Smooth Muscle Contractile Responses Research Articles

Functional anticholinergic activity of drugs classified as strong and moderate on the anticholinergic burden scale on bladder and ileum.

Several medications are commonly administered to older Japanese patients. Since some of them have not been included in previously developed scales to estimate the anticholinergic burden, we have developed a new muscarinic receptor binding-based anticholinergic burden scale. This study aimed to investigate the functional inhibitory effects of 60 medications, classified as anticholinergic burden scales 3 and 2 by the anticholinergic burden scale, on muscarinic receptor-mediated contractions in the bladder and ileum. The relaxation response induced by these drugs on isolated rat bladders and ileum smooth muscles constricted by carbachol was assessed using the organ bath method. All drugs inhibited smooth muscle contractile responses induced by the muscarinic receptor activation in a concentration-dependent manner in the rat bladder and ileum. Notably, variations were observed in the relaxation responses of the drugs, and the function EC50 values were positively correlated with the binding IC50 values in the bladder and ileum. The results of this study provide functional pharmacological evidence for the muscarinic receptor binding-based anticholinergic burden scale. Implementation of this scale may help reduce the risk of constipation and urinary retention, which are common side effects associated with anticholinergic drugs.

Ex Vivo Study of Colon Health, Contractility and Innervation in Male and Female Rats after Regular Exposure to Instant Cascara Beverage.

Instant Cascara (IC) is a sustainable beverage made from dried coffee cherry pulp, a by-product of coffee processing. It is rich in nutrients and bioactive compounds and has a high concentration of antioxidants. This study explored the impact of regular IC consumption on colonic motor function and innervation. Over a period of 4 weeks, male and female healthy rats were given drinking water containing 10 mg/mL of IC. Thereafter, colon samples were obtained to evaluate the longitudinal (LM) and circular (CM) smooth muscle contractile response to acetylcholine (ACh) and electrical field stimulation (EFS) in an organ bath, before and after atropine administration (10-6 M). Histological and immunohistochemical analyses assessed colon damage, muscle thickness, and immunoreactivity to substance P (SP) and neuronal nitric oxide synthase (nNOS). ACh and EFS induced similar responses across groups, but the CM response to EFS was greater in females compared with males, despite their lower body weight. Atropine completely blocked the response to ACh but only partially antagonized the neural response to EFS, particularly that of CM in females treated with IC, which had a greater liquid intake than those exposed to water. However, in the myenteric ganglia, no statistically significant differences were observed in SP or nNOS. Our results suggest that regular IC exposure may enhance specific neural pathway functions, particularly in females, possibly due to their increased IC consumption.

Spasmolytic Activity of 1,3-Disubstituted 3,4-Dihydroisoquinolines.

This article concerns the spasmolytic activities of some novel 1,3-disubstituted 3,4-dihydroisoquinolines. These compounds can be evaluated as potential therapeutic candidates according to Lipinski's rule of five, showing high gastrointestinal absorption and the ability to cross the blood-brain barrier, which is a very important parameter in the drug discovery processes. In silico simulation predicted smooth muscle relaxant activity for all the compounds. Since smooth muscle contractile failure is a characteristic feature of many disorders, in the current paper, we concentrate on the parameters of the spontaneous contractile responses of smooth muscle (SM) cells compared to the well-known drug mebeverine. Two of the newly synthesized substances can be identified as essential modulating regulators and potentially used as therapeutic molecules. One of these molecules also showed significant DPPH antioxidant activity compared to rutin.

Bile acids differentially regulate longitudinal smooth muscle contractility in everted mouse ileum.

Bile acids regulate gastrointestinal motility by mechanisms that are poorly understood. Standard isolated tissue bath assays might not recapitulate invivo physiology if contractile responses to certain bile acids require direct application to the intestinal mucosa. We sought to determine the feasibility of quantifying longitudinal smooth muscle contractile responses to bile acids from intact segments of everted mouse ileum. Ileum from adult female C57BL/6J mice was isolated, gently everted over a notched metal rod, and mounted in tissue baths. Individual bile acids and agonists of bile acid receptors were added to the baths, and longitudinal smooth muscle contractile responses were quantified by isometric force transduction. Ursodeoxycholic acid robustly increased contractile responses in a dose-dependent manner. Deoxycholic acid stimulated contractility at low doses but inhibited contractility at high doses. Chenodeoxycholic acid, glycocholic acid, and lithocholic acid did not alter contractility. The dose-dependent increase in contractility resulting from the application of ursodeoxycholic acid was recapitulated by INT-777, an agonist of the Takeda G protein-coupled receptor 5 (TGR5), and by cevimeline, a muscarinic acetylcholine receptor agonist. Agonists to the nuclear receptors farnesoid X receptor, glucocorticoid receptor, pregnane X receptor, vitamin D receptor, and to the plasma membrane epidermal growth factor receptor did not modify baseline contractile patterns. These results demonstrate that gentle eversion of intact mouse ileum facilitates the quantification of longitudinal smooth muscle contractile responses to individual bile acids. Prokinetic effects of ursodeoxycholic acid and low-dose deoxycholic acid are replicated by agonists to TGR5 and muscarinic acetylcholine receptors.

Smooth muscle contractile responses to bile acids in mouse ileum require TGR5 but not ASBT.

Many disorders of gut-brain interaction (DGBIs) are more prevalent in women than men and feature alterations in gastrointestinal motility and bile acid homeostasis. Mechanisms by which bile acids regulate gastrointestinal motility are poorly characterized. We recently validated an adapted tissue bath technique using everted mouse ileum, which revealed differential contractile responses to ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA). Here, we aimed to determine whether these responses are dependent on host sex, the plasma membrane bile acid receptor TGR5, or the apical sodium-dependent bile acid transporter ASBT. Ileal segments from male and female mice were everted and suspended in tissue baths. Contractile responses to physiologic concentrations of UDCA and DCA were quantified with or without TGR5 or ASBT inhibitors. Phosphorylation of extracellular signal-regulated kinase (ERK) and myosin light chain (MLC), markers of TGR5 activation and smooth muscle contraction, respectively, were assessed with western blot. There were no sex differences in the dose-dependent contractile responses to bile acids. At 100 μmol/L, UDCA but not DCA increased MLC phosphorylation and increased contractility. TGR5 inhibition decreased ERK phosphorylation and led to decreases in contractility, phosphorylated MLC, and surprisingly, total MLC. ASBT inhibition did not affect contractile responses. Differential effects of UDCA and DCA on ileal smooth muscle contractility are not dependent on host sex or ASBT-mediated transport. Bile acids signal through mucosal TGR5, which regulates smooth muscle contractility by complex mechanisms. Understanding how bile acids differentially regulate gastrointestinal motility could facilitate new therapeutic options for specific DGBIs.

Measurement of Endothelium-dependent Vasorelaxation in the Mouse Thoracic Aorta using Tensometric Small Volume Chamber Myography.

Small volume chamber tensometric myography is a commonly used technique to evaluate the vascular contractility of small and large blood vessels in laboratory animals and small arteries isolated from human tissue. The technique allows researchers to maintain isolated blood vessels in a tightly controlled and standardized (near-physiological) setting, with the option of adjusting to various environmental factors, while challenging the isolated vessels with different pharmacological agents that can induce vasoconstriction or vasodilation. The myograph chamber also provides a platform to measure vascular reactivity in response to various hormones, inhibitors, and agonists that may impact the function of smooth muscle and endothelial layers separately or simultaneously. The blood vessel wall is a complex structure consisting of three different layers: the intima (endothelial layer), media (smooth muscle and elastin fibers), and adventitia (collagen and other connective tissue). To gain a clear understanding of the functional properties of each layer, it is critical to have access to an experimental platform and system that would allow for a combinational approach to study all three layers simultaneously. Such an approach demands access to a semi-physiological condition that would mimic the in vivo environment in an ex vivo setting. Small volume chamber tensometric myography has provided an ideal environment to evaluate the impact of environmental cues, experimental variables, or pharmacological agonists and antagonists on vascular properties. For many years, scientists have used the tensometric myograph technique to measure endothelial function and smooth muscle contractility in response to different agents. In this report, a small volume chamber tensometric myograph system is used to measure endothelial function in the isolated mouse aorta. This report focuses on how small volume chamber tensometric myography can be used to evaluate the functional integrity of the endothelium in small segments of a large artery such as the thoracic aorta.

Uncovering the Effects of Estrogen in an iPSC Derived‐Vascular Smooth Muscle Model of Marfan Syndrome

The phenotype and severity of cardiovascular diseases, such as a thoracic aortic aneurysm and dissection and atherosclerosis, differ between females and males. Notably, coronary artery dissection risk increases four‐fold in women during late pregnancy and postpartum, suggesting that female sex hormones such as estrogen may play a role in vascular pathophysiology, yet are considered protective under most other circumstances. This paradox underscores the fact that the effect of estrogen on the vasculature is not fully understood at a molecular level, and most previous work in this area has been done using immortalized cell lines or animal models, which may not accurately model human responses. Furthermore, examining these effects in genetic diseases such as Marfan syndrome (MFS) allows us to view these effects in an aneurysm phenotypic model. Marfan syndrome is a connective tissue disorder caused by a mutation in the fibrillin‐1 gene. Individuals typically develop aortic root aneurysms with aortic disruption and rupture that remains a leading cause of death. Here, we used Healthy and MFS iPSC‐derived vascular smooth muscle cells (iVSMCs) to examine the effects of estrogen. Healthy and MFS IPSC’s were differentiated into iVSMCs for 21 days then treated with estrogen for 72hrs. Immunofluorescence of smooth muscle markers and contractility assays were conducted to confirm a contractile vascular smooth muscle phenotype. A proteomic profile was established to evaluate molecular differences. Peptide data was collected using a Thermo Fusion Lumos Orbitrap mass spectrometer. Data were analyzed using the DIA‐Neural networks software. We showed expression of smooth muscle markers and contractile response to stimuli. MFS iVSMCs showed decreased contractility response and a decrease in FBN1 expression compared to healthy control (MFS iVSMC vs Control iVMSC of‐1.4). The proteomic profile revealed an upregulation in proteins related to elastin fiber formation and cell adhesion in both Healthy and Marfan iVSMCs following estrogen treatment. FBN1 expression increased in response to estrogen in both Healthy and Marfan iVSMC. As well as increases in Lysyl Oxidase and TGFB2 expression in MFS iVSMC following treatment. We also see a downregulation of collagen expression and RNA synthesis in healthy iVSMCs. These findings will allow us to gain a greater understanding of the complex interaction estrogen has on the vasculature and to determine their role in cardiovascular disease risk.

Comparison of 2 single incision slings on the vagina in an ovine model

Stress urinary incontinence carries a significant healthcare burden for women worldwide. Single incision slings are minimally invasive mesh devices designed to treat stress urinary incontinence. For prolapse repair, meshes with higher porosity and lower structural stiffness have been associated with improved outcomes. In this study, we compared the higher stiffness, lower porosity Altis sling with the lower stiffness, higher porosity Solyx sling in an ovine model. We hypothesized that SIS-B would have a negative impact on the host response. A total of Altis and Solyx single incision slings were implanted suburethrally into sheep according to the manufacturer's instructions on minimal tension. The mesh-urethral-vaginal complex and adjacent ungrafted vagina (no mesh control) were harvested en bloc at 3 months. Masson's trichrome and picrosirius red staining of 6 μm thin sections was performed to measure interfiber distance and tissue integration. Smooth muscle contractility to a 120 mM KCl stimulus was performed in an organ bath to measure myofiber-driven contractions. Standard biochemical assays were used to quantify glycosaminoglycan, total collagen, and elastin content, and collagen subtypes. Bending stiffness was performed in response to a uniaxial force to define susceptibility to folding/buckling. Statistical analysis was performed using Mann-Whitney, Gabriel's pairwise post hoc, Wilcoxon matched-pairs, and chi-square tests. The animals had similar ages (3-5 years), parity (multiparous), and weights (45-72 kg). Trichrome cross sections showed that the Altis sling buckled in a "C" or "S" shape in most samples (8 of 11), whereas buckling after Solyx sling implantation was observed in only a single sample (1 of 13; P=.004). Tissue integration, as measured by the presence of collagen or smooth muscle between the mesh fibers on trichrome 4× imaging, was increased in samples implanted with the Solyx sling compared with the Altis sling (P<.05). Total collagen content decreased significantly with both products when compared with the ungrafted vagina consistent with stress shielding. There was no difference in the 2 groups with regard to glycosaminoglycan or elastin content. The Altis sling mesh tissue complex demonstrated significantly higher amounts of both collagen types I and III than the Solyx sling-implanted tissue and the ungrafted control. Smooth muscle contractility in response to 120 mM KCl was decreased after implantation of both slings compared with the sham (P=.011 and P<.01), with no difference between mesh types (P=.099). Bending stiffness in the Altis sling was more than 4 times lower than in the Solyx, indicating an increased propensity to buckle (0.0186 vs 0.0883). The structurally stiffer Altis sling had decreased tissue integration and increased propensity to buckle after implantation. Increased collagen types I and III after the implantation of this device suggests that these changes may be associated with a fibrotic response. In contrast, the Solyx sling largely maintained a flat configuration and had improved tissue integration. The deformation of the Altis sling is not an intended effect and is likely caused by its lower bending stiffness. Both meshes induced a decrease in collagen content and smooth muscle contractility similar to previous findings for prolapse meshes and consistent with stress shielding. The long-term impact of buckling warrants further investigation.

Thymoquinone reduces ischemia and reperfusion-induced intestinal injury in rats, through anti-oxidative and anti-inflammatory effects.

The aim of the present study was to investigate the effect of thymoquinone on ischemia/reperfusion (I/R) injury at 150 min or/and 24 h of reperfusion in male Wistar Rats. The therapeutic value of thymoquinone on cellular damage caused by reactive oxygene species or inflammatory processes during intestinal ischemia/reperfusion was investigated using pharmacological function studies on smooth muscle contractile responses of acetylcholine (Ach) and KCl, along with myeloperoxidase activity, malondialdehyhde, glutathione and cytokine levels such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β in serum and ileum tissue of rats. Thymoquinone was administered at a dose of 50 mg/kg orally for three times: 30 min, 24 h and 48 h prior to the surgical procedure. Soon after reperfusion timing (150 min or 24 h), the contractility traces to KCl and acetylcholine of the ileum smooth muscle were recorded through isolated organ bath. Pretreatment with thymoquinone reversed the disrupted contractility of the ileum smooth muscle at the 24 h reperfusion. Increased malondialdehyde and depleted glutathione levels and high myeloperoxidase activity determined in the ileum I/R tissue returned to reasonable amounts by pretreatment of Thymoquinone, which attenuated malondialdehyde quantity, restored glutathione level and inhibited myeloperoxidase activity. In addition, both serum and tissue TNF-α and IL-1β activities were modulated by thymoquinone at 24 h of intestinal I/R. The results indicate that thymoquinone may have therapeutic value due to its immunomodulating, radical scavenging and/or antioxidant effects in intestinal I/R injury including oxidant damage mechanisms.

Simulated vaginal delivery causes transients vaginal smooth muscle hypersensitivity and urethral sphincter dysfunction.

Although pelvic floor dysfunction (PFD) has a multifactorial etiology, pregnancy and childbirth are considered crucial events predisposing to urinary incontinence as well as pelvic organ prolapse, which are highly prevalent. Rats are the most frequently used animal model and pudendal nerve crush (PNC) and vaginal distension (VD) are often used to mimic vaginal delivery. To document the time course of events after simulated vaginal delivery (SVD) on the urethral sphincter and the vaginal smooth muscle layer. Virgin female Sprague-Dawley rats were subjected to SVD (PNC + VD) or sham surgery and evaluated at 7, 14, 21, and 42 days after the injury. Urethral function was determined in vivo by microultrasound during cystometry and vaginal smooth muscle layer was harvested for in vitro pharmacologic investigation by isometric tension recording. Furthermore, vaginal and urethral samples were investigated by immunohistochemistry and real-timequantitative polymerase chain reaction. Microultrasound showed no bursting of the urethral sphincter in the SVD group at 7 days with a functional recovery starting at 14 days, and normal bursting at 21 and 42 days. Vaginal smooth muscle showed higher sensitivity to carbachol at 14 and 21 days after injury; however, at 42 days, its sensitivity decreased when compared with sham. SVD induces urethral dysfunction and a shift in vaginal smooth muscle contractile responses to carbachol.

Особливості спазмолітичної дії препарату «Беластезин»

Мета дослідження – порівняльне вивчення спазмолітичної активності препарату «Беластезин», таблетки та атропіну на моделі ацетилхолін-залежної скоротливої активності (міогенної та нейрогенної) ізольованих смужок гладеньких м’язів кишківника (ГМК) мурчаків. Дослідження було проведене на 10 мурчаках-самцях (масою 300–350 г). Для запобігання можливості інтерферуючого впливу бензокаїну на скоротливі реакції гладеньких м’язів вилучення алкалоїдів з таблеткової маси проводили у водному розчині, а на смужках ГМК тестували лише фракції водорозчинних сполук. Зважаючи на загальну подібність механізму дії та фізіологічних ефектів, специфічну активність Беластезину порівнювали з атропіну сульфатом. Введення досліджуваного препарату та атропіну в діапазоні концентрацій (10–9–10–6 моль/л) у камеру зі смужками одночасно з ацетилхоліном (АХ) (10–6 моль/л) викликало зниження амплітуди та інтегральної інтенсивності АХ-стимульованої скоротливої активності ГМК. Беластезин відрізнявся від атропіну за характером антагоністичної взаємодії з М-холінорецепторами, про що свідчили зміни в нахилі кривої «концентрація-ефект» (рівняння Больцмана). Беластезин у діапазоні низьких концентрацій (10–10–10–7 моль/л) підвищував інтенсивність скоротливої діяльності смужок у разі електростимуляції, тоді як атропін знижував скоротливу активність ГМК. Відмінності в дії препарату, які були відмічені за умов функціонального тесту, дозволяють висловити припущення, що Беластезин виявляв активність більш характерну для скополаміну (гіосцину), ніж для атропіну (гіосціаміну). В умовах дозозалежної АХ-стимуляції скоротливої активності ГМК Беластезин викликав суттєве зрушення кривих «концентрація-ефект» порівняно з помірним ефектом атропіну. Досліджуваний препарат також зменшував амплітуду максимальної відповіді на відміну від атропіну. Отримані результати свідчать про те, що природна суміш алкалоїдів у складі Беластезину більш ефективна, ніж атропін щодо усунення спастичної діяльності гладеньких м’язів, яка спричинена стимуляцією М-холінорецепторів.

Smoothelin-like 1 deletion enhances myogenic reactivity of mesenteric arteries with alterations in PKC and myosin phosphatase signaling

The role of the smoothelin-like 1 (SMTNL1) protein in mediating vascular smooth muscle contractile responses to intraluminal pressure was examined in resistance vessels. Mesenteric arterioles from wild type (WT) and SMTNL1 global knock-out (KO) mice were examined with pressure myography. SMTNL1 deletion was associated with enhanced myogenic tone in vessels isolated from male, but not female, mice. Intraluminal pressures greater than 40 mmHg generated statistically significant differences in myogenic reactivity between WT and KO vessels. No overt morphological differences were recorded for vessels dissected from KO animals, but SMTNL1 deletion was associated with loss of myosin phosphatase-targeting protein MYPT1 and increase in the myosin phosphatase inhibitor protein CPI-17. Additionally, we observed altered contractile responses of isolated arteries from SMTNL1 KO mice to phenylephrine, KCl-dependent membrane depolarization and phorbol 12,13-dibutyrate (PDBu). Using pharmacological approaches, myogenic responses of both WT and KO vessels were equally affected by Rho-associated kinase (ROCK) inhibition; however, augmented protein kinase C (PKC) signaling was found to contribute to the increased myogenic reactivity of SMTNL1 KO vessels across the 60–120 mmHg pressure range. Based on these findings, we conclude that deletion of SMTNL1 contributes to enhancement of pressure-induced contractility of mesenteric resistance vessels by influencing the activity of myosin phosphatase.

The modulation of ureteral smooth muscle contractile responses by α1- and α2-adrenoceptor activation.

This study was performed to investigate the influence of α-adrenoceptor subtypes upon ureteral smooth muscle contractile responses. Rat ureters were challenged in vitro with noradrenaline (NA), the α1-adrenoceptor agonist phenylephrine (PE), and the α2-adrenoceptor agonist clonidine (CLON). The influences of the agonists on the magnitude and frequency of acetylcholine (ACh)-stimulated phasic contractile responses were recorded. The magnitude of the phasic contractile responses effected by ACh was not significantly influenced by the adrenoceptor agonists, but the frequency of the response was significantly enhanced by all three agonists (p < 0.05). Idazoxan and prazosin abolished the rise in frequency effected by CLON and PE, respectively, whereas both antagonists in combination were required to abolish the increase in frequency effected by NA. It has been demonstrated that α1- and α2-adrenoceptors modulate the contractile function of rat ureteral smooth muscle by increasing the frequency, but not the magnitude, of phasic contractile responses. The enhancement of contractile function by NA is mediated by mechanisms dependent upon both α1- and α2-adrenoceptors.

Phosphodiestarase‐9 Inhibitor (BAY 73‐6691) Reduces the Cavernous Smooth Muscle Contractile Response and Improves the Effect of Chronic Treatment with Tadalafil in Heart Failure

INTRODUCTIONPatients with heart failure (HF) display erectile dysfunction (ED). Currently a pharmacological option for the ED treatment are the phosphodiesterase (PDE) 5 inhibitors, however, these has no complete satisfactory response in all patients. The role of other PDEs to treat the ED remains poorly investigated. PDE‐9 specifically hydrolyzes cyclic‐GMP, and was detected in mice and human corpus cavernosum (CC) moreover, the PDE9 inhibitor selective (BAY 73‐6691) amplifies the NO‐cGMP‐mediated cavernosal responses, may be of therapeutic value for ED treatment associated with HF.OBJECTIVEThe aims of the present study were to characterize the effect of BAY 73‐6691, both on the contractile response of rats CC submitted to aortocaval fistula (ACF) model, as in the CC contractile response of rats with HF submitted to chronic treatment with tadalafil.METHODSMale Sprague‐Dawley rats (250g) were submitted to the ACF. After 48 hours, the animals were divided in SHAM and ACF groups (acute protocol). After 8 weeks, to validation of HF, the animals underwent to echocardiogram and were divided into 4 groups: SHAM and HF groups (0.9% saline solution, daily), and TADALAFIL and HF TADALAFIL groups (treated with Tadalafil 5 mg/daily), the treatment was maintained for 4 weeks (chronic protocol). Echocardiogram were used to evaluate the final cardiac function. In all protocols, cumulative concentration‐response curve to phenylephrine (PE; 10 nM‐100μM) and neurogenic contractile responses induced by electrical‐field stimulation (EFS; 1‐32 Hz) were obtained in cavernous smooth muscle, both in the presence or absence of BAY 73‐6691.RESULTSAfter 48 hours, both the neurogenic contraction as well contractile response to PE were increased in the ACF group when compared to the Sham group (p &lt;0.05). However, BAY 73‐6691 restored adrenergic hypercontractility in the ACF group (p &lt;0.05). After 12 weeks, there was an increase in LV mass and a decrease in ejection fraction in HF group when compared to the Sham group (p &lt;0.05). However, in the HF Tadalafil group, the treatment was able to restore the ventricular function and decrease LV mass (p &lt;0.05). Moreover, the neurogenic contraction and contractile response to PE in CC were increased in the HF group when compared to the Sham group (p &lt;0.05) and the treatment with tadalafil restored adrenergic hypercontractility of CC in the HF Tadalafil group (p &lt;0.05). In addition, the CC hypercontractility, observed in the HF group, in response to EFS and PE were reduced with the addition of BAY 73‐6691 (P&lt;0.05). Moreover, BAY 73‐6691 shown an additive in the reduction of contractile mechanism in the rats CC with HF treated with tadalafil (p &lt;0.05).CONCLUSIONBAY 73‐6691 reduces the adrenergic hypercontractility in the CC of ACF rat. Treatment with Tadalafil improve the cardiac function and restore the increased of contractile response of rat CC with HF. Moreover, BAY 73‐6691 (in vitro) promote additive effect associated with treatment of tadalafil and restore the adrenergic hypercontractility in the CC of rats with HF.Support or Funding InformationFAPESP ‐ Grant number: 2011/21095‐4This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Beneficial effects of Rifaximin in post‐infectious irritable bowel syndrome mouse model beyond gut microbiota

Rifaximin is a minimally absorbed antibiotic, which has shown efficacy in irritable bowel syndrome (IBS) patients. However, the mechanism on how it effects in IBS is still incompletely defined. In this study, Trichinella spiralis-infected post-infectious (PI) IBS mouse model was used, to assess the action of rifaximin on visceral hypersensitivity, barrier function, gut inflammation, and microbiota. Post-infectious IBS model was established by T.spiralis infection in mice. Rifaximin were administered to PI-IBS mice for seven consecutive days. The abdominal withdrawal reflex and threshold of colorectal distention were employed to evaluate visceral sensitivity. Smooth muscle contractile response was recorded in the organ bath. Intestinal permeability was measured by Ussing chamber. Expression of tight junction protein and cytokines were measured by Western blotting. Ilumina miseq platform was used to analyze bacterial 16S ribosomal RNA. Post-infectious IBS mice treated with rifaximin exhibited decreased abdominal withdrawal reflex score, increased threshold, reduced contractile response, and intestinal permeability. Rifaximin also suppressed the expression of interleukin-12 and interleukin-17 and promoted the expression of the major tight junction protein occludin. Furthermore, rifaximin did not change the composition and diversity, and the study reavealed that rifaximin had a tiny effect on the relative abundance of Lactobacillus and Bifidobacterium in this PI-IBS model. Rifaximin alleviated visceral hypersensitivity, recovered intestinal barrier function, and inhibited low-grade inflammation in colon and ileum of PI-IBS mouse model. Moreover, rifaximin exerts anti-inflammatory effects with only a minimal effect on the overall composition and diversity of the gut microbiota in this model.

Regulation of voltage-gated potassium channels in vascular smooth muscle during hypertension and metabolic disorders.

Voltage-gated potassium (KV ) channels are key regulators of vascular smooth muscle contractility and vascular tone, and thus have major influence on the microcirculation. KV channels are important determinants of vascular smooth muscle membrane potential (Em ). A number of KV subunits are expressed in the plasma membrane of smooth muscle cells. Each subunit confers distinct kinetics and regulatory properties that allow for fine control of Em to orchestrate vascular tone. Modifications in KV subunit expression and/or channel activity can contribute to changes in vascular smooth muscle contractility in response to different stimuli and in diverse pathological conditions. Consistent with this, a number of studies suggest alterations in KV subunit expression and/or function as underlying contributing mechanisms for small resistance artery dysfunction in pathologies such as hypertension and metabolic disorders, including diabetes. Here, we review our current knowledge on the effects of these pathologies on KV channel expression and function in vascular smooth muscle cells, and the repercussions on (micro)vascular function.

CD34 Differentially Regulates Contractile and Noncontractile Elements of Airway Reactivity.

Airway hyperresponsiveness (AHR), a major hallmark of asthma, results from alterations of contractile and noncontractile elements of airway reactivity. CD34 is a sialomucin that is expressed on various cells involved in asthma, such as eosinophils and airway smooth muscle precursors, highlighting its potential influence in AHR. To study the role of CD34 in regulating the contractile and noncontractile elements of AHR, AHR was induced by chronic exposure to house dust mite (HDM) antigen. To assess the role of CD34 on the contractile elements of AHR, airway reactivity and airway smooth muscle contractility in response to methacholine were measured. To assess CD34's role in regulating the noncontractile elements of AHR, a chimeric mouse model was used to determine the impact of CD34 expression on inflammatory versus microenvironmental cells in AHR development. Extracellular matrix production, mucus production, and mast cell degranulation were also measured. Whereas wild-type mice developed AHR in response to HDM, a loss of airway reactivity was observed in Cd34-/- mice 24 hours after the last exposure to HDM compared with naive controls. This was reversed when airway reactivity was measured 1 week after the last HDM exposure. Additionally, mast cell degranulation and mucus production were altered in the absence of CD34 expression. Importantly, simultaneous expression of CD34 on cells originating from the hematopoietic compartment and the microenvironment was needed for expression of this phenotype. These results provide evidence that CD34 is required for AHR and airway reactivity maintenance in the early days after an inflammatory episode in asthma.

The Inhibitory Effect of Botulinum Toxin Type A on Rat Pyloric Smooth Muscle Contractile Response to Substance P In Vitro.

A decrease in pyloric myoelectrical activity and pyloric substance P (SP) content following intrasphincteric injection of botulinum toxin type A (BTX-A) in free move rats have been demonstrated in our previous studies. The aim of the present study was to investigate the inhibitory effect of BTX-A on rat pyloric muscle contractile response to SP in vitro and the distributions of SP and neurokinin 1 receptor (NK1R) immunoreactive (IR) cells and fibers within pylorus. After treatment with atropine, BTX-A (10 U/mL), similar to [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-SP (APTL-SP, 1 μmol/L) which is an NK1R antagonist, decreased electric field stimulation (EFS)-induced contractile tension and frequency, whereas, subsequent administration of APTL-SP did not act on contractility. Incubation with BTX-A at 4 and 10 U/mL for 4 h respectively decreased SP (1 μmol/L)-induced contractions by 26.64% ± 5.12% and 74.92% ± 3.62%. SP-IR fibers and NK1R-IR cells both located within pylorus including mucosa and circular muscle layer. However, fewer SP-fibers were observed in pylorus treated with BTX-A (10 U/mL). In conclusion, BTX-A inhibits SP release from enteric terminals in pylorus and EFS-induced contractile responses when muscarinic cholinergic receptors are blocked by atropine. In addition, BTX-A concentration- and time-dependently directly inhibits SP-induced pyloric smooth muscle contractility.

Protein kinase C modulates frequency of micturition and non-voiding contractions in the urinary bladder via neuronal and myogenic mechanisms.

BackgroundProtein Kinase C (PKC) dysfunction is implicated in a variety of smooth muscle disorders including detrusor overactivity associated with frequency and urgency of micturition. In this study, we aimed to evaluate the modulatory effects of endogenous PKC-dependent pathways on bladder storage and emptying function.MethodsWe utilized in vivo cystometry and in vitro organ bath studies using isolated bladder muscle strips (BMS) from rats to measure contractility, intravesical pressure, and voided volume. Both in vitro and in vivo results were statistically analyzed using one-way repeated measures ANOVA between the groups followed by Bonferroni’s post-test, as appropriate (Systat Software Inc., San Jose, CA).ResultsEffects of PKC activators, phorbol-12,13-dibutyrate (PDBu), and phorbol-12,13-myristate (PMA), were concentration-dependent, with high concentrations increasing frequency of micturition, and sensitivity of intramural nerves to electrical field stimulation (EFS), in vitro, while lower concentrations had no effect on BMS sensitivity to EFS. The PKC inhibitors, bisindolylmaleimide1 (Bim-1), (28 nM), and Ro318220 (50 μM) triggered an increase in the number of non-voiding contractions (NVC), and a decrease in the voided volume associated with reduced ability to maintain contractile force upon EFS, but did not affect peak force in vitro. Both low (50 nM) and high PDBu 1 micromolar (1uM) decreased the sensitivity of BMS to carbachol. Application of a low concentration of PDBu inhibited spontaneous contractions, in vitro, and Bim-1-induced NVC, and restored normal voiding frequency during urodynamic recordings in vivo.ConclusionsIn summary, the effects of low PKC stimulation include inhibition of smooth muscle contractile responses, whereas high levels of PKC stimulation increased nerve-mediated contractions in vitro, and micturition contractions in vivo. These results indicate that endogenous PKC signaling displays a concentration-dependent contraction profile in the urinary bladder via both smooth muscle and nerve-mediated pathways.

Brilliant blue FCF is a nontoxic dye for saphenous vein graft marking that abrogates response to injury

Injury to saphenous vein grafts during surgical preparation may contribute to the subsequent development of intimal hyperplasia, the primary cause of graft failure. Surgical skin markers currently used for vascular marking contain gentian violet and isopropanol, which damage tissue and impair physiologic functions. Brilliant blue FCF (FCF) is a nontoxic dye alternative that may also ameliorate preparation-induced injury. Porcine saphenous vein (PSV) was used to evaluate the effect of FCF on physiologic responses in a muscle bath. Cytotoxicity of FCF was measured using human umbilical venous smooth muscle cells. Effect of FCF on the development of intimal hyperplasia was evaluated in organ culture using PSV. Intracellular calcium fluxes and contractile responses were measured in response to agonists and inhibitors in rat aorta and human saphenous vein. Marking with FCF did not impair smooth muscle contractile responses and restored stretch injury-induced loss in smooth muscle contractility of PSV. Gentian violet has cytotoxic effects on human umbilical venous smooth muscle cells, whereas FCF is nontoxic. FCF inhibited intimal thickening in PSV in organ culture. Contraction induced by 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate and intracellular calcium flux were inhibited by FCF, oxidized adenosine triphosphate, KN-62, and brilliant blue G, suggesting that FCF may inhibit the purinergic receptor P2X7. Our studies indicated that FCF is a nontoxic marking dye for vein grafts that ameliorates vein graft injury and prevents intimal thickening, possibly due to P2X7 receptor inhibition. FCF represents a nontoxic alternative for vein graft marking and a potentially therapeutic approach to enhance outcome in autologous transplantation of human saphenous vein into the coronary and peripheral arterial circulation.