β2- and β3-adrenergic receptors drive COMT-dependent pain by increasing production of nitric oxide and cytokines

Abstract

Decreased activity of catechol-O-methyltransferase (COMT), an enzyme that metabolizes catecholamines, contributes to pain in humans and animals. Previously, we demonstrated that development of COMT-dependent pain is mediated by both β2- and β3-adrenergic receptors (β2ARs and β3ARs). Here we investigated molecules downstream of β2- and β3ARs driving pain in animals with decreased COMT activity. Based on evidence linking their role in pain and synthesis downstream of β2- and β3AR stimulation, we hypothesized that nitric oxide (NO) and proinflammatory cytokines drive COMT-dependent pain. To test this, we measured plasma NO derivatives and cytokines in rats receiving the COMT inhibitor OR486 in the presence or absence of the β2AR antagonist ICI118,551+β3AR antagonist SR59320A. We also assessed whether the NO synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) and cytokine-neutralizing antibodies block the development of COMT-dependent pain. Results showed that animals receiving OR486 exhibited higher levels of NO derivatives, tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2) in a β2- and β3AR-dependent manner. Additionally, inhibition of NO synthases and neutralization of the innate immunity cytokines TNFα, IL-1β, and IL-6 blocked the development of COMT-dependent pain. Finally, we found that NO influences TNFα, IL-1β, IL-6, and CCL2 levels, whereas TNFα and IL-6 influence NO levels. Altogether, these results demonstrate that β2- and β3ARs contribute to COMT-dependent pain, at least partly, by increasing NO and cytokines. Furthermore, they identify β2- and β3ARs, NO, and proinflammatory cytokines as potential therapeutic targets for pain patients with abnormalities in COMT physiology.

Keywords: Allodynia; Catecholamines; Chemokine (C-C motif) ligand 2 (CCL2); Epinephrine; Hyperalgesia; Inflammation; Interleukin 1 beta (IL-1β); Interleukin 6 (IL-6); Monocyte chemotactic protein 1 (MCP-1); Nitrate; Nitrite; Norepinephrine; Tumor necrosis factor alpha (TNFα).

Copyright © 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Figures

Fig. 1. Timeline of administered treatments used in this study

The COMT inhibitor OR486 or vehicle was administered in the presence or absence of the β2- and β3-adrenergic receptor antagonists ICI118,551 and SR59320A, the NO synthase inhibitor L-NAME, or neutralizing antibodies against TNFα, IL-1β , IL-6, or CCL2.

Fig. 2. COMT inhibition increases pain, NO derivatives, and cytokines via β2,3ARs

Animals receiving OR486 (30 mg/kg) exhibit (A) mechanical allodynia, (B) mechanical hyperalgesia, and (C) thermal hyperalgesia, as well as increased circulating levels of (D) nitrite, (E) TNFα, (F) IL-1β, (G) IL-6, and (H) CCL2. COMT-dependent increases in pain, nitrite, and cytokines were completely blocked by co-administration of ICI118,551 (0.5 mg/kg) and SR59320A (5.0mg/kg). N=6-10 per group. Data are mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 different from Veh/Veh, #P<0.05 different ICI+SR/Veh and ICI+SR/OR486.

Fig. 3. Inhibition of NO synthesis prevents COMT-dependent pain

Administration of the universal nitric oxide synthase inhibitor L-NAME (30 mg/kg) prior to OR486 (30 mg/kg) normalized (A) mechanical allodynia, (B) mechanical hyperalgesia, and (C) thermal hyperalgesia. N=8-10 per group. Data are mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 different from Veh/Veh.

Fig. 4. Neutralization of TNFα, IL-1β, and IL-6, but not CCL2, blocks COMT-dependent pain

Administration of α-TNFα (75 μg), α-IL-1β (75 μg), or α-IL-6 (75 μg) prior to OR486 (30 mg/kg) normalized (A, D, G) mechanical allodynia, (B, E, H) mechanical hyperalgesia, and (C, F, I) thermal hyperalgesia. (J-L) Administration of α-CCL2 failed to block OR486-induced increases in mechanical and thermal pain. N=6-8 per group. Data are mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 different from Control IgG/Veh. #P<0.05 different from α-TNFα/Veh and α-TNFα/OR486.

Fig. 5. Inhibition of NO synthesis prevents COMT-dependent increases in cytokines

Administration of the nitric oxide synthase inhibitor L-NAME (30 mg/kg) prior to OR486 (30 mg/kg) blocked increases in circulating levels of (A) TNFα, (B) IL-1β, (C) IL-6, and (D) CCL2. N=6-10 per group. *P<0.05 different from Veh/Veh.

Fig. 6. Neutralization of TNFα and IL-6 prevents COMT- dependent increases in NO

OR486-induced increases in total nitrite (nitrite and nitrate) were blocked by pretreatment with (A) α-TNFα (75 μg) or (C) α-IL-6 (75 μg), but not (B) α-IL-1β (75 μg) or (D) α-CCL2 (75 μg). N=6-8 per group. Data are mean ± SEM. %P<0.05 different from α-TNFα/Veh, #P< 0.05 different from α-IL-6/Veh and α-IL-6/OR486.