| Background:IBS is a kind of functional bowl disease that has the highest incidence rate in the word. The etiology and pathogenesis of IBS is extremely complicated. As the increase of social stress and competition, more attentions have been paid on the effect of acute and chronic stresses on the pathogenesis of IBS. Along with the researches of the bioactive substances like the brain-gut peptides and gastrointestinal hormones, some researches found the level change of the bioactive substances in the IBS patients, like calcitionin gene-related peptide (CGRP), cholecystokinin (CCK), motilin (MTL), melatonin (MT),substance P (SP),etc. These bioactive substances are related to the preprandial and postprandial gastrointestinal movements and participate in the secretion, absorption and the sensation of the gastrointestinal tract. Whether the aberrant level of the bioactive substances is related to the genesis and development of IBS remains unknown and needs to be researched. Gastrointestinal motility disorder is directly related to the genesis of IBS. Some researches revealed that the rangeability (increase or reduction) of colonic contraction frequency, amplitude and pike potential are significantly enhanced in IBS patients, especially after the meal and psychological stimulations. So the purpose of this research is to investigate the level change of the brain-gut peptides and gastrointestinal hormones in the acute and chorionic stresses, and its effect on the colonic motility.Part IObjectives:To investigate the chronic water avoidance stress (cWAS) on the frequencies of fecal output, the basal fecal pellet output and the fecal output in the acute stress situation, to investigate the cWAS on the brain-gut peptides and gastrointestinal hormones, and discuss the mechanisms involved in.Methods:20rats were divided in to chronic water avoidance stress (cWAS) and sham cWAS group. The changes in the frequencies of fecal output and the levels of brain-gut peptides and gastrointestinal hormone levels in plasma induced by chronic water avoidance stress were observed.Results:The fecal pellet output in all animals was significantly increased by WAS with exposure to the water for1hour, and was more for the first five days (FFD) than for the last five days (LFD)(6.14+0.69vs.4.88+0.50, n=10, p<0.01,). However, there was no such effect with exposure to sham stress (2.46±0.67vs.2.42±0.23, n=10, p>0.05). The basal fecal pellet output of the WAS rats at day eleven showed no significant difference from that of the SWAS rats (3.1±0.99vs.2.5±0.84, n=10, p=0.164, Figure1). When exposed to the acute stress situation, the WAS rats had a greater fecal pellet output than the SWAS rats (9.5±1.72vs.7.8±1.68, n=10, p<0.05). The concentrations of SP, TRH, CCK and MTL were significantly increased in the WAS rats compared to those in the SWAS rats, while the concentrations of VIP, CGRP and CRH showed no significant difference between the two groups. Furthermore, the plasma levels of PPY in WAS rats were significantly lower than those in SWAS rats.Conclusions:The relative levels of gastrointestinal hormones and the brain-gut peptide possibly changes the way of colonic motility and the magnitude in the acute and chronic stress of the outside world; In addition, the central nervous mechanism about expression of receptors may be involved in chronic stress. Objectives:To investigate the effects and the mechanisms of cWAS on the contractile activity in rat proximal colonic smooth muscle strips and to investigate the effects and the mechanisms of WAS rat plasma on the contractile activity in normal rat proximal colonic smooth muscle strips. And discuss the mechanisms involved in.Methods:Rats were anesthetized with ether, followed by sacrifice along the ventral midline. The proximal colon was removed, cleaned and opened along the mesenteric border and then placed in Ca2+-free physiological saline solution (PSS) bubbled with carbogen (95%02/5%CO2). The smooth muscle strips (3mm*8mm) were obtained after the mucosa and submucosa were excised. When studying longitudinal smooth muscles (LM), the strips parallel to the mesenteric were used and when studying circular smooth muscles (CM), the strips vertical to the mesenteric were used. Each fresh smooth muscle strip was mounted in an organ bath and connected to an isometric force transducer (JZJOIH, Chengdu, China). The organ baths contained6mL Tyrode’s buffer at37℃and were constantly warmed by a circulating water jacket at37℃and bubbled with carbogen (95%O2/5%CO2). One end of the strip was fixed to a hook on the bottom of the chamber, while the other end was connected by a thread to an external isometric force transducer at the top. Each muscle strip was placed under a resting preload of1.0g to obtain a maximum response to1M ACh and allowed to equilibrate for60min. During the equilibration period, the sections were washed every20min with Tyrode’s and the basal tension was maintained. To obtain a stable and acceptable level of sensitivity before the experimental procedure began, the colonic section was challenged with40mM KC1until reproducible responses were obtained. The frequencies of contraction were calculated by counting the contraction waves per20minutes. The mean contractile amplitude and frequency of spontaneous contractions were recorded (WAS and sham WAS values) and compared with the mean contractile amplitude and frequency when the control strips were exposed to100ml WAS plasma or sham WAS plasma.Results:The baseline amplitude of spontaneous contractile activities from the WAS rat colons was significantly increased when compared to that of the SWAS rats. The mean active tension of longitudinal smooth muscle (LM) in WAS rats was significantly higher than that in SWAS rats (1.41+0.2g vs.1.12±0.27g, n=10, P<0.01) and the mean active tension of circular smooth muscle (CM) in WAS rats was significantly higher than that in SWAS rats (0.46±0.11g vs.0.35±0.07g, n=10, P <0.05). However, the frequency of spontaneous contractions of CM or LM from WAS rats did not change significantly when compared to that of SWAS rats. When tetrodotoxin (TTX,100nM) was added into the organ bath for30min to block the influence of the neuronal factors in the enteric nervous system on smooth muscle contraction, the spontaneous contractions decreased in the two groups, but the difference between them still persisted. The frequency of spontaneous muscle strip contractions was again not significantly different between the two groups. The amplitudes of contractile activities of LM and CM with added WAS rat plasma (WP100ul/6ml) were significantly decreased (1.07±0.29g vs.0.83±0.31g and0.31±0.1g vs.0.23±0.82g, respectively, n=10, P<0.05) in the presence of100nM TTX in the organ bath. However, the amplitude of contractile activities with added SWAS rat plasma (SWP100ul/6ml) was not significantly changed (p>0.05). The frequency of spontaneous contraction activities of CM or LM from WAS rats was not significantly changed in the absence and presence of WP or SWP.Conclusions:cWAS increased spontaneous contractile activity of proximal colonic smooth muscle strips in vitro, but cWAS plasma decreased spontaneous contractile amplitude of proximal colonic smooth muscle (PCSM) strips. These effects may be through the relative levels of gastrointestinal hormones and the brain-gut peptide and the smooth muscle cell. Objectives:To investigate the effect of cWAS on voltage-dependent potassium currents and calcium-activated potassium currents, and investigate the effect of cWAS rat plasma on voltage-dependent potassium currents and calcium-activated potassium currents. And discuss the mechanisms involved in.Methods:Single SMCs were isolated by enzymatic digestion. The strips of proximal colon were pinned to the base of the Sylgard surface of a Petri dish and the mucosa and submucosa was carefully dissected away under an anatomical microscope. The tissue was cut into small strips (about2mm-5-6mm) and placed in Ca2+-free PSS solution that contained0.12%(w/v) collagenase II supplemented with0.2%soybean trypsin inhibitor and0.2%BSA, and incubated for19-34min at37℃. After completion of digestion, the segments were washed five times in a Ca2+-free PSS solution and then triturated gently with a fire-polished Pasteur pipette to create a cell suspension. Cells were stored at0～4℃and used within8h. Several drops of cell suspensions were placed in a recording chamber that was mounted with an inverted microscope (Olympus, Japan). After adhering to the coverslip, the cells were infused with Tyrode’s buffer (2mL/min). Pipettes were made using a micropipette puller (P-97; Sutter, USA). Typical pipette resistances were3-5MV. A gigaseal was formed with negative suction. Capacitance was compensated for and the residual capacitance current was removed digitally. Whole-cell currents were recorded with an EPC-10amplifier (HEKA, Germany). Acquisition and analysis of data were accomplished by using PulseFit (HEKA Instrument, Germany). The values of WAS and sham WAS IBKca and IKv currents were recorded and the effects of WAS plasma or sham WAS plasma at different concentrations were investigated on IBKca and IKv when added to control rats.Results:With whole-cell voltage-clamp recordings, a delayed rectifier IK was found to be the predominant Kv current in freshly isolated rat PCSMCs (n=28/30). IK was activated slowly at membrane potentials from positive to220mV with no inactivating kinetics over the stimulation period (600ms). The transient and fastinactivating IA was rare in these cells (2/30). The IKv of WAS rats is decreased when compared to that of SWAS rats (8.05+0.86pA/pF vs.12.86+0.91pA/pF, n=10, p<0.01) at+60mv). The large conductance IBKCa current was detected by using a depolarizing step pulse from a holding potential of-60mV to+80mV for400ms. The IBKca current density of WAS rats was decreased when compared to that of SWAS rats (21.49+1.75pA/pF vs.27.20+1.30pA/pF, n=10, p<0.01) at+80mv. To explore the effect of plasma on IKV and IBKca current in proximal colon SMCs, we took the plasma of the WAS or SWAS rats to perfuse the PCSMCs of normal Wistar rats. The plasma of the WAS or SWAS rats activated the IKv current in freshly isolated normal rat PCSMCs (from13.05+0.66pA/pF to17.10+1.27pA/pF, n=10, p<0.01, or to13.69+0.78pA/pF, respectively) at+60mv. As shown in Figure7, the plasma of the WAS or SWAS rats also activated BKCa current in freshly isolated control rat proximal colon SMCs (from27.55+1.33pA/pF to32.54+2.02pA/pF, n=10, p<0.01, or to28.59+1.49pA/pF, respectively).Conclusions:cWAS increased the voltage-dependent potassium currents and calcium-activated potassium currents. But cWAS rat plasma inhibited the voltage-dependent potassium currents and calcium-activated potassium currents of normal rats. It can be considered that the voltage-dependent potassium currents and calcium-activated potassium currents of the smooth muscle cells of the colon result in the level of openness of iron channels in the cells> hormone receptor on the cell membrane and the level Hormone of plasma. |
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