Endomorphin-1 Reduces Nitric Oxide Production in LPS-treated Rat ( Murine Macrophage Cell Line)

Employment of opiod-derived drugs that bind to mu-opioid receptors (MOR) expressed on neurons and non-neuronal cell types in the spinal cord are shown to be an effective treatment for chronic neuropathic pain. Recently, infiltrating macrophages into pain-relevant spinal sites have been observed in animal models of neuropathic pain. Additionally, immune-like glial cells play a critical role in mediating neuropathic pain via actions of proinflammatory signaling molecules such as nitric oxide. Endomorphin-1(EM-1) is highly selective for the MOR, and when bound causes receptor internalization which initiates a cascade leading to potent analgesic effects. One measure of EM-1 efficacy in the context of chronic pain is to examine its effects on the proinflammatory production of NO. Lipopolysaccharide (LPS) is a molecule that stimulates NO production in macrophages and can be used as a model for examining inflammatory signaling using peptides such as EM-1. The goal of our study is to examine the effects of EM-1 on the potential reduction of NO production in a mouse macrophage cell line, RAW 264.7. We pretreated mouse macrophage with 100 pM, 1 nM, 5nM, 50 nM or 500 nM of EM-1 for 10 minutes followed by a 10 minute LPS (10 ng) co-treatment with EM-1. Results revealed a dose-dependent effect of EM-1 on NO production. The studies are the foundation for a series of experiments characterizing second-generation EM-1 peptides delivered to the spinal cord.