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. 2012 Jul;166(6):1936–1945. doi: 10.1111/j.1476-5381.2012.01894.x

Characterization of the hypothermic effects of imidazoline I2 receptor agonists in rats

David A Thorn 1, Xiao-Fei An 1,4, Yanan Zhang 2, Maria Pigini 3, Jun-Xu Li 1
PMCID: PMC3402816  PMID: 22324428

Abstract

BACKGROUND AND PURPOSE

Imidazoline I2 receptors have been implicated in several CNS disorders. Although several I2 receptor agonists have been described, no simple and sensitive in vivo bioassay is available for studying I2 receptor ligands. This study examined I2 receptor agonist-induced hypothermia as a functional in vivo assay of I2 receptor agonism.

EXPERIMENTAL APPROACH

Different groups of rats were used to examine the effects of I2 receptor agonists on the rectal temperature and locomotion. The pharmacological mechanisms were investigated by combining I2 receptor ligands and different antagonists.

KEY RESULTS

All the selective I2 receptor agonists examined (2-BFI, diphenyzoline, phenyzoline, CR4056, tracizoline, BU224 and S22687, 3.2–56 mg·kg–1, i.p.) dose-dependently and markedly decreased the rectal temperature (hypothermia) in rats, with varied duration of action. Pharmacological mechanism of the observed hypothermia was studied by combining the I2 receptor agonists (2-BFI, BU224, tracizoline and diphenyzoline) with imidazoline I2 receptor/ α2 adrenoceptor antagonist idazoxan, selective I1 receptor antagonist efaroxan, α2 adrenoceptor antagonist/5-HT1A receptor agonist yohimbine. Idazoxan but not yohimbine or efaroxan attenuated the hypothermic effects of 2-BFI, BU224, tracizoline and diphenyzoline, supporting the I2 receptor mechanism. In contrast, both idazoxan and yohimbine attenuated hypothermia induced by the α2 adrenoceptor agonist clonidine. Among all the I2 receptor agonists studied, only S22687 markedly increased the locomotor activity in rats.

CONCLUSIONS AND IMPLICATIONS

Imidazoline I2 receptor agonists can produce hypothermic effects, which are primarily mediated by I2 receptors. These data suggest that I2 receptor agonist-induced hypothermia is a simple and sensitive in vivo assay for studying I2 receptor ligands.

Keywords: imidazoline I2 receptor, hypothermia, locomotion, drug combination, rats

Introduction

Imidazoline receptors are a group of heterogeneous receptors that are widely distributed and recognize prevalently imidazoline compounds (Regunathan and Reis, 1996; Head and Mayorov, 2006). Three different imidazoline receptors have been described: I1 receptors are critically involved in central control of hypertension (Head and Mayorov, 2006; Nikolic and Agbaba, 2011); two I1 receptor preferring agonists, moxonidine and rilmenidine, are clinically used to control hypertension (Sica, 2007; Edwards et al., 2011); I2 receptors are thought to be involved in neuroprotection, pain and several CNS disorders (Garcia-Sevilla et al., 1999; Li and Zhang, 2011); I3 receptors are involved in pancreatic insulin secretion (Eglen et al., 1998).

Imidazoline I2 receptors have been suggested as a potential therapeutic target for certain brain disorders. Autoradiographical studies reveal that I2 receptors are widely distributed in the CNS, with high bindings to the area postrema, interpeduncular nucleus, arcuate nucleus, mammillary peduncle, ependyma and pineal gland (Lione et al., 1998). The density of I2 receptors in humans is dynamically altered under some disease conditions (Garcia-Sevilla et al., 1999). For example, the I2 receptor density is decreased in victims of suicide, heroin addicts and Huntington's disease patients, unaltered in Parkinson's disease patients, and markedly increased in Alzheimer's disease and glial tumour patients (Garcia-Sevilla et al., 1999). In rats, chronic treatment with an antidepressant imipramine increases while treatment with heroin decreases the brain I2 receptor density (Sastre et al., 1996; Zhu et al., 1997). In addition, a recently renewed interest is to target I2 receptors for the treatment of pain conditions (Li and Zhang, 2011). For example, a selective I2 receptor agonist, CR4056, shows promising antihyperalgesic activity for inflammatory and neuropathic pain in preclinical studies and is currently seeking phase I clinical trial (Ferrari et al., 2011). This evidence points to the possibility that I2 receptors may be functionally involved in these disorders and continued research efforts may eventually lead to novel treatment strategies.

Although I2 receptors have not been cloned, recent studies suggest a link between I2 receptors and AMP-activated protein kinase and PI3K-AKT signalling pathways (Lui et al., 2010; Zhang et al., 2012). These new developments may eventually facilitate the understanding of the I2 receptor system. Nevertheless, currently, the identification of I2 receptor ligands is still reliant primarily on receptor-binding studies, and receptor binding data cannot predict the in vivo activity of I2 receptor ligands. Attempts have been made to develop in vivo bioassays for the study of I2 receptor ligands. For example, it has been suggested that enhancement of morphine antinociception could be used to differentiate I2 receptor agonists and antagonists (Sanchez-Blazquez et al., 2000). However, given the relatively modest effects of I2 receptor agonists on the action of morphine, it is difficult to interpret the effects of the I2 receptor ligands in a quantitative manner with this assay. Moreover, this assay has limited sensitivity in capturing the I2 receptor agonism activity, as only I2 receptor agonists with high efficacy can be recognized and ligands with lower efficacy such as BU224 may be erroneously tagged as an ‘antagonist’ (Li and Zhang, 2011). A simple and sensitive in vivo assay for I2 receptor ligands will help increase the understanding of the functional role of I2 receptors and facilitate the rapid development of novel I2 receptor ligands. This study reports that I2 receptor agonists reliably decreased body temperature in a highly quantitative manner in rats, which can be used as a sensitive in vivo assay for studying I2 receptor ligands.

Methods

Subjects

A total of 57 adult male Sprague–Dawley rats (Harlan, Indianapolis, IN, USA) were used in this study. Rats were housed individually on a 12/12-h light/dark cycle (behavioural experiments were conducted during the light period) with free access to water and food except during experimental sessions. Animals were maintained and experiments were conducted in accordance with the Institutional Animal Care and Use Committee, University at Buffalo, the State University of New York, and with the 1996 Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, Washington DC).

Body temperature measurement

Body temperature was measured in a quiet procedure room maintained under identical environmental controls (temperature, humidity and lighting) with the animal colony room. Rats were habituated to the procedure room for at least 30 min before each test. Body temperature was measured by gently inserting a rectal probe (5.0 cm) and recording temperature from the digital thermometer (BAT7001H, Physitemp Instruments Inc., Clifton, NJ, USA) (Li et al., 2009). Rats were handled for at least 3 days before testing drugs in order to habituate rats to the procedure.

Forty-six rats were used in the hypothermia studies. Rats were randomly assigned to eight groups with five to six rats in each. Each group of rats was generally only used for studying one agonist alone and/or in combination with antagonists, and testing was conducted no more than once per week. One group of rats was used to study the effects of phenyzoline and tracizoline, another group to study the effects of CR4056 and clonidine. During a test session, a baseline body temperature measurement was immediately followed by the injection of a dose of a drug, and the follow-up measurements were conducted every 15 min until the effect of the drug dissipated or until 3 h had passed by. A notable exception was testing the effect of high doses of tracizoline for which the measurement was continued for a total of 5 h. When a drug combination was studied, the first drug was administered 10 min before the first measurement, which was immediately followed by the administration of a second drug.

Locomotor activity

The locomotor activity of the rats was monitored by a video surveillance camera mounted on the ceiling and connected to the corresponding software (Smart Junior, Panlab SL, Barcelona, Spain). Four black acrylic boxes (40 × 40 × 30 cm, L × W × H) were used as test arena throughout the study. Eleven rats were randomly assigned to two groups (five and six each, respectively) and were used for all the studies. Because it has been shown that a selective I2 receptor ligand, S23229 and its stereoisomer S23230 both markedly increased the locomotor activity in rats, accompanied by the overshoot of dopamine release in the brain, it has been proposed that I2 receptor activation stimulates locomotor activity in rats (Barrot et al., 2000). Thus, this study was designed to examine the potential locomotor-stimulating effects of drugs. To fulfil this purpose, rats were habituated to the test environment for at least three sessions to minimize novelty-induced hyperlocomotion. One saline injection session was followed to allow rats to be familiar with the injection procedure and further confirm the low baseline activity. Rats were generally tested once per week. During a test session, the rats were allowed 20 min to explore the test arena, which was followed by the injection of a drug. The locomotor activity was then recorded for 2 h.

Data analyses

For the body temperature data, the relative body temperature changes (°C, mean ± SEM) were calculated by subtracting the baseline body temperature readings (first measurement of each test session) from all the subsequent measurements and plotted as a function of time or dose. The significance of the drug effects was compared with saline treatment sessions and analysed using two-way repeated measures anova (time × treatment) followed by Bonferroni's post hoc test. The maximal changes in body temperature for each test session were also used to construct the dose–effect curves of the test drugs. The effects were analysed using one-way repeated measure anova followed by Bonferroni's post hoc test where appropriate.

For the locomotor activity studies, the data (total locomotion counts within 2 h) were converted into percentage of saline control using the follow formula: control % = (locomotion after drug/locomotion after saline) × 100. The data were considered significantly different from saline control if the 95% confidence limits do not include 100 (Li et al., 2011a).

Drugs

2-BFI hydrochloride, BU224 hydrochloride, S22687, diphenyzoline oxalate, phenyzoline oxalate, tracizoline oxalate and CR4056 were synthesized according to standard procedures (Jarry et al., 1997; Pigini et al., 1997; Gentili et al., 2006; Ishihara and Togo, 2007; Giordani et al., 2008). Clonidine hydrochloride, idazoxan hydrochloride, efaroxan hydrochloride and yohimbine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). WAY100135 hydrochloride was purchased from Tocris Bioscience (Ellisville, MO, USA). Unless otherwise noted, all drugs were dissolved in physiological saline and administered i.p. CR4056 was suspended in 5% Tween 80 and sonicated before use. It has been shown that up to 16% Tween 80 in saline does not alter the locomotor activity in rodents (Castro et al., 1995). Doses are expressed as mg of the form indicated earlier kg-1 body weight. Injection volumes were 1 mL·kg−1.

Results

All the I2 receptor agonists with a wide range of selectivity at I2 receptors over I1 receptors (8- to 4917-fold) and α2 adrenoceptors (45- to 7431-fold, Table 1) dose-dependently and significantly decreased the body temperature (Figure 1). 2-BFI (Figure 1A), diphenyzoline (Figure 1B) and phenyzoline (Figure 1C) produced a similar hypothermic effect, with smaller doses showing little effect and larger doses (32 mg·kg−1 for 2-BFI and diphenyzoline, and 56 mg·kg−1 for phenyzoline) progressively reaching the nadir (−3.56 ± 0.17, −2.82 ± 0.31 and −3.08 ± 0.24°C for 2-BFI, diphenyzoline and phenyzoline, respectively), and the effect lasting for at least 180 min. Although tracizoline showed a similar pattern for the hypothermic effect (nadir at −2.72 ± 0.12°C; Figure 1G), the duration of action was longer than 300 min. In contrast, BU224 showed an atypical dose–effect function (Figure 1D). At smaller doses (3.2–17.8 mg·kg−1), the effect of BU224 was strikingly similar to that of 2-BFI. For example, at a dose of 17.8 mg·kg−1, both 2-BFI and BU224 reached the nadir (−2.36 ± 0.22 and −2.18 ± 0.12°C for 2-BFI and BU224, respectively) 45–60 min after drug administration and the effect gradually dissipated 3 h later. However, unlike 2-BFI, 32 mg·kg−1 BU224 did not further decrease the body temperature but reached a similar nadir after a much longer period of time (nadir at −2.08 ± 0.36°C 135 min after drug administration). CR4056 also dose-dependently decreased the body temperature (Figure 1E); however, 32 mg·kg−1 CR4056 reached the nadir (−2.13 ± 0.14°C 90 min after drug administration) and increasing the dose did not further decrease the body temperature (−1.90 ± 0.14°C at a dose of 56 mg·kg−1). S22687 demonstrated a different dose–effect function from any of the other I2 receptor agonists (Figure 1F). At 10 mg·kg−1, S22687 reached the hypothermic nadir (−1.64 ± 0.10°C) 45 min after drug administration and the effect lasted nearly 120 min (Figure 1F). However, at 17.8 mg·kg−1, the body temperature was quickly increased (peak of 0.84 ± 0.14°C 30 min after drug injection) followed by a slight decrease (nadir of −0.60 ± 0.14°C 60 min after drug injection). The body temperature returned to pre-drug level 120 min after drug administration.

Table 1.

Binding affinities and selectivities of I2 receptor agonists at I1, I2 receptors and α2 adrenoceptors

Drug I1 (Ki or IC50, nM) I2 (Ki, nM) α2 (Ki, nM) I2/I1 I2/α2 References
Tracizoline 19.1 1.9 14,118 10 7431 Polidori et al. (2000); Gentili et al. (2006)
2-BFI 6392* 1.3 3,736 4917 2874 Hudson et al. (1997)
BU224 1747 2.1 2,231 832 1062 Hudson et al. (1999)
Phenyzoline 3697 2.5 1,985 1479 794 Gentili et al. (2006; 2008)
S22687 370 45 11,000 8 244 Barrot et al. (2000)
Diphenyzoline 6340 158.5 7,079 40 45 Gentili et al. (2006; 2008)
CR4056 ND 596 (IC50) N.D, but inactive at 10 µM – Ferrari et al. (2011)
Idazoxan 1259 10.6 55.4 119 5 Hudson et al. (1997); Eglen et al. (1998)
Efaroxan 52 >10,000 13 <0.005 <0.001 Eglen et al. (1998)
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Selectivity (I2/I1 or I2/α2) was determined by comparing the Ki or IC50 values of the compounds on both receptors.

*

Personal communication with Dr Alan L. Hudson, University of Alberta.

ND, not determined.

Figure 1.

Figure 1

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Effects of imidazoline I2 receptor agonists on the body temperature in rats. Ordinates, body temperature changes (°C); Abscissa, time after drug administration (min). Each panel represents data from one compound with the drug name shown on top left of the panel.

The maximal body temperature changes of the different doses were used to construct the dose–effect functions of the respective I2 receptor agonists to facilitate visual inspection of the hypothermic effects (Figure 2). 2-BFI, diphenyzoline, phenyzoline and tracizoline produced a monotonic dose–effect function in decreasing the body temperature, with 2-BFI and diphenyzoline being somewhat more potent than phenyzoline and tracizoline. However, because the maximal effects of the drugs were unknown, the ED50 values could not be determined. Responses to BU224 and CR4056 reached the plateau at doses of 17.8 mg·kg−1 and 32 mg·kg−1, respectively; while S22687 showed a clear bi-phasic dose–effect function. All the doses significantly decreased the body temperature except 3.2 mg·kg−1 diphenyzoline.

Figure 2.

Figure 2

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Dose–response functions for body temperature changes induced by I2 receptor agonists. Each data point represents the maximal body temperature change from the tested dose of the drug. Filled symbols indicated significantly different from saline control. See Figure 1 for other details.

In order to understand the pharmacological mechanisms of the observed hypothermic effects induced by I2 receptor agonists, the non-selective I2 receptor/α2 adrenoceptor antagonist idazoxan, the non-selective I1 receptor/α2 adrenoceptor antagonist efaroxan, and the selective 5-HT1A receptor antagonist WAY100135 were combined with selected doses of I2 receptor agonists. At a dose of 3 mg·kg−1, idazoxan significantly attenuated the hypothermic effects induced by 10 mg·kg−1 2-BFI (Figure 3A). Two-way anova revealed significant main effects of time [F (6, 48) = 29.05, P < 0.0001] and idazoxan treatment [F (1, 48) = 46.68, P < 0.01]. In contrast, 2 mg·kg−1 yohimbine significantly potentiated the hypothermic effects of 2-BFI (Figure 3A). Two-way anova revealed significant main effects of time [F (6, 54) = 34.35, P < 0.0001] and yohimbine treatment [F (1, 54) = 38.04, P < 0.0001]. Similar interactions were observed for BU224 (10 mg·kg−1) and tracizoline (32 mg·kg−1) in combination with 3 mg·kg−1 idazoxan or 2 mg·kg−1 yohimbine. For BU224, in combination with idazoxan, two-way anova revealed significant main effects of time [F (7, 63) = 42.08, P < 0.0001] and idazoxan treatment [F (1, 63) = 34.60, P < 0.01]. For BU224, in combination with yohimbine, two-way anova revealed significant main effects of time [F (11, 99) = 45.86, P < 0.0001] and yohimbine treatment [F (1, 99) = 38.05, P < 0.0001]. For tracizoline, in combination with idazoxan, two-way anova revealed significant main effects of time [F (15, 135) = 13.77, P < 0.0001], idazoxan treatment [F (1, 135) = 61.48, P < 0.001], and time × idazoxan treatment interaction [F (15, 135) = 5.72, P < 0.0001]. For tracizoline, in combination with yohimbine, two-way anova revealed significant main effects of time [F (15, 135) = 53.30, P < 0.0001] and time × yohimbine treatment interaction [F (15, 135) = 2.99, P < 0.0001]. Idazoxan (3 mg·kg−1) also significantly attenuated the hypothermic effects of diphenyzoline (17.8 mg·kg−1) (Figure 3D, solid triangles); however, a combination of 2 mg·kg−1 yohimbine with diphenyzoline induced the hypothermia that was not different from that produced by diphenyzoline alone (Figure 3D). Two-way anova revealed significant main effects of time [F ([7, 63) = 24.75, P < 0.0001], idazoxan treatment [F (1, 63) = 36.30, P < 0.01), and time × idazoxan treatment interaction [F (7, 63) = 12.84, P < 0.0001] for the combination of diphenyzoline and idazoxan. For the combination of diphenyzoline and yohimbine, two-way anova only indicated a significant main effect of time [F (11, 99) = 67.46, P < 0.0001].

Figure 3.

Figure 3

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Effects of idazoxan (3 mg·kg−1) and yohimbine (2 mg·kg−1) on the hypothermic activities of 2-BFI (A), BU224 (B), tracizoline (C), diphenyzoline (D) and clonidine (E). Idazoxan and yohimbine were administered 10 min before each I2 receptor agonist. Filled symbols indicated significantly different from the effect of I2 receptor agonist alone. See Figure 1 for other details.

Clonidine (1 mg·kg−1) significantly decreased the body temperature and this reached a nadir (−3.48 ± 0.42°C) 210 min after drug administration. Both 3 mg·kg−1 idazoxan and 2 mg·kg−1 yohimbine significantlty attenuated the hypothermic effect of clonidine (Figure 3E). For clonidine, in combination with idazoxan, two-way anova revealed significant main effects of time [F (15, 15) = 16.22, P < 0.0001], idazoxan treatment [F (1, 150) = 28.36, P < 0.01], and time × idazoxan treatment interaction [F (15, 150) = 28.32, P < 0.0001]. For clonidine, in combination with yohimbine, two-way anova revealed significant main effects of time [F (15, 150) = 20.39, P < 0.0001], yohimbine treatment [F (1, 150) = 10.98, P < 0.05], and time × yohimbine treatment interaction [F (15, 150) = 41.25, P < 0.0001].

2-BFI and BU224 were also studied in combination with non-selective I1 receptor/α2 adrenoceptor antagonist efaroxan or selective 5-HT1A receptor antagonist WAY100135 (Figure 4). Efaroxan at a dose of 1 mg·kg−1 slightly but significantly potentiated the hypothermic effects of 10 mg·kg−1 2-BFI (Figure 4A). Two-way anova revealed significant main effect of time [F (6, 54) = 69.19, P < 0.0001] and time × efaroxan treatment interaction [F (6, 54) = 6.03, P < 0.0001], but the main effect of efaroxan treatment [F (1, 54) = 3.09, P > 0.05] did not reach statistical significance. However, for the WAY100135 (2 mg·kg−1) + 2-BFI combination, two-way anova only found statistical significance for time [F (6, 54) = 80.37, P < 0.0001]. Similarly, 1 mg·kg−1 efaroxan also significantly potentiated the hypothermic effects of 10 mg·kg−1 BU224 (Figure 4B). Two-way anova revealed significant main effect of time [F (11, 99) = 79.78, P < 0.0001], eraroxan treatment [F (1, 99) = 5.01, P < 0.05], and time × efaroxan treatment interaction [F (1, 99) = 1.33, P < 0.01]. However, for the WAY100135 + BU224 combination, two-way anova only found statistical significance for time [F (11, 99) = 85.93, P < 0.0001].

Figure 4.

Figure 4

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Effects of 1 mg·kg−1 efaroxan or 2 mg·kg−1 WAY100135 on the hypothermic activities of 2-BFI (A) and BU224 (B). See Figures 1 and 3 for other details.

Yohimbine (2 mg·kg−1) significantly decreased the body temperature in rats (Figure 5). This effect was significantly attenuated by 2 mg·kg−1 WAY100135. Two-way anova revealed significant main effects of time [F (11, 110) = 52.66, P < 0.0001], WAY100135 treatment [F (1, 110) = 20.12, P < 0.01], and time × WAY100135 treatment interaction [F (11, 110) = 2.80, P < 0.001].

Figure 5.

Figure 5

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Effects of 2 mg·kg−1 yohimbine alone or in combination with 2 mg·kg−1 WAY100135 on the body temperature in rats. All data points for yohimbine alone were significantly different from saline control. Filled symbols indicated significantly different from the effect of yohimbine alone. See Figure 1 for other details.

Although 0.32 mg·kg−1 haloperidol alone did not alter the body temperature (data not shown), it significantly decreased the body temperature changes induced by 17.8 mg·kg−1 S22687 (Figure 6, filled squares). Thus, in the presence of 0.32 mg·kg−1 haloperidol, the bi-phasic nature of 17.8 mg·kg−1 S22687-induced body temperature changes was no longer evident. In fact, S22687 produced a monotonic hypothermic effect, similar to that evoked by 10 mg·kg−1 S22687 (compare triangles in Figure 1F with squares in Figure 6). Two-way anova revealed significant main effects of time [F (7, 56) = 41.76, P < 0.0001], haloperidol treatment [F (1, 56) = 31.39, P < 0.01], and time × haloperidol treatment interaction [F (7, 56) = 2.58, P < 0.01].

Figure 6.

Figure 6

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Effects of 17.8 mg·kg−1 S22687 alone or in combination with 0.32 mg·kg−1 haloperidol on the body temperature in rats. Filled symbols indicated significantly different from the effect of S22687 alone. See Figure 1 for other details.

At the doses that significantly altered the body temperature (10 and 17.8 mg·kg−1), S22687 markedly increased the locomotor activity (Table 2). Further increasing the dose of S22687 to 32 mg·kg−1 produced a hyperlocomotive effect that was lower than that of 17.8 mg·kg−1, thus demonstrating a typical bell-shaped dose–effect curve. At the doses that significantly decreased the body temperature, 2-BFI, BU224, phenyzoline and diphenzyline did not significantly alter the locomotor activity in rats (Table 2). Although tracizoline slightly increased the locomotor activity at a dose of 10 mg·kg−1, this effect was not dose-dependent as after a larger dose (32 mg·kg−1) of tracizoline, the locomotor activity was not different from that of vehicle treatment. CR4056 significantly decreased the locomotor activity at doses that markedly decreased the body temperature.

Table 2.

Effects of I2 receptor agonists on the locomotor activity in rats

Drug dose (mg·kg−1) Locomotion (% vehicle ± 95% confidence limit)
2-BFI
 10 86.2 (65.7, 106.7)
 17.8 95.7 (50.2, 141.2)
Diphenyzoline
 10 111.1 (66.1, 156.1)
 32 83.3 (76.6, 89.9)
Phenyzoline
 10 126.6 (76.2, 177.0)
 32 92.7 (71.3, 114.1)
Tracizoline
 10 159.2 (125.7, 192.8)*
 32 109.0 (85.4, 132.5)
BU224
 10 107.7 (80.9, 134.5)
 17.8 73.4 (39.7, 107.1)
CR4056
 10 71.2 (57.3, 85.0)*
 32 65.6 (44.8, 86.5)*
S22687
 10 269.4 (133.3, 405.5)*
 17.8 755.4 (429.2, 1081.6)*
 32 239.1 (152.7, 325.5)*
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*

Indicates data significantly different from saline control.

Discussion

The primary findings of the current studies were that compounds selectively binding to imidazoline I2 receptors consistently and dose-dependently decreased the body temperature in rats, and the effects were antagonized by the imidazoline I2 receptor antagonist/α2 adrenoceptor antagonist idazoxan, but not by the I1 receptor antagonist/α2 adrenoceptor antagonist efaroxan or 5-HT1A receptor antagonist WAY100135. The hypothermic effects were further enhanced by the α2 adrenoceptor antagonist/5-HT1A receptor agonist yohimbine. The hypothermic effects of yohimbine were blocked by the selective 5-HT1A receptor antagonist WAY100135, which most likely accounted for the observed enhancement of I2 receptor ligands-induced hypothermia, therefore, the effects of idazoxan can only be interpreted as I2 receptor antagonism. Collectively, these results suggest that activation of imidazoline I2 receptors produces hypothermia, and consequently, this offers a validated and simple in vivo assay for understanding the neuropharmacology of the I2 receptor system and facilitating the development of new I2 receptor ligands.

The concept of imidazoline receptors has been proposed and studied for nearly two decades (Eglen et al., 1998). Major progress has been made in understanding the I1 receptor system and drugs that primarily act on I1 receptors are clinically used for the treatment of hypertension and other chronic disorders (Nikolic and Agbaba, 2011). However, the understanding of I2 receptors has long been hampered by the lack of valid functional assays and selective ligands. Over the years, several selective I2 receptor ligands have been developed such as RS-45041-190, 2-BFI, BU224, tracizoline and LSL60101 (Alemany et al., 1995; Brown et al., 1995; Hudson et al., 2003; Gentili et al., 2006). These compounds have been valuable research tools to facilitate the better understanding of I2 receptors. However, pharmacological selectivity of those compounds has only been demonstrated in in vitro receptor binding studies and has not been verified in in vivo assays, primarily because reliable in vivo functional bioassays related to I2 receptor agonism are lacking. I2 receptor activation has been suggested to produce hyperphagia in rats (Brown et al., 1995; Polidori et al., 2000). This effect waits to be elucidated as no pharmacological antagonism was attempted in previous studies and there are no data to confirm that the observed hyperphagic effects were truly mediated by I2 receptors. Studies with selective I2 receptor ligands on antidepressant-like effects have yielded mixed results (O'Neill et al., 2001; Hudson et al., 2003). It has been suggested that modulation of morphine analgesia may be used as an assay for the detection of ligands with I2 receptor activity (Sanchez-Blazquez et al., 2000). Although this assay is useful, the reading of any effect has to rely on the pharmacological effect of another drug, which complicates the interpretation of the results. Moreover, the modest interaction preludes generating orderly and highly quantitative data. Collectively, there is no in vivo functional assay that can easily capture compounds with imidazoline I2 receptor activity.

In this study, several imidazoline I2 receptor agonists with varied pharmacological selectivity for I2 receptors over I1 receptors (range of selectivity: 8- to 4917-fold) and α2 adrenoceptors (range of selectivity: 45- to 7431-fold) were examined for their effects in body temperature (Table 1). Without exception, all the compounds showed robust and dose-dependent hypothermic effects, although the maximal effect and duration of action seemed to vary across the drugs. It was apparent that BU224 and CR4056 reached the plateau of the hypothermic effect that was smaller than the effect produced by 2-BFI and phenyzoline. This could be due to either limited efficacy at I2 receptors, or drug actions on another mechanism that counteract their effect on I2 receptors (e.g. a second mechanism produces hyperthermic activity), or both. Although it was unclear which mechanisms accounted for the effects of BU224 and CR4056, the unexpected pattern of the duration of action of 32 mg·kg−1 BU224 (Figure 1D) suggested that at this high dose BU224 may act on another unidentified receptor that partially reversed its hypothermic effect. Another unexpected finding was that S22687 at a dose of 17.8 mg·kg−1 first increased the body temperature followed by a slight decrease of the body temperature. This dose of S22687 also markedly increased the locomotor activity in the rats (Table 2), an effect secondary to central dopamine release (Barrot et al., 2000). It was postulated that the hyperlocomotion may increase the body temperature, which in turn counteracts S22687-induced hypothermia. A dose of 0.32 mg·kg−1 of the non-selective dopamine D1/D2 receptor antagonist haloperidol completely reversed the biphasic pattern of S22687-induced body temperature changes (Figure 5). This dose of haloperidol is sufficient to block a large population of dopamine D1/D2 subtype receptors and inhibits behavioural (e.g. hyperlocomotion, discriminative stimulus) effects of indirect-acting dopamine receptor agonists such as methamphetamine and cocaine (Costanza et al., 2001; Steed et al., 2011). This effect is unlikely to be a common mechanism of I2 receptor drugs but rather mediated by non-imidazoline receptor mechanisms, as S22687 was the only I2 receptor ligand that increased locomotor activity and other drugs with higher selectivity on I2 receptors did not change the locomotor activity up to doses that markedly decreased the body temperature in rats (Table 2).

The I2 receptor mechanism of the hypothermia induced by the compounds examined was confirmed by drug combination studies. There are currently no selective I2 receptor antagonists available and idazoxan is frequently used as an I2 receptor antagonist. Idazoxan non-selectively binds to both I2 receptors and α2 adrenoceptors (Table 1) and previous studies have demonstrated that idazoxan can block the antinociception induced by 2-BFI and BU224 (Li et al., 2011b), attenuate the effects of 2-BFI, LSL60101 and phenyzoline for their potentiation of morphine-induced antinociception (Sanchez-Blazquez et al., 2000; Gentili et al., 2006), and inhibit CR4056-induced antinociception (Ferrari et al., 2011). Consistent with previous studies, this study found that idazoxan significantly prevented the hypothermic effects of 2-BFI, BU224, tracizoline and diphenyzoline. However, because idazoxan also binds to α2 adrenoceptors and is widely used as an α2 adrenoceptor antagonist (Bill et al., 1989; Dekeyne and Millan, 2006; Gamo et al., 2010), the effects of a selective α2 adrenoceptor antagonist yohimbine were also studied in combination with the I2 receptor ligands to exclude the potential α2 adrenoceptor mechanism. Surprisingly, yohimbine markedly potentiated the hypothermic effects of 2-BFI, BU224 and tracizoline (Figure 3). Yohimbine also binds to 5-HT1A receptors and has been reported to produce hypothermia in rats (Dilsaver and Davidson, 1989; Winter and Rabin, 1992). Because activation of 5-HT1A receptors produces hypothermia (Li et al., 2009), we reasoned that yohimbine may produce hypothermia by activating 5-HT1A receptors and the observed potentiation of the hypothermia induced by the I2 receptor ligands might be due to the concurrent activation of I2 receptors and 5-HT1A receptors. Indeed, yohimbine alone markedly decreased body temperature, and the effect was antagonized by a selective 5-HT1A receptor antagonist, WAY100135 (Przegalinski et al., 1994). Interestingly, the hypothermic effect induced by yohimbine in combination with diphenyzoline was not different from that induced by diphenyzoline alone, which indicates that yohimbine partially blocked the hypothermic effects of diphenyzoline. Given that diphenyzoline only has 45-fold selectivity for I2 receptors over α2 adrenoceptors (Table 1), it is conceivable that the hypothermic effect of diphenyzoline was a congruent effect of activating both receptors. The α2 adrenoceptor agonist clonidine was studied for comparison purposes. Many effects of clonidine, including its hypothermic effects, are blocked by α2 adrenoceptor antagonists (Junnarkar and Singh, 1988; Halliday et al., 1991). In contrast to the I2 receptor ligands, the hypothermic effect of clonidine was markedly inhibited by both idazoxan and yohimbine, confirming the α2 adrenoceptor mechamism.

Activation of I1 receptors has also been shown to decrease body temperature (Cambridge and Robinson, 2005). However the hypothermic effects observed in this study are unlikely to be due to I1 receptor agonism both because most I2 receptor ligands have low affinity at I1 receptors (nM vs. µM, Table 1) and because the I1 receptor antagonist/α2 adrenoceptor antagonist efaroxan, at a dose that significantly blocks the antinociceptive effects of a selective I1 receptor agonist moxonidine (Shannon and Lutz, 2000), did not attenuate the hypothermic effects of I2 receptor agonists (Figure 4). In fact, efaroxan slightly potentiated the hypothermic effects, which could be due to its 5-HT1A receptor partial agonist property (Kleven et al., 2005). Although it has been shown that I2 receptor ligands also modulate brain 5-HT turnover (Hudson et al., 1999) and 5-HT1A receptor agonism decreases body temperature, the observed effects cannot be attributed to 5-HT1A receptor activation, because the selective 5-HT1A receptor antagonist WAY100135 did not attenuate 2-BFI- and BU224-induced hypothermia (Figure 4).

In the present study, BU224 decreased the body temperature to a maximum of −2.18°C, which was significantly lower than that produced by 2-BFI (−3.56°C). This is consistent with the literature suggesting the low-efficacy nature of BU224. Previous studies suggest that the effect of BU224 is assay-dependent. For example, BU224 similar to 2-BFI induces acute nociception in a writhing test and increases locomotion in nigrostriatal-lesioned rats (Macinnes and Duty, 2004; Li et al., 2011b). However, BU224 prevents 2-BFI-induced enhancement of morphine antinociception in tail flick tests (Sanchez-Blazquez et al., 2000; Thorn et al., 2011), demonstrating I2 receptor antagonist effects. This assay-dependency is in parallel with the profile of a partial agonist (or preferably low-efficacy agonist), and suggests that the efficacy demand of these assays is different. In this regard, body temperature change seems to have a moderate efficacy demand such that BU224 produces an effect that is smaller than a higher-efficacy agonist 2-BFI.

In summary, this study reported firstly that compounds selective for I2 receptors produced hypothermic effects by activating I2 receptors in rats. In combination with antagonism studies, this assay can be a useful and simple in vivo functional assay for studying I2 receptor ligands and furthering the understanding of the functional significance of I2 receptor systems. This study also demonstrated that activation of I2 receptors does not consistently produce hyperlocomotion in rats and suggests that the previous assertion that I2 receptor agonists may have abuse liability (Barrot et al., 2000) requires further evaluation. This is particularly relevant as drugs acting on I2 receptors may have important therapeutic potential for several neuropsychiatric disorders including pain and neuroprotection for ischaemia and brain injury (Qiu and Zheng, 2006; Li and Zhang, 2011).

Acknowledgments

The authors thank Dr Jerrold Winter, Department of Pharmacology and Toxicology, University at Buffalo, for the constructive discussions during the course of this study.

Glossary

2-BFI

2-(2-benzofuranyl)-2-imidazoline

BU224

2-(4, 5-dihydroimidazol-2-yl) quinolone

CR4056

2-phenyl-6-(1H imidazol-1yl) quinazoline

diphenyzoline

2-(2-[1,1′-biphenyl]-2ylethyl)- 4,5-dihidro-1H-imidazole

efaroxan

hydrochloride, 2-ethyl-2-(imidazolin-2-yl)-2,3-dihydrobenzofuran hydrochloride

phenyzoline

4,5-dihidro-2-(2-phenylethyl)-1H-imidazole

S22687

5-[2-methyl phenoxy methyl] 1, 3-oxazolin-2-yl) amine

tracizoline

2-styryl-4,5-dihydro-lH-imidazole

WAY100135

(S)-N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide

Conflict of interest

None.

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