Significance: Activated G protein-coupled receptors (GPCRs) internalize and can continue to signal from endosomes. The contribution of endosomal signaling to human disease is unknown. Proteases that are generated in the colon of patients with irritable bowel syndrome (IBS) can cleave protease-activated receptor-2 (PAR ₂ ) on nociceptors to cause pain. We evaluated whether PAR ₂ generates signals in endosomes of nociceptors that mediate persistent hyperexcitability and pain. Biopsies of colonic mucosa from IBS patients released proteases that induced PAR ₂ endocytosis, endosomal signaling, and persistent hyperexcitability of nociceptors. When conjugated to the transmembrane lipid cholestanol, PAR ₂ antagonists accumulated in endosomes and suppressed persistent hyperexcitability. The results reveal the therapeutic potential of endosomally targeted PAR ₂ antagonists for IBS pain, and expand the contribution of endosomal GPCR signaling to encompass processes that are relevant to disease.

Nociceptors, i.e. sensory neurons tuned to detect noxious stimuli, are found in numerous phyla of the Animalia kingdom and are often polymodal, responding to a variety of stimuli, e.g. heat, cold, pressure and chemicals, such as acid. Owing to the ability of protons to have a profound effect on ionic homeostasis and damage macromolecular structures, it is no wonder that the ability to detect acid is conserved across many species. To detect changes in pH, nociceptors are equipped with an assortment of different acid sensors, some of which can detect mild changes in pH, such as the acid-sensing ion channels, proton-sensing G protein-coupled receptors and several two-pore potassium channels, whereas others, such as the transient receptor potential vanilloid 1 ion channel, require larger shifts in pH. This review will discuss the evolution of acid sensation and the different mechanisms by which nociceptors can detect acid. This article is part of the Theo Murphy meeting issue ‘Evolution of mechanisms and behaviour important for pain’.

Pain is a principal contributor to the global burden of arthritis with peripheral sensitization being a major cause of arthritis-related pain. Within the knee joint, distal endings of dorsal root ganglion neurons (knee neurons) interact with fibroblast-like synoviocytes (FLS) and the inflammatory mediators they secrete, which are thought to promote peripheral sensitization. Correspondingly, RNA sequencing has demonstrated detectable levels of proinflammatory genes in FLS derived from arthritis patients. This study confirms that stimulation with tumor necrosis factor (TNF-α) results in expression of proinflammatory genes in mouse and human FLS (derived from osteoarthritis and rheumatoid arthritis patients), as well as increased secretion of cytokines from mouse TNF-α-stimulated FLS (TNF-FLS). Electrophysiological recordings from retrograde labelled knee neurons cocultured with TNF-FLS, or supernatant derived from TNF-FLS, revealed a depolarized resting membrane potential, increased spontaneous action potential firing, and enhanced TRPV1 function, all consistent with a role for FLS in mediating the sensitization of pain-sensing nerves in arthritis. Therefore, data from this study demonstrate the ability of FLS activated by TNF-α to promote neuronal sensitization, results that highlight the importance of both nonneuronal and neuronal cells to the development of pain in arthritis.

Objective: Joint pain is the major clinical symptom of arthritis that affects millions of people. Controlling the excitability of knee‐innervating dorsal root ganglion (DRG) neurons (knee neurons) could potentially provide pain relief. We undertook this study to evaluate whether the newly engineered adeno‐associated virus (AAV) serotype, AAV‐PHP.S, can deliver functional artificial receptors to control knee neuron excitability following intraarticular knee injection.Methods: The AAV‐PHP.S virus, packaged with dTomato fluorescent protein and either excitatory (G_q) or inhibitory (G_i) designer receptors exclusively activated by designer drugs (DREADDs), was injected into the knee joints of adult mice. Labeling of DRG neurons with AAV‐PHP.S from the knee was evaluated using immunohistochemistry. The functionality of G_q‐ and G_i‐DREADDs was evaluated using whole‐cell patch clamp electrophysiology on acutely cultured DRG neurons. Pain behavior in mice was assessed using a digging assay, dynamic weight bearing, and rotarod performance, before and after intraperitoneal administration of the DREADD activator, Compound 21.Results: We showed that AAV‐PHP.S can deliver functional genes into ~7% of lumbar DRG neurons when injected into the knee joint in a similar manner to the well‐established retrograde tracer, fast blue. Short‐term activation of AAV‐PHP.S–delivered G_q‐DREADD increased excitability of knee neurons in vitro (P = 0.02 by unpaired t‐test), without inducing overt pain in mice when activated in vivo. By contrast, in vivo G_i‐DREADD activation alleviated digging deficits induced by Freund's complete adjuvant–mediated knee inflammation (P = 0.0002 by repeated‐measures analysis of variance [ANOVA] followed by Holm‐Sidak multiple comparisons test). A concomitant decrease in knee neuron excitability was observed in vitro (P = 0.005 by ANOVA followed by Holm‐Sidak multiple comparisons test).Conclusion: We describe an AAV‐mediated chemogenetic approach to specifically control joint pain, which may be utilized in translational arthritic pain research.

Ongoing, spontaneous pain is characteristic of inflammatory joint pain and reduces an individual's quality of life. To understand the neural basis of inflammatory joint pain, we made a unilateral knee injection of complete Freund's adjuvant (CFA) in mice, which reduced their natural digging behavior. We hypothesized that sensitization of knee-innervating dorsal root ganglion (DRG) neurons underlies this altered behavior. To test this hypothesis, we performed electrophysiological recordings on retrograde labeled knee-innervating primary DRG neuron cultures and measured their responses to a number of electrical and chemical stimuli. We found that 24-h after CFA-induced knee inflammation, knee neurons show a decreased action potential generation threshold, as well as increased GABA and capsaicin sensitivity, but have unaltered acid sensitivity. The inflammation-induced sensitization of knee neurons persisted for 24-h in culture, but was not observed after 48-h in culture. Through immunohistochemistry, we showed that the increased knee neuron capsaicin sensitivity correlated with enhanced expression of the capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) in knee-innervating neurons of the CFA-injected side. We also observed an increase in the co-expression of TRPV1 with tropomyosin receptor kinase A (TrkA), which is the receptor for nerve growth factor (NGF), suggesting that NGF partially induces the increased TRPV1 expression. Lastly, we found that systemic administration of the TRPV1 antagonist, A-425619, reversed the decrease in digging behavior induced by CFA injection, further confirming the role of TRPV1, expressed by knee neurons, in acute inflammatory joint pain.

Enteroendocrine cells are specialised sensory cells located in the intestinal epithelium and generate signals in response to food ingestion. Whilst traditionally considered hormone-producing cells, there is evidence that they also initiate activity in the afferent vagus nerve and thereby signal directly to the brainstem. We investigate whether enteroendocrine L-cells, well known for their production of the incretin hormone glucagon-like peptide-1 (GLP-1), also release other neuro-transmitters/modulators. We demonstrate regulated ATP release by ATP measurements in cell supernatants and by using sniffer patches that generate electrical currents upon ATP exposure. Employing purinergic receptor antagonists, we demonstrate that evoked ATP release from L-cells triggers electrical responses in neighbouring enterocytes through P2Y₂ and nodose ganglion neurones in co-cultures through P2X_2/3-receptors. We conclude that L-cells co-secrete ATP together with GLP-1 and PYY, and that ATP acts as an additional signal triggering vagal activation and potentially synergising with the actions of locally elevated peptide hormone concentrations.