Opioids act by binding to G-protein receptors that are highly conserved across species. In mammalian species, 3 opioid receptors (OPRM1, OPRK1, and OPRD1) have been identified, each of which is encoded by a separate gene. There is notable individual variability within species in the effective dose, adverse effects, and behavioral responses evident after administration of opioids; in several species, this variability is associated with opioid receptor polymorphisms.1,2 Splice variants of OPRM1 have been identified that account for some of the variability; of these variants, at least 31 in mice and 19 in humans have been described.1 Further examination of the human OPRM1 gene has led to the discovery of > 700 SNPs.2 The most common of these is the A118G variant (rs1799971), which results in replacement of asparagine with aspartate at position 40 in the amino acid sequence of OPRM1.3 The A118G variant has been evaluated extensively for its role in the perception of pain, analgesic treatment, adverse effects associated with opioid treatment, and addiction to opioids and other substances (eg, alcohol and nicotine).3,4 It has been concluded from these studies3,4 that to achieve adequate pain control, patients with the G allele require more opioid medication than patients with the A allele.
Administration of μ-opioid receptor agonists to horses is associated with a decreased response to noxious stimuli,5 but as the dose increases, there is an increase in locomotor activity.6 A number of studies7,8,a in horses have found substantial individual variability in the responses to these drugs. It has been considered possible that this variability might be attributable to mutations in the equine OPRM1. In 1 studyb DNA from 48 horses was examined for variations in exon 1 of the OPRM1 gene, and 4 SNPs were discovered. However, only one of these variations resulted in a change in the amino acid sequence in the receptor (the G57C SNP), wherein histidine replaces glutamine at position 19 in the amino acid sequence of the horse OPRM1 gene.
The objective of the study reported here was to examine the locomotor effect of fentanyl on horses heterozygous for the G57C polymorphism of the μ-opioid receptor, compared with effects in a control group that did not have this mutation. Our hypothesis was that there would be no difference in response to fentanyl administration between horses with and without the mutation.
Materials and Methods
Animals
Twenty healthy mature horses that had previously been genotyped with regard to OPRM1 for another project were used in the study. Horses were deemed to be healthy on the basis of results of physical examination and a CBC. Ten horses were heterozygous for the G57C polymorphism of the μ-opioid receptor, and 10 control horses did not have this mutation. Control horses were matched on the basis of breed, sex, and age as closely as possible with the horses with the mutation. The study protocol was approved by the Institutional Animal Care and Use Committee at the University of California-Davis.
Procedures
All experiments were conducted at the same time of day (morning) in a quiet area. Horses were housed in paddocks. The evening prior to each experiment, horses were moved to a stall (3.7 × 3.7 m) with access to a yard (3.7 × 6.4 m) to allow for acclimatization. On the morning of an experiment, the door to the yard was closed 20 to 30 minutes before the beginning of an experiment. A 14-gauge, 140-mm catheterc was placed in 1 jugular vein, and the left forefoot was marked with white zinc tape. Hay was provided several hours before the beginning of each experiment, and horses had ad libitum access to hay and water at all times during the locomotor experiments.
Locomotor response
Locomotor activity in response to fentanyl was quantified by recording the number of steps (ie, times the taped forefoot of a horse left the ground) in each 2-minute period. Each horse was observed by 1 of 3 investigators (LAW, PJP, or YSB). The number of steps for each 2-minute period was recorded on a manually operated counter for 20 consecutive minutes (baseline measurement). Fentanyld was dissolved in water to create a solution with a concentration of 1 mg/mL, and a bolus of the fentanyl solution was administered (20 μg/kg, IV). The number of steps for each 2-minute period was then counted for 60 consecutive minutes immediately after drug administration. Other activities of the horses were monitored, and stereotypical behaviors (eg, snatching at food, head bobbing, and crib biting), sedation, and ataxia were recorded but not quantified.
Assessment of the locomotor response was repeated for some horses that were considered nonresponders. A higher dose of fentanyl (30 μg/kg, IV) was administered to horses that had no increase in locomotor response and to breed- and age-matched horses from the other group. Number of steps was counted as described previously. There was a minimum washout period of 3 weeks between administrations of fentanyl doses.
Statistical analysis
Characteristics of the horses were compared by use of Student t tests for continuous measures and Fisher exact tests for categorical measures. The mean baseline step count per 2-minute period was calculated for each horse, and this value was subtracted from each step count per 2-minute period after fentanyl administration. Adjusted counts with negative values were assigned a value of 0. Data were analyzed by use of a repeated-measures ANOVA to account for the multiple observations on each horse; the model initially included main effects of sex, breed (combining Arabians and Paints), time, and polymorphism status and the interaction between time and polymorphism status. Terms were removed from the model sequentially when they were not significant at P < 0.05. Analyses were performed with commercially available statistical software.e
Results
One control horse showed a stereotypical behavior (weaving) during the baseline period; this behavior increased in response to the fentanyl administration. Data for this horse were removed from the analysis. Thus, there were data available for 10 horses with the G57C polymorphism and for 9 control horses without the G57C polymorphism. For the 19 horses included in the analyses, there were no significant differences between the 2 groups with regard to body weight; breed, age, and sex did not differ significantly because these variables were matched on the basis of the study design (Table 1).
Characteristics of 10 horses heterozygous for the G57C polymorphism of the μ-opioid receptor and 9 control horses* without this polymorphism.
| Variable | Horses with the G57C polymorphism | Horses without the G57C polymorphism | P value† |
|---|---|---|---|
| Breed | 5 Thoroughbreds, 3 Quarter Horses, 1 Arabian, and 1 Paint | 4 Thoroughbreds, 3 Quarter Horses, 1 Arabian, and 1 Paint | 1.00‡ |
| Sex | 5 mares, 5 geldings | 6 mares, 3 geldings | 0.65‡ |
| Age (y)§ | 15.5 ± 3.7 | 15.0 ± 4.6 | 0.80‖ |
| Body weight (kg)§ | 591 ± 50 | 559 ± 36 | 0.13‖ |
Data were collected for 10 control horses; however, data for 1 control horse were removed because the horse had abnormal behavior.
Values of P < 0.05 were considered significant.
Reported value is the result of a Fisher exact test.
Value is reported as mean ± SD.
Reported value is the result of a Student t test.
The number of steps was counted during the 60-minute period after administration of fentanyl (Figure 1). Horses with the G57C polymorphism had higher median step counts, compared with step counts for horses without the polymorphism, from 2 to 44 minutes after fentanyl administration. Results for the repeated-measures ANOVA indicated that sex and breed were not significantly correlated with step count; therefore, they were removed from the model. The final model included terms for polymorphism status, time, and the interaction between polymorphism status and time. There was a significant (P < 0.001) interaction between polymorphism status and time (higher mean step counts over time for horses with the G57C polymorphism than for the control horses).
Median (first to third quartile) adjusted step counts determined after administration of a bolus of fentanyl for horses with (white diamonds; n = 10) and without (black circles; 9) the G57C polymorphism of the μ-opioid receptor. The number of steps each horse took was counted over consecutive 2-minute periods for 20 minutes to determine a baseline value. The horse then received a bolus of fentanyl (20 μg/kg, IV; time 0), and the number of steps was again counted during consecutive 2-minute periods for 60 minutes. The mean baseline value was subtracted from each 2-minute period after fentanyl administration; step counts with negative values were assigned a value of 0.
Citation: American Journal of Veterinary Research 77, 8; 10.2460/ajvr.77.8.828
Median (first to third quartile) adjusted step counts determined after administration of a bolus of fentanyl for horses with (white diamonds; n = 10) and without (black circles; 9) the G57C polymorphism of the μ-opioid receptor. The number of steps each horse took was counted over consecutive 2-minute periods for 20 minutes to determine a baseline value. The horse then received a bolus of fentanyl (20 μg/kg, IV; time 0), and the number of steps was again counted during consecutive 2-minute periods for 60 minutes. The mean baseline value was subtracted from each 2-minute period after fentanyl administration; step counts with negative values were assigned a value of 0.
Citation: American Journal of Veterinary Research 77, 8; 10.2460/ajvr.77.8.828
Median (first to third quartile) adjusted step counts determined after administration of a bolus of fentanyl for horses with (white diamonds; n = 10) and without (black circles; 9) the G57C polymorphism of the μ-opioid receptor. The number of steps each horse took was counted over consecutive 2-minute periods for 20 minutes to determine a baseline value. The horse then received a bolus of fentanyl (20 μg/kg, IV; time 0), and the number of steps was again counted during consecutive 2-minute periods for 60 minutes. The mean baseline value was subtracted from each 2-minute period after fentanyl administration; step counts with negative values were assigned a value of 0.
Citation: American Journal of Veterinary Research 77, 8; 10.2460/ajvr.77.8.828
Two Quarter Horses in the control group were considered to be nonresponders; thus, the experiment was repeated by administration of a higher dose of fentanyl (30 μg/kg, IV) to these 2 horses and 2 Quarter Horses that had the G57C polymorphism. In 3 of these 4 horses, there was an increase in the step count during the first 20 minutes after fentanyl administration, although the 2 horses with the lowest step counts had an extremely short duration (approx 10 minutes) of increased step counts.
Discussion
In the study reported here, there was a significant difference in the locomotor response to fentanyl between horses with the G57C polymorphism and control horses. Similar to results for other studies in which this method was used, the increase in step count associated with opioid administration was highly variable in that some horses had no response at the initial dose, whereas others had an increase in step count of > 80 steps/2 min. In this study, some of the nonresponders were retested, and we found that the nonresponse appeared to be a dose-related phenomenon in that these horses did not respond to the original dose of fentanyl but had an increase in the step count when a higher dose of fentanyl was administered. However, even at the higher dose, the duration of the response in these 2 horses was much shorter than the duration of the response in the other horses. No testing was conducted with fentanyl doses > 30 μg/kg because it has been reported6 that the end point for higher doses is recumbency, and the authors did not want to risk a horse falling down in a concrete stall.
The approach used in the present study to examine differences in locomotor response between horses with and without the G57C SNP has been used extensively during the past 3 decades as a means of examining the effects of various opioids.6 The dose of fentanyl used in the study reported here was deliberately chosen to induce a significant increase in step count for 20 to 30 minutes in most horses. In an earlier study,9 a dose of 16 μg/kg increased locomotor activity for approximately 30 minutes in Thoroughbred mares. We chose to count the steps by means of visual observation, although more sophisticated methods have been used to automate the counting process.10 Both methods are subject to errors. An observer can only stand in 1 place, and horses may move rapidly or the marked foot may not be visible at all times; thus, the count may not be accurate. An automated system only counts when the strategically placed infrared light beams are crossed, so it is possible that a horse moving slowly with many small steps would be counted in the same manner as a horse moving more quickly with fewer larger steps. The observers in the study reported here were all experienced with horses, and systematic errors attributable to observer bias would likely have been compensated for by the fact that the observers were unaware of the group identity for each horse.
Opioid administration is followed by an increase in locomotor activity in a number of species. Extensive experiments have been performed with mice to examine this effect. In that species, it appears the effects are mediated by an increase in dopamine in the mesolimbic area of the brain.11 The increase in activity can be blocked by antagonists of dopamine-2 and 3 receptors (eg, eticlopride or sulpiride) at doses that do not affect spontaneous activity when administered alone.12–14 Neither the dopamine-1–receptor antagonist NNC 01-0756 nor the dopamine-2– and 3–receptor antagonist eticlopride was able to decrease locomotor activity stimulated by alfentanil in horses,15 which suggests that the mechanisms involved in horses may not be the same as those involved in mice. Acepromazine, which also has dopamine-receptor antagonist properties, has been used to reduce adverse effects of opioids in horses16,17; however, acepromazine could also exert its effects via other mechanisms. Although there are distinct differences in the locomotor response to morphine among various strains of mice,18 there have been no tests of differences in opioid-stimulated locomotor activity among breeds of horses, to our knowledge. In the present study, the 2 nonresponders were both Quarter Horses, but the number of horses of each breed was insufficient to draw any conclusions about breed differences.
A mutation of the OPRM1 gene could have an effect on the locomotor response to opioids in a number of ways. There could be an alteration in affinity of the resulting receptor or potency of agonists at the opioid receptor. Such effects have been detected for the A118G OPRM1 polymorphism expressed in hamster tumor cells and rat sympathetic ganglion cells.19,20 However, experiments conducted with other cell lines have failed to yield similar findings.21–23 It is also possible that SNPs affect receptor expression at different levels in the nociceptive pathway, which causes a decrease in receptor numbers or a decrease of signal efficiency in coupling with G-proteins. Evidence based on functional MRI evaluations and binding of agonists to opioid receptors in brain slices of humans and mice supports a reduction in receptor expression and signaling efficiency as mechanisms of action.24–26 The A118G OPRM1 mutation has also been found to produce a receptor that is less stable than the unmutated receptor as a result of reduced N-glycosylation.27
One of the weaknesses of the study reported here was that we were unable to include horses homozygous for the G57C polymorphism. In a study28 in which investigators assessed the dose of morphine required to manage pain in humans with cancer, it was found that the doses of morphine required to relieve pain in patients who were homozygous for the A118G SNP were higher than the doses required for patients without the polymorphism or patients who were heterozygous for the SNP. Analysis of results for the present study suggested that although there was a significant difference in locomotor activity between horses without the G57C polymorphism and horses heterozygous for the SNP, assessment of horses homozygous for the SNP may have more clearly revealed locomotor effects that are a result of the G57C polymorphism.
In the study reported here, there was a difference in the locomotor response to fentanyl for horses heterozygous for the G57C polymorphism of the μ-opioid receptor, compared with the locomotor response for horses without this mutation. Further studies are needed to determine the clinical relevance of this finding with regard to opioid-induced analgesia in this species.
Acknowledgments
Supported by the Cummings School of Veterinary Medicine extramural activities program.
Presented in abstract form at the American College of Veterinary Anesthesia and Analgesia Annual Meeting, San Diego, September 2013.
ABBREVIATIONS
| SNP | Single nucleotide polymorphism |
Footnotes
Pascoe PJ, Taylor PM. Is opioid-induced locomotor activity in horses dopamine mediated? (abstr), in Proceedings. 6th Int Cong Vet Anaesthesiol 1997;131.
Wetmore LA, Hawley A, Meola D. Identification of single nucleotide polymorphisms (SNPS) within exon 1 of the horse mu opioid receptor gene (abstr). Vet Anesth Analg 2011;38:17.
Abbocath-T, Hospira, Sligo, Ireland.
Fentanyl citrate powder, Covidien, Mallinckrodt, St Louis, Mo.
SAS, version 9.2, SAS Institute Inc, Cary, NC.
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