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).

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 β_2,3ARs

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.