Desensitization to the Behavioral Effects of α²-Adrenergic Agonists in Rats

Male Sprague-Dawley rats weighing 220-270 g were used in the current study after approval of the experimental protocol from the Animal Care and Use Committee of the Department of Veterans Affairs Medical Center, Palo Alto. The rats for the control and treatment groups originated from the same litter and were stratified to match weight distribution as closely as possible.

Continuous Drug Administration. Osmotic minipumps (mean pumping rate 0.48 plus/minus 0.02 micro liter *symbol* h sup -1; Alzet 2002, Alza, Palo Alto, CA) were inserted subcutaneously in the dorsal thoracic region during halothane anesthesia. In preliminary experiments osmotic pumps containing the vehicle only were implanted into control animals. This control group did not differ in hypnotic or analgesic responsiveness from sham-operated control animals; therefore the sham-operated animals were used.

Depletion of alpha²Adrenoceptor Reserve. Depletion of alpha²adrenoceptors was produced with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), which covalently binds and permanently inactivates alpha²adrenoceptors; therefore, responsiveness to alpha²agonists is dependent on the recovery of the newly expressed receptor population. [23]Rats were administered 0.1, 0.2, or 0.3 mg *symbol* kg sup -1 EEDQ subcutaneously.

Analgesic Testing. Analgesic response was measured by the tail-flick latency response. A high-intensity light was focused on the rat's tail, and the time required for the rat to move its tail out of the light beam was automatically recorded (Tail-flick apparatus, Columbus instruments, Columbus, OH) and referred as "tail-flick latency." A different patch of the tail was exposed to the light beam on each trial to minimize the risk of tissue damage. The animals were placed on the heating blanket to maintain body and tail temperature during the experiment. [24]At a defined cutoff time of 10 s, the trial was terminated if no response had occurred. Data were expressed as a percentage of the maximal effect according to the following formula: Equation 1.

Dexmedetomidine and clonidine were used in the analgesic test at doses of 50 micro gram *symbol* kg sup -1 and 150 micro gram *symbol* kg sup -1, intraperitoneally, respectively, and the tail-flick test was performed 40 min after treatment of drugs. From pilot studies these doses and this time interval produced approximately 50% of maximal effect.

Hypnotic Response. The hypnotic response to dexmedetomidine, 100 micro gram *symbol* kg sup -1 intraperitoneally, was defined by the loss of the rat's righting reflex, and its duration was measured in minutes and referred to as "sleep-time." The sleep-time was measured as the time from the rat's inability to right itself when placed on its back until the time when it spontaneously and completely reverted to the prone position.

Receptor Density. alpha²Receptor density was measured in the locus ceruleus and spinal cord 3 days after EEDQ administration. (The two behavioral responses were different at this juncture; vide infra). Animals were killed by decapitation during CO²narcosis, and the locus ceruleus (two per rat) and spinal cord were harvested. Tissue was homogenized in volumes (locus ceruleus in 500 micro liter and spinal cord in 10 ml) of icecold Tris, 50 mM, ethylenediamine tetraacetic acid 0.8 mM buffer pH 7.5. After centrifugation at 500 g (RC-5B, rotor SS-34; Sorvall, Newtown, CT) for 5 min, the supernatants were collected and centrifuged at 44,000 g (20 min). The pellets were then washed once with the same buffer. The final pellets were stored at -80 degrees Celsius in ice-cold potassium phosphate 50 mM buffer, pH 7.65, for no longer than 3 weeks.

On the day of the assay, aliquots of membranes were thawed at room temperature and resuspended in Tris 50 mM buffer, pH 7.6, containing 10 mM MgCl². The membrane preparation was incubated for 15 min at 37 degrees Celsius and centrifuged at 44,000g for 20 min. This washing procedure was repeated, and the final membrane pellet was resuspended in 150 micro liter (locus ceruleus) or 10 ml (spinal cord) of 50 mM Tris-HCl, 10 mM MgCl², I mM ethyleneglycol bis (beta-aminoethyl ether)-tetraacetic acid, pH 7.6 (buffer A). The binding procedures on the locus ceruleus and spinal cord were adapted from Baron and Siegel [25]and Uhlen and Wikberg, [26]respectively.

Tolerance and Cross-tolerance to the Analgesic Actions of Clonidine or Dexmedetomidine. Pumps set to deliver clonidine, 15 micro gram *symbol* kg sup -1 *symbol* h sup -1, or dexmedetomidine, 5 micro gram *symbol* kg sup -1 + h - 1, for 7 days were inserted. These doses were based on pilot studies that revealed that a threefold larger dose of clonidine was required to exert an anesthetic and analgesic action comparable to that produced with dexmedetomidine. After 7 days rats were administered median effective analgesic doses of clonidine, 150 micro gram *symbol* kg sup -1, or dexmedetomidine, 50 micro gram *symbol* kg sup -1, and their tail-flick latency response was measured.

Receptor Reserve and Analgesic Responses to Dexmedetomidine and Clonidine. After 0.1, 0.2, or 0.3 mg subcutaneous EEDQ, separate cohorts of rats were tested for their analgesic response to dexmedetomidine, 50 micro gram *symbol* kg sup -1, or clonidine, 150 micro gram *symbol* kg sup -1, at 1, 3, or 8 days.

Receptor Reserve and Hypnotic Response to Dexmedetomidine. After 0.1, 0.2, or 0.3 mg subcutaneous EEDQ, separate cohorts of rats were tested for their hypnotic response to dexmedetomidine, 100 micro gram *symbol* kg sup -1, at 1, 3, or 8 days.

The results were analyzed by one-way analysis of variance, and post hoc comparisons between two groups were assessed by Scheffe's test. A P value of < 0.05 was considered statistically significant.

The analgesic effects of acute clonidine and dexmedetomidine were not significantly affected by chronic treatment of dexmedetomidine at 5 micro gram *symbol* kg sup -1 *symbol* min sup -1, although there was a trend toward an attenuated analgesic response to acute clonidine administration (Figure 1). However, the analgesic effect of acute clonidine was significantly decreased after chronic treatment of clonidine (15 micro gram *symbol* kg sup -1 *symbol* min sup -1), whereas the analgesic response to acute dexmedetomidine administration was not affected (Figure 2).

The analgesic response to dexmedetomidine was significantly decreased by each dose of EEDQ on day 1 (Figure 3). This response had recovered to normal on day 3 in the 0.1- and 0.2-mg *symbol* kg sup -1 EEDQ treatment groups but was still attenuated after 0.3 mg *symbol* kg sup -1 EEDQ treatment (Figure 3). The analgesic response to clonidine was attenuated on day 1 after EEDQ treatment for all doses and remained attenuated on day 3 after 0.2 and 0.3 mg *symbol* kg sup -1 EEDQ. Full recovery of the analgesic response was still not observed by day 8 in the 0.3-mg *symbol* kg sup -1 EEDQ treatment group (Figure 4). Treatment with EEDQ produced a dose-dependent reduction of alpha²adrenoceptor binding sites (B^max) in the spinal cord (Figure 5) without affecting the binding affinity (dissociation constant).

The hypnotic response to dexmedetomidine was attenuated on days 1 and 3 after EEDQ treatment regardless of the dose and full recovery of the hypnotic response was still not observed by day 8 in the 0.3-mg *symbol* kg sup -1 EEDQ treatment group (Figure 6). Three days after treatment with EEDQ, there was a reduction of alpha²adrenoceptor binding sites (B^max) in locus ceruleus (Figure 7) similar to that observed in the spinal cord (Figure 5).

Because of the faster recovery of the analgesic response to dexmedetomidine (Figure 4) compared with the hypnotic response (Figure 6) after EEDQ treatment, we performed additional experiments to determine whether the development of tolerance to these two responses was different. Previously we reported that the hypnotic response to dexmedetomidine was significantly attenuated after chronic treatment of dexmedetomidine at 3 and 10 micro gram *symbol* kg sup -1 *symbol* min sup -1. [1-6]In contrast, chronic administration of dexmedetomidine at 3 micro gram *symbol* kg sup -1 *symbol* min sup -1 did not affect the analgesic potency of acute dexmedetomidine (Figure 8), although the attenuation of the analgesic effect of dexmedetomidine was observed in the large-dose (10 micro gram *symbol* kg sup -1 *symbol* min sup -1) group.

After 7 days of treatment of clonidine and dexmedetomidine the analgesic response to the acute administration of clonidine was more attenuated than that seen after dexmedetomidine. This difference reached statistical significance in the clonidine-treated rats (Figure 1and Figure 2). Similary, the recovery of the analgesic effect of dexmedetomidine (Figure 3) was more rapid than that observed with clonidine (Figure 4) after EEDQ administration.

Before considering the reasons for the differences in the analgesic responses of these alpha²agonists, it is prudent to consider the limitations in the study design. Rather than defining a full dose-response curve for the analgesic response, only single dose effects were tested. Although the selection of the median effective dose allows assessment of whether the sensitivity is altered, this technique does not allow determination of whether the maximal analgesic response is also altered. Furthermore, because drug concentrations were not measured in the effect compartment it cannot be established whether the altered analgesic responses are attributable to pharmacokinetic or pharmacodynamic interactions. Also, because relatively small groups of animals were tested, there exists the possibility of a type II error in our data analysis. Notwithstanding these limitations, this study does reveal quantitative differences in the manner by which the analgesic responses to clonidine and dexmedetomidine are altered after chronic treatment with clonidine or dexmedetomidine and also in the recovery of the analgesic response after EEDQ treatment.

The tail-flick latency response test of analgesia is dependent mainly on a spinal segmental reflex in which the alpha²adrenoceptors in the dorsal horn of the spinal cord interrupts the nociceptive pathway to the ventral root. [27]To compare the number of receptors required to produce an analgesic effect in response to dexmedetomidine or clonidine, one can correlate recovery of receptors in the spinal cord and analgesic responsiveness after EEDQ treatment. Three days after EEDQ treatment receptor recovery was 77, 44 and 37%, completed after 0.1, 0.2, and 0.3 mg *symbol* kg sup -1 EEDQ treatment, respectively (Figure 5). From these values one can extrapolate that only 44% receptor recovery is required to transduce the analgesic effect of dexmedetomidine, whereas 77% receptor recovery is required to restore the analgesic action of clonidine. Current radiolabeled ligands cannot distinguish between the three alpha²adrenoceptor subtypes (alpha sub 2A, alpha^2B, and alpha^2C) that are known to be expressed in mammals. Thus it is possible that EEDQ treatment may exert greater antagonism at the subtype mediating the analgesic response to clonidine compared with the subtype responsible for the response to dexmedetomidine. However, this possiblity seems unlikely: Takano and Yaksh documented that dexmedetomidine and clonidine act on a similar spinal receptor population, which they suggested was the alpha^2Asubtype, [12]In addition, only the alpha^2Aadrenoceptor subtype has been shown to exist in the spinal cord. [28].

Chronic administration of alpha²agonists produces receptor down-regulation [29]and uncoupling of the receptor from G proteins. [8]Both processes effectively decrease the number of receptors capable of transducing the pharmacologic response to alpha²agonists. Because dexmedetomidine requires fewer alpha²adrenoceptors to be occupied to exert a given analgesic effect than is the case of clonidine (vide supra), the analgesic response to dexmedetomidine is preserved in an experimental paradigm in which the analgesic response to clonidine is attenuated. Thus, cross-tolerance to dexmedetomidine did not occur after chronic clonidine treatment because a sufficient number of receptors remained to transduce the analgesic response to this highly efficacious agonist.

Recovery of the hypnotic response to dexmedetomidine after EEDQ (Figure 6) occurred more slowly than did the analgesic response (Figure 3) even though the adrenoceptor recovery was similar (Figure 5and Figure 7). Recovery of the behavioral responses can be more easily compared in Figure 9, in which the data are represented as the percentage recovery in the absence of EEDQ.

The analgesic response is more difficult to desensitize (Figure 8) than was reported earlier for the hypnotic response. [6]It is notable that the two studies were performed in a similar manner but on different rat litters. Therefore, it is possible that the differences could be the result of biologic variation. However, this possibility appears to be unlikely because of the inbred nature of the rat strain and because data from the EEDQ experiments confirmed the differences. Another possible reason why these behavioral responses are differently affected could be that the analgesic response is measured by sensitivity at a single time point, whereas the hypnotic response is measured as the duration of a quantal response.

Because the possibility exists that EEDQ treatment may exert greater antagonism at the alpha²adrenoceptors in certain behavioral effect sites, the recovery of the alpha²adrenoceptor populations was examined in the locus ceruleus (hypnotic response) and the spinal cord (analgesic response). Essentially, EEDQ treatment antagonized both sites (Figure 5and Figure 7) although after EEDQ, 0.1 mg *symbol* kg sup -1, the decrease in alpha²adrenoceptors reaches statistical significance only for the spinal cord. There are probably supraspinal, in addition to spinal, sites at which alpha²agonists can act to produce its antinociceptive action. Because the alpha²adrenoceptors was not assessed at these putative sites, it is possible that EEDQ spared these sites, providing a reason for the differences in recovery of responsiveness for the two behaviors. Until these putative sites are clearly identified and examined after EEDQ treatment, we are unable to exclude this remote possibility. Current radiolabeled ligands cannot distinguish among the three alpha²adrenoceptor subtypes (alpha²C10, alpha²C4, and alpha²C2) that are known to be expressed in mammals. Thus it is possible that although the overall alpha²adrenoceptor population was similarly affected, EEDQ treatment may exert more blockade at the subtype mediating the hypnotic response compared with the subtype that mediates the analgesic response. However, the same subtype, alpha²C10 (also known was alpha²) appears to be present in the locus ceruleus [20]and the spinal cord, [26,28]although there may also be an additional non-alpha^2Asubtype present in the spinal cord. [12].

Whereas the analgesic response to dexmedetomidine has normalized on day 3 after this dose of EEDQ (Figure 1), the hypnotic response to dexmedetomidine is still significantly depressed (Figure 6). In fact even when the alpha²adrenoceptors in the spinal cord is reduced by more than 50% (after 0.2 mg/kg EEDQ), the analgesic response is no different than that seen in the control animals. In contrast, a less than 25% reduction in the locus ceruleus alpha²adrenoceptor population, significantly attenuates the hypnotic response. Thus, recovery of more than 75% of the receptors is required to normalize the hypnotic response of dexmedetomidine, but approximately 44% of receptors are enough for recovery of the analgesic response. Therefore, the analgesic response to dexmedetomidine recovers more promptly (Figure 1) than does the hypnotic response (Figure 6) because fewer alpha²adrenoceptors must be occupied to produce the analgesic response than must be occupied for the hypnotic response. These results strongly suggest that the transduction pathway for alpha sub 2 -mediated analgesia is more efficiently coupled than the hypnotic action.

We have now begun to elucidate the molecular mechanism for the pharmacodynamic change in sensitivity in the locus ceruleus of rats chronically infused with alpha²agonists. [8]There appears to be an uncoupling between the alpha²adrenoceptors and the pertussis toxin-sensitive G proteins in the locus ceruleus. Although we have not yet investigated whether the same findings are present in the spinal cord, it is possible that a similar uncoupling in the spinal cord would be less disruptive because of the reserve present in the analgesic response.

Recently, alpha²agonists have been applied to clinical settings throughout perioperative period. Analgesia was measured by a spinal reflex (tail-flick latency) in response to the systemic administration of alpha²agonists, and it may not be appropriate to extrapolate these data to the antinociceptive effects produced by these compounds when administered directly into the neuraxis in other settings of pain. However, our data do suggest that dexmedetomidine may be a more desirable drug than clonidine to control chronic pain. Also, dexmedetomidine may continue to be an effective analgesic agent after tolerance has developed to the analgesic effects of clonidine. Furthermore, alpha²agonists can be expected to sustain their analgesic effect while the unwanted sedative properties may dissipate more rapidly. In addition, the current results suggest that the search for a novel alpha²agonist that retains the ability to exert a potent antinociceptive action without causing sedation is a viable proposition.

The authors thank Dr. Risto Lammintausta (Research and Development, Orion Farmos, Turku, Finland) for providing dexmedetomidine.