STIM1 is a core trigger of airway smooth muscle remodeling and hyperresponsiveness in asthma.

Airway remodeling and airway hyperresponsiveness are central drivers of asthma severity. Airway remodeling is a structural change involving the dedifferentiation of airway smooth muscle (ASM) cells from a quiescent to a proliferative and secretory phenotype. Here, we show up-regulation of the endoplasmic reticulum Ca2+ sensor stromal-interacting molecule 1 (STIM1) in ASM of asthmatic mice. STIM1 is required for metabolic and transcriptional reprogramming that supports airway remodeling, including ASM proliferation, migration, secretion of cytokines and extracellular matrix, enhanced mitochondrial mass, and increased oxidative phosphorylation and glycolytic flux. Mechanistically, STIM1-mediated Ca2+ influx is critical for the activation of nuclear factor of activated T cells 4 and subsequent interleukin-6 secretion and transcription of pro-remodeling transcription factors, growth factors, surface receptors, and asthma-associated proteins. STIM1 drives airway hyperresponsiveness in asthmatic mice through enhanced frequency and amplitude of ASM cytosolic Ca2+ oscillations. Our data advocates for ASM STIM1 as a target for asthma therapy.

Long-term follow-up of TREK-1 KO mice reveals the development of bladder hypertrophy and impaired bladder smooth muscle contractility with age.

Stretch-activated two-pore domain K+ (K2P) channels play important roles in many visceral organs, including the urinary bladder. The TWIK-related K+ channel TREK-1 is the predominantly expressed K2P channel in the urinary bladder of humans and rodents. Downregulation of TREK-1 channels was observed in the urinary bladder of patients with detrusor overactivity, suggesting their involvement in the pathogenesis of voiding dysfunction. This study aimed to characterize the long-term effects of TREK-1 on bladder function with global and smooth muscle-specific TREK-1 knockout (KO) mice. Bladder morphology, bladder smooth muscle (BSM) contractility, and voiding patterns were evaluated up to 12 mo of age. Both sexes were included in this study to probe the potential sex differences. Smooth muscle-specific TREK-1 KO mice were used to distinguish the effects of TREK-1 downregulation in BSM from the neural pathways involved in the control of bladder contraction and relaxation. TREK-1 KO mice developed enlarged urinary bladders (by 60.0% for males and by 45.1% for females at 6 mo; P < 0.001 compared with the age-matched control group) and had a significantly increased bladder capacity (by 137.7% at 12 mo; P < 0.0001) and compliance (by 73.4% at 12 mo; P < 0.0001). Bladder strips isolated from TREK-1 KO mice exhibited decreased contractility (peak force after KCl at 6 mo was 1.6 ± 0.7 N/g compared with 3.4 ± 2.0 N/g in the control group; P = 0.0005). The lack of TREK-1 channels exclusively in BSM did not replicate the bladder phenotype observed in TREK-1 KO mice, suggesting a strong neurogenic origin of TREK-1-related bladder dysfunction.NEW & NOTEWORTHY This study compared voiding function and bladder phenotypes in global and smooth muscle-specific TREK-1 KO mice. We found significant age-related changes in bladder contractility, suggesting that the lack of TREK-1 channel activity might contribute to age-related changes in bladder smooth muscle physiology.

Inhibition of human prostate smooth muscle contraction by the inhibitors of protein kinase C, GF109203X, and Go6983.

INTRODUCTION: Prostate smooth muscle contraction is promoted by receptor-induced activation of intracellular signaling pathways. The presumed involvement in etiology and medical treatment of lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH) imparts a high clinical relevance to prostate smooth muscle contraction, which is contrasted by incomplete understanding at the molecular level. Involvement of protein kinase C (PKC) has been commonly assumed, but available studies were limited to nonhuman prostate smooth muscle or cell cultures. Here, we examined the effects of the PKC inhibitors Go6983 and GF109203x on contractions of human prostate tissues. METHODS: Prostate tissues were obtained from radical prostatectomy. Contractions were induced by electric field stimulation (EFS), α1 -adrenergic agonists (noradrenaline, phenylephrine, methoxamine), thromboxane A2 analog U46619, endothelin-1, or calcium chloride in an organ bath. RESULTS: GF109203X (500 nM) and Go6983 (300 nM) reduced EFS-, noradrenaline-, phenylephrine-, methoxamine-, and U46619-induced contractions of human prostate tissues, with maximum inhibitions approaching up to 55%. Using concentrations of 3 µM, GF109203X and Go6983 inhibited EFS- and noradrenaline-induced contractions, with similar effect sizes as 500 and 300 nM, respectively. Endothelin-1-induced contractions were not inhibited by GF109203X, and to neglectable extent by Go6983. After depolarization in calcium-free solution, calcium chloride-induced concentration-dependent contractions, which were inhibited by GF109203X and Go6983. CONCLUSIONS: GF109203X and Go6983 inhibit neurogenic, α1 -adrenergic, and thromboxane A2 -induced smooth muscle contractions in the human prostate, suggesting a role of PKC for human prostate smooth muscle contraction. The inhibition may by be imparted by inhibition of calcium sensitivity.

Gastrointestinal dysfunction in autism displayed by altered motility and achalasia in Foxp1+/- mice.

Gastrointestinal dysfunctions in individuals with autism spectrum disorder are poorly understood, although they are common among this group of patients. FOXP1 haploinsufficiency is characterized by autistic behavior, language impairment, and intellectual disability, but feeding difficulties and gastrointestinal problems have also been reported. Whether these are primary impairments, the result of altered eating behavior, or side effects of psychotropic medication remains unclear. To address this question, we investigated Foxp1+/- mice reflecting FOXP1 haploinsufficiency. These animals show decreased body weight and altered feeding behavior with reduced food and water intake. A pronounced muscular atrophy was detected in the esophagus and colon, caused by reduced muscle cell proliferation. Nitric oxide-induced relaxation of the lower esophageal sphincter was impaired and achalasia was confirmed in vivo by manometry. Foxp1 targets (Nexn, Rbms3, and Wls) identified in the brain were dysregulated in the adult Foxp1+/- esophagus. Total gastrointestinal transit was significantly prolonged due to impaired colonic contractility. Our results have uncovered a previously unknown dysfunction (achalasia and impaired gut motility) that explains the gastrointestinal disturbances in patients with FOXP1 syndrome, with potential wider relevance for autism.

Airway smooth muscle pathophysiology in asthma.

The airway smooth muscle (ASM) cell plays a central role in the pathogenesis of asthma and constitutes an important target for treatment. These cells control muscle tone and thus regulate the opening of the airway lumen and air passage. Evidence indicates that ASM cells participate in the airway hyperresponsiveness as well as the inflammatory and remodeling processes observed in asthmatic subjects. Therapeutic approaches require a comprehensive understanding of the structure and function of the ASM in both the normal and disease states. This review updates current knowledge about ASM and its effects on airway narrowing, remodeling, and inflammation in asthma.

Prostate-to-bladder cross-sensitization in a model of zymosan-induced chronic pelvic pain syndrome in rats.

BACKGROUND: The aim of the present study was to investigate the effects of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) on bladder function and pathophysiology. METHODS: To create a model for CPPS, rats were intraprostatically injected with zymosan or saline, serving as control. Metabolic cage experiments were performed 7, 14, or 21 days after zymosan injection and after 14 days in the control group. Thereafter, cystometry was performed in which simulated micturition cycles were induced by saline infusion and contractile responses to the cholinergic agonist methacholine and the purinergic agonist ATP were measured. Following cystometry, the prostate and urinary bladder were excised and assessed histopathologically for possible inflammatory changes. RESULTS: Metabolic cage data revealed a significantly increased urinary frequency in zymosan treated rats. Likewise, the volume per micturition was significantly lower in all CPPS groups compared to controls. Cystometry showed a significant increase in the number of nonvoiding contractions, longer voiding time, and a trend towards lower compliance in CPPS rats compared to controls. Induction of CPPS led to significantly reduced cholinergic and purinergic contractile responses. Histopathological analysis demonstrated prostatic inflammation in all CPPS groups, in particular in later stage groups. Both the extent and grade of bladder inflammation were significantly higher in CPPS groups compared to controls. CONCLUSIONS: The current findings demonstrate a potential prostate-to-bladder cross-sensitization leading to symptoms of bladder overactivity and signs of bladder inflammation. Future clinical studies are required to verify the outcomes of the current study and enable advancement of patient care.

Investigating Helicobacter pylori-related pyloric hypomotility: functional, histological, and molecular alterations.

Multiple theories have been proposed describing the pathogenic mechanisms of Helicobacter pylori (H. pylori)-associated gastric motility disorders. We assessed ex vivo pyloric activity in H. pylori-infected rats, and tried to explore the associated ghrelin hormone alteration and pyloric fibrogenesis. In addition, miR-1 was assessed in pyloric tissue samples, being recently accused of having a role in smooth muscle dysfunction. Ninety adult male Wistar albino rats were assigned into nine groups: 1) control group, 2) sterile broth (vehicle group), 3) amoxicillin control, 4) omeperazole control, 5) clarithromycin control, 6) triple therapy control, 7) H. pylori- group, 8) H. pylori-clarithromycin group, and 9) H. pylori-triple therapy group. Urease enzyme activity was applied as an indicator of H. pylori infection. Ex vivo pyloric contractility was evaluated. Serum ghrelin was assessed, and histological tissue evaluation was performed. Besides, pyloric muscle miR-1 expression was measured. The immunological epithelial to mesenchymal transition (EMT) markers; transforming growth factor ß (TGFß), α-smooth muscle actin (α-SMA), and E-cadherin-3 were also evaluated. By H. pylori infection, a significant (P < 0.001) reduced pyloric contractility index was recorded. The miR-1 expression was decreased (P < 0.001) in the H. pylori-infected group, associated with reduced serum ghrelin, elevated TGFß, and α-SMA levels and reduced E-cadherin levels. Decreased miR-1 and disturbed molecular pattern were improved by treatment. In conclusion, H. pylori infection was associated with reduced miR-1, epithelial to mesenchymal transition, and pyloric hypomotility. The miR-1 may be a target for further studies to assess its possible involvement in H. pylori-associated pyloric dysfunction, which might help in the management of human H. pylori manifestations and complications.NEW & NOTEWORTHY This work is investigating functional, histopathological, and molecular changes underlying Helicobacter pylori hypomotility and is correlating these with miR-1, whose disturbance is supposed to be involved in smooth muscle dysfunction and cell proliferation according to literature. Epithelial to mesenchymal transition and reduced ghrelin hormone may contribute to H. pylori infection-associated hypomotility. H. pylori infection was associated with reduced pyloric miR-1 expression. Targeting miR-1 could be valuable in the clinical management of pyloric hypofunction.

Nerve transfer for restoration of lower motor neuron-lesioned bladder function. Part 2: correlation between histological changes and nerve evoked contractions.

We determined the effect of pelvic organ decentralization and reinnervation 1 yr later on urinary bladder histology and function. Nineteen canines underwent decentralization by bilateral transection of all coccygeal and sacral (S) spinal roots, dorsal roots of lumbar (L)7, and hypogastric nerves. After exclusions, eight were reinnervated 12 mo postdecentralization with obturator-to-pelvic and sciatic-to-pudendal nerve transfers, then euthanized 8-12 mo later. Four served as long-term decentralized only animals. Before euthanasia, pelvic or transferred nerves and L1-S3 spinal roots were stimulated and maximum detrusor pressure (MDP) recorded. Bladder specimens were collected for histological and ex vivo smooth muscle contractility studies. Both reinnervated and decentralized animals showed less or denuded urothelium, fewer intramural ganglia, and more inflammation and collagen, than controls, although percent muscle was maintained. In reinnervated animals, pgp9.5+ axon density was higher compared with decentralized animals. Ex vivo smooth muscle contractions in response to KCl correlated positively with submucosal inflammation, detrusor muscle thickness, and pgp9.5+ axon density. In vivo, reinnervated animals showed higher MDP after stimulation of L1-L6 roots compared with their transected L7-S3 roots, and reinnervated and decentralized animals showed lower MDP than controls after stimulation of nerves (due likely to fibrotic nerve encapsulation). MDP correlated negatively with detrusor collagen and inflammation, and positively with pgp9.5+ axon density and intramural ganglia numbers. These results demonstrate that bladder function can be improved by transfer of obturator nerves to pelvic nerves at 1 yr after decentralization, although the fibrosis and inflammation that developed were associated with decreased contractile function.

Constitutive LH receptor activity impairs NO-mediated penile smooth muscle relaxation.

Timely activation of the luteinizing hormone receptor (LHCGR) is critical for fertility. Activating mutations in LHCGR cause familial male-limited precocious puberty (FMPP) due to premature synthesis of testosterone. A mouse model of FMPP (KiLHRD582G), expressing a constitutively activating mutation in LHCGR, was previously developed in our laboratory. KiLHRD582G mice became progressively infertile due to sexual dysfunction and exhibited smooth muscle loss and chondrocyte accumulation in the penis. In this study, we tested the hypothesis that KiLHRD582G mice had erectile dysfunction due to impaired smooth muscle function. Apomorphine-induced erection studies determined that KiLHRD582G mice had erectile dysfunction. Penile smooth muscle and endothelial function were assessed using penile cavernosal strips. Penile endothelial cell content was not changed in KiLHRD582G mice. The maximal relaxation response to acetylcholine and the nitric oxide donor, sodium nitroprusside, was significantly reduced in KiLHRD582G mice indicating an impairment in the nitric oxide (NO)-mediated signaling. Cyclic GMP (cGMP) levels were significantly reduced in KiLHRD582G mice in response to acetylcholine, sodium nitroprusside and the soluble guanylate cyclase stimulator, BAY 41-2272. Expression of NOS1, NOS3 and PKRG1 were unchanged. The Rho-kinase signaling pathway for smooth muscle contraction was not altered. Together, these data indicate that KiLHRD582G mice have erectile dysfunction due to impaired NO-mediated activation of soluble guanylate cyclase resulting in decreased levels of cGMP and penile smooth muscle relaxation. These studies in the KiLHRD582G mice demonstrate that activating mutations in the mouse LHCGR cause erectile dysfunction due to impairment of the NO-mediated signaling pathway in the penile smooth muscle.

7,8-Dihydroxyflavone Enhanced Colonic Cholinergic Contraction and Relieved Loperamide-Induced Constipation in Rats.

BACKGROUND: Whether 7,8-dihydroxyflavone (7,8-DHF), a tyrosine kinase receptor B (TrkB) agonist, modulates colonic smooth muscle motility and/or alleviates constipation has not yet been studied. AIMS: Here, we aimed to determine how 7,8-DHF influences carbachol (CCh)-stimulated contraction of colonic strips and the in vivo effect of 7,8-DHF on constipation. METHODS: Muscle strips were isolated from rat colons for recording contractile tension and performing western blotting. Constipation was induced in rats with loperamide. RESULTS: Although it specifically activated TrkB, 7,8-DHF applied alone neither activated PLCγ1 in the colonic strips nor induced colonic strip contraction. However, 7,8-DHF enhanced CCh-stimulated PLCγ1 activation and strip contraction. The PLCγ1 antagonist U73122 suppressed both CCh-stimulated and 7,8-DHF-enhanced/CCh-stimulated contraction. While clarifying the underlying mechanism, we revealed that 7,8-DHF augmented muscarinic M3 receptor expression in the colonic strips. The M3-selective antagonist tarafenacin specifically inhibited the 7,8-DHF-enhanced/CCh-stimulated contraction of the colonic strips. Since 7,8-DHF increased Akt phosphorylation, and LY294002 (an antagonist of PI3K upstream of Akt) dramatically inhibited both 7,8-DHF-augmented M3 expression and 7,8-DHF-enhanced/CCh-stimulated contractions, we assumed that 7,8-DHF/TrkB/Akt was associated with the modulation of M3 expression in the colonic strips. ANA-12, a specific TrkB antagonist, not only inhibited TrkB activation by 7,8-DHF but also suppressed 7,8-DHF-enhanced cholinergic contraction, 7,8-DHF/CCh-mediated activation of PLCγ1/Akt, and M3 overexpression in colonic strips. In vivo 7,8-DHF, also by promoting intestinal motility and M3 expression, significantly alleviated loperamide-induced functional constipation in rats. CONCLUSIONS: Our results suggest that 7,8-DHF regulates colonic motility possibly via a TrkB/Akt/M3 pathway and may be applicable for alleviating constipation.

A missing distal complex of the external and internal anal sphincters: a macroscopic and histologic study using Japanese and German elderly cadavers.

The lower margin of the internal anal sphincter (IAS) is considered to lie on a J-shaped, subcutaneous part (SCP) of the external anal sphincter (EAS). The lower IAS is united with the J-shaped SCP to form a smooth-striated muscle complex. In the first part of this study, we ensured the presence of the J-shaped EAS in the lateral wall of the anal canal from 12 near-term fetuses. Second, in the lateral anal wall, the examination of the longitudinal section from 20 male and 24 female Japanese cadavers (72-95 years-old) demonstrated that the J-shaped EAS was lost in 15 (34%) due to the very small SCP. Third, we demonstrated that the J-shaped EAS was restricted in the latera anal wall using longitudinal histological sections of the anal canal from 11 male Japanese cadavers (75-89 years-old). Therefore, a site-dependent difference in the IAS-EAS configuration was evident. Finally, we compared a frequency of the lost J-shape between human populations using 10 mm-thick frontal slices from 36 Japanese and 28 German cadavers. The two groups of cadavers were compatible in age (a 0.2-years' difference in males). The macroscopic observations revealed that the J-shaped EAS was absent from 13 (36%) Japanese and six (20%) German specimens, suggesting that the SCP degeneration occurred more frequent in elderly Japanese than elderly German individuals (p < 0.05). The distal IAS-EAS complex seemed to push residual feces out of the anal canal at a transient phase from evacuation to closure. The absence might be the first sigh of anal dysfunction.

Protein-elongating mutations in MYH11 are implicated in a dominantly inherited smooth muscle dysmotility syndrome with severe esophageal, gastric, and intestinal disease.

Gastrointestinal motility disorders include a spectrum of mild to severe clinical phenotypes that are caused by smooth muscle dysfunction. We investigated the genetic etiology of severe esophageal, gastric, and colonic dysmotility in two unrelated families with autosomal dominant disease presentation. Using exome sequencing, we identified a 2 base pair insertion at the end of the myosin heavy chain 11 (MYH11) gene in all affected members of Family 1 [NM_001040113:c.5819_5820insCA(p.Gln1941Asnfs*91)] and a 1 base pair deletion at the same genetic locus in Proband 2 [NM_001040113:c.5819del(p.Pro1940Hisfs*91)]. Both variants are predicted to result in a similarly elongated protein product. Heterozygous dominant negative MYH11 pathogenic variants have been associated with thoracic aortic aneurysm and dissection while biallelic null alleles have been associated with megacystis microcolon intestinal hypoperistalsis syndrome. This report highlights heterozygous protein-elongating MYH11 variants affecting the SM2 isoforms of MYH11 as a cause for severe gastrointestinal dysmotility, and we hypothesize that the mechanistic pathogenesis of this disease, dominant hypercontractile loss-of-function, is distinct from those implicated in other diseases involving MYH11 dysfunction.

Subacute TGFß Exposure Drives Airway Hyperresponsiveness in Cystic Fibrosis Mice through the PI3K Pathway.

Cystic fibrosis (CF) is a lethal genetic disease characterized by progressive lung damage and airway obstruction. The majority of patients demonstrate airway hyperresponsiveness (AHR), which is associated with more rapid lung function decline. Recent studies in the neonatal CF pig demonstrated airway smooth muscle (ASM) dysfunction. These findings, combined with observed CF transmembrane conductance regulator (CFTR) expression in ASM, suggest that a fundamental defect in ASM function contributes to lung function decline in CF. One established driver of AHR and ASM dysfunction is transforming growth factor (TGF) ß1, a genetic modifier of CF lung disease. Prior studies demonstrated that TGFß exposure in CF mice drives features of CF lung disease, including goblet cell hyperplasia and abnormal lung mechanics. CF mice displayed aberrant responses to pulmonary TGFß, with elevated PI3K signaling and greater increases in lung resistance compared with controls. Here, we show that TGFß drives abnormalities in CF ASM structure and function through PI3K signaling that is enhanced in CFTR-deficient lungs. CF and non-CF mice were exposed intratracheally to an adenoviral vector containing the TGFß1 cDNA, empty vector, or PBS only. We assessed methacholine-induced AHR, bronchodilator response, and ASM area in control and CF mice. Notably, CF mice demonstrated enhanced AHR and bronchodilator response with greater ASM area increases compared with non-CF mice. Furthermore, therapeutic inhibition of PI3K signaling mitigated the TGFß-induced AHR and goblet cell hyperplasia in CF mice. These results highlight a latent AHR phenotype in CFTR deficiency that is enhanced through TGFß-induced PI3K signaling.

Rhinovirus C15 Induces Airway Hyperresponsiveness via Calcium Mobilization in Airway Smooth Muscle.

Rhinovirus (RV) exposure evokes exacerbations of asthma that markedly impact morbidity and mortality worldwide. The mechanisms by which RV induces airway hyperresponsiveness (AHR) or by which specific RV serotypes differentially evoke AHR remain unknown. We posit that RV infection evokes AHR and inflammatory mediator release, which correlate with degrees of RV infection. Furthermore, we posit that rhinovirus C-induced AHR requires paracrine or autocrine mediator release from epithelium that modulates agonist-induced calcium mobilization in human airway smooth muscle. In these studies, we used an ex vivo model to measure bronchoconstriction and mediator release from infected airways in human precision cut lung slices to understand how RV exposure alters airway constriction. We found that rhinovirus C15 (RV-C15) infection augmented carbachol-induced airway narrowing and significantly increased release of IP-10 (IFN-γ-induced protein 10) and MIP-1ß (macrophage inflammatory protein-1ß) but not IL-6. RV-C15 infection of human airway epithelial cells augmented agonist-induced intracellular calcium flux and phosphorylation of myosin light chain in co-cultured human airway smooth muscle to carbachol, but not after histamine stimulation. Our data suggest that RV-C15-induced structural cell inflammatory responses are associated with viral load but that inflammatory responses and alterations in agonist-mediated constriction of human small airways are uncoupled from viral load of the tissue.

Antagonism of proNGF or its receptor p75NTR reverses remodelling and improves bladder function in a mouse model of diabetic voiding dysfunction.

AIMS/HYPOTHESIS: Although 80% of diabetic patients will suffer from voiding difficulties and urinary symptoms, defined as diabetic voiding dysfunction (DVD), therapeutic targets and treatment options are limited. We hypothesise that the blockade of the pro-nerve growth factor (NGF)/p75 neurotrophin receptor (p75NTR) axis by an anti-proNGF monoclonal antibody or by a small molecule p75NTR antagonist (THX-B) can restore bladder remodelling (represented by bladder weight) in an animal model of DVD. Secondary outcomes of the study include improvements in bladder compliance, contractility and morphology, as well as in voiding behaviour, proNGF/NGF balance and TNF-α expression. METHODS: In a streptozotocin-induced mouse model of diabetes, diabetic mice received either a blocking anti-proNGF monoclonal antibody or a p75NTR antagonist small molecule as weekly systemic injections for 4 weeks. Animals were tested at baseline (at 2 weeks of diabetes induction), and after 2 and 4 weeks of treatment. Outcomes measured were voiding function with voiding spot assays and cystometry. Bladders were assessed by histological, contractility and protein expression assays. RESULTS: Diabetic mice showed features of DVD as early as 2 weeks after diabetes diagnosis (baseline) presented by hypertrophy, reduced contractility and abnormal cystometric parameters. Following treatment initiation, a twofold increase (p < 0.05) in untreated diabetic mouse bladder weight and thickness compared with non-diabetic controls was observed, and this change was reversed by p75NTR antagonism (37% reduction in bladder weight compared with untreated diabetic mice [95% CI 14%, 60%]) after 4 weeks of treatment. However, blocking proNGF did not help to reverse bladder hypertrophy. While diabetic mice had significantly worse cystometric parameters and contractile responses than non-diabetic controls, proNGF antagonism normalised bladder compliance (0.007 [Q1-Q3; 0.006-0.009] vs 0.015 [Q1-Q3; 0.014-0.029] ml/cmH2O in untreated diabetic mice, representing 62% reduction [95% CI 8%, 110%], p < 0.05) and contractility to KCl, carbachol and electrical field stimulation (p < 0.05 compared with the diabetic group) after 2 weeks of treatment. These effects were not observed after 4 weeks of treatment with proNGF antagonist. p75NTR antagonism did not show important improvements in cystometric parameters after 2 weeks of treatment. Slightly improved bladder compliance (0.01 [Q1-Q3; 0.009-0.012] vs 0.013 [Q1-Q3; 0.011-0.016] ml/cmH2O for untreated diabetic mice) was seen in the p75NTR antagonist-treated group after 4 weeks of treatment with significantly stabilised contractile responses to KCl, carbachol and electric field stimulation (p < 0.05 for each) compared with diabetic mice. Bladder dysfunction observed in diabetic mice was associated with a significant increase in bladder proNGF/NGF ratio (3.1 [±1.2] vs 0.26 [±0.04] ng/pg in control group, p < 0.05 at week 2 of treatment) and TNF-α (p < 0.05). The proNGF/NGF ratio was partially reduced (about 60% reduction) with both treatments (1.03 [±0.6] ng/pg for proNGF antibody-treated group and 1.4 [±0.76] ng/pg for p75NTR blocker-treated group after 2 weeks of treatment), concomitant with a significant decrease in the bladder levels of TNF-α (p < 0.05), despite persistent hyperglycaemia. CONCLUSIONS/INTERPRETATION: Our findings indicate that blockade of proNGF and the p75NTR receptor in diabetes can impede the development and progression of DVD. The reported improvements in morphological and functional features in our DVD model validates the proNGF/p75NTR axis as a potential therapeutic target in this pathology. Graphical abstract.

Chlamydia muridarum infection differentially alters smooth muscle function in mouse uterine horn and cervix.

Chlamydia trachomatis infection is a primary cause of reproductive tract diseases including infertility. Previous studies showed that this infection alters physiological activities in mouse oviducts. Whether this occurs in the uterus and cervix has never been investigated. This study characterized the physiological activities of the uterine horn and the cervix in a Chlamydia muridarum (Cmu)-infected mouse model at three infection time points of 7, 14, and 21 days postinfection (dpi). Cmu infection significantly decreased contractile force of spontaneous contraction in the cervix (7 and 14 dpi; P < 0.001 and P < 0.05, respectively), but this effect was not observed in the uterine horn. The responses of the uterine horn and cervix to oxytocin were significantly altered by Cmu infection at 7 dpi (P < 0.0001), but such responses were attenuated at 14 and 21 dpi. Cmu infection increased contractile force to prostaglandin (PGF2α) by 53-83% in the uterine horn. This corresponded with the increased messenger ribonucleic acid (mRNA) expression of Ptgfr that encodes for its receptor. However, Cmu infection did not affect contractions of the uterine horn and cervix to PGE2 and histamine. The mRNA expression of Otr and Ptger4 was inversely correlated with the mRNA expression of Il1b, Il6 in the uterine horn of Cmu-inoculated mice (P < 0.01 to P < 0.001), suggesting that the changes in the Otr and Ptger4 mRNA expression might be linked to the changes in inflammatory cytokines. Lastly, this study also showed a novel physiological finding of the differential response to PGE2 in mouse uterine horn and cervix.

Extrinsic Primary Afferent Neurons Link Visceral Pain to Colon Motility Through a Spinal Reflex in Mice.

BACKGROUND & AIMS: Proper colon function requires signals from extrinsic primary afferent neurons (ExPANs) located in spinal ganglia. Most ExPANs express the vanilloid receptor TRPV1, and a dense plexus of TRPV1-positive fibers is found around myenteric neurons. Capsaicin, a TRPV1 agonist, can initiate activity in myenteric neurons and produce muscle contraction. ExPANs might therefore form motility-regulating synapses onto myenteric neurons. ExPANs mediate visceral pain, and myenteric neurons mediate colon motility, so we investigated communication between ExPANs and myenteric neurons and the circuits by which ExPANs modulate colon function. METHODS: In live mice and colon tissues that express a transgene encoding the calcium indicator GCaMP, we visualized levels of activity in myenteric neurons during smooth muscle contractions induced by application of capsaicin, direct colon stimulation, stimulation of ExPANs, or stimulation of preganglionic parasympathetic neuron (PPN) axons. To localize central targets of ExPANs, we optogenetically activated TRPV1-expressing ExPANs in live mice and then quantified Fos immunoreactivity to identify activated spinal neurons. RESULTS: Focal electrical stimulation of mouse colon produced phased-locked calcium signals in myenteric neurons and produced colon contractions. Stimulation of the L6 ventral root, which contains PPN axons, also produced myenteric activation and contractions that were comparable to those of direct colon stimulation. Surprisingly, capsaicin application to the isolated L6 dorsal root ganglia, which produced robust calcium signals in neurons throughout the ganglion, did not activate myenteric neurons. Electrical activation of the ganglia, which activated even more neurons than capsaicin, did not produce myenteric activation or contractions unless the spinal cord was intact, indicating that a complete afferent-to-efferent (PPN) circuit was necessary for ExPANs to regulate myenteric neurons. In TRPV1-channel rhodopsin-2 mice, light activation of ExPANs induced a pain-like visceromotor response and expression of Fos in spinal PPN neurons. CONCLUSIONS: In mice, ExPANs regulate myenteric neuron activity and smooth muscle contraction via a parasympathetic spinal circuit, linking sensation and pain to motility.

TAS-303 Ameliorates Carbachol-Induced Detrusor Overactivity in Rats, Revealing Its Therapeutic Potential for Overactive Bladder.

Urinary incontinence is defined as an involuntary leakage of urine and is categorized into three types: stress urinary incontinence (SUI), urge urinary incontinence (UUI), and mixed urinary incontinence, which includes symptoms of SUI and UUI. As the underlying mechanisms of SUI and UUI are different, no drug is approved to treat all three types of urinary incontinence. TAS-303 is a selective norepinephrine reuptake inhibitor and has therapeutic potential for patients with SUI. In this report, we describe newly discovered pharmacological properties of TAS-303 and its effects on bladder function. Radioligand binding studies showed that TAS-303 inhibits M3 muscarinic receptor binding, with a Ki value of 547 nM. TAS-303 at 1, 3, and 10 mg/kg dose-dependently prolonged the intercontraction interval of carbachol-induced detrusor overactivity in rats, exhibiting a maximal effect that was comparable to tolterodine. These effects may result from coordinated regulation of bladder afferent activity via M3 muscarinic inhibition and ß3 adrenoreceptor activation by norepinephrine elevation due to norepinephrine transporter inhibition. Moreover, TAS-303 at the effective dose for bladder function did not induce dry mouth or constipation in rats, showing that this compound may have a lower risk of antimuscarinic side effects. Thus, TAS-303 is expected to be a new profile agent with therapeutic potential for all types of urinary incontinence. SIGNIFICANCE STATEMENT: Urinary incontinence is categorized into stress, urge, and mixed urinary incontinence, but because the underlying mechanisms of each differ, no drugs are available that treat all three. TAS-303 has therapeutic potential for stress urinary incontinence. This study describes newly discovered pharmacological properties of TAS-303, which ameliorated bladder afferent activity partly via M3 muscarinic inhibition, indicating improvement in urge urinary incontinence, and highlights the potential of TAS-303 as a new therapeutic agent for all types of urinary incontinence.

FFAR1 activation attenuates histamine-induced myosin light chain phosphorylation and cortical tension development in human airway smooth muscle cells.

BACKGROUND: Activation of free fatty acid receptors (FFAR1 and FFAR4) which are G protein-coupled receptors (GPCRs) with established (patho)physiological roles in a variety of obesity-related disorders, induce human airway smooth muscle (HASM) cell proliferation and shortening. We reported amplified agonist-induced cell shortening in HASM cells obtained from obese lung donors. We hypothesized that FFAR1 modulate excitation-contraction (EC) coupling in HASM cells and play a role in obesity-associated airway hyperresponsiveness. METHODS: In HASM cells pre-treated (30 min) with FFAR1 agonists TAK875 and GW9508, we measured histamine-induced Ca2+ mobilization, myosin light chain (MLC) phosphorylation, and cortical tension development with magnetic twisting cytometry (MTC). Phosphorylation of MLC phosphatase and Akt also were determined in the presence of the FFAR1 agonists or vehicle. In addition, the effects of TAK875 on MLC phosphorylation were measured in HASM cells desensitized to ß2AR agonists by overnight salmeterol treatment. The inhibitory effect of TAK875 on MLC phosphorylation was compared between HASM cells from age and sex-matched non-obese and obese human lung donors. The mean measurements were compared using One-Way ANOVA with Dunnett's test for multiple group comparisons or Student's t-test two-group comparison. For cortical tension measurements by magnetic twisted cytometry, mixed effect model using SAS V.9.2 was applied. Means were considered significant when p ≤ 0.05. RESULTS: Unexpectedly, we found that TAK875, a synthetic FFAR1 agonist, attenuated histamine-induced MLC phosphorylation and cortical tension development in HASM cells. These physiological outcomes were unassociated with changes in histamine-evoked Ca2+ flux, protein kinase B (AKT) activation, or MLC phosphatase inhibition. Of note, TAK875-mediated inhibition of MLC phosphorylation was maintained in ß2AR-desensitized HASM cells and across obese and non-obese donor-derived HASM cells. CONCLUSIONS: Taken together, our findings identified the FFAR1 agonist TAK875 as a novel bronchoprotective agent that warrants further investigation to treat difficult-to-control asthma and/or airway hyperreactivity in obesity.

EphrinB2/ephB2-mediated myenteric synaptic plasticity: mechanisms underlying the persistent muscle hypercontractility and pain in postinfectious IBS.

Patients with irritable bowel syndrome (IBS) show pain hypersensitivity and smooth muscle hypercontractility in response to colorectal distension (CRD). Synaptic plasticity, a key process of memory formation, in the enteric nervous system may be a novel explanation. This study aimed to explore the regulatory role of ephrinB2/ephB2 in enteric synaptic plasticity and colonic hyperreactive motility in IBS. Postinfectious (PI)-IBS was induced by Trichinella spiralis infection in rats. Isometric contractions of colonic circular muscle strips, particularly neural-mediated contractions, were recorded ex vivo. Meanwhile, ephrinB2/ephB2-mediated enteric structural and functional synaptic plasticity were assessed in the colonic muscularis, indicating that ephrinB2 and ephB2 were located on enteric nerves and up-regulated in the colonic muscularis of PI-IBS rats. Colonic hypersensitivity to CRD and neural-mediated colonic hypercontractility were present in PI-IBS rats, which were correlated with increased levels of cellular homologous fos protein (c-fos) and activity-regulated cystoskeleton-associated protein (arc), the synaptic plasticity-related immediate early genes, and were ameliorated by ephB2Fc (an ephB2 receptor blocker) or MK801 (an NMDA receptor inhibitor) exposure. EphrinB2/ephB2 facilitated synaptic sprouting and NMDA receptor-mediated synaptic potentiation in the colonic muscularis of PI-IBS rats and in the longitudinal muscle-myenteric plexus cultures, involving the Erk-MAPK and PI3K-protein kinase B pathways. In conclusion, ephrinB2/ephB2 promoted the synaptic sprouting and potentiation of myenteric nerves involved in persistent muscle hypercontractility and pain in PI-IBS. Hence, ephrinB2/ephB2 may be an emerging target for the treatment of IBS.-Zhang, L., Wang, R., Bai, T., Xiang, X., Qian, W., Song, J., Hou, X. EphrinB2/ephB2-mediated myenteric synaptic plasticity: mechanisms underlying the persistent muscle hypercontractility and pain in postinfectious IBS.