Longitudinal Muscle Strips Research Articles

Nitric oxide and ion channels mediate L-cysteine-induced inhibition of colonic smooth muscle contraction.

Previous studies have suggested that L-cysteine regulates gut motility through hydrogen sulfide. However, the mechanisms involved in the L-cysteine-induced response have not been extensively studied. This study aimed to investigate the underlying mechanisms of action of L-cysteine on spontaneous contraction of rat colon. Longitudinal and circular muscle strips from rat middle colon were prepared to measure the spontaneous contractile activities of colon in an organ bath system. Whole-cell voltage-clamp techniques were applied to record the currents of L-type voltage-dependent Ca2+ channels (VDCCs) and voltage-gated K+ channels (Kv) in isolated smooth muscle cells (SMCs) from colon. L-cysteine inhibited the spontaneous contraction of longitudinal and circular muscle strips from the rat colon in a concentration-dependent manner. The inhibition induced by L-cysteine was significantly decreased by inhibitors of H2S synthesis (p < 0.05). Furthermore, the suppression induced by L-cysteine was partially attenuated by tetrodotoxin, L-NNA and glibenclamide (p < 0.05). Whole-cell voltage-clamp recordings showed that L-cysteine caused a remarkable reduction in the peak currents of VDCCs and significantly increased the membrane currents of Kv channels in isolated SMCs (p < 0.05). We concluded that L-cysteine inhibits the contractile activities of smooth muscle strips from the rat colon. The relaxation in response to L-cysteine may be in part mediated by a nitrergic pathway and by inhibiting the VDCCs in combination with a direct activation of the KV channels and KATP channels.

Contractility of isolated colonic smooth muscle strips from rats treated with cancer chemotherapy: differential effects of cisplatin and vincristine.

Certain antineoplastic drugs cause gastrointestinal disorders even after the end of treatment. Enteric neuropathy has been associated with some of these alterations. Our goal was to assess the impact of repeated treatment with cisplatin and vincristine on the contractility of circular and longitudinal muscle strips isolated from the rat colon. Two cohorts of male rats were used: in cohort 1, rats received one intraperitoneal (ip) injection of saline or cisplatin (2 mg kg-1 week-1) on the first day of weeks 1-5; in cohort 2, rats received two cycles of five daily ip injections (Monday to Friday, weeks 1-2) of saline or vincristine (0.1 mg kg-1 day-1). Body weight and food and water intake were monitored throughout the study. One week after treatment, responses of colonic smooth muscle strips to acetylcholine (10-9-10-5 M) and electrical field stimulation (EFS, 0.1-20 Hz), before and after atropine (10-6 M), were evaluated in an organ bath. Both drugs decreased body weight gain. Compared to saline, cisplatin significantly decreased responses of both longitudinal and circular smooth muscle strips to EFS, whereas vincristine tended to increase them, although in a non-significant manner. No differences were observed in the muscle response to acetylcholine. Atropine abolished the contractile responses induced by acetylcholine, although those induced by EFS were only partially reduced in the presence of atropine. The findings suggest that although both drugs cause the development of enteric neuropathy, this seems to have a functional impact only in cisplatin-treated animals. Understanding the effects of chemotherapy on gastrointestinal motor function is vital for enhancing the quality of life of cancer patients.

Effects and underlying mechanisms of L-arginine on spontaneous muscle contraction of rat colon.

Arginine (Arg), as a basic amino acid, has been reported to be involved in regulation of gut motility. However, the evidence is limited and the underlying mechanism is not fully understood. Our study was conducted to investigate the effects of L-Arg on spontaneous contraction of the longitudinal muscle strip (LMS) of the rat colon and the relevant mechanisms. An organ bath system was used to detect the contractile force of the LMS. Whole-cell voltage-clamp techniques were applied to observe alterations in the currents of large conductance Ca2+-activated K+ (KCa) channels, voltage-dependent potassium (KV) channels, and L-type Ca2+ channels (LTCCs) in smooth muscle cells (SMCs) of the colon. We found that L-Arg within the physiological concentration had no effect on contraction of LMS, while 1mM L-Arg significantly increased both the amplitude and frequency of LMS contractility. And the increase in force was mucosa-dependent, whereas changes in frequency as well as in amplitude were inhibited by atropine. In addition, L-Arg (1mM) activated the LTTCs and inhibited both KCa channels and KV channels on SMCs. Thus, L-Arg above the physiological concentration exerted an excitatory effect on colonic LM contraction, and stimulation by L-Arg was mediated by ACh. In addition, LTCCs, KCa channels, and KV channels on SMCs were involved in the action of L-Arg.

6-OHDA-Induced Changes in Colonic Segment Contractility in the Rat Model of Parkinson's Disease.

Gastrointestinal dysfunction is one of the most common non-motor symptoms in Parkinson's disease (PD). The exact mechanisms behind these symptoms are not clearly understood. Studies in the well-established 6-hydroxydopamine (6-OHDA) lesioned rats of PD have shown altered contractility in isolated circular and longitudinal smooth muscle strips of distal colon. Contractile changes in proximal colon and distal ileum are nevertheless poorly studied. Moreover, segments may serve as better tissue preparations to understand the interplay between circular and longitudinal smooth muscle. This study aimed to compare changes in contractility between isolated full-thickness distal colon muscle strips and segments, and extend the investigation to proximal colon and distal ileum in the 6-OHDA rat model. Spontaneous contractions and contractions induced by electrical field stimulation (EFS) and by the non-selective muscarinic agonist methacholine were investigated in strip and/or segment preparations of smooth muscle tissue from distal and proximal colon and distal ileum in an in vitro organ bath comparing 6-OHDA-lesioned rats with Sham-operated animals. Key Results. Our data showed increased contractility evoked by EFS and methacholine in segments, but not in circular and longitudinal tissue strips of distal colon after central 6-OHDA-induced dopamine denervation. Changes in proximal colon segments were also displayed in high K+ Krebs-induced contractility and spontaneous contractions. This study further confirms changes in smooth muscle contractility in distal colon and to some extent in proximal colon, but not in distal ileum in the 6-OHDA rat model of PD. However, the changes depended on tissue preparation.

The effects of Saccharomyces boulardii on rat colonic hypermotility induced by repeated water avoidance stress and the potential mechanism.

Saccharomyces boulardii (Sb) has been reported to have the potential to regulate gut motility. The aim of this experiment was to explore the possible function of Sb in gut hypermotility elicited by repeated water avoidance stress (WAS). Adult male Wistar rats (N=24) were divided into one of the following three groups: control (C), NS (normal saline) + WAS group (N), and Sb + WAS group (S). A diarrhea-predominant irritable bowel syndrome (IBS-D) model in rats was induced using the WAS method. Gut motility was evaluated by stool pellet expulsion per hour. The contractile activity of the colonic muscle strips was measured using an RM6240 multichannel physiological signal instrument. qRT-PCR and immunohistochemistry were used to assess Toll-like receptor 4 (TLR4) expression in colon tissue. ELISA was used to measure the level of cytokines in the serum and colonic tissue. Also, the microbiota composition was determined using high-throughput 16S rRNA sequencing. The results showed that oral Sb decreased the WAS-induced increased defecation and colonic hypermotility in vivo. Furthermore, Sb also decreased the contractile amplitude of colonic circular muscle (CM) and longitudinal muscle (LM) strips in a dose-dependent manner in vitro. Repeated WAS increased TLR4 expression, but Sb reversed it. Sb also reduced interleukin-6 (IL-6), IL-1β, and interferon-γ (IFN-γ) levels in serum and colonic tissue, while increasing IL-10 levels in colonic tissue. Meanwhile, the rats from the NS + WAS group had decreased microbiota diversity and had lower relative abundances of Patescibacteria, Epsilonbacteraeota, Cyanobacteria, and Turicibacter compared with controls. The rats in the Sb + WAS group showed a tendency to increase the relative abundance of Blautia when compared to control rats and had lower relative abundances of Acidobacteria and Anaerostipes compared with the NS + WAS group. Our findings demonstrated that Sb improved colonic hypermotility in rats, reversed the high-expression of TLR4 in the colon caused by repeated WAS, modulated cytokines in the colon and serum, and altered the gut microbiota, indicating that Sb may be useful for IBS-D.

In Vivo Evaluation of Anti-Nociceptive Effects of Silver Nanoparticles.

Silver nanoparticles (AgNPs) are widely used commercially due to their antimicrobial effects. Little is known about the effect of AgNPs on neural transmission and pain response. The aim of this study was to assess the anti-nociceptive activity of AgNPs. AgNPs were prepared at 16 ug/mL, white albino rats were injected with various doses of AgNPs, and challenged using a hot-plate test and paw withdrawal latency (PWL) was measured. The chronic constriction injury (CCI) model was utilized to evaluate the pedal withdrawal reflex and tail withdrawal reflex. An electrophysiological study was conducted utilizing colon longitudinal muscle strips. AgNPs increased the latency of PWL in a dose-dependent matter over the duration of 6 h. The paw withdrawal threshold in animals with CCI significantly increased after AgNPs administration. In isolated colon longitudinal muscle strips, AgNPs significantly reduced the colonic migrating motor complexes (MMCs) and contraction. This action was completely reversed after removing the AgNPs and adding acetylcholine to the preparation. In this study, AgNPs showed significant anti-nociception properties. To our knowledge, this is the first report to describe this pharmacological action of AgNPs.

Intravenous administration of xenin-25 accelerates cyclic ruminal contractions in healthy conscious sheep

The present study aimed to determine the effect and mode of action of the intravenous injection of xenin-25 on cyclic contractions of the rumen in healthy conscious sheep and mode of its action. Clinically healthy male sheep were equipped with a rumen cannula by surgery under anesthesia, and ruminal contractions were recorded with manometry in conscious animals after the recovery period. Intravenous xenin-25 injection induced a cluster of premature ruminal phasic contractions in a dose-dependent manner between 0.03 and 1 nmol/kg, and the change at the highest dose was statistically significant. In contrast, intravenous neurotensin injection inhibited the amplitude of cyclic rumen contractions. The xenin-25 effect was not significantly altered by prior injection of the neurotensin receptor subtype-1 antagonist SR 48692 at 30 and 100 nmol/kg. After euthanasia the ruminal muscles were excised for in vitro experiments. A single xenin-25 application (0.3–10 μM) to the longitudinal and circular muscle strips of the rumen did not induce any change in tension or electric field stimulation-induced phasic contractions of the muscle strips. These results demonstrated that circulating xenin-25 stimulates rumen contractions by acting on sites except the intramural intrinsic nerve plexus or smooth muscles of the rumen, implying that xenin-25 acts on the gastric center and/or cholinergic efferent nerve innervated to the ovine rumen.

Oxytocin, Prostaglandin F2α, and Prostaglandin E2 Induced Contractions in Isolated Uterine Smooth Muscle Strips from Non‐pregnant, Late Pregnant, and Postpartum Rats

Objective/IntroductionNumerous studies have investigated uterotonic agents (e.g., oxytocin) in uterine smooth muscle (USM) from non‐pregnant (NP) and late‐pregnant (LP) women and/or animal models of pregnancy. Surprisingly, few studies have investigated USM from the post‐partum period to determine whether USM contractility returns to its pre‐pregnant state. We designed a study in rodents to investigate whether USM contractility to oxytocin (OXY), prostaglandin F2α (PGF2α), and prostaglandin E2 (PGE2) returned to pre‐pregnant values within 30 days postpartum.MethodsStudies were performed in NP (n=12), LP (21d gestation, n=4), 3 days post‐partum (PP3, n=2), and 30 days post‐partum (PP30, n=4) Sprague Dawley rats. Uterine horns were cleaned of connective tissue and cut along the longitudinal axis allowing isolation of USM (width: 2mm, length: 4±1mm) oriented in the longitudinal muscle (LM) or circular muscle (CM) direction. CM or LM strips were mounted in tissue baths containing Krebs and equilibrated @ 37°C for 60 min prior to starting one of the two following protocols.Protocol 1 identified the optimal resting tension (in grams: 0.25, 0.50, 1.0, and 1.5) to measure spontaneous contraction (SCA), K+‐induced contraction (60mM K+), and cumulative OXY‐induced contraction (10‐11 to 10‐7) in CM and LM of NP rats (N=4‐6).Protocol 2 was designed assess SCA, K+‐induced contraction, cumulative concentration‐response relationships to OXY (10‐11 to 10‐7), PGF2α (10‐8 to 10‐4), or PGE2 (10‐8 to 10‐4) in CM and LM, of NP, LP, PP3, and PP30 rats (N=2‐6).Statistical comparisons were made using either a t‐test or two‐way ANOVA, and considered statistically different at P&lt;0.05.ResultsProtocol 1‐ LM SCA was greater than CM SCA at all resting tensions. K+‐induced constrictions in LM and CM were similar at each resting tension. OXY‐induced contraction in LM was generally greater than CM (P&lt;0.05 at 0.25g, 0.5g, and 1.5g tension, comparable at 0.5, 1.0, and 1.5g tension).Protocol 2 ‐ SCA in NP, LM was greater than NP, CM (P&lt;0.05). There was no difference in SCA in CM vs LM in USM from LP, PP3, and PP30 animals. K+‐induced constrictions in CM and LM were similar in USM from NP, LP, PP3 and PP30 animals. OXY‐induced contractions were greater in LM than CM for all pregnancy states (P&lt;0.05). PGE2 contractions were greater in LM vs CM in USM from NP rats (P&lt;0.05), but were similar in USM from LP, PP3, and PP30 animals. In contrast, PGF2α‐contractions were similar in LM and CM for all pregnancy states.Summary/ConclusionOur preliminary data suggests that uterotonic agents be assessed at either 0.5g or 1.0g resting tension. In addition, uterotonic agents exhibit significantly greater contraction in longitudinal vs circular orientation. Finally, contractility to uterotonic agents returns to pre‐pregnant values by PP30.

Therapeutic Effects of Berberine Hydrochloride on Stress-Induced Diarrhea-Predominant Irritable Bowel Syndrome Rats by Inhibiting Neurotransmission in Colonic Smooth Muscle.

The etiology of diarrhea-predominant irritable bowel syndrome (IBS-D) is complicated and closely related to neurotransmission in the gastrointestinal (GI) tract. Developing new strategies for treating this disease is a major challenge for IBS-D research. Berberine hydrochloride (BBH), the derivative of berberine, is a herbal constituent used to treat IBS. Previous studies have shown that BBH has potential anti-inflammatory, antibacterial, analgesic, and antidiarrheal effects and a wide range of biological activities, especially in regulating the release of some neurotransmitters. A modified IBS-D rat model induced by chronic restraint stress was used in all experiments to study the effects of BBH on the GI tract. This study measured the abdominal withdrawal reflex (AWR) response to graded colorectal distention (CRD; 20, 40, 60, and 80 mmHg) and observed the fecal areas of stress-induced IBS-D model. Experiments were conducted using organ bath techniques, which were performed in vitro using strips of colonic longitudinal smooth muscle. Inhibitory and excitatory neurotransmitter agents were added to each organ bath to observe contractile responses on the strips and the treatment effect exerted by BBH. The IBS-D rat model was successfully induced by chronic restraint stress, which resulted in an increased defecation frequency and visceral hypersensitivity similar to that of humans. BBH could reduce 4-h fecal areas and AWR response to CRD in IBS-D. The stress-induced IBS-D model showed upregulated colonic mRNA expression levels of 5-hydroxytryptamine-3A receptor and downregulated expression levels of neuronal nitric oxide synthase. Meanwhile, BBH could reverse this outcome. The responses of substances that regulate the contraction induced by related neurotransmission in the longitudinal smooth muscle of IBS-D colon (including the agonist of acetylcholine, carbachol; NOS inhibitor, L-NAME; and P2Y1 receptor antagonist, MRS2500) can be inhibited by BBH. In summary, BBH promotes defecation frequency and visceral hypersensitivity in IBS-D and exerts inhibitory effects on contractile responses in colonic longitudinal smooth muscle. Thus, BBH may represent a new therapeutic approach for treating IBS-D.

Region-specific effects of the cysteine protease papain on gastric motility.

Papaya is a traditional remedy for gastrointestinal complaints in the folk medicine. On this basis, papain, a cysteine protease of the fruit, is sold as a nutritional supplement, although scientific data on its effects in the gastrointestinal tract are lacking. We aimed to explore the effect of papain on gastric motility in vitro. Guinea pig antrum and corpus strips were mounted in organ bath. Papain reversibly increased the amplitude of ongoing phasic contractions in both circular and longitudinal antrum strips without having an effect on the frequency or on the muscle tone. All three tested doses of papain (end cc.: 12.5mgL-1 , 50mgL-1 , 100mgL-1 ) were similarly effective. Contrarily, in the corpus circular and longitudinal muscle strips, papain caused a dose-dependent relaxation, which was preceded by a transient contraction in most tissues. The effect was resistant to tetrodotoxin (1µM), but diminished by the cysteine protease inhibitor E64 (4.5µM) in both regions. In the corpus, L-NAME (100µM) and the protease-activated receptor (PAR)-1 antagonist SCH79797 (5µM) or the PAR-2 antagonist GB 83 (3µM) did not change the effect of papain significantly. This demonstrates that the effects of papain are not neurally mediated and nitrergic pathways are not involved in the mechanism. The effects are linked to the enzymatic activity, but not executed via PAR-1 or 2. Papain alters gastric motility in a region-specific manner, which could at least partly explain its claimed beneficial effects in functional gastrointestinal disorders.

Effects of the herbal preparation STW 5-II on in vitro muscle activity in the guinea pig stomach.

STW 5 is a combination of nine medicinal herbal extracts and used to treat functional gastrointestinal disorders including functional dyspepsia. It has a region-specific effect by relaxing the proximal and contracting the distal stomach. The research combination STW 5-II (Iberogast® Advance) lacks three herbal extracts but seems clinically as effective as STW 5. However, the action of STW 5-II on gastric motility is unknown. In vitro circular and longitudinal muscle tone and contractility were recorded from guinea pig gastric fundus and antrum with isometric force transducers. STW 5-II decreased concentration-dependently (64-512µg/ml) the tone of circular and longitudinal muscle strips from the fundus. In contrast, STW 5-II increased concentration-dependently contraction amplitude in antral circular and longitudinal muscle. The effects were region-dependent but comparable in the two muscle layers. Application of 512µg STW 5 or STW 5-II revealed comparable effects in the fundus and antrum circular and longitudinal muscle strips. STW 5-II had a region-specific effect and relaxed the proximal stomach but increased the contractility in the antrum. It was as effective as STW 5 which may explain its comparable clinical effects in treating functional dyspepsia. Impaired accommodation may be normalized through relaxation of the fundus, while the motility-promoting effects leading to an increase in antral motility may activate the gastric pump.

High efficiency preparation of skinned mouse cardiac muscle strips from cryosections for contractility studies.

What is the central question of this study? Can frozen cardiac papillary muscles and cryosectioning be used to reliably obtain uniform cardiac muscle strips with high yields? What is the main finding and its importance? A new method was developed using frozen cardiac papillary muscles and cryosectioning to reliably obtain uniform cardiac muscle strips with high yields. Experimental results demonstrate that this new methodology significantly increases the efficiency and application of quantitative biomechanical studies using skinned muscle fibres with an additional advantage of no need for transferring live animals. Skinned cardiac muscle preparations are widely used to study contractile function of myofilament proteins and pathophysiological changes. The current methods applied in these biomechanical studies include detergent permeabilization of freshly isolated papillary muscle, ventricular trabeculae, surgically dissected ventricular muscle strips, mechanically blended cardiac muscle bundles or myocytes, and enzymatically isolated single cardiomyocytes. To facilitate and expand the skinned cardiac muscle approach, we have developed an efficient and readily practical method for mechanical studies of skinned mouse cardiac papillary muscle strips prepared from cryosections. Longitudinal papillary muscle strips of 120-150µm width cut from 35-70µm-thick cryosections are mounted to a force transducer and chemically skinned for the studies of force-pCa and sarcomere length-tension relationship and rate of tension redevelopment. In addition to more effective skinning and perfusion than with whole papillary muscle and much higher yield of useful preparations than that from trabeculae, this new methodology has two more major advantages. One is to allow for the use of frozen cardiac muscle in storage to maximize the value of muscle samples, facilitating resource sharing among research institutions without the need of transferring live animals or fresh biopsies. The other is that the integrity of the muscle strips is well preserved during the preparation and mechanical studies, allowing coupled characterization of myofilament proteins. The combined power of biomechanics and protein biochemistry can provide novel insights into integrative physiological and pathophysiological mechanisms of cardiac muscle contraction while the high yield of high-quality muscle strips also provides an efficient platform for development of therapeutic reagents.

Characterization of gut contractility and microbiota in patients with severe chronic constipation.

Chronic constipation (CC) is one of the most common gastrointestinal disorders worldwide. Its pathogenesis, however, remains largely unclear. The purpose of the present work was to gain an insight into the role of contractility and microbiota in the etiology of CC. To this end, we studied spontaneous and evoked contractile activity of descending colon segments from patients that have undergone surgery for refractory forms of CC. The juxta-mucosal microbiota of these colon samples were characterized with culture-based and 16S rRNA sequencing techniques. In patients with CC the spontaneous colonic motility remained unchanged compared to the control group without dysfunction of intestinal motility. Moreover, contractions induced by potassium chloride and carbachol were increased in both circular and longitudinal colonic muscle strips, thus indicating preservation of contractile apparatus and increased sensitivity to cholinergic nerve stimulation in the constipated intestine. In the test group, the gut microbiota composition was assessed as being typically human, with four dominant bacterial phyla, namely Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, as well as usual representation of the most prevalent gut bacterial genera. Yet, significant inter-individual differences were revealed. The phylogenetic diversity of gut microbiota was not affected by age, sex, or colonic anatomy (dolichocolon, megacolon). The abundance of butyrate-producing genera Roseburia, Coprococcus, and Faecalibacterium was low, whereas conventional probiotic genera Lactobacillus and Bifidobacteria were not decreased in the gut microbiomes of the constipated patients. As evidenced by our study, specific microbial biomarkers for constipation state are absent. The results point to a probable role played by the overall gut microbiota at the functional level. To our knowledge, this is the first comprehensive characterization of CC pathogenesis, finding lack of disruption of motor activity of colonic smooth muscle cells and insufficiency of particular members of gut microbiota usually implicated in CC.

Mechanisms Associated to Nitroxyl (HNO)-Induced Relaxation in the Intestinal Smooth Muscle.

The pharmacological properties of nitroxyl (HNO) donors in the gastrointestinal tract are unknown. We investigated the properties of this molecule in the regulation of gastrointestinal contractility focusing on its possible interaction with other gaseous signaling molecules such as NO and H2S. Organ bath, Ca2+ imaging, and microelectrode recordings were performed on rat intestinal samples, using Angeli’s salt as HNO donor. Angeli’s salt caused a concentration-dependent relaxation of longitudinal or circular muscle strips of the ileum and the proximal colon. This relaxation was strongly inhibited by the Rho-kinase inhibitor Y-27632 (10 μM), by the reducing agent DTT or by the inhibitor of soluble guanylate cyclase (sGC) ODQ (10 μM) alone or in combination with the inhibitors of the endogenous synthesis of H2S β-cyano-L-alanine (5 mM) and amino-oxyacetate (5 mM). Preventing endogenous synthesis of NO by the NO synthase inhibitor L-NAME (200 μM) did not affect the relaxation induced by HNO. HNO induced an increase in cytosolic Ca2+ concentration in colonic myocytes. It also elicited myocyte membrane hyperpolarization that amounted to −10.6 ± 1.1 mV. ODQ (10 μM) and Apamin (1 μM), a selective inhibitor of small conductance Ca2+-activated K+ channels (SKca), strongly antagonized this effect. We conclude that HNO relaxes the gastrointestinal tract musculature by hyperpolarizing myocytes via activation of the sGC/cGMP pathway similarly to NO, not only inhibiting the RhoK and activating MLCP as do both NO and H2S but also increasing cytosolic Ca2+ for activation of SKCa contributing to hyperpolarization.

Opposite effects of dopamine on the mechanical activity of circular and longitudinal muscle of human colon.

Because dopamine (DA) has gained increasing evidence as modulator of gut motility, we aimed to characterize dopaminergic response in human colon, evaluating function and distribution of dopamine receptors in circular vs longitudinal muscle strips. Mechanical responses to DA and dopaminergic agonists on slow phasic contractions and on basal tone were examined in vitro as changes in isometric tension. RT-PCR was used to reveal the distribution of dopaminergic receptors. In spontaneous active circular muscle, DA induced an increase in the amplitude of slow phasic contractions and of the basal tone, via activation of D1-like receptors. DA contractile responses were insensitive to neural blockers or to atropine and inhibited by phospholipase C (PLC) pathway inhibitors. In precontracted circular muscle strips, DA, at the higher concentrations tested, caused a relaxant response via activation of D2-like receptors. In the longitudinal muscle, DA caused only muscular relaxation due to activation of D2-like receptors. DA relaxant responses were insensitive to neural blockers or to nitric oxide synthase inhibitor and reduced by a wide-spectrum K+ channel blockers. Transcripts encoding for all the dopaminergic receptor subtypes was observed in both circular and longitudinal preparations. Dopamine is able to modulate contractile activity of the human colon. In the circular muscle layer, DA induces mainly muscular contraction activating non-neural D1-like receptors, coupled to PLC/IP3 pathway. In the longitudinal muscle layer, DA induces muscular relaxation acting on non-neural D2-like receptors leading to the increase in K+ conductance.

Colonic dysmotility and inflammation associated with high fat diet-induced obesity: role of the enteric glia

AbstractIntroductionEnteric glial cells (EGCs) contribute to the regulation of bowel motility, and have been implicated in the onset and development of several digestive disorders. However, the involvement of EGCs in obesity-related intestinal dysmotility is unknown. Accordingly, this study examined the role of EGCs in colonic neuromuscular dysfunctions in a mouse model of diet-induced obesity.Materials and MethodsC57BL/6 male mice (n = 6 per group) were fed with standard diet (SD) or high fat diet (HFD) for 8 weeks. Body and epididymal fat weight, and blood fasting glucose levels were evaluated the day before sacrifice. Colonic longitudinal muscle strips were set up in organ baths with Krebs solution and connected to isometric transducers. The effects of fluorocitrate (FC, gliotoxin) were tested on contractile responses mediated by NK1 tachykininergic receptors upon application of electrical stimuli (0.5 ms, 28 V, 10 Hz) [incubation with atropine, guanethidine, L-NAME, GR159897 and SB218795 (NK2 and NK3 antagonists, respectively)] or exogenous substance P (SP). Colonic levels of interleukin (IL)-1β, IL-6, malondialdehyde (MDA) and occludin (a tight junction protein involved the maintenance of mucosal barrier) were measured. Cultured rat EGCs were exposed to palmitate and lipopolysaccharide (LPS), either alone or in combination, to mimic the exposure to HFD. IL-1β and SP levels were then assessed in cell supernatants, while toll-like receptor 4 (TLR4) expression was evaluated in cell lysates.ResultsHFD-mice displayed increments of body weight, epididymal fat weight and blood glucose levels. In in vitro experiments, electrically induced colonic tachykininergic contractions were enhanced in HFD mice, as compared with SD animals. No differences were observed when comparing contractions to exogenous SP. The increase in electrically evoked tachykininergic contractions was blunted upon incubation with the gliotoxin FC. Exogenous SP-induced contractions were not affected by FC. HFD mice displayed an increase in colonic IL-1β, IL-6 and MDA levels and a reduced occludin expression, as compared with SD mice. Exposure of EGCs to palmitate, alone or in combination with LPS, resulted in a significant increase in TLR4 expression, while LPS alone was without effects. The combination of palmitate and LPS increased significantly IL-1β and SP levels in cell supernatants, while single treatments were without effects.DiscussionHFD is characterized by colonic dysmotility along with bowel inflammation, oxidative stress, and an impairment of mucosal barrier integrity. In this setting, the hyperactivation of EGCs, likely via TLR4, appears to contribute to inflammation and colonic tachykininergic motor dysfunctions.

The Effect of Vitamin D3 on the Contractile Response of Isolated Rat Uterus

AbstractBackground: Vitamin D receptors (VDR) are expressed in many reproductive tissues indicating a potential role of vitamin D3-VDR in the regulation of reproductive functions. Nevertheless, the data about the effect of vitamin D3 in the uterus are scarce.Aim of Study: This study was designed to evaluate the potential effects of vitamin D3 on spontaneous, KCl-induced and oxytocin-induced contractions in the rat uterus in vitro.Material and Methods: The study was conducted on 10 healthy adult female albino non-pregnant rats. Full-thickness longitudinal muscle strips were dissected from each non-pregnant rat and then myometrial tension was recorded. The strips were mounted vertically in organ baths and exposed to vitamin D3 and different uterotonic agents to delineate the potential action of vitamin D3 on the rat myometrial contrac-tility. Spontaneous contractions were recorded using mechan-ical activity recording system. We evaluated the effects of 3 different dosed of vitamin D3 on spontaneous uterine contrac-tions; then on concentrated KCl-induced uterine contractions and oxytocin (OT)-induced uterine contractions. Furthermore, the effects of vitamin D3 on spontaneous uterine contractions pretreated with nifedipine; a voltage-gated L-type Ca2+ channel antagonist were investigated.Results: Vitamin D3 significantly inhibited the spontaneous uterine contractions in a dose dependent manner. Moreover, vitamin D3 inhibited the uterine contractions whether induced by oxytocin or concentrated KCl. Administration of nifedipine resulted in a significant decline in the force amplitude of spontaneous uterine contractions. In the presence of vitamin D3, the uterine relaxant effect of nifedipine was significantly augmented.Conclusions: The inhibitory effects of the in vitro admin-istration of vitamin D3 on rat uterus may yield novel insights into its therapeutic use. Further, it may be recommended to be used as a novel tocolytic agent for preventing unwanted uterine contractions in early pregnancy and relieving pain related to dysmenorrhea.

Amphibian toxins from the skin of &lt;i&gt;Bufo regularis&lt;/i&gt; have inhibitory effect on electrically invoked contractile response and bimodal effects on tone of longitudinal muscle of guinea pig ileum

The skins of some amphibians contain potentially bioactive principles that may have pharmaceutical, medicinal, toxicological or chemical importance. In addition, such active principles can be used as tools in biomedical research. The present study aims at isolating and purifying bioactive principles from the skin of Bufo regularis and studying their effect on isolated longitudinal smooth muscle strip of guinea pig ileum. High performance liquid chromatography (HPLC) was used to isolate toad toxins. The effects of crude, semi-purified and purified extracts were tested on longitudinal smooth muscle of guinea pig ileum using organ bath method. Effect of the toxins was studied on electrically-induced contractile response and the basal tone of the longitudinal muscle strip. HPLC purification resulted in four different bioactive components with a λmax UV absorbance pattern of around 295 nm. When tested on guinea pig ileum they had persistent inhibitory effect on the electrically-induced contractile responses. The pattern of effect was initial excitatory followed by long lasting inhibitory effect on tone of longitudinal muscle. The HPLC eluate at 79th min in methanol preparative run corresponding to the eluate at 40th min in the acetonitrile run had the maximum bioactivity. Hence, it was concluded that the skin of B. regularis contains four different components which vary in their potency on isolated smooth muscle strip of guinea pig ileum.Keywords: Bufo regularis, organ bath, longitudinal muscle of ileum, toad toxin, electrical field stimulation

The Characterization of Circular and Longitudinal Uterine Smooth Muscle Function from Non‐Pregnant and Late‐Pregnant Rats

Objective/IntroductionThe rodent uterus is a hollow organ that increases approximately 10 fold in size with pregnancy. Myometrial smooth muscle orients longitudinally and circularly allowing flexibility in size and shape during pregnancy. To date most studies have focused on changes in longitudinal smooth muscle with pregnancy. Here we studied how circular smooth muscle of the uterus changes with pregnancy in comparison with longitudinal muscle.MethodsStudies were performed in age‐matched (~77days) non‐pregnant (NP, n=5) and late‐pregnant (LP, d21g±1d, n=4) Sprague‐Dawley rats. Uterine horns from NP and LP rats were rapidly removed, placed in 4°C PSS, and cleaned of all connective tissue. Uterine tissue was cut (ovary to uterine body) allowing both longitudinal muscle (LM) and circular muscle (CM) strips (width: 1.5mm, length: ~4mmt) to be isolated. Muscle strips were mounted in vertical organ chamber baths containing PSS and maintained @ 37°C. After ~45 min. equilibration, strips were challenged with 60mM [K+]o. Tissues not responding to 60mM [K+]o were excluded. Strips were assessed using one of two protocols. Protocol 1: Optimal stretch (L0) ‐ determined by comparing spontaneous contractile activity (e.g., contraction amplitude, area under the curve (AUC), and frequency), along with 60mM [K+]o ‐induced contraction (contraction amplitude and AUC) at 250mg, 500mg, and 1000mg resting tension. Protocol 2: Passive mechanical characteristics ‐ Stress vs strain (SvS) relationships were assessed in CM and LM strips. Tissue equilibrated in Ca2+ free PSS to eliminate spontaneous contractility. Tissue was then stretched in 500 μm steps until tissue failure.ResultsOur data indicates pregnancy significantly increases K+‐induced constriction amplitude, and K+‐induced AUC of CM (P&lt;0.05, NP n=5; LP n=4). Interestingly, neither pregnancy status nor resting tension significantly altered spontaneous contractile frequency, amplitude or contractile AUC in CM or LM. Finally, SvS values were significantly different between CM and LM in both NP and LP (P&lt;0.05).Summary/ConclusionOur preliminary SvS data suggests that CM is “stiffer” than LM in both NP and LP rats. Furthermore, SvS values of CM and LM from LP rats has a significant rightward shift compared to NP suggesting greater compliance in pregnancy. Finally, the effect of pregnancy on spontaneous contractile activity and K+‐induced constrictions appear to be directed at CM vs LM, suggesting that CM remodeling may play a key role in healthy pregnancy. These studies will serve as the foundation for future studies assessing rodent models of complicated pregnancies.Support or Funding InformationFunding: Biomedical Sciences Program (RM, JVE, DME) and Biomedical Sciences Start‐up Funds (DME)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of neurotensin in the regulation of gastric motility in healthy conscious sheep

The goal of present study was to determine the effects of the intravenous (i.v.) administration of neurotensin (NT) on the ovine forestomach and abomasal motility in conscious sheep. NT injection at 0.3 nmol/kg slightly raised abomasal pressure, although the effect was not dose-dependent. A bolus i.v. injection of NT at 1 or 3 nmol/kg significantly inhibited the amplitude of cyclic ruminal contractions. NT injection did not alter omasal motility. Pre-injection of an NT receptor subtype-1 antagonist, SR 48692, at 60 nmol/kg immediately before NT injection did not block the inhibitory effect of NT. In an in vitro study using smooth muscle strips of the rumen dorsal sac, NT application at 0.3–10 μmol/L did not inhibit the bethanechol (BCh, 10 μmol/L)-induced tonic contractions of either the longitudinal and circular muscle strips, nor did NT inhibit the electrical field stimulation (EFS)-induced phasic contractions of the muscle strips. The results suggest that circulating NT selectively inhibits the amplitude of cyclic rumen contractions presumably by inhibiting the gastric center in the medulla oblongata and/or the vagus nerves, but not through its peripheral action. An elevation in the plasma concentration of NT appears able to exert the ileal brake-like effect on ruminal motility in sheep.