Purinergic inhibitory regulation of esophageal smooth muscle is mediated by P2Y receptors and ATP-dependent potassium channels in rats.

Purines such as ATP are regulatory transmitters in motility of the gastrointestinal tract. The aims of this study were to propose functional roles of purinergic regulation of esophageal motility. An isolated segment of the rat esophagus was placed in an organ bath, and mechanical responses were recorded using a force transducer. Exogenous application of ATP (10-100 µM) evoked relaxation of the esophageal smooth muscle in a longitudinal direction under the condition of carbachol (1 µM) -induced precontraction. Pretreatment with a non-selective P2 receptor antagonist, suramin (500 µM), and a P2Y receptor antagonist, cibacron blue F3GA (200 µM), inhibited the ATP (100 µM) -induced relaxation, but a P2X receptor antagonist, pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (50 µM), did not affect it. A blocker of ATP-dependent potassium channels (KATP channels), glibenclamide (200 µM), inhibited the ATP-induced relaxation and application of an opener of KATP channels, nicorandil (50 µM), produced relaxation. The findings suggest that ATP is involved in inhibitory regulation of the longitudinal smooth muscle in the muscularis mucosae of the rat esophagus via activation of P2Y receptors and then opening of KATP channels.

New insights into the characterization of the mechanism of action of hyoscine butylbromide in the human colon ex vivo.

INTRODUCTION: Hyoscine butylbromide (HBB) is one of the most used antispasmodics in clinical practice. Recent translational consensus has demonstrated a similarity between human colonic motor patterns studied ex vivo and in vivo, suggesting ex vivo can predict in vivo results. It is unclear whether the mechanism of action of antispasmodics can predict different use in clinical practice. The aim of the present study is to bridge this gap dissecting HBB's role in excitatory and inhibitory neural pathways. METHODS: 309 colon samples from 48 patients were studied in muscle bath experiments. HBB was tested on: 1-spontaneous phasic contractions (SPCs); 2-carbachol-induced contractility; electrical field stimulation (EFS)-induced selective stimulation of 3-excitatory and 4-inhibitory pathways and 5- SPCs and EFS-induced contractions enhanced by neostigmine. Atropine, AF-DX116 (M2 blocker) and DAU-5884 (M3 blocker) were used as comparators. RESULTS: In the presence of tetrodotoxin (TTX), HBB and atropine 1 µM reduced SPCs. HBB and atropine concentration-dependently reduced carbachol- and EFS-induced contractions. Inhibitory effects of DAU-5884 on EFS-induced contractions were more potent than of AF-DX116. HBB did not affect the off-response associated to neural inhibitory responses. Neostigmine enhanced both SPCs and EFS-induced contractions. In the presence of TTX and ω-conotoxin (GVIA), neostigmine still enhanced SPCs. Addition of HBB and atropine reduced these responses. CONCLUSIONS: This study demonstrates that HBB inhibits neural cholinergic contractions associated to muscarinic (mainly M3) receptors. HBB has a potential role in reducing colonic spasm induced by the release of acetylcholine from enteric motor neurons and from an atypical source including a potential non-neuronal origin.

Sensitivity of the airway smooth muscle in terms of force, shortening and stiffness.

Eight pig tracheal strips were stimulated to contract with log increments of methacholine from 10-8 to 10-5 M. For each strip, the concentration-response was repeated four times in a randomized order to measure isometric force, isotonic shortening against a load corresponding to either 5 or 10 % of a reference force, and average force, stiffness, elastance and resistance over one cycle while the strip length was oscillating sinusoidally by 5 % at 0.2 Hz. For each readout, the logEC50 was calculated and compared. Isotonic shortening with a 5 % load had the lowest logEC50 (-7.13), yielding a greater sensitivity than any other contractile readout (p<0.05). It was followed by isotonic shortening with a 10 % load (-6.66), elastance (-6.46), stiffness (-6.46), resistance (-6.38), isometric force (-6.32), and average force (-6.30). Some of these differences were significant. For example, the EC50 with the average force was 44 % greater than with the elastance (p=0.001). The methacholine sensitivity is thus affected by the contractile readout being measured.

Carrageenan maintains the contractile phenotype of vascular smooth muscle cells by increasing macromolecular crowding in vitro.

BACKGROUND: The contractile phenotype of vascular smooth muscle cells (VSMCs) results in good diastolic and contractile capacities, and its altered function is the main pathophysiological basis for diseases such as hypertension. VSMCs exist as a synthetic phenotype in vitro, making it challenging to maintain a contractile phenotype for research. It is widely recognized that the common medium in vitro is significantly less crowded than in the in vivo environment. Additionally, VSMCs have a heightened sense for detecting changes in medium crowding. However, it is unclear whether macromolecular crowding (MMC) helps maintain the VSMCs contractile phenotype. PURPOSE: This study aimed to explore the phenotypic, behavioral and gene expression changes of VSMCs after increasing the crowding degree by adding carrageenan (CR). METHODS: The degree of medium crowding was examined by a dynamic light scattering assay; VSMCs survival and activity were examined by calcein/PI cell activity and toxicity and CCK-8 assays; VSMCs phenotypes and migration were examined by WB and wound healing assays; and gene expression was examined by transcriptomic analysis and RT-qPCR. RESULTS: Notably, 225 µg/mL CR significantly increased the crowding degree of the medium and did not affect cell survival. Simultaneously, CR significantly promoted the contraction phenotypic marker expression in VSMCs, shortened cell length, decreased cell proliferation, and inhibited cell migration. CR significantly altered gene expression in VSMCs. Specifically, 856 genes were upregulated and 1207 genes were downregulated. These alterations primarily affect the cellular ion channel transport, microtubule movement, respiratory metabolism, amino acid transport, and extracellular matrix synthesis. The upregulated genes were primarily involved in the cytoskeleton and contraction processes of VSMCs, whereas the downregulated genes were mainly involved in extracellular matrix synthesis. CONCLUSIONS: The in vitro study showed that VSMCs can maintain the contractile phenotype by sensing changes in the crowding of the culture environment, which can be maintained by adding CR.

Taurine activates the AKT-mTOR axis to restore muscle mass and contractile strength in human 3D in vitro models of steroid myopathy.

Steroid myopathy is a clinically challenging condition exacerbated by prolonged corticosteroid use or adrenal tumors. In this study, we engineered a functional three-dimensional (3D) in vitro skeletal muscle model to investigate steroid myopathy. By subjecting our bioengineered muscle tissues to dexamethasone treatment, we reproduced the molecular and functional aspects of this disease. Dexamethasone caused a substantial reduction in muscle force, myotube diameter and induced fatigue. We observed nuclear translocation of the glucocorticoid receptor (GCR) and activation of the ubiquitin-proteasome system within our model, suggesting their coordinated role in muscle atrophy. We then examined the therapeutic potential of taurine in our 3D model for steroid myopathy. Our findings revealed an upregulation of phosphorylated AKT by taurine, effectively countering the hyperactivation of the ubiquitin-proteasomal pathway. Importantly, we demonstrate that discontinuing corticosteroid treatment was insufficient to restore muscle mass and function. Taurine treatment, when administered concurrently with corticosteroids, notably enhanced contractile strength and protein turnover by upregulating the AKT-mTOR axis. Our model not only identifies a promising therapeutic target, but also suggests combinatorial treatment that may benefit individuals undergoing corticosteroid treatment or those diagnosed with adrenal tumors.

Generation of Chicken Contractile Skeletal Muscle Structure Using Decellularized Plant Scaffolds.

Cultured meat is a meat analogue produced by in vitro cell culture, which can replace the conventional animal production system. Tissue engineering using myogenic cells and biomaterials is a core technology for cultured meat production. In this study, we provide an efficient and economical method to produce skeletal muscle tissue-like structures by culturing chicken myoblasts in a fetal bovine serum (FBS)-free medium and plant-derived scaffolds. An FBS-free medium supplemented with 10% horse serum (HS) and 5% chick embryo extract (CEE) was suitable for the proliferation and differentiation of chicken myoblasts. Decellularized celery scaffolds (Decelery), manufactured using 1% sodium dodecyl sulfate (SDS), were nontoxic to cells and supported myoblast proliferation and differentiation. Decelery could support the 3D culture of chicken myoblasts, which could adhere and coagulate to the surface of the Decelery and form MYH1E+ and F-actin+ myotubes. After 2 weeks of culture on Decelery, fully grown myoblasts completely covered the surface of the scaffolds and formed fiber-like myotube structures. They further differentiated to form spontaneously contracting myofiber-like myotubes on the scaffold surface, indicating that the Decelery scaffold system could support the formation of a functional mature myofiber structure. In addition, as the spontaneously contracting myofibers did not detach from the surface of the Decelery, the Decelery system is a suitable biomaterial for the long-term culture and maintenance of the myofiber structures.

Paradoxical potentiation of the exercise pressor reflex by endomorphin 2 in the presence of naloxone.

When contracting muscles are freely perfused, the acid-sensing ion channel 3 (ASIC3) on group IV afferents plays a minor role in evoking the exercise pressor reflex. We recently showed in isolated dorsal root ganglion neurons innervating the gastrocnemius muscles that two mu opioid receptor agonists, namely endomorphin 2 and oxycodone, potentiated the sustained inward ASIC3 current evoked by acidic solutions. This in vitro finding prompted us to determine whether endomorphin 2 and oxycodone, when infused into the arterial supply of freely perfused contracting hindlimb muscles, potentiated the exercise pressor reflex. We found that infusion of endomorphin 2 and naloxone in decerebrated rats potentiated the pressor responses to contraction of the triceps surae muscles. The endomorphin 2-induced potentiation of the pressor responses to contraction was prevented by infusion of APETx2, an ASIC3 antagonist. Specifically, the peak pressor response to contraction averaged 19.3 ± 5.6 mmHg for control (n = 10), 27.2 ± 8.1 mmHg after naloxone and endomorphin 2 infusion (n = 10), and 20 ± 8 mmHg after APETx2 and endomorphin 2 infusion (n = 10). Infusion of endomorphin 2 and naloxone did not potentiate the pressor responses to contraction in ASIC3 knockout rats (n = 6). Partly similar findings were observed when oxycodone was substituted for endomorphin 2. Oxycodone infusion significantly increased the exercise pressor reflex over its control level, but subsequent APETx2 infusion failed to restore the increase to its control level (n = 9). The peak pressor response averaged 23.1 ± 8.6 mmHg for control (n = 9), 33.2 ± 11 mmHg after naloxone and oxycodone were infused (n = 9), and 27 ± 8.6 mmHg after APETx2 and oxycodone were infused (n = 9). Our data suggest that after opioid receptor blockade, ASIC3 stimulation by the endogenous mu opioid, endomorphin 2, potentiated the exercise pressor reflex.NEW & NOTEWORTHY This paper provides the first in vivo evidence that endomorphin 2, an endogenous opioid peptide, can paradoxically increase the magnitude of the exercise pressor reflex by an ASIC3-dependent mechanism even when the contracting muscles are freely perfused.

PTHrP attenuates spontaneous contractions in detrusor smooth muscle of the rat bladder by activating spontaneous transient outward potassium currents.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca2+-activated K+ channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca2+ dynamics were visualized using Cal520 AM -loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10 nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30 mV but not - 40 mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20 mV in a manner sensitive to iberiotoxin (100 nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (CPA 10 µM), L-type voltage-dependent Ca2+ channel (LVDCC) (nifedipine 3 µM) or adenylyl cyclase (SQ22536 100 µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100 nM)-sensitive manner. PTHrP also reduced the number of Ca2+ spikes during each burst of spontaneous Ca2+ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca2+ release which prematurely terminates Ca2+ transient bursts resulting in the attenuation of SPCs.

FLECS technology for high-throughput screening of hypercontractile cellular phenotypes in fibrosis: A function-first approach to anti-fibrotic drug discovery.

The pivotal role of myofibroblast contractility in the pathophysiology of fibrosis is widely recognized, yet HTS approaches are not available to quantify this critically important function in drug discovery. We developed, validated, and scaled-up a HTS platform that quantifies contractile function of primary human lung myofibroblasts upon treatment with pro-fibrotic TGF-ß1. With the fully automated assay we screened a library of 40,000 novel small molecules in under 80 h of total assay run-time. We identified 42 hit compounds that inhibited the TGF-ß1-induced contractile phenotype of myofibroblasts, and enriched for 19 that specifically target myofibroblasts but not phenotypically related smooth muscle cells. Selected hits were validated in an ex vivo lung tissue models for their inhibitory effects on fibrotic gene upregulation by TGF-ß1. Our results demonstrate that integrating a functional contraction test into the drug screening process is key to identify compounds with targeted and diverse activity as potential anti-fibrotic agents.

Does L-carnitine prevent cadmium-induced damage in gastrointestinal contractility and histological changes in prepubertal rat / ¿La L-carnitina previene el daño inducido por cadmio en la contractilidad gastrointestinal y los cambios histológicos en ratas prepúberes?

SUMMARY: Cadmium (Cd) is the industrial and environmental toxic heavy metal which is found in air, water and soil. Cd, adversely affects many organs in humans such as kidney, intestine, liver, testis and lungs. L-carnitine (LC) is an important agent that plays essential role in energy metabolism. In our study, we aimed to work out whether LC application has any protective effect on intestinal contractility and morphologic damage of prepubertal rat duodenum on Cd-induced toxicity. Twenty eight prepubertal female Wistar rats were divided into four groups. The first group is control (C), second group; Cd group; Cadmium chloride was given 2 mg/kg 28 days with a one-day break by i.p. The third group; Cd+LC, which cadmium chloride was given 2 mg/kg i.p. and LC was given orally by gastric lavage. The LC dose was given as 75 mg/kg. The fourth group; LC, which only LC was given orally. The intestinal segments were isolated and suspended in tissue bath. Contractile responses were induced by acetylcholine (ACh) and relaxation was achieved with phenylephrine. Also the segments were examined for histological changes by light microscopy. Ach-induced contractions were higher in Cd+LC, LC, and control group compared to the Cd group in duodenal segments. The phenylephrine-induced relaxations were lower in Cd groups as compared with Control, Cd+LC and LC group in duodenal segments. In Cd group intestinal morphology was observed to be severely damaged whereas in Cd+LC group the damage was noticeably lower. Cd administration caused severe cellular damage and decreased gastrointestinal motility. Treatment with the LC has affected the gastrointestinal contractility and reduced the damage in intestinal morphology, which occured after Cd application.

El cadmio (Cd) es el metal pesado tóxico industrial y ambiental que se encuentra en el aire, el agua y el suelo. El Cd afecta negativamente a muchos órganos humanos, como los riñones, los intestinos, el hígado, los testículos y los pulmones. La L-carnitina (LC) es un agente importante que juega un rol esencial en el metabolismo energético. El objetivo de este estudio fue determinar si la aplicación de LC tiene algún efecto protector sobre la contractilidad intestinal y el daño morfológico del duodeno de rata prepuberal sobre la toxicidad inducida por Cd. Veintiocho ratas Wistar hembras prepúberes se dividieron en cuatro grupos. El primer grupo control (C), segundo grupo; grupo cd; Se administró cloruro de cadmio 2 mg/kg durante 28 días con un descanso de un día por vía i.p. El tercer grupo; Cd+LC, al que se administró cloruro de cadmio 2 mg/kg i.p. y LC se administró por vía oral mediante lavado gástrico. La dosis de LC se administró como 75 mg/kg. El cuarto grupo; LC, al cual solo LC se administraba por vía oral. Los segmentos intestinales fueron aislados y suspendieron en baño de tejido. Las respuestas contráctiles fueron inducidas por acetilcolina (ACh) y la relajación se logró con fenilefrina. También se examinaron los segmentos en busca de cambios histológicos mediante microscopía óptica. Las contracciones inducidas por Ach fueron mayores en Cd+LC, LC y el grupo control en comparación con el grupo Cd en los segmentos duodenales. Las relajaciones inducidas por fenilefrina fueron menores en los grupos Cd en comparación con el grupo Control, Cd+LC y LC en los segmentos duodenales. En el grupo Cd se observó que la morfología intestinal estaba severamente dañada mientras que en el grupo Cd+LC el daño fue notablemente menor. La administración de Cd causó daño celular severo y disminución de la motilidad gastrointestinal. El tratamiento con LC afectó la contractilidad gastrointestinal y redujo el daño en la morfología intestinal, que ocurría después de la aplicación de Cd.

Comparison of Rheological Properties of Healthy versus Dupuytren Fibroblasts When Treated with a Cell Contraction Inhibitor by Atomic Force Microscope.

Mechanical properties of healthy and Dupuytren fibroblasts were investigated by atomic force microscopy (AFM). In addition to standard force curves, rheological properties were assessed using an oscillatory testing methodology, in which the frequency was swept from 1 Hz to 1 kHz, and data were analyzed using the structural damping model. Dupuytren fibroblasts showed larger apparent Young's modulus values than healthy ones, which is in agreement with previous results. Moreover, cell mechanics were compared before and after ML-7 treatment, which is a myosin light chain kinase inhibitor (MLCK) that reduces myosin activity and hence cell contraction. We employed two different concentrations of ML-7 inhibitor and could observe distinct cell reactions. At 1 µM, healthy and scar fibroblasts did not show measurable changes in stiffness, but Dupuytren fibroblasts displayed a softening and recovery after some time. When increasing ML-7 concentration (3 µM), the majority of cells reacted, Dupuytren fibroblasts were the most susceptible, not being able to recover from the drug and dying. These results suggested that ML-7 is a potent inhibitor for MLCK and that myosin II is essential for cytoskeleton stabilization and cell survival.

Trans-ε-viniferin as an inhibitor of TMEM16A preventing intestinal smooth muscle contraction.

TMEM16A regulator is an important tool to study the physiological functions and pathogenesis related to TMEM16A. In the present study, trans-ε-viniferin (TV) was identified as a TMEM16A inhibitor with inhibitory activity against TMEM16A mediated Cl- currents, which was reversible, without affecting intracytoplasmic Ca2+ concentration and TMEM16A protein expression. TV inhibited intestinal peristalsis and prolonged gastrointestinal transport time. TV could inhibit autonomic and Eact-stimulated intestinal contractility, and was equally effective in ACh- and HA-induced high contractile states. The results indicate that TV significantly inhibits the intestinal smooth muscle contraction, which may be applied in the treatment of TMEM16A-related intestinal dynamic abnormalities.

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.

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.

Effects of Cardiotoxins from Naja oxiana Cobra Venom on Rat Heart Muscle and Aorta: A Comparative Study of Toxin-Induced Contraction Mechanisms.

Cardiotoxins (CaTxs) are a group of snake toxins that affect the cardiovascular system (CVS). Two types (S and P) of CaTxs are known, but the exact differences in the effects of these types on CVS have not been thoroughly studied. We investigated cellular mechanisms of action on CVS for Naja oxiana cobra CaTxs CTX-1 (S-type) and CTX-2 (P-type) focusing on the papillary muscle (PM) contractility and contraction of aortic rings (AR) supplemented by pharmacological analysis. It was found that CTX-1 and CTX-2 exerted dose-dependent effects manifested in PM contracture and AR contraction. CTX-2 impaired functions of PM and AR more strongly than CTX-1. Effects of CaTxs on PM were significantly reduced by nifedipine, an L-type Ca2+ channel blocker, and by KB-R7943, an inhibitor of reverse-mode Na+/Ca2+ exchange. Furthermore, 2-aminoethoxydiphenyl borate, an inhibitor of store-operated calcium entry, partially restored PM contractility damaged by CaTxs. The CaTx influence on AR contracture was significantly reduced by nifedipine and KB-R7943. The involvement of reverse-mode Na+/Ca2+ exchange in the effect of CaTxs on the rat aorta was shown for the first time. The results obtained indicate that CaTx effects on CVS are mainly associated with disturbance of transporting systems responsible for the Ca2+ influx.

Balanced modulation of neuromuscular synaptic transmission via M1 and M2 muscarinic receptors during inhibition of cholinesterases.

Organophosphorus (OP) compounds that inhibit acetylcholinesterase are a common cause of poisoning worldwide, resulting in several hundred thousand deaths each year. The pathways activated during OP compound poisoning via overstimulation of muscarinic acetylcholine receptors (mAChRs) play a decisive role in toxidrome. The antidotal therapy includes atropine, which is a nonspecific blocker of all mAChR subtypes. Atropine is efficient for mitigating depression in respiratory control centers but does not benefit patients with OP-induced skeletal muscle weakness. By using an ex vivo model of OP-induced muscle weakness, we studied the effects of the M1/M4 mAChR antagonist pirenzepine and the M2/M4 mAChR antagonist methoctramine on the force of mouse diaphragm muscle contraction. It was shown that weakness caused by the application of paraoxon can be significantly prevented by methoctramine (1 µM). However, neither pirenzepine (0.1 µM) nor atropine (1 µM) was able to prevent muscle weakness. Moreover, the application of pirenzepine significantly reduced the positive effect of methoctramine. Thus, balanced modulation of neuromuscular synaptic transmission via M1 and M2 mAChRs contributes to paraoxon-induced muscle weakness. It was shown that methoctramine (10 µmol/kg, i.p.) and atropine (50 µmol/kg, i.p.) were equieffective toward increasing the survival of mice poisoned with a 2xLD50 dose of paraoxon.

Lippia origanoides essential oil induces tocolytic effect in virgin rat uterus and inhibits writhing in a dysmenorrhea mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: The species Lippia origanoides Kunth, popularly known as "salva-de-marajó", is used in Brazilian traditional "quilombola" communities to treat menstrual cramps and uterine inflammation. AIM OF THE STUDY: Evaluate the spasmolytic activity of Lippia origanoides essential oil (LOO) on experimental models of uterine conditions related to menstrual cramps and investigate its mechanism of action. MATERIALS AND METHODS: Virgin rat-isolated uterus was mounted in the organ bath apparatus to evaluate the spasmolytic effect of LOO on basal tonus and contractions induced by carbachol, KCl, or oxytocin. We used pharmacological agents to verify the relaxation mechanism of LOO. The evaluation of uterine contractility in virgin rats, after treatment with LOO for three consecutive days, was carried out by the construction of a concentration-response curve with oxytocin or carbachol. The primary dysmenorrhea animal model was replicated with an injection of estradiol cypionate in female mice for three consecutive days, followed by intraperitoneal application of oxytocin. RESULTS: LOO relaxed the rat uterus precontracted with 10-2 IU/mL oxytocin (logEC50 = 1.98 ± 0.07), 1 µM carbachol (logEC50 = 1.42 ± 0.07) or 60 mM KCl (logEC50 = 1.53 ± 0.05). It was also able relax uterus on spontaneous contractions (logEC50 = 0.41 ± 0.05). Preincubation with glibenclamide, propranolol, phentolamine or L-NAME in contractions induced by carbachol did not alter significantly the relaxing effect of LOO. However, in the presence of 4-aminopyridine, CsCl or tetraethylammonium there was a reduction of LOO potency, whereas the blockers methylene blue, ODQ, aminophylline and heparin potentiated the LOO relaxing effect. Preincubation with LOO in a Ca2+ free medium at concentrations of 27 µg/mL or 81 µg/mL reduced the contraction induced by carbachol. The administration of LOO for 3 days did not alter uterus contractility. The treatment with LOO at 30 or 100 mg/kg intraperitoneally, or 100 mg/kg orally, inhibited writhing in female mice. The association of LOO at 10 mg/kg with nifedipine or mefenamic acid potentiated writhing inhibition in mice. CONCLUSIONS: The essential oil of L. origanoides has tocolytic activity in rat isolated uterus pre-contracted with KCl, oxytocin, or carbachol. This effect is possibly related to the opening of potassium channels (Kir, KV, and KCa), cAMP increase, and diminution of intracellular Ca2+. This relaxant effect, probably, contributed to reduce the number of writhings in an animal model of dysmenorrhea being potentiated by nifedipine or mefenamic acid. Taken together, the results here presented indicate that this species has a pharmacological potential for the treatment of primary dysmenorrhea, supporting its use in folk medicine.

Platelet-activating factor (PAF) strongly enhances contractile mechanical activities in guinea pig and mouse urinary bladder.

In this study, we investigated the effects of platelet-activating factor (PAF) on the basal tone and spontaneous contractile activities of guinea pig (GP) and mouse urinary bladder (UB) smooth muscle (UBSM) tissues to determine whether PAF could induce UBSM tissue contraction. In addition, we examined the mRNA expression of the PAF receptor, PAF-synthesizing enzyme (lysophosphatidylcholine acyltransferase, LPCAT), and PAF-degrading enzyme (PAF acetylhydrolase, PAF-AH) in GP and mouse UB tissues using RT-qPCR. PAF (10-9-10-6 M) strongly enhanced the basal tone and spontaneous contractile activities (amplitude and frequency) of GP and mouse UBSM tissues in a concentration-dependent manner. The enhancing effects of PAF (10-6 M) on both GP and mouse UBSM contractile activities were strongly suppressed by pretreatment with apafant (a PAF receptor antagonist, GP: 10-5 M; mouse: 3 × 10-5 M). The PAF receptor (Ptafr), LPCAT (Lpcat1, Lpcat2), and PAF-AH (Pafah1b3, Pafah2) mRNAs were detected in GP and mouse UB tissues. These findings reveal that PAF strongly enhances the contractile mechanical activities of UBSM tissues through its receptor and suggest that the PAF-synthesizing and -degrading system exists in UBSM tissues. PAF may serve as both an endogenous UBSM constrictor and an endogenous mediator leading to detrusor overactivity.

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.

BoNT/A in the Urinary Bladder-More to the Story than Silencing of Cholinergic Nerves.

Botulinum neurotoxin (BoNT/A) is an FDA and NICE approved second-line treatment for overactive bladder (OAB) in patients either not responsive or intolerant to anti-cholinergic drugs. BoNT/A acts to weaken muscle contraction by blocking release of the neurotransmitter acetyl choline (ACh) at neuromuscular junctions. However, this biological activity does not easily explain all the observed effects in clinical and non-clinical studies. There are also conflicting reports of expression of the BoNT/A protein receptor, SV2, and intracellular target protein, SNAP-25, in the urothelium and bladder. This review presents the current evidence of BoNT/A's effect on bladder sensation, potential mechanisms by which it might exert these effects and discusses recent advances in understanding the action of BoNT in bladder tissue.