Integration of nutritional, microbial and inflammatory events along the gut-brain axis can alter bowel physiology and organism behaviour. Colonic sensory neurons activate reflex pathways and give ...rise to conscious sensation, but the diversity and division of function within these neurons is poorly understood. The identification of signalling pathways contributing to visceral sensation is constrained by a paucity of molecular markers. Here we address this by comprehensive transcriptomic profiling and unsupervised clustering of individual mouse colonic sensory neurons.
Unbiased single-cell RNA-sequencing was performed on retrogradely traced mouse colonic sensory neurons isolated from both thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglia associated with lumbar splanchnic and pelvic spinal pathways, respectively. Identified neuronal subtypes were validated by single-cell qRT-PCR, immunohistochemistry (IHC) and Ca
-imaging.
Transcriptomic profiling and unsupervised clustering of 314 colonic sensory neurons revealed seven neuronal subtypes. Of these, five neuronal subtypes accounted for 99% of TL neurons, with LS neurons almost exclusively populating the remaining two subtypes. We identify and classify neurons based on novel subtype-specific marker genes using single-cell qRT-PCR and IHC to validate subtypes derived from RNA-sequencing. Lastly, functional Ca
-imaging was conducted on colonic sensory neurons to demonstrate subtype-selective differential agonist activation.
We identify seven subtypes of colonic sensory neurons using unbiased single-cell RNA-sequencing and confirm translation of patterning to protein expression, describing sensory diversity encompassing all modalities of colonic neuronal sensitivity. These results provide a pathway to molecular interrogation of colonic sensory innervation in health and disease, together with identifying novel targets for drug development.
Dysmenorrhea is a common chronic pelvic pain syndrome affecting women of childbearing potential. Family studies suggest that genetic background influences the severity of dysmenorrhea, but genetic ...predisposition and molecular mechanisms underlying dysmenorrhea are not understood. In this study, we conduct the first genome-wide association study to identify genetic factors associated with dysmenorrhea pain severity. A cohort of females of European descent (n = 11,891) aged 18 to 45 years rated their average dysmenorrhea pain severity. We used a linear regression model adjusting for age and body mass index, identifying one genome-wide significant (P < 5 × 10) association (rs7523086, P = 4.1 × 10, effect size 0.1 95% confidence interval, 0.074-0.126). This single nucleotide polymorphism is colocalising with NGF, encoding nerve growth factor. The presence of one risk allele corresponds to a predicted 0.1-point increase in pain intensity on a 4-point ordinal pain scale. The putative effects on NGF function and/or expression remain unknown. However, genetic variation colocalises with active epigenetic marks in fat and ovary tissues, and expression levels in aorta tissue of a noncoding RNA flanking NGF correlate. Participants reporting extreme dysmenorrhea pain were more likely to report being positive for endometriosis, polycystic ovarian syndrome, depression, and other psychiatric disorders. Our results indicate that dysmenorrhea pain severity is partly genetically determined. NGF already has an established role in chronic pain disorders, and our findings suggest that NGF may be an important mediator for gynaecological/pelvic pain in the viscera.
Agonists at the µ-opioid receptor are known to produce potent analgesic responses in the clinical setting, therefore, an increased understanding of the molecular interactions of ligands at this ...receptor could lead to improved analgesics. As historically morphine has been shown to be a poor recruiter of β-arrestin in recombinant cell systems and this can be overcome by the co-expression of GRK2, we investigated the effects of GRK2 co-expression, in a recombinant µ-opioid receptor cell line, on ligand affinity and intrinsic activity in both β-arrestin recruitment and (35)SGTPγS binding assays. We also investigated the effect of receptor depletion in the β-arrestin assay. GRK2 co-expression increased both agonist Emax and potency in the β-arrestin assay. The increase in agonist potency could not be reversed using receptor depletion, supporting that the effects were due to a novel receptor conformation not system amplification. We also observed a small but significant effect on agonist KL values. Potency values in the (35)SGTPγS assay were unchanged; however, inverse agonist activity became evident with GRK2 co-expression. We conclude that this is direct evidence that the µ-opioid receptor is an allosteric protein and the co-expression of signalling molecules elicits changes in its conformation and thus ligand affinity. This has implications when describing how ligands interact with the receptor and how efficacy is determined.
Patients with overactive bladder often exhibit abnormal bladder contractions in response to intravesical cold saline (positive ice-water test). The molecular entity involved in cold sensation within ...the urinary bladder is unknown, but a potential candidate is the ion channel, transient receptor potential (melastatin)-8 (TRPM8). The objective of the present study was to investigate the role of TRPM8 in a bladder-cooling reflex evoked in anaesthetised guinea-pigs that is comparable to the positive ice-water test seen in patients. Guinea-pig TRPM8 was cloned from L6 dorsal root ganglia (DRG) and expressed in HEK293 cells. Functional agonist- and cold-induced Ca2+ influx and electrophysiology assays were performed in these cells, and for comparison in HEK293 cells expressing human TRPM8, using a novel TRPM8 antagonist, the S-enantiomer of 1-phenylethyl 4-(benzyloxy)-3-methoxybenzyl (2-aminoethyl) carbamate hydrochloride (PBMC). Potency data from these assays was used to calculate intravenous infusion protocols for targeted plasma concentrations of PBMC in studies on micturition reflexes evoked by intravesical infusion of menthol or cold saline in anaesthetised guinea-pigs. Tissue expression of TRPM8 in guinea-pig bladder, urethra and in dorsal root ganglia neurones traced from the bladder was also investigated. TRPM8 mRNA and protein were detected in L6 dorsal root ganglia, bladder urothelium and smooth muscle. PBMC antagonised in vitro activation of human and guinea-pig TRPM8 and reversed menthol and cold-induced facilitation of the micturition reflex at plasma concentrations consistent with in vitro potencies. The present data suggest that the bladder-cooling reflex in the guinea-pig involves TRPM8. The potential significance of TRPM8 in bladder disease states deserves future investigation.
Several populations of interstitial cells of Cajal (ICC) exist in the bladder, associated with intramural nerves. Although ICC respond to exogenous agonists, there is currently no evidence of their ...functional innervation. The objective was to determine whether bladder ICC are functionally innervated. Guinea-pig bladder tissues, loaded with fluo-4AM were imaged with fluorescent microscopy and challenged with neurogenic electrical field stimulation (EFS). All subtypes of ICC and smooth muscle cells (SMC) displayed spontaneous Ca(2+)-oscillations. EFS (0.5 Hz, 2 Hz, 10 Hz) evoked tetrodotoxin (1 µM)-sensitive Ca(2+)-transients in lamina propria ICC (ICC-LP), detrusor ICC and perivascular ICC (PICC) associated with mucosal microvessels. EFS responses in ICC-LP were significantly reduced by atropine or suramin. SMC and vascular SMC (VSM) also responded to EFS. Spontaneous Ca(2+)-oscillations in individual ICC-LP within networks occurred asynchronously whereas EFS evoked coordinated Ca(2+)-transients in all ICC-LP within a field of view. Non-correlated Ca(2+)-oscillations in detrusor ICC and adjacent SMC pre-EFS, contrasted with simultaneous neurogenic Ca(2+) transients evoked by EFS. Spontaneous Ca(2+)-oscillations in PICC were little affected by EFS, whereas large Ca(2+)-transients were evoked in pre-EFS quiescent PICC. EFS also increased the frequency of VSM Ca(2+)-oscillations. In conclusion, ICC-LP, detrusor ICC and PICC are functionally innervated. Interestingly, Ca(2+)-activity within ICC-LP networks and between detrusor ICC and their adjacent SMC were synchronous under neural control. VSM and PICC Ca(2+)-activity was regulated by bladder nerves. These novel findings demonstrate functional neural control of bladder ICC. Similar studies should now be carried out on neurogenic bladder to elucidate the contribution of impaired nerve-ICC communication to bladder pathophysiology.
There are several conditions associated with dysfunction of the lower urinary tract or which result in a reduction in the ability to engage in satisfactory sexual function and result in significant ...bother to sufferers, partners and/or carers. This review describes some of the animal models that may be used to discover safe and effective medicines with which to treat them. While alpha adrenoceptor antagonists and 5‐alpha‐reductase inhibitors deliver improvement in symptom relief in benign prostatic hyperplasia sufferers, the availability of efficacious and well‐tolerated medicines to treat incontinence is less well served. Stress urinary incontinence (SUI) has no approved medical therapy in the United States and overactive bladder (OAB) therapy is limited to treatment with muscarinic antagonists (anti‐muscarinics). SUI and OAB are characterised by high prevalence, a growing ageing population and a strong desire from sufferers and physicians for more effective treatment options. High patient numbers with low presentation rates characterizes sexual dysfunction in men and women. The introduction of Viagra™ in 1998 for treating male erectile dysfunction and the success of the phosphodiesterase type 5 inhibitor class (PDE5 inhibitor) have indicated the willingness of sufferers to seek treatment when an effective alternative to injections and devices is available. The main value of preclinical models in discovering new medicines is to predict clinical outcomes. This translation can be established relatively easily in areas of medicine where there are a large number of drugs with different underlying pharmacological mechanisms in clinical usage. However, apart from, for example, the use of PDE5 inhibitors to treat male erectile dysfunction and the use of anti‐muscarinics to treat OAB, this clinical information is limited. Therefore, current confidence in existing preclinical models is based on our understanding of the biochemical, physiological, pathophysiological and psychological mechanisms underlying the conditions in humans and how they are reflected in preclinical models. Confidence in both the models used and the pharmacological data generated is reinforced if different models of related aspects of the same disorder generate confirmatory data. However, these models will only be fully validated in retrospect once the pharmacological agents they have helped identify are tested in humans.
British Journal of Pharmacology (2006) 147, S62–S79. doi:10.1038/sj.bjp.0706630
What's known on the subject? and What does the study add?
Detrusor underactivity is highly prevalent, particularly in the elderly. It is assumed to result from detrusor failure, although detrusor ...contractility is often derived from urodynamics studies. Given that detrusor pressure and force are not proportional and urodynamics cannot identify the basis of the pathology, we produced a neurogenic animal model with a highly‐compliant bladder and studied detrusor muscle properties, aiming to increase our understanding of the underlying pathology.
Highly compliant bladders were characterized by reduced passive wall stiffness and stretched detrusor muscle strips exhibited an enhanced rate of relaxation. These detrusor strips displayed spontaneous contractions that were of greater amplitude (expressed as a ratio of bladder wall stiffness) than those of strips from sham‐operated animals; spontaneous contractions increased in amplitude when stimulated by an agonist. These data imply that compliance is not the result of a reduction of detrusor contractility; we hypothesize that altered matrix properties reduce the magnitude with which force can be generated to void the bladder.
Objective
To characterize passive and active changes in detrusor activity in a highly compliant bladder.
Materials and Methods
Bladders from adult female Sprague–Dawley rats were used 5 weeks after lower thoracic (T8) spinal cord transection or a sham‐operation.
Passive wall properties were assessed by pressure–volume relationships from whole bladders and the tensile response of bladder strips after a rapid (<0.5 s) stretch.
Active properties were assessed from the frequency and amplitude of spontaneous contractions of bladder strips, and their response to the inotropic TRPV4 agonist GSK1016790A.
Results
Passive bladder wall stiffness of SCT bladders was significantly reduced compared to that of the sham‐operated control group (N = 6 and 8, respectively) and SCT bladder strips relaxed more quickly than those from sham‐operated rats.
The frequency of spontaneous contractions was reduced in SCT rats, and their amplitude, expressed as a ratio of bladder wall stiffness, was greater than in sham‐operated rats.
GSK1016790A (0.1 μM) significantly increased amplitude in strips from both sham‐operated and SCT groups.
Conclusions
There is no evidence of contractile failure in a highly‐compliant bladder. The observations of reduced passive bladder wall stiffness and an enhanced rate of stress relaxation lead to the conclusion that increased compliance is marked by altered matrix properties that dissipate muscle force, thereby generating low pressures.
Contractile agonists may be effective for improving bladder function in detrusor underactivity.
Idiopathic detrusor instability (IDI) is a common cause of lower urinary tract storage symptoms, such as urgency, frequency and urge incontinence. We have investigated the in vitro properties and ...pattern of innervation of the detrusor from patients with this condition.
Full thickness bladder specimens were obtained perioperatively from 14 patients with IDI and from 14 cadaveric controls undergoing transplant organ retrieval. Isolated detrusor smooth muscle strips were mounted in organ baths for isometric tension recording. Frequency-response curves to electrical field stimulation (EFS) (1 Hz to 50 Hz) and concentration response curves for carbachol (10
−7 M to 5 × 10
−4 M) and potassium (12 mM to 120 mM) were constructed. Acetylcholinesterase histochemistry and immunohistochemistry for both phosphorylated and non-phosphorylated neurofilaments was carried out on frozen sections of control and IDI bladders.
IDI strips developed greater spontaneous tone (0.25 gm./mg. versus 0.12 gm./mg.; p <0.0001) and more spontaneous fused tetanic contractions (16.8% versus 6.8%; p <0.005) during an initial 90 minutes equilibration period. The IDI strips were less responsive than controls to nerve stimulation (max. response to EFS 0.79 gm./mg. versus 1.23 gm./mg.; p <0.0001) and were supersensitive to potassium (EC
50 39.7 mM versus 45.7 mM; p = 0.003) but not to carbachol (EC
50 7.3 × 10
−6 M versus 6.6 × 10
−6 M; p = 0.48). Morphometric studies revealed reduced staining of presumed cholinergic nerves, with 34.7% of IDI smooth muscle bundles appearing denervated compared with 1.5% of controls (p <0.0001).
Our study supports the notion that there is a fundamental abnormality in IDI at the level of the bladder wall, with evidence of altered spontaneous contractile activity consistent with an increased electrical coupling of cells, a patchy denervation of the detrusor and a potassium supersensitivity.
The development of G protein-biased agonists for the μ-opioid receptor (MOR) offers a clear drug discovery rationale for improved analgesia and reduced side-effects of opiate pharmacotherapy. ...However, our understanding of the molecular mechanisms governing ligand bias is limited, which hinders our ability to rationally design biased compounds. We have investigated the role of MOR binding site residues W320 and Y328 in controlling bias, by receptor mutagenesis. The pharmacology of a panel of ligands in a cAMP and a β-arrestin2 assay were compared between the wildtype and mutated receptors, with bias factors calculated by operational analysis using ΔΔlog(τ/KA) values. 3Hdiprenorphine competition binding was used to estimate affinity changes. Introducing the mutations W320A and Y328F caused changes in pathway bias, with different patterns of change between ligands. For example, DAMGO increased relative β-arrestin2 activity at the W320A mutant, whilst its β-arrestin2 response was completely lost at Y328F. In contrast, endomorphin-1 gained activity with Y328F but lost activity at W320A, in both pathways. For endomorphin-2 there was a directional shift from cAMP bias at the wildtype towards more β-arrestin2 bias at W320A. We also observe clear uncoupling between mutation-driven changes in function and binding affinity. These findings suggest that the mutations influenced the balance of pathway activation in a ligand-specific manner, thus identifying residues in the MOR binding pocket that govern ligand bias. This increases our understanding of how ligand/receptor binding interactions can be translated into agonist-specific pathway activation.
•Mutating μ-opioid receptor binding site changes ligand bias.•Ligand-specific patterns of change were observed across mutations.•Residues W320A and Y328F qualitatively control how receptor is activated.•This gains insights into how binding is decoded into pathway-specific receptor activation.
Key points
Voltage‐gated sodium channels play a fundamental role in determining neuronal excitability.
Specifically, voltage‐gated sodium channel subtype NaV1.7 is required for sensing acute and ...inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown.
Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling.
These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality‐specific manner and help to direct drug discovery efforts towards novel visceral analgesics.
Voltage‐gated sodium channel NaV1.7 is required for acute and inflammatory pain in mice and humans but its significance for visceral pain is unknown. Here we examine the role of NaV1.7 in visceral pain processing and the development of referred hyperalgesia using a conditional nociceptor‐specific NaV1.7 knockout mouse (NaV1.7Nav1.8) and selective small‐molecule NaV1.7 antagonist PF‐5198007. NaV1.7Nav1.8 mice showed normal nociceptive behaviours in response to intracolonic application of either capsaicin or mustard oil, stimuli known to evoke sustained nociceptor activity and sensitization following tissue damage, respectively. Normal responses following induction of cystitis by cyclophosphamide were also observed in both NaV1.7Nav1.8 and littermate controls. Loss, or blockade, of NaV1.7 did not affect afferent responses to noxious mechanical and chemical stimuli in nerve–gut preparations in mouse, or following antagonism of NaV1.7 in resected human appendix stimulated by noxious distending pressures. However, expression analysis of voltage‐gated sodium channel α subunits revealed NaV1.7 mRNA transcripts in nearly all retrogradely labelled colonic neurons, suggesting redundancy in function. By contrast, using comparative somatic behavioural models we identify that genetic deletion of NaV1.7 (in NaV1.8‐expressing neurons) regulates noxious heat pain threshold and that this can be recapitulated by the selective NaV1.7 antagonist PF‐5198007. Our data demonstrate that NaV1.7 (in NaV1.8‐expressing neurons) contributes to defined pain pathways in a modality‐dependent manner, modulating somatic noxious heat pain, but is not required for visceral pain processing, and advocate that pharmacological block of NaV1.7 alone in the viscera may be insufficient in targeting chronic visceral pain.
Key points
Voltage‐gated sodium channels play a fundamental role in determining neuronal excitability.
Specifically, voltage‐gated sodium channel subtype NaV1.7 is required for sensing acute and inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown.
Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling.
These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality‐specific manner and help to direct drug discovery efforts towards novel visceral analgesics.
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