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Comparative Study
. 1997 Jan 15;17(2):735-44.
doi: 10.1523/JNEUROSCI.17-02-00735.1997.

Multiple receptors involved in peripheral alpha 2, mu, and A1 antinociception, tolerance, and withdrawal

K O Aley  1 , J D Levine
Affiliations
Comparative Study

Multiple receptors involved in peripheral alpha 2, mu, and A1 antinociception, tolerance, and withdrawal

K O Aley et al. J Neurosci. .

Abstract

We examined the interactions among three classes of peripherally-acting antinociceptive agents (mu-opioid, alpha 2-adrenergic, and A1-adenosine) in the development of tolerance and dependence to their antinociceptive effects. Antinociception was determined by assessing the degree of inhibition of prostaglandin E2 (PGE2)-induced mechanical hyperalgesia, using the Randall-Selitto paw-withdrawal test. Tolerance developed within 4 hr to the antinociceptive effect of the alpha 2-adrenergic agonist clonidine; dependence also occurred at that time, demonstrated as a withdrawal hyperalgesia that was precipitated by the alpha 2-receptor antagonist yohimbine. These findings are similar to those reported previously for tolerance and dependence to mu and A1 peripheral antinociception (Aley et al., 1995). Furthermore, cross-tolerance and cross-withdrawal between mu, A1, and alpha 2 agonists occurred. The observations of cross-tolerance and cross-withdrawal suggest that all three receptors are located on the same primary afferent nociceptors. In addition, the observations suggest that the mechanisms of tolerance and dependence to the antinociceptive effects of mu, A1, and alpha 2 are mediated by a common mechanism. Although any of the agonists administered alone produce antinociception, we found that mu, A1, and alpha 2 receptors may not act independently to produce antinociception, but rather may require the physical presence of the other receptors to produce antinociception by any one agonist. This was suggested by the finding that clonidine (alpha 2-agonist) antinociception was blocked not only by yohimbine (alpha 2-antagonist) but also by PACPX (A1-antagonist) and by naloxone (mu-antagonist), and that DAMGO (mu-agonist) antinociception and CPA (A1-agonist) antinociception were blocked not only by naloxone (mu-antagonist) and PACPX (A1-antagonist), respectively, but also by yohimbine (alpha 2-antagonist). This cross-antagonism of antinociception occurred at the ID50 dose for each antagonist at its homologous receptor. To test the hypothesis that the physical presence of mu-opioid receptor is required not only for mu antinociception but also for alpha 2 antinociception, antisense oligodeoxynucleotides (ODNs) for the mu-opioid and alpha 2C-adrenergic receptors were administered intrathecally to reduce the expression of these receptors on primary afferent neurons. These studies demonstrated that mu-opioid ODN administration decreased not only mu-opioid but also alpha 2-adrenergic antinociception; A1 antinociception was unaffected. In contrast, alpha 2C-adrenergic ODN decreased antinociception induced by all three classes of antinociceptive agents. In conclusion, these data suggest that peripheral antinociception induced by mu, alpha 2, and A1 agonists requires the physical presence of multiple receptors. We propose that there is a mu, A1, alpha 2 receptor complex mediating antinociception in the periphery. In addition, there is cross-tolerance and cross-dependence between mu, A1, and alpha 2 antinociception, suggesting that their underlying mechanisms are related.

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Figures

Fig. 1.
Fig. 1.
A, Repeated administration of clonidine produces tolerance to antinociception. Effect of PGE2 (E2), clonidine plus PGE2(Cl+E2), clonidine once hourly for 3 hr (Clx3), clonidine once hourly for 3 hr, and at the fourth hour clonidine plus PGE2 (Clx3, Cl+E2) on mechanical paw withdrawal threshold in the rat.B, Bidirectional cross-tolerance develops among A1, α2, and μ antinociception. Effect of clonidine plus PGE2 (Cl+E2), DAMGO once hourly for 3 hr (Dx3), DAMGO once hourly for 3 hr and at the fourth hour clonidine plus PGE2(Dx3,Cl+E2), CPA once hourly for 3 hr and at the fourth hour clonidine plus PGE2 (CPAx3, Cl+E2), CPA once hourly for 3 hr (CPAx3), clonidine once hourly for 3 hr and at the fourth hour DAMGO plus PGE2 (Clx3, D+E2), and clonidine once hourly for 3 hr and at the fourth hour CPA plus PGE2 (Clx3, CPA+E2) on mechanical paw withdrawal threshold in the rat.
Fig. 2.
Fig. 2.
A, Yohimbine precipitates withdrawal hyperalgesia in clonidine tolerant paws. Effect of PGE2 (E2), clonidine plus PGE2(Cl+E2), clonidine once hourly for 3 hr (Clx3), clonidine once hourly for 3 hr (Clx3), clonidine once hourly for 3 hr, and at the fourth hour yohimbine (Clx3, Yo) on mechanical paw-withdrawal threshold in the rat. B, Bidirectional cross-withdrawal develops among A1, α2, and μ antinociception. Effect of clonidine once hourly for 3 hr and at the fourth hour naloxone (Clx3, N), clonidine once hourly for 3 hr and at the fourth hour PACPX (Clx3, PACPX), clonidine once hourly for 3 hr (Clx3), DAMGO once hourly for 3 hr and at the fourth hour yohimbine (Dx3, Yo), DAMGO once hourly for 3 hr (Dx3), CPA once hourly for 3 hr (CPAx3), CPA once hourly for 3 hr and at the fourth hour yohimbine (CPAx3, Yo), and CPA once hourly for 3 hr (CPAx3) on mechanical paw withdrawal threshold in the rat.
Fig. 3.
Fig. 3.
μ, α2, and A1antagonists dose-dependently block μ, α2, and A1 antinociception, respectively. A, Naloxone dose-dependently blocks DAMGO antinociception. Effect of PGE2 (E2), DAMGO plus PGE2(D+E2) and various doses of naloxone (1 ng to 1 μg), and DAMGO plus PGE2 (N+D+E2), on mechanical paw withdrawal threshold in the rat. B, Yohimbine dose-dependently blocks clonidine antinociception. Effect of PGE2 (E2), clonidine plus PGE2(Cl+E2), and various doses of yohimbine (1 ng to 1 μg) and clonidine plus PGE2 (Yo+Cl+E2) on mechanical paw withdrawal threshold in the rat. C, PACPX dose-dependently blocks CPA antinociception. Effect of PGE2(E2), DAMGO plus PGE2(CPA+E2), and various doses of PACPX (1 ng to 1 μg) and CPA plus PGE2 (PACPX+CPA+E2) on mechanical paw withdrawal threshold in the rat.
Fig. 4.
Fig. 4.
Multiple receptors are involved in μ, α2, and A1 antinociception. A, Clonidine α2 antinociception is blocked not only by yohimbine but also by naloxone and PACPX. Effect of PGE2(E2), clonidine plus PGE2(Cl+E2), yohimbine plus clonidine plus PGE2(Yo+Cl+E2), naloxone plus clonidine plus PGE2 (N+Cl+E2), and PACPX plus clonidine plus PGE2 (PACPX+Cl+E2) on mechanical paw withdrawal threshold in the rat. B, DAMGO μ antinociception is blocked not only by naloxone but also by yohimbine. Effect of PGE2 (E2), DAMGO plus PGE2 (D+E2), naloxone plus DAMGO plus PGE2 (N+D+E2), yohimbine plus DAMGO plus PGE2 (Yo+D+E2), and PACPX plus DAMGO plus PGE2 (PACPX+D+E2) on mechanical paw withdrawal threshold in the rat. C, CPA A1antinociception is blocked not only by PACPX but also by yohimbine. Effect of PGE2 (E2), CPA plus PGE2 (CPA+E2), PACPX plus CPA plus PGE2 (PACPX+CPA+E2), naloxone plus CPA plus PGE2 (N+CPA+E2), and yohimbine plus CPA plus PGE2 (Yo+CPA+E2) on mechanical paw withdrawal threshold in the rat.
Fig. 5.
Fig. 5.
Multiple receptors are involved in α2, μ, and A1 tolerance and withdrawal.A, Yohimbine withdrawal is blocked not only by clonidine but also by DAMGO and CPA. Effect of clonidine once hourly for 3 hr and at the fourth hour yohimbine (Clx3,Yo), clonidine once hourly for 3 hr and at the fourth hour clonidine plus yohimbine (Clx3,Cl+Yo), clonidine once hourly for 3 hr and at the fourth hour DAMGO plus yohimbine (Clx3,D+Yo), and clonidine once hourly for 3 hr and at the fourth hour CPA plus yohimbine (Clx3,CPA+Yo) on mechanical paw withdrawal threshold in the rat. B, Naloxone withdrawal is blocked not only by DAMGO but also by clonidine. Effect of DAMGO once hourly for 3 hr and at the fourth hour naloxone (Dx3,N), DAMGO once hourly for 3 hr and at the fourth hour DAMGO plus naloxone (Dx3,D+N), DAMGO once hourly for 3 hr and at the fourth hour clonidine plus naloxone (Dx3,Cl+N), and DAMGO once hourly for 3 hr and at the fourth hour CPA plus naloxone (Dx3,CPA+N) on mechanical paw withdrawal threshold in the rat. C, PACPX withdrawal is blocked not only by CPA but also by clonidine. Effect of CPA once hourly for 3 hr and at the fourth hour PACPX (CPAx3,PACPX), CPA once hourly for 3 hr and at the fourth hour CPA plus PACPX (CPAx3,CPA+PACPX), CPA once hourly for 3 hr and at the fourth hour clonidine plus PACPX (CPAx3,Cl+PACPX), and CPA once hourly for 3 hr and at the fourth hour DAMGO plus naloxone (CPAx3,D+PACPX) on mechanical paw withdrawal threshold in the rat.
Fig. 6.
Fig. 6.
Antisense μ ODN treatment blocks not only μ antinociception but also α2 antinociception. A1 antinociception is unaffected. A, Effect of PGE2 (E2), DAMGO plus PGE2(D+E2), μ-antisense (AS) ODN 1 μg intrathecally on alternate days × 3 and DAMGO plus PGE2 [μ-(AS)x3,D+E2], μ-sense (S) ODN 1 μg intrathecally on alternate days × 3, and DAMGO plus PGE2 [μ-(S)x3,D+E2] on mechanical paw-withdrawal threshold. B, Effect of PGE2 (E2), clonidine plus PGE2(Cl+E2), μ-(AS) ODN 1 μg intrathecally on alternate days × 3, and clonidine plus PGE2[μ-(AS)x3,Cl+E2], μ-(S) ODN 1 μg intrathecally on alternate days × 3, and clonidine plus PGE2[μ-(S)x3,Cl+E2] on mechanical paw-withdrawal threshold. C, Effect of PGE2(E2), CPA plus PGE2 (CPA+E2), μ-(AS) ODN 1 μg intrathecally on alternate days × 3, CPA plus PGE2 [μ-(AS)x3,CPA+E2], μ-(S) ODN 1 μg intrathecally on alternate days × 3, and CPA plus PGE2 [μ-(S)x3,CPA+E2] on mechanical paw withdrawal threshold in the rat.
Fig. 7.
Fig. 7.
Antisense α2C ODN treatment blocks not only α2 antinociception but also μ and A1 antinociception. A, Effect of PGE2 (E2), clonidine plus PGE2(Cl+E2), α2-(AS) ODN 1 μg intrathecally on alternate days × 3, and clonidine plus PGE2[α2-(AS)x3,CCl+E2], α2-(S) ODN 1 μg intrathecally on alternate days × 3, and clonidine plus PGE2 [α2-(S)x3,Cl+E2] on mechanical paw withdrawal threshold in the rat. B, Effect of PGE2 (E2), DAMGO plus PGE2 (D+E2), α2-(AS) ODN 1 μg intrathecally on alternate days × 3, and DAMGO plus PGE2 [α2-(AS)x3,D+E2], α2-(S) ODN 1 μg intrathecally on alternate days × 3, and DAMGO plus PGE2[α2-(S)x3,D+E2] on mechanical paw withdrawal threshold in the rat. C, Effect of PGE2(E2), CPA plus PGE2 (CPA + E2), α2-(AS) ODN 1 μg intrathecally on alternate days × 3, and CPA plus PGE2 [α2-(AS)x3,CPA+E2], α2-(S) ODN 1 μg intrathecally on alternate days × 3, and CPA plus PGE2[α2-(S)x3,CPA+E2] on mechanical paw withdrawal threshold in the rat.
Fig. 8.
Fig. 8.
Schematic diagram of hypothesized topological/physical arrangement of the three receptors for peripheral antinociception in the cell membrane. μ (DAMGO), α2C (Clonidine), and A1(CPA) agonism all result in peripheral antinociception mediated through a common second messenger pathway, leading to complete symmetrical cross-tolerance and cross-dependence. However, the asymmetrical interactions are proposed to be a result of the central position of the α2C receptor leading to bidirectional interactions between this receptor and the two other receptors but no interaction between the μ and A1 receptors.
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