The beta2 adrenergic receptor regulates morphine tolerance and physical dependence

Abstract

Adaptations to the chronic administration of opioids reduce the utility of these drugs in treating pain and support addiction. Recent genetics-based approaches have implicated the beta2 adrenergic receptor (beta2-AR) in controlling some of these responses. We do not know, however, whether this receptor can modulate tolerance, dependence or changes in gene expression caused by chronic opioid administration. For our studies we used C57BL/6 mice and beta2-AR knockout mice in the FVB background. Morphine dose-response relationships were established both prior to and after chronic morphine treatment. In some cases, the selective beta2-AR antagonist butoxamine was administered along with or after morphine. Physical dependence was assessed using naloxone-precipitated withdrawal. The expression of calcitonin gene related peptide (CGRP) and substance P (SP) were measured in spinal cord and dorsal root ganglion (DRG) tissues using both real-time PCR and enzyme-linked immunoassay (ELISA). Both the co-administration of butoxamine with morphine and the administration of butoxamine after chronic morphine reversed morphine tolerance. Morphine failed to cause tolerance in beta2-AR knockout mice. Physical dependence was reduced under the same circumstances. The chronic administration of butoxamine with morphine reduced or eliminated the normally observed up-regulation of CGRP and SP in spinal cord and DRG tissues. Our results suggest that the beta2-AR modulates both opioid tolerance and physical dependence. Activation of beta2-ARs appears to be required for some of the key neurochemical changes which characterize chronic opioid administration. Therefore, beta2-AR antagonists show some promise as agents to enhance chronic opioid analgesic therapy.

Figure 1

Morphine dose-response relationships in the tail flick assay for mice treated with a β2-AR antagonist. Panel A presents the dose-response relationships of opioid naïve mice given escalating doses of morphine along with saline pretreatment or pretreatment with 2mg/kg butoxamine. Panel B presents the dose-response relationship for morphine naïve mice, mice treated chronically with morphine prior to dose-response assessment, or mice treated chronically with morphine and then pretreated with butoxamine immediately before morphine dose-response assessment. Panel C presents the dose-response relationship for morphine naïve mice, mice treated chronically with morphine prior to dose-response assessment, or mice treated chronically with both morphine and butoxamine. For each group, N=8 mice.

Figure 2

Morphine dose-response relationships for FVB wild type and FVB-β2-AR null mutant mice. Panel A shows the dose-response FVB wild type relationships in opioid naïve mice and mice treated chronically with morphine. Panel B demonstrates these dose-response relationships for FVB-β2-AR null mutant mice. For each group, N=8 mice.

Figure 3

Assessment of physical dependence on morphine after chronic administration. In panel A data are provided showing the number of jumps made by the groups of morphine dependent mice after the injection of 10mg/kg naloxone. The leftmost column contains data from C57BL/6 mice treated chronically with morphine prior to naloxone injection. Also presented in Panel A are data from mice treated chronically with morphine and given butoxamine one time prior to naloxone administration, and mice treated chronically with both morphine and butoxamine. In panel B, FVB wild type and FVB-β2-AR null mutant mice were treated chronically with morphine followed by naloxone administration. For each group, N=8 mice. Data are expressed as the means ± S.E.M. **P<0.01; ***P<0.001.

Figure 4

Immunohistochemical localization of CGRP and SP in DRG neurons. Panel A presents the results of DRG staining for β2-AR and CGRP. Panel B presents results from separate sections in which staining was carried out for both β2-AR and SP. In both panels the sections were viewed under 20X magnification.

Figure 5

The expression of α-CGRP and SP precursor (preprotachykinin-A, PPT-A) mRNA in dorsal root ganglia (DRGs) and spinal cord tiussue. In these experiments C57BL/6 mice were opioid naïve or treated chronically with morphine or morphine and butoxamine. Panel A provides the data from assays of αCGRP. Panel B provides data of PPT-A. N=6 mice for each group. Data are expressed as the means ± S.E.M. *P<0.05; **P<0.01.

Figure 6

The expression of CGRP and SP peptides in spinal cord tissue. In these experiments C57BL/6 mice were opioid naïve or treated chronically with morphine or morphine and butoxamine. Spinal cord peptide levels were measured with ELISA assays. N=6 mice for each group. Data are expressed as the means ± S.E.M. *P<0.05; **P<0.01.