[精品论文]Hydrogen sulfide modulated duodenal motility in rat via activating TRPV1 and KATP channels.doc

精品论文Hydrogen sulfide modulated duodenal motility in rat via activating TRPV1 and KATP channelsLU Wen, LI Jing, YE Yanfang, CHE Tongtong, LUO Yan, LI Jingxin, ZHAN Renzhi,5YAO Wei, LIU Kejing, CUI Shuang, LIU Chuanyong(Department of Physiology, Shandong University School of Medicine, JiNan 250012) Abstract: The aim of this study was to investigate the effect of hydrogen sulfide (H2S) on duodenal motility in rats and the underlying mechanism. The isometric contraction of longitudinal smooth muscle strips of duodenum was monitored by polygraph. The expression of H2S-producing enzymes,10cystathionine -synthase (CBS) and cystathionine -lyase (CSE) in duodenum were located byimmunofluorescence staining. NaHS (0.5 mM, 1 mM, 2 mM) exerts early transient excitation and late long-lasting inhibition on the contraction of the duodenal muscle strips. The excitatory effect was attenuated by pretreatment of Capsazepine (10 µM, TRPV1 antagonists), L703606 (10 µM, NK1 receptor antagonists) and NEM (0.1 mM, N-ethylmaleimide, sulfhydryl alkylating agent). The15inhibitory effect was reversed by Glibenclamide (a KATP channel blocker) and NEM pretreatment.NaHS caused isolated smooth muscle cells relaxation which was completely abolished by pretreatmentwith Glib (10 µM) and NEM (0.1 mM). The TRPV1 channel was expressed in sensory nerve terminals of myenteric plexus and the KATP channel was expressed in smooth muscle cells. All the neurons in rat duodenal myenteric plexuses expressed CBS and CSE. L-cysteine (0.5 mM, 1 mM), a substrate20for CBS and CSE, and SAM (1 mM), the activator of CBS, increased the contraction of duodenal muscle strips. AOAA (0.05 mM, the CBS inhibitor) and PAG (0.5 mM, the CSE inhibitor) attenuated the excitatory responses evoked by L-cysteine (1mM). Both Capsazepine and L703606 significantly attenuated the excitatory effects induced by L-cysteine or SAM. In summary, our results suggest thatexogenous H2S has a dual effect on the duodenal motility in rat. The excitatory effect is mediated by25activation of TRPV1 channels in sensory nerve terminals with the consequent release of SP, which inturn increases smooth muscle contraction. The inhibitory effect is mediated by direct activation of KATP channels on the smooth muscle cells. Both effects of exogenous H2S might be through protein s-sulfhydration. Endogenous H2S excited the motility of duodenal muscle strips with the same mechanism of NaHS but did not inhibit it. The findings of the present study may reveal a novel30mechanism of the excitatory effect of H2S on gastrointestinal motility.Keywords: H2S; duodenum; motility; TRPV1; KATP0IntroductionThe presence of hydrogen sulfide (H2S) in our environment is easily recognizable for its35peculiar rotten-egg smell. However, recent studies have established that H2S is a biologically relevant signaling molecule in mammals. H2S is generated in mammalian cells mainly by two pyridoxal phosphate dependent enzymes, cystathionine -synthase (CBS) and cystathionine-lyase (CSE), using L-cysteine as the main substrate. CBS and CSE are expressed in many organs, such as the brain, the liver, the kidney and the enteric nervous system (ENS) (1-7). Like nitric40oxide (NO) and carbon monoxide (CO), H2S has a prominent role in smooth muscle relaxation (8-9). It acts as a physiologic vasorelaxant and dilates vascular smooth muscle mainly by opening ATP-sensitive K+ (KATP) channels (9-13). In addition, H2S opens KATP channels in cardiomyocytes, pancreatic -cells, neurons and gastrointestinal smooth muscle cells, thereby regulating myocardial contractility, insulin secretion, neurotransmission and gastrointestinal45contractility (14).Although the role of H2S as a vasodilator by opening KATP channels had been recognized, the effects of H2S in modulating other ion channels has only recently begun to be addressed. H2SFoundations: Specialized Research Fund for the Doctoral Program of Higher Education (No. 20090131110046) Brief author introduction:Lu Wen, (1987-), female, BSc. and PhD., Research interest: gastrointestinal physiology.Correspondance author: LIU Chuanyong, (1969-), male, Prof., research area: gastrointestinal physiology. E-mail:liucy- 19 -stimulates capsaicin-sensitive sensory afferent fibers in rat urinary bladder and transient receptor potentials vanilloid 1 (TRPV1) channels in the lung (15-16). Marcello Trevisani et al. found that50H2S provokes tachykinin-mediated neurogenic inflammatory responses in guinea-pig airways and this effect is mediated by stimulation of TRPV1 channels on sensory nerves endings (17). Moreover, Schicho et al. demonstrated that sodium hydrosulfide (NaHS, the donor of H2S, caused secretion in the guinea-pig and human colon via activating TRPV1 located on afferent neurons, causing local release of substance P (SP) (7). However, whether the similar mechanism of H2S is55involved in regulation of gastrointestinal motility is still unclear.3A lot of studies indicated that H2S is involved in the regulation of gastrointestinal motility but the results were controversy. H2S suppresses colonic motility in mouse, human and rat but exerts dual effect on the gastric motility in guinea-pig (18-20). Ise F et al. reported that H2S stimulates HCO - secretion in rat duodenum (21), but as far as we know, there is no report about the effect of60H2S on duodenal motility.In our preliminary experiments, we found that NaHS exerted a dual effect on rat duodenal motility in a manner of early transient excitation and late long-lasting inhibition. Based on this preliminary data and the references, we hypothesized that the early transient excitation of the duodenal motility might be mediated by opening TRPV1 channels on the sensory fibers followed65by the release of SP and the late long-lasting inhibition is caused by opening KATP channels on smooth muscle.In order to test this hypothesis, we monitored the isometric tension of the longitudinal muscle strips of duodenum and the length of the isolated smooth muscle cells from duodenum in rats to investigate the role of exogenous and endogenous H2S on the smooth muscle contraction. TRPV170and KATP channels and the two key H2S-producing enzymes, CBS and CSE, were localized byimmunofluorescence staining. The effects of the H2S donor NaHS on afferent nerve activity was monitored by afferent nerve recordings. The results support our hypothesis and reveal a new mechanism of H2S in the regulation of duodenal motility.1Materials and Methods751.1 AnimalsWistar male rats (250 300 g) were provided by the Experimental Animal Center of Shandong University. All the procedures described below were approved by the Ethics Committee for Research on Animals, Shandong University School of Medicine.1.2 Muscle strips preparation80After the rat was sacrificed by cervical dislocation, a segment of duodenum (1 cm from pylorus) was removed immediately and put into a glass beaker containing 4 °C Krebs solution. The segment was cut along the mesenteric border and the content of the duodenum was washed. The mucosal and submucosal layers were carefully striped using fine forceps under a microscope in a silica gel dish filled with oxygenated Krebs solution. The longitudinal muscle strips (4 mm85wide, 10 mm long) were prepared by cutting along parallel direction of the longer axis of theduodenal segment. Both ends of the muscle strips were attached to a silk thread.1.3 Recording of the contraction of muscle stripsThe isolated duodenal smooth muscle strips were suspended in tissue chambers containing 5 ml Krebs solution bubbled with 5% CO2/95% O2. The chambers were maintained at 37 °C using a90water pump （ZH-Z,Zhenghua Biological Equipment limited company，Huaibei, China）. One95100105110115120125130end of the muscle strip was connected to a fixed hook and the other end was attached to an external isometric force transducer (JH-2B, Chengdu Instrument Factory, Chengdu, China). A polygraph system (ML785-PowerLab, ADI, Sydney, Australia) was used to record the tension of the duodenal strips. The muscle strips was incubated at a 0.5 1 g preload. Once the spontaneous contraction was stable for more than 15 min, NaHS (0.1 mM, 0.5 mM, 1 mM and 2 mM), L-cysteine (0.1 mM, 0.5 mM and 1 mM) or S-adenosyl-L-methionine (SAM, 0.1 mM, 0.5 mM and 1 mM) were administrated to the chambers. Each muscle strip was exposed to the reagent only once. The tension was recorded continuously for at least 30 min after treatment with the reagent. In other experiments, the muscle strips were pretreated with Carbachol (2 µM), Capsazepine (10µM), L703606 (10 µM), Glibenclamide (Glib, 10 µM), Tetrodotoxin (TTX, 10 µM), N-ethylmaleimide (NEM, 0.1 mM), D, L-propargylglycine (PAG, 0.5 mM) and amino-oxyacetic acid (AOAA, 0.05 mM) 10 20 min before NaHS, L-cysteine or SAM administration. Control strips were exposed to normal solution (NS) or dimethyl sulfoxide (DMSO) for the same time period.1.4 Preparation of smooth muscle cellsSmooth muscle cells were prepared from duodenum as described by Zhao et al. with some modifications (18, 22). Briefly, the muscle strips were placed into Ca2+-free physiologic saline solution (PSS) and cut into small segments (2 × 2 mm), digested at 37 °C in 1 ml Ca2+-free solution which containing 1.7 1.9 mg collagenase II (Worthington Biochemicals, Lakewood, NJ, USA), 500 700 g papain (Sigma-Aldrich, St. Louis, MO, USA), 1 mg Dithiothreitol (Sigma-Aldrich, St. Louis, MO, USA), 0.5 mg trypsin inhibitor (Sigma-Aldrich, St. Louis, MO, USA), 1 mg bovine serum albumin (Roche Diagnostics GmbH, Mannhein, Germany) for 20 30min.Andthen,themusclesegmentswerewashedseveraltimeswiththe modified Kraft-Bruhe solution (KB solution) followed by gently triturated using a wide-bore fire-polished glass pipette (2 mm bore diameter) to obtain a cell suspension. Dispersed cells were kept in KB solution at 4 °C for experiments.1.5 Measurement of the length of isolated smooth muscle cellsDrops of the cell suspension prepared above were transferred to a dish and cultured at room temperature for 10 15 min in order to make the cells to adhere to the bottom of the dish. ThenPSS （2 ml）was added into the cell dish. An inverted microscope (IX51, Olympus, Tokyo, Japan)was used to observe the smooth muscle cells and take photographs.Image Pro Plus 6.0 (MediaCybernetics, Leiden, The Netherlands) was used to measure the length of smooth muscle cells.1.6 Immunofluorescence on LMMPLongitudinal muscle myenteric plexus (LMMP) was prepared as our previous report (23). In the experiments of double-labeling two proteins in myenteric plexus, including CBS and NeuN (neuron-specific nuclear protein), CSE and NeuN, CBS and CSE, LMMP was incubated in Krebs solution containing papain (10 mg/ml) for 50 min to digest the smooth muscle cells. Thereafter, the specimen was stretched in order to expose the myenteric plexus more clearly. The preparations were washed three times with phosphate-buffered saline (PBS) and soaked in 4% paraformaldehyde for 30 min. Subsequently, they were incubated with hydrogen peroxide (3%) for 15 min to quench endogenous peroxidase activity, tritonX-100 (0.2%) for 20 min to improve permeability of cell membranes and donkey serum (10%) for 1 hour to block non-specific binding. All these procedures were conducted at room temperature. In the case of double labeling of TRPV1 and calcitonin gene-related peptide (CGRP), the specimens were incubated with 2.5%135140145150155160165170175donkey serum which contained rabbit polyclonal anti-TRPV1 antibody (1:1000, Neuromics Minneapolis, MN, USA) and mouse monoclonal anti-CGRP antibody (1: 500, Abcam, Cambridge, UK) for about 12 16 h at 4 °C. For Kir6.2(a subunit of KATP channels) and SM22 (a smooth muscle marker) double staining, the tissue was incubated with a primary antibody mixture including rabbit polyclonal anti-Kir6.2 antibody (1: 500, Abcam, Cambridge, UK) and goat polyclonal anti-SM22 antibody (1: 200, Abcam, Cambridge, UK) at 4 °C overnight. In triple-labeling studies, the LMMP specimens were incubated with either rabbit anti-CBS (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-CSE (1: 200, Abcam, Cambridge,UK) polyclonal antibody mixed with mouse monoclonal anti-NeuN (1: 200, Chemicon，CA, USA)and goat polyclonal anti-SM22 antibody (1: 200, Abcam, Cambridge, UK) at 4 °C overnight. In the experiment of double staining of CBS and NeuN, CSE and NeuN, CBS and CSE, the primary antibody had been made the appropriate changes. After several washes for 10 min each in PBS, tissues were incubated with corresponding secondary antibody, Alexa Fluor 350-conjugated donkey anti-goat (1: 300, Invitrogen, Carlsbad, CA), Alexa Fluor 488-conjugated donkey anti-mouse (1: 300, Invitrogen, Carlsbad, CA) and Alexa Fluor 568-conjugated donkey anti-rabbit (1: 300, Invitrogen, Carlsbad, CA) IgG for 2 h at room temperature followed by three times washes with PBS. Additional controls during the staining procedure were performed by omitting the primary antibodies from the incubation solution. The specimens were examined under confocal microscopy (LSM780; Carl Zeiss, Jena, Germany). The protocol for localization of Kir6.2 and SM22 on smooth muscle cells was same to that for LMMP. The smooth muscle cells used in this experiment was purchased from Chi Scientific (Jiangsu, China).1.7 Mesenteric afferent nerve recordingsAfter rats were anaesthetized with pentobarbitone sodium (60 mg/kg), a segment of duodenum with a clear artery projection and the mesentery attached was excised carefully and immersed in Krebs solution quickly. The tissue was moved to a purpose-built organ bath and fixed the both ends to the organ chamber, which was continuously perfused with Krebs solution bubbledwith 5% CO2/95%O2 at a rate of 10 ml/min at 34 . Intraluminal pressure was recorded by apressure transducer. The mesenteric bundle was pinned out onto the sylgard base of the recording chamber and one of the two mesenteric nerves was carefully dissected out of the neurovascular mesenteric bundle. The nerve bundle was wrapped around one arm of a bipolar wire platinum recording electrode, while one strip of connective tissue was attached to the other electrode. The signa