Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome.

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 (PAR2), 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 PAR2-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 PAR2, which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR2, which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR2-dependent hyperexcitability of nociceptors, and PAR2 association with ß-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR2 A cholestanol-conjugated PAR2 antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR2 signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR2 antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases.

Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission.

G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8-37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8-37-cholestanol, but not unconjugated CGRP8-37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8-37-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP8-37 Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.

Therapeutic Targeting of Endosomal G-Protein-Coupled Receptors.

G-protein-coupled receptors (GPCRs) are conventionally considered to function at the plasma membrane, where they detect extracellular ligands and activate heterotrimeric G proteins that transmit intracellular signals. Consequently, drug discovery efforts have focused on identification of agonists and antagonists of cell surface GPCRs. However, ß-arrestin (ARR)-dependent desensitization and endocytosis rapidly terminate G protein signaling at the plasma membrane. Emerging evidence indicates that GPCRs can continue to signal from endosomes by G-protein- and ßARR-dependent processes. By regulating the duration and location of intracellular signaling events, GPCRs in endosomes control critically important processes, including gene transcription and ion channel activity. Thus, GPCRs in endosomes, in addition to at the cell surface, have emerged as important therapeutic targets.

Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.

Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) control many pathophysiological processes and are the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NK1R) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NK1R in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane-targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and ß-arrestin blocked SP-induced NK1R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal-regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NK1R antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NK1R antagonists. These results reveal a critical role for endosomal signaling of the NK1R in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.

The selective mu opioid receptor antagonist, alvimopan, improves delayed GI transit of postoperative ileus in rats.

Postoperative ileus (POI) is often exacerbated by opioid analgesic use during and following surgery, since mu opioid receptor activation results in a further delay of gastrointestinal (GI) transit. The effects of alvimopan, a novel, selective, and peripherally acting mu opioid receptor antagonist, and the reference compound methylnaltrexone, upon POI were investigated in rats. Under isoflurane anesthesia, POI was induced by laparotomy with intestinal manipulation. Immediately after the surgery, the rats received (51)Cr by gavage. Three hours after the surgery, the rats were sacrificed and GI transit was estimated using the geometric center (GC) of (51)Cr. Alvimopan (0.1-3 mg/kg) or methylnaltrexone (100 mg/kg) were administered by gavage either before or after the surgery, with or without morphine administration (1 mg/kg). GI transit was delayed by intestinal manipulation (GC = 2.92 +/- 0.17). Alvimopan (1 and 3 mg/kg) significantly reversed this delayed GI transit when administered 45 min prior to surgery. However, the effects of alvimopan were less pronounced when administered following surgery. Morphine administration further delayed GI transit induced by intestinal manipulation (GC = 1.97 +/- 0.11). Under these conditions, alvimopan (1 and 3 mg/kg) also significantly improved delayed GI transit when administered before surgery. Methylnaltrexone was inactive under all experimental conditions. These data suggest that mu opioid receptors play a role in the pathogenesis of POI, and that the clinical benefit reported to be afforded by alvimopan may be in part mediated via inhibition of an endogenous opioid release as well as blockade of the unwanted GI actions of analgesic agents.

An animal model of chronic inflammatory pain: pharmacological and temporal differentiation from acute models.

Clinically, inflammatory pain is far more persistent than that typically modelled pre-clinically, with the majority of animal models focussing on short-term effects of the inflammatory pain response. The large attrition rate of compounds in the clinic which show pre-clinical efficacy suggests the need for novel models of, or approaches to, chronic inflammatory pain if novel mechanisms are to make it to the market. A model in which a more chronic inflammatory hypersensitivity phenotype is profiled may allow for a more clinically predictive tool. The aims of these studies were to characterise and validate a chronic model of inflammatory pain. We have shown that injection of a large volume of adjuvant to the intra-articular space of the rat knee results in a prolonged inflammatory pain response, compared to the response in an acute adjuvant model. Additionally, this model also results in a hypersensitive state in the presence and absence of inflammation. A range of clinically effective analgesics demonstrate activity in this chronic model, including morphine (3mg/kg, t.i.d.), dexamethasone (1mg/kg, b.i.d.), ibuprofen (30mg/kg, t.i.d.), etoricoxib (5mg/kg, b.i.d.) and rofecoxib (0.3-10mg/kg, b.i.d.). A further aim was to exemplify the utility of this chronic model over the more acute intra-plantar adjuvant model using two novel therapeutic approaches; NR2B selective NMDA receptor antagonism and iNOS inhibition. Our data shows that different effects were observed with these therapies when comparing the acute model with the model of chronic inflammatory joint pain. These data suggest that the chronic model may be more relevant to identifying mechanisms for the treatment of chronic inflammatory pain states in the clinic.

Human native kappa opioid receptor functions not predicted by recombinant receptors: Implications for drug design.

If activation of recombinant G protein-coupled receptors in host cells (by drugs or other ligands) has predictive value, similar data must be obtained with native receptors naturally expressed in tissues. Using mouse and human recombinant κ opioid receptors transfected into a host cell, two selectively-acting compounds (ICI204448, asimadoline) equi-effectively activated both receptors, assessed by measuring two different cell signalling pathways which were equally affected without evidence of bias. In mouse intestine, naturally expressing κ receptors within its nervous system, both compounds also equi-effectively activated the receptor, inhibiting nerve-mediated muscle contraction. However, whereas ICI204448 acted similarly in human intestine, where κ receptors are again expressed within its nervous system, asimadoline was inhibitory only at very high concentrations; instead, low concentrations of asimadoline reduced the activity of ICI204448. This demonstration of species-dependence in activation of native, not recombinant κ receptors may be explained by different mouse/human receptor structures affecting receptor expression and/or interactions with intracellular signalling pathways in native environments, to reveal differences in intrinsic efficacy between receptor agonists. These results have profound implications in drug design for κ and perhaps other receptors, in terms of recombinant-to-native receptor translation, species-dependency and possibly, a need to use human, therapeutically-relevant, not surrogate tissues.

Drugs targeting functional bowel disorders: insights from animal studies.

Increased understanding of gastrointestinal pathophysiology in functional bowel disorders is leading to improved focus on relevant enteric, vagal and spinal afferent nervous systems, and targets operating on these systems. These targets include receptors for tachykinins, motilin, ghrelin, corticotropin-releasing factor and somatostatin.

Asimadoline and its potential for the treatment of diarrhea-predominant irritable bowel syndrome: a review.

Irritable bowel syndrome (IBS) is a multifactorial condition with principal symptoms of pain and altered bowel function. The kappa-opioid agonist asimadoline is being evaluated in Phase III as a potential treatment for IBS. Asimadoline, to date, has shown a good safety profile and the target Phase III population - diarrhea-predominant IBS patients with at least moderate pain - was iteratively determined in a prospective manner from a Phase II dose-ranging study. The clinical data in support of this population are reviewed in this article. Furthermore, the scientific rationale for the use of asimadoline in the treatment of IBS is reviewed. Considering the high patient and societal burdens of IBS, new treatments for IBS represent therapeutic advances.

Dual role of 5-HT3 receptors in a rat model of delayed stress-induced visceral hyperalgesia.

Despite its beneficial effect in IBS patients, the mechanism of action of the 5-HT3 receptor (5-HT3R) antagonist alosetron is still incompletely understood. We aimed to characterize the effect and site(s) of action in a model of stress-induced sensitization of visceral nociception in rats. Adult male Wistar rats were equipped for recording of visceromotor response (VMR) to phasic colorectal distension (CRD; 10-60 mmHg). VMR to CRD was recorded 24 h after an acute session of water avoidance (WA) stress (post-WA). Baseline and post-WA responses were measured in rats exposed to WA or sham-WA, treated with alosetron at 0.3 mg/kg subcutaneously (s.c.) 25 nmol intrathecally (i.t.) or vehicle before post-WA CRD. Some rats were treated with capsaicin/vehicle on the cervical vagus nerve and received alosetron (0.3 mg/kg, s.c.) 15 min before post-WA CRD. WA stress led to visceral hyperalgesia 24 h later. Alosetron (0.3 mg/kg, s.c.), failed to inhibit WA-induced exacerbation of VMR to CRD. Stress-induced visceral hyperalgesia was abolished when alosetron was injected intrathecally (P<0.05) in intact rats or subcutaneously (0.3 mg/kg) in capsaicin-pretreated animals (P<0.05). Capsaicin-pretreatment did not affect the exacerbating effect of stress on visceral sensitivity. Alosetron had no inhibitory effect on normal visceral pain responses when administered subcutaneously or intrathecally. We demonstrated that 5-HT3Rs on central terminals of spinal afferents are engaged in the facilitatory effect of stress on visceral sensory information processing. In addition, we showed that stress-induced sensitization of visceral nociception is independent of 5-HT3R activation on vagal afferents.

Inhibition of BK channel activity by association with calcineurin in rat brain.

Large conductance calcium-activated potassium (BK(Ca)) channels are regulated by a number of different protein kinases and phosphatases. The close association of enzymes and channel have been shown to underlie many examples of modulation. However, only the association of protein kinase A with the BK(Ca) channel has been detailed [Tian et al. (2003)J. Biol. Chem., 278, 8669-8677]. We have found using reciprocal immunoprecipitations that the BK(Ca) channel associates with the calcium/calmodulin-dependent phosphatase calcineurin, in Wistar rat brain. A HA-tagged construct of the carboxyl terminus of rSlo(27), a variant of the BK(Ca) channel that is abundant in the hippocampus [Ha et al. (2000)Eur. J. Biochem., 267, 910-9218], was found to associate only with the B subunit of calcineurin. This data suggests that the majority of the interaction of the BK(Ca) channel with calcineurin is mediated by the B subunit of the phosphatase. This was confirmed by using glutathione-S-transferase (GST) fusion proteins of the linker regions between the S7-S10 hydrophobic domains in the carboxyl terminus of rSlo(27), where only the B subunit of calcineurin interacted with regions between S7 and S9 of the channel. Addition of a constitutively active calcineurin (CaN(420)) to inside-out membrane patches excised from cultured hippocampal neurons resulted in a dramatic reduction in BK(Ca) channel open probability, with only very short-duration events being apparent. These data suggest that BK(Ca) channel activity is inhibited by calcineurin, an effect mediated by the association of the calcineurin B subunit with the carboxyl terminus of the channel.

Differential chemosensory function and receptor expression of splanchnic and pelvic colonic afferents in mice.

Lumbar splanchnic (LSN) and sacral pelvic (PN) nerves convey different mechanosensory information from the colon to the spinal cord. Here we determined whether these pathways also differ in their chemosensitivity and receptor expression. Using an in vitro mouse colon preparation, individual primary afferents were tested with selective P2X and transient receptor potential vanilloid receptor 1 (TRPV1) receptor ligands. Afferent cell bodies in thoracolumbar and lumbosacral dorsal root ganglia (DRG) were retrogradely labelled from the colon and analysed for P2X3- and TRPV1-like immunoreactivity (LI). Forty per cent of LSN afferents responded to alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-meATP; 1 mm), an effect that was concentration dependent and reversed by the P2X antagonist pyridoxyl5-phosphate 6-azophenyl-2',4'-disulphonic acid (PPADS) (100 microm). Significantly fewer PN afferents (7%) responded to alpha,beta-meATP. Correspondingly, 36% of colonic thoracolumbar DRG neurones exhibited P2X3-LI compared with only 19% of colonic lumbosacral neurones. Capsaicin (3 microm) excited 61% of LSN afferents and 47% of PN afferents; 82% of thoracolumbar and 50% of lumbosacral colonic DRG neurones displayed TRPV1-LI. Mechanically insensitive afferents were recruited by alpha,beta-meATP or capsaicin, and were almost exclusive to the LSN. Capsaicin-responsive LSN afferents displayed marked mechanical desensitization after responding to capsaicin, which did not occur in capsaicin-responsive PN afferents. Therefore, colonic LSN and PN pathways differ in their chemosensitivity to known noxious stimuli and their corresponding receptor expression. As these pathways relay information that may relate to symptoms in functional gastrointestinal disease, these results may have implications for the efficacy of therapies targeting receptor modulation.

Somatostatin sst(2) receptors inhibit peristalsis in the rat and mouse jejunum.

Somatostatin [somatotropin release-inhibitory factor (SRIF)] has widespread actions throughout the gastrointestinal tract, but the receptor mechanisms involved are not fully characterized. We have examined the effect of selective SRIF-receptor ligands on intestinal peristalsis by studying migrating motor complexes (MMCs) in isolated segments of jejunum from rats, mice, and sst(2)-receptor knockout mice. MMCs were recorded in 4- to 5-cm segments of jejunum mounted horizontally in vitro. MMCs occurred in rat and mouse jejunum with intervals of 104.4 +/- 10 and 131.2 +/- 8 s, respectively. SRIF, octreotide, and BIM-23027 increased the interval between MMCs, an effect fully or partially antagonized by the sst(2)-receptor antagonist Cyanamid154806. A non-sst(2) receptor-mediated component was evident in mouse as confirmed by the observation of an inhibitory action of SRIF in sst(2) knockout tissue. Blocking nitric oxide generation abolished the response to SRIF in rat but not mouse jejunum. sst(2) Receptors mediate inhibition of peristalsis in both rat and mouse jejunum, but a non-sst(2) component also exists in the mouse. Nitrergic mechanisms are differentially involved in rat and mouse jejunum.

Expression of intermediate conductance potassium channel immunoreactivity in neurons and epithelial cells of the rat gastrointestinal tract.

Recent functional evidence suggests that intermediate conductance calcium-activated potassium channels (IK channels) occur in neurons in the small intestine and in mucosal epithelial cells in the colon. This study was undertaken to investigate whether IK channel immunoreactivity occurs at these and at other sites in the gastrointestinal tract of the rat. IK channel immunoreactivity was found in nerve cell bodies throughout the gastrointestinal tract, from the esophagus to the rectum. It was revealed in the initial segments of the axons, but not in axon terminals. The majority of immunoreactive neurons had Dogiel type II morphology and in the myenteric plexus of the ileum all immunoreactive neurons were of this shape. Intrinsic primary afferent neurons in the rat small intestine are Dogiel type II neurons that are immunoreactive for calretinin, and it was found that almost all the IK channel immunoreactive neurons were also calretinin immunoreactive. IK channel immunoreactivity also occurred in calretinin-immunoreactive, Dogiel type II neurons in the caecum. Epithelial cells of the mucosal lining were immunoreactive in the esophagus, stomach, small and large intestines. In the intestines, the immunoreactivity occurred in transporting enterocytes, but not in mucous cells. Immunoreactivity was at both the apical and basolateral surfaces. A small proportion of mucosal endocrine cells was immunoreactive in the duodenum, ileum and caecum, but not in the stomach, proximal colon, distal colon or rectum. There was immunoreactivity of vascular endothelial cells. It is concluded that IK channels are located on cell bodies and proximal parts of axons of intrinsic primary afferent neurons, where, from functional studies, they would be predicted to lower neuronal excitability when opened in response to calcium entry. In the mucosa of the small and large intestine, IK channels are probably involved in control of potassium exchange, and in the esophageal and gastric mucosa they are possibly involved in control of cell volume in response to osmotic challenge.

Excitation of rat colonic afferent fibres by 5-HT(3) receptors.

The gastrointestinal tract contains most of the body's 5-hydroxytryptamine (5-HT) and releases large amounts after meals or exposure to toxins. Increased 5-HT release occurs in patients with irritable bowel syndrome (IBS) and their peak plasma 5-HT levels correlate with pain episodes. 5-HT(3) receptor antagonists reduce symptoms of IBS clinically, but their site of action is unclear and the potential for other therapeutic targets is unexplored. Here we investigated effects of 5-HT on sensory afferents from the colon and the expression of 5-HT(3) receptors on their cell bodies in the dorsal root ganglia (DRG). Distal colon, inferior mesenteric ganglion and the lumbar splanchnic nerve bundle (LSN) were placed in a specialized organ bath. Eighty-six single fibres were recorded from the LSN. Three classes of primary afferents were found: 70 high-threshold serosal afferents, four low-threshold muscular afferents and 12 mucosal afferents. Afferent cell bodies were retrogradely labelled from the distal colon to the lumbar DRG, where they were processed for 5-HT(3) receptor-like immunoreactivity. Fifty-six percent of colonic afferents responded to 5-HT (between 10(-6) and 10(-3) M) and 30 % responded to the selective 5-HT(3) agonist, 2-methyl-5-HT (between 10(-6) and 10(-2) M). Responses to 2-methyl-5-HT were blocked by the 5-HT(3) receptor antagonist alosetron (2 x 10(-7) M), whereas responses to 5-HT were only partly inhibited. Twenty-six percent of L1 DRG cell bodies retrogradely labelled from the colon displayed 5-HT(3) receptor-like immunoreactivity. We conclude that colonic sensory neurones expressing 5-HT(3) receptors also functionally express the receptors at their peripheral endings. Our data reveal actions of 5-HT on colonic afferent endings via both 5-HT(3) and non-5-HT(3) receptors.