ABSTRACT
Background: Neuromedin B (Nmb) is implicated in the regulation of nociception of sensory neurons. However, the underlying cellular and molecular mechanisms remain unknown. Methods: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of Nmb on the sensory neuronal excitability and peripheral pain sensitivity mediated by Cav3.2 T-type channels. Results: Nmb reversibly and concentration-dependently increased T-type channel currents (IT) in small-sized trigeminal ganglion (TG) neurons through the activation of neuromedin B receptor (NmbR). This NmbR-mediated IT response was Gq protein-coupled, but independent of protein kinase C activity. Either intracellular application of the QEHA peptide or shRNA-mediated knockdown of Gß abolished the NmbR-induced IT response. Inhibition of protein kinase A (PKA) or AMP-activated protein kinase (AMPK) completely abolished the Nmb-induced IT response. Analysis of phospho-AMPK (p-AMPK) revealed that Nmb significantly activated AMPK, while AMPK inhibition prevented the Nmb-induced increase in PKA activity. In a heterologous expression system, activation of NmbR significantly enhanced the Cav3.2 channel currents, while the Cav3.1 and Cav3.3 channel currents remained unaffected. Nmb induced TG neuronal hyperexcitability and concomitantly induced mechanical and thermal hypersensitivity, both of which were attenuated by T-type channel blockade. Moreover, blockade of NmbR signalling prevented mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain, and this effect was attenuated by siRNA knockdown of Cav3.2. Conclusions: Our study reveals a novel mechanism by which NmbR stimulates Cav3.2 channels through a Gßγ-dependent AMPK/PKA pathway. In mouse models, this mechanism appears to drive the hyperexcitability of TG neurons and induce pain hypersensitivity.
Subject(s)
Calcium Channels, T-Type/metabolism , Pain/metabolism , Receptors, Bombesin/metabolism , Action Potentials , Animals , Calcium Channels, T-Type/physiology , Cyclic AMP-Dependent Protein Kinases/metabolism , Female , Freund's Adjuvant/pharmacology , Ganglia, Spinal/metabolism , Male , Mice , Mice, Inbred ICR , Neurokinin B/analogs & derivatives , Neurokinin B/metabolism , Pain/physiopathology , Receptors, Bombesin/physiology , Receptors, G-Protein-Coupled/metabolism , Sensory Receptor Cells/metabolism , Sensory Receptor Cells/physiology , Signal Transduction/drug effects , Trigeminal Ganglion/cytology , Trigeminal Ganglion/metabolismABSTRACT
Recent studies implicate melatonin in the antinociceptive activity of sensory neurons. However, the underlying mechanisms are still largely unknown. Here, we identify a critical role of melatonin in functionally regulating Cav3.2 T-type Ca2+ channels (T-type channel) in trigeminal ganglion (TG) neurons. Melatonin inhibited T-type channels in small TG neurons via the melatonin receptor 2 (MT2 receptor) and a pertussis toxin-sensitive G-protein pathway. Immunoprecipitation analyses revealed that the intracellular subunit of the MT2 receptor coprecipitated with Gαo . Both shRNA-mediated knockdown of Gαo and intracellular application of QEHA peptide abolished the inhibitory effects of melatonin. Protein kinase C (PKC) antagonists abolished the melatonin-induced T-type channel response, whereas inhibition of conventional PKC isoforms elicited no effect. Furthermore, application of melatonin increased membrane abundance of PKC-eta (PKCη ) while antagonism of PKCη or shRNA targeting PKCη prevented the melatonin-mediated effects. In a heterologous expression system, activation of MT2 receptor strongly inhibited Cav3.2 T-type channel currents but had no effect on Cav3.1 and Cav3.3 current amplitudes. The selective Cav3.2 response was PKCη dependent and was accompanied by a negative shift in the steady-state inactivation curve. Furthermore, melatonin decreased the action potential firing rate of small TG neurons and attenuated the mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain. These actions were inhibited by T-type channel blockade. Together, our results demonstrated that melatonin inhibits Cav3.2 T-type channel activity through the MT2 receptor coupled to novel Gßγ -mediated PKCη signaling, subsequently decreasing the membrane excitability of TG neurons and pain hypersensitivity in mice.