The Effect of Imidazoline Receptors and α²-adrenoceptors on the Anesthetic Requirement (MAC) for Halothane in Rats 

The protocol of this study was approved by the Animal Care Committee of Osaka University, Faculty of Medicine.

Male Sprague-Dawley rats were housed in temperature-controlled environment under 12-h light:12-h dark cycle with lighting at 8:00–20:00 for at least 1 week. We performed all experiments between 10:00–16:00.

The rats were anesthetized with 2% halothane in oxygen in a 60–1 glass chamber. For the administration of chemicals, a 20-gauge catheter (Angiocath, Becton Dickinson, Sandy, UT) was placed in the abdominal cavity after loss of lighting reflex. The concentration of halothane in the chamber was thereafter maintained at 1%, which was monitored continuously by an infrared gas analyzer (Datex model AA 102–30–00, Helsinki, Finland) and intermittently by a mass spectrometer (Marquet Gas Analysis, Medical gas analyzer Model 1100, Perkin Elmer). After a 120-min stabilization period, the MAC for halothane was determined by the tail-clamp method. [14] The tail of each rat was clamped with a full-length hemostat (20 cm) to the first ratchet position for 60 s. If purposeful movements to this supramaximal stimulus was elicited, halothane concentration was increased by 10% of the initial concentration, and it was decreased by 10% if the response was not noted. Additional halothane concentration was maintained for 40 min, and the tail was reclamped. We defined the MAC as the median concentration of halothane between the highest concentration with a positive response and the lowest concentration with a negative response. Rectal temperature were maintained at 37.0 +/- 0.5 [degree sign] Celsius with heating lamps throughout experiments.

The rats (n = 72) were divided into the following groups, and the MAC for halothane was determined in each group.

1. Control group (n = 8); the vehicle (0.5 ml) was administered intraperitoneally (IP).

2. Dexmedetomidine groups; 10 (n = 8), 20 (n = 8), and 30 (n = 8) micro gram/kg of dexmedetomidine were administered same as control group, respectively.

3. Rauwolscine group (n = 8); 30 micro gram/kg of dexmedetomidine and 20 mg/kg of rauwolscine were coadministered same as control group.

4. Rilmenidine groups; 20 (n = 8), 50 (n = 8), 100 (n = 8), and 1,000 (n = 8) micro gram/kg of rilmenidine were administered same as control group, respectively.

In these groups, a different rat was used for each experiment.

All chemicals were dissolved in saline (Termo, Tokyo, Japan) to the appropriate concentration, with each rat receiving a 0.5-ml injection, and administered 20 min before the first clamp.

Date are expressed as mean +/- SD. The results of multiple groups were analyzed by one-way analysis of variance (ANOVA), and comparisons between groups were analyzed by Scheffe's test. The statistical significance was defined as P < 0.05.

The MAC for halothane in each group was shown in Figure 1.

Dexmedetomidine (10, 20, 30 micro gram/kg, IP) decreased the MAC for halothane by 11.5%, 19.6%, and 42.9%, respectively. The coadministration of rauwolscine (20 mg/kg, IP) completely reversed the MAC-reducing effect of dexmedetomidine (30 micro gram/kg, IP). Rilmenidine (20, 50, 100, 1,000 micro gram/kg, IP) did not change the MAC for halothane.

The present study indicate that the MAC-reducing action of dexmedetomidine is mediated through alpha²-adrenoceptors and that rilmenidine, a selective imidazoline receptor agonist, did not change the MAC for halothane.

The alpha²-adrenoceptor agonists such as clonidine are known to have sedative, anxiolytic, hypnotic, and analgesic actions. [15,16] In addition, these agents decrease the sympathetic neural activity and circulating catecholamines. [17] With these properties, alpha²-adrenoceptor agonists reduce the anesthetic requirement and attenuate the hemodynamic responses against the surgical stimuli during general anesthesia. [18,19] These beneficial actions of alpha²-adrenoceptor agonists had been considered to be mediated via activation of alpha²-adrenoceptors. However, Bousquet et al. [4,20] reported that a certain alpha²-adrenoceptor agonist with an imidazole, imidazoline, oxazoline, or guanidine moiety in the molecule binds not only to alpha²-adrenoceptors but also to imidazoline receptors. Thus, it is important to clarify the role of both receptors on the anesthesia.

Previously, Segal et al. [6] reported that dexmedetomidine, a selective alpha²-adrenoceptor agonist, reduced the MAC for halothane, and this effect of dexmedetomidine was reversed by idazoxan, an alpha²-adrenoceptor antagonist. However, dexmedetomidine has a weak affinity for imidazoline receptors, and idazoxan is also known to have an affinity for imidazoline receptors. [21,22] Thus, one cannot conclude that alpha²-adrenoceptors mediate the anesthetic-sparing action of dexmedetomidine. In our experiments, dexmedetomidine reduced the MAC for halothane as shown in Figure 1, and this effect of dexmedetomidine was completely reversed by coadministration of rauwolscine, a classical alpha²-adrenoceptor antagonist with little affinity for imidazoline receptors (Figure 1). [7,8] These findings confirm that the anesthetic-sparing effect of dexmedetomidine is mediated via activation of alpha²-adrenoceptors.

Alpha²-Adrenoceptors are located presynaptically and regulate the release of norepinephrine (NE) by autoinhibition. [23] Depletion of NE in the CNS decreased the anesthetic requirement for halothane. [24,25] The presynaptic inhibition of NE might be responsible for anesthetic-sparing action of alpha²-adrenoceptor agonists. However, alpha²-adrenoceptor agonists induced further decrease in the anesthetic requirement of NE-depleted animals. [6] These findings imply that postsynaptic alpha²-adrenoceptors also play an important role in the action of dexmedetomidine. In the CNS, alpha²-adrenoceptors are widely distributed in cerebrum cortex, brain stem, and spinal cord. [11] It has been demonstrated that the alpha²-adrenoceptors in the locus coeruleus (LC) mediate hypnotic and analgesic actions. [16,26] In addition, activation of alpha²-adrenoceptors in the spinal cord exerts analgesia. [27] Considering that the hypnosis and analgesia are important factors for general anesthesia, alpha²-adrenoceptors in these regions might be responsible for the anesthetic-sparing effect of dexmedetomidine.

In the present study, rilmenidine did not change the MAC for halothane. Rilmenidine, a centrally acting antihypertensive agent, binds selectively to imidazoline receptors in the CNS, [9,11,28] and this property was considered to be responsible for the antihypertensive action of this agent. [5,10,29] The signal transduction of imidazoline receptors is not clear, [30,31] and the imidazoline receptors ligands have been classified based on the functional properties. [5,20,29] Previous binding studies indicate that imidazoline receptors are located predominantly in the rostral ventrolateral medulla oblongata (RVLM). [28] Rilmenidine decreased arterial blood pressure or sympathetic neural activity, when microinjected to RVLM, is considered to be an imidazoline receptor agonist. [10] It has been demonstrated that intravenous administration of rilmenidine (100 to 1,000 micro gram/kg)-induced hypotension lasted more than 120 min via activation of imidazoline receptors. [10] In our study, the MAC value in rilmenidine groups almost equal to the MAC in control group, and determination of MAC in rilmenidine groups were completed within 100 min. These findings indicate that the imidazoline receptors are activated during MAC determinations in rilmenidine groups, and that the stimulation of imidazoline receptors did not affect the MAC for halothane.

The imidazoline receptors in the C1 area of the RVLM are considered to be responsible for the hypotensive action of alpha²adrenoceptor agonists such as clonidine. [10] The neurons of the C1 area project to excite sympathetic preganglionic neurons of the spinal cord. [32] Nucleus tractus solitarii (NTS) and C1 area are functionally connected to control the sympathovagal activity. [33] The NTS contains the afferent terminals of the vagus nerve and regulates activity of the dorsal motor nucleus of the vagus, wherein efferent parasympathetic nerves originate. [33] Thus, C1 area is considered to be an important area for sympathovagal vasomotor control. Previously, we have reported that rilmenidine prevents epinephrine-induced dysrhythmias during halothane anesthesia in the rats, [13] and inhibition of sympathetic neural activity seems to be important for this action of rilmenidine. [12] In our experiment, rilmenidine did not change the anesthetic requirement for halothane. Thus, rilmenidine may prevent such dysrhythmias and stabilize the hemodynamic parameters without changing the depth of anesthesia. These properties of rilmenidine would be useful for the treatment of patients such as pheochromocytoma and hypertension.

In conclusion, dexmedetomidine reduced anesthetic requirement for halothane, and this effect of dexmedetomidine was mediated via activation of alpha²-adrenoceptors. On the other hand, activation of imidazoline receptors did not change the anesthetic requirement for halothane.

The authors thank Dr. Mervyn Maze for his helpful comments on the manuscript. They also thank Institut de Recherches Internationales Servier (Courbevoie Cedex, France) and Abbott Laboratories (North Chicago, IL) for supplying rilmenidine and dexmedetomidine, respectively. The authors thank Miss Akama, Miss Ohta, and Miss Yamamoto for their assistance throughout this study.