Gastric Smooth Muscle Cells Research Articles

Effects of β‑hydroxybutyric acid and ghrelin on the motility and inflammation of gastric antral smooth muscle cells involving the regulation of growth hormone secretagogue receptor.

Ghrelin is an orexigenic hormone that is produced by gastric cells. Ghrelin stimulates food intake and increases gastric movement. In rat model, injected β-hydroxybutyric acid (β-HB) leads to a decrease in body weight. It has been reported that patients with gastric erosions are slower to evacuate the stomach. The aim of the present study was to investigate the effects of ghrelin and β-HB on motility and inflammation in rat gastric antral smooth muscle cells (GASMCs). GASMCs were extracted from rat gastric antrum. Cell viability was determined using the Cell Counting Kit-8 assay. A reactive oxygen species (ROS) assay kit was used to analyze the levels of ROS using flow cytometry. Protein levels were determined using western blotting, and the expression levels of mRNAs were evaluated using reverse transcription-quantitative PCR. β-HB inhibited the expression of myosin regulatory light polypeptide 9 (MYL9), myosin light chain kinase (MLCK), transforming protein RhoA (RhoA), Rho-associated protein kinase-1 (ROCK-1) and growth hormone secretagogue receptor (GHS-R). By contrast, ghrelin increased the expression of MYL9, MLCK, RhoA, ROCK-1 and GHS-R in β-HB-treated GASMCs. β-HB increased the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and ROS, and decreased the levels of manganese (Mn) superoxide dismutase (SOD), copper/zinc (Cu/Zn)SOD and catalase. Ghrelin decreased the expression of TNF-α, IL-6, ROS and catalase, whereas ghrelin promoted the expression of MnSOD and Cu/ZnSOD in β-HB-treated GASMCs. Short interfering RNA targeting GHS-R inhibited the expression of MYL9, MLCK, RhoA and ROCK-1, and increased the levels of TNF-α, IL-6 and ROS in β-HB-treated or ghrelin-treated GASMCs. The present study provided preliminary evidence that β-HB inhibits the motility of GASMCs and promotes inflammation in GASMCs, whereas ghrelin decreases these effects. GHS-R acted as a primary regulator of motility and inflammation in GASMCs treated with β-HB and ghrelin.

Adiponectin Decreases Gastric Smooth Muscle Cell Excitability in Mice

Some adipokines known to regulate food intake at a central level can also affect gastrointestinal motor responses. These are recognized to be peripheral signals able to influence feeding behavior as well. In this view, it has been recently observed that adiponectin (ADPN), which seems to have a role in sending satiety signals at the central nervous system level, actually affects the mechanical responses in gastric strips from mice. However, at present, there are no data in the literature about the electrophysiological effects of ADPN on gastric smooth muscle. To this aim, we achieved experiments on smooth muscle cells (SMCs) of gastric fundus to find out a possible action on SMC excitability and on membrane phenomena leading to the mechanical response. Experiments were made inserting a microelectrode in a single cell of a muscle strip of the gastric fundus excised from adult female mice. We found that ADPN was able to hyperpolarize the resting membrane potential, to enhance the delayed rectifier K+ currents and to reduce the voltage-dependent Ca2+ currents. Our overall results suggest an inhibitory action of ADPN on gastric SMC excitation–contraction coupling. In conclusion, the depressant action of ADPN on the gastric SMC excitability, here reported for the first time, together with its well-known involvement in metabolism, might lead us to consider a possible contribution of ADPN also as a peripheral signal in the hunger–satiety cycle and thus in feeding behavior.

Correction to: Effects of AMPK on Apoptosis and Energy Metabolism of Gastric Smooth Muscle Cells in Rats with Diabetic Gastroparesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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 AMPK on Apoptosis and Energy Metabolism of Gastric Smooth Muscle Cells in Rats with Diabetic Gastroparesis.

This study aimed to investigate the effect of AMPK on apoptosis and energy metabolism of gastric smooth muscle cells in diabetic rats and to explore the role of AMPK in the pathogenesis of diabetic gastroparesis (DGP). After establishment of a diabetic rat model, rats were divided into normal control (NC), 4-week (DM4W), 6-week (DM6W), and 8-week (DM8W) diabetic model groups. The gastric residual pigment ratio, intestinal transit rate, and intestinal propulsion rate in each group were detected to confirm the successful establishment of the DGP model. The spontaneous contraction in isolated gastric smooth muscle strips of the NC and DM8W groups was experimentally observed. The expression of phospho-AMPK, AMPK, phospho-LKB1, LKB1, phospho-TAK1, TAK1, and CaMMKβ in rat gastric smooth muscle tissues was detected by western blot analysis; ADP, AMP, ATP contents, and the energy charge were detected using Elisa; and apoptosis of gastric smooth muscle cells was detected by flow cytometry. The rat gastric smooth muscle cells were cultured in vitro, and treated with an AMPK inhibitor and an agonist. At 24 and 48 h, the effects of AMPK on apoptosis and energy metabolism of gastric smooth muscle cells were observed. Reduced spontaneous contractions, AMPK activation, cell apoptosis, and energy metabolism disorders were observed in gastric smooth muscle tissues of a diabetic rat, and AMPK activation was associated with an increased ratio of ADP/ATP, AMP/ATP, LKB1 activity, and CaMMKβ expression. From in vitro cell culture experiments, we found that AMPK activation of high-glucose conditions promoted cell apoptosis. Inhibition of AMPK had no obvious effect on apoptosis at the early stage with high glucose, but the inhibitory effect was significant at the late stage with high glucose. AMPK can regulate both mitochondrial metabolism and glycolysis pathways under high-glucose conditions. During the early stage with high glucose, AMPK was the main promotion factor of the mitochondrial metabolism pathway, but did not increase the ATP production, AMPK also promoted the glycolysis pathway. During the late stage with high glucose, AMPK was a major inhibitor of the mitochondrial pathway, and still played a role in promoting the glycolytic pathway, which acted as the main regulator. Apoptosis and energy metabolism disorders were present in gastric smooth muscle cells during the occurrence of DGP. Under high-glucose condition, AMPK was activated, which can promote apoptosis, change the energetic metabolism pathway of cells, inhibit mitochondrial energy metabolism, and promote glycolysis.

Changes in Gastric Smooth Muscle Cell Contraction during Pregnancy: Effect of Estrogen.

It is well known that pregnancy is associated with frequent gastrointestinal (GI) disorders and symptoms. Moreover, previous reports have shown that estrogen, which changes in levels during pregnancy, participates in the regulation of GI motility and is involved in the pathogenesis of various functional disorders in the stomach. The aim of the current study was to explore the changes in the expression of estrogen receptors (ERs) and examine the effect of estrogen on nitric oxide- (NO-) cyclic guanosine monophosphate (cGMP) pathway and thus relaxation in gastric smooth muscle cells (GSMC) during pregnancy. Single GSMC from early-pregnant and late-pregnant Sprague-Dawley rats were used. Protein and mRNA expression levels of ERs were measured via specifically designed enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), respectively. NO and cGMP levels were measured via specifically designed ELISA kits. Effect of estrogen on acetylcholine- (ACh-) induced contraction of single GSMC was measured via scanning micrometry in the presence or absence of the NO synthase inhibitor, N-nitro-L-arginine (L-NNA), or guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Estrogen increased both NO and cGMP levels and their levels were greater in early compared to late pregnancy. Expression of ERs was greater in early compared to late pregnancy. ACh induced greater contraction of GSMC in late pregnancy compared to early pregnancy. Estrogen inhibited ACh-induced contraction in both periods of pregnancy. Importantly, pretreatment of GSMC with either L-NNA or ODQ abolished estrogen inhibitory action on muscle contraction. In conclusion, GSMC contractile behavior undergoes drastic changes in response to estrogen during pregnancy and this might explain some of the pregnancy-associated gastric disorders.

Effects of Short‐Term and Long‐Term Dexamethasone Action on Expression of Glucocorticoid and Mineralocorticoid Receptors in the Gastric Mucosa

Glucocorticoids may have dual action on the stomach: gastroprotective and proulcerogenic one. Our previous study was designed to investigate how physiological gastroprotective action of glucocorticoids can be transformed to pathological ulcerogenic effect. The results obtained demonstrate that single injection of dexamethasone at the dose of 1 mg/kg may attenuate or aggravate indomethacin‐induced gastric erosions depending on the time of its injection before indomethacin (prolongation of dexamethasone action). At the present study to elucidate the mechanisms of transformation of gastroprotective action of dexamethasone into ulcerogenic one we tested the hypothesis that disbalance between the glucocorticoid and mineralocorticoid receptors (GR and MR) may contribute to this transformation. To test the hypothesis, effect ща dexamethasone at the dose of 1 mg /kg, (i.p.) on the expression of GR and MR in the gastric mucosa was studied 1 h (a short‐term action when the gastroprotective action was observed) and 24 h (long‐term action when the proulcerogenic effect was observed) after its administration. The expression of GR and MR was detected by a immunohistochemical staining technique using α‐glucocorticoid (ab3580; 1:500. Abcam) and mineralоcorticoid (ab41912; 1:500. Abcam) receptor antibodies. The results of the staining were assessed by a morphometric study of microscopic images in five fields of view using the ImajeJ software. A distribution of cytoplasmic staining was estimated with relative stained area (%) in gastric glands and smooth muscle cells of muscularis mucosae. We found that GR are presented in the parietal cells, whereas MR are ubiquitous. Dexamethasone provoked translocation of both GR and MR into nuclei. The short‐term effect of dexamethasone resulted in a decrease of cytoplasmic GR and their translocation into the cell nuclei. The long‐term dexamethasone exposure increased cytoplasmic GR expression not accompanied by nuclear translocation. The short‐term dexamethasone action caused an increase of cytoplasmic MR level, but did not lead to nuclear translocation of these receptors. The long‐term action of dexamethasone induced nuclear translocation of MR, without significant changes of MR expression in cytoplasm. The results suggest that disbalance between GR and MR may contribute to transformation of gastroprotective action of dexamethasone to proulcerogenic effect and support the hypothesis.Support or Funding InformationThe study was supported by grant of Russian Science Foundation (RSF) N 14‐15‐00790.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Developmental changes in contraction of gastric smooth muscle cells in rats correlate with their differences in RhoA/ROCK pathway

Previous research has shown that smooth muscle of the stomach undergoes developmental changes in the intracellular regulatory mechanism responsible for the contractile process. Whether these developmental changes relate to differences in the expression and/or activity of the key enzymes regulating smooth muscle contraction has not been previously evaluated. Therefore, we aimed to examine the expression and activation of the small monomeric G protein and Rho kinase (ROCK) as well as their correlation with the contraction of gastric smooth muscle cells (GSMCs) in newborn vs. adult rats. Freshly isolated single GSMCs from Sprague-Dawley rats at 1 week (newborn) and 3 months (adult) of age were used in the study. Protein and mRNA expression levels of both ROCK2 and total RhoA were higher in adult compared to newborn rats. Moreover, acetylcholine (ACh)-induced contractions of GSMCs in adult rats were significantly higher than that in newborn animals. Meanwhile, ROCK and Rho activation was higher in adult stomach cells compared to newborn ones. Pretreatment of GSMCs with Y-27632, the ROCK inhibitor, significantly reduced ACh-induced contraction in both groups of cells and greatly abolished contractile differences. In conclusion, our results indicate that RhoA/ROCK pathway and contraction of stomach muscle cells are under developmental regulation.

Erratum to: Role of Potassium Channels in the Effects of Hydrogen Sulfide on Contractility of Gastric Smooth Muscle Cells in Rats

Erratum to: Role of Potassium Channels in the Effects of Hydrogen Sulfide on Contractility of Gastric Smooth Muscle Cells in Rats

Insulin-like growth factor-1 inhibits the apoptosis of rat gastric smooth muscle cells cultured under high glucose condition through PI3K-Akt-PKC-Ca2+ pathway

This study investigated the effect of insulin-like growth factor 1 (IGF-1) on rat gastric smooth muscle cell apoptosis under high glucose conditions, and explored the involvement of the PI3K-Akt-PKC-Ca2+ pathway. Rat gastric smooth muscle cells were cultured in vitro under normal and high glucose conditions and treated with IGF-1. Related protein expression, PKC activity, changes of intracellular Ca2+ concentration and cell apoptosis were detected at 24 and 48 h by western blotting, enzyme-linked immunosorbent assay, confocal laser-scanning microscopy and flow cytometry, respectively. Compared with the non-treated group, PKCβ1, p-PKCβ1, PI3K and p-Akt expression in IGF-1-treated cells in normal and high glucose conditions at 24 and 48 h were increased; PKCα expression was increased in the 24-h high glucose + IGF-1 group, and decreased in the 48-h normal glucose + IGF-1 group; p-PKCα expression was decreased in the 24-h normal glucose + IGF-1 group, and increased in the 24-h and 48-h high glucose + IGF-1 group. PKC activity was increased in the 24-h normal glucose + IGF-1, 24-h and 48-h high glucose + IGF-1 groups compared with the non-treated group. After 24 and 48 h of IGF-1 treatment, the Ca2+ concentration was significantly increased in the normal glucose group, and decreased in the high glucose group compared with the non-treated group. The apoptosis rate in the 48-high glucose + IGF-1 group was significantly lower than that in the 48-h normal glucose + IGF-1 and 24-h high glucose + IGF-1 groups. Under high glucose conditions, IGF-1 can inhibit apoptosis in rat gastric smooth muscle cells through activating the PI3K-Akt-PKC pathway, and decreasing intracellular Ca2+ concentration.

Regulation of gastric smooth muscle contraction via Ca2+-dependent and Ca2+-independent actin polymerization.

In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-β/Ca2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-β1 via Gαq, IP3 formation, Ca2+ release and Ca2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr402. The sustained Ca2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr118 and association of phosphorylated paxillin with the GEF proteins p21-activated kinase (PAK) interacting exchange factor α, β (α and β PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC20 phosphorylation.

Muscarinic m2 receptor-mediated actin polymerization via PI3 kinase γ and integrin-linked kinase in gastric smooth muscle.

Actin polymerization plays an important role in smooth muscle contraction. Integrin-linked kinase (ILK) was shown to mediate actin polymerization in airway smooth muscle. The role of ILK in actin polymerization in response to m2 receptor activation was not in gastric smooth muscle. Phosphorylation of paxillin, neuronal Wiskott-Aldrich syndrome protein (N-WASp), and association of paxillin with GEF proteins (Cool2/αPix [Cool2/PAK-interacting exchange factor alpha], Cool1/βPix [Cool1/PAK-interacting exchange factor beta], and DOCK 180 [Dedicator of cytokinesis]) and N-WASp with Arp2/3 complex were measured by western blot. Activation of Cdc42 was determined using an antibody for activated Cdc42. Actin polymerization was measured as an increase in F-actin/G-actin ratio. Phosphorylation of paxillin, an association of paxillin with GEF proteins, Cdc42 activity, and actin polymerization were increased in response to m2 receptor activation in gastric smooth muscle cells. The increases in paxillin phosphorylation, Cdc42 activity, and actin polymerization were inhibited by a PI3Kγ inhibitor (AS-605240), ILK siRNA, and ILK dominant negative mutant (ILK [R211]). Increase in actin polymerization was also inhibited by Cdc42 dominant negative mutant (Cdc42 [T17N]). Increases in the association of paxillin with GEF proteins, phosphorylation of N-WASp and its association with Arp2/3 complex were inhibited by ILK (R211). In gastric smooth muscle cells, activation of PI3Kγ by muscarinic m2 receptors causes ILK-dependent phosphorylation of paxillin, an association of paxillin with Cdc42 GEF proteins and activation of Cdc42, which, in turn, causes phosphorylation of N-WASp and its association with Arp2/3 complex leading to actin polymerization.

The role of CNP-mediated PKG/PKA-PLCβ pathway in diabetes-induced gastric motility disorder

Our previous work demonstrated that the C-type natriuretic peptide (CNP)/cyclic guanosine monophosphate (cGMP)/cyclic adenosine monophosphate (cAMP) pathway in gastric antrum smooth muscle of rats with diabetes was upregulated and played an important role in the development of diabetic gastric dysmotility. Our goal for this study was to explore the downstream signaling pathways of CNP. We found that the expressions of protein kinase G (PKG) and protein kinase A (PKA) in gastric smooth muscle tissue of rats with diabetes were significantly upregulated. The expressions of β-type phospholipase C 3(PLCβ3) and β-type phospholipase C 1(PLCβ1) protein were reduced, whereas Phosphor-PLCβ3Ser1105 (P-PLCβ3Ser1105) was increased. The inhibitory effect of CNP on gastric antral smooth muscle in diabetic rats was significantly greater than in the normal group. The content of trisphosphate inositol (IP3) in the gastric antral smooth muscle of rats with diabetes was significantly lower than that of the normal group. After blocking PKA with N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89, a blockage PKA), the effect of CNP on the production of IP3 was decreased, while blocking PKG with KT5823 (a blockage PKG) simultaneously, and CNP can no longer reduce the IP3 production. CNP promoted the phosphorylation of PLCβ3Ser1105, thereby inhibiting the activity of PLCβ3 in gastric smooth muscle tissue of rats with diabetes; this effect can be abolished by blocking PKA and PKG. These results suggested that CNP can decrease IP3 level in gastric smooth muscle cells and thus inhibit gastric smooth muscle contraction through PKG/PKA-PLCβ pathway, which may play an important role in the development of diabetic gastroparesis.

Effect of progesterone on nitric oxide/cyclic guanosine monophosphate signaling and contraction in gastric smooth muscle cells.

Previous studies have shown that progesterone could inhibit muscle contraction in various sites of the gastrointestinal tract. The underlying mechanisms responsible for these inhibitory effects of progesterone are not fully known. The aim of the current study was to investigate the effect of progesterone on the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway and muscle contraction in the stomach. Single gastric smooth muscle cells from female Sprague-Dawley rats were used. The expression of progesterone receptor (PR) mRNA was analyzed by reverse transcription polymerase chain reaction. NO and cGMP levels were measured via specific ELISAs. Acetylcholine (ACh)-induced contraction of single gastric muscle cells preincubated with progesterone was measured via scanning micrometry in the presence or absence of the NO synthase inhibitor, Nω-Nitro-L-arginine (L-NNA), or guanylyl cyclase inhibitor, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and expressed as percent shortening from resting cell length. PR expression was detected in the stomach muscle cells. Progesterone inhibited ACh-induced gastric muscle cell contraction. Furthermore, progesterone increased NO and cGMP levels in single gastric muscle cells. Most notably, pre-incubation of muscle cells with either L-NNA or ODQ abolished the inhibitory action of progesterone on muscle contraction. These present observations suggest that progesterone promotes muscle cell relaxation in the stomach potentially via the NO/cGMP pathway.

Role of Potassium Channels in the Effects of Hydrogen Sulfide on Contractility of Gastric Smooth Muscle Cells in Rats

The effect of sodium hydrosulfide (NaHS), a hydrogen sulfide (H2S) donor, on spontaneous contractile activity of rat gastric smooth muscle cells was analyzed. Experiments were conducted on gastric stripes under conditions of isometric contraction. It was shown that NaHS has a biphasic effect on spontaneous contractile activity, increasing tonic tension and the amplitude of phasic contractions within the first minutes since application. This initial phase is followed by a decrease in amplitude, basal tone, and frequency of spontaneous contractions. The inhibitory effect of NaHS was dose-dependent at concentrations from 10 to 600 μM. Preliminary application of tetraethylammonium and 4-aminopirydine, inhibitors of voltage-gated and calciumactivated potassium channels, prevented a NaHS-induced initial increase in basal tone and phasic contraction amplitude. Activation of ATP-dependent potassium channels (KATP-channels) by diazoxide prevented in part a NaHS-induced decrease in basal tone and amplitude of spontaneous contractions. Glibenclamide, an inhibitor of KATP-channels, decreased the inhibitory effect of NaHS on amplitude, basal tone and frequency of spontaneous contractions. It was concluded that in rat gastric smooth muscles the excitatory effect of H2S is mediated by the inhibition of voltagegated and calcium-activated potassium channels, while its inhibitory effect involves the activation of KATP-channels.

Effects of insulin-like growth factor-1 on endoplasmic reticulum stress and autophagy in rat gastric smooth muscle cells cultured at different glucose concentrations in vitro.

The purpose of the study was to observe changes in endoplasmic reticulum stress (ERS)- and autophagy-related proteins in gastric smooth muscle tissues of diabetic rats with gastroparesis, investigate the effect of insulin-like growth factor 1 (IGF-1) on ERS and autophagy in rat gastric smooth muscle cells cultured under different glucose concentrations, and explore the influence of IGF-1 on development of diabetic gastroparesis (DGP). After establishing a rat model of DGP, rats were divided into normal control (NC) and 6-week diabetic model (DM6W) groups. Expression of ERS-related and autophagy-related proteins was detected by western blot analysis and immunofluorescence assay in rat gastric smooth muscle tissue and in vitro-cultured rat gastric smooth muscle cells exposed to different glucose concentrations and treatment with IGF-1 for 24 or 48h. Changes in glucose-regulated-protein-78 (GRP78), growth arrest and DNA damage-inducible gene 153 (CHOP), and microtubule-associated protein 1A/1B light chain 3B (LC3) expression levels were detected by western blot analysis, and GRP78 and LC3 expression were examined by confocal laser-scanning microscopy. In vivo expression levels of GRP78, CHOP, and LC3 were significantly higher in the DM6W group compared with the NC group (p < 0.001). Twenty-four hours after cells were cultured at different glucose concentrations in vitro, expression of GRP78, CHOP, and LC3II/Iwas significantly higher in the high glucose-treated group compared with the normal glucose group (p < 0.05). After IGF-1 intervention, CHOP and GRP78 expression were significantly higher in the normal glucose + IGF-1 group compared with the normal glucose group (p < 0.01), while no significant difference was found between high glucose and high glucose + IGF-1 groups. LC3II/Iexpression was significantly lower in the normal glucose + IGF-1 group compared with the normal glucose group, and was significantly lower in the high glucose and high glucose + IGF-1 groups (p < 0.05). After 48h of culture, CHOP expression was significantly higher and LC3II/I expression was significantly lower in the high glucose group compared with the normal glucose group (p < 0.05), but no significant change in GRP78 expression was observed between these two groups. After IGF-1 intervention, there was no difference in CHOP or GRP78 expression between normal glucose + IGF-1 and normal glucose groups. However, CHOP and GRP78 expression were significantly lower in the high glucose + IGF-1 group compared with the high glucose group (p < 0.05). There was no significant difference in LC3II/I expression between normal glucose + IGF-1 and normal glucose groups, or high glucose + IGF-1 and high glucose groups. Results of confocal laser-scanning microscopy showed significantly lower expression of LC3II/Iin the high glucose + IGF-1 group compared with the high glucose group (p < 0.05). ERS and autophagy were involved in the occurrence of DGP. IGF-1 exerted an inhibitory effect on ERS in rat gastric smooth muscle cells cultured under high glucose conditions, and this inhibitory effect increased with time. IGF-1 inhibited the level of autophagy in rat gastric smooth muscle cells cultured under high glucose conditions at early stages, which may be achieved through inhibition of ERS.

Estrogen relaxes gastric muscle cells via a nitric oxide- and cyclic guanosine monophosphate-dependent mechanism: A sex-associated differential effect.

Various gastrointestinal (GI) disorders have a higher prevalence in women than in men. In addition, estrogen has been demonstrated to have an inhibitory effect on the contractility of GI smooth muscle. Although increased plasma estrogen levels have been implicated in GI disorders, the role of gastric estrogen receptor (ER) in these sex-specific differences remains to be fully elucidated. The present study was designed to investigate the sex-associated differences in the expression of the two ER isoforms, ERα and ERβ, and the effect of estrogen on gastric muscle contraction via the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway. Experiments were performed on single gastric smooth muscle cells (GSMCs) isolated from male and female Sprague Dawley rats. The effect of acetylcholine (ACh), a muscarinic agonist, on the contraction of GSMCs was measured via scanning micrometry in the presence or absence of 1 µM 17β-estradiol (E2), an agonist to the majority of ERs, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), an ERα agonist, or diarylpropionitrile (DPN), an ERβ agonist. The protein expression levels of ER subtypes in GSMCs were measured using a specifically designed ELISA. GSMCs from female rats had a higher expression of ERα and ERβ protein compared with GSMCs from males. ACh induced less contraction in female that in male GSMCs. Pre-treatment of GSMCs with E2 reduced the contraction of GSMCs from both sexes, but to a greater extent in those from females. PPT and DPN inhibited ACh-induced contraction in GSMCs from females. Furthermore, E2 increased NO and cGMP levels in GSMCs from males and females; however, higher levels were measured in females. Of note, pre-incubation of female GSMCs with Nω-nitro-L-arginine, a NO synthase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a guanylyl cyclase inhibitor, reduced the inhibitory effect of estrogen on GSMC contraction. In conclusion, estrogen relaxes GSMCs via an NO/cGMP-dependent mechanism, and the reduced contraction in GSMCs from females by estrogen may be associated with the sex-associated increased expression of ERα and ERβ, and greater production of NO and cGMP, compared with that in GSMCs from males.

Gender differences in the regulation of MLC20 phosphorylation and smooth muscle contraction in rat stomach.

Evidence of sex-related differences in gastrointestinal (GI) functions has been reported in the literature. In addition, various GI disorders have disproportionate prevalence between the sexes. An essential step in the initiation of smooth muscle contraction is the phosphorylation of the 20-kDa regulatory myosin light chain (MLC20) by the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK). However, whether male stomach smooth muscle inherits different contractile signaling mechanisms for the regulation of MLC20 phosphorylation from that in females has not been established. The present study was designed to investigate sex-associated differences in the regulation of MLC20 phosphorylation and thus muscle contraction in gastric smooth muscle cells (GSMCs). Experiments were performed on GSMCs freshly isolated from male and female rats. Contraction of the GSMCs in response to acetylcholine (ACh), a muscarinic agonist, was measured via scanning micrometry in the presence or absence of the MLCK inhibitor, ML-7. Additionally, the protein levels of MLC20, MLCK and phosphorylated MLC20 were measured by ELISA. The protein levels of MLC20 and MLCK were indifferent between the sexes. ACh induced greater contraction (P<0.05) as well as greater MLC20 phosphorylation (P<0.05) in male GSMCs compared with female. Pretreatment of GSMCs with ML-7 significantly reduced the ACh-induced contraction (P<0.05) and MLC20 phosphorylation (P<0.05) in the male and female cells, and notably, abolished the contractile differences between the sexes. In conclusion, MLC20 phosphorylation and thus muscle contraction may be activated to a greater extent in male rat stomach compared with that in females.

LncRNA MALAT1 is up-regulated in diabetic gastroparesis and involved in high-glucose-induced cellular processes in human gastric smooth muscle cells

Recent years, widespread long non-coding RNAs (lncRNAs) were identified and known as regulator of gene expression. Diabetic gastroparesis (DGP) is one of the most common chronic complications of diabetes mellitus. There was no research reported the role of lncRNAs in DGP. In this study, we firstly established a rat model of DGP by STZ injection. Then, we detected the expression of MALAT1 and found that expression of MALAT1 was up-regulated in rat model of DGP, comparing to the control group (P < .01). Furthermore, we revealed that MALAT1 expression was increased in the samples from diabetic patients with DGP symptoms, in comparison with the control. In addition, we demonstrated that the inhibition of MALAT1 increased the expression of α-SMA and SM myosin heavy chains, reduced the cell viability, inhibited the potential of cell migration and induced cell apoptosis in human gastric smooth muscle cells (SMCs). Ultimately, we found that the regulation of MALAT1 expression modulated the function of high-glucose stimulation in human gastric SMCs. Therefore, our study firstly indicated that MALAT1 was up-regulated in DGP and played an important role in the pathogenesis of DGP.

Higenamine inhibits apoptosis and maintains survival of gastric smooth muscle cells in diabetic gastroparesis rat model via activating the β2-AR/PI3K/AKT pathway

Diabetic gastroparesis (DGP) is a common complication of diabetes mellitus (DM). The numerous clinical symptoms of DGP and the great cost on the treatment of DGP seriously lowered the patients’ life quality. However, the pathogenic mechanism of DGP is still elusive till now. In this study, we aimed to explore the effect of higenamine on the proliferation and apoptosis of gastric smooth muscle cells (SMCs) in DGP rat model. The DGP rat model was built by intraperitoneal injection of Streptozotocin (STZ) into male Sprague-Dawley (SD) rats. Compared with the healthy control group, the level of DGP indicator c-kit was strongly suppressed and the level of Gsα was largely elevated in the STZ-induced model group. By contrast, the addition of higenamine obviously counteracted the effect of STZ on the expression of c-kit and Gsα. Besides that, higenamine improved the decreased emptying ability of the stomach. In addition, the number of gastric SMCs was strongly decreased and cell morphology became irregular in STZ-induced model group. The treatment of higenamine weakened the harm of STZ on the number and morphology of gastric SMCs. Beyond that, higenamine promoted gastric SMCs proliferation and inhibited gastric SMCs apoptosis in DGP model. Further research revealed that higenamine regulated cell proliferation and apoptosis via activating the β2-AR/PI3K/AKT pathway. Taken together, our research revealed that higenamine maintained the survival of gastric SMCs in DGP rat model via the β2-AR/PI3K/AKT pathway, providing a new sight for the treatment of DGP.