Gastric Smooth Muscle Cells Research Articles

Significance of dynamic changes in gastric smooth muscle cell apoptosis, PI3K-AKT-mTOR and AMPK-mTOR signaling in a rat model of diabetic gastroparesis.

The aim of the present study was to investigate the significance of cell apoptosis, the phosphoinositide-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway, and the 5' adenosine monophosphate-activated protein kinase (AMPK)‑mTOR pathways in the process of diabetic gastroparesis. Changes in gastric smooth muscle cells of diabetic rats with induced gastroparesis were examined. The diabetic rat model was established by dividing animals into a normal control group and diabetic model groups examined at 2, 4 and 6weeks. Diabetic gastroparesis was evaluated by examining the rates of gastric residual pigment, whereas flow cytometry was used to detect the apoptosis of gastric smooth muscle cells. The expression levels of PI3K and phosphorylated (p‑) AKT, AMPK, mTOR, tuberous sclerosis complex 2, p70 ribosomal S6 kinase, and eukaryotic translation initiation factor 4‑binding protein 1 were determined in gastric muscles using western blot analysis. Diabetic gastroparesis was confirmed in models at 6weeks. The apoptosis of gastric smooth muscle cells gradually increased in all diabetic groups, and significant changes were observed in key proteins involved in PI3K‑AKT‑mTOR and AMPK‑mTOR signaling. The results indicated that apoptosis was important in the occurrence of diabetic gastroparesis, and the PI3K‑AKT‑mTOR and AMPK‑mTOR pathways were activated during the apoptotic processes, but were incapable of regulating apoptosis.

Serotonin and its Functions as Gastrointestinal Hormone

Background: Serotonin plays the role of critical signal molecule in great number of physiologic processes and pathologic symptoms of gastrointestinal tract. As a gastrointestinal hormone serotonin takes place in the regulation of smooth muscle motor activity and glandular secretion. It affects the gastrointestinal sensation of pain, nausea and vomiting. The aim of our study is to determine the presence and localization of serotonin-producing EC cells and distribution of serotonin receptor 5-HTR3 in the human stomach.Material and methods: Biopsy specimens from 25 patients aged 40-70 years: body and antral part of the stomach are studied by immunohistochemical reactions for serotonin, serotonin receptor 5-HTR3; and transmission electron microscopy.Results: The serotonin-producing cells are found between columnar cells of gastric epithelium, in the fundic and pyloric glands of the gastric mucosa. Their ultrastructural characteristics show the presence of EC1 and EC2 enterochromaffin cells. The expression of serotonin receptor 5-HTR3 in covering epithelium, glandular and interstitial cells shows the possibility serotonin to affect gastric mucosa secretion. The intensive expression of 5-HTR3 receptors in gastric wall smooth muscle cells demonstrates the key role of serotonin in gastrointestinal motility.Conclusion: In order to obtain a better understanding of the functional role of serotonin, we investigate the localization of serotonin-producing EC cells, their ultrastructural characteristics and the presence and distribution of serotonin receptor 5HTR3 in the stomach. Enzymes needed for serotonin secretion and degradation, its transporters, as it great numbers of receptors are the base new drugs to be synthesized aiding clinical practice in gastroenterology.

Atractylodin Induces Myosin Light Chain Phosphorylation and Promotes Gastric Emptying through Ghrelin Receptor.

Atractylodin is one of the main constituents in the rhizomes of Atractylodes lancea Thunb., being capable of treating cancer cachexia-anorexia and age-related diseases as an agonist of growth hormone secretagogue receptor (GHSR). GHSR was herein expressed in human gastric smooth muscle cells (HGSMCs) and activated by ghrelin receptor agonist L-692,585. Like L-692,585, atractylodin also increased Ca2+ and enhanced the phosphorylation of myosin light chain (MLC) through GHSR in HGSMCs. In addition, atractylodin promoted gastric emptying and MLC phosphorylation in the gastric antrum of mice also through GHSR. Collectively, atractylodin can activate GHSR in gastric smooth muscle, as a potential target in clinical practice.

Advanced glycation end products interfere with gastric smooth muscle contractile marker expression via the AGE/RAGE/NF-κB pathway

Excessive production of advanced glycation end products (AGE) has been implicated in the pathogenesis of diabetic complications. Smooth muscle (SM) phenotype transition is involved in diabetes-associated gastric motility dysfunction. We investigated whether AGE interfere with gastric antral SM contractile marker expression. Sixteen Sprague-Dawley rats were randomly divided into control and streptozotocin-induced diabetic groups. Sixteen weeks after streptozotocin administration, gastric antral SM strip contractility in the groups were measured. The gastric tissue expression of AGE was tested. Primary cultured gastric smooth muscle cells (SMCs) were used in complementary in vitro studies. In the presence and absence of AGE, SMCs were transfected with myocardin plasmid or treated with nuclear factor-κB (NF-κB) inhibitor or anti-RAGE antibody. Diabetic rats showed weakness of SM strip contractility and decreased expression of SM contractile marker genes (myosin heavy chains [MHC], α-actin, calponin) as compared with the control group. Gastric antral SM layer Nε-(carboxymethyl) lysine (CML) level, the major AGE compound, were increased in the diabetic rats. AGE downregulated SM contractile markers and myocardin expression in a concentration-dependent manner. Myocardin overexpression prevented these results. AGE treatment activated NF-κB in SMCs. The NF-κB inhibitor BAY 11–7082 and anti-RAGE antibody blocked the effects of AGE on myocardin downregulation. AGE may induce the development of gastric dysmotility by downregulating SM contractile proteins and myocardin expression via the AGE/RAGE/NF-κB pathway.

α-Terpineol Induces Gastric Retention of Liquids by Inhibiting Vagal Parasympathetic Pathways in Rats

α-Terpineol is a monoterpene with smooth muscle relaxant properties. In this study, its effects on the gastric emptying rate of awake rats were evaluated with emphasis on the mode by which it induces gastrointestinal actions. Administered by gavage, α-terpineol (50 mg/kg) delayed gastric emptying of a liquid test meal at 10 min postprandial. Hexamethonium or guanethidine did not interfere with the retarding effect induced by α-terpineol, but atropine and L-NG-nitroarginine methyl ester abolished it. In vagotomized rats, α-terpineol did not delay gastric emptying. In isolated strips of gastric fundus, concentration-effect curves in response to carbamylcholine were higher in magnitude after treatment with the monoterpene. α-Terpineol (1 to 2000 µM) relaxed sustained contractions induced by carbamylcholine or a high K+ concentration in a concentration-dependent manner. This relaxing effect was not affected by the presence of L-NG-nitroarginine methyl ester, 1 H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one, tetraethylammonium, or atropine. Smooth muscle contractions induced by electrical field stimulation were inhibited by α-terpineol. In conclusion, α-terpineol induced gastric retention in awake rats through mechanisms that depended on intact vagal innervation to the stomach, which involved cholinergic/nitrergic signalling. Such a retarding effect induced by α-terpineol appears not to result from a direct action of the monoterpene on gastric smooth muscle cells.

Gastric and pyloric sphincter muscle function and the developmental-dependent regulation of gastric content emptying in the rat.

Feeding intolerance is a common issue in the care of preterm neonates. The condition manifests as delayed emptying of gastric contents and represents a therapeutic challenge, since the factors accounting for its manifestations are unknown. The main goal of this study was to comparatively investigate the age-related function of rat gastric and pyloric smooth muscle and their putative regulators. We hypothesized that a reduced gastric muscle contraction potential early in life contributes to the delayed gastric emptying of the newborn. Newborn and adult rat gastric (fundus) and pyloric sphincter tissues were comparatively studied in vitro. Shortening of the tissue-specific dissociated smooth muscle cell was evaluated, and expression of the key regulatory proteins Rho-associated kinase 2 and myosin light chain kinase was determined. Gastric and pyloric smooth muscle cell shortening was significantly greater in the adult than the respective newborn counterpart. Expression of myosin light chain kinase and Rho-associated kinase 2 was developmentally regulated and increased with age. Pyloric sphincter muscle expresses a higher neuronal nitric oxide synthase and phosphorylated vasodilator-stimulated phosphoprotein content in newborn than adult tissue. Compared with later in life, the newborn rat gastropyloric muscle has a Ca(2+)-related reduced potential for contraction and the pyloric sphincter relaxation-dependent modulators are overexpressed. To the extent that these rodent data can be extrapolated to humans, the delayed gastric emptying in the newborn reflects reduced stomach muscle contraction potential, as opposed to increased pyloric sphincter tone.

NPs/NPRs Signaling Pathways May Be Involved in Depression-Induced Loss of Gastric ICC by Decreasing the Production of mSCF.

It is well known that natriuretic peptides (NPs) are involved in the regulation of gastrointestinal motility. Interstitial cells of Cajal (ICC) are the pacemaker cells of gastrointestinal motility and gastrointestinal dyskinesia is one of the important digestive tract symptoms of depression. However, it is unclear whether they are involved in depression-induced loss of ICC. The aim of the present study was to investigate the relationship between the natriuretic peptide signaling pathway and depression-induced loss of gastric ICC in depressed rats. These results showed that the expression of c-kit and stem cell factor (SCF) in smooth muscle layers of stomach were down-regulated in depressed rats at the mRNA and protein levels. The expression of natriuretic peptide receptor (NPR)-A, B and C were up-regulated in the stomach of depressed rats at the mRNA and protein levels. NPR-A, B and C can significantly decrease the expression of SCF to treat cultured gastric smooth muscle cells (GSMCs) obtained from normal rats with different concentrations of C-type natriuretic peptide (CNP). Pretreatment of cultured GSMCs with 8-Brom-cGMP (8-Br-cGMP, a membrane permeable cGMP analog), cANF (a specific NPR-C agonist) and CNP (10−6 mol/L) demonstrated that 8-Br-cGMP had a similar effect as CNP, but treatment with cANF did not. The results of the methyl thiazolyl tetrazolium bromide (MTT) assay indicated that high concentrations of cANF (10−6 mol/L) restrained the proliferation of cultured GSMCs. Taken together, these results indicate that the up-regulation of the NPs/NPR-C and NPs/NPR-A, B/cGMP signaling pathways may be involved in depression-induced loss of gastric ICC.

Up-regulation of the Ang II/AT1 receptor may compensate for the loss of gastric antrum ICC via the PI3k/Akt signaling pathway in STZ-induced diabetic mice

The classic renin–angiotensin system (RAS) is a complex system in which angiotensin II (Ang II) has been identified as an important endogenous regulator that influences both smooth muscle contraction and cell growth. Although a local RAS is known to exist in the gastrointestinal tract, it is unclear whether Ang II is involved in the loss of gastric interstitial cells of Cajal (ICC) in diabetic mice. The present study was designed to investigate the effect of Ang II on ICC survival in streptozotocin (STZ)-induced diabetic mice. Western blot, immunofluorescence, isometric muscle recording, enzyme-linked immunosorbent assay (ELISA) and a cell counting kit-8 were used in this research. Our results demonstrate that the c-Kit and membrane-bound stem cell factor (mSCF) protein expression levels in gastric smooth muscle were decreased in STZ-induced diabetic mice. However, the angiotensin receptor type 1 (AT1R) expression levels in gastric smooth muscle and angiotensin-converting enzyme (ACE) expression levels in gastric mucosa were increased. The effect of Ang II on the tonic contraction of gastric smooth muscle was potentiated in diabetic mice, and the plasma Ang II level was enhanced. Ang II increased mSCF expression, cell proliferation, and Akt-Ser473 phosphorylation in cultured gastric smooth muscle cells (GSMCs). These effects were reduced by specific inhibitors ZD7155 (an AT1R antagonist) and LY294002 (a PI3-kinase inhibitor). Our results suggest that Ang II increases mSCF expression and cell proliferation in cultured GSMCs in a PI3K/Akt signaling-dependent manner. ACE and AT1R up-regulation in the stomach may help compensate for ICC loss in STZ-induced diabetic mice.

Targeting extra-oral bitter taste receptors modulates gastrointestinal motility with effects on satiation.

Bitter taste receptors (TAS2Rs) are present in extra-oral tissues, including gut endocrine cells. This study explored the presence and mechanism of action of TAS2R agonists on gut smooth muscle in vitro and investigated functional effects of intra-gastric administration of TAS2R agonists on gastric motility and satiation. TAS2Rs and taste signalling elements were expressed in smooth muscle tissue along the mouse gut and in human gastric smooth muscle cells (hGSMC). Bitter tastants induced concentration and region-dependent contractility changes in mouse intestinal muscle strips. Contractions induced by denatonium benzoate (DB) in gastric fundus were mediated via increases in intracellular Ca2+ release and extracellular Ca2+-influx, partially masked by a hyperpolarizing K+-efflux. Intra-gastric administration of DB in mice induced a TAS2R-dependent delay in gastric emptying. In hGSMC, bitter compounds evoked Ca2+-rises and increased ERK-phosphorylation. Healthy volunteers showed an impaired fundic relaxation in response to nutrient infusion and a decreased nutrient volume tolerance and increased satiation during an oral nutrient challenge test after intra-gastric DB administration. These findings suggest a potential role for intestinal TAS2Rs as therapeutic targets to alter gastrointestinal motility and hence to interfere with hunger signalling.

Effect of Electro-acupuncture on Ultrastructure in Diabetic Gastroparesis Model Rats

Abstract Objective: To observe the effects of electro-acupuncture (EA) on ultrastructure of gastric antrum smooth muscle cells (GASMCs) in diabetic gastroparesis (DGP) model rats, and to explore the possible mechanism underlying the therapeutic effects of EA. Methods: Sixty Sprague Dawley rats were randomly divided into 5 groups: blank control (group A), DGP model (group B), EA point (group C), EA non-point (group D), Metoclopramide control (group E). DGP rat model was established by 2% STZ intraperitoneal injection at once, and then being fed with high-sugar-high-fat (HSHF) for 8wks. Phenol red lavage method was employed to measure gastric emptying rate (GER) and intestinal migration rate (IMR). The ultrastructure of GASMCs was observed under the electronic microscope. Results: Compared with the blank control group, the GER and IMR in model group were decreased significantly (P＜0.05 or P＜0.01). Compared with the model group, the GER and IMR in EA point group were increased significantly(P＜0.05 or P＜0.01). Electro-acupuncture on “Zu San Li”(ST36) point, etc. has been observed improving the ultrastructure of GASMCs, as well as increasing the number of interstitial cells of Cajal (ICCs). Conclusion: Electro-acupuncture can improve the GI motility promisingly, based on a potential underlying mechanism that the electro-acupuncture can improve the ultrastructure of gastric smooth muscle, and increase the number of ICCs.

The role of the RhoA/ROCK pathway in gender-dependent differences in gastric smooth muscle contraction.

Gender-related differences in various gastric functions and diseases have been reported, with women having a higher prevalence of gastrointestinal disturbances than men. The aim of this study was to investigate sex-dependent differences in activation of the Rho-associated protein kinase (ROCK; RhoA/Rho kinase) pathway and muscle contraction in the stomach using single gastric smooth muscle cells (GSMC) from male and female Sprague-Dawley rats. Expression of ROCK1 and ROCK2 protein and acetylcholine (ACh)-induced activation of RhoA and ROCK were measured using a specifically designed enzyme-linked immunosorbent assay and activity assay kits, respectively. Contraction of a single GSMC was measured by scanning micrometry in the presence or absence of the ROCK inhibitor Y27632 dihydrochloride. ACh-induced activation of RhoA and ROCK and subsequent contraction were greater in male rats than in female rats but neither was related to differences in the expression of ROCK1 or ROCK2 or total RhoA amount. Most important, Y27632 inhibited and abolished differences in ACh-induced contraction in both sexes. In conclusion, increased ACh-induced contraction in the GSMC of male rats is attributable to greater RhoA/ROCK activation independent of differences in the expression of ROCK isoforms or total RhoA.

Inhibition of RhoA-dependent pathway and contraction by endogenous hydrogen sulfide in rabbit gastric smooth muscle cells.

Inhibitory neurotransmitters, chiefly nitric oxide and vasoactive intestinal peptide, increase cyclic nucleotide levels and inhibit muscle contraction via inhibition of myosin light chain (MLC) kinase and activation of MLC phosphatase (MLCP). H2S produced as an endogenous signaling molecule synthesized mainly from l-cysteine via cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) regulates muscle contraction. The aim of this study was to analyze the expression of CSE and H2S function in the regulation of MLCP activity, 20-kDa regulatory light chain of myosin II (MLC20) phosphorylation, and contraction in isolated gastric smooth muscle cells. Both mRNA expression and protein expression of CSE, but not CBS, were detected in smooth muscle cells of rabbit, human, and mouse stomach. l-cysteine, an activator of CSE, and NaHS, a donor of H2S, inhibited carbachol-induced Rho kinase and PKC activity, Rho kinase-sensitive phosphorylation of MYPT1, PKC-sensitive phosphorylation of CPI-17, and MLC20 phosphorylation and sustained muscle contraction. The inhibitory effects of l-cysteine, but not NaHS, were blocked upon suppression of CSE expression by siRNA or inhibition of its activity by dl-propargylglycine (PPG) suggesting that the effect of l-cysteine is mediated via activation of CSE. Glibenclamide, an inhibitor of KATP channels, had no effect on the inhibition of contraction by H2S. Both l-cysteine and NaHS had no effect on basal cAMP and cGMP levels but augmented forskolin-induced cAMP and SNP-induced cGMP formation. We conclude that both endogenous and exogenous H2S inhibit muscle contraction, and the mechanism involves inhibition of Rho kinase and PKC activities and stimulation of MLCP activity leading to MLC20 dephosphorylation and inhibition of muscle contraction.

Exogenous H2 S enhances mice gastric smooth muscle tension through S-sulfhydration of KV 4.3, mediating the inhibition of the voltage-dependent potassium current.

Hydrogen sulfide (H2 S) has been shown to have an excitatory effect on gastric motility, but the underlying molecular mechanism is unclear. In this study, we aimed to investigate the possible targets of H2 S and determine how H2 S affects its target proteins during H2 S-induced contraction. Patch-clamp and potentiometric fluorescence dye were utilized to measure the electrophysiological changes. The Biotin-switch assay was utilized to detect the protein S-sulfhydration. The isometric tension measurement was conducted too. Exogenous H2 S enhanced the tonic contraction of gastric antral smooth muscle, and voltage-dependent potassium channel (KV ) blocker and Dithiothreitol (DTT, a reducing agent) abolished the excitatory effect of NaHS. Exogenous H2 S inhibited the fast inactivation component of the voltage-dependent potassium channel current (IKVfast ) in isolated gastric antral smooth muscle cells. H2 S inhibited the KV 4.3 current in H293 cells with heterologous expression of KV 4.3, but did not inhibit the KV 4.1 and KV 4.2 currents, which together contribute greatly to IKVfast . NaHS significantly decreased the membrane potential in cultured gastric smooth muscle cells, but the NaHS-induced depolarization was suppressed by knockdown of KV 4.3 and N-ethylamaleimide (NEM), a free thiol group blocker. In addition, NaHS sulfhydrated KV 4.3 in H293 cells and in gastric smooth muscle tissue. However, this S-sulfhydration was inhibited by NEM and DTT. Meanwhile the NaHS-induced inhibition of IKVfast and KV 4.3 was also blocked by NEM and DTT. These results suggest that exogenous H2 S sulfhydrates KV 4.3 to decrease the membrane potential, thereby enhancing the basal tension of gastric antral smooth muscle.

NAADP-sensitive two-pore channels are present and functional in gastric smooth muscle cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as an important modulator of Ca2+ release from the endo-lysosomal system in a variety of cells by a new and ubiquitous class of endo-lysosomal ion channels known as the two-pore channels (TPCs). However, the role of TPCs in NAADP action in smooth muscle is not known. In the present work, we investigated the effects of NAADP in gastric smooth muscle cells and its ability to release Ca2+ by TPCs. We show that Ca2+ signals mediated by NAADP were inhibited by disrupting Ca2+ handling by either acidic organelles (using bafilomycin A1) or the Endoplasmic Reticulum (using thapsigargin, ryanodine or 2-APB). Transcripts for endogenous TPC1 and TPC2 were readily detected and recombinant TPCs localized to the endosomes and/or lysosomes. Overexpression of wild-type TPCs but not pore mutants enhanced NAADP-mediated cytosolic Ca2+ signals. Desensitizing the NAADP pathway inhibited Ca2+-responses to extracellular stimulation with carbachol but not ATP. Taken together, these results indicate that NAADP likely induces Ca2+ release from the endolysosomal system through TPCs which is subsequently amplified via the ER in an agonist-specific manner. Thus, we suggest a second messenger role for NAADP in smooth muscle cells.

Effect of Oxidative Stress on the Expression of Thin Filament-Associated Proteins in Gastric Smooth Muscle Cells

Thin filament-associated proteins such as calponin, caldesmon, and smoothelin are believed to regulate acto-myosin interaction and thus, muscle contraction. Oxidative stress has been found to affect the normal contractile behavior of smooth muscle and is involved in the pathogenesis of a number of human diseases such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the expression of smooth muscle contractile proteins. The aim of the current study is to investigate the effect of oxidative stress on the expression of thin filament-associated proteins in rat gastric smooth muscle. Single smooth muscle cells of the stomach obtained from Sprague-Dawley rats were used. Muscle cells were treated with hydrogen peroxide (H2O2) (500 μM) for 30 min or the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) (1 mM) for 90 min to induce oxidative stress. Calponin, caldesmon, and smoothelin expressions were measured via specifically designed enzyme-linked immunosorbent assay. We found that exposure to exogenous H2O2 or incubation of dispersed gastric muscle cells with SIN-1 significantly increased the expression of calponin, caldesmon, and smoothelin proteins. In conclusion: oxidative stress increases the expression of thin filament-associated proteins in gastric smooth muscle, suggesting an important role in gastrointestinal motility disorders associated with oxidative stress.

ER stress and ER stress-induced apoptosis are activated in gastric SMCs in diabetic rats.

To investigate the gastric muscle injury caused by endoplasmic reticulum (ER) stress in rats with diabetic gastroparesis. Forty rats were randomly divided into two groups: a control group and a diabetic group. Diabetes was induced by intraperitoneal injection of 60 mg/kg of streptozotocin. Gastric emptying was determined at the 4(th) and 12(th) week. The ultrastructural changes in gastric smooth muscle cells (SMCs) were investigated by transmission electron microscopy. TdT-mediated dUTP nick end labeling (TUNEL) assay was performed to assess apoptosis of SMCs. Expression of the ER stress marker, glucose-regulated protein 78 (GRP78), and the ER-specific apoptosis mediator, caspase-12 protein, was determined by immunohistochemistry. Gastric emptying was significantly lower in the diabetic rats than in the control rats at the 12(th) wk (40.71% ± 2.50%, control rats vs 54.65% ± 5.22%, diabetic rats; P < 0.05). Swollen and distended ER with an irregular shape was observed in gastric SMCs in diabetic rats. Apoptosis of gastric SMCs increased in the diabetic rats in addition to increased expression of GRP78 and caspase-12 proteins. ER stress and ER stress-mediated apoptosis are activated in gastric SMCs in diabetic rats with gastroparesis.

Effect of Yiqi Yangyin Huayu Tongluo Recipe on apoptosis of gastric smooth muscle cells in rats with diabetic gastroparesis

Effect of Yiqi Yangyin Huayu Tongluo Recipe on apoptosis of gastric smooth muscle cells in rats with diabetic gastroparesis

Different regulatory effects of hydrogen sulfide and nitric oxide on gastric motility in mice

NO and H2S are gaseous signaling molecules that modulate smooth muscle motility. We aimed to identify expressions of enzymes that catalyze H2S and NO generation in mouse gastric smooth muscle, and determine relationships between endogenous H2S and NO in regulation of smooth muscle motility. Western blotting and immunocytochemistry methods were used to track expressions of neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in gastric smooth muscles. Smooth muscle motility was recorded by isometric force transducers. cGMP production was measured by a specific radioimmunoassay. We found that CBS, CSE, eNOS, and nNOS were all expressed in mice gastric antral smooth muscle tissues, and in cultured gastric antral smooth muscle cells. AOAA significantly inhibited smooth muscle contractions in the gastric antrum, which was significantly recovered by NaHS, while PAG had no significant effect. l-NAME enhanced contractions. NaHS at low concentrations increased basal tension but decreased it at high concentrations. SNP significantly inhibited the contractions, which could be recovered by NaHS both in the absence and presence of CuSO4. ODQ did not block NaHS-induced excitatory effect, while IBMX partially blocked this effect. cGMP production in smooth muscle was significantly increased by SNP but was not affected by NaHS. All these results suggest that endogenous H2S and NO appear to play opposite roles in regulating gastric motility and their effects may be via separate signal transduction pathways. Intracellular H2S/NO levels may be maintained in a state of balance to warrant normal smooth muscle motility.

Influence of obestatin on the gastric longitudinal smooth muscle from mice: mechanical and electrophysiological studies

Obestatin is a hormone released from the stomach deriving from the same peptide precursor as ghrelin. It is known to act as an anorectic hormone decreasing food intake, but contrasting results have been reported about the effects of obestatin on gastrointestinal motility. The aim of the present study was to investigate whether this peptide may act on the gastric longitudinal smooth muscle by using a combined mechanical and electrophysiological approach. When fundal strips from mice were mounted in organ baths for isometric recording of the mechanical activity, obestatin caused a tetrodotoxin-insensitive decrease of the basal tension and a reduction in amplitude of the neurally induced cholinergic contractile responses, even in the presence of the nitric oxide synthesis inhibitor N(G)-nitro-l-arginine. Obestatin reduced the amplitude of the response to the ganglionic stimulating agent dimethylphenyl piperazinium iodide but did not influence that to methacholine. In nonadrenergic, noncholinergic conditions, obestatin still decreased the basal tension of the preparations without influencing the neurally induced relaxant responses. For comparison, in circular fundal strips, obestatin had no effects. Notably, in the longitudinal antral ones, obestatin only caused a decrease of the basal tension. Electrophysiological experiments, performed by a single microelectrode inserted in a gastric longitudinal smooth muscle cell, showed that obestatin had similar effects in fundal and antral preparations: it decreased the resting specific membrane conductance, inhibited Ca(2+) currents, and positively shifted their voltage threshold of activation. In conclusion, the present results indicate that obestatin influences gastric smooth muscle exerting site-specific effects.

Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1

Caveolae act as scaffolding proteins for several G protein-coupled receptor signaling molecules to regulate their activity. Caveolin-1, the predominant isoform in smooth muscle, drives the formation of caveolae. The precise role of caveolin-1 and caveolae as scaffolds for G protein-coupled receptor signaling and contraction in gastrointestinal muscle is unclear. Thus the aim of this study was to examine the role of caveolin-1 in the regulation of Gq- and Gi-coupled receptor signaling. RT-PCR, Western blot, and radioligand-binding studies demonstrated the selective expression of M2 and M3 receptors in gastric smooth muscle cells. Carbachol (CCh) stimulated phosphatidylinositol (PI) hydrolysis, Rho kinase and zipper-interacting protein (ZIP) kinase activity, induced myosin phosphatase 1 (MYPT1) phosphorylation (at Thr(696)) and 20-kDa myosin light chain (MLC20) phosphorylation (at Ser(19)) and muscle contraction, and inhibited cAMP formation. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activity, phosphorylation of MYPT1 and MLC20, and muscle contraction in response to CCh were attenuated by methyl β-cyclodextrin (MβCD) or caveolin-1 small interfering RNA (siRNA). Similar inhibition of PI hydrolysis, Rho kinase, and ZIP kinase activity and muscle contraction in response to CCh and gastric emptying in vivo was obtained in caveolin-1-knockout mice compared with wild-type mice. Agonist-induced internalization of M2, but not M3, receptors was blocked by MβCD or caveolin-1 siRNA. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activities in response to other Gq-coupled receptor agonists such as histamine and substance P was also attenuated by MβCD or caveolin-1 siRNA. Taken together, these results suggest that caveolin-1 facilitates signaling by Gq-coupled receptors and contributes to enhanced smooth muscle function.