Amplitude Of Spontaneous Contractions Research Articles

Extracellular acidification increases uterine contraction in pregnant mouse by increasing intracellular calcium.

As uterine extracellular pH decreases during the ischemic conditions of labor, but its effects on myometrial contraction are largely unknown, there is a need to elucidate its physiological effects and mechanisms of action. Furthermore, it is not known if any of the effects of extracellular acidification are affected by pregnancy, thus we also determined how gestation affects the response to acidification. Nonpregnant, mid-, and term-pregnant myometrial strips were obtained from humanely killed mice. Contractions were recorded under spontaneous, depolarized, and oxytocin-stimulated conditions. The extracellular pH of the perfusate was changed from 7.4 to 6.9 or 7.9 in HEPES-buffered physiological saline. Intracellular pH was measured using SNARF, and intracellular calcium was measured using Indo-1. Statistical differences were tested using the appropriate t-test. Extracellular acidification significantly increased the frequency and amplitude of spontaneous contractions in pregnant, but not nonpregnant, myometrium, whereas alkalinization decreased contractions. Intracellular acidification, via Na-butyrate, transiently increased force in pregnant tissue. Intracellular pH was gradually acidified when extracellular pH was acidified, but extracellular acidification increased contractility before any significant change in intracellular pH. If myometrial force was driven by oxytocin or high-K depolarization, then extracellular pH did not further increase force. Intracellular calcium changes mirrored those of force in the spontaneously contracting pregnant myometrium, and if calcium entry was prevented by nifedipine, extracellular acidification could not induce a rise in force. Extracellular acidification increases excitability, calcium entry, and thus force in pregnant mouse myometrium, and this may contribute to increasing contractions during labor when ischemic conditions and acidemia occur.

The P2Y1 receptor in the colonic myenteric plexus of rats and its correlation with opioid-induced constipation

This study was designed to explore the expression changes of P2Y1 receptors in the distal colonic myenteric layer of rats. An opioid induced constipation(OIC) rat model was generated by intraperitoneal (i.p) injection of loperamide. At 7 days post-treatment, the model rats were assessed by calculating the fecal water content and the gastrointestinal transit ratio. The immunofluorescence (IF)-based histochemical study was used to observe the distribution of P2Y1 receptors in the distal colonic myenteric plexus. Western blotting (WB) was performed to evaluate the expression changes of P2Y1 proteins in the myenteric layer, and the electrophysiological approaches were carried out to determine the regulatory roles of P2Y1 receptors on distal colonic motor function. IF showed that P2Y1 receptors are co-expressed MOR in the enteric nerve cells of the distal colonic myenteric plexus. Moreover, the WB revealed that the protein levels of P2Y1 were significantly decreased in the distal colonic myenteric layer of OIC rats. In vitro tension experiments exhibited that the P2Y1 receptor antagonist MRS2500 enhanced the spontaneous contraction amplitude, adding EM2 and β-FNA did not have any effect on MRS2500. Therefore, P2Y1 receptor expression could be associated with the occurrence of OIC in this rat model and the regulation of colonic motility by MOR may be related to the release of purine neurotransmitters such as ATP in the colonic nervous system.

THU264 Enteric Activity Enhanced Ileal GLP-1 Secretion

Abstract Disclosure: Y. Chen: None. G. Jiao: None. Y. Lei: None. Y. Li: None. L. Sha: None. GLP-1 is a multifunctional endocrine hormone secreted by intestinal L cells in gastrointestinal tract contributing to the maintenance of glucose homeostasis. The distal part of small intestine such as ileum has a great density of L cells. In this study, we provided a direct evidence that enteric activity modulated GLP-1 secretion in rat ileum. Methods: A segment of rat ileum was dissected. The proximal section of the ileum segment was used to pace the enteric activity with an intestinal electrical stimulation(IES) designed from our previous intestinal myoelectric activity analyzing mimicking the ileum activity in feeding and the adjacent distal section was perfused vascularly to collect and measure GLP-1 secretion. Ileal motility in the distal section of the ileum segment was also recorded to monitor the enteric activity. The lumen of the ileum segment was filled with saline. Results: The frequency and the amplitude of spontaneous contraction in the distal ileum section was 14.14±2.82 cycles/min and 1.58±0.46 g (n=5). The area under curve (AUC) was 180.40±61.68 g·s. The GLP-1 secretion level in the distal ileum section was 15.47±0.46 pg/mL. With IES, The frequency was increased to 18.27±2.23 cycles/min (P<0.05, n = 5) and the AUC was increased to 303.64±79.75 (p<0.05). The amplitude had no significant change (P>0.05). The GLP-1 secretion was significantly increased to 24.71±2.47 pg/mL(p<0.05) with enhanced enteric activity. we provided a direct evidence to show the enhancement of GLP-1 secretion with enteric activity. Our result indicated that with feeding-simulated enteric activity, GLP-1 secretion could be elevated by around 60 percent in rat ileum. (supported by the National Natural Science Foundation of China, Grant No.81670492) Presentation: Thursday, June 15, 2023

Inhibitory effect and underlying mechanism of essential oil of Prangos ferulacea Lindl (L.) on spontaneous and induced uterine contractions in non-pregnant rats

Evidence suggests the use of natural compounds as support in the management of uterine contractility disorders. We recently demonstrated that the essential oil of Apiacea Prangos ferulacea (L.) (Prangoil) modulates intestinal smooth muscle contractility. Thus, we aimed to evaluate if Prangoil could also affect the contractility of uterine muscle in non-pregnant rat and to investigate the related action mechanism/s. The effects of the aromatic monoterpenes, β-ocimene and carvacrol, constituents of Prangoil, were also evaluated. Spontaneous contractions and contraction-induced by K+-depolarization and oxytocin in rat uterus were recorded in vitro, using organ bath technique. Prangoil reduced the amplitude of spontaneous contractions as well as responses to KCl and oxytocin. β-ocimene and carvacrol matched oil inhibitory effects. Prangoil effects were not affected by nitrergic and adenylyl cyclase inhibitors or non-specific potassium channel blocker, but they were reduced by nifedipine, L-type calcium channel inhibitor, or 2-aminoethoxydiphenylborate (2-APB), membrane-permeant inositol 1,4,5-triphosphate receptor inhibitor. The response to β-ocimene was reduced by nifedipine and by 2-APB (20 μM), whilst carvacrol inhibitory effect was attenuated only by nifedipine. In conclusion, Prangoil, and its components, β-ocimene and carvacrol, reduced spontaneous and KCl or oxytocin-induced contractions of rat myometrium, mainly modulating extracellular Ca2+ influx through L-Type channels and Ca2+ release from the intracellular store. Further studies could contribute to evaluate the potential use of Prangoil against disorders characterized by abnormal uterine contractions.

Characterization of rat gastric myogenic contractions and modulation by oxytocin and arginine-vasopressin

BackgroundInterstitial cells of Cajal generate slow wave gastric electrical activity, initiating spontaneous muscle contractions. This becomes dysrhythmic during nausea when [Arg8]-vasopressin (AVP) is also released. In human stomach AVP increased spontaneous contraction activity and muscle tone, not neuronally-mediated contractions. Rodents cannot vomit, releasing the related hormone, oxytocin (OT) instead. We hypothesised that rat stomach would behave differently. Experimental approachSpontaneous and electrically-evoked (EFS) contractions were measured in rat forestomach and antrum circular muscle. Custom software defined spontaneous contractions by analysing eight motility parameters. ResultsThe forestomach was quiescent. Irregular antrum contractions became regular adjacent to the pylorus (1.7 ± 0.4 mN; 1.2 ± 0.1 contractions/min, n = 12). These were unaffected by tetrodotoxin (10−6 M), atropine (10−6 M) and L-NAME (3 × 10−4 M). In both regions, AVP (pEC50∼9.0) and OT (∼0.5 log10-unit less potent) caused contraction (greater in antrum), competitively antagonized by, respectively, SR49059 (pKB∼9.5) and L371257 (pKB∼9.0), reduced by tetrodotoxin but unaffected by atropine. In the antrum, AVP and OT (∼2 log10-units less potent/efficacious) regularized and increased spontaneous contraction amplitude, frequency, rates of contraction/decay. In both regions, EFS-evoked contractions, abolished by atropine/tetrodotoxin, were reduced by AVP and OT, with AVP more potent and efficacious, particularly in forestomach. ConclusionIrregular spontaneous contractions of gastric antrum suggest variable ICC-muscle coupling. AVP and less potently, OT, enhanced frequency and force of contractions via V1A and OT receptors. Compared with human, differences in contraction regularity, potency and ability of AVP/OT to affect neuronal function suggests caution when using rat stomach to model ICC functions and nauseagenic stimuli.

Inhibition of uterine contractility by guanine-based purines in non-pregnant rats.

Growing evidence pointed out that guanine-based purines are able to modulate smooth muscle contractile activity of blood vessels and gastrointestinal tract. Since, so far, possible guanine-based purine modulation of uterine musculature is unknown, the aim of the present study was to investigate in vitro, using organ bath technique, guanosine and guanine effects on spontaneous uterine contraction, and uterine contraction induced by K+-depolarization and oxytocin in a non-pregnant rat. Guanosine, but not guanine, reduced the amplitude of spontaneous contraction of the uterine muscle in a dose-dependent manner. The inhibitory response was antagonized by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a membrane nucleoside transporter inhibitor, but persisted in the presence of theophylline, a nonselective adenosine receptor antagonist, or propanolol, β1/β2 adrenoreceptor antagonist or blockers of a nitrergic pathway. In addition, potassium channel blockers did not influence guanosine-induced effects. Guanosine was able to inhibit the external calcium (Ca2+) influx-induced contraction, but it did not affect the contraction induced by high-KCl solution, indicating that guanosine does not interact with L-type voltage-gated calcium channel. Guanosine prevented/reduced uterine contractions induced by oxytocin, even in the absence of external calcium. In conclusion, guanosine is able to reduce both spontaneous and oxytocin-induced contractions of rat myometrium, likely subsequently to its intracellular intake. The blockade of extracellular Ca2+ influx and reduction of Ca2+ release from the intracellular store are the mechanisms involved in the guanosine-induced tocolytic effects.

5-HT3 receptors modulate changes in voiding pattern and bladder contractility in water avoidance stress-induced bladder overactivity in male mice.

Chronic psychological stress aggravates painful bladder syndrome symptoms. Previous studies suggest roles of 5-HT3 receptors in regulating micturition and bladder hypersensitivity. This study aimed to investigate the roles of 5-HT3 receptors in modulating voiding patterns and spontaneous bladder contractile properties in water avoidance stress-induced mice. Voiding patterns in sham stress (SS), water avoidance stress (WS), and water avoidance stress with daily oral gavage of ondansetron (1mg/kg BW) (WA) groups were analyzed after exposure to repeated water avoidance stress for 10days. Changes in contractile activity of isolated bladder in response to KCl, carbachol, and 5-hydroxytryptamine were determined. Bladder mast cell quantification was examined using toluidine blue staining. Urine voided area was significantly decreased in WS group after exposure to 10days of the stress protocol, which was reversed in the WA group. The WS group had a higher number of urine spots than the SS group. Increased mast cell degranulation was observed in the stressed mice. Bladder strips of the WS group showed higher tonic and amplitude of spontaneous contraction than the SS group, which were normalized by ondansetron administration. Increased response to carbachol-induced bladder contraction was observed in the bladder of stressed mice, which was attenuated with ondansetron pre-incubation. Water avoidance stress-induced mice exhibited changes in voiding pattern, which was reversed by oral administration with a 5-HT3 receptor antagonist (ondansetron). Enhanced contractile response to cholinergic stimulation in the urinary bladder of the psychological stress-induced bladder overactivity was mediated through 5-HT3 receptors.

Downregulation of Ca2+-Activated Cl- Channel TMEM16A Mediated by Angiotensin II in Cirrhotic Portal Hypertensive Mice.

Portal hypertension is defined as an increased pressure in the portal venous system and occurs as a major complication in chronic liver diseases. The pathological mechanism underlying the pathogenesis and development of portal hypertension has been extensively investigated. Vascular tone of portal vein smooth muscles (PVSMs) is regulated by the activities of several ion channels, including Ca2+-activated Cl− (ClCa) channels. TMEM16A is mainly responsible for ClCa channel conductance in vascular smooth muscle cells, including portal vein smooth muscle cells (PVSMCs). In the present study, the functional roles of TMEM16A channels were examined using two experimental portal hypertensive models, bile duct ligation (BDL) mice with cirrhotic portal hypertension and partial portal vein ligation (PPVL) mice with non-cirrhotic portal hypertension. Expression analyses revealed that the expression of TMEM16A was downregulated in BDL-PVSMs, but not in PPVL-PVSMs. Whole-cell ClCa currents were smaller in BDL-PVSMCs than in sham- and PPVL-PVSMCs. The amplitude of spontaneous contractions was smaller and the frequency was higher in BDL-PVSMs than in sham- and PPVL-PVSMs. Spontaneous contractions sensitive to a specific inhibitor of TMEM16A channels, T16Ainh-A01, were reduced in BDL-PVSMs. Furthermore, in normal PVSMs, the downregulation of TMEM16A expression was mimicked by the exposure to angiotensin II, but not to bilirubin. This study suggests that the activity of ClCa channels is attenuated by the downregulation of TMEM16A expression in PVSMCs associated with cirrhotic portal hypertension, which is partly mediated by increased angiotensin II in cirrhosis.

A-Effects of Drugs on Spontaneous Contractions of Rabbit Jejunum

Aim: To find out possible to determine effect of C. reflexa extract on spontaneous contractions of rabbit jejunum. Methods: Animal experimental in vitro study from 1st October 2017 to 30 September 2018 at Pharmacology experimental lab, Wah Medical College Wah Cantt. Study model was Isolated pieces of rabbit jejunum and ileum. Sampling Technique used was Simple Random. Tissue considered fit for experiment after initial equilibration and stabilization. Result: Three increasing concentrations (0.1mg/ml, 0.2mg/ml and 0.4mg/ml) of C. reflexa extract decreased the response to 3.83±0.40, 2.33±0.33 and 1.33± 0.21mm. Decrease in response was significant with p- value 0.04, 0.008 and 0.003 respectively. Conclusion: Freshly prepared aqueous solution of crude extract of C. reflexa decreases the amplitude of spontaneous contractions of isolated rabbit jejunum. Keywords: Rabbit jejunum, effect of drug

Isolation and characterization of FMRFamide-like peptides in the venoms of solitary sphecid wasps

Purification of small peptide components in the venoms of the solitary sphecid wasps, Sphex argentatus argentatus and Isodontia harmandi, led to the isolation of several major peptides. Analysis of MS/MS spectra by MALDI-TOF/TOF revealed the sequence of a new peptide Sa112 (EDVDHVFLRF-NH2), which is structurally very similar to leucomyosupressin (pQDVDHVFLRF-NH2) and SchistoFLRFamide (PDVDHVFLRF-NH2), the FMRFamide-like peptides from cockroach and locust, respectively. Indeed, this new peptide, like SchistoFLRFamide, inhibited the frequency and amplitude of spontaneous contractions of the locust oviduct in a dose-dependent manner. A non-amidated peptide Sa12b (EDVDHVFLRF) was also isolated, but this peptide had no effect on spontaneous locust oviduct contraction. This is the first example of a FMRF-like peptide to be found in solitary wasp venom. Additionally, a truncated form of the myosuppressins, which has previously been synthesized and tested for biological activity, DVDHVFLRF-NH2 (Sh5b), was found for the first time as a natural product. Four other novel peptides were isolated and characterized as Sa81 (EDDLEDFNPTVS), Sa10 (EDDLEDFNPTIA), Sh41 (DDLSDFNPKV), and Sh42 (EDDLSDFNPKV). They are structurally related to each other, having a high content of acidic amino acids, but no structural similarity to any known peptides. Ion channel associated activities of Sh41 and Sh42 were tested, but did not show any activity for Na+, K+, Ca2+ channels.

Fetuin-A exerts a protective effect against experimentally induced intestinal ischemia/reperfusion by suppressing autophagic cell death.

Intestinal tissue is highly susceptible to ischemia/reperfusion injury in many hazardous health conditions. The anti-inflammatory and antioxidant glycoprotein fetuin-A showed efficacy in cerebral ischemic injury; however, its protective role against intestinal ischemia/reperfusion remains elusive. Therefore, this study investigated the protective role of fetuin-A supplementation against intestinal structural changes and dysfunction in a rat model of intestinal ischemia/reperfusion. We equally divided 72 male rats into control, sham, ischemia/reperfusion, and fetuin-A-pretreated ischemia/reperfusion (100 mg/kg/day fetuin-A intraperitoneally for three days prior to surgery and a third dose 1 h prior to the experiment) groups. After 2 h of reperfusion, the jejunum was dissected and examined for spontaneous contractility. A jejunal homogenate was used to assess inflammatory and oxidative stress enzymes. Staining of histological sections was carried out with hematoxylin, eosin and Masson's trichrome stain for evaluation. Immunohistochemistry was performed to detect autophagy proteins beclin-1, LC3, and p62. This study found that fetuin-A significantly improved ischemia/reperfusion-induced mucosal injury by reducing the percentage of areas of collagen deposition, increasing the amplitude of spontaneous contraction, decreasing inflammation and oxidative stress, and upregulating p62 expression, which was accompanied by beclin-1 and LC3 downregulation. Our findings suggest that fetuin-A treatment can prevent ischemia/reperfusion-induced jejunal structural and functional changes by increasing antioxidant activity and regulating autophagy disturbances observed in the ischemia/reperfusion rat model. Furthermore, fetuin-A may provide a protective influence against intestinal ischemia/reperfusion complications.

Short chain fatty acids and colon motility in a mouse model of irritable bowel syndrome

BackgroundIrritable bowel syndrome (IBS) is defined as a multifactorial disorder associated with visceral hypersensitivity, altered gut motility and dysfunction of the brain-gut axis. Gut microbiota and its metabolites are proposed as possible etiological factors of IBS. Short chain fatty acids (SCFAs) induce both inhibitory and stimulatory action on colon motility, however, their effects on the IBS model were not investigated. The aim of our study was to investigate the level of SFCAs in feces and their effects on colon motility in a mouse model of IBS.MethodsIBS model was induced in mice by intracolonic infusion of 1% acetic acid during the early postnatal period. Mice colon hypersensitivity was assessed by the threshold of the abdominal withdrawal reflex in response to colorectal distention. Colon contractility was studied using proximal colon specimens in isometric conditions. Transit rates were assessed by the pellet propulsion in the isolated colon. Concentrations of SCFAs in feces were measured using gas–liquid chromatography.ResultsThe concentration of SCFAs in feces of IBS model mice was higher compared to the control group. Visceral sensitivity to colorectal distension and colonic transit rate were increased indicating IBS with predominant diarrhea. The frequency and amplitude of spontaneous contractions of proximal colon segments from IBS mice were higher, but carbachol induced contractions were lower compared to control. During acute application of SCFAs (sodium propionate, sodium acetate or butyric acid) dose-dependently (0.5–30 mM) decreased tonic tension, frequency and amplitude of spontaneous and carbachol-evoked contractions. In the mouse IBS group the inhibitory effects SCFAs on spontaneous and carbachol-evoked contractions were less pronounced. At the same time intraluminal administration of butyrate (5 mM) increased the transit rate in the colon of both groups, but its stimulatory effect was more pronounced in mouse IBS model group.ConclusionOur data indicate that the increased transit rate in the mouse IBS model group is associated with a disbalance of activating and inhibiting action of SCFAs due to chronically elevated SCFA levels, which may impact the pathogenesis of IBS with predominant diarrhea syndrome.

Chemical and functional analyses of Rhinella icterica (Spix, 1824) toad secretion screened on contractions of the heart and oviduct in Locusta migratoria

Rhinella icterica is a Brazilian toad with a parotoid secretion that is toxic to insects. In this work, we examined the entomotoxicity of this secretion in locust (Locusta migratoria) semi-isolated heart and oviduct preparations in vitro. The parotoid secretion caused negative chronotropism in semi-isolated heart preparations (at the highest dose tested: 500 μg) and markedly enhanced the amplitude of spontaneous contractions and tonus of oviduct muscle (0.001–100 μg). In addition, the secretion enhanced neurally-evoked contractions of oviduct muscle, which was more sensitive to low concentrations of secretion than the semi-isolated heart. The highest dose of secretion (100 μg) caused neuromuscular blockade. In zero calcium-high magnesium saline, the secretion still enhanced muscle tonus, suggesting the release of intracellular calcium to stimulate contraction. Reverse-phase HPLC of the secretion yielded eight fractions, of which only fractions 4 and 5 affected oviduct muscle tonus and neurally-evoked contractions. No phospholipase A2 activity was detected in the secretion or its chromatographic fractions. The analysis of fractions 4 and 5 by LC-DAD-MS/MS revealed the following chemical compounds: suberoyl arginine, hellebrigenin, hellebrigenin 3-suberoyl arginine ester, marinobufagin 3-pimeloyl arginine ester, telocinobufagin 3-suberoyl arginine ester, marinobufagin 3-suberoyl arginine ester, bufalin 3-adipoyl arginine, marinobufagin, bufotalinin, and bufalitoxin. These findings indicate that R. icterica parotoid secretion is active in both of the preparations examined, with the activity in oviduct possibly being mediated by bufadienolides.

Shugan Decoction Alleviates Colonic Dysmotility in Female SERT-Knockout Rats by Decreasing M3 Receptor Expression.

BackgroundIrritable bowel syndrome (IBS) is a functional gut disease characterized by visceral hypersensitivity and gut motor dysfunction. Serotonin (5-hydroxytryptamine, 5-HT) is an important enteric neurotransmitter. High levels of 5-HT aggravate IBS symptoms. The serotonin reuptake transporter (SERT) is a membrane-embedded transporter involved in IBS pathogenesis that plays an important role in regulating 5-HT signaling.AimWe investigated whether gut motor function was altered in SERT-knockout (SERT-KO) rats. Additionally, we sought to determine whether Shugan decoction (SGD), a clinically experienced prescription for the treatment of IBS, exerts regulatory effects on intestinal motility in SERT-KO rats, and attempted to identify the mechanisms involved.MethodSERT-KO rats were produced by transcription activator-like effector nuclease (TALEN) technology. Fecal pellet output was measured for ten consecutive days to estimate distal colonic motility. Small intestinal motility was measured by charcoal-meal experiments. The colonic and small intestinal muscle contractile activities were measured by organ bath study. Western blot was used to analyze the muscarinic receptor expression in colon tissue.ResultCompared with that in wild-type (WT) rats, the defecation amount, amplitude of spontaneous contraction, and the tension of ACh-induced contraction of colonic longitudinal smooth muscle in SERT-KO rats were significantly increased. The expression of muscarinic receptor subtype-3 (M3R) in the colons of SERT-KO rats was also elevated. SGD can decrease defecation of SERT-KO rats. Moreover, SGD reduced the amplitude of spontaneous contraction, the frequency and tension of ACh-induced contraction of colonic longitudinal smooth muscle, and the expression of M3R in the colon in SERT-KO rats.ConclusionsSERT-KO rats showed increased defecation accompanied by enhanced colonic motility and M3R expression. The findings suggest that SGD modifies colonic dysmotility and reduces defecation in SERT-KO rats by down-regulating M3R expression in the colon.

TRPM8 Channel Activation Reduces the Spontaneous Contractions in Human Distal Colon.

The transient receptor potential-melastatin 8 (TRPM8) is a non-selective Ca2+-permeable channel, activated by cold, membrane depolarization, and different cooling compounds. TRPM8 expression has been found in gut mucosal, submucosal, and muscular nerve endings. Although TRPM8 plays a role in pathological conditions, being involved in visceral pain and inflammation, the physiological functions in the digestive system remain unclear as yet. The aims of the present study were: (i) to verify the TRPM8 expression in human distal colon; (ii) to examine the effects of TRPM8 activation on colonic contractility; (iii) to characterize the mechanism of action. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting were used to analyze TRPM8 expression. The responses of human colon circular strips to different TRPM8 agonists [1-[Dialkyl-phosphinoyl]-alkane (DAPA) 2–5, 1-[Diisopropyl-phosphinoyl]-alkane (DIPA) 1–7, DIPA 1–8, DIPA 1–9, DIPA 1–10, and DIPA 1–12) were recorded using a vertical organ bath. The biomolecular analysis revealed gene and protein expression of TRPM8 in both mucosal and smooth muscle layers. All the agonists tested, except-DIPA 1–12, produced a concentration-dependent decrease in spontaneous contraction amplitude. The effect was significantly antagonized by 5-benzyloxytryptamine, a TRPM8 antagonist. The DIPA 1–8 agonist resulted in the most efficacious and potent activation among the tested molecules. The DIPA 1–8 effects were not affected by tetrodotoxin, a neural blocker, but they were significantly reduced by tetraethylammonium chloride, a non-selective blocker of K+ channels. Moreover, iberiotoxin, a blocker of the large-conductance Ca2+-dependent K+-channels, but not apamin, a blocker of small-conductance Ca2+-dependent K+ channels, significantly reduced the inhibitory DIPA 1–8 actions. The results of the present study demonstrated that TRPM8 receptors are also expressed in human distal colon in healthy conditions and that ligand-dependent TRPM8 activation is able to reduce the colonic spontaneous motility, probably by the opening of the large-conductance Ca2+-dependent K+-channels.

The effects of intra-stomach obestatin administration on intestinal contractility in neonatal piglets fed milk formula

A 23-amino acid peptide named obestatin is derived from the ghrelin gene. The aim of the experiment was to study the effects of enteral obestatin administration for a 6-day period on intestinal contractility in piglets fed milk formula. Pigs were treated with 0.9% NaCl (group C) or varying doses of obestatin: 2 μg/kg body weight (BW) (group O2), 10 μg/kg BW (O10) or 15 μg/kg BW (O15) every 8 hours via a stomach tube. Blood was sampled for assessment of obestatin concentration. Duodenal and middle jejunum whole-thickness preparations were studied in an organ bath for isometric recording under electric field stimulation (EFS) and increasing doses of acetylcholine (ACh), and in the presence of atropine and tetrodotoxin (TTX). Additionally, the measurement of intestinal muscularis layer and the immunodetection of Muscarinic Acetylcholine Receptors (M1 and M2) were performed. In comparison to C animals, the obestatin concentration in blood plasma was significantly increased in groups O10 and O15. In both studied intestinal segments, significant increases in the frequency and amplitude of spontaneous contractions were observed in O15 and C groups. In the duodenum and middle jejunum significant differences in responsiveness to EFS (0.5, 5 and 50 Hz) were observed between the groups. The addition of 10−4 M ACh to the duodenum significantly increased the responsiveness in tissues. In contrast, in the middle jejunum a significant increase in the amplitude of contraction was observed after the addition of 10−9 and 10−6 M ACh (groups O15 and O10, respectively). Pretreatment with atropine and TTX resulted in a significant decrease in the responsiveness of the intestinal preparations from all groups, in both studied segments. The increased contractility was not dependent on the expression of muscarinic receptors. Results indicate the importance of enteral obestatin administration in the regulation of intestinal contractility in neonatal piglets.

T-type, but not L-type, voltage-gated calcium channels are dispensable for lymphatic pacemaking and spontaneous contractions

The spontaneous contractions of collecting lymphatic vessels provide an essential propulsive force to return lymph centrally. These contractions are driven by an intrinsic electrical pacemaker, working through an unknown underlying ionic mechanism that becomes compromised in some forms of lymphedema. In previous studies, T-type voltage-gated Ca2+ channels (VGCCs) were implicated in this pacemaking mechanism, based on the effects of the reputedly selective T-type VGCC inhibitors mibefradil and Ni2+. Our goal was to test this idea in a more definitive way using genetic knock out mice. First, we demonstrated through both PCR and immunostaining that mouse lymphatic muscle cells expressed Cav3.1 and Cav3.2 and produced functional T-type VGCC currents when patch clamped. We then employed genetic deletion strategies to selectively test the roles of each T-type VGCC isoform in the regulation of lymphatic pacemaking. Surprisingly, global deletion of either, or both, isoform(s) was without significant effect on either the frequency, amplitude, or fractional pump flow of lymphatic collectors from two different regions of the mouse, studied ex vivo. Further, both WT and Cav3.1−/−; 3.2−/− double knock-out lymphatic vessels responded similarly to mibefradil and Ni2+, which substantially reduced contraction amplitudes and slightly increased frequencies at almost all pressures in both strains: a pattern consistent with inhibition of L-type rather than T-type VGCCs. Neither T-type VGCC isoform was required for ACh-induced inhibition of contraction, a mechanism by which those channels in smooth muscle are thought to be targets of endothelium-derived nitric oxide. Sharp intracellular electrode measurements in lymphatic smooth muscle revealed only subtle, but not significant, differences in the resting membrane potential and action potential characteristics between vessels from wild-type and Cav3.1−/−; 3.2−/− double knock-out mice. In contrast, smooth-muscle specific deletion of the L-type VGCC, Cav1.2, completely abolished all lymphatic spontaneous contractions. Collectively our results suggest that, although T-type VGCCs are expressed in mouse lymphatic smooth muscle, they do not play a significant role in modulating the frequency of the ionic pacemaker or the amplitude of spontaneous contractions. We conclude that the effects of mibefradil and Ni2+ in other lymphatic preparations are largely or completely explained by off-target effects on L-type VGCCs, which are essential for controlling both the frequency and strength of spontaneous contractions.

Contractile Activity of Lymph Nodes in Young, Middle-Aged, and Old Rats.

We studied age-associated changes in contractile activity of isolated cervical and mesenteric lymph nodes in rats. In young and middle-aged rats, no significant changes in contractile activity of lymph nodes were revealed. In old rats, the amplitude of spontaneous contractions of lymph nodes was lower and their rate was higher than in young and middle-aged animals; the sensitivity of lymph nodes to vasoactive agents was reduced and the threshold of the response was higher in old rats.

Altered detrusor contractility and voiding patterns in mice lacking the mechanosensitive TREK-1 channel

BackgroundPreviously published results from our laboratory identified a mechano-gated two-pore domain potassium channel, TREK-1, as a main mechanosensor in the smooth muscle of the human urinary bladder. One of the limitations of in vitro experiments on isolated human detrusor included inability to evaluate in vivo effects of TREK-1 on voiding function, as the channel is also expressed in the nervous system, and may modulate micturition via neural pathways. Therefore, in the present study, we aimed to assess the role of TREK-1 channel in bladder function and voiding patterns in vivo by using TREK-1 knockout (KO) mice.MethodsAdult C57BL/6 J wild-type (WT, N = 32) and TREK-1 KO (N = 33) mice were used in this study. The overall phenotype and bladder function were evaluated by gene and protein expression of TREK-1 channel, in vitro contractile experiments using detrusor strips in response to stretch and pharmacological stimuli, and cystometry in unanesthetized animals.ResultsTREK-1 KO animals had an elevated basal muscle tone and enhanced spontaneous activity in the detrusor without detectable changes in bladder morphology/histology. Stretch applied to isolated detrusor strips increased the amplitude of spontaneous contractions by 109% in the TREK-1 KO group in contrast to a 61% increase in WT mice (p ≤ 0.05 to respective baseline for each group). The detrusor strips from TREK-1 KO mice also generated more contractile force in response to electric field stimulation and high potassium concentration in comparison to WT group (p ≤ 0.05 for both tests). However, cystometric recordings from TREK-1 KO mice revealed a significant increase in the duration of the intermicturition interval, enhanced bladder capacity and increased number of non-voiding contractions in comparison to WT mice.ConclusionsOur results provide evidence that global down-regulation of TREK-1 channels has dual effects on detrusor contractility and micturition patterns in vivo. The observed differences are likely due to expression of TREK-1 channel not only in detrusor myocytes but also in afferent and efferent neural pathways involved in regulation of micturition which may underly the “mixed” voiding phenotype in TREK-1 KO mice.

Effects of ajmaline on contraction patterns of isolated rat gastric antrum and portal vein smooth muscle strips and on neurogenic relaxations of gastric fundus.

Class-I-antiarrhythmics like ajmaline are known to alter smooth muscle function, which may cause alterations in gastrointestinal motility. The effects of ajmaline on isolated gastric and portal vein smooth muscle and the underlying mechanisms are unknown. We studied the effects of ajmaline on the contractile patterns of isolated preparations of gastric antrum and portal vein from Wistar rats. The organ bath technique was used to measure spontaneous or pharmacologically induced isometric contractions. Changes in force observed after application of ajmaline or under control conditions are reported as % of the amplitude of an initial K+-induced contraction. Electric field stimulation was used to study neurogenic relaxations of gastric fundus smooth muscle. Ajmaline increased the amplitude of spontaneous contractions of muscle strips (portal vein: control 31.1 ± 15.2%, with 100μM ajmaline 76.6 ± 32.3%, n = 9, p < 0.01; gastric antrum: control 9.5 ± 1.6%, with 100μM ajmaline 63.9 ± 9.96%, n = 14, p < 0.01). The frequency of spontaneous activity was reduced in portal vein, but not in gastric antrum strips. The effects of ajmaline were not blocked by tetrodotoxin, L-nitroarginine methyl ester, or atropine. Ajmaline abolished coordinated neurogenic relaxations triggered by electric field stimulation and partly reversed the inhibition of GA spontaneous activity caused by the gap junction blocker carbenoxolone. Ajmaline enhances the amplitude of spontaneous contractions in rat gastric and portal vein smooth muscle. This effect may be accompanied, but not caused by an inhibition of enteric neurotransmission. Enhanced syncytial coupling as indicated by its ability to antagonize the effects of carbenoxolone is likely to underlie the enhancement of contractility.