To determine if NO production was required for the development of COMT-dependent pain, individual groups of animals received i

To determine if NO production was required for the development of COMT-dependent pain, individual groups of animals received i.p L-NAME (30 mg/kg) or vehicle 30 min before i.p. Results showed that animals receiving OR486 exhibited higher levels of NO derivatives, tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2) in a 2-and 3AR-dependent manner. Additionally, inhibition of NO synthases and neutralization of the innate immunity cytokines TNF, IL-1, and IL-6 blocked the development of COMT-dependent pain. Finally, we found that NO influences TNF, IL-1, IL-6 and CCL2 levels, Src Inhibitor 1 while TNF and IL-6 influence NO levels. Altogether, these results demonstrate that 2- and 3ARs contribute to COMT-dependent pain, at least partly, by increasing NO and cytokines. Furthermore, they identify 2- and 3ARs, NO, and pro-inflammatory cytokines as potential therapeutic targets for pain patients with abnormalities in COMT physiology. Src Inhibitor 1 2- and 3-adrenergic receptors (2- and 3ARs). Antagonism of both 2- and 3ARs are required to Src Inhibitor 1 completely block acute COMT-dependent pain, as antagonism of either 2- or 3ARs alone Src Inhibitor 1 only produces a partial blockade [53]. 2ARs and 3ARs are G-protein coupled receptors expressed in peripheral, spinal, and supraspinal sites involved in pain transmission. Stimulation of 2- or 3ARs on peripheral afferents sensitizes nociceptors [2,37] and produces allodynia [35] through activating intracellular kinases. Additionally, stimulation of 2- or 3ARs indirectly enhance pain transmission through the release of pro-inflammatory molecules including nitric oxide and cytokines [1,7,21-23,28,49,75,77]. Nitric oxide (NO) is usually a gaseous molecule whose production by NO synthases can be induced by stimulation of 2ARs on endothelial cells, easy muscle, sympathetic afferent neurons, and macrophages [1,21,28] or stimulation of 3ARs on adipocytes and fibroblasts [7,23]. Following release, NO lowers nociceptor firing thresholds [3,5] to enhance experimental inflammatory and neuropathic pain [29,41,59]. Furthermore, NO can stimulate release of additional molecules involved in nociception, including pro-inflammatory cytokines [9,29]. Pro-inflammatory cytokines linked to pain include tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2, MCP-1). 2- and 3AR stimulation promotes the production and release of TNF, IL-1, IL-6, and CCL2 [22,49,63,75,77], which act to lower nociceptor firing thresholds and Mouse monoclonal to CD152(FITC) enhance pain [4,14,57,58][33,73]. Of note, NO and cytokines influence one another’s release. NO drives the production and release of cytokines including TNF and IL-1 [9,13,32,83], while cytokines upregulate NO synthase expression and promote NO release [25,42,74,78]. This positive feedback loop may contribute to the development and/or maintenance of pain [13]. While NO and cytokines are released following 2- and 3AR stimulation and linked with pain, their role in COMT-dependent pain has not been established. To investigate the role of NO and cytokines in COMT-dependent pain mediated by 2- and 3ARs, we measured plasma NO and cytokines following administration of a COMT inhibitor in the presence or absence of 2- and 3AR antagonists. Additionally, we measured mechanical and thermal pain sensitivity following COMT inhibition in the presence or absence of a NO synthase inhibitor or TNF, IL-1, IL-6, or CCL2 neutralizing antibodies. Results demonstrate that (1) COMT-dependent pain is accompanied by increases in peripheral NO derivatives and cytokines mediated by 2- and 3ARs, (2) inhibition of NO synthesis and neutralization of the innate immunity cytokines TNF, IL-1, IL-6 block COMT-dependent pain, and (3) NO and cytokines potentiate one another’s biosynthesis: NO promotes TNF, IL-1, IL-6, and CCL2 release while TNF and IL-6 promote NO release. 2. Materials and Methods 2.1 Subjects Adult male Sprague Dawley rats (Charles River Laboratories, Raleigh, NC) were used in all experiments. Rats weighed between 215-265 g for 2- and 3AR antagonism and NO synthase inhibition experiments and between 315-360 g for cytokine neutralization experiments. 2.2 Drugs and chemicals As described in Nackley et al., 2007 [53], OR486 was dissolved in DMSO and diluted in 0.9% saline (3:2). ICI18551, SR59230A, and L-NAME were dissolved in DMSO and 0.9% saline (1:4). Functional.