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Anesth Analg 1999;88:204-208
© 1999 International Anesthesia Research Society

GENERAL ARTICLES

Naloxone Inhibits Gastric Emptying in the Rat

Takashi Asai, MD, PhD*,*, and Ian Power, BSc(Hons), MD, FRCA{dagger}

*Department of Anesthesiology, Kansai Medical University, Moriguchi City, Osaka, Japan; *Department of Anaesthetics and Intensive Care Medicine, University of Wales College of Medicine, Cardiff, United Kingdom; and {dagger}Department of Anaesthesia and Pain Management, Royal North Shore Hospital and University of Sydney, St. Leonards, New South Wales, Australia

Address correspondence and reprint requests to Takashi Asai, Department of Anesthesiology, Kansai Medical University, 10–15 Fumizono-cho, Moriguchi City, Osaka, 570-8507, Japan. Address e-mail to asait{at}takii.kmu.ac.jp


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Naloxone is generally considered to be a pure antagonist, but it may produce several behavioral effects, such as hyperalgesia or stimulation of respiration. We studied the effect of naloxone on gastric emptying and gastrointestinal transit in rats. Six to eight Wistar rats (200–250 g) were used for each experiment. Either saline or naloxone (0.01–10 mg/kg) was injected intraperitoneally at 0 min. At 30 min, radiolabeled saline or milk 1 mL was infused into the stomach. At 60 min, gastric emptying and gastrointestinal transit were calculated by measuring the radioactivity in the gastrointestinal tract. Naloxone significantly inhibited gastric emptying of saline (P = 0.002) and of milk (P < 0.05), but not the gastrointestinal transit of either (P > 0.05). Gastric emptying of saline showed a significant peak (P < 0.05) in the dose-response curve at 0.7 mg/kg. Therefore, naloxone significantly inhibits gastric emptying of saline and milk, but not the gastrointestinal transit of either.

Implications: Although naloxone is generally considered to be a pure opioid receptor antagonist, it delays gastric emptying of saline or milk, as does morphine in the rat. However, it is uncertain from our results whether naloxone inhibited gastric emptying by antagonizing the effects of endogenous opioids.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Naloxone is generally considered to be a pure opioid receptor antagonist (1,2). However, it may cause several behavioral effects, such as hyperalgesia or stimulation of respiration (3–5). One possible explanation for this is that naloxone inhibits the effect of endogenous opioids (6–10). However, it also produces morphine-like effects (11–17).

The abundance of endogenous opioids in the gastrointestinal tract (18) suggests that these opioids are involved in controlling gastrointestinal motility. Both synthetic and endogenous opioids delay gastric emptying and intestinal transit (19,20). Therefore, naloxone may increase gastric emptying and intestinal transit by antagonizing possible inhibitory effects of endogenous opioids. However, in our earlier experiments, it seemed that naloxone significantly delayed the gastric emptying of saline (20). The main aim of the current study was to determine whether naloxone affected gastric emptying and gastrointestinal transit of liquids.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Male Wistar rats weighing 200–250 g were used. The study was conducted under the Animal (Scientific Procedures) Act of 1986 with approval by an institutional ethics committee. Before the experiment, rats were housed under standard, controlled environmental conditions, with a 12-h light/dark cycle. The animals were fasted for 24 h, but they were allowed free access to water until 20–30 min before the start of the experiment. Each animal was kept individually in a wire mesh cage to prevent coprophagy during fasting. All experiments were started between 10:00 and 11:00 AM. Naloxone was freshly prepared on the day of each experiment.

A group of six rats was allocated for each set of experiments. If more than one rat was excluded (see below), another group of six rats was used until a group in which no more than one rat was excluded completed the experiment. Data from the nonexcluded rats from all the groups for each set of circumstances (the number of rats ranged from six to eight) were used.

Gastric emptying and gastrointestinal transit of liquids containing no nutrients (saline) and nutrients (milk) were used because the mechanisms for gastric emptying differ for these liquids (21,22) and thus may be affected differently by naloxone. Nonskim, pasteurized milk (containing 3.2 g of protein, 4.7 g of carbohydrate, and 4.0 g of fat in 100 mL) was used.

Either naloxone (0.1–10.0 mg/kg) or saline was given intraperitoneally in a volume of 1.0 mL/kg. These doses were based on the reported doses that produced the antinociceptive effect, hyperalgesic effect, or both (3,4).

Thirty minutes later, the rats were lightly anesthetized with halothane. Once the rat had become unconscious, 1.0 mL of either saline or milk containing a radioactive marker (0.5 µCi [18.5 kBq] of chromium as the sodium salt [51Cr as Na2CrO4]) was given through a metal cannula into the stomach. The rat was allowed to recover from anesthesia, which usually occurred within a few minutes.

Another 30 min later, the rats were killed, the esophagus just proximal to the gastric fundus and the duodenum just distal to the pylorus were cross-clamped, and the stomach and small intestine were removed. A dozen clamps were applied to the small intestine during gentle removal of the intestine to minimize the movement of contents. The intestine was placed on a ruled template and divided into 10 equal segments. The stomach and the segments of intestine were placed into individual counting tubes.

The radioactivity in each segment was measured using an automatic {gamma}-sample counter, and counts per minute (cpm) were obtained. Each sample was counted twice, and the mean cpm was obtained. To obtain the control total count, 1.0 mL of radiolabeled saline or milk was placed into a tube, and the radioactivity was counted. If the recovered total cpm from the gastrointestinal tract was <90% of the control total count or if there was chyme in either the stomach or small intestine, the data were not used.

Gastric emptying of liquids was calculated as the percentage of radioactivity (cpm) that had entered the small intestine: Go


From these values, the percent inhibition of gastric emptying (%GE) was calculated from Go


where control %GE = the mean %GE in the rats that received intraperitoneal saline injection, and test %GE = the %GE in each rat that received naloxone.

Gastrointestinal transit was assessed using the "geometric center" (the center of gravity; GC), which was calculated by a modification of a method described by Miller et al. (23) (Figure 1). The major difference between their method and ours was that they infused radiolabeled liquids directly into the duodenum, whereas we infused them into the stomach. With their method, it is possible to eliminate a possible influence of gastric emptying on the transit in the small intestine, but it is impossible to examine gastric emptying simultaneously. In addition, a considerably high proportion of radiolabeled marker is absorbed from the small intestine when the marker is infused directly into the duodenum (24,25). With our method, it is possible to simultaneously observe gastric emptying and distribution of a marker in the small intestine in each rat, although accelerated or delayed gastric emptying may alter small intestinal transit. In addition, the loss of the radiolabeled marker from the gastrointestinal tract is minimal because the marker is hardly absorbed from the gastrointestinal tract when it is infused into the stomach (24,25). Bearing this disadvantage of our method in mind, we used the term gastrointestinal transit rather than small intestinal transit: Go


The percent inhibition of gastrointestinal transit, expressed in terms of the GC, was calculated from Go


The Mann-Whitney U-test was used to compare median gastric emptying and gastrointestinal transit between rats injected with saline and those injected with naloxone; the values for all doses of naloxone were pooled for this analysis. A P value <0.05 was considered significant. Confidence intervals for the median difference in gastric emptying between saline and naloxone were calculated. The variation of percent inhibition with the dose of naloxone on gastric emptying and gastrointestinal transit was modeled by fitting a quadratic equation to fractional inhibition against dose. The significance of the curvature of the resulting parabola was tested.



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  		Figure 1. Three hypothetical examples of the distribution of marker in the 10 segments of the small intestine. The distribution is expressed as geometric center (the center of gravity) = {Sigma}(CiSi)/{Sigma}Ci, where Ci = count in segment Si (23). a, All the marker is in Segment 1; therefore, the geometric center is 1 (1 x 1 + 0 x 2 + ... + 0 x 10 = 1). b, All the marker is in Segment 10; therefore, the geometric center is 1 (0 x 1 + ...+ 0 x 9 + 1 x 10 = 10). c, The marker is evenly distributed throughout the ten segments; therefore, the geometric center is 5.5 (0.1 x [1 + 2 + ... + 10]/1 = 5.5). Reproduced from Reference (20).

 

   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Naloxone significantly inhibited gastric emptying of saline (P = 0.002) (Table 1, Figure 2) but not its gastrointestinal transit (Table 1). There was significant curvature of the dose-response curve for gastric emptying, with a peak effect at 0.7 mg/kg, but none for gastrointestinal transit (Figure 2).


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  		Table 1. Effect of Naloxone on Gastric Emptying and Gastrointestinal Transit
 


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  		Figure 2. Effect of naloxone on gastric emptying of saline (upper panel) or milk (lower panel). Either saline or milk 1 mL, containing a radiolabeled marker (51Cr), was infused into the stomach 30 min after an intraperitoneal injection of naloxone. Gastric emptying was examined 30 min later. A fitted parabola is shown, together with the value obtained from each rat ({circ}). Symbols are slightly displaced if there are similar values for any given dose. The values at 0 dose are for the saline control. Naloxone significantly inhibited gastric emptying of both saline (P = 0.002) and milk (P < 0.05). There was significant curvature of the dose-response curve for gastric emptying of saline (with a peak effect at 0.7 mg/kg), but not for emptying of milk (the quadratic equation for gastric emptying of saline: y = 66.4 - 2.24z + 8.80z2, where y is percent gastric emptying and z is log dose; the SE of the coefficient for z2 is 4.23, t = 2.09, df 29, P < 0.05; the quadratic equation for gastric emptying of milk: y = 52.0 - 5.81z + 10.47z2; the SE of the coefficient for z2 is 6.02, t = 1.74, P > 0.05).

 
Similarly, naloxone significantly inhibited gastric emptying of milk (P < 0.05) (Table 1, Figure 2) but not its gastrointestinal transit (Table 1). However, the curvature of the dose-response curve for gastric emptying was not significant (Figure 2).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In our study, naloxone significantly inhibited gastric emptying of saline and milk. In most previous studies using rodents, naloxone, which was used as a control, did not significantly affect gastric emptying (26–28). However, in one study, it significantly inhibited gastric emptying of a solid meal (29). In contrast, in another study, a small dose of naloxone (0.1 mg/kg) accelerated gastric emptying of both milk and liquids containing fat, although it did not significantly affect the emptying of liquids containing no fat (30).

In humans, one study showed a significant inhibition (31). In addition, a close analysis of other studies reveals that, in all studies in which no significant effect of naloxone was detected, the mean residual gastric volume was always larger in subjects in whom naloxone had been injected than in those who had received saline (11,32–35). Therefore, it is possible that the number of subjects used in the previous studies was too small to detect a significant effect of naloxone on gastric emptying (ß error).

The inhibitory effect of naloxone on gastric emptying of saline or milk was not progressively dose-dependent: the effect decreased at larger doses (Table 1). There was a significant peak in the dose-response curve for gastric emptying of saline at 0.7 mg/kg. A similar quadratic relationship has been reported for the effect of naloxone on nociception (antinociception or hyperalgesia), with the maximal effect at 1–3 mg/kg in rodents (3,4). No explanation has been given as to why naloxone loses its nociceptive effect at larger doses, but common mechanisms may be involved in producing a nociceptive effect and the inhibitory effect on gastric emptying.

In our study, naloxone did not significantly affect gastrointestinal transit. This result is consistent with previous studies, which have shown no significant effect of naloxone on gastrointestinal transit (36,37). The difference in the effect on gastric emptying and intestinal transit produced by naloxone and several other drugs (20,38,39) indicates that the mechanisms for controlling gastric emptying and intestinal transit are different.

In our previous (20) and current studies, both naloxone and morphine inhibited gastric emptying; however, the effect of naloxone was much weaker than that of morphine, and naloxone antagonized the effect of morphine. There are several situations in which both morphine and naloxone produce similar effects, but, when they are given together, naloxone antagonizes the effect of morphine. In humans, both naloxone and opioid agonists may inhibit gastric acid secretion (11,12). In isolated axons of the squid, both naloxone and morphine inhibited membrane ionic currents to a similar degree (13). In cats, both morphine and naloxone, which were applied iontophoreti-caly to the spinal cord, depressed the activity of spinal interneurones (14).

Naloxone and morphine also inhibit contractions of feline gastrocnemius muscles; however, when both drugs are injected together, naloxone antagonizes the inhibitory effect of morphine (15). In mice that lack ß-endorphin by site-directed mutagenesis, both morphine and naloxone produced antinociception; naloxone antagonized the effect of morphine (16). In pigeons that had been trained to respond to a specific light (pecking a box that allows access to grain), these two drugs inhibited the response. When given together, naloxone antagonized the effect of morphine (17).

Two possible mechanisms have been suggested for the behavioral effects of naloxone. One is that naloxone produces behavioral effects by antagonizing the intrinsic effect of endogenous opioids. Naloxone inhibits antinociception induced by acupuncture (6), electroconvulsive shock (7), electrical stimulation of a supraspinal site (8), electrical stimulation of the foot (9), or thermal stimulation of the tail (10). The other possibility is that naloxone produces behavioral effects through the {gamma}-aminobutyrate receptor (40,41).

In conclusion, we have shown that naloxone inhibits gastric emptying, which indicates that naloxone may produce behavioral effects similar to those of opioid agonists. It is unclear whether naloxone inhibits gastric emptying by antagonizing the effects of endogenous opioids.


   Acknowledgments
 
We thank W. W. Mapleson, Department of Anesthetics and Intensive Care Medicine, University of Wales College of Medicine, for his critical review of the manuscript.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication October 13, 1998.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press