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.

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.

Towards automated assessment of drug efficacy on cardiomyopathic human heart function.

Structural and functional abnormalities of ventricular myocardium related to Hypertrophic and Dilated Cardiomyopathies (HCM and DCM) are commonly caused by inherited mutations in sarcomeric proteins. Automating predictions of mutation and drug effects on human cardiac muscle function could enhance early diagnosis and evaluation of response to treatments for cardiomyopathies. However, the development of automation tools that integrate results from multiple experiments, conducted under various experimental conditions at different time and spatial scales from multiple species, is a significant challenge. Such automation tools usually require large sets of training data, which are extremely limited or even nonexistent from humans, thus any information generated could be significantly inaccurate or even misleading. Recent advances in multiscale computational tools, coupled with multiple experiments, for the translation of data concerning sarcomere level molecular interactions, including the effect of drugs, to the level of cardiac muscle fibers and tissues, might provide the most relevant approach for generating databases for automation algorithms. Using the MUSICO platform for precise modeling of protein-protein interactions and Ca2+ regulation in cardiac muscle cells, coupled with the finite element (FE) solver Mexie via Mijailovich-Prodanovic surrogate model, we can simulate experimental data from various sources. These tools collectively enable quantitative assessment of the changes in human cardiac muscle contractile responses caused by HCM and DCM extending from the molecular level to whole organ function, as well as prediction of improvements of cardiac muscle function by drugs. Our simulations successfully predicted pressure and volume changes due to troponin-C mutations and the effect of drugs, disopyramide and digoxin, in improving heart function in HCM and DCM, respectively, during multiple heartbeats in human heart. These simulations can potentially provide sufficient information for physicians to evaluate the progress of disease and the efficacy of administrated therapeutics.

OR07-5 PARP1 Contributes to the Glucocorticoid Receptor Transcriptional Response And Influences the Skeletal Muscle Phenotype

Abstract Background NAD+-dependent PARP1 (Poly-(ADP-ribosyl)polymerase 1) generates the post-translational modification ADP-Ribosylation (ADPR). Given PARP1-ADPR determines cellular NAD+ availability, which in turn impacts a range of hormonally governed actions, we sought to identify the molecular roles of PARP1 in relation to glucocorticoids in the endocrine sensitive tissue skeletal muscle. Importance: Activity of the glucocorticoid receptor (GR) is dictated by molecular partners whose actions skew the frequency with which GR produces meaningful transcriptional outcomes. As a result, our molecular understanding of this steroid hormone receptor remains incomplete. Hypothesis PARP1 through ADPR actions impacts skeletal muscle phenotype and co-regulates glucocorticoid-mediated transcriptional responses. Experimental Design: Murine muscle myoblasts (C2C12) or primary murine muscle cultures were treated with PARP inhibitors (PJ34(10uM) or BYK204165(1uM)) or transfected with silencing RNA targeting PARP1. Cells were treated with ± dexamethasone (1uM). Major Results Analysis of differentiating muscle revealed PARP1 and ADPR are dynamic at days 0-5 (measured by Western immunoblot. p<0.001; n=6). Chemical inhibition of PARP1 significantly decreased ADPR (Western immunoblot. p<0.001; n=9) but did not prevent myotube formation. To identify if initial ADPR dynamics held consequence for fully differentiated myotubes, cells were treated with a single dose of the PARP1 inhibitor BYK204165 at induction of differentiation. Myotube lysates were collected 5 days later and were subjected to unlabelled SWATH-MS. This measured the abundance of 180 proteins as significantly different in PARP inhibitor-treated cells (n=7). Analyses of these proteins revealed over-representation of pathways governing muscle development (FDR 8.99-08), contraction (FDR 9.47-08, myofiber assembly (FDR 8.06-05), and energy metabolism (FDR 2.93-06). Indicating PARP1-ADPR regulates myogenesis and plays a role in the striated muscle contractile response. ChIP assays for PARP1 binding in cells showed a (30-fold enrichment ±3.2SEM) at GR response elements and detectable ADPR was reduced when exposed to dexamethasone (Western immunoblot n=5). These data support ADPR as responsive to glucocorticoid exposure and PARP1 influences GR recruitment to the genome. RNAseq of siPARP1 (n=4) versus scrambled myoblasts (n=5) identified differential expression of 165 genes. Gene ontology revealed over-representation in siPARP1 cells of those pathways regulating muscle differentiation, including actin-binding, cytoskeletal structure, and NAD+ binding genes. SiRNA of PARP1 had no influence over the ability of glucocorticoids to activate archetype target genes (DUSP1, MURF1, ATROGIN1 & GILZ), but differentially impacted the expression of 434 other genes. Conclusions PARP1 mediated ADPR is critical to the 'early phase' events of muscle cell differentiation. PARP1 influences short-term transcriptional behaviors of the glucocorticoid receptor; in turn, glucocorticoids themselves impact PARP1-genome interaction events. These findings are important for the understanding of general GR mechanisms and have relevance for the widespread functional decline observed during tissue glucocorticoid excess. Presentation: Saturday, June 11, 2022 12:30 p.m. - 12:45 p.m.

The Effects of 1,1-Dimethylbiguanide Hydrochloride (Metformin) on Detrusor Muscle Contractile Response in Ovariectomized Female Rats

Aim: Menopause is defined as the depletion of the ovarian follicular reserve followed by the cessation of menstrual cycles. It has been
 reported that gonadal steroid hormones play an important role in bladder function in women. Changes in urine pattern including
 overactive bladder, stress incontinence and recurrent urinary tract infections occur as a result of menopause. 1,1-dimethylbiguanide
 hydrochloride, metformin, (MET) is an oral anti-diabetic drug used to reduce hepatic glucose production and peripheral insulin
 resistance. Recent studies have revealed that MET has a protective effects in diabetes induced bladder dysfunction. The aim of this
 study was to test the therapeutic potential of MET in detrusor contractile function of ovariectomized (OVX) female rats.
 Material and Methods: Bilateral ovariectomy was performed to eliminate endogenous gonadal steroids secretion. Four groups are
 designed with 8 animals in each group: Control, MET-administered control, OVX, and MET-administered OVX groups. MET (25 mg/
 kg) was administered daily by oral gavage for 14 days. Contractile activity of isolated bladder muscle strips were evaluated in vitro organ
 bath. The contractile responses of detrusor strips were determined using different doses of carbachol (10-8-10-2M) and purinergic
 agonist ATP. The relaxation response of strips were determined by isoproterenol
 Results: The contractile responses of detrusor muscle strips to carbachol at doses 10-5-10-2 M were decreased in the OVX group
 compared to control and MET treated control groups. MET treatment partially reversed the reduction in OVX-induced contractile
 responses at 10-2 and 10 -3 M carbachol doses. There were no statistically significant difference in relaxation response between the
 experimental groups.
 Conclusion: Our findings suggest that treatment with MET could be the new potential therapeutic agent against bladder dyfunction
 in postmenopausal women. Further studies are needed for the therapeutic potential of MET in detrusor dysfunction induced by
 menopause.

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.

Effect of Myosin Isoforms on Cardiac Muscle Twitch of Mice, Rats and Humans.

To understand how pathology-induced changes in contractile protein isoforms modulate cardiac muscle function, it is necessary to quantify the temporal-mechanical properties of contractions that occur under various conditions. Pathological responses are much easier to study in animal model systems than in humans, but extrapolation between species presents numerous challenges. Employing computational approaches can help elucidate relationships that are difficult to test experimentally by translating the observations from rats and mice, as model organisms, to the human heart. Here, we use the spatially explicit MUSICO platform to model twitch contractions from rodent and human trabeculae collected in a single laboratory. This approach allowed us to identify the variations in kinetic characteristics of α- and β-myosin isoforms across species and to quantify their effect on cardiac muscle contractile responses. The simulations showed how the twitch transient varied with the ratio of the two myosin isoforms. Particularly, the rate of tension rise was proportional to the fraction of α-myosin present, while the β-isoform dominated the rate of relaxation unless α-myosin was >50%. Moreover, both the myosin isoform and the Ca2+ transient contributed to the twitch tension transient, allowing two levels of regulation of twitch contraction.

Tensiomyographic Neuromuscular Response of the Peroneus Longus and Tibialis Anterior with Chronic Ankle Instability.

This study aimed to investigate the muscle contractile response of the peroneus longus (PL) and tibialis anterior (TA) in groups with and without chronic ankle instability (CAI) using tensiomyography. Twenty-three adults, 12 with CAI and 11 healthy participants, participated in this study. All subjects underwent a tensiomyographic assessment of the PL and TA to measure delay time, contraction time and maximal displacement. The ankle evertor and invertor normalized peak torques, maximum work done and muscle thickness of the PL and TA were calculated. The delay time and contraction time of the PL in the CAI side were significantly higher than those in the healthy group (p < 0.05); however, no significant difference could be detected in the TA between groups. Furthermore, there was no significant difference in the normalized peak torques, maximum work done and muscle thickness of the PL and TA between groups. The CAI side demonstrated a delayed muscle contractile response of the PL when compared with the healthy group although there was no difference in muscle strength and muscle size. Clinicians should consider the muscle contractile response of the PL for rehabilitation of the ankle evertor with CAI.

The Sex-Specific VC Neurons Are Mechanically Activated Motor Neurons That Facilitate Serotonin-Induced Egg Laying in C. elegans.

Successful execution of behavior requires coordinated activity and communication between multiple cell types. Studies using the relatively simple neural circuits of invertebrates have helped to uncover how conserved molecular and cellular signaling events shape animal behavior. To understand the mechanisms underlying neural circuit activity and behavior, we have been studying a simple circuit that drives egg-laying behavior in the nematode worm Caenorhabditis elegans Here we show that the sex-specific, ventral C (VC) motor neurons are important for vulval muscle contractility and egg laying in response to serotonin. Ca2+ imaging experiments show the VCs are active during times of vulval muscle contraction and vulval opening, and optogenetic stimulation of the VCs promotes vulval muscle Ca2+ activity. Blocking VC neurotransmission inhibits egg laying in response to serotonin and increases the failure rate of egg-laying attempts, indicating that VC signaling facilitates full vulval muscle contraction and opening of the vulva for efficient egg laying. We also find the VCs are mechanically activated in response to vulval opening. Optogenetic stimulation of the vulval muscles is sufficient to drive VC Ca2+ activity and requires muscle contractility, showing the presynaptic VCs and the postsynaptic vulval muscles can mutually excite each other. Together, our results demonstrate that the VC neurons facilitate efficient execution of egg-laying behavior by coordinating postsynaptic muscle contractility in response to serotonin and mechanosensory feedback.SIGNIFICANCE STATEMENT Many animal motor behaviors are modulated by the neurotransmitters, serotonin and ACh. Such motor circuits also respond to mechanosensory feedback, but how neurotransmitters and mechanoreceptors work together to coordinate behavior is not well understood. We address these questions using the egg-laying circuit in Caenorhabditis elegans where we can manipulate presynaptic neuron and postsynaptic muscle activity in behaving animals while recording circuit responses through Ca2+ imaging. We find that the cholinergic VC motoneurons are important for proper vulval muscle contractility and egg laying in response to serotonin. Muscle contraction also activates the VCs, forming a positive feedback loop that promotes full contraction for egg release. In all, mechanosensory feedback provides a parallel form of modulation that shapes circuit responses to neurotransmitters.

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.

Medications used to treat bladder disorders may alter effects of neuromodulation.

Neuromodulation (nerve stimulation) can produce analgesia. One form, bilateral pudendal nerve stimulation (bPNS), suppresses responses to urinary bladder distension (UBD) in hypersensitive rats. Drugs can modify this effect (eg, benzodiazepines, but not opioids, suppress bPNS effects). Prior to a clinical trial of bPNS effects on bladder pain, we felt it was prudent to survey the effects of medications commonly used in patients with bladder disorders. Bladder hypersensitivity was produced by neonatal bladder inflammation in rat pups coupled with a second inflammatory insult as an adult. Antimuscarinic (oxybutynin), β3 -adrenoceptor agonist (mirabegron, CL316243), α1 -adrenoceptor antagonist (tamsulosin), antidepressant (amitriptyline), muscle relaxing (baclofen), and sedative (propofol) agents were administered and effects of bPNS on responses to UBD assessed. bPNS consisted of bilateral biphasic electrical stimulation of the mixed motor/sensory component of the pudendal nerves. Visceromotor responses (VMRs; abdominal muscle contractile responses) were used as nociceptive endpoints. Many of these drugs directly inhibited the VMRs to UBD, but only mirabegron, at the doses employed, significantly reduced inhibitory effects of bPNS. In the presence of the other drugs, bPNS continued to produce statistically significant inhibition of VMRs to UBD. This study suggests that concurrent therapy with drugs used to treat bladder disorders could affect assessment of the effects of bPNS on bladder hypersensitivity. This study gives guidance to clinical trials using bPNS for the treatment of painful bladder syndromes and suggests potential clinical use of some of these medications in the treatment of these same disorders.

Benzodiazepines Suppress Neuromodulatory Effects of Pudendal Nerve Stimulation on Rat Bladder Nociception.

Neuromodulation, as a therapeutic modality for pain treatment, is an alternative to opioid therapies and therefore receiving increased interest and use. Neuromodulation at a peripheral nerve target, in the form of bilateral electrical pudendal nerve stimulation (bPNS), has been shown to reduce bladder hypersensitivity in rats and anecdotally reduces pain in humans with pelvic pain of urological origin. Recent studies have identified a role for spinal γ-aminobutyric acid (GABA) receptors in this effect. Concomitant medication use, such as benzodiazepines, could alter responses to neuromodulation, and so before the development of a clinical trial to confirm translation of this potential therapy, the potential interactions between acute and chronic use of benzodiazepines and bPNS were examined in a preclinical model. Bladder hypersensitivity was produced by neonatal bladder inflammation in rat pups coupled with a second inflammatory insult as an adult. Diazepam (1-5 mg/kg intraperitoneal [i.p.]) or vehicle was administered acutely (with or without bPNS) and chronically (5 mg/kg subcutaneous [s.c.] daily for 2 weeks before the final experiment). bPNS was delivered as bilateral biphasic electrical stimulation of the mixed motor/sensory component of the pudendal nerves. Visceromotor responses (VMRs; abdominal muscle contractile responses to urinary bladder distension [UBD]) were used as nociceptive end points. Due to the profound effects of diazepam, the effect of midazolam (0.5-1.0 mg/kg i.p.) on VMRs and bPNS effects was also studied. Diazepam and midazolam both produced a dose-dependent, flumazenil-reversible inhibition of VMRs to UBD. bPNS resulted in statistically significant inhibition of VMRs to UBD in hypersensitive rats that had received vehicle injections. Select doses of diazepam and midazolam suppressed the inhibitory effect of bPNS on VMRs. This study suggests that inhibitory effects of bPNS on bladder pain could be suppressed in subjects receiving benzodiazepine therapy, suggesting that potential clinical testing of pudendal nerve stimulation for the treatment of painful bladder syndromes may be confounded by the use of benzodiazepines. Clinical assessment of other forms of neuromodulation should also be screened for impacts of benzodiazepines.

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.

Neuromodulatory effects of pudendal nerve stimulation on bladder hypersensitivity are present in opioid-pretreated rats

Background and objectivesBilateral electrical pudendal nerve stimulation (bPNS) reduces bladder hypersensitivity in rat models and anecdotally reduces pain in humans with pelvic pain of urologic origin. Concomitant opioids are known...

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.