Pharmacology/Drug Development

The Sigma-1 receptor (σ1R) has emerged as an interesting pharmacological target because it inhibits analgesia mediated by mu-opioid receptors (MOR), and also facilitates the development of neuropathic pain. Based on these findings, the recent cloning of the Sigma-2 receptor (σ2R) led us to investigate its potential role as a regulator of opioid analgesia and of pain hypersensitivity in σ2R knockout mice. In contrast to σ1R deficient mice, σ2R knockout mice developed mechanical allodynia following establishment of chronic constriction injury-induced neuropathic pain, which was alleviated by the σ1R antagonist S1RA. The analgesic effects of morphine, [D-Ala, N-MePhe, Gly-ol]-encephalin (DAMGO) and β-endorphin increased in σ1R mice and diminished in σ2R mice. The analgesic effect of morphine was increased in σ2R mice by treatment with S1RA. However, σ2R mice and wild-type mice exhibited comparable antinociceptive responses to the delta receptor agonist [D-Pen2,5]-encephalin (DPDPE), the cannabinoid type 1 receptor agonist WIN55,212-2 and the α2-adrenergic receptor agonist clonidine. Therefore, while σR1 inhibits and σ2R facilitates MOR-mediated analgesia these receptors exchange their roles when regulating neuropathic pain perception. Our study may help identify new pharmacological targets for diminishing pain perception and improving opioid detoxification therapies.

The peripheral mu-opioid receptor (MOR) has been recognized as a potential target to provide safer analgesia with reduced central side effects. Although analgesic incompetence of the peripheral MOR in the absence of inflammation was initially identified more than a decade ago, there has been very limited investigation into the underlying signaling mechanisms. Here we identify that G protein-coupled receptor kinase 2 (GRK2) constitutively interacts with the MOR in peripheral sensory neurons to suppress peripheral MOR activity. Brief exposure to bradykinin (BK) causes uncoupling of GRK2 from the MOR and subsequent restoration of MOR functionality in dorsal root ganglion (DRG) neurons. Interestingly, prolonged BK treatment induces constitutive activation of the MOR through a mechanism that involves protein kinase C (PKC) activation. After silencing Raf kinase inhibitory protein (RKIP) by RNA interference, BK-induced constitutive MOR activation is completely abrogated, which agrees with previous findings that BK activates PKC signaling to initiate GRK2 sequestration by RKIP. Furthermore, we demonstrate that constitutive, peripheral MOR activity requires GRK2 uncoupling and that the FDA-approved SSRI paroxetine promotes this state of uncoupling. Collectively, these results indicate that GRK2 tightly regulates MOR functional states and controls constitutive MOR activity in peripheral sensory neurons, supporting the potential for targeting the kinase to provide safer analgesia.

Oral squamous cell carcinoma (OSCC) is one of the most painful cancers, which interferes with orofacial function including talking and eating. We report that legumain (Lgmn) cleaves protease-activated receptor-2 (PAR) in the acidic OSCC microenvironment to cause pain. Lgmn is a cysteine protease of late endosomes and lysosomes that can be secreted; it exhibits maximal activity in acidic environments. The role of Lgmn in PAR-dependent cancer pain is unknown. We studied Lgmn activation in human oral cancers and oral cancer mouse models. Lgmn was activated in OSCC patient tumors, compared to matched normal oral tissue. After intraplantar, facial or lingual injection, Lgmn evoked nociception in wild-type (WT) female mice but not in female mice lacking PAR in Na1.8-positive neurons (), nor in female mice treated with a Lgmn inhibitor, LI-1. Inoculation of an OSCC cell line caused mechanical and thermal hyperalgesia that was reversed by LI-1. and deletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR-dependent hyperexcitability of trigeminal neurons from WT female mice. deletion, LI-1 and inhibitors of adenylyl cyclase or protein kinase A prevented the effects of Lgmn. Under acidified conditions, Lgmn cleaved within the extracellular N-terminus of PAR at Asn↓Arg, proximal to the canonical trypsin activation site. Lgmn activated PAR by biased mechanisms in HEK293 cells to induce Ca mobilization, cAMP formation and protein kinase A/D activation, but not β-arrestin recruitment or PAR endocytosis. Thus, in the acidified OSCC microenvironment Lgmn activates PAR by biased mechanisms that evoke cancer pain.Oral squamous cell carcinoma (OSCC) is one of the most painful cancers. We report that legumain (Lgmn), which exhibits maximal activity in acidic environments, cleaves protease-activated receptor-2 (PAR) on neurons to produce OSCC pain. Active Lgmn was elevated in OSCC patient tumors, compared to matched normal oral tissue. Lgmn evokes pain-like behavior through PAR Exposure of pain-sensing neurons to Lgmn decreased the current required to generate an action potential through PAR Inhibitors of adenylyl cyclase and protein kinase A prevented the effects of Lgmn. Lgmn activated PAR to induce calcium mobilization, cAMP formation and activation of protein kinase D and A, but not β-arrestin recruitment or PAR endocytosis. Thus, Lgmn is a biased agonist of PAR that evokes cancer pain.