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The present study focused on lithocholic acid (LCA), a secondary bile acid that contributes to cholestatic pruritus. Although recent studies have found that LCA acts on MAS-related G protein-coupled receptor family member X4 (MRGPRX4) in humans, it is unclear which subtypes of MRGPRs are activated by LCA in mice since there is no precise ortholog of human MRGPRX4 in the mouse genome. Using calcium imaging, we found that LCA could activate mouse Mrgpra1 when transiently expressed in HEK293T cells. Moreover, LCA similarly activates mouse Mrgprb2. Importantly, LCA-induced responses showed dose-dependent effects through Mrgpra1 and Mrgprb2. Moreover, treatment with QWF (an antagonist of Mrgpra1 and Mrgprb2), YM254890 (Gα inhibitor), and U73122 (an inhibitor of phospholipase C) significantly suppressed the LCA-induced responses, implying that the LCA-induced responses are indeed mediated by Mrgpra1 and Mrgprb2. Furthermore, LCA activated primary cultures of mouse sensory neurons and peritoneal mast cells, suggesting that Mrgpra1 and Mrgprb2 contribute to LCA-induced pruritus. However, acute injection of LCA did not induce noticeable differences in scratching behavior, implying that the pruritogenic role of LCA may be marginal in non-cholestatic conditions. In summary, the present study identified for the first time that LCA can activate Mrgpra1 and Mrgprb2. The current findings provide further insight into the similarities and differences between human and mouse MRGPR families, paving a way to understand the complex roles of these pruriceptors.
Learn More >Voltage-gated sodium (Na) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4-S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4-S5 linker and S6 helix in a conformation-dependent manner. Using F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4-S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic Na channels seems to have a mechanism similar to the recently proposed "door-wedge" action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic Na channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.
Learn More >A remarkable molecular and functional heterogeneity of the primary sensory neurons and dorsal horn interneurons transmits pain- and or itch-relevant information, but the molecular signature of the projection neurons that convey the messages to the brain is unclear. Here, using retro-TRAP (translating ribosome affinity purification) and RNA sequencing, we reveal extensive molecular diversity of spino- and trigeminoparabrachial projection neurons. Among the many genes identified, we highlight distinct subsets of -, -, -, and -expressing projection neurons. By combining in situ hybridization of retrogradely labeled neurons with Fos-based assays, we also demonstrate significant functional heterogeneity, including both convergence and segregation of pain- and itch-provoking inputs into molecularly diverse subsets of NK1R- and non-NK1R-expressing projection neurons.
Learn More >Opioid drugs are widely used analgesics that activate the G protein-coupled µ-opioid receptor, whose endogenous neuropeptide agonists, endorphins and enkephalins, are potent pain relievers. The therapeutic utility of opioid drugs is hindered by development of tolerance to the analgesic effects, requiring dose escalation for persistent pain control and leading to overdose and fatal respiratory distress. The prevailing hypothesis is that the intended analgesic effects of opioid drugs are mediated by µ-opioid receptor signaling to G protein, while the side-effects of respiratory depression and analgesic tolerance are caused by engagement of the receptor with the arrestin-3 protein. Consequently, opioid drug development has focused exclusively on identifying agonists devoid of arrestin-3 engagement. Here, we challenge the prevailing hypothesis with a panel of six clinically relevant opioid drugs and mice of three distinct genotypes with varying abilities to promote morphine-mediated arrestin-3 engagement. With this genetic and pharmacological approach, we demonstrate that arrestin-3 recruitment does not impact respiratory depression, and effective arrestin-3 engagement reduces, rather than exacerbates, the development of analgesic tolerance. These studies suggest that future development of safer opioids should focus on identifying opioid ligands that recruit both G protein and arrestin-3, thereby mimicking the signaling profile of most endogenous µ-opioid receptor agonists.
Learn More >Migraine is a complex neurological disorder that is characterized by throbbing head pain, increased sensitivity to light, sound, and touch, as well as nausea and fatigue. It is one of the most common and most disabling disorders globally but mechanisms causing migraine are poorly understood. While head pain is a typical feature of attacks, they also often present with cutaneous hypersensitivity in the rest of the body. In contrast, primary pain conditions in the lower parts of the body are less commonly associated with cephalic hypersensitivity. Previous studies indicate that application of stimuli to the meninges of rodents causes cutaneous facial as well as hindpaw hypersensitivity. In the present study, we asked whether widespread hypersensitivity is a unique feature of dural stimulation or whether body-wide responses occur similarly when the same stimulus is given in other locations.
Learn More >Reactive nitroxidative species, such as nitric oxide but particularly peroxynitrite, have been strongly implicated in pain mechanisms. Targeting peroxynitrite is anti-nociceptive in pain models, but little is known about its role in migraine mechanisms. Given the need to validate novel targets for migraine headache, our objective was to study the potential of reactive nitroxidative species, particularly peroxynitrite, as novel targets for drug discovery and their role in migraine mechanisms.
Learn More >Nociceptors detect noxious capsaicin (CAPS) via the transient receptor potential vanilloid 1 (TRPV1) ion channel, but coding mechanisms for relaying CAPS concentration [CAPS] remain obscure. Prolonged (up to 1h.) exposure to CAPS is used clinically to desensitise sensory fibres for treatment of neuropathic pain, but its signalling has typically been studied in cultures of dissociated sensory neurons employing low cell numbers and very short exposure times. Thus, it was pertinent to examine responses to longer CAPS exposures in large populations of adult neurons.
Learn More >The opioid receptors are important regulators of pain, reward, and addiction. Limited evidence suggests the mu and delta opioid receptors form a heterodimer (MDOR), which may act as a negative feedback brake on opioid-induced analgesia. However, evidence for the MDOR in vivo is indirect and limited, and there are few selective tools available. We recently published the first MDOR-selective antagonist, D24M, allowing us to test the role of the MDOR in mice. We thus co-treated CD-1 mice with D24M and opioids in tail flick, paw incision, and chemotherapy-induced peripheral neuropathy pain models. D24M treatment enhanced oxymorphone anti-nociception in all models by 54.7%-628%. This enhancement could not be replicated with the mu and delta selective antagonists CTAP, naltrindole, and naloxonazine, and D24M had a mild transient effect in the Rotarod test, suggesting this increase is selective to the MDOR. However, D24M had no effect on morphine or buprenorphine, suggesting that only specific opioids interact with the MDOR. To find a mechanism we performed phosphoproteomic analysis on brainstems of mice. We found that the kinases Src and CaMKII were repressed by oxymorphone, which was restored by D24M. We were able to confirm the role of Src and CaMKII in D24M-enhanced anti-nociception using small molecule inhibitors (KN93, Src-I1). Together these results provide direct in vivo evidence that the MDOR acts as an opioid negative feedback brake, which occurs via the repression of Src and CaMKII signal transduction. These results further suggest that MDOR antagonism could be a means to improve clinical opioid therapy.
Learn More >Clinically, posttraumatic stress disorder (PTSD) and chronic pain are highly comorbid conditions, but the underlying mechanisms of and therapeutic strategies against PTSD-related pain remain unclear. Our previous studies suggested that dysregulation of neuroinflammation contributes to the development of stress-induced hyperalgesia. Recent studies reported that angiotensin II was a 'stress-related hormone', and could induce glial activation by stimulating the type 1 receptor (AT1R). In the present study, we aimed to investigate whether AT1R blockade could attenuate mechanical allodynia induced by PTSD-like stress. Adult male rats were exposed to single prolonged stress (SPS) to establish a model of PTSD-pain comorbidity. Our results showed that SPS exposure increased the levels of angiotensin II in the hippocampus, prefrontal cortex (PFC) and spinal cord; intraperitoneal injection of losartan attenuated SPS-induced mechanical allodynia, and suppressed SPS-induced glial activation (both microglia and astrocytes) and proinflammatory cytokine expression in the PFC and spinal cord, but not in the hippocampus. We further showed that intrathecal injection of losartan also exerted anti-hyperalgesic effect and suppressed SPS-induced glial activation and proinflammatory cytokine expression in the spinal cord. These results indicated that AT1R blockade by losartan attenuated mechanical allodynia induced by PTSD-like stress, and this may be attributed to the suppression of glial activation and proinflammatory cytokine expression in the spinal cord. Although further research is warranted to verify our findings in female rodents and to assess pharmacological effects of AT1R blockade in PFC and hippocampus, our study suggested the therapeutic potential of targeting AT1R in the treatment of PTSD-related chronic pain.
Learn More >Neuropathic pain is a major health problem that affects up to 7-10% of the population worldwide. Currently, neuropathic pain is difficult to treat due to its elusive mechanisms. Here we report that orphan G protein-coupled receptor 151 (GPR151) in nociceptive sensory neurons controls neuropathic pain induced by nerve injury. GPR151 was mainly expressed in nonpeptidergic C-fiber dorsal root ganglion (DRG) neurons and highly upregulated after nerve injury. Importantly, conditional knockout of Gpr151 in adult nociceptive sensory neurons significantly alleviated chronic constriction injury (CCI)-induced neuropathic pain-like behavior but did not affect basal nociception. Moreover, GPR151 in DRG neurons was required for CCI-induced neuronal hyperexcitability and upregulation of colony-stimulating factor 1 (CSF1), which is necessary for microglial activation in the spinal cord after nerve injury. Mechanistically, GPR151 coupled with P2X3 ion channels and promoted their functional activities in neuropathic pain-like hypersensitivity. Knockout of Gpr151 suppressed P2X3-mediated calcium elevation and spontaneous pain behavior in CCI mice. Conversely, overexpression of Gpr151 significantly enhanced P2X3-mediated calcium elevation and DRG neuronal excitability. Furthermore, knockdown of P2X3 in DRGs reversed CCI-induced CSF1 upregulation, spinal microglial activation, and neuropathic pain-like behavior. Finally, the co-expression of GPR151 and P2X3 was confirmed in small-diameter human DRG neurons, indicating the clinical relevance of our findings. Together, our results suggest that GPR151 in nociceptive DRG neurons plays a key role in the pathogenesis of neuropathic pain and could be a potential target for treating neuropathic pain.
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