Proton-sensing G protein-coupled receptor signalling in inflammation
Inflammation, the body’s response to tissue damage or infection, involves a number of cell types and is often associated with localised tissue acidosis. The resulting decrease in extracellular pH can exacerbate inflammation, additionally protons may directly depolarise or sensitise sensory neurones to drive the heightened nociception associated with chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Extracellular protons can coordinate cellular responses through a number of proton-sensitive receptors including both ion channels and G protein-coupled receptors, many of which are expressed by immune cells and sensory neurones, the principal drivers of inflammation and nociception respectively. While proton-sensitive ion channels, such as the acid-sensing ion channel family have established roles in inflammatory pain, the contribution of proton-sensing G protein-coupled receptors (PS-GPCRs) is less clear. The overarching aim of this work was to further basic pharmacological understanding of the PS-GPCR family and assess their role in inflammatory processes and nociception.
The pain associated with chronic inflammation is severely debilitating and represents major unmet clinical need worldwide, there is thus considerable interest in elucidating the molecular mechanisms underlying inflammatory pain. Animal studies represent a valuable tool for studying the complexities of inflammation, which involves a concert of cell types. However, the interplay between changes at the cellular level and the manifestation of hypersensitive states in rodent models is often poorly assessed. Here, combining evoked and spontaneous behavioural assays, target innervating electrophysiology and gene expression studies, a comprehensive characterisation of sensory neurones and pain behaviour was made for the intraplantar carrageenan model of inflammatory pain in mice. Results highlighted a dynamic relationship between physical inflammation, changes in sensory neurone function and rodent pain experience, and suggested a potential role of PS-GPCRs in the observed pain.
To further explore the possibility that PS-GPCRs might mediate proton-induced pain, attention was turned to the naked mole-rat (Heterocephalus glaber), a species which does not perceive acid as painful. Cloning and characterisation of PS-GPCRs from naked mole-rats and mice revealed differences in the abilities of protons to coordinate signalling at PS-GPCRs between the two species, furthering the idea that PS-GPCRs might contribute to proton-induced pain.
The lack of pharmacological and physiological understanding of PS-GPCRs may be attributed to the fact that protons activate a number of other receptors, as well as technical difficulties arising from the high sensitivity of many pharmacological assays to perturbations in pH. Using a uniform cellular background, the signalling and intracellular trafficking of the PS-GPCR GPR65 was interrogated in response to stimulation with protons as well as two other reported agonists. These studies highlighted that a synthetic agonist (BTB09089), with higher selectivity for GPR65 over other PS-GPCRs, was able to recapitulate many of the proton- induced signalling responses of GPR65. Injecting BTB09089 into the knee joint of mice, resulted in joint swelling and a concomitant increase in nociceptive behaviours. Electrophysiological studies of knee-innervating neurones isolated from mice injected with BTB09089 revealed higher excitability, offering some explanation to the observed pain. However, acute stimulation of naïve neurones with BTB09089 alone could not reproduce the changes in excitability, suggesting the involvement of another cell type. Fibroblast-like synoviocytes, non-neuronal cells resident in the joint, were shown to express GPR65 and secrete pro-inflammatory mediators in response to BTB09089 stimulation, which might be responsible for the sensory neurone sensitisation observed.
Taken together data presented here support a central hypothesis that PS-GPCRs, in particular GPR65, are critical integrators of neuroimmune responses to tissue acidosis in inflammation.
Biotechnology and Biological Sciences Research Council (1943916)