Fisetin, a natural flavonoid found in a variety of vegetables and fruits, has been shown to possess many biological functions. The present study was undertaken to investigate the influence of fisetin on vascular smooth muscle contractility and to determine the mechanism involved. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Fisetin significantly relaxed fluoride-, thromboxane A2- or phorbol ester-induced vascular contraction suggesting as a possible anti-hypertensive on the agonist-induced vascular contraction regardless of endothelial nitric oxide synthesis. Furthermore, fisetin significantly inhibited fluoride-induced increases in pMYPT1 levels and phorbol ester-induced increases in pERK1/2 levels suggesting the mechanism involving the inhibition of Rho-kinase activity and the subsequent phosphorylation of MYPT1 and MEK activity and the subsequent phosphorylation of ERK1/2. This study provides evidence regarding the mechanism underlying the relaxation effect of fisetin on agonist-induced vascular contraction regardless of endothelial function.
Animals ; Fluorides ; Fruit ; Humans ; Isometric Contraction ; Male ; Muscle, Smooth, Vascular ; Nitric Oxide ; Phosphorylation ; Rats ; Relaxation ; rho-Associated Kinases ; Vegetables

Contraction of smooth muscle is initiated by an increase in cytosolic Ca2+ leading to activation of Ca2+/calmodulin-dependnet myosin light chain (MLC) kinase and phosphorylation of MLC. The types of contraction and signaling mechanisms mediating contraction differ depending on the region. The involvement of these different mechanisms varies depending on the source of Ca2+ and the kinetic of Ca2+ mobilization. Ca2+ mobilizing agonists stimulate different phospholipases (PLC-beta, PLD and PLA2) to generate one or more Ca2+ mobilizing messengers (IP3 and AA), and diacylglycerol (DAG), an activator of protein kinase C (PKC). The relative contributions of PLC-beta, PLA2 and PLD to generate second messengers vary greatly between cells and types of contraction. In smooth muscle cell derived form the circular muscle layer of the intestine, preferential hydrolysis of PIP2 and generation of IP3 and IP3-dependent Ca2+ release initiate the contraction. In smooth muscle cells derived from longitudinal muscle layer of the intestine, preferential hydrolysis of PC by PLA2, generation of AA and AA-mediated Ca2+ influx, cADP ribose formation and Ca2+/-induced Ca2+ release initiate the contraction. Sustained contraction, however, in both cell types is mediated by Ca2+/-independent mechanism involving activation of PKC- epsilon by DAG derived form PLD. A functional linkage between G13, RhoA, ROCK, PKC- epsilon, CPI-17 and MLC phosphorylation in sustained contraction has been implicated. Contraction of normal esophageal circular muscle (ESO) in response to acetylcholine (ACh) is linked to M2 muscarinic receptors activating at least three intracellular phospholipases, i.e. phosphatidylcholine-specific phospholipase C (PC-PLC), phospholipase D (PLD) and the high molecular weight (85 kDa) cytosolic phospholipase A2 (cPLA2) to induce phosphatidylcholine (PC) metabolism, production of diacylglycerol (DAG) and arachidonic acid (AA), resulting in activation of a protein kinase C (PKC)-dependent pathway. In contrast, lower esophageal sphincter (LES) contraction induced by maximally effective doses of ACh is mediated by muscarinic M3 receptors, linked to pertussis toxin-insensitive GTP-binding proteins of the Gq/11 type. They activate phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate (PIP2), producing inositol 1, 4, 5-trisphosphate (IP3) and DAG. IP3 causes release of intracellular Ca2+ and formation of a Ca2+/-calmodulin complex, resulting in activation of myosin light chain kinase and contraction through a calmodulin-dependent pathway.
Acetylcholine ; Arachidonic Acid ; Cyclic ADP-Ribose ; Cytosol ; Esophageal Sphincter, Lower ; GTP-Binding Proteins ; Hydrolysis ; Inositol ; Intestines ; Metabolism ; Molecular Weight ; Muscle, Smooth* ; Myocytes, Smooth Muscle ; Myosin Light Chains ; Myosin-Light-Chain Kinase ; Negotiating ; Phosphatidylcholines ; Phosphatidylinositols ; Phospholipase D ; Phospholipases ; Phospholipases A2 ; Phosphorylation ; Phosphotransferases ; Protein Kinase C ; Receptor, Muscarinic M3 ; Receptors, Muscarinic ; Second Messenger Systems ; Type C Phospholipases ; Whooping Cough

We investigated inhibitory effects of extract containing quercetin-3-O-beta-D-glucuronopyranoside (ECQ) extracted from Rumex Aquaticus Herba on indomethacin-induced gastric damage in Rats. Gastritis was induced in male Sprague-Dawley rats (200~220 g) by oral administration of indomethacin at a dose of 40 mg/kg. One hour before administration of indomethacin, animals were orally pretreated with ECQ at doses of 0.3, 1, 3 or 10 mg/kg. Six hours after indomethacin administration, the rats were sacrificed and the stomach was excised and opened along the greater curvature, and the surface area of gastric lesion was measured using optical microscope. Superoxide dismutase (SOD), catalase (CAT), myeloperoxidase (MPO) activities and malondialdehyde (MDA) levels were measured by ELISA. Western blot analysis was performed to detect protein expression of SOD-2. Linear hemorrhagic mucosal lesions were observed in the stomach 6 hours after oral administration of indomethacin. Pretreatment with ECQ significantly reduced the severity of the lesions in a dose-dependent manner. It also inhibited the reductions in SOD and CAT activities and SOD expression by the indomethacin-induced gastric damage. In addition, the pretreatment with ECQ significantly suppressed the elevation of the MPO activity and the MDA levels induced by indomethacin. These results suggest that ECQ has the inhibitory effects via antioxidative action against indomethacin-induced gastritis in rats.
Administration, Oral ; Animals ; Blotting, Western ; Catalase ; Cats ; Enzyme-Linked Immunosorbent Assay ; Gastritis ; Humans ; Indomethacin ; Male ; Malondialdehyde ; Peroxidase ; Quercetin ; Rats ; Rats, Sprague-Dawley ; Rumex ; Stomach ; Superoxide Dismutase

The precise mechanism of set-point regulation in hypothalamus was not elucidated. Nitric oxide synthases (NOS) were detected in hypothalamus, however, the roles of NO in hypothalamus was not fully studied. So, we tested the effects of NO on body temperature because preoptic-anterior hypothalamus was known as the presumptive primary fever-producing site. NO donor sodium nitroprusside (SNP, 4 nmol, i.c.v.) elicited marked febrile response, and this febrile response was completely blocked by indomethacin (a cyclooxygenase inhibitor). But, ODQ (selective guanylate cyclase inhibitor, 50 microgram, i.c.v.) did not inhibit fever induced by SNP. The cyclic GMP analogue dibutyryl-cGMP (100 microgram, i.c.v.) induced significant pyreses, which is blocked by indomethacin. NG-nitro-L-arginine methyl ester (L-NAME, non selective NOS inhibitor) inhibited fever induced by interleukin-1 beta (IL-1 beta, 10 ng, i.c.v.), one of endogenous pyrogens. These results indicate that NO may have an important role, not related to stimulation of soluble guanylate cyclase, in the signal pathway of thermoregulation in hypothalamus.
Body Temperature ; Body Temperature Regulation ; Central Nervous System* ; Cyclic GMP ; Fever ; Guanylate Cyclase ; Humans ; Hypothalamus ; Indomethacin ; Interleukin-1beta ; NG-Nitroarginine Methyl Ester ; Nitric Oxide* ; Nitroprusside ; Prostaglandin-Endoperoxide Synthases ; Pyrogens ; Signal Transduction ; Tissue Donors

Carbon monoxide (CO) binds to soluble guanylate cyclase to lead its activation and elicits smooth muscle relaxation. The vascular tissues have a high capacity to produce CO, since heme oxygenase-2 (HO-2) is constitutively expressed in endothelial and smooth muscle cells, and HO-1 can be greatly up-regulated by oxidative stress. Moreover, the substrate of HO, heme, is readily available for catalysis in vascular tissue. Although the activation of heme oxygenase pathway under various stress conditions may provide a defence mechanism in compromised tissues, the specific role of HO-1-derived CO in the control of aortic contractility still remains to be elucidated. The present study was done to determine the effect of HO-1 induction on the aortic contractility. Thus, the effects of incubation of aortic tissue with S-nitroso-N-acetylpenicillamine (SNAP) for 1 hr on the aortic contractile response to phenylephrine were studied. The preincubation with SNAP resulted in depression of the vasoconstrictor response to phenylephrine. This effect was restored by HO inhibitor or methylene blue but not by NOS inhibitor. The attenuation of vascular reactivity by preincubation with SNAP was also revealed in endothelium-free rings. AlF4--evoked contraction in control did not differ from that in SNP-treated group. These results suggest that increased production of CO was responsible for the reduction of the contractile response to phenylephrine in aortic ring preincubated with SNAP and this effect of SNAP was independent on endothelium.
Carbon Monoxide ; Catalysis ; Depression ; Endothelium ; Guanylate Cyclase ; Heme Oxygenase (Decyclizing)* ; Heme* ; Humans ; Methylene Blue ; Muscle, Smooth ; Myocytes, Smooth Muscle ; Oxidative Stress ; Phenylephrine ; Relaxation ; S-Nitroso-N-Acetylpenicillamine ; Tissue Donors*

We previously shown that LES contraction depends on M3 receptors linked to PTX insensitive Gq protein and activation of PLC. This results in production of IP3, which mediates calcium release, and contraction through a CaM dependent pathway. In the esophagus ACh activates M2 receptors linked to PTX sensitive Gi3 protein, resulting in activation of PLD, presumably, production of DAG. We investigated the role of PLC isozymes which can be activated by Gq or Gbeta protein on ACh-induced contraction in LES and esophagus. Immunoblot analysis showed the presence of 3 types of PLC isozymes, PLC-beta1, PLC-beta3, and PLC-gamma1, but not PLC-beta2, PLC-beta4, PLC-gamma2, PLC-delta1, and PLC-delta2 from both LES and esophageal muscle. ACh produced contraction in a dose dependent manner in LES and esophageal muscle cells obtained by enzymatic digestion with collagenase. PLC-beta1 or PLC-beta3 antibody incubation reduced contraction in response to ACh in LES but not in esophageal permeabilized cells, but PLC-gamma1 antibody incubation did not have an inhibitory effect. The inhibition by PLC-beta1 or PLC-beta3 antibody on Ach-induced contraction was antibody concentration dependent. The combination with PLC-beta1 and PLC-beta3 antibody completely abolished the contraction, suggesting that PLC-beta1 and PLC-beta3 have a synergism to inhibit the contraction in LES. PLC-beta1, -beta3 or -gamma1 antibody did not reduce the contraction of LES cells in response to DAG (10 (-6) M), suggesting that this isozyme of PLC may not activate PKC. When Gq/11 antibody was incubated, the inhibitory effect of the incubation of PLC beta3, but not of PLC beta1 was additive (Fig. 6). In contrast, when Gbeta antibody was incubated, the inhibitory effect of the incubation of PLC beta1, but not of PLC beta3 was additive. This data suggest that Gq/11 or Gbeta may activate cooperatively different PLC isozyme, PLCbeta1 or PLCbeta3 respectively.
Animals ; Calcium ; Cats* ; Collagenases ; Digestion ; Esophageal Sphincter, Lower* ; Esophagus ; GTP-Binding Protein alpha Subunits, Gq-G11* ; Isoenzymes ; Muscle Cells ; Phospholipase C beta ; Type C Phospholipases

The gastrointestinal (GI) tract is a series of hollow organs that is responsible for the digestion and absorption of ingested foods and the excretion of waste. Any changes in the GI tract can lead to GI disorders. GI disorders are highly prevalent in the population and account for substantial morbidity, mortality, and healthcare utilization. GI disorders can be functional, or organic with structural changes. Functional GI disorders include functional dyspepsia and irritable bowel syndrome. Organic GI disorders include inflammation of the GI tract due to chronic infection, drugs, trauma, and other causes. Recent studies have highlighted a new explanatory mechanism for GI disorders. It has been suggested that autophagy, an intracellular homeostatic mechanism, also plays an important role in the pathogenesis of GI disorders. Autophagy has three primary forms: macroautophagy, microautophagy, and chaperone-mediated autophagy. It may affect intestinal homeostasis, host defense against intestinal pathogens, regulation of the gut microbiota, and innate and adaptive immunity. Drugs targeting autophagy could, therefore, have therapeutic potential for treating GI disorders. In this review, we provide an overview of current understanding regarding the evidence for autophagy in GI diseases and updates on potential treatments, including drugs and complementary and alternative medicines.

The gastrointestinal (GI) tract is a series of hollow organs that is responsible for the digestion and absorption of ingested foods and the excretion of waste. Any changes in the GI tract can lead to GI disorders. GI disorders are highly prevalent in the population and account for substantial morbidity, mortality, and healthcare utilization. GI disorders can be functional, or organic with structural changes. Functional GI disorders include functional dyspepsia and irritable bowel syndrome. Organic GI disorders include inflammation of the GI tract due to chronic infection, drugs, trauma, and other causes. Recent studies have highlighted a new explanatory mechanism for GI disorders. It has been suggested that autophagy, an intracellular homeostatic mechanism, also plays an important role in the pathogenesis of GI disorders. Autophagy has three primary forms: macroautophagy, microautophagy, and chaperone-mediated autophagy. It may affect intestinal homeostasis, host defense against intestinal pathogens, regulation of the gut microbiota, and innate and adaptive immunity. Drugs targeting autophagy could, therefore, have therapeutic potential for treating GI disorders. In this review, we provide an overview of current understanding regarding the evidence for autophagy in GI diseases and updates on potential treatments, including drugs and complementary and alternative medicines.

Postoperative ileus (POI), a symptom that occurs after abdominal surgery, reduces gastrointestinal motility. Although its mechanism is unclear, POI symptoms are known to be caused by inflammation 6 to 72 h after surgery. As proton pump inhibitors exhibit protective effect against acute inflammation, the purpose of this study was to determine the effect of ilaprazole on a POI rat model. POI was induced in rats by abdominal surgery. Rats were divided into six groups: control: normal rat + 0.5% CMC-Na, vehicle: POI rat + 0.5% CMC-Na, mosapride: POI rat + mosapride 2 mg/kg, ilaprazole 1 mg/kg: POI rat + ilaprazole 1 mg/kg, ilaprazole 3 mg/kg: POI rat + ilaprazole 3 mg/kg, and ilaprazole 10 mg/kg: POI rat + ilaprazole 10 mg/kg. Gastrointestinal motility was confirmed by measuring gastric emptying (GE) and gastrointestinal transit (GIT). In the small intestine, inflammation was confirmed by measuring TNF-α and IL-1β; oxidative stress was confirmed by SOD, GSH, and MDA levels; and histological changes were observed by H&E staining. Based on the findings, GE and GIT were decreased in the vehicle group and improved in the ilaprazole 10 mg/kg group. In the ilaprazole 10 mg/kg group, TNF-α and IL-1β levels were decreased, SOD and GSH levels were increased, and MDA levels were decreased. Histological damage was also reduced in the ilaprazole-treated groups. These findings suggest that ilaprazole prevents the decrease in gastrointestinal motility, a major symptom of postoperative ileus, and reduces inflammation and oxidative stress.

In this investigation, we made a study of the efficacy of luteolin (a flavonoid found in plants such as vegetables, herbs and fruits) on vascular contractibility and to elucidate the mechanism underlying the relaxation. Isometric contractions of denuded muscles were stored and combined with western blot analysis which was conducted to assess the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to examine the effect of luteolin on the RhoA/ROCK/CPI-17 pathway. Luteolin significantly alleviated phorbol ester-, fluoride- and thromboxane mimetic-elicited contractions regardless of endothelial nitric oxide synthesis, implying its direct effect on smooth muscle. It also significantly alleviated the fluoride-elicited elevation in pCPI-17 and pMYPT1 levels and phorbol 12,13-dibutyrate-elicited in-crease in pERK1/2 level, suggesting depression of ROCK and PKC/MEK activity and ensuing phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that luteolin-elicited relaxation includes myosin phosphatase reactivation and calcium desensitization, which seems to be arbitrated by CPI-17 dephosphorylation via ROCK/PKC inhibition.