RESUMO
A general approach to design proteins that bind tightly and specifically to intrinsically disordered regions (IDRs) of proteins and flexible peptides would have wide application in biological research, therapeutics, and diagnosis. However, the lack of defined structures and the high variability in sequence and conformational preferences has complicated such efforts. We sought to develop a method combining biophysical principles with deep learning to readily generate binders for any disordered sequence. Instead of assuming a fixed regular structure for the target, general recognition is achieved by threading the query sequence through diverse extended binding modes in hundreds of templates with varying pocket depths and spacings, followed by RFdiffusion refinement to optimize the binder-target fit. We tested the method by designing binders to 39 highly diverse unstructured targets. Experimental testing of â¼36 designs per target yielded binders with affinities better than 100 nM in 34 cases, and in the pM range in four cases. The co-crystal structure of a designed binder in complex with dynorphin A is closely consistent with the design model. All by all binding experiments for 20 designs binding diverse targets show they are highly specific for the intended targets, with no crosstalk even for the closely related dynorphin A and dynorphin B. Our approach thus could provide a general solution to the intrinsically disordered protein and peptide recognition problem.
RESUMO
The crypt-villus structure of the small intestine serves as an essential protective barrier, with its integrity monitored by the gut's sensory system. Enterochromaffin (EC) cells, which are rare sensory epithelial cells that release serotonin (5-HT), surveil the mucosal environment and signal both within and outside the gut. However, it remains unclear whether EC cells in intestinal crypts and villi respond to different stimuli and elicit distinct responses. In this study, we introduce a new reporter mouse model to observe the release and propagation of serotonin in live intestines. Using this system, we show that crypt EC cells exhibit two modes of serotonin release: transient receptor potential A1 (TRPA1)-dependent tonic serotonin release that controls basal ionic secretion, and irritant-evoked serotonin release that activates gut sensory neurons. Furthermore, we find that a thick protective mucus layer prevents TRPA1 receptors on crypt EC cells from responding to luminal irritants such as reactive electrophiles; if this mucus layer is compromised, then crypt EC cells become susceptible to activation by luminal irritants. On the other hand, villus EC cells detect oxidative stress through TRPM2 channels and co-release serotonin and ATP to activate nearby gut sensory fibers. Our work highlights the physiological importance of intestinal architecture and differential TRP channel expression in sensing noxious stimuli that elicit nausea and/or pain sensations in the gut.
RESUMO
The distal colon and rectum (colorectum) are innervated by spinal and vagal afferent pathways. The central circuits into which vagal and spinal afferents relay colorectal nociceptive information remain to be comparatively assessed. To address this, regional colorectal retrograde tracing and colorectal distension (CRD)-evoked neuronal activation were used to compare the circuits within the dorsal vagal complex (DVC) and dorsal horn (thoracolumbar [TL] and lumbosacral [LS] spinal levels) into which vagal and spinal colorectal afferents project. Vagal afferent projections were observed in the nucleus tractus solitarius (NTS), area postrema (AP), and dorsal motor nucleus of the vagus (DMV), labeled from the rostral colorectum. In the NTS, projections were opposed to catecholamine and pontine parabrachial nuclei (PbN)-projecting neurons. Spinal afferent projections were labeled from rostral through to caudal aspects of the colorectum. In the dorsal horn, the number of neurons activated by CRD was linked to pressure intensity, unlike in the DVC. In the NTS, 13% ± 0.6% of CRD-activated neurons projected to the PbN. In the dorsal horn, at the TL spinal level, afferent input was associated with PbN-projecting neurons in lamina I (LI), with 63% ± 3.15% of CRD-activated neurons in LI projecting to the PbN. On the other hand, at the LS spinal level, only 18% ± 0.6% of CRD-activated neurons in LI projected to the PbN. The collective data identify differences in the central neuroanatomy that support the disparate roles of vagal and spinal afferent signaling in the facilitation and modulation of colorectal nociceptive responses.
Assuntos
Neoplasias Colorretais , Nervo Vago , Camundongos , Animais , Vias Aferentes/fisiologia , Neurônios , Corno Dorsal da Medula Espinal , Neoplasias Colorretais/metabolismo , Medula Espinal/metabolismo , Neurônios Aferentes/fisiologiaRESUMO
Chronic pelvic pain (CPP) is the most debilitating symptom of gynaecological disorders such as endometriosis. However, it remains unclear how sensory neurons from pelvic organs affected by endometriosis, such as the female reproductive tract, detect and transmit nociceptive events and how these signals are processed within the central nervous system (CNS). Using a previously characterized mouse model of endometriosis, we investigated whether the increased pain sensitivity occurring in endometriosis could be attributed to (i) changes in mechanosensory properties of sensory afferents innervating the reproductive tract, (ii) alterations in sensory input from reproductive organs to the spinal cord or (iii) neuroinflammation and sensitization of spinal neural circuits. Mechanosensitivity of vagina-innervating primary afferents was examined using an ex vivo single-unit extracellular recording preparation. Nociceptive signalling from the vagina to the spinal cord was quantified by phosphorylated MAP kinase ERK1/2 immunoreactivity. Immunohistochemistry was used to determine glial and neuronal circuit alterations within the spinal cord. We found that sensory afferents innervating the rostral, but not caudal portions of the mouse vagina, developed mechanical hypersensitivity in endometriosis. Nociceptive signalling from the vagina to the spinal cord was significantly enhanced in mice with endometriosis. Moreover, mice with endometriosis developed microgliosis, astrogliosis and enhanced substance P neurokinin-1 receptor immunoreactivity within the spinal cord, suggesting the development of neuroinflammation and sensitization of spinal circuitry in endometriosis. These results demonstrate endometriosis-induced neuroplasticity occurring at both peripheral and central sites of sensory afferent pathways. These findings may help to explain the altered sensitivity to pain in endometriosis and provide a novel platform for targeted pain relief treatments for this debilitating disorder.
RESUMO
Gastrointestinal (GI) discomfort is a hallmark of most gut disorders and represents an important component of chronic visceral pain1. For the growing population afflicted by irritable bowel syndrome, GI hypersensitivity and pain persist long after tissue injury has resolved2. Irritable bowel syndrome also exhibits a strong sex bias, afflicting women three times more than men1. Here, we focus on enterochromaffin (EC) cells, which are rare excitable, serotonergic neuroendocrine cells in the gut epithelium3-5. EC cells detect and transduce noxious stimuli to nearby mucosal nerve endings3,6 but involvement of this signalling pathway in visceral pain and attendant sex differences has not been assessed. By enhancing or suppressing EC cell function in vivo, we show that these cells are sufficient to elicit hypersensitivity to gut distension and necessary for the sensitizing actions of isovalerate, a bacterial short-chain fatty acid associated with GI inflammation7,8. Remarkably, prolonged EC cell activation produced persistent visceral hypersensitivity, even in the absence of an instigating inflammatory episode. Furthermore, perturbing EC cell activity promoted anxiety-like behaviours which normalized after blockade of serotonergic signalling. Sex differences were noted across a range of paradigms, indicating that the EC cell-mucosal afferent circuit is tonically engaged in females. Our findings validate a critical role for EC cell-mucosal afferent signalling in acute and persistent GI pain, in addition to highlighting genetic models for studying visceral hypersensitivity and the sex bias of gut pain.
Assuntos
Ansiedade , Células Enterocromafins , Dor Visceral , Feminino , Humanos , Masculino , Ansiedade/complicações , Ansiedade/fisiopatologia , Sistema Digestório/inervação , Sistema Digestório/fisiopatologia , Células Enterocromafins/metabolismo , Síndrome do Intestino Irritável/complicações , Síndrome do Intestino Irritável/fisiopatologia , Síndrome do Intestino Irritável/psicologia , Caracteres Sexuais , Dor Visceral/complicações , Dor Visceral/fisiopatologia , Dor Visceral/psicologia , Inflamação/complicações , Inflamação/fisiopatologia , Serotonina/metabolismo , Reprodutibilidade dos TestesRESUMO
Chronic pelvic pain (CPP) is the primary symptom of endometriosis patients, but adequate treatments are lacking. Modulation of ion channels expressed by sensory nerves innervating the viscera has shown promise for the treatment of irritable bowel syndrome and overactive bladder. However, similar approaches for endometriosis-associated CPP remain underdeveloped. Here, we examined the role of the voltage-gated sodium (NaV ) channel NaV 1.7 in (i) the sensitivity of vagina-innervating sensory afferents and investigated whether (ii) NaV 1.7 inhibition reduces nociceptive signals from the vagina and (iii) ameliorates endometriosis-associated CPP. The mechanical responsiveness of vagina-innervating sensory afferents was assessed with ex vivo single-unit recording preparations. Pain evoked by vaginal distension (VD) was quantified by the visceromotor response (VMR) in vivo. In control mice, pharmacological activation of NaV 1.7 with OD1 sensitised vagina-innervating pelvic afferents to mechanical stimuli. Using a syngeneic mouse model of endometriosis, we established that endometriosis sensitised vagina-innervating pelvic afferents to mechanical stimuli. The highly selective NaV 1.7 inhibitor Tsp1a revealed that this afferent hypersensitivity occurred in a NaV 1.7-dependent manner. Moreover, in vivo intra-vaginal treatment with Tsp1a reduced the exaggerated VMRs to VD which is characteristic of mice with endometriosis. Conversely, Tsp1a did not alter ex vivo afferent mechanosensitivity nor in vivo VMRs to VD in Sham control mice. Collectively, these findings suggest that NaV 1.7 plays a crucial role in endometriosis-induced vaginal hyperalgesia. Importantly, NaV 1.7 inhibition selectively alleviated endometriosis-associated CPP without the loss of normal sensation, suggesting that selective targeting of NaV 1.7 could improve the quality of life of women with endometriosis.
RESUMO
The management of visceral pain in patients with disorders of gut-brain interaction, notably irritable bowel syndrome, presents a considerable clinical challenge, with few available treatment options. Patients are increasingly using cannabis and cannabinoids to control abdominal pain. Cannabis acts on receptors of the endocannabinoid system, an endogenous system of lipid mediators that regulates gastrointestinal function and pain processing pathways in health and disease. The endocannabinoid system represents a logical molecular therapeutic target for the treatment of pain in irritable bowel syndrome. Here, we review the physiological and pathophysiological functions of the endocannabinoid system with a focus on the peripheral and central regulation of gastrointestinal function and visceral nociception. We address the use of cannabinoids in pain management, comparing them to other treatment modalities, including opioids and neuromodulators. Finally, we discuss emerging therapeutic candidates targeting the endocannabinoid system for the treatment of pain in irritable bowel syndrome.
Assuntos
Canabinoides , Cannabis , Síndrome do Intestino Irritável , Humanos , Endocanabinoides/uso terapêutico , Endocanabinoides/metabolismo , Síndrome do Intestino Irritável/complicações , Síndrome do Intestino Irritável/tratamento farmacológico , Canabinoides/uso terapêutico , Canabinoides/metabolismo , Dor Abdominal/tratamento farmacológico , Dor Abdominal/etiologia , Cannabis/metabolismoRESUMO
ABSTRACT: The bladder wall is innervated by a complex network of afferent nerves that detect bladder stretch during filling. Sensory signals, generated in response to distension, are relayed to the spinal cord and brain to evoke physiological and painful sensations and regulate urine storage and voiding. Hyperexcitability of these sensory pathways is a key component in the development of chronic bladder hypersensitivity disorders including interstitial cystitis/bladder pain syndrome and overactive bladder syndrome. Despite this, the full array of ion channels that regulate bladder afferent responses to mechanical stimuli have yet to be determined. Here, we investigated the role of low-voltage-activated T-type calcium (Ca V 3) channels in regulating bladder afferent responses to distension. Using single-cell reverse-transcription polymerase chain reaction and immunofluorescence, we revealed ubiquitous expression of Ca V 3.2, but not Ca V 3.1 or Ca V 3.3, in individual bladder-innervating dorsal root ganglia neurons. Pharmacological inhibition of Ca V 3.2 with TTA-A2 and ABT-639, selective blockers of T-type calcium channels, dose-dependently attenuated ex-vivo bladder afferent responses to distension in the absence of changes to muscle compliance. Further evaluation revealed that Ca V 3.2 blockers significantly inhibited both low- and high-threshold afferents, decreasing peak responses to distension, and delayed activation thresholds, thereby attenuating bladder afferent responses to both physiological and noxious distension. Nocifensive visceromotor responses to noxious bladder distension in vivo were also significantly reduced by inhibition of Ca V 3 with TTA-A2. Together, these data provide evidence of a major role for Ca V 3.2 in regulating bladder afferent responses to bladder distension and nociceptive signalling to the spinal cord.
Assuntos
Canais de Cálcio Tipo T , Cistite Intersticial , Humanos , Bexiga Urinária/inervação , Neurônios Aferentes/fisiologia , Canais de Cálcio Tipo T/metabolismo , Vias Aferentes/fisiologia , Cistite Intersticial/metabolismo , Gânglios Espinais/metabolismoRESUMO
Our understanding of how abdominal organs (like the gut) communicate with the brain, via sensory nerves, has been limited by a lack of techniques to selectively activate or inhibit populations of spinal primary afferent neurons within dorsal root ganglia (DRG), of live animals. We report a survival surgery technique in mice, where select DRG are surgically removed (unilaterally or bilaterally), without interfering with other sensory or motor nerves. Using this approach, pain responses evoked by rectal distension were abolished by bilateral lumbosacral L5-S1 DRG removal, but not thoracolumbar T13-L1 DRG removal. However, animals lacking T13-L1 or L5-S1 DRG both showed reduced pain sensitivity to distal colonic distension. Removal of DRG led to selective loss of peripheral CGRP-expressing spinal afferent axons innervating visceral organs, arising from discrete spinal segments. This method thus allows spinal segment-specific determination of sensory pathway functions in conscious, free-to-move animals, without genetic modification.
Assuntos
Encéfalo , Gânglios Espinais , Animais , Colo , Gânglios Espinais/metabolismo , Camundongos , DorRESUMO
The mechanisms underlying chronic bladder conditions such as interstitial cystitis/bladder pain syndrome (IC/BPS) and overactive bladder syndrome (OAB) are incompletely understood. However, targeting specific receptors mediating neuronal sensitivity to specific stimuli is an emerging treatment strategy. Recently, irritant-sensing receptors including the bile acid receptor TGR5, have been identified within the viscera and are thought to play a key role in neuronal hypersensitivity. Here, in mice, we identify mRNA expression of TGR5 (Gpbar1) in all layers of the bladder as well as in the lumbosacral dorsal root ganglia (DRG) and in isolated bladder-innervating DRG neurons. In bladder-innervating DRG neurons Gpbar1 mRNA was 100% co-expressed with Trpv1 and 30% co-expressed with Trpa1. In vitro live-cell calcium imaging of bladder-innervating DRG neurons showed direct activation of a sub-population of bladder-innervating DRG neurons with the synthetic TGR5 agonist CCDC, which was diminished in Trpv1-/- but not Trpa1-/- DRG neurons. CCDC also activated a small percentage of non-neuronal cells. Using an ex vivo mouse bladder afferent recording preparation we show intravesical application of endogenous (5α-pregnan-3ß-ol-20-one sulphate, Pg5α) and synthetic (CCDC) TGR5 agonists enhanced afferent mechanosensitivity to bladder distension. Correspondingly, in vivo intravesical administration of CCDC increased the number of spinal dorsal horn neurons that were activated by bladder distension. The enhanced mechanosensitivity induced by CCDC ex vivo and in vivo was absent using Gpbar1-/- mice. Together, these results indicate a role for the TGR5 receptor in mediating bladder afferent hypersensitivity to distension and thus may be important to the symptoms associated with IC/BPS and OAB.
Assuntos
Cistite Intersticial , Retenção Urinária , Animais , Cistite Intersticial/metabolismo , Gânglios Espinais/metabolismo , Camundongos , Neurônios Aferentes/fisiologia , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Bexiga Urinária/metabolismoRESUMO
ABSTRACT: Endometriosis is a chronic and debilitating condition, commonly characterised by chronic pelvic pain (CPP) and infertility. Chronic pelvic pain can be experienced across multiple pelvic organs, with comorbidities commonly effecting the bowel, bladder, and vagina. Despite research efforts into endometriosis pathophysiology, little is known about how endometriosis induces CPP, and as such, therapeutic interventions are lacking. The aim of this study was to characterise a syngeneic mouse model of endometriosis that mimics naturally occurring retrograde menstruation, thought to precede endometriosis development in patients, and determine whether these mice exhibit signs of CPP and altered behaviour. We characterised the development of endometriosis over 10 weeks following uterine tissue inoculation, measured in vivo and ex vivo hypersensitivity to mechanical stimuli across multiple visceral organs, and assessed alterations in animal spontaneous behaviour. We confirmed that inoculated uterine horn tissue formed into endometriosis lesions throughout the peritoneal cavity, with significant growth by 8 to 10 weeks post inoculation. Additionally, we found that mice with fully developed endometriosis displayed hypersensitivity evoked by (1) vaginal distension, (2) colorectal distension, (3) bladder distension, and (4) cutaneous thermal stimulation, compared to their sham counterparts. Moreover, endometriosis mice displayed alterations in spontaneous behaviour indicative of (5) altered bladder function and (6) anxiety. This model creates a foundation for mechanistical studies into the diffuse CPP associated with endometriosis and the development of targeted therapeutic interventions to improve the quality of life of women with endometriosis.
Assuntos
Dor Crônica , Endometriose , Animais , Dor Crônica/complicações , Modelos Animais de Doenças , Endometriose/complicações , Feminino , Humanos , Camundongos , Dor Pélvica/diagnóstico , Qualidade de VidaRESUMO
Irritable bowel syndrome (IBS) is a chronic gastrointestinal disorder characterized by abdominal pain and altered bowel habit that affects ~11% of the global population. Over the past decade, preclinical and clinical studies have revealed a variety of novel mechanisms relating to the visceral analgesic effects of guanylate cyclase-C (GC-C) agonists. Here we discuss the mechanisms by which GC-C agonists target the GC-C/cyclic guanosine-3',5'-monophosphate (cGMP) pathway, resulting in visceral analgesia as well as clinically relevant relief of abdominal pain and other sensations in IBS patients. Due to the preponderance of evidence we focus on linaclotide, a 14-amino acid GC-C agonist with very low oral bioavailability that acts within the gut. Collectively, the weight of experimental and clinical evidence supports the concept that GC-C agonists act as peripherally acting visceral analgesics.
Assuntos
Dor Crônica , Agonistas da Guanilil Ciclase C , Síndrome do Intestino Irritável , Dor Visceral , Dor Abdominal/tratamento farmacológico , Dor Crônica/tratamento farmacológico , Guanilato Ciclase/metabolismo , Guanilato Ciclase/uso terapêutico , Agonistas da Guanilil Ciclase C/farmacologia , Agonistas da Guanilil Ciclase C/uso terapêutico , Humanos , Síndrome do Intestino Irritável/tratamento farmacológico , Dor Visceral/tratamento farmacológicoRESUMO
Three Orai (Orai1, Orai2, and Orai3) and two stromal interaction molecule (STIM1 and STIM2) mammalian protein homologues constitute major components of the store-operated Ca2+ entry mechanism. When co-expressed with STIM1, Orai1, Orai2 and Orai3 form highly selective Ca2+ channels with properties of Ca2+ release-activated Ca2+ (CRAC) channels. Despite the high level of homology between Orai proteins, CRAC channels formed by different Orai isoforms have distinctive properties, particularly with regards to Ca2+ -dependent inactivation, inhibition/potentiation by 2-aminoethyl diphenylborinate and sensitivity to reactive oxygen species. This study characterises and compares the regulation of Orai1, Orai2- and Orai3-mediated CRAC current (ICRAC ) by intracellular pH (pHi ). Using whole-cell patch clamping of HEK293T cells heterologously expressing Orai and STIM1, we show that ICRAC formed by each Orai homologue has a unique sensitivity to changes in pHi . Orai1-mediated ICRAC exhibits a strong dependence on pHi of both current amplitude and the kinetics of Ca2+ -dependent inactivation. In contrast, Orai2 amplitude, but not kinetics, depends on pHi , whereas Orai3 shows no dependence on pHi at all. Investigation of different Orai1-Orai3 chimeras suggests that pHi dependence of Orai1 resides in both the N-terminus and intracellular loop 2, and may also involve pH-dependent interactions with STIM1. KEY POINTS: It has been shown previously that Orai1/stromal interaction molecule 1 (STIM1)-mediated Ca2+ release-activated Ca2+ current (ICRAC ) is inhibited by intracellular acidification and potentiated by intracellular alkalinisation. The present study reveals that CRAC channels formed by each of the Orai homologues Orai1, Orai2 and Orai3 has a unique sensitivity to changes in intracellular pH (pHi ). The amplitude of Orai2 current is affected by the changes in pHi similarly to the amplitude of Orai1. However, unlike Orai1, fast Ca2+ -dependent inactivation of Orai2 is unaffected by acidic pHi . In contrast to both Orai1 and Orai2, Orai3 is not sensitive to pHi changes. Domain swapping between Orai1 and Orai3 identified the N-terminus and intracellular loop 2 as the molecular structures responsible for Orai1 regulation by pHi . Reduction of ICRAC dependence on pHi seen in a STIM1-independent Orai1 mutant suggested that some parts of STIM1 are also involved in ICRAC modulation by pHi .
Assuntos
Canais de Cálcio , Canais de Cálcio Ativados pela Liberação de Cálcio , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Proteína ORAI1/genética , Proteína ORAI2/metabolismo , Molécula 1 de Interação Estromal/metabolismoRESUMO
ABSTRACT: Abdominal pain is a key symptom of inflammatory bowel disease and irritable bowel syndrome, for which there are inadequate therapeutic options. We tested whether olorinab-a highly selective, full agonist of the cannabinoid receptor 2 (CB2)-reduced visceral hypersensitivity in models of colitis and chronic visceral hypersensitivity (CVH). In rodents, colitis was induced by intrarectal administration of nitrobenzene sulfonic acid derivatives. Control or colitis animals were administered vehicle or olorinab (3 or 30 mg/kg) twice daily by oral gavage for 5 days, starting 1 day before colitis induction. Chronic visceral hypersensitivity mice were administered olorinab (1, 3, 10, or 30 mg/kg) twice daily by oral gavage for 5 days, starting 24 days after colitis induction. Visceral mechanosensitivity was assessed in vivo by quantifying visceromotor responses (VMRs) to colorectal distension. Ex vivo afferent recordings determined colonic nociceptor firing evoked by mechanical stimuli. Colitis and CVH animals displayed significantly elevated VMRs to colorectal distension and colonic nociceptor hypersensitivity. Olorinab treatment significantly reduced VMRs to control levels in colitis and CVH animals. In addition, olorinab reduced nociceptor hypersensitivity in colitis and CVH states in a concentration- and CB2-dependent manner. By contrast, olorinab did not alter VMRs nor nociceptor responsiveness in control animals. Cannabinoid receptor 2 mRNA was detected in colonic tissue, particularly within epithelial cells, and dorsal root ganglia, with no significant differences between healthy, colitis, and CVH states. These results demonstrate that olorinab reduces visceral hypersensitivity through CB2 agonism in animal models, suggesting that olorinab may provide a novel therapy for inflammatory bowel disease- and irritable bowel syndrome-associated abdominal pain.
Assuntos
Colite , Síndrome do Intestino Irritável , Dor Visceral , Animais , Colite/induzido quimicamente , Colite/complicações , Colite/tratamento farmacológico , Colo , Modelos Animais de Doenças , Síndrome do Intestino Irritável/complicações , Síndrome do Intestino Irritável/tratamento farmacológico , Camundongos , Receptores de Canabinoides , Roedores , Dor Visceral/tratamento farmacológico , Dor Visceral/etiologiaRESUMO
The human nociceptor-specific voltage-gated sodium channel 1.7 (hNaV1.7) is critical for sensing various types of somatic pain, but it appears not to play a primary role in acute visceral pain. However, its role in chronic visceral pain remains to be determined. We used assay-guided fractionation to isolate a novel hNaV1.7 inhibitor, Tsp1a, from tarantula venom. Tsp1a is 28-residue peptide that potently inhibits hNaV1.7 (IC50 = 10 nM), with greater than 100-fold selectivity over hNaV1.3-hNaV1.6, 45-fold selectivity over hNaV1.1, and 24-fold selectivity over hNaV1.2. Tsp1a is a gating modifier that inhibits NaV1.7 by inducing a hyperpolarizing shift in the voltage-dependence of channel inactivation and slowing recovery from fast inactivation. NMR studies revealed that Tsp1a adopts a classical knottin fold, and like many knottin peptides, it is exceptionally stable in human serum. Remarkably, intracolonic administration of Tsp1a completely reversed chronic visceral hypersensitivity in a mouse model of irritable bowel syndrome. The ability of Tsp1a to reduce visceral hypersensitivity in a model of irritable bowel syndrome suggests that pharmacological inhibition of hNaV1.7 at peripheral sensory nerve endings might be a viable approach for eliciting analgesia in patients suffering from chronic visceral pain.
RESUMO
The rising opioid crisis has become a worldwide societal and public health burden, resulting from the abuse of prescription opioids. Targeting the κ-opioid receptor (KOR) in the periphery has emerged as a powerful approach to develop novel pain medications without central side effects. Inspired by the traditional use of sunflower (Helianthus annuus) preparations for analgesic purposes, we developed novel stabilized KOR ligands (termed as helianorphins) by incorporating different dynorphin A sequence fragments into a cyclic sunflower peptide scaffold. As a result, helianorphin-19 selectively bound to and fully activated the KOR with nanomolar potency. Importantly, helianorphin-19 exhibited strong KOR-specific peripheral analgesic activity in a mouse model of chronic visceral pain, without inducing unwanted central effects on motor coordination/sedation. Our study provides a proof of principle that cyclic peptides from plants may be used as templates to develop potent and stable peptide analgesics applicable via enteric administration by targeting the peripheral KOR for the treatment of chronic abdominal pain.
Assuntos
Dor Abdominal/tratamento farmacológico , Analgésicos/farmacologia , Peptídeos Cíclicos/farmacologia , Extratos Vegetais/farmacologia , Receptores Opioides kappa/antagonistas & inibidores , Analgésicos/síntese química , Analgésicos/química , Animais , Células Cultivadas , Doença Crônica , Relação Dose-Resposta a Droga , Desenho de Fármacos , Células HEK293 , Helianthus/química , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Extratos Vegetais/síntese química , Extratos Vegetais/química , Receptores Opioides kappa/metabolismo , Sementes/química , Relação Estrutura-AtividadeRESUMO
Chronic abdominal pain is a common clinical condition experienced by patients with irritable bowel syndrome (IBS). A general lack of suitable treatment options for the management of visceral pain is the major contributing factor to the debilitating nature of the disease. Understanding the underlying causes of chronic visceral pain is pivotal to identifying new effective therapies for IBS. This review provides the current evidence, demonstrating that mediators and receptors that induce itch in the skin also act as "gut irritants" in the gastrointestinal tract. Activation of these receptors triggers specific changes in the neuronal excitability of sensory pathways responsible for the transmission of nociceptive information from the periphery to the central nervous system leading to visceral hypersensitivity and visceral pain. Accumulating evidence points to significant roles of irritant mediators and their receptors in visceral hypersensitivity and thus constitutes potential targets for the development of more effective therapeutic options for IBS.
Assuntos
Colo/metabolismo , Hiperalgesia/metabolismo , Síndrome do Intestino Irritável/metabolismo , Dor Visceral/metabolismo , Histamina/metabolismo , Humanos , Mastócitos/metabolismoRESUMO
Endometriosis is a painful inflammatory disorder affecting ~10% of women of reproductive age. Although chronic pelvic pain (CPP) remains the main symptom of endometriosis patients, adequate treatments for CPP are lacking. Animal models that recapitulate the features and symptoms experienced by women with endometriosis are essential for investigating the etiology of endometriosis, as well as developing new treatments. In this study, we used an autologous mouse model of endometriosis to examine a combination of disease features and symptoms including: a 10 week time course of endometriotic lesion development; the chronic inflammatory environment and development of neuroangiogenesis within lesions; sensory hypersensitivity and altered pain responses to vaginal, colon, bladder, and skin stimulation in conscious animals; and spontaneous animal behavior. We found significant increases in lesion size from week 6 posttransplant. Lesions displayed endometrial glands, stroma, and underwent neuroangiogenesis. Additionally, peritoneal fluid of mice with endometriosis contained known inflammatory mediators and angiogenic factors. Compared to Sham, mice with endometriosis displayed: enhanced sensitivity to pain evoked by (i) vaginal and (ii) colorectal distension, (iii) altered bladder function and increased sensitivity to cutaneous (iv) thermal and (v) mechanical stimuli. The development of endometriosis had no effect on spontaneous behavior. This study describes a comprehensive characterization of a mouse model of endometriosis, recapitulating the clinical features and symptoms experienced by women with endometriosis. Moreover, it delivers the groundwork to investigate the etiology of endometriosis and provides a platform for the development of therapeutical interventions to manage endometriosis-associated CPP.
Assuntos
Doenças do Colo/etiologia , Endometriose/patologia , Dermatopatias/etiologia , Doenças da Bexiga Urinária/etiologia , Doenças Vaginais/etiologia , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Feminino , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora , DorRESUMO
Understanding the sensory mechanisms innervating the bladder is paramount to developing efficacious treatments for chronic bladder hypersensitivity conditions. The contribution of Mas-gene-related G protein-coupled receptors (Mrgpr) to bladder signaling is currently unknown. Using male and female mice, we show with single-cell RT-PCR that subpopulations of DRG neurons innervating the mouse bladder express MrgprA3 (14%) and MrgprC11 (38%), either individually or in combination, with high levels of coexpression with Trpv1 (81%-89%). Calcium imaging studies demonstrated MrgprA3 and MrgprC11 agonists (chloroquine, BAM8-22, and neuropeptide FF) activated subpopulations of bladder-innervating DRG neurons, showing functional evidence of coexpression between MrgprA3, MrgprC11, and TRPV1. In ex vivo bladder-nerve preparations, chloroquine, BAM8-22, and neuropeptide FF all evoked mechanical hypersensitivity in subpopulations (20%-41%) of bladder afferents. These effects were absent in recordings from Mrgpr-clusterΔ-/- mice. In vitro whole-cell patch-clamp recordings showed that application of an MrgprA3/C11 agonist mixture induced neuronal hyperexcitability in 44% of bladder-innervating DRG neurons. Finally, in vivo instillation of an MrgprA3/C11 agonist mixture into the bladder of WT mice induced a significant activation of dorsal horn neurons within the lumbosacral spinal cord, as quantified by pERK immunoreactivity. This MrgprA3/C11 agonist-induced activation was particularly apparent within the superficial dorsal horn and the sacral parasympathetic nuclei of WT, but not Mrgpr-clusterΔ-/- mice. This study demonstrates, for the first time, functional expression of MrgprA3 and MrgprC11 in bladder afferents. Activation of these receptors triggers hypersensitivity to distension, a critically valuable factor for therapeutic target development.SIGNIFICANCE STATEMENT Determining how bladder afferents become sensitized is the first step in finding effective treatments for common urological disorders such as overactive bladder and interstitial cystitis/bladder pain syndrome. Here we show that two of the key receptors, MrgprA3 and MrgprC11, that mediate itch from the skin are also expressed on afferents innervating the bladder. Activation of these receptors results in sensitization of bladder afferents, resulting in sensory signals being sent into the spinal cord that prematurely indicate bladder fullness. Targeting bladder afferents expressing MrgprA3 or MrgprC11 and preventing their sensitization may provide a novel approach for treating overactive bladder and interstitial cystitis/bladder pain syndrome.