Opiate receptors in the periaqueductal gray region and alpha2 adrenoceptors in the spinal cord of the rat mediate the antinociceptive properties of nitrous oxide (N2O). The availability of genetically altered mice facilitates the detection of the precise protein species involved in the transduction pathway. In this study, the authors establish the similarity between rats and mice in the antinociceptive action of N2O and investigate which alpha2 adrenoceptor subtypes mediate this response.
After obtaining institutional approval, antinociceptive dose-response and time-course to N2O was measured in wild-type and transgenic mice (D79N), with a nonfunctional alpha2A adrenoceptor using tail-flick latency. The antinociceptive effect of N2O was tested after pretreatment systemically with yohimbine (nonselective alpha2 antagonist), naloxone (opiate antagonist), L659,066 (peripheral alpha2-antagonist) and prazosin (alpha2B- and alpha2C-selective antagonist). The tail-flick latency to dexmedetomidine (D-med), a nonselective alpha2 agonist, was tested in wild-type and transgenic mice.
N2O produced antinociception in both D79N transgenic and wild-type litter mates, although the response was less pronounced in the transgenic mice. Antinociception from N2O decreased over time with continuing exposure, and the decrement was more pronounced in the transgenic mice. The antinociceptive response could be dose dependently antagonized by opiate receptor and selective alpha2B-/alpha2C-receptor antagonists but not by a central nervous system-impermeant alpha2 antagonist (L659,066). Whereas dexmedetomidine exhibited no antinociceptive response in the D79N mice, the robust antinociceptive response in the wild-type litter mates could not be blocked by a selective alpha2B-/alpha2C-receptor antagonist.
These data confirm that the antinociceptive response to an exogenous alpha2-agonist is mediated by an alpha2A adrenoceptor and that there appears to be a role for the alpha2B- or alpha2C-adrenoceptor subtypes, or both, in the analgesic response to N2O.
ALMOST 200 years ago, Sir Humphrey Davy first demonstrated the analgesic properties of nitrous oxide (N2O); however, the mechanism for this action still has not been defined. Inhalation of 20-25% N2O in oxygen can produce an analgesic effect equivalent to that produced by 15 mg morphine sulfate in humans and similar to morphine, N2O analgesia is in part reversed by opiate-receptor antagonists in humans and animals. [3-7]Furthermore, there is unilateral cross-tolerance between morphine and N2O. [4,8]
Based on these observations, a mechanism of action for N2O has been proposed in which opiate receptors are activated through the release of endogenous ligands. Neurochemical studies have supported such a mechanism. [9-12]The periaqueductal gray has long been known to be an important site for the analgesic action of opiates (Cf); therefore, we and others demonstrated that the analgesic properties of N2O could be blocked by the discrete introduction of opiate antagonists directly into the periaqueductal gray. Furthermore, because Camarata et al. demonstrated that opiate-induced analgesia at the periaqueductal gray could be blocked by intrathecal administration of [small alpha, Greek]2antagonists; we followed this up by demonstrating that the antinociceptive effects of N2O too could be prevented by blockade of [small alpha, Greek]2adrenoceptors in the spinal cord.
Molecular genetic cloning studies in humans, rats, and mice have shown that, in each of these species three genes on separate chromosomes encode distinct [small alpha, Greek]2-adrenergicreceptor subtypes. Pharmacologic studies have defined four subtypes named [small alpha, Greek]2A, [small alpha, Greek]2B, [small alpha, Greek]2C, and [small alpha, Greek]2D, with the [small alpha, Greek]2Drepresenting a rodent species homologue of human [small alpha, Greek]2A(for review see reference ). Radiolabeled ligand and in situ hybridization studies with mRNA probes reveal that the receptor subtypes are nonhomogenously distributed in the central nervous system. [18-26]In the central nervous system, the [small alpha, Greek]2Asubtype is the most prevalent and ubiquitous of the three, whereas [small alpha, Greek]2Bis only present in a few discrete sites, principally the thalamic nuclei, and then in very small amounts. Because of the absence of selective pharmacologic probes, we and others [28,29]resorted to genetically altered animals to ascribe the various properties of [small alpha, Greek]2agonists to the different receptor subtypes. Using transgenic mice (D79N; provided by Dr. Lee Limbird) in which a single amino acid residue at position 79 has been mutated from aspartic acid (D) to asparagine (N) to render the [small alpha, Greek]2A-receptorsubtype dysfunctional, we reported that the antinociceptive properties of a nonselective [small alpha, Greek]2agonist were mediated by the [small alpha, Greek]2A-adrenoceptorsubtype . We now investigate whether the [small alpha, Greek](2A-adrenoceptor) subtype is exclusively responsible for the antinociceptive properties of N2O.
The experimental protocol was approved by the Animal Care and Use Committee at the Veterans Administration Palo Alto Health Care System. Male 129/svj wild-type mice (Jackson Labs, Bar Harbor, ME) and D79N transgenic mice (bred at Vanderbilt University, Nashville, TN) weighing 20-30 g (8-10 weeks old) at the time of testing were used. A total of 152 mice were used. Each mouse may have been tested on more than one occasion with a period of 1 week allowed to elapse between successive testings. (In preliminary studies we established that responsiveness does not differ week to week.)
Antinociceptive Testing. The antinociceptive response was assessed using an analgesiometer for measurement of the tail-flick latency (TFL) response. A high-intensity light was focused on the middle third of the mouse's tail, and the time for the mouse to move its tail out of the light beam was automatically recorded (Tail-Flick Apparatus; Columbus Instruments, Columbus, OH) and referred to as TFL. 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. A cut-off time of 10 s was predetermined, at which time the trial was discontinued if no response occurred. Each TFL data point consisted of an average of two trials on an individual animal. From the TFL the percent maximal possible antinociceptive effect (%MPE) was calculated as follows:
-(postdrug latency)-(basal latency)/(cut-off latency)-(basal latency) x 100%.
Our decision to use this “spinal reflex” to investigate the antinociceptive response to N2O is predicated by its widespread application and validation to define the pharmacology of spinally mediated nociception. Alterations in these reflexes may also reflect changes in the activity of supraspinally located neurons that regulate the afferent transmission of nociceptive information by means of direct or indirect projections to the dorsal horn of the spinal cord (for review see reference ).
Gas Exposures. All gas exposures were performed in an acrylic chamber (81 x 43 x 34 cm) with a sliding door on one side (for insertion of the mice). This airtight chamber was large enough to contain the analgesiometer device. Fresh test gases (10 l/min) were introduced into the chamber via an inflow port, circulated throughout the chamber by a small fan, and purged by vacuum set to aspirate at the same rate as the fresh gas inflow. Oxygen concentration in the chamber was maintained between 22 and 45%, whereas N2O concentration was varied between 0, 25, 50, and 75% by adjusting the flow rates of N2O, air, and nitrogen (AirLiquide, Houston, TX). Gas concentrations were measured continuously and flow rates were adjusted appropriately to maintain the desired concentrations.
To determine the dependence of N2O-induced antinociception on [small alpha, Greek]2adrenoceptors, cohorts of mice (n = 8) were pretreated with yohimbine (Sigma Chemical, St Louis, MO), 1 and 2 mg/kg intraperitoneally 30 min before 70% N2O exposure, or cohorts of mice (n = 6) were treated with atipamezole (Orion-Farmos, Turku Finland), 1 mg/kg 15 min before dexmedetomidine (Orion-Farmos) 100 [micro sign]g/kg intraperitoneally.
To determine the dependence on peripheral [small alpha, Greek]2adrenoceptors, cohorts of mice (n = 8) were pretreated with L659,066 (Merck, Sharp and Dhome Laboratories, Rahway, NJ), 1 and 10 mg/kg intraperitoneally 30 min before N2O exposure, or cohorts of mice (n = 6) were treated with intraperitoneal L659,066, 10 mg/kg 15 min before intraperitoneal dexmedetomidine 100 [micro sign]g/kg.
To determine the dependence of [small alpha, Greek]2A-or [small alpha, Greek]2B-adrenoceptorsubtypes, or both, cohorts of mice (n = 8) were pretreated with prazosin (Sigma Chemical), 1, 5, and 10 mg/kg intraperitoneally 30 min before 70% N2O exposure, or cohorts of mice (n = 6) were treated with prazosin, 10 mg/kg intraperitoneally 15 min before intraperitoneal dexmedetomidine, 100 [micro sign]g/kg.
At the doses used, the antagonists had no independent effect on TFL.
Statistical Analysis. In most cases, data were analyzed using a single-factor analysis of variance and expressed as a mean +/− standard error of the mean. To assess whether the response curves for the effect of concentration and time of exposure were different, two-way analysis of variance was performed.
Exposure to N2O produced a dose-dependent antinociceptive response in both the wild-type and the transgenic mice (Figure 1). The time-course of antinociception during continuous exposure to 70% N2O (Figure 2) reveals a maximum response at 30 min, which gradually returned to baseline over the next 60 min. When individual concentrations and time points were compared, no significant difference was noted between transgenic and wild-type mice by single-factor analysis of variance. However when data from all concentrations were simultaneously analyzed by two-factor analysis of variance, the response of the transgenic animals was found to be less than observed in the wild type. In both types of mice, the antinociceptive response was blocked dose dependently by the opiate antagonist naloxone (Figure 3) and the nonselective [small alpha, Greek]2antagonist yohimbine (Figure 4). However, the peripherally active [small alpha, Greek]2antagonist L659,066 (Figure 5) was without effect in either the transgenic or the wild-type mice. Prazosin, the selective [small alpha, Greek]2Band [small alpha, Greek]2Cantagonist, dose dependently blocked the antinociceptive effect in both the wild-type and the transgenic mice (Figure 6). Dexmedetomidine, the potent nonselective [small alpha, Greek]2agonist, exhibits a dose-dependent antinociceptive effect in wild-type animals (Figure 7A); unlike N2O (Figure 1), dexmedetomidine does not produce antinociception in transgenic animals (Figure 7A). The antinociceptive effect of dexmedetomidine in wild-type mice was not blocked by doses of prazosin (Figure 7B), which significantly attenuated the antinociceptive response to N2O (Figure 6). Similarly, the antinociceptive effect of dexmedetomidine in wild-type mice was not blocked by L659,066, the peripheral [small alpha, Greek]2antagonist.
From these data we may deduce that, similar to the rat, the antinociceptive response to N2O in mice is dependent on opiate and [small alpha, Greek]2-adrenergicreceptor pathways. In D79N mice, a genetically altered strain with dysfunctional [small alpha, Greek](2A) adrenoceptors, N2O can still exert a significant antinociceptive response. Because this antinociceptive response can be blocked by a nonselective [small alpha, Greek]2antagonist, these data indicate that receptor subtypes other than [small alpha, Greek]2Atransduce the N2O effect. This is supported by the experiment in which prazosin ([small alpha, Greek]2B-and [small alpha, Greek]2C-subtypeantagonist with extremely low affinity for the [small alpha, Greek]2Asubtype)[33,34]blocked the antinociceptive effect of N2O. Although dexmedetomidine and N2O both produce their antinociceptive responses via [small alpha, Greek]2adrenoceptors (blocked by either yohimbine [Figure 4] or atipamezole [Figure 7B]), there are two major differences. Prazosin does not attenuate the antinociceptive response to dexmedetomidine (Figure 7B), whereas it dose dependently blocks the antinociceptive response to N2O (Figure 6). Secondly, dexmedetomidine is ineffective in D79N transgenic mice, whereas our current report shows that the response to N2O is still present in these mice.
Definition of the molecular components involved in a behavioral response to a drug has relied heavily on the use of pharmacologic probes. Unfortunately, in the field of [small alpha, Greek]2-adrenoceptorpharmacology this has proven to be unhelpful because of the lack of subtype-selective ligands. The only compound that has proven to be at all useful is the prototype [small alpha, Greek]1antagonist prazosin, which has between a 10- and 100-fold higher affinity for the [small alpha, Greek]2Band [small alpha, Greek]2Csubtype than for the [small alpha, Greek]2Asubtype, respectively. [33,34]Yohimbine is used to distinguish pharmacologic actions at the [small alpha, Greek]2adrenoceptor (which it blocks) from the [small alpha, Greek]1(at which it has extremely low affinity) adrenoceptors. Because yohimbine is an effective blocker of the response (Figure 4), it is clear that the antinociceptive response to N2O is cause by [small alpha, Greek]2and not [small alpha, Greek]1adrenoceptors. Therefore, the attenuation by prazosin of the antinociceptive action of N2O is caused by its activity at [small alpha, Greek]2B/Cadrenoceptors and not by its well-described ability to block [small alpha, Greek]1adrenoceptors.
Gene targeting has provided a novel approach to study the functional role of identified proteins in response to hormones, neurotransmitters, and pharmacons (for review see reference ). In studies related to anesthesia, Matthes et al. demonstrated that the [micro sign]-opioid receptor gene product is the molecular target of morphine in vivo. Addressing the [small alpha, Greek]2-adrenergicreceptor, we reported that the entire repertoire of anesthetic and analgesic actions of dexmedetomidine, the [small alpha, Greek]2agonist, was blocked in D79N mice. The hypnotic response to barbiturates, the anesthetic-sparing action of adenosine, and the antinociceptive response to morphine were each unaltered in the D79N mice, demonstrating the specificity of the altered molecular component. Actions mediated by the [small alpha, Greek]2B-receptorsubtype, including hypertension, remain unaffected in mice with the dysfunctional [small alpha, Greek]2A-receptorsubtype. Therefore, D79N transgenic mice have a discrete and selective defect only in the signaling pathway involving the [small alpha, Greek]2A-receptorsubtype. Although the data from the studies cited are relatively unambiguous, there are caveats that need to be addressed. It is possible that other genetic loci in the strain of mice from which the embryonic stem cells were derived can account for some of the changes. This can be addressed by back-crossing through multiple generations to yield a congenic strain that differs in only one genetic locus from its wild type. Also, the genetic alteration may induce a compensatory change that could affect the behavioral phenotype. This can be resolved by performing “rescue” experiments in which the genetically altered mice are mated with a strain that overexpresses the original gene, thereby diluting the contribution of the altered gene.
One issue that requires resolution is the fact that an exogenously administered [small alpha, Greek]2agonist exhibits no analgesic response in D79N mice, whereas the N2O-provoked release of an endogenous [small alpha, Greek]2agonist still produces analgesia in D79N, albeit less pronounced than that found in the litter mates. Thus, N (2) O does not depend, exclusively, on the [small alpha, Greek]2Aadrenoceptor to transduce its antinociceptive response, as is the case for the exogenously administered [small alpha, Greek]2agonist (Figure 7A); rather, N2O can use non-[small alpha, Greek]2A-adrenoceptorsubtypes. The reason for this difference between endogenously and exogenously generated [small alpha, Greek]2signaling is unlikely to be caused by the agonists themselves, because dexmedetomidine (the exogenous agonist) and norepinephrine (the putative endogenous agonist) both are relatively nonselective with respect to the [small alpha, Greek]2-adrenoceptorsubtypes. In our previous report with dexmedetomidine, we used a ramping hot-plate nociceptive paradigm rather than the TFL response in which only a spinal reflexive nociceptive pathway is involved. However, we subsequently showed that dexmedetomidine has no antinociceptive properties in the D79N when the TFL paradigm was used (Figure 7A). Another pertinent difference between the two studies is the fact that dexmedetomidine was administered systemically whereas, during N2O exposure, norepinephrine was presumably released at the terminals of the descending noradrenergic pathway in the dorsal horn of the spinal cord. However, it has been shown recently that even intrathecally administered dexmedetomidine is ineffective in the tail-flick test in D79N mice. The current findings are consistent with our previous observation that systemic morphine is as effective in the D79N as in the wild-type mice, because previous studies showed that morphine works, in part, through the supraspinal activation in the periaqueductal gray of a descending noradrenergic pathway, which is presumably similar to the action of N2O.
The results from our study have potentially far-reaching consequences because they illustrate that the [small alpha, Greek]2B-or [small alpha, Greek]2C-receptorsubtypes, or both, are capable of mediating an analgesic response. These proteins represent a potential target for subsequent novel drug design and synthesis for analgesia and provide the hope that the sedative effects of [small alpha, Greek](2) agonists may be avoided because we showed that this property is transduced exclusively at the [small alpha, Greek]2A-receptorsubtype. [27,39]
In conclusion, we showed that N2O antinociception involves opiate and [small alpha, Greek]2-adrenergicreceptor transduction pathways in mice, which represent an important species in which to further investigate the molecular mechanism because of its accessibility to gene transfer technology. In a transgenic mice strain with dysfunctional [small alpha, Greek]2Aadrenoceptors, we are still able to demonstrate a significant antinociceptive response to N2O, indicating that [small alpha, Greek]2Band [small alpha, Greek]2Csubtypes also participate in this response. Studies are ongoing to develop mice lacking [small alpha, Greek]2Bor [small alpha, Greek]2Csubtypes, or both, to determine whether their involvement is sufficient to produce the antinociceptive response to N2O.