Uy Dong Sohn

Signal transduction pathways in esophageal and lower esophageal sphincter circular muscle.

Esophageal reflux is a common condition that affects children and 1 in 10 adults, and if untreated may result in chronic esophagitis, aspiration pneumonia, esophageal strictures, and Barretts esophagus, a premalignant condition. Although esophagitis is a multifactorial disease that may depend on transient lower esophageal sphincter (LES) relaxation, speed of esophageal clearance, mucosal resistance, and other factors, impairment of LES pressure is a common finding in patients complaining of chronic heartburn. Our data suggest that esophageal and LES circular muscle utilize distinct Ca2+ sources, phospholipid pools, and signal transduction pathways to contract in response to acetylcholine (ACh): (1) In esophageal muscle ACh-induced contraction requires influx of extracellular Ca2+ and may be linked to phosphatidylcholine metabolism, production of diacylglycerol (DAG) and arachidonic acid, and activation of a protein kinase C (PKC)-dependent pathway. (2) In LES muscle ACh-induced contraction utilizes intracellular Ca2+ release arising from metabolism of phosphatidylinositol (PI), and a calmodulin-myosin light chain kinase-dependent pathway. Resting LES tone, on the other hand, may be due to relatively low basal PI hydrolysis resulting in submaximal levels of inositol triphosphate (IP3)-induced calcium release and interaction with DAG to activate PKC. (3) After induction of experimental esophagitis, basal levels of PI hydrolysis and intracellular calcium stores are substantially reduced, resulting in a reduction of resting tone. In addition the signal transduction pathway responsible for LES contraction in response to ACh changes from one that depends on IP3 production, calcium release, and calmodulin activation to one that relies on influx of extracellular calcium and activation of PKC.

The intracellular pathway of the acetylcholine‐induced contraction in cat detrusor muscle cells

The present study was aimed to investigate intracellular pathways involved in acetylcholine (ACh)‐induced contraction in cat detrusor muscle cells Contraction was expressed as per cent shortening of length of individually isolated smooth muscle cells obtained by enzymatic digestion. Dispersed intact and permeabilized cells were prepared for the treatment of drugs and antibody to enzymes, respectively. Using Western blot, we confirmed the presence of related proteins. The maximal contraction to ACh was generated at 10−11 M. This response was preferentially antagonized by M3 muscarinic receptor antagonist ρ‐fluoro‐hexahydrosiladifenidol (ρF‐HSD) but not by the M1 antagonist pirenzepine and the M2 muscarinic receptor antagonist methoctramine. We identified G‐proteins Gq/11, Gs, G0, Gi1, Gi2 and Gi3 in the bladder detrusor muscle. ACh‐induced contraction was selectively inhibited by Gq/11 antibody but not to other G subunit. The phosphatidylinositol‐specific phospholipase C (PI‐PLC) inhibitor neomycin reduced ACh‐induced contraction. However, the inhibitors of the phospholipase D, the phospholipase A2 and protein kinase C did not attenuate the ACh‐induced contraction. ACh‐induced contraction was inhibited by antibody to PLC‐β1 but not PLC‐β3 and PLC‐γ. Thapsigargin or strontium, which depletes or blocks intracellular calcium release, inhibited ACh‐induced contraction. Inositol 1,4,5‐triphosphate (IP3) receptor inhibitor heparin reduced ACh‐induced contraction. These results suggest that in cat detrusor muscle contraction induced by ACh is mediated via M3 muscarinic receptor‐dependent activation of Gq/11 and PLC‐β1 and IP3‐dependent Ca2+ release.

The protective effect of eupatilin on indomethacin-induced cell damage in cultured feline ileal smooth muscle cells: Involvement of HO-1 and ERK

Chronic users of non-steroidal anti-inflammatory drugs frequently develop ulcerative lesions in their intestines. The purpose of the present study was to investigate whether eupatilin, an active ingredient derived from Artemisia plants, prevents this side effect in vitro. Extracts of the whole herb of Artemisia asiatica Nakai have been used in oriental medicine for the treatment of inflammation. As measured by the MTT assay, the treatment of cultured feline ileal smooth muscle cells (ISMCs) with 2.5mM indomethacin for 2h decreased the cell viability to 43%. Pretreatment with eupatilin resulted in dose-dependent inhibition on indomethacin-induced cell damage. This cytoprotective effect of eupatilin required concentrations of more than 150 microM and incubation periods of longer than 16 h. Pretreatment of ISMC with cycloheximide, an inhibitor of protein synthesis, attenuated the cytoprotective effect of eupatilin, suggesting that eupatilin induces proteins that are responsible for the cytoprotection. Heme oxygenase-1 (HO-1), which is known as a cytoprotective enzyme due to its anti-inflammatory actions, is a candidate protein since ZnPP, an HO-1 inhibitor, repressed the protective effect of eupatilin on indomethacin-induced cell damage in a concentration-dependent manner. Western blot analysis revealed that eupatilin-mediated HO-1 induction occurred in a concentration- and time-dependent manner. We also found that PD98059, a MEK (MAPK/ERK kinase) inhibitor, attenuated the eupatilin-induced HO-1 expression and nuclear translocation of transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). Taken together, the data imply that eupatilin protects ISMC from cell damage caused by indomethacin, and that its cytoprotective action could be attributed to eupatilin-mediated HO-1 induction via ERK and Nrf2 signaling in ISMC.

The Effect of Caffeic Acid on Wound Healing in Skin-incised Mice

This study was carried out to investigate the wound healing effect of caffeic acid in skin-incised mice. Caffeic acid showed significant effects on anti-inflammatory activity and wound healing, such as myeloperoxidase activity, lipid peroxidation, phospholipase A(2) activity and collagen-like polymer synthesis, in incised-wound tissue. On the other hand, it significantly stimulated collagen-like polymer synthesis in NIH 3T3 fibroblast cells, while inhibited both silica-induced reactive oxygen species generation and melittin-induced arachidonic acid release and PGE(2) production in Raw 264.7 cells, and histamine release in RBL 2H3 cells stimulated by melittin or arachidonic acid. Therefore, caffeic acid appears to have a potent antioxidant and anti-inflammatory effect in cell culture system, which may be related to wound healing in skin-incised mice.

Signal transduction mechanism via adenosine A1 receptor in the cat esophageal smooth muscle cells

We investigated what adenosine receptor type exists and the signaling pathways on the contraction of circular muscle cells isolated by enzymatic digestion from the cat esophagus. Adenosine or the selective A1 receptor agonist R-PIA causes a concentration-dependent contraction. After pretreatment with A1 receptor antagonist, DPCPX, adenosine-mediated contraction was abolished. Adenosine-induced contraction was significantly increased when A1 receptors were preserved by pretreatment with DPCPX followed by inactivation of all unprotected receptors with N-ethylmaleimide. Adenosine- or R-PIA-induced contraction was significantly augmented in the preserved cells and the increase was abolished in the presence of the A1 receptor antagonist DPCPX. PTX abolished contraction induced by adenosine or R-PIA, implying that contraction activated by A1 receptor was coupled to a pertussis toxin (PTX)-sensitive G(i) protein. After permeabilization, contraction was inhibited by G(i2), but not by G(i1) and G(i3), antibodies. These data suggest that adenosine-induced contraction of esophagus depends on PTX-sensitive G(i2.) Adenosine- or R-PIA-induced contraction of esophageal smooth muscle cells was not affected by the phospholipase D (PLD) inhibitor rho-chloromercuribenzoic acid (rhoCMB), phospholipase A(2) (PLA(2)) inhibitor DEDA or PKC antagonist chelerythrine, but was significantly abolished by phospholipase C (PLC) inhibitor, neomycin. PLC-beta3 antibody inhibited R-PIA-induced contraction. R-PIA-induced contraction of esophageal muscle cells was inhibited by IP(3) receptor antagonist heparin, which suggests that the contraction of esophageal smooth muscle cells is dependent on phosphatidylinositol-specific phospholipase (PI-PLC) and IP(3). In conclusion, adenosine- and R-PIA-induced contraction in cat esophageal smooth muscle cell was mediated by A1 receptor. A1 receptor is coupled to PTX-sensitive G protein G(i2), which results in the activation of PI-PLC-beta3. PI hydrolysis by PI-PLC forms IP(3), which binds to IP(3) receptor on endoplasmic reticulum, resulting in the release of intracellular Ca(2+).

Quercetin-3-O-β-d-glucuronopyranoside (QGC)-induced HO-1 expression through ERK and PI3K activation in cultured feline esophageal epithelial cells

Heme oxygenase-1 (HO-1) is one of the antioxidant enzymes which help protect against cellular damage. The present study examined the ability of Quercetin-3-O-beta-D-glucuronopyranoside (QGC), flavonoid glucoside extracted from Rumex Aquaticus Herba, to induce expression of HO-1 and analyzed its signaling mechanism in cultured feline esophageal epithelial cells (EEC). Culture of the esophageal epithelial cells from cat was prepared. The data suggested that QGC could result in enhanced antioxidant enzyme defense system via HO-1 expression and Nrf2 translocation involving both the ERK and PI3K-Akt pathways as well as partly PKC pathways in EEC.

The Inhibitory Effect of Quercetin-3-O-β-D-Glucuronopyranoside on Gastritis and Reflux Esophagitis in Rats

It was evaluated the inhibitory action of quercetin-3-O-beta-D-glucuronopyranoside (QGC) on reflux esophagitis and gastritis in rats. QGC was isolated from the herba of Rumex Aquaticus. Reflux esophagitis or gastritis was induced surgically or by administering indomethacin, respectively. Oral QGC decreased ulcer index, injury area, gastric volume, and acid output and increased gastric pH as compared with quercetin. Furthermore, QGC significantly decreased gastric lesion sizes induced by exposing the gastric mucosa to indomethacin. Malondialdehyde levels were found to increase significantly after inducing reflux esophagitis, and were reduced by QGC, but not by quercetin or omeprazole. These results show that QGC can inhibit reflux esophagitis and gastritis in rats.

Beneficial effects of phosphatidylcholine on high-fat diet-induced obesity, hyperlipidemia and fatty liver in mice.

AIMS Soybean-derived PC is an essential cell membrane phospholipid that is composed of unsaturated fatty acids, including oleic acid. The present study aimed to evaluate the potential alleviation effects of soybean PC on high fat diet (HFD)-induced obesity and its related complications. MAIN METHODS We fed C57BL/6 mice a HFD for 12 weeks and administered PC orally for 8 or 12 weeks at different doses. At the end of the experiment, blood was prepared for biochemical analysis and leptin ELISA. Aorta, epididymal and mesenteric fat and liver were removed surgically, weighed and observed for histological or immunohistochemical changes. KEY FINDINGS PC significantly prevented body weight gain and lipid accumulation and alleviated hyperlipidemia by decreasing triglyceride (TG) and total cholesterol (TC) levels and the atherogenic index in serum or by increasing the HDL/TC ratio. Aortic apoE expression and serum leptin levels were suppressed by PC treatment in the HFD-induced obese mouse model. Elevated serum aspartate aminotransferase and alanine aminotransferase levels in HFD-fed mice were decreased in the PC groups. PC treatment significantly decreased HFD-induced liver weight and hepatic lipid accumulation. SIGNIFICANCE PC treatment alleviated HFD-induced obese status and obesity-related complications such as hyperlipidemic changes that induce cardiovascular disease and NAFLD.

Leukotrienes in acetylcholine-induced contraction of esophageal circular smooth muscle in experimental esophagitis

BACKGROUND & AIMS Phospholipase A2 (PLA2) participates in acetylcholine (ACh)-induced contraction of esophageal circular smooth muscle. Because PLA2, arachidonic acid, and its metabolites are involved in inflammatory responses, their role after induction of experimental esophagitis was examined. METHODS Experiments were performed in esophageal smooth muscle cells (ESO) isolated by enzymatic digestion from the circular layer of normal and esophagitis animals. Content of peptidoleukotrienes (leukotriene [LT] C4, LTD4, and LTE4) was measured in esophageal circular muscle tissue. RESULTS The cytosolic PLA2 antagonist trifluoromethyl ketone analogue of arachidonic acid inhibited ACh-induced contraction of normal and esophagitis ESO. Inhibition by secreted PLA2 antagonists AM5 and MJ33 was significantly greater in esophagitis ESO. The lipoxygenase inhibitor nordihydro-guaiaretic acid and the LTD4 antagonist ICI 198,615 inhibited ACh-induced contraction of esophagitis but not of normal ESO. Secreted PLA2 and LTD4 contracted normal ESO more than esophagitis ESO. However, in esophagitis, ESO contraction was increased by threshold diacylglycerol concentration. Resting levels of LTs were greater in esophagitis than in normal circular esophageal muscle and increased in response to ACh in esophagitis but not in normal esophageal muscle. CONCLUSIONS Esophagitis shifts the signal transduction pathway activated by ACh. Esophagitis increased the contribution of secreted PLA2 and of LTs to ACh-induced contraction.

The role of nitric oxide and prostaglandin E2 on the hyperalgesia induced by excitatory amino acids in rats.

The present study was designed to investigate the role of nitric oxide (NO), N‐methyl‐D‐aspartate (NMDA) receptor and prostaglandins on hyperalgesia induced in rats by excitatory amino acids and the possibility that prostaglandins may act as the retrograde messenger in the spinal cord like NO.