand, the CRD test is a reliable, reproducible, and useful method for study of visceral pain in the rat. In addition, CRD produces visceral pain sensation in humans. [15-18]Although all of these stimuli are assumed to elicit visceral pain it is likely that they do so by very different mechanisms. It is possible that the short-duration mechanical stimulus associated with CRD activates neuronal systems different from those activated by the chemical stimuli associated with writhing tests. In the skin, silent chemoreceptors have been postulated as an explanation for the response to some forms of noxious stimuli. Chemical stimulation, because of the long duration of the stimulus (30-60 min), may also induce inflammation that contributes to the sensory experience. Short-term CRD does not appear to cause inflammation.
The current study was designed to examine the relative contribution of the three spinal opioid receptor subtypes, especially delta and kappa receptors, in visceral antinociception induced by the CRD test. We used a primary micro agonist (morphine), a highly selective delta agonist (DPDPE), and a highly selective kappa agonist (U50,488H).
Materials and Methods
The protocol of this study was approved by the Yale Animal Care and Use Committee. Experiments were conducted on adult male Sprague-Dawley rats weighing 300-400 g. Animals were housed in individual cages, given free access to food and water, and kept on a 12-h light-dark cycle, with light beginning at 6:00 AM.
In all animals in this study an intrathecal catheter was implanted for drug administration. During halothane (1-2%) anesthesia, a catheter (polyethylene [PE]-10 tube, 8.5-9 cm long) was inserted through a slit in the atlantooccipital membrane into the subarachnoid space, and the tip of the catheter was placed near the lumbar enlargement of the spinal cord according to a method described previously. The rostral end of the catheter was connected to another catheter (PE-50 tube, 3 cm long) for drug administration. The catheter was anchored to the surrounding musculature to maintain its position. Total dead space of the whole catheter was 12 micro liter. At the termination of the surgery, animals were treated with 15,000-21,000 U intramuscular penicillin G to prevent infection. Animals were observed for at least 10 days before experimental use. Animals exhibiting neurologic impairments or infection as a result of the surgical procedure (10-15% of the animals) were excluded from experimental use. The location of the tip of the catheter was verified at the end of the terminal experiment by dye injection.
In this study, visceromotor response (VMR) (a contraction of abdominal musculature) to CRD was used as a measure of visceral nociception. CRD was achieved by pressure-controlled air inflation of a latex distension balloon (5 cm long) as illustrated in Figure 1. The distension balloon was connected to a pressure-controlled balloon inflator through a distension balloon catheter and was inflated continuously at a rate of 6 mmHg/s beginning at 0 mmHg until a clear VMR was evoked or until a maximum pressure of 80 mmHg was reached. The distending pressure was limited to 80 mmHg to avoid tissue damage. The pressure control device was modeled after one described previously. Pressure within the distension balloon was monitored continuously through an in-line pressure transducer.
In this study, a small latex balloon (1.5 cm long) was used to detect the VMR objectively. Figure 1illustrates the device used for both the CRD and the detection of VMR. The detection balloon was attached distal to the distension balloon and was used to detect the increase in intraabdominal pressure when the VMR was evoked. The detection balloon was connected to a pressure transducer through a smaller catheter (PE-160 tube), and pressure within the detection balloon was continuously monitored along with the distending pressure on a chart recorder. The detection balloon was completely isolated from the pressure control device and mounted far enough away (1.5 cm) from the distension balloon so as not to interfere with or be affected by the distension balloon. The detection balloon was filled with 0.6 ml air after placement to ensure that it would reliably detect changes in intraluminal pressure. The distension balloon was not inflated before each test.
Because gastrointestinal distension can evoke reflex contraction and relaxation in other parts of the gastrointestinal tract, it was important that we validated the use of the detection balloon. In pilot studies* we demonstrated that there was no significant difference between balloon-detected and visually detected thresholds and that thresholds defined by this technique were comparable to those reported by others. According to the original report by Ness and Gebhart, the minimum distending pressure necessary to evoke a VMR was defined as the visceromotor threshold. In this study, the distending pressure corresponding to the onset of a sudden and sustained increase in the detection pressure was defined as the visceromotor threshold. The increase in detection pressure was associated with a visible contraction of abdominal musculature.
Testing was done 10-19 days after surgery. All data (visceromotor thresholds) were obtained from 87 awake rats. Fifty-nine of 87 rats were used again 3-5 days after the initial experiment but never received the same drug twice.
In the case of intrathecal administration, the animal was lightly anesthetized with halothane for insertion of the distension and detection balloons. Both balloons, lightly coated with petroleum jelly, were inserted intraanally. The distension balloon was positioned in the descending colon and rectum such that the end of the balloon was 1-2 cm inside the anus. The detection balloon was positioned in the descending colon proximal to the distension balloon. Both balloons were kept in position by taping the balloon catheter to the base of the tail. After balloon insertion, the rats were allowed to recover from anesthesia for 10-20 min. For 20-60 min after full recovery, baseline visceromotor thresholds were repeatedly (5-8 times) measured every 5-10 min. The average of the last three values was defined as a control threshold. After baseline measurements, drugs were administered through the chronically implanted catheter. Postdrug thresholds were measured 5, 10, 15, 20, 30, and 45 min after administration. Each postdrug measurement was done only once at each time point. Five rats received intrathecal vehicle (physiologic saline 5 micro liter) for control trials and evaluation of the reliability of the detection balloon technique.
In the case of intravenous administration, a catheter (PE-50 tube) was inserted into the external jugular vein during halothane (1-2%) anesthesia, and both the distension and detection balloons were inserted intraanally as for intrathecal administration. After insertion, the rats were allowed to recover from anesthesia for 30-60 min. Within 20-60 min after full recovery, baseline visceromotor thresholds were measured repeatedly (five to eight times) every 5-10 min. The average of last three values was defined as a control threshold. Postdrug thresholds were measured 2.5, 5, 10, 15, and 20 min after administration. Each measurement was done only once at each time point. Four rats received intravenous vehicle (physiologic saline 0.5 ml) for control trials and evaluation of the reliability of the detection balloon method.
The following drugs were used: morphine sulfate, DPDPE, U50,488H, naloxone hydrochloride, and naltrindole hydrochloride. All drugs except naltrindole were purchased from Sigma Chemical (St. Louis, MO). Naltrindole was purchased from Research Biochemicals (Natick, MA). Morphine, U50,488H, and naloxone were dissolved in sterile physiologic saline (0.9% sodium chloride). DPDPE and naltrindole were dissolved in distilled water. DPDPE was frozen in aliquots at -80 degrees Celsius until use. The compound was not used again after thawing.
Drugs were administered intrathecally in a volume of 5 micro liter. The dead space (12 micro liter) of the catheter was cleared by a slow flush (30-60 s) of physiologic saline. Doses of agonists were as follows: morphine, 0.5, 1.0, 2.5, and 5.0 micro gram; DPDPE, 2.5, 5.0, 10.0, and 25.0 micro gram; and U50,488H 5.0, 10.0, 50.0, and 100.0 micro gram. Naloxone (2.5 micro gram) was coadministered with morphine (2.5 micro gram) in four animals. Naltrindole (5 micro gram) was coadministered with morphine (2.5 micro gram) in four animals and with DPDPE (10 micro gram) in five animals.
U50,488H was also administered intravenously in a volume of 0.5 ml by slow injection (over a 30-s period). Doses were 0.5, 1.0, 2.5, and 5.0 mg/kg (n = 7 for each).
To evaluate the time course and dose dependence of the antinociception, the thresholds after drug administration were compared with those of vehicle and the predrug thresholds by one-way and two-way analyses of variance for repeated measures. Antagonism by naloxone or naltrindole of morphine or DPDPE was analyzed by one-way analysis of variance. Fisher's least-significant difference test was used for post hoc comparisons of individual mean values. P values < 0.05 were deemed statistically significant. To draw dose-effect lines for drugs, all threshold values were converted to percentage maximal possible effect (MPE) by the following equation: percentage MPE = 100 x (postdrug threshold - control threshold)/(80 - control threshold). By least-squares linear regression analysis, doses producing 50% MPE and its 95% confidence intervals were calculated.
Visceromotor Thresholds in Vehicle-treated Animals
Visceromotor thresholds in both intrathecal (n = 5) and intravenous (n = 4) vehicle-treated rats were constant over the 60-min observation period (Table 1). The mean value of all control visceromotor thresholds determined in this study was 22 mmHg.
Antinociceptive Effects of Intrathecal Morphine and DPDPE in Response to Colorectal Distension
As shown in Figure 2, the intrathecal administration of morphine (0.5-5.0 micro gram) significantly (P < 0.05 or P < 0.01) increased the visceromotor thresholds in a dose-dependent manner. The peak effects were observed approximately 15 min after administration. At this time point, the thresholds (means plus/minus SEM) for 0.5, 1.0, 25, and 5.0 micro gram morphine were 34 plus/minus 5, 41 plus/minus 6, 56 plus/minus 6, and 71 plus/minus 5 mmHg, respectively. Figure 3shows the effects of 2.5-25 micro gram intrathecal DPDPE. The visceromotor thresholds increased significantly (P < 0.05 or P < 0.01) in a dose-dependent manner. The time for peak effect was approximately 15 min after administration. At this time point, the thresholds for 2.5, 5.0, 10, and 25 micro gram DPDPE were 28 plus/minus 3, 34 plus/minus 2, 46 plus/minus 5, and 61 plus/minus 5 mmHg, respectively. With the exception of the largest dose (25 micro gram), the thresholds returned to control levels within 45 min of administration.
As shown in Figure 4, the antinociceptive effect of morphine (2.5 micro gram) was significantly (P < 0.01) antagonized by naloxone (2.5 micro gram) but not naltrindole (5 micro gram) at 15 min after administration. The antinociceptive effect of DPDPE (10 micro gram) was significantly (P < 0.01) antagonized by naltrindole (5 micro gram) at 15 min after administration.
Antinociceptive Effects of Intrathecal and Intravenous U50,488H in Response to Colorectal Distension
In contrast to morphine and DPDPE, intrathecal U50,488H increased the visceromotor thresholds significantly (P < 0.05) at only 5 and 10 min after administration of 100 micro gram, a maximum dose that could be dissolved in 5 micro liter saline (Figure 5). The thresholds at each time point were 32 plus/minus 3 and 33 plus/minus 2 mmHg, respectively. Other doses of U50,488H had no significant effect on the threshold at any time. In contrast to intrathecal U50,488H, intravenous U50,488H (0.5-5.0 mg/kg) increased the visceromotor thresholds significantly (P < 0.05 or P < 0.01) in a dose-dependent manner (Figure 6). The time of peak effect was approximately 2.5 min after administration. At this time point, the thresholds for 0.5, 1.0, 2.5, and 5.0 mg/kg were 29 plus/minus 3, 42 plus/minus 3, 53 plus/minus 7, and 65 plus/minus 7 mmHg, respectively. The dose producing 50% MPE at 2.5 min after administration was 2.6 mg/kg (95% confidence interval 1.9-3.6 mg/kg). The thresholds returned to control levels by 20 min at all doses tested.
Potencies of Intrathecal Morphine, DPDPE and U50,488H for Producing 50% Maximum Possible Effect
In Figure 7, dose-effect curves for intrathecal morphine, DPDPE, and U50,488H are displayed. In contrast to morphine and DPDPE, intrathecal U50,488H did not produce 50% MPE even at the maximal dose used in this study. Fifty percent-MPE doses of intrathecal morphine and DPDPE at the time of peak effect were 2.2 micro gram (95% confidence interval 1.5-3.2 micro gram) and 16.4 micro gram (95% confidence interval 13.2-21.6 micro gram), respectively. From the values of nanomoles for the 50% MPE, the rank order of potencies of individual drugs was morphine > DPDPE > > U50,488H. Morphine was 7.6 times more potent than DPDPE. Whereas slopes of regression lines for morphine and DPDPE were not significantly different, the regression line for U50,488H was not parallel with those for morphine and DPDPE.
In the current study, we have demonstrated that intrathecal micro (morphine) and delta (DPDPE) but not kappa (U50,488H) opioid receptor agonists produce potent antinociceptive effects when tested against a visceral pain model that relies on mechanical distension of a hollow organ. kappa-Opioid receptors seem to also be involved in attenuating a VMR to CRD but only when the agonists are administered systemically. These results are in agreement with a recent report of a similar study by Danzebrink and colleagues. .
The evidence that intrathecal morphine produces powerful visceral antinociception in the CRD test is consistent with previous reports. [2,6]Ness and Gebhart reported that the median effective doses of intrathecal morphine for inhibiting cardiovascular responses in the rat were 6.2 micro gram (pressor response) and 5.2 micro gram (tachycardia response). Maves and Gebhart reported that the 50% MPE dose of intrathecal morphine in inhibiting VMR was 1.5 micro gram in the rat. In the current study, the VMR was inhibited with a 50% MPE dose of 2.2 micro gram. That intrathecal morphine exerted visceral antinociceptive effects was also demonstrated using the writhing test in the rat. [10,13]These reports suggest that spinal micro-opioid receptors are likely to be involved in modulating visceral nociception.
Although morphine preferentially interacts with micro receptors, it has been reported that it may affect somatic and visceral responses through interactions with delta receptors. In the current study, the antinociceptive effect of morphine was antagonized by a nonselective opioid receptor antagonist, naloxone, but not by a highly selective delta receptor antagonist, naltrindole. Our results suggest that intrathecal morphine produces visceral antinociception against mechanical stimuli mainly through the micro- rather than delta-opioid receptors.
Our finding that intrathecal DPDPE is effective in the modulation of visceral nociceptive information is in agreement with experiments in mice reported by Porreca et al. In addition, we demonstrated that the visceral antinociceptive effect of intrathecal DPDPE was antagonized by the selective delta receptor antagonist, naltrindole. Therefore, it is likely that spinal delta-opioid receptors mediate visceral antinociception induced by mechanical distension of hollow visceral.
Our results are not in complete agreement with the work of Schmauss and Yaksh. They reported a significant reduction of writhing by U50,488H at doses tested in the current study. In their study 100 micro gram of U50,488H caused a 70% reduction in the cumulative writhing score. They also reported that delta agonists were without effect at the doses studied. The most obvious difference in the studies is the method for eliciting a noxious visceral stimulus. We propose that the differences in pharmacologic effect between the two studies is a result of differences between the stimuli. CRD provides a selective physiologic stimulus that activates mechanoreceptors in the muscle layer of the colon and rectum. In contrast, intraperitoneal injection of an irritant substance is thought to activate unknown receptors mainly on the serosal side of visceral structures but also those associated with nonvisceral structures. It is clear that different pathways are likely to be activated by selective mechanical versus nonselective chemical stimuli. In addition, even though visceral afferent nerves may be activated by chemical stimuli, it is important to remember that they carry information about both noxious and nonnoxious stimuli. A third possibility is that the chronic nature of the chemical stimulus (data collected over 60 min) elicits different sensory experience than the phasic CRD (balloon inflation for 30 s). It is likely that 60 min of ongoing stimulation may induce an inflammatory response that contributes to the sensory experience. Long-term but not less intense CRD has been shown to produce an inflamed colon. .
In this study, although we have not examined the direct antinociceptive effects of U50,488H at supraspinal sites, evidence that intravenous U50,488H is effective in inhibiting VMR to CRD suggests that the supraspinal or peripheral kappa receptors can be involved in antinociception against CRD. Similar results have been reported from work that examined both behavior and c-fos expression. .
Two recent studies [26,27]point to possible peripheral sites of action for kappa-opioid analgesia against noxious responses to distension of hollow viscera (duodenum and colon). Evidence for a peripheral site of action is of importance. Twenty years ago opioid analgesia was assumed to be attributable to actions limited to the brain. In the past two decades, emphasis has been placed on spinal pharmacologic mechanisms. We must now begin to evaluate the peripheral pharmacologic mechanisms that may also be involved.
The effect of systemically administered U50,488H raises an issue that must be considered when drug studies are conducted with CRD as the test stimulus. It is well established that opioids can influence gastrointestinal motility. We need to recognize that such an effect may influence responses to CRD in at least two different ways. If the resting tone of the smooth muscle is changed, then the resistance against which the stimulus is presented would be altered. We observed no such change in pressure records in the detecting balloon. A second possible change would be a change in the responsiveness of the smooth muscle or mechanoreceptors to mechanical stimulation. However, Diop and colleagues reported no significant change in volume of air required to distend the colon, suggesting that the compliance of the colon was not altered.
It is important to keep in mind that all pain is not the same and that the pharmacologic control of pain may depend in part on the nature of the stimulus and the neurochemical events that are activated. A challenge we face is to define more precisely the nature of clinical pain and the neurotransmitter systems involved in communicating information about clinical pain.
* Harada Y, Nishioka, Kitahata, LM, Collins JG: Additional technique for detecting visceromotor response to colorectal distension in awake and lightly pentobarbital anesthetized rats (abstract). Society for Neuroscience Abstracts 17:1010, 1991.