Once activated at the surface of cells, G protein-coupled receptors (GPCRs) redistribute to endosomes, where they can continue to signal. Whether GPCRs in endosomes generate signals that contribute to human disease is unknown. We evaluated endosomal signaling of protease-activated receptor-2 (PAR), which has been proposed to mediate pain in patients with irritable bowel syndrome (IBS). Trypsin, elastase, and cathepsin S, which are activated in the colonic mucosa of patients with IBS and in experimental animals with colitis, caused persistent PAR-dependent hyperexcitability of nociceptors, sensitization of colonic afferent neurons to mechanical stimuli, and somatic mechanical allodynia. Inhibitors of clathrin- and dynamin-dependent endocytosis and of mitogen-activated protein kinase kinase-1 prevented trypsin-induced hyperexcitability, sensitization, and allodynia. However, they did not affect elastase- or cathepsin S-induced hyperexcitability, sensitization, or allodynia. Trypsin stimulated endocytosis of PAR, which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR, which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR-dependent hyperexcitability of nociceptors, and PAR association with β-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR A cholestanol-conjugated PAR antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases.

The endocannabinoid system (ECS) plays a critical role in obesity development. The pharmacological blockade of cannabinoid receptor type 1 (CB(1)) has been shown to reduce body weight and to alleviate obesity-related metabolic disorders. An unsolved question is at which anatomical level CB(1) modulates energy balance and the mechanisms involved in its action. Here, we demonstrate that CB(1) receptors expressed in forebrain and sympathetic neurons play a key role in the pathophysiological development of diet-induced obesity. Conditional mutant mice lacking CB(1) expression in neurons known to control energy balance, but not in nonneuronal peripheral organs, displayed a lean phenotype and resistance to diet-induced obesity. This phenotype results from an increase in lipid oxidation and thermogenesis as a consequence of an enhanced sympathetic tone and a decrease in energy absorption. In conclusion, CB(1) signaling in the forebrain and sympathetic neurons is a key determinant of the ECS control of energy balance.

Background Specialized endoderm derived epithelial cells, i.e. enteroendocrine cells (EECs), are widely distributed throughout the gastrointestinal (GI) tract. EECs form the largest endocrine organ in the body and play a key role in the control of GI secretion and motility, the regulation of food intake, postprandial glucose levels and metabolism. EECs sense luminal content and release signaling molecules that can enter the circulation to act as classic hormones on distant targets, act locally on neighboring cells and on distinct neuronal pathways including enteric and extrinsic neurons. Recent studies have shed light on EEC sensory transmission by showing direct connections between EECs and the nervous system via axon-like processes that form a well-defined neuroepithelial circuits through which EECs can directly communicate with the neurons innervating the GI tract to initiate appropriate functional responses. Purpose This review will highlight the role played by the EECs in the complex and integrated sensory information responses, and discuss the new findings regarding EECs in the brain-gut axis bidirectional communication.

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Efficacy of monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor (calcitonin receptor-like receptor/receptor activity modifying protein-1, CLR/RAMP1) implicates peripherally-released CGRP in migraine pain. However, the site and mechanism of CGRP-evoked peripheral pain remain unclear. By cell-selective RAMP1 gene deletion, we reveal that CGRP released from mouse cutaneous trigeminal fibers targets CLR/RAMP1 on surrounding Schwann cells to evoke periorbital mechanical allodynia. CLR/RAMP1 activation in human and mouse Schwann cells generates long-lasting signals from endosomes that evoke cAMP-dependent formation of NO. NO, by gating Schwann cell transient receptor potential ankyrin 1 (TRPA1), releases ROS, which in a feed-forward manner sustain allodynia via nociceptor TRPA1. When encapsulated into nanoparticles that release cargo in acidified endosomes, a CLR/RAMP1 antagonist provides superior inhibition of CGRP signaling and allodynia in mice. Our data suggest that the CGRP-mediated neuronal/Schwann cell pathway mediates allodynia associated with neurogenic inflammation, contributing to the algesic action of CGRP in mice.

Whether G protein-coupled receptors signal from endosomes to control important pathophysiological processes and are therapeutic targets is uncertain. We report that opioids from the inflamed colon activate δ-opioid receptors (DOPr) in endosomes of nociceptors. Biopsy samples of inflamed colonic mucosa from patients and mice with colitis released opioids that activated DOPr on nociceptors to cause a sustained decrease in excitability. DOPr agonists inhibited mechanically sensitive colonic nociceptors. DOPr endocytosis and endosomal signaling by protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways mediated the sustained inhibitory actions of endogenous opioids and DOPr agonists. DOPr agonists stimulated the recruitment of Gαi/oand β-arrestin1/2 to endosomes. Analysis of compartmentalized signaling revealed a requirement of DOPr endocytosis for activation of PKC at the plasma membrane and in the cytosol and ERK in the nucleus. We explored a nanoparticle delivery strategy to evaluate whether endosomal DOPr might be a therapeutic target for pain. The DOPr agonist DADLE was coupled to a liposome shell for targeting DOPr-positive nociceptors and incorporated into a mesoporous silica core for release in the acidic and reducing endosomal environment. Nanoparticles activated DOPr at the plasma membrane, were preferentially endocytosed by DOPr-expressing cells, and were delivered to DOPr-positive early endosomes. Nanoparticles caused a long-lasting activation of DOPr in endosomes, which provided sustained inhibition of nociceptor excitability and relief from inflammatory pain. Conversely, nanoparticles containing a DOPr antagonist abolished the sustained inhibitory effects of DADLE. Thus, DOPr in endosomes is an endogenous mechanism and a therapeutic target for relief from chronic inflammatory pain.