Fundus Smooth Muscle Research Articles

Inhibitory mechanisms of docosahexaenoic acid on carbachol-, angiotensin II-, and bradykinin-induced contractions in guinea pig gastric fundus smooth muscle

We studied the inhibitory actions of docosahexaenoic acid (DHA) on the contractions induced by carbachol (CCh), angiotensin II (Ang II), and bradykinin (BK) in guinea pig (GP) gastric fundus smooth muscle (GFSM), particularly focusing on the possible inhibition of store-operated Ca2+ channels (SOCCs). DHA significantly suppressed the contractions induced by CCh, Ang II, and BK; the inhibition of BK-induced contractions was the strongest. Although all contractions were greatly dependent on external Ca2+, more than 80% of BK-induced contractions remained even in the presence of verapamil, a voltage-dependent Ca2+ channel inhibitor. BK-induced contractions in the presence of verapamil were not suppressed by LOE-908 (a receptor-operated Ca2+ channel (ROCC) inhibitor) but were suppressed by SKF-96365 (an SOCC and ROCC inhibitor). BK-induced contractions in the presence of verapamil plus LOE-908 were strongly inhibited by DHA. Furthermore, DHA inhibited GFSM contractions induced by cyclopiazonic acid (CPA) in the presence of verapamil plus LOE-908 and inhibited the intracellular Ca2+ increase due to Ca2+ addition in CPA-treated 293T cells. These findings indicate that Ca2+ influx through SOCCs plays a crucial role in BK-induced contraction in GP GFSM and that this inhibition by DHA is a new mechanism by which this fatty acid inhibits GFSM contractions.

Effects of Chinese ginger Alpinia officinarum-based phytopharmaceutical compound on gastric motility

BackgroundAlpinia officinarum, better known as Chinese ginger, is used for the treatment of gastrointestinal disorders in traditional Chinese and Ayurveda medicine. However, scientific evidence of an effect on gastrointestinal motility is missing. PurposeThis study aimed to estimate the effect of a phytopharmaceutical compound (PPC) based on Alpinia officinarum on gastric motility in vitro in order to assess its potential use in the treatment of functional dyspepsia. Study DesignWe investigated to which extent the compound affects the activity of gastric smooth muscle and we identified involved mediating cellular mechanisms. MethodsBasal and stimulated muscle activity were recorded from muscle-myenteric plexus preparations from three different regions of the guinea pig stomach before and after incubation with different concentrations of the PPC in an organ bath setup. Effect-mediating mechanisms were investigated by pharmacology. ResultsPPC exhibited a smooth muscle relaxation in a concentration-dependent manner. This was region- and muscle type specific as it was greater in fundus and corpus than in antrum and more pronounced in the circular than in the longitudinal muscle layer. The effect was primarily myogenic and at least partly mediated by TRPA1. ConclusionThe investigated PPC reduces basal tone particularly of fundus smooth muscle in a non-neuronal manner by inhibiting calcium influx via TRPA1, without affecting antral motility. Based on these data, Alpinia officinarum could be considered for phytopharmaceutical treatment of functional dyspepsia.

Electrophysiology, molecular modelling, and functional analysis of the effects of dietary quercetin and flavonoid analogues on Kir6.1 channels in rat stomach fundus smooth muscle

Flavonoids, ubiquitously distributed in the plant world, are regularly ingested with diets rich in fruit, vegetables, wine, and tea. During digestion, they are partially absorbed in the stomach. The present work aimed to assess the in vitro effects of quercetin and ten structurally related flavonoids on the rat gastric fundus smooth muscle, focussing on ATP-dependent K+ (Kir6.1) channels, which play a central role in the regulation of resting membrane potential, membrane excitability and, consequently, of gastric motility. Whole-cell currents through Kir6.1 channels (IKir6.1) were recorded with the patch-clamp technique and the mechanical activity of gastric fundus smooth muscle strips was studied under isometric conditions. Galangin ≈ tamarixetin > quercetin > kaempferol > isorhamnetin ≈ luteolin ≈ fisetin > (±)-taxifolin inhibited pinacidil-evoked, glibenclamide-sensitive IKir6.1 in a concentration-dependent manner. Morin, rutin, and myricetin were ineffective. The steric hindrance of the molecule and the number and position of hydroxyl groups on the B ring played an important role in the activity of the molecule. Molecular docking simulations revealed a possible binding site for flavonoids in the C-terminal domain of the Kir6.1 channel subunit SUR2B, in a flexible loop formed by residues 251 to 254 of chains C and D. Galangin and tamarixetin, but not rutin relaxed both high K+- and carbachol-induced contraction of fundus strips in a concentration-dependent manner. Furthermore, both flavonoids shifted to the right the concentration-relaxation curves to either pinacidil or L-cysteine constructed in strips pre-contracted by high K+, rutin being ineffective. In conclusion, IKir6.1 inhibition exerted by dietary flavonoids might counterbalance their myorelaxant activity, affect gastric accommodation or, at least, some stages of digestion.

Defining the Molecular Mechanisms of the Relaxant Action of Adiponectin on Murine Gastric Fundus Smooth Muscle: Potential Translational Perspectives on Eating Disorder Management.

Adiponectin (ADPN), a hormone produced by adipose tissue, facilitates gastric relaxation and can be a satiety signal in the network connecting peripheral organs and the central nervous system for feeding behavior control. Here, we performed preclinical research by morpho-functional analyses on murine gastric fundus smooth muscle to add insights into the molecular mechanisms underpinning ADPN action. Moreover, we conducted a clinical study to evaluate the potential use of ADPN as a biomarker for eating disorders (ED) based on the demonstrated gastric alterations and hormone level fluctuations that are often associated with ED. The clinical study recruited patients with ED and healthy controls who underwent blood draws for ADPN dosage and psychopathology evaluation tests. The findings of this basic research support the ADPN relaxant action, as indicated by the smooth muscle cell membrane pro-relaxant effects, with mild modifications of contractile apparatus and slight inhibitory effects on gap junctions. All of these actions engaged the ADPN/nitric oxide/guanylate cyclase pathway. The clinical data failed to unravel a correlation between ADPN levels and the considered ED, thus negating the potential use of ADPN as a valid biomarker for ED management for the moment. Nevertheless, this adipokine can modulate physiological eating behavior, and its effects deserve further investigation.

Docosahexaenoic acid and eicosapentaenoic acid strongly inhibit prostanoid TP receptor-dependent contractions of guinea pig gastric fundus smooth muscle.

The inhibitory effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and linoleic acid (LA) on the contractions induced by five prostanoids and U46619 (a TP receptor agonist) were examined in guinea pig gastric fundus smooth muscle (GFSM). Tension changes were isometrically measured, and the mRNA expression of prostanoid receptors was measured by RT‐qPCR. DHA and EPA significantly inhibited contractions induced by the prostanoids and U46619, whereas LA inhibited those induced by prostaglandin D2 and U46619. The mRNA expression levels of the prostanoid receptors were TP ≈ EP3 >> FP > EP1. The inhibition by DHA, EPA, and LA was positively correlated with that by SQ 29,548 (a TP receptor antagonist) but not with that by L‐798,106 (an EP3 receptor antagonist). DHA and EPA suppressed high KCl‐induced contractions by 35% and 25%, respectively, and the contractions induced by the prostanoids and U46619 were suppressed by verapamil, a voltage‐dependent Ca2+ channel (VDCC) inhibitor, by 40%–85%. Although LA did not suppress high KCl‐induced contractions, it suppressed U46619‐induced contractions in the presence of verapamil. However, LA did not show significant inhibitory effects on U46619‐induced Ca2+ increases in TP receptor‐expressing cells. In contrast, LA inhibited U46619‐induced contractions in the presence of verapamil, which was also suppressed by SKF‐96365 (a store‐operated Ca2+ channel [SOCC] inhibitor). These findings suggest that the TP receptor and VDCC are targets of DHA and EPA to inhibit prostanoid‐induced contractions of guinea pig GFSM, and SOCCs play a significant role in LA‐induced inhibition of U46619‐induced contractions.

Adiponectin and the regulation of gastric content volume in the newborn rat

BACKGROUNDOral intake is dependent on the gastric ability to accommodate the food bolus. Comparatively, neonates have a smaller gastric capacity than adults and this may limit the volume of their milk intake. Yet, we previously reported that the newborn rat gastric milk volume is greatest after birth and, when normalized to body weight, decreases with postnatal age. Such age-dependent changes are not the result of intake differences, but greater gastric accommodation and reduced emptying rate. AIMHypothesizing that breastmilk-derived adiponectin is the factor regulating gastric accommodation in neonates, we comparatively evaluated its effects on the rat fundic muscle tone at different postnatal ages. METHODSIn freshly dispersed smooth muscle cells (SMC), we measured the adiponectin effect on the carbachol-induced length changes.RESULTSAdiponectin significantly reduced the carbachol-stimulated SMC shortening independently of age. In the presence of the inhibitor iberiotoxin, the adiponectin effect on SMC shortening was suppressed, suggesting that it is mediated via large-conductance Ca2+ sensitive K+ channel activation. Lastly, we comparatively measured the newborn rat gastric milk curd adiponectin content in one- and two-week-old rats and found a 50% lower value in the latter. CONCLUSIONAdiponectin, a major component of breastmilk, downregulates fundic smooth muscle contraction potential, thus facilitating gastric volume accommodation. This rodent’s adaptive response maximizes breastmilk intake volume after birth.

Characterization of the A-type potassium current in murine gastric fundus smooth muscles.

Transient outward, or "A-type," currents are rapidly inactivating voltage-gated potassium currents that operate at negative membrane potentials. A-type currents have not been reported in the gastric fundus, a tonic smooth muscle. We used whole cell voltage clamp to identify and characterize A-type currents in smooth muscle cells (SMCs) isolated from murine fundus. A-type currents were robust in these cells with peak amplitudes averaging 1.5 nA at 0 mV. Inactivation was rapid with a time constant of 71 ms at 0 mV; recovery from inactivation at -80 mV was similarly rapid with a time constant of 75 ms. A-type currents in fundus were blocked by 4-aminopyridine (4-AP), flecainide, and phrixotoxin-1 (PaTX1). Remaining currents after 4-AP and PaTX1 displayed half-activation potentials that were shifted to more positive potentials and showed incomplete inactivation. Currents after tetraethylammonium (TEA) displayed half inactivation at -48.1 ± 1.0 mV. Conventional microelectrode and contractile experiments on intact fundus muscles showed that 4-AP depolarized membrane potential and increased tone under conditions in which enteric neurotransmission was blocked. These data suggest that A-type K+ channels in fundus SMCs are likely active at physiological membrane potentials, and sustained activation of A-type channels contributes to the negative membrane potentials of this tonic smooth muscle. Quantitative analysis of Kv4 expression showed that Kcnd3 was dominantly expressed in fundus SMCs. These data were confirmed by immunohistochemistry, which revealed Kv4.3-like immunoreactivity within the tunica muscularis. These observations indicate that Kv4 channels likely form the A-type current in murine fundus SMCs.

Signaling pathways underlying changes in the contractility of the stomach fundus smooth muscle in diabetic rats.

Dysfunction of gastrointestinal (GI) motility is a common complication in patients with diabetes mellitus (DM). Studies related to changes in fundus contraction induced by inhibitors in DM are not well known. Therefore, this study aimed to investigate the signaling pathways involved in the changes in the contraction of fundus smooth muscle obtained from control and DM rats. DM was induced by injecting streptozotocin (65 mg/kg) into Sprague-Dawley rats. The rats were sacrificed after 14 days. Fundus smooth muscle contraction was stimulated using electrical field stimulation (amplitude, 50 V; duration, 1 min; frequency, 2-20 Hz) and acetylcholine (0.1 mM). The inhibitor-mediated cell membrane was pre-treated with atropine, verapamil, methysergide, ketanserin, ondansetron, and GR 113808. Inhibitors related to intracellular signaling, such as U73122, chelerythrine, L-NNA, were also used. ML-9 and Y-27632 were identified as inhibitors of factors of myosin light chain (MLC). The contractility was observed to be lower in the DM group than in the control group. Further, the activities of phospholipase C (PLC), protein kinase C (PKC), and myosin light chain kinase (MLCK) were decreased in the DM group. DM reduced the activity of PLC, PKC, and MLCK, which resulted in a decrease in the contractility of the fundus smooth muscle. Therefore, our results present the mechanism of this DM-mediated GI disorder.

The Selective Rat Toxicant Norbormide Blocks KATP Channels in Smooth Muscle Cells But Not in Insulin-Secreting Cells.

Norbormide is a toxicant selective for rats to which it induces a widespread vasoconstriction. In a recent paper, we hypothesized a role of ATP-sensitive potassium (KATP) channels in norbormide-induced vasoconstriction. The current study was undertaken to verify this hypothesis by comparing the effects of norbormide with those of glibenclamide, a known KATP channel blocker. The whole-cell patch-clamp method was used to record KATP currents in myocytes freshly isolated from the rat and mouse caudal artery and from the rat gastric fundus, as well as in insulin-secreting pancreatic beta cells (INS-1 cells). Smooth muscle contractile function was assessed on either rat caudal artery rings or gastric fundus strips. Molecular modeling and docking simulation to KATP channel proteins were investigated in silico. Both norbormide (a racemic mixture of endo and exo isomers) and glibenclamide inhibited KATP currents in rat and mouse caudal artery myocytes, as well as in gastric fundus smooth muscle cells. In rat INS-1 cells, only glibenclamide blocked KATP channels, whereas norbormide was ineffective. The inhibitory effect of norbormide in rat caudal artery myocytes was not stereo-specific as both the endo isomers (active as vasoconstrictor) and the exo isomers (inactive as vasoconstrictor) had similar inhibitory activity. In rat caudal artery rings, norbormide-induced contraction was partially reverted by the KATP channel opener pinacidil. Computational approaches indicated the SUR subunit of KATP channels as the binding site for norbormide. KATP channel inhibition may play a role in norbormide-induced vasoconstriction, but does not explain the species selectivity, tissue selectivity, and stereoselectivity of its constricting activity. The lack of effect in INS-1 cells suggests a potential selectivity of norbormide for smooth muscle KATP channels.

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.

Quantitative in situ proximity ligation assays examining protein interactions and phosphorylation during smooth muscle contractions

Antibody-based in situ proximity ligation assays (isPLA) have the potential to study protein phosphorylation and protein interactions with spatial resolution in intact tissues. However, the application of isPLA at the tissue level is limited by a lack of appropriate positive and negative controls and the difficulty in accounting for changes in tissue shape. Here we demonstrate a set of experimental and computational approaches using gastric fundus smooth muscles to improve the validity of quantitative isPLA. Appropriate positive and negative biological controls and PLA technical controls were selected to ensure experimental rigor. To account for changes in morphology between relaxed and contracted smooth muscles, target PLA spots were normalized to smooth muscle myosin light chain 20 PLA spots or the cellular cross-sectional areas. We describe the computational steps necessary to filter out false-positive improperly sized spots and set the thresholds for counting true positive PLA spots to quantify the PLA signals. We tested our approach by examining protein phosphorylation and protein interactions in smooth muscle myofilament Ca2+ sensitization pathways from resting and contracted gastric fundus smooth muscles. In conclusion, our tissue-level isPLA method enables unbiased quantitation of protein phosphorylation and protein-protein interactions in intact smooth muscle tissues, suggesting the potential for quantitative isPLA applications in other types of intact tissues.

The sphingosine analog fingolimod (FTY720) enhances tone and contractility of rat gastric fundus smooth muscle.

Sphingosine and its metabolite sphingosine phosphate (S1P) regulate a multitude of biological functions, including the contractile state of smooth. Gastrointestinal side effects have been reported in patients treated with FTY720, a sphingosine analog that is approved for the treatment of multiple sclerosis. The aim of this study was to characterize the effects of FTY720 on rat gastric fundus smooth muscle under basal conditions and during activation induced by high-K+ solution. Isometric contractions of isolated circular strips of gastric fundus smooth muscle were recorded using the organ bath method. The effects of FTY720 or vehicle were recorded under control conditions and in the presence of indomethacin, L-NAME, HA-1100, nifedipine, JTE-013, and suramin. Tone and contractions recorded in the presence of FTY720 or vehicle are reported as % of the amplitude of an initial high-K+ contraction obtained under control conditions. From a concentration of 10μmol L-1 onwards, FTY720 increased the tone, reaching 8.9%±7.5% at 100μmol L-1 (P<.05). With indomethacin in the solution, the effects of FTY720 were enhanced (32.1%±7.7%; P<.001). The FTY720-induced increase in tone was abolished in the absence of extracellular Ca2+ and reduced by nifedipine, HA-1100, JTE-013, and suramin. Furthermore, FTY720 increased high-K+ contractions in the presence of indomethacin. FTY720 increases tone and contractile responses to depolarization in gastric fundus smooth muscle by triggering calcium entry and calcium sensitization in a S1P receptor-dependent manner. Taken together, the experimental results presented in this work suggest that FTY720 may increase gastric tone and contractility in patients.

A role for focal adhesion kinase in facilitating the contractile responses of murine gastric fundus smooth muscles.

Activation of focal adhesion kinase (FAK) by integrin signalling facilitates smooth muscle contraction by transmitting the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here we report that electrical field stimulation (EFS) of cholinergic motor neurons activates FAK in gastric fundus smooth muscles, and that FAK activation by EFS is atropine-sensitive but nicardipine-insensitive. PDBu and calyculin A contracted gastric fundus muscles Ca2+ -independently and also activated FAK. Inhibition of FAK activation inhibits the contractile responses evoked by EFS, and inhibits CPI-17 phosphorylation at T38. This study indicates that mechanical force or tension is sufficient to activate FAK, and that FAK appears to be involved in the activation of the protein kinase C-CPI-17 Ca2+ sensitization pathway in gastric fundus smooth muscles. These results reveal a novel role for FAK in gastric fundus smooth muscle contraction by facilitating CPI-17 phosphorylation. Smooth muscle contraction involves regulating myosin light chain phosphorylation and dephosphorylation by myosin light chain kinase and myosin light chain phosphatase. C-kinase potentiated protein phosphatase-1 inhibitor of 17kDa (CPI-17) and myosin phosphatase targeting subunit of myosin light-chain phosphatase (MYPT1) are crucial for regulating gastrointestinal smooth muscle contraction by inhibiting myosin light chain phosphatase. Integrin signalling involves the dynamic recruitment of several proteins, including focal adhesion kinase (FAK), to focal adhesions. FAK tyrosine kinase activation is involved in cell adhesion to the extracellular matrix via integrin signalling. FAK participates in linking the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here, we show that cholinergic stimulation activates FAK in gastric fundus smooth muscles. Electrical field stimulation in the presence of Nω -nitro-l-arginine methyl ester and MRS2500 contracted gastric fundus smooth muscle strips and increased FAK Y397 phosphorylation (pY397). Atropine blocked the contractions and prevented the increase in pY397. The FAK inhibitor PF-431396 inhibited the contractions and the increase in pY397. PF-431396 also inhibited the electrical field stimulation-induced increase in CPI-17 T38 phosphorylation, and reduced MYPT1 T696 and T853, and myosin light chain S19 phosphorylation. Ca2+ influx was unaffected by PF-431396. Nicardipine inhibited the contractions but had no effect on the increase in pY397. Phorbol 12,13-dibutyrate or calyculin A contracted gastric fundus smooth muscle strips Ca2+ independently and increased pY397. Our findings suggest that FAK is activated by mechanical forces during contraction and reveal a novel role of FAK in the regulation of CPI-17 phosphorylation.

Calcium‐dependent relaxant effect of carvacrol on gastric fundus smooth muscle

Carvacrol is a monoterpene that relaxes smooth muscle on several tissues. It has been largely studied due its huge biomedical applications such as antioxidant, antibacterial, hepatoprotective, anti‐inflammatory, antispasmodic and gastroprotective. However, mechanisms involved in those pharmacological actions, specially that antispasmodic, are not well known. Thus, we aimed to evaluate the myorelaxant effect of carvacrol on the gastric fundus and its possible underlying mechanisms of action. We used male Wistar rats weighing between 150 and 350 g, kept at 22 °C, by day/night cycle, with water and food ad libitum. The rats were sacrificed after Thiopental Sodium 40 mg/kg anesthesy. Stomach was removed after laparothomy and placed in Tyrode's nutrient solution at 37°C under aeration with carbogen solution containing 95% O2 and 5% CO2. Thereafter, stomach was sectioned into longitudinal strips of length 1 cm in length each, which were individually suspended in glass chambers and stabilized for 60 minutes, when nutrient solution was changed every 15 minutes. Gastric tissues were previously contracted at different times by both 60 mM isotonic KCl (K60) or 1 μM carbachol (CCh). The inhibition of the submaximal contractile response induced by CCh or K60 was evaluated by comparing the responses before (control) and after by the addition of carvacrol in different concentrations (3 to 1000 μM) into the organs bath after a period of 10 minutes. To evaluate the role of extracellular calcium on the relaxant effects, tissue was pre‐incubated with carvacrol exposed to calcium‐free K60, and contracted by K60. Statistical analysis was performed by mean ± SEM of the measures, by two‐way ANOVA analyse with Bonferroni pos‐hoc test, with significance level p&lt;0.05. Carvacrol relaxed gastric fundus previously contracted by CCh, a muscarinic agonist type M3 (1000 μM relaxed 46%, 600 μM relaxed 35% and 300 μM relaxed 22%). In the experiment performed with 60 mM KCl, the contractions were reduced in the proportions of 26%, 33%, 42%, 56%, and 70% at 30, 100, 300, 600 and 1000 μM, respectively.At the concentration of 1000 μM of carvacrol in medium at cumulative concentrations of Ca2+ in Ca2+‐free solution, the concentration 0.5 mM inhibited contraction in 61% (11.75 ± 112). However, at 1 mM concentration we had a contraction reduction of 86% (35.92 ± 136.2) and 2mM concentration inhibited the contraction in 71% (21.12 ± 121.4 μg/ml). Carvacrol relaxed 29% of the CCh‐induced contraction in the presence of nifedipine. In presence of TEA, a K+ channels inhibitor, it was possible to observe that carvacrol in the concentration 1000μM was able to induce the relaxation about 14% (−85,23 ± 6,755) in the contraction maintained by CCh. Carvacrol showed relaxant effect on gastric fundus, mainly by extracellular calcium blockage.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aerobic metabolism on muscle contraction in porcine gastric smooth muscle.

Exposure to chronic hypoxic conditions causes various gastric diseases, including gastric ulcers. It has been suggested that gastric smooth muscle contraction is associated with aerobic metabolism. However, there are no reports on the association between gastric smooth muscle contraction and aerobic metabolism, and we have investigated this association in the present study. High K+- and carbachol (CCh)-induced muscle contractions involved increasing O2 consumption. Aeration with N2 (hypoxia) and NaCN significantly decreased high K+- and CCh-induced muscle contraction and O2 consumption. In addition, hypoxia and NaCN significantly decreased creatine phosphate (PCr) contents in the presence of high K+. Moreover, decrease in CCh-induced contraction and O2 consumption was greater than that of high K+. Our results suggest that hypoxia and NaCN inhibit high K+- and CCh-induced contractions in gastric fundus smooth muscles by decreasing O2 consumption and intracellular PCr content. However, the inhibition of CCh-induced muscle contraction was greater than that of high K+-induced muscle contraction.

Synthesis and photodegradation studies of analogues of muscle relaxant 1,4-dihydropyridine compounds.

This paper describes the synthesis of 1,4-dihydropyridine compounds (DHPs) endowed with good muscle relaxant activity and stability to light. Six new condensed DHPs were synthesized by the microwave irradiation method. A long-chain ester moiety [2-(methacryloyloxy)ethyl] and various substituents on the phenyl ring were demonstrated to affect the muscle relaxant activity occurring in isolated rabbit gastric fundus smooth muscle strips. Forced photodegradation conditions were applied to the molecules according to the ICH rules. The degradation profile of the drugs was monitored by spectrophotometry coupled with the multivariate curve resolution technique. Formation of the oxidized pyridine derivative was observed for all the studied DHPs, except for one compound, which showed very fast degradation and formation of a second photo-product. Pharmacological tests on the molecules showed a good muscle relaxing effect, with a mechanism similar to that of nifedipine, however, proving to be more stable to light.

H2 S-induced gastric fundus smooth muscle tension potentiation is mediated by the phosphoinositide 3-kinase/Akt/endothelial nitric oxide synthase pathway.

What is the central question of this study? The present study investigated the relationship between H2 S and NO in regulation of gastric fundus tension. What is the main finding and its importance? Endogenous or exogenous H2 S and NO have opposite effects on fundus tension, and H2 S-induced gastric fundus tension enhancements are mediated by inhibition of NO generation through the phosphoinositide 3-kinase/Akt pathway. These results are very important in exploring the mechanism of physiological accommodation and accommodation disorder. Hydrogen sulphide (H2 S) is considered a new gasotransmitter, along with NO and CO. It was recently confirmed that H2 S and NO play important roles in the regulation of gastrointestinal smooth muscle tension. The present study was designed to elucidate the interactions between H2 S and NO with respect to the regulation of gastric fundus smooth muscle tension using Western blotting, physiological and electrochemical techniques. Real-time H2 S and NO generation was detected in gastric smooth muscle tissue. NaHS, an H2 S donor, enhanced fundus smooth muscle tension, whereas SNP, an NO donor, decreased fundus smooth muscle tension in a dose-dependent manner. NaHS-induced increases in fundus smooth muscle tension were suppressed by l-NAME, an NO synthase inhibitor. Aminooxyacetic acid (AOAA), a cystathionine β-synthase inhibitor, exerted inhibitory effects on fundus smooth muscle tension; these effects were also suppressed by l-NAME. Real-time NO generation was significantly potentiated by AOAA. Endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 and Akt phosphorylation at serine308 and threonine473 were significantly inhibited by NaHS. LY294002, a phosphoinositide 3-kinase inhibitor, blocked these NaHS-mediated effects. However, eNOS phosphorylation at serine1177 and Akt phosphorylation at serine308 and threonine473 were significantly potentiated by AOAA. Cystathionine β-synthase siRNA interference significantly increased eNOS phosphorylation at serine 1177 and Akt phosphorylation at serine 308 and threonine 473. Cystathionine β-synthase siRNA interference also increased total eNOS protein expression levels but did not significantly change total Akt kinase protein expression levels. These results suggest that H2 S-induced enhancement of gastric fundus tension is mediated by inhibition of NO generation through the phosphoinositide 3-kinase/Akt pathway.

A role for focal adhesion kinase in the contraction of murine gastric fundus smooth muscle evoked by cholinergic neurotransmission

Cholinergic neurotransmission‐induced smooth muscle contraction involves the regulation of myosin light chain (LC20) phosphorylation and dephosphorylation by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). C‐kinase potentiated protein phosphatase‐1 inhibitor protein of 17 kDa (CPI‐17), and myosin phosphatase target subunit 1 (MYPT1) are crucial for gastrointestinal smooth muscle contractile regulation via their inhibition of MLCP activity. Besides these mechanisms, integrin‐mediated cell‐matrix adhesions connect the cytoskeleton with the extracellular matrix to facilitate cohesion and to generate tension throughout the whole muscle in response cholinergic neurotransmission. Cell‐matrix adhesions and integrin signaling involve the dynamic recruitment and assembly of several proteins, primarily focal adhesion kinase (FAK), at the adhesive site. FAK is a tyrosine kinase whose phosphorylation state and activity is tightly linked with cell adhesion to the extracellular matrix through integrin receptors. FAK participates in transmitting the force generated by the activation of contractile proteins in smooth muscle cells (SMC), to the extracellular matrix filaments and throughout the smooth muscle tissue.Here, we address the hypothesis that FAK activation is involved in the contraction of gastric fundus smooth muscle in response to cholinergic stimulation. FAK (not Pyk2) was identified as a major tyrosine‐phosphorylated protein in response to electric field stimulation (EFS). EFS in the presence of L‐NNA and MRS2500 contracted gastric fundus smooth muscle strips and significantly increased FAK phosphorylation at Tyr397. In addition, EFS increased the association of FAK with Beta‐1 integrin. The FAK inhibitor PF‐431396 inhibited the EFS‐stimulated contractions, the increase in FAK Tyr397 phosphorylation, and the association of FAK with Beta‐1 integrin. PF‐431396 also blocked the EFS‐induced increase in MYPT1 Thr853, CPI‐17 Thr38, and LC20 Ser19 phosphorylation. The muscarinic acetylcholine receptor antagonist atropine, but not the L‐type Ca2+ channel blocker nicardipine, prevented the EFS‐induced increase in FAK Tyr397 phosphorylation. However, FAK Tyr397 phosphorylation was increased during high K+‐induced contraction, and both were inhibited by PF‐431396. Our findings demonstrate that FAK Tyr397 phosphorylation and association with integrin receptors in SMC plays a role in the contractile response of gastric fundus smooth muscles to cholinergic transmission.Support or Funding InformationThis project was supported by a Takeda Innovation Center Grant, Takeda Pharmaceuticals, Takeda California, San Diego, CA 92121

Role of Telokin in Regulating Murine Gastric Fundus Smooth Muscle Tension.

Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/W V mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased telokin phosphorylation and reduced MLC phosphorylation. In telokin-/- gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted telokin-/- gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8-Bromo-cGMP, SNP, and nitrergic neurotransmission. However, basal telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased telokin Ser13 phosphorylation in both wild-type and W/W V gastric fundus smooth muscles. Our findings indicate that telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to augment gastric fundus mechanical responses to inhibitory neurotransmission.

Proximity Ligation Assay of Interactions between β‐1 Integrin and Ca2+ Sensitization Proteins During the Contractile Response of Gastric Fundus Smooth Muscles to Cholinergic Stimuli

Contraction and relaxation of smooth muscle involves the regulation of myosin light chain (LC20) phosphorylation by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Rho‐associated, coiled‐coil containing protein kinase 2 (ROCK2), and myosin phosphatase target subunit 1 (MYPT1) are crucial for gastrointestinal smooth muscle contractile regulation via their inhibition of MLCP activity. β‐1 integrin is a trans‐membrane receptor that forms focal contacts and mediates the attachment of smooth muscle cells to the extracellular matrix and therefore plays an important role in cell adhesion, tension generation, and mechanotransduction in smooth muscle cells.Here we use proximity ligation assays (PLA) to examine the role of β‐1 integrin in gastric fundus smooth muscle contraction. Muscle contraction with bath‐applied carbachol (CCh) or neurally‐released ACh by atropine‐sensitive electrical field stimulation (EFS) significantly increased the interaction of MYPT1, ROCK2 and S19 phosphorylated LC20 with β‐1 integrin. Furthermore, PLA analysis of intact gastric fundus smooth muscle tissue shows that both bath‐applied CCh and neurally‐released ACh increase MYPT1‐T853 phosphorylation, in contrast to our previous findings obtained by western blot analysis of whole cell lysates showing that only CCh treatment increased MYPT1‐T853 phosphorylation. These results indicate that Ca2+ sensitization and contractile proteins interact with β‐1 integrin in focal contacts in response to cholinergic stimuli, indicating a potentially important role of β‐1 integrin in contributing to the contractile responses of fundus smooth muscles to cholinergic stimuli.