Neuropilin 2 Is a Novel Regulator of Distal Colon Contractility.

Appropriate coordination of smooth muscle contraction and relaxation is essential for normal colonic motility. The impact of perturbed motility ranges from moderate, in conditions such as colitis, to potentially fatal in the case of pseudo-obstruction. The mechanisms underlying aberrant motility and the extent to which they can be targeted pharmacologically are incompletely understood. This study identified colonic smooth muscle as a major site of expression of neuropilin 2 (Nrp2) in mice and humans. Mice with inducible smooth muscle-specific knockout of Nrp2 had an increase in evoked contraction of colonic rings in response to carbachol at 1 and 4 weeks following initiation of deletion. KCl-induced contractions were also increased at 4 weeks. Colonic motility was similarly enhanced, as evidenced by faster bead expulsion in Nrp2-deleted mice versus Nrp2-intact controls. In length-tension analysis of the distal colon, passive tension was similar in Nrp2-deficient and Nrp2-intact mice, but at low strains, active stiffness was greater in Nrp2-deficient animals. Consistent with the findings in conditional Nrp2 mice, Nrp2-null mice showed increased contractility in response to carbachol and KCl. Evaluation of selected proteins implicated in smooth muscle contraction revealed no significant differences in the level of α-smooth muscle actin, myosin light chain, calponin, or RhoA. Together, these findings identify Nrp2 as a novel regulator of colonic contractility that may be targetable in conditions characterized by dysmotility.

Tas2R activation relaxes airway smooth muscle by release of Gαt targeting on AChR signaling.

Both chronic obstructive pulmonary disease (COPD) and asthma are severe respiratory diseases. Bitter receptor-mediated bronchodilation is a potential therapy for asthma, but the mechanism underlying the agonistic relaxation of airway smooth muscle (ASM) is not well defined. By exploring the ASM relaxation mechanism of bitter substances, we observed that pretreatment with the bitter substances nearly abolished the methacholine (MCh)-induced increase in the ASM cell (ASMC) calcium concentration, thereby suppressing the calcium-induced contraction release. The ASM relaxation was significantly inhibited by simultaneous deletion of three Gαt proteins, suggesting an interaction between Tas2R and AChR signaling cascades in the relaxation process. Biochemically, the Gαt released by Tas2R activation complexes with AChR and blocks the Gαq cycling of AChR signal transduction. More importantly, a bitter substance, kudinoside A, not only attenuates airway constriction but also significantly inhibits pulmonary inflammation and tissue remodeling in COPD rats, indicating its modulation of additional Gαq-associated pathological processes. Thus, our results suggest that Tas2R activation may be an ideal strategy for halting multiple pathological processes of COPD.

Osteomodulin attenuates smooth muscle cell osteogenic transition in vascular calcification.

RATIONALE: Vascular calcification is a prominent feature of late-stage diabetes, renal and cardiovascular disease (CVD), and has been linked to adverse events. Recent studies in patients reported that plasma levels of osteomodulin (OMD), a proteoglycan involved in bone mineralisation, associate with diabetes and CVD. We hypothesised that OMD could be implicated in these diseases via vascular calcification as a common underlying factor and aimed to investigate its role in this context. METHODS AND RESULTS: In patients with chronic kidney disease, plasma OMD levels correlated with markers of inflammation and bone turnover, with the protein present in calcified arterial media. Plasma OMD also associated with cardiac calcification and the protein was detected in calcified valve leaflets by immunohistochemistry. In patients with carotid atherosclerosis, circulating OMD was increased in association with plaque calcification as assessed by computed tomography. Transcriptomic and proteomic data showed that OMD was upregulated in atherosclerotic compared to control arteries, particularly in calcified plaques, where OMD expression correlated positively with markers of smooth muscle cells (SMCs), osteoblasts and glycoproteins. Immunostaining confirmed that OMD was abundantly present in calcified plaques, localised to extracellular matrix and regions rich in α-SMA+ cells. In vivo, OMD was enriched in SMCs around calcified nodules in aortic media of nephrectomised rats and in plaques from ApoE-/- mice on warfarin. In vitro experiments revealed that OMD mRNA was upregulated in SMCs stimulated with IFNγ, BMP2, TGFß1, phosphate and ß-glycerophosphate, and by administration of recombinant human OMD protein (rhOMD). Mechanistically, addition of rhOMD repressed the calcification process of SMCs treated with phosphate by maintaining their contractile phenotype along with enriched matrix organisation, thereby attenuating SMC osteoblastic transformation. Mechanistically, the role of OMD is exerted likely through its link with SMAD3 and TGFB1 signalling, and interplay with BMP2 in vascular tissues. CONCLUSION: We report a consistent association of both circulating and tissue OMD levels with cardiovascular calcification, highlighting the potential of OMD as a clinical biomarker. OMD was localised in medial and intimal α-SMA+ regions of calcified cardiovascular tissues, induced by pro-inflammatory and pro-osteogenic stimuli, while the presence of OMD in extracellular environment attenuated SMC calcification.

Docosahexaenoic Acid Selectively Suppresses U46619- and PGF2α-Induced Contractions in Guinea Pig Tracheal Smooth Muscles.

We investigated the potential inhibitory effects of docosahexaenoic acid (DHA) on the contractions of guinea pig tracheal smooth muscles in response to U46619 (a thromboxane A2 (TXA2) mimetic) and prostaglandin F2α (PGF2α) to examine whether this n-3 polyunsaturated fatty acid suppresses prostanoid-induced tracheal contractions. DHA (3 × 10-5 M) significantly suppressed tracheal contractions elicited by lower concentrations of U46619 (10-8 M) and PGF2α (5 × 10-7 M) (vs. control), although it did not suppress the contractions induced by higher concentrations (U46619: 10-7 M; PGF2α: 10-5 M). Supporting these findings, DHA (4 × 10-5 M/6 × 10-5 M) shifted the concentration-response curves for U46619 (10-9-10-6 M) and PGF2α (10-8-10-5 M) to the right. However, the slope of the regression line in the Schild plot of DHA vs. U46619/PGF2α was larger than unity. The tracheal contractions induced by U46619 (10-8 M) and PGF2α (5 × 10-7 M) were significantly suppressed by the prostanoid TP receptor antagonist SQ 29,548 (10-6 M) (vs. ethanol-treated). In contrast, DHA (4 × 10-5 M) did not show significant inhibitory effects on the contractions induced by acetylcholine (10-8-10-4 M), histamine (10-8-10-4 M), and leukotriene D4 (10-11-10-7 M) (vs. ethanol-treated). These findings indicate that DHA selectively suppresses tracheal contractions induced by U46619 and PGF2α. Therefore, DHA may be a useful therapeutic agent against asthma associated with tracheal/bronchial hyper-constriction caused by prostanoids including TXA2 and PGF2α.

Magnolol and honokiol target TRPC4 to regulate extracellular calcium influx and relax intestinal smooth muscle.

ETHNOPHARMACOLOGICAL RELEVANCE: Magnolia officinalis Cortex (M. officinalis) is a classical traditional Chinese medicine (TCM) widely used to treat digestive system diseases. It effectively regulates gastrointestinal motility to improve abdominal pain, abdominal distension and other symptoms. Magnolol (MAG) and honokiol (HON) are the main pharmacodynamic components responsible for the gastrointestinal activity of M. officinalis. AIM OF THE STUDY: The transient receptor potential (TRP) family is highly expressed in the gastrointestinal tract and participates in the regulation of gastrointestinal motility, visceral hypersensitivity, visceral secretion and other physiological activities. In this study, the calcium-lowering mechanisms of MAG and HON contributing to the smooth muscle relaxation associated with TRP are discussed. MATERIALS AND METHODS: The relaxation smooth muscle effects of MAG and HON were tested by the isolated intestine tone tests. A synthetic MAG probe (MAG-P) was used to target fishing for their possible target. The distribution of MAG on the smooth muscle was identified by a molecular tracer based on chemical biology. Ca2+ imaging and dual-luciferase reporter assays were used to determine the effects on the target proteins. Finally, the calcium-mediating effects of MAG and HON on smooth muscle cells and TRPC4-knockdown cells were compared to verify the potential mechanism. RESULTS: After confirming the smooth muscle relaxation in the small intestine induced by MAG and HON, the relaxation effect was identified mainly due to the downregulation of intracellular calcium by controlling external calcium influx. Although MAG and HON inhibited both TRPV4 and TRPC4 channels to reduce calcium levels, the inhibitory effect on TRPC4 channels is an important mechanism of their smooth muscle relaxation effect, since TRPC4 is widely expressed in the small intestinal smooth muscle cells. CONCLUSIONS: The inhibition of MAG and HON on TRPC4 channels contributes to the relaxation of intestinal smooth muscle.

Histamine receptors rapidly desensitize without altering nerve-evoked contractions in murine urinary bladder smooth muscle.

Histamine has been implicated in urinary bladder dysfunction as an inflammatory mediator driving sensory nerve hypersensitivity. However, the direct influence of histamine on smooth muscle has not been thoroughly investigated. We hypothesized that histamine directly contracts urinary bladder smooth muscle (UBSM) independent of effects on nerves. Single cell quantitative RT-PCR determined that only histamine H1 and H2 receptors were expressed on UBSM cells. In isolated tissue bath experiments, histamine (200 µM) caused a highly variable and rapidly desensitizing contraction that was completely abolished by the H1 receptor antagonist fexofenadine (5 µM) and the Gq/11 inhibitor YM254890 (1 µM). Neither the muscarinic receptor antagonist atropine (1 µM), the Na+ channel blocker tetrodotoxin (1 µM), nor the transient receptor potential vanilloid type 1 antagonist capsazepine (10 µM) altered responses to histamine, suggesting that nerve activation was not involved. UBSM desensitization to histamine was not due to receptor internalization, as neither the cholesterol-depleting agent methyl-ß-cyclodextrin (10 mM), the dynamin-mediated endocytosis inhibitor dynasore (100 µM), nor the clathrin-mediated endocytosis inhibitor pitstop2 (15 µM) augmented or prolonged histamine contractions. Buffer from desensitized tissues still contracted histamine-naïve tissues, revealing that histamine was not metabolized. Prolonged exposure to histamine also had no effect on contractions due to electrical field stimulation, suggesting that both efferent nerve and UBSM excitability were unchanged. Together, these data suggest that histamine, although able to transiently contract UBSM, does not have a lasting effect on UBSM excitability or responses to efferent nerve input. Thus, any acute effects of histamine directly on UBSM contractility are unlikely to alter urinary bladder function.NEW & NOTEWORTHY Histamine is commonly associated with inflammatory bladder pathologies. We sought to investigate the role of histamine on urinary bladder contractility. Histamine contracts the bladder, but this response is highly variable and desensitizes completely in minutes. This desensitization is not due to internalization of the receptor or metabolism of histamine. Because nerve-evoked contractions are also not increased in the presence of histamine, our findings suggest that histamine is not directly acting to change contractility.

Protective role of activating PPARγ in advanced glycation end products-induced impairment of coronary artery vasodilation via inhibiting p38 phosphorylation and reactive oxygen species production.

Advanced glycation end products (AGEs) can damage voltage-gated K+ (Kv) channels and attenuate coronary artery vasodilation, but the underlying mechanisms remain unclear. The aim of this study was to investigate the role and potential mechanism of PPARγ in AGEs-induced Kv 1 channels impairment. We used both primary rat coronary smooth muscle cells (CSMCs) in vitro and Zucker Diabetic Fatty (ZDF) rat model in vivo. Overexpression of the Pparg gene by lentivirus vector (LV-Pparg) was used to transfect CSMCs for upregulation PPARγ. Kv 1.2 and Kv 1.5 currents were measured by patch clamp. The vascular tone of coronary artery was evaluated by isometric force measurements. The proteins expression of Kv1.2 and Kv1.5 channel were detected by western blot. PPARγ was detected by immunofluorescence and western blot. Oxidative stress markers including superoxide dismutase (SOD), glutathione peroxidase (GPx) and malondialdehyde (MDA) were detected by enzyme linked immunosorbent assay (ELISA). The phosphorylation of p38 mitogen-activated protein kinase (MAPK) and total p38 expression were detected by western blot. The intracellular ROS levels were measured by the fluorescent dye 2',7'- dichlorofluorescein diacetate (DCFDA) and a cellular ROS assay kit. We found that activating PPARγ via LV-Pparg (100 MOI, 5 × 108 TU/mL) prevented AGEs (100 µg/mL) -mediated impairment of Kv 1.2 and Kv 1.5 channels activity and improved the reduction of Kv 1.2 and Kv 1.5 protein expression in CSMCs. Isometric force measurements showed that activating PPARγ by pioglitazone (10 mg/kg/d, intragastric administration) improved the impairment of coronary artery vasodilation, and western blot analysis showed that activating PPARγ increased the Kv 1.2 and Kv 1.5 protein expression, while inhibiting PPARγ by GW9662 (10 mg/kg/d, intraperitoneal injection) attenuated these effects in ZDF rats. Furthermore, LV-Pparg overexpression PPARγ attenuated NADPH oxidase activity, which was shown as the reduction of the NOX2 and p22phox expression by western blot analysis, decreased the MDA production and increased the SOD and GPx activities by ELISA, finally led to reduce AGEs-mediated ROS production. Moreover, activating PPARγ by LV-Pparg inhibited AGEs-induced phosphorylation of p38 MAPK, by which could reduce AGEs-mediated NOX2, p22phox expression and ROS production, while CSMCs treatment with SB203580 (10 µmol/L), a p38 MAPK inhibitor, attenuated these effects. Activating PPARγ plays a protective role in AGEs-induced impairment of coronary artery vasodilation by inhibiting p38 phosphorylation to attenuate NOX2 and p22phox expression and further decrease oxidative stress induced by ROS overproduction.

Cellular Mechanism Underlying the Facilitation of Contractile Response Induced by Tumor Necrosis Factor-α in Mouse Tracheal Smooth Muscle.

The proinflammatory cytokine tumor necrosis factor-α (TNF-α) augments intracellular Ca2+ signaling and contractile responses of airway smooth muscles, leading to airway hyperresponsiveness. However, the underlying mechanism has not been fully elucidated. This study aimed to investigate the cellular mechanism of the potentiated contraction of mouse tracheal smooth muscle induced by TNF-α. The results showed that TNF-α triggered facilitation of mouse tracheal smooth muscle contraction in an epithelium-independent manner. The TNF-α-induced hypercontractility could be suppressed by the protein kinase C inhibitor GF109203X, the tyrosine kinase inhibitor genistein, the Src inhibitor PP2, or the L-type voltage-dependent Ca2+ channel blocker nifedipine. Following TNF-α incubation, the α1C L-type Ca2+ channel (CaV1.2) was up-regulated in cultured primary mouse tracheal smooth muscle cells. Pronounced phosphotyrosine levels were observed in mouse tracheas. In conclusion, this study shows that TNF-α enhanced airway smooth muscle contraction via protein kinase C-Src-CaV1.2 pathways, which provides novel insights into the pathologic role of proinflammatory cytokines in mediating airway hyperresponsiveness.

Validation of Fenoterol to Study ß2-Adrenoceptor Function in the Rat Urinary Bladder.

Fenoterol is a ß2-adrenoceptor (AR)-selective agonist that is commonly used to investigate relaxation responses mediated by ß2-AR in smooth muscle preparations. Some data have questioned this because fenoterol had low potency in the rat urinary bladder when a muscarinic agonist was used as a pre-contraction agent and because some investigators proposed that fenoterol may act in part via ß3-AR. We designed the present study to investigate whether fenoterol is a proper pharmacological tool to study ß2-AR-mediated relaxation responses in the rat urinary bladder. Firstly, we have compared the effect of pre-contraction agents on fenoterol potency and found that fenoterol potency was about 1.5 log units greater against KCl than carbachol (pEC50 7.19 ± 0.66 and 5.62 ± 1.09 of KCl and of carbachol, respectively). To test the selectivity of fenoterol, we have determined the effects of the ß2-AR antagonist ICI 118,551 and the ß3-AR antagonist L 748,337 on relaxation responses to fenoterol. While 300 nM L 748,337 had little effect on the potency of fenoterol (pEC50 6.56 ± 0.25 and 6.33 ± 0.61 in the absence and presence of L 748,337, respectively), the relaxation curve for fenoterol was right-shifted in the presence 300 nM ICI 118,551 (pEC50 5.03 ± 0.18). Thus, we conclude that fenoterol is a proper pharmacological tool to assess ß2-AR-mediated responses in the rat urinary bladder and most likely in other smooth-muscle preparations containing multiple subtypes of the ß-AR.

The inhibitory effect of trimetazidine on detrusor contractility - a potential repositioning of trimetazidine for the treatment of overactive bladder.

OBJECTIVES: This study aimed to identify the effect of trimetazidine (TMZ), an antianginal drug, on detrusor smooth muscle (DSM) contractility and its possible mechanisms of action. METHODS: We performed in-vitro contractility studies on isolated mouse DSM strips and investigated the effect of TMZ on Ca2+ levels in fura-2-loaded A7r5 cells. KEY FINDINGS: TMZ (300 or 1000 µM) inhibited carbachol (CCh)- and KCl-induced contractions and produced a concentration-dependent (10-1000 µM) relaxation in KCl-precontracted DSM strips. TMZ-induced relaxation was markedly decreased by BaCl2, an inward-rectifying K+ channel blocker, but was not altered by preincubation with tetraethylammonium, glibenclamide, 4-aminopyridine, propranolol, L-NAME or methylene blue. TMZ (300 or 1000 µM) reduced both the CaCl2-induced contraction of depolarized DSM strips under Ca2+-free conditions and the CCh-induced contraction of DSM strips preincubated with nifedipine in Ca2+-containing Krebs solution. Furthermore, TMZ (1000 µM) significantly decreased the Ca2+ levels in fura-2-loaded A7r5 cells. CONCLUSIONS: TMZ decreased DSM contractility and caused a concentration-dependent relaxation of the tissue possibly through its actions on Ca2+ transients and K+ channels. Our results provide preclinical evidence that TMZ would be a potential candidate to treat disorders related to the overactivity of the bladder.

Identification and characterization of an atypical Gαs-biased ß2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis.

G protein-coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For ß2-adrenergic receptors (ß2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and ß-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable ß-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling-a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: ß2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from ß-arrestin, in contrast to albuterol and C5-S C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from ß-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of ß2AR actions favorable for treating obstructive lung disease.

Early phase of pupil dilation is mediated by the peripheral parasympathetic pathway.

Pupil diameter fluctuates in association with changes in brain states induced by the neuromodulator systems. However, it remains unclear how the neuromodulator systems control the activity of the iris sphincter (constrictor) and dilator muscles to change the pupil size. The present study compared temporal patterns of pupil dilation during movement when each muscle was pharmacologically manipulated in the human eye. When the iris sphincter muscle was blocked with tropicamide, the latency of pupil dilation was delayed and the magnitude of pupil dilation was reduced during movement. In contrast, when the iris dilator muscle was continuously stimulated with phenylephrine, the latency and magnitude of rapid pupil dilation did not differ from the untreated control eye, but sustained pupil dilation was reduced until the end of movement. These results suggest that the iris sphincter muscle, which is under the control of the parasympathetic pathway, is quickly modulated by the neuromodulator system and plays a major role in rapid pupil dilation. However, the iris dilator muscle receives signals from the neuromodulator system with a slow latency and is involved in maintaining sustained pupil dilation.NEW & NOTEWORTHY By pharmacologically manipulating the pupil dilator and constrictor muscles of human eye separately, we found that the pupil constrictor muscle is a primary controller of rapid pupil dilation upon brain arousal. However, the pupil dilator muscle, which is innervated by the sympathetic nervous system and is generally considered as a major regulator of pupil dilation, is not involved in rapid pupil dilation, but was involved in long-lasting pupil dilation.

New Modified Recombinant Botulinum Neurotoxin Type F with Enhanced Potency.

Botulinum neurotoxins (BoNTs) are notorious toxins and powerful agents and can be lethal, causing botulism, but they are also widely used as therapeutics, particularly to treat neuromuscular disorders. As of today, the commercial BoNT treatments available are from native A or B serotypes. Serotype F has shown efficacy in a clinical trial but has scarcely been used, most likely due to its medium duration of effect. Previously, the uniqueness of the light chain of the F7 subtype was identified and reported, showing an extended interaction with its substrates, VAMPs 1, 2 and 3, and a superior catalytic activity compared to other BoNT/F subtypes. In order to more extensively study the properties of this neurotoxin, we engineered a modified F7 chimera, mrBoNT/F7-1, in which all the regions of the neurotoxin were identical to BoNT/F7 except the activation loop, which was the activation loop from BoNT/F1. Use of the activation loop from BoNT/F1 allowed easier post-translational proteolytic activation of the recombinant protein without otherwise affecting its properties. mrBoNT/F7-1 was expressed, purified and then tested in a suite of in vitro and in vivo assays. mrBoNT/F7-1 was active and showed enhanced potency in comparison to both native and recombinant BoNT/F1. Additionally, the safety profile remained comparable to BoNT/F1 despite the increased potency. This new modified recombinant toxin F7 could be further exploited to develop unique therapeutics to address unmet medical needs.

Repeated Low-Dose Acrolein Triggers Irreversible Lamina Propria Edema in Urinary Bladder, Transient Voiding Behavior and Widening of Eyes to Mechanical Stimuli.

Acrolein is a metabolite of cyclophosphamide (CYP), an alkylating agent used for a wide range of benign and malignant diseases. CYP treatments are known to trigger hemorrhagic cystitis in patients and animals. Significant effort has been made to prevent CYP/acrolein-induced cystitis, while still maintaining its therapeutic benefits. As a result, supplementary therapeutic options to mediate the protective role against CYP/acrolein and lower doses of CYP are currently given to targeted patients, as compared to past treatments. There is still a need to further study the effects of the repeated low-dose CYP/acrolein on the pathophysiology of the urinary bladder. In our study, a one-time treatment of acrolein and repeated low-dose acrolein triggered the thickening of the smooth muscle and lamina propria in the urinary bladder of C57BL/6J mice, respectively. The first dose of acrolein did not trigger voiding dysfunction, but the second dose triggered high-volume low-frequency voiding. Interestingly, our new scoring criteria and concurrent behavioral assessment revealed that mice with repeated low-dose acrolein had a wider opening of eyes in response to mechanical stimuli. Our study suggests that clinical symptoms among patients undergoing prolonged low-dose CYP may differ from previously reported symptoms of CYP-induced hemorrhagic cystitis.

Aberrant enteric neuromuscular system and dysbiosis in amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis is a neuromuscular disease characterized by the progressive death of motor neurons and muscle atrophy. The gastrointestinal symptoms in ALS patients were largely ignored or underestimated. The relationship between the enteric neuromuscular system and microbiome in ALS progression is unknown. We performed longitudinal studies on the enteric neuron system (ENS) and microbiome in the ALS human-SOD1G93A (Superoxide Dismutase 1) transgenic mice. We treated age-matched wild-type and ALS mice with butyrate or antibiotics to investigate the microbiome and neuromuscular functions. We examined intestinal mobility, microbiome, an ENS marker GFAP (Glial Fibrillary Acidic Protein), a smooth muscle marker (SMMHC, Smooth Muscle Myosin Heavy Chain), and human colonoids. The distribution of human-G93A-SOD1 protein was tested as an indicator of ALS progression. At 2-month-old before ALS onset, SOD1G93A mice had significantly lower intestinal mobility, decreased grip strength, and reduced time in the rotarod. We observed increased GFAP and decreased SMMHC expression. These changes correlated with consistent increased aggregation of mutated SOD1G93A in the colon, small intestine, and spinal cord. Butyrate or antibiotics treated SOD1G93A mice had a significantly longer latency to fall in the rotarod test, reduced SOD1G93A aggregation, and enhanced enteric neuromuscular function. Feces from 2-month-old SOD1G93A mice significantly enhanced SOD1G93A aggregation in human colonoids transfected with a SOD1G93A-GFP plasmid. Longitudinal studies of microbiome data further showed the altered bacterial community related to autoimmunity (e.g., Clostridium sp. ASF502, Lachnospiraceae bacterium A4), inflammation (e.g., Enterohabdus Muris,), and metabolism (e.g., Desulfovibrio fairfieldensis) at 1- and 2-month-old SOD1G93A mice, suggesting the early microbial contribution to the pathological changes. We have demonstrated a novel link between the microbiome, hSOD1G93A aggregation, and intestinal mobility. Dysbiosis occurred at the early stage of the ALS mice before observed mutated-SOD1 aggregation and dysfunction of ENS. Manipulating the microbiome improves the muscle performance of SOD1G93A mice. We provide insights into the fundamentals of intestinal neuromuscular function and microbiome in ALS.

Prostanoid TP receptor stimulation enhances contractile activities in guinea pig urinary bladder smooth muscle through activation of Ca2+ entry channels: Potential targets in the treatment of urinary bladder contractile dysfunction.

AIMS: We examined the potential stimulatory effects of U46619 (a prostanoid TP receptor agonist) and five prostanoids on the contractile activities of urinary bladder smooth muscle (UBSM), focusing on the role of the TP receptor and its associated Ca2+ influx routes to understand the roles of prostanoids in the regulation of UB contractile activity. MAIN METHODS: Changes in the basal tone and spontaneous contractile activity (amplitude and frequency) of isolated guinea pig UBSM were measured isotonically. The presence of TP receptors in UBSM was examined by RT-qPCR and immunofluorescence. KEY FINDINGS: U46619, prostaglandin (PG) E2, PGF2α, and PGA2 enhanced UBSM basal tone and spontaneous contractile activities, which were measured as amplitudes and frequencies. The enhancing effects of U46619 were completely suppressed by SQ 29,548 (a TP receptor antagonist), which also partially suppressed the stimulating effects of other prostanoids. The expression of TP receptors in UBSMs was verified at the mRNA and protein level. The enhancing effects of U46619 completely disappeared in Ca2+-free solution. U46619-enhanced basal tone was completely suppressed by verapamil, an inhibitor of voltage-dependent Ca2+ channels (VDCCs), and verapamil strongly decreased the spontaneous contraction frequency. The spontaneous contractions remaining in the presence of verapamil were strongly suppressed by SKF-96365 (an inhibitor of receptor-operated Ca2+ channels (ROCCs)/store-operated Ca2+ channels (SOCCs)), but not by LOE-908 (an inhibitor of ROCCs). SIGNIFICANCE: Prostanoids can enhance UBSM contractile activities and thus may be endogenous candidates for induction of detrusor overactivity. The TP receptor and TP-receptor-activated VDCCs/SOCCs are key molecules responsible for these effects.

Spasmolytic Effects of Aphanizomenon Flos Aquae (AFA) Extract on the Human Colon Contractility.

The blue-green algae Aphanizomenon flos aquae (AFA), rich in beneficial nutrients, exerts various beneficial effects, acting in different organs including the gut. Klamin® is an AFA extract particularly rich in ß-PEA, a trace-amine considered a neuromodulator in the central nervous system. To date, it is not clear if ß-PEA exerts a role in the enteric nervous system. The aims of the present study were to investigate the effects induced by Klamin® on the human distal colon mechanical activity, to analyze the mechanism of action, and to verify a ß-PEA involvement. The organ bath technique, RT-PCR, and immunohistochemistry (IHC) were used. Klamin® reduced, in a concentration-dependent manner, the amplitude of the spontaneous contractions. EPPTB, a trace-amine receptor (TAAR1) antagonist, significantly antagonized the inhibitory effects of both Klamin® and exogenous ß-PEA, suggesting a trace-amine involvement in the Klamin® effects. Accordingly, AphaMax®, an AFA extract containing lesser amount of ß-PEA, failed to modify colon contractility. Moreover, the Klamin® effects were abolished by tetrodotoxin, a neural blocker, but not by L-NAME, a nitric oxide-synthase inhibitor. On the contrary methysergide, a serotonin receptor antagonist, significantly antagonized the Klamin® effects, as well as the contractility reduction induced by 5-HT. The RT-PCR analysis revealed TAAR1 gene expression in the colon and the IHC experiments showed that 5-HT-positive neurons are co-expressed with TAAR1 positive neurons. In conclusion, the results of this study suggest that Klamin® exerts spasmolytic effects in human colon contractility through ß-PEA, that, by activating neural TAAR1, induce serotonin release from serotoninergic neurons of the myenteric plexus.

Influence of Tilia tomentosa Moench Extract on Mouse Small Intestine Neuromuscular Contractility.

Functional gastrointestinal disorders (FGIDs) are characterized by abdominal pain, bloating and bowel disturbances. FGID therapy is primarily symptomatic, including treatment with herbal remedies. Flower extract of Tilia tomentosa Moench (TtM) is occasionally used as an anti-spasmodic in popular medicine. Since its effect on intestinal response is unknown, we evaluated the influence of TtM extract on small intestine contractility. Ileal preparations from C57BL/6J mice were mounted in organ baths to assess changes in muscle tension, following addition of TtM extract (0.5-36 µg/mL) or a vehicle (ethanol). Changes in contractile response to receptor- and non-receptor-mediated stimuli were assessed in ileal preparations pretreated with 12 µg/mL TtM. Alterations in the enteric nervous system neuroglial network were analyzed by confocal immunofluorescence. Increasing addition of TtM induced a marked relaxation in ileal specimens compared to the vehicle. Pretreatment with TtM affected cholinergic and tachykininergic neuromuscular contractions as well as K+-induced smooth muscle depolarization. Following incubation with TtM, a significant reduction in non-adrenergic non-cholinergic-mediated relaxation sensitive to Nω-Nitro-L-arginine methyl ester hydrochloride (pan-nitric oxide synthase inhibitor) was found. In vitro incubation of intestinal specimens with TtM did not affect the myenteric plexus neuroglial network. Our findings show that TtM-induced intestinal relaxation is mediated by nitric oxide pathways, providing a pharmacological basis for the use of TtM in FGIDs.

Mechanisms underlying spontaneous phasic contractions and sympathetic control of smooth muscle in the rat caudal epididymis.

Here we investigate mechanisms underlying spontaneous phasic contractions (SPCs) and sympathetic control of contractility in the rat epididymis, a long tubular duct involved in transportation and maturation of sperm. Longitudinal contractions of short segments (~ 1.5 mm) of rat proximal and distal caudal epididymal duct were measured + / - nerve stimulation. The extent of sympathetic innervation of these duct regions was determined by immunohistochemistry. Proximal caudal duct segments (150-300 µm dia.) exhibited SPCs, while distal segments (350-500 µm) were quiescent in ~ 80% of preparations. SPC amplitude and frequency were reduced by the L-type voltage-dependent Ca2+ channel (LVDCC) blocker nifedipine (1 µM), with the T-type voltage-dependent Ca2+ channel (TVDCC) blocker ML218 (1 µM) specifically decreasing SPC frequency. SPCs were inhibited upon blockade of the SR/ER Ca2+-ATPase (CPA 10 µM). SPCs were also inhibited by caffeine (1 µM), 2-APB (100 µM), niflumic acid (100 µM), or by lowering extracellular [Cl-] from 134.4 to 12.4 mM but not by ryanodine (25 µM) or tetracaine (100 µM). Electrical field stimulation (EFS) at 2 Hz for 60 s caused a sustained α1-adrenoceptor-sensitive contraction in distal segments and enhanced and/or induced α2-adrenoceptor-sensitive oscillatory phasic contractions in proximal and distal segments, the latter mimicked by application of the α2-adrenoceptor agonist clonidine. We hypothesise that SPCs in the proximal cauda are triggered by pacemaker mechanisms involving rhythmic IP3 receptor-operated SR/ER store Ca2+ release and resultant activation of CaCC with TVDCCs and possibly LVDCCs subserving in this process. Sympathetic nerve-released noradrenaline induces α2-adrenoceptor-mediated phasic contractions in the proximal and distal cauda. These findings provide new pharmacological targets for male infertility and contraception.

Combined agonists act synergistically to increase mucociliary clearance in a cystic fibrosis airway model.

Mucus clearance, a primary innate defense mechanism of airways, is defective in patients with cystic fibrosis (CF) and CF animals. In previous work, the combination of a low dose of the cholinergic agonist, carbachol with forskolin or a ß adrenergic agonist, isoproterenol synergistically increased mucociliary clearance velocity (MCCV) in ferret tracheas. Importantly, the present study shows that synergistic MCCV can also be produced in CF ferrets, with increases ~ 55% of WT. Synergistic MCCV was also produced in pigs. The combined agonists increased MCCV by increasing surface fluid via multiple mechanisms: increased fluid secretion from submucosal glands, increased anion secretion across surface epithelia and decreased Na+ absorption. To avoid bronchoconstriction, the cAMP agonist was applied 30 min before carbachol. This approach to increasing mucus clearance warrants testing for safety and efficacy in humans as a potential therapeutic for muco-obstructive diseases.