Further Characterization of the Receptor Mechanism Involved in the Antidysrhythmic Effect of Dexmedetomidine on Halothane/Epinephrine Dysrhythmias in Dogs

Methods: Adult mongrel dogs were anesthetized with halothane (1.3%) and monitored continuously for systemic arterial pressure and premature ventricular contractions. The dysrhythmogenic dose of epinephrine was defined as the smallest dose producing four or more premature ventricular contractions within 15-s period. We examined the antidysrhythmic action of dexmedetomidine in the presence of two kinds of alpha²antagonists, that is, agents that label imidazoline receptors and exert a pharmacologic action through imidazoline receptors (idazoxan and atipamezole) and agents that are nonimidazoline compounds and are lacking in pharmacologic action through imidazoline receptors (rauwolscine and L-659,066). They were given cerebroventricularly.

Results: Idazoxan and atipamezole significantly inhibited the antidysrhythmic action of dexmedetomidine, whereas rauwolscine and L-659,066 did not.

Conclusions: Because alpha²antagonists having imidazoline or imidazole structures inhibited the antidysrhythmic action of dexmedetomidine, and the inhibition produced by the nonimidazoline alpha sub 2 antagonists was not significant, imidazoline receptors in the central nervous system are more responsible for the antidysrhythmic action of dexmedetomidine than are alpha²adrenoceptors.

ADMINISTRATION of alpha²-adrenergic agonists during anesthesia is associated with sedation, analgesia, hemodynamic stability, and reduced anesthetic requirements. Although these effects are believed to be mediated through activation of alpha²adrenoceptors that are widely distributed in the brain and involved in regulation of several physiologic processes, investigations have documented that some of the above agonists also interact with the imidazoline receptor, which binds agents, such as imidazoles (e.g., medetomidine), imidazolines (e.g., clonidine, idazoxan), some guanidines (e.g., guanabenz, amiloride), and oxazoles (rilmenidine) but have little affinity for agonists or antagonists without the above structures, such as epinephrine, yohimbine, and rauwolscine. We reported that dexmedetomidine, a highly selective alpha²agonist, prevents the halothane/epinephrine-induced dysrhythmias via an action within the central nervous system, and a later study suggested the involvement of imidazoline receptors in the modulation of halothane-epinephrine dysrhythmias. Dexmedetomidine contains an imidazoline in its chemical structure and has an affinity for imidazoline receptors despite weak affinity compared with that for alpha²adrenoceptors. Thus, it may be possible that the imidazoline receptor contributes to the antidysrhythmic effect of dexmedetomidine. In the current study, using alpha²antagonists with or without affinity for imidazoline receptors, we sought to identify the receptor in the central nervous system involved in the antidysrhythmic property of dexmedetomidine.

The studies were conducted under guidelines provided by the Animal Care Committee of Osaka University Faculty of Medicine.

Eighty-seven adult mongrel dogs of either sex, weighing 7.3–12.5 kg, were used in 113 experiments. Whenever different experiments were performed with the same dog, more than 7 days elapsed between experiments. The same dog was not used more than once in the same experimental group. Two dysrhythmogenic doses obtained in the same dog in separate experiments were considered two different observations. Anesthesia was induced with halothane alone and maintained at an end-tidal concentration of 1.3%, which was monitored continuously by an anesthetic gas analyzer (Datex model AA-102–30–00, Helsinki, Finland). The trachea of each dog was intubated with a cuffed tracheal tube, and the lungs were mechanically ventilated (Aika R60, Tokyo, Japan). The end-tidal carbon dioxide concentration was continuously monitored with an expired gas monitor (Minato 1H 21A, Osaka, Japan) and maintained at a level of 35–40 mmHg. A heating lamp and circulating water blanket were used to maintain the esophageal temperature at 36.5–38.5 degrees Celsius.

Lead II of the electrocardiogram was monitored continuously. A femoral artery catheter was inserted for pressure monitoring and blood gas and serum electrolyte sampling. A right femoral vein was cannulated for the administration of drugs and of lactated Ringer's solution, which was infused at a rate of 10 ml *symbol* kg sup -1 *symbol* h sup -1. Serum Potassium sup + was maintained between 3.5 and 4.5 mEq *symbol* 1 sup -1 by infusing Potassium sup + at a rate of 1–10 mEq *symbol* h sup -1. Arterial pH, oxygen tension, and serum Sodium sup + were maintained within the ranges of 7.35–7.45, 85–100 mmHg, and 135–150 mEq *symbol* 1 sup -1, respectively.

The dysrhythmia threshold was achieved when four or more premature ventricular contractions occurred within 15 s. The dysrhythmogenic dose of epinephrine was defined as the smallest dose that produced dysrhythmias. According to our previous method, the dysrhythmogenic dose of epinephrine was determined with logarithmically spaced infusions of epinephrine lasting 3 min. Recovery periods of 10–30 min were allowed between infusions until the hemodynamic parameters (arterial blood pressure and heart rate) returned to the basal levels. The infusion was started at the minimum dose of 0.67 micro gram *symbol* kg sup -1 *symbol* min sup -1, and the dose was increased by e⁰.4 (e = 2.72) until the dysrhythmia threshold was achieved. If dysrhythmias occurred at one of these doses, a smaller dysrhythmic dose, divided by e sup 0.2, was tested. When the criterion for dysrhythmogenic dose had been satisfied, an arterial blood sample was collected to measure the plasma concentration of epinephrine. The epinephrine concentration was measured in a fully automated high-performance liquid chromatography-fluorometric system (Tosoh model HLC-8030 Catecholamine Analyzer, Tokyo, Japan) using a diphenylethylenediamine condensation method. This assay method has a limit of sensitivity of 10 pg *symbol* ml sup -1 for epinephrine. The inter- and intraassay variations were less than 3%.

Our previous study demonstrated that continuous intravenous administration of dexmedetomidine, 0.5 micro gram *symbol* kg sup -1 *symbol* min sup -1, significantly prevents epinephrine-induced dysrhythmias during halothane anesthesia. Therefore, in the current study, we first reconfirmed the antidysrhythmic effect of continuous intravenous administration of dexmedetomidine at this dose in the presence of cerebroventricular saline, using the method in our previous report and examined whether the property of dexmedetomidine was inhibited by four alpha²antagonists. The antagonists were diluted in 0.9% saline and administered into the cisterna magna through a 20-G needle percutaneously to elucidate the comparative involvement of alpha²adrenoceptors and imidazoline receptors in the central nervous system as the site of action of antidysrhythmic effect of dexmedetomidine. The four alpha²antagonists used in this study were as follows:(1) idazoxan, an imidazoline compound, which has almost same affinity for imidazoline receptors as for alpha²adrenoceptors and exerts alpha²antagonistic potency similar to that of rauwolscine and yohimbine ;(2) atipamezole, an imidazoline compound, which also has some activity at imidazoline receptors * and whose alpha²antagonistic potency is 10 times as potent as idazoxan ;(3) rauwolscine, a nonimidazoline alpha²antagonist that has little affinity for imidazoline receptors and whose alpha²antagonistic potency is similar to that of yohimbine ; and (4) L-659,066, a nonimidazoline alpha²antagonist that is lacking in pharmacologic action through imidazoline receptors and whose alpha²antagonistic activity is about a tenth of that of atipamezole and almost equivalent to that of rauwolscine. According to the previous data above described, the doses of each antagonist were determined to share roughly equieffective alpha²antagonistic potency, that is, the dose ranges tested of idazoxan, atipamezole, rauwolscine, and L-659,066 were 5–20, 0.5–2.0, 10–40, and 10–40 micro gram *symbol* kg sup -1, respectively. The cerebroventricular administration of these antagonists, including the saline and the intravenous dexmedetomidine infusion, were scheduled 25 and 30 min before starting epinephrine infusions, respectively. In these antagonistic studies, the dysrhythmogenic threshold of epinephrine in the presence of dexmedetomidine, 0.5 micro gram *symbol* kg sup -1 *symbol* min sup -1, and with no antagonist (saline only) was shared as a value at dose 0 in each experiment.

Hemodynamic parameters (heart rate and systolic and diastolic arterial blood pressures) were recorded at the time the dysrhythmogenic dose was achieved under the different treatment conditions. The data were expressed as mean plus/minus SEM. The results of multiple groups were analyzed by one-way analysis of variance, followed by post hoc Scheffe's test for multiple comparison. A comparison of two groups was assessed by Student's t test. We considered P < 0.05 to be statistically significant.

Dexmedetomidine affected basal hemodynamic variables (the data just before the epinephrine infusion), i.e., it reduced basal heart rate and increased basal blood pressure. However, pretreatment of various cerebroventricular alpha²antagonist did not significantly modify these hemodynamic effects of dexmedetomidine. Dexmedetomidine treatment (0.5 micro gram *symbol* kg sup -1 *symbol* min sup -1) significantly increased both the dysrhythmogenic dose and the plasma concentration of epinephrine at which dysrhythmias occurred (Figure 1). The effects of idazoxan, atipamezole, rauwolscine, and L-659,066 on the antidysrhythmic action of dexmedetomidine are shown in Figure 2, Figure 3, Figure 4, Figure 5, respectively. Idazoxan significantly blocked the antidysrhythmic action of dexmedetomidine at all doses examined (F2-22). Similarly, atipamezole did so dose-dependently and achieved a significant inhibition at the medium dose, whereas the high dose of atipamezole showed a slight reversal of the effect instead of further inhibition (F3-22). On the other hand, the inhibition of nonimidazole alpha²antagonists (rauwolscine and L-659,066) were not significant (F4-22and F5-22). The hemodynamic parameters at the genesis of the dysrhythmias are shown in Table 1, Table 2, Table 3, Table 4. These antagonists, except L-659,066, decreased systolic arterial pressure significantly at the onset of the dysrhythmias, whereas the changes of diastolic blood pressure and heart rate were not so remarkable.

The current results demonstrate that dexmedetomidine prevents epinephrine-induced dysrhythmias in halothane-anesthetized dogs, and this action is significantly inhibited by an alpha²antagonist with an imidazoline or a related structure and an affinity for imidazoline receptors (i.e., idazoxan and atipamezole). The inhibition produced by a nonimidazoline alpha²antagonist (L-659,066 and rauwolscine) is not significant (F2-22, F3-22, F4-22, F5-22).

Two reports by Bousquet's group demonstrated that the imidazoline receptor is involved in the hypotensive effect of clonidine, which was previously believed to be exerted through activation of alpha sub 2 adrenoceptors. Later, the imidazoline receptor was documented to be responsible for other several pharmacologic properties of alpha²agonists. More recently, the protein of this receptor has been isolated from bovine adrenal chromaffin cells, and evidence has accumulated suggesting that this type of receptor is distinct from an alpha²adrenoceptor and its signal transduction is different from that of alpha²adrenoceptors. Considering that dexmedetomidine has an imidazole structure and possesses an affinity for imidazoline receptor, we thought it important to identify the receptor mechanism involved in the antidysrhythmic actions of dexmedetomidine. The current findings suggest that the imidazoline receptors play a more important role than alpha²adrenoceptors in inhibition by dexmedetomidine of the genesis of halothane-epinephrine dysrhythmias.

When we compared the inhibitory property of the four alpha²antagonists we tested, idazoxan had the most definite inhibitory action. Idazoxan has an imidazoline in its chemical structure, and previous work demonstrated that idazoxan bound to imidazoline receptor with high affinity and acts as an antagonist at the receptors. In comparison, atipamezole is a imidazole compound. Wikberg and Uhen reported that imidazoles have affinities for imidazoline receptors, and Miettinen et al.** suggested that atipamezole has an affinity for imidazoline receptors. Therefore, some of the pharmacologic actions of atipamezole may be mediated through these receptors. In fact, we reported that atipamezole alone significantly potentiated dysrhythmogenicity of epinephrine by blocking imidazoline receptors, not alpha²receptors. In contrast with these antagonists, neither L-659,066 nor rauwolscine contains imidazoline or a related structure, and imidazoline receptors may not be involved in their pharmacologic actions. In the current study, these alpha²antagonists were used in roughly equieffective dose ranges based on previous data, although nonimidazoline antagonists might have a slightly greater alpha²antagonistic potency. However, pharmacokinetic data of the four antagonists in the cerebrospinal fluid have not been published; thus, it might be possible that larger doses of nonimidazoline compounds may be required to exert equipotent alpha²antagonistic action to idazoxan and atipamezole. However, it may be unlikely. As shown in F4-22and F5-22, rauwolscine and L-659,066 tended to inhibit the effect of dexmedetomidine, but the inhibition of each agent was reversed at the highest dose, suggesting that more concentrated nonimidazoline antagonists in the cerebrospinal fluid are not effective to exert further inhibition of the antidysrhythmic action of dexmedetomidine. Because the order of the inhibitory potency was idazoxan > atipamezole > rauwolscine = L-659,066 in the current results, we may deduce that the potency differences are dependent on the interaction with imidazoline receptors. Recent radioligand binding studies have suggested the pharmacologic classification of two subtypes of imidazoline receptors, namely I¹and I², although one binding study suggested another nonadrenergic binding site that has high affinity for atipamezole in neonatal rat lung. Because the differences in the affinity of dexmedetomidine, idazoxan, and atipamezole are not well elucidated, we cannot identify the receptor subtype involved in the antidysrhythmic action of dexmedetomidine. However, if idazoxan and atipamezole differ in their receptor subtype selectivity, then differences in their inhibitory potency of the antidysrhythmic effect of dexmedetomidine might be due to blockade of separate subtypes. In addition, although neither the structure nor the physiologic role of the imidazoline receptors has been well clarified, previous biochemical data indicate that imidazoline receptors are separated from alpha²adrenoceptors and facilitate different transduction passways. Thus, further studies are required to clarify the detailed intracellular mechanism following imidazoline receptors involved in the antidysrhythmic action of dexmedetomidine.

The precise site in the central nervous system involved in the antidysrhythmic action of dexmedetomidine has not been elucidated. Although the locus ceruleus is known to be a principal site of the hypnotic response of alpha²agonists and the involvement of alpha^2Aadrenoceptors, not imidazoline receptors, was reported, the nucleus reticularis lateralis in the ventrolateral medulla, a region relatively rich in imidazoline receptors, was reported to be a site of the hypotensive action of clonidine. In the current results, imidazoline receptors are more responsible than alpha²adrenoceptors for the antidysrhythmic action of dexmedetomidine, so the nucleus reticularis lateralis might be one region originating this action. This brain area is connected functionally with nucleus tractus solitarius, which modulates autonomic control, including vagal activity, which plays a critical role in the antidysrhythmic property of dexmedetomidine. .

Hemodynamic parameters have been known to be an important factor in modulating the genesis of halothane-epinephrine dysrhythmias. Considering that imidazoline receptors are involved in central hypotensive action of imidazoline agents, including clonidine, the different profile concerning pharmacologic activity through imidazoline receptors between imidazoline and nonimidazoline compounds might affect the hemodynamic variables in the current study. However, this possibility is unlikely, because the strong hemodynamic action of the intravenous epinephrine used in this study is likely to mask any hemodynamic difference. In fact, as shown in T1-22, T2-22, T3-22, T4-22, systolic blood pressures at the onset of the dysrhythmias correlated with dysrhythmogenic doses of epinephrine, indicating that the hemodynamic parameters were dependent on the epinephrine infusion doses in the current study.

In conclusion, the current data suggest that imidazoline receptors in the central nervous system may be involved in the antidysrhythmic effect of dexmedetomidine, and a major contribution of alpha²adrenoceptors in this action is unlikely.

The authors thank Farmos Pharmaceutica (Turku, Finland), for supplying dexmedetomidine and atipamezole, and Merck Sharp & Dohme Research Laboratories (West Point, PA), for giving L-659,066.

* Miettinen T, Voutilainen R, Savola J-M, Sjoholm B, Ahtee L, Scheinin M:³H-atipamezole, a new alpha²-adrenoceptor radioligand for receptor binding studies (abstract). Soc Neurosci Abs 16:384, 1990.