| PartⅠHydrogen sulfide modulates duodenal motility in male rats via activating TRPV1and KATP channelsObjective:Hydrogen sulfide (H2S) in our environment is easily recognized for its peculiar rotten-egg smell. However, recent studies have established that H2S is a biological relevant signaling molecule in mammals. H2S is generated in mammalian cells mainly by two pyridoxal phosphate dependent enzymes, including cystathionine β-synthase (CBS) and cystathionine γ-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). A lot of studies indicate that H2S is involved in the regulation of gastrointestinal motility, but the results are controversy. H2S suppresses colonic motility in mouse, human and rats but exerts a dual effect on the gastric motility in guinea-pig. As far as we know, there is no report about the effect of H2S on duodenal motility. In our preliminary experiments, we found that the two key H2S-producing enzymes, CBS and CSE, were localized in rat duodenal myenteric plexuses, but the role of H2S in regulating duodenal motility and the underlying mechanism are still unclear. The aim of this study was to investigate the effect of exogenous and endogenous H2S on duodenal motility in rats and the underlying mechanism.Methods: Recording of the contraction of muscle stripsAfter the rat was sacrificed by cervical dislocation, a segment of duodenum (1cm from pylorus) was removed immediately and put into a glass beaker containing4℃Krebs solution. The segment was cut along the mesenteric border and the content of the segment was washed away. The mucosal and submucosal layers were striped carefully using fine forceps under a microscope in a silica gel dish filled with oxygenated Krebs solution. The longitudinal muscle strips (4mm wide,10mm long) were prepared by cutting parallel to the longer axis of the duodenal segment. Both ends of the muscle strips were attached to silk thread. One end of the muscle strip was connected to a fixed hook and the other end was attached to an external isometric force transducer. A polygraph system was used to record the tension of the duodenal muscle strips.Measurement of the length of isolated smooth muscle cellsThe muscle strips above were placed into Ca2+-free physiological saline solution (PSS), cut into small segments (2×2mm), and then digested at37℃in1ml Ca2+-free solution which containing1.7-1.9mg collagenase II,500-700μg papain,1mg dithiothreitol,0.5mg trypsin inhibitor,1mg bovine serum albumin for20-30min. Next, the muscle segments were washed several times with the modified Kraft-Bruhe solution (KB solution) followed by gently triturated using a wide-bore fire-polished glass pipette (2mm bore diameter) to obtain a cell suspension. Dispersed cells were kept in KB solution at4℃for experiments. An inverted microscope was used to observe the smooth muscle cells and take photographs. Image Pro Plus6.0was used to measure the length of smooth muscle cells.Immunofluorescence in LMMPLongitudinal muscle myenteric plexus (LMMP) was prepared by striping circular muscle layer of the muscle strips. The preparations were washed three times with phosphate-buffered saline (PBS) and soaked in4%paraformaldehyde for30min. Subsequently, they were incubated with hydrogen peroxide (3%) for15min to quench endogenous peroxidase activity, tritonX-100(0.2%) for20min to improve permeability of cell membrane and donkey serum (10%) for1hr to block non-specific binding. All these procedures were conducted at room temperature. The tissues were incubated with the primary antibody mixture at4℃overnight. After several washes for10min each in PBS, the tissues were incubated with corresponding secondary antibodies for2hr at room temperature followed by three times washes with PBS. The specimens were examined under confocal microscopy.Data analysisThe motility index of muscle strips was calculated by integrating the recording traces of the muscle strip tension by a software program (Chart5). The mean motility index for3min before treatment with NaHS was taken as baseline and the values at different time points after each reagent (NaHS, L-cysteine or SAM) treatment were normalized to R by dividing the baseline. The R value before NaHS treatment was equal to1.The basal tension of muscle strips was measured by chart5. The average basal tension for3min before NaHS treatment was taken as baseline. The change of basal tension after NaHS treatment was calculated by subtracting the baseline, so the change of basal tension before NaHS application was equal to0g.The cell length of isolated smooth muscle cells before NaHS treatment was taken as baseline. The change of cell length after NaHS treatment was calculated by subtracting the baseline, so the change in each experiment before NaHS application was equal to0μm.Values were expressed as mean±SEM. One-way analysis of variance (ANOVA) followed by Dunnett’s test was used to compare several treatment groups with one control group, t test was used to determine the difference between two groups. P<0.05was considered to be significant difference. The n means the number of rats or the number of smooth muscle cells in a group.Results:1. Effects of NaHS on the spontaneous contraction of duodenal muscle stripsNaHS (0.5mM,1mM and2mM) caused a marked biphasic effect on the spontaneous contraction of the duodenal longitudinal muscle strips of rats. Shortly after NaHS (1mM) administration, the contraction of muscle strips was increased significantly, reached the highest level at1-2min and returned to normal within5min. Immediately after NaHS (1mM) administration, the R value of motility index was increased to4.8±0.5(n=10), much bigger than that of the control group (1.01±0.03, n=6, P<0.05). Following this excitatory effect, there was a long-lasting inhibition of the contraction of muscle strips. Unlike the early transient excitation, the inhibition of the spontaneous contraction was more than30min. Fifteen minutes after1mM NaHS application, the R value of motility index was decreased to0.2±0.05(n=10), much smaller than that of the control group (0.8±0.04, n=6,P<0.05). The contractile activities of the muscle strips partially recovered at45min.2. Effect of NaHS on carbachol-induced contraction of the duodenal longitudinal muscle stripsCarbachol (2μM) induced a long-lasting increase of the tension of the muscle strips. This effect persisted for more than20min. NaHS was administrated at10min after carbachol administration. Immediately after NaHS treatment, the basal tension of the muscle strips was decreased in a dose-dependent manner. Five minutes after NaHS (0.2mM,0.5mM and1mM) administration, the basal tension of the muscle strips was reduced by0.8±0.2g,0.9±0.2g and1.3±0.2g respectively, significantly different from that of the control group (0.03±0.01g, n=6, P<0.05). Low concentration of NaHS (0.05mM) did not exert significant effect.3. Involvement of TRPV1channels and substance P in NaHS-induced transient excitatory effect on the duodenal muscle stripsBoth Capsazepine (10μM), the TRPV1antagonist, and L703606(10μM), the NK1receptor antagonist, attenuated the excitatory effect of NaHS (1mM) on the contraction of duodenal muscle strips. One minute after NaHS administration, the R values of motility index in Capsazepine+NaHS and L703606+NaHS groups were1.7±0.2(n=10, P<0.05) and1.2±0.1(n=8, P<0.05), much lower than that of DMSO+NaHS group (4.8±0.5, n=11). However, the excitatory effect of NaHS on the contraction of the muscle strips was not altered by pretreatment with Glib (10μM), a KATP channel blocker or TTX (10μM), a blocker of voltage sensitive sodium channels.4. Involvement of KATP channels in NaHS-induced long-lasting inhibition on the contraction of muscle stripsAlthough Glib did not influence the early excitatory effect of NaHS, it mainly abolished the late long-lasting inhibition. At15min after NaHS administration, the R value of motility index was0.8±0.1(n=8, P<0.05) in Glib+NaHS group, much higher than that of the control group (0.3±0.05, n=11). Capsazepine, L703606and TTX did not influence the long-lasting inhibitory effect of NaHS on the contraction of muscle strips.5. Involvement of protein s-sulfhydration in NaHS-induced biphasic effects of duodenal muscle stripsPretreatment with NEM (0.1mM), a wildly used sulfhydryl alkylation compound, significantly attenuated the transient excitatory effect induced by NaHS on the contraction of muscle strips. One minute after NaHS (1mM) administration, the R value of the motility index was reduced from4.6±0.4(n=13) to1.6±0.3(n=9, P <0.05). The long-lasting inhibition of the contraction of muscle strips was partially but significantly reversed by pretreatment with NEM. Fifteen minutes after NaHS (1mM) administration, the R value of motility index of muscle contraction was reversed from0.2±0.05(NS+NaHS group, n=13) to0.5±0.1(NEM+NaHS group, n=9, P<0.05).6. Expression of KATP channels in the smooth muscle of rat duodenumSM22is a marker of smooth muscle and Kir6.2is a subunit of KATP channels. Double labeling of Kir6.2and SM22in isolated smooth muscle cells and LMMP showed that KATP channels were expressed in smooth muscle cells.7. The direct effect of NaHS on the isolated smooth muscle cells Administration of NaHS (0.5mM and1mM) induced a prompt relaxation of the cells and the effect lasted for more than5min. Low dose of NaHS (0.1mM) had no effect on the cell length. Five minutes after administration of NaHS (1mM), the change of cell length was increased to3±0.5μm (n=18, P<0.05) while the control group was0.1±0.2μm. The relaxation induced by NaHS (1mM) was completely abolished by pretreatment with Glib (10μM) or NEM (0.1mM). Five minutes after NaHS (1mM) administration, the changes of cell length were reduced from2.8±0.4μm (DMSO+NaHS) to-0.2±0.3μm (Glib+NaHS, n=18, P<0.001) and from2.9±0.5μm (PSS+NaHS) to0.1±0.4μm (NEM+NaHS, n=12,P<0.001) respectively.8. Expression of H2S-producing enzymes in rat duodenal enteric nervous systemBoth CBS and CSE were present in the duodenal myenteric plexus neurons while neither CBS nor CSE was expressed in smooth muscle cells. CSE-immunoreactive cells were also positive for CBS in rat myenteric plexus, indicating that both H2S-producing enzymes were expressed in the same cells.9. Effects of L-cysteine and SAM on the contraction of duodenal muscle stripsL-cysteine (0.5mM and1mM), a substrate of the H2S-producing enzymes, significantly increased the motility of the muscle strips in a dose-dependent manner. After L-cysteine (1mM) administration, the motility of muscle strips was increased significantly, reaching the highest level at2min, and returned to normal within15min. Two minutes after L-cysteine (1mM) administration, the R value of motility index was increased to1.9±0.1(n=9), much higher than that of the control group (0.9±0.05, n=6,P<0.05). Pretreatment with PAG (0.5mM, an inhibitor of CSE) and AOAA (0.05mM, an inhibitor of CBS) significantly attenuated the excitatory effect of L-cysteine (1mM) on the contraction of muscle strips. Two minutes after L-cysteine administration, the R value of motility index was decreased from1.9±0.1(NS+L-cysteine, n=8) to1.3±0.1(PAG+AOAA+L-cysteine, n=7,P=0.009). The excitatory effect was also blocked by Capsazepine (10μM) or L703606(10μM) pretreatment. The R value were decreased from1.9±0.1(DMSO+L-cysteine, n=7) to1.4±0.07(Capsazepine+L-cysteine, n=13, P<0.05) and1±0.1(L703606+L-cysteine, n=6, P<0.05). SAM (1mM), a CBS activator, increased the contraction of duodenal muscle strips, but lower concentrations of SAM (0.1mM,0.5mM) had no effect. The manner of the excitatory effect of SAM was similar to that of L-cysteine. The response to SAM (1mM) was significantly reduced in the presence of Capsazepine (10μM) or L703606(10μM). Unlike NaHS, neither L-cysteine nor SAM inhibited the contraction of muscle strips at15min after the chemicals administration.Conclusions:Our results suggest that both exogenous and endogenous H2S excite duodenal spontaneous contraction in vitro and this effect is mediated by activating TRPV1channels in afferent nerve terminals with the consequent release of SP which bonds with NK1receptors in duodenal smooth muscle. In addition to this excitatory effect, exogenous H2S also exerts a long-lasting inhibitory effect which is mediated by directly opening KATP channels in the smooth muscle cells. Both effects of exogenous H2S might act through protein s-sulfhydration. H2S might involve in the efferent like function of capsaicin-sensitive afferent nerve terminals. PartⅡH2S hyperpolarized myenteric plexus S neurons by activating KATP channelsObjective:The enteric nervous system (ENS) is composed of submucosal plexus and myenteric plexus. The neurons in ENS, including AH neurons and S neurons, play an important role in regulating the feeling, movement, secretion and blood flow of the gastrointestinal tract. But the effect of H2S on ENS neurons was not known yet. The objective of this study is to investigate the effect of H2S on the electrophysiological properties of the neurons in the ENS.MethodsIsolation of myenteric neuronsThe LMMP preparation was then incubated in Krebs saline containing papain (10mg/mL) at37℃for50min. The LMMP was then cut into pieces which were placed in Dulbecco’s modified eagle’s medium containing collagenase Ⅱ (1mg/mL) at37℃for55min, then triturated with a fire-polished glass pipette to obtain a cell suspension After centrifuged at1000r/mim for6min, the sediment was collected and re-suspended in1ml DMEM containing10%fetal bovine serum. Cells were then cultured at37℃in a5%CO2incubator.Whole-cell patch clamp recordingSignals were detected in voltage-clamp or current-clamp modes using a computer amplifier and a Digidata. Glass pipettes with a resistance of5-7MΩ were used to form GΩ seals. Signals were low-pass Bessel filtered at4kHz, and then digitized at10kHz. Patch pipettes were filled with an intracellular saline. All recordings were conducted at30℃. Identification of AH and S cellsAction potentials were evoked by injecting three50ms-duration depolarizing current pulses spaced at100ms intervals. The stimulus intensity of the first pulse was two times threshold and for each subsequent pulse the intensity was increased by50pA. The triple pulse protocol always evoked a sAHP of3-20s duration and2-15mV amplitude in neurons. These neurons were AH cells. Neurons that did not have sAHPs were S cells.Immunofluo rescenceThe protocol for localization of Kir6.2and NeuN in isolated myenteric plexus neurons and LMMP was same with the method in part Ⅰ.Statistical analysisFor patch clamp recording, the membrane potential and outward current before treatment with NaHS were taken as the baseline. The change of membrane potential or outward current after NaHS treatment was calculated by subtracting the baseline. The analysis method followed that of part ⅠResults1. Effect of NaHS on the resting membrane potential of S neuronsThe resting membrane potential of S cells was about45-55mV. Immediately after NaHS (0.1mM,0.5mM and1mM) administration, the membrane potential was hyperpolarized significantly in a dose-dependent manner, reaching maximal effect at1min and returning to normal after washing the cells with extracellular saline. One minute after administration of NaHS, the membrane potential was hyperpolarized by3.9±0.7mV (P<0.05, n=9, NaHS0.1mM),7.6±1.3mV (P<0.05, n=15, NaHS0.5mM) and13.8±1.8mV (P<0.05, n=13, NaHS1mM) respectively. Low concentration of NaHS (0.01mM) did not exert significant effect.2. Effects of NaHS on the outward currents of S neurons The cell was initially held at a-80mV command potential to close voltage sensitive outwardly rectifying conductance. Outward current was then activated using a100ms-voltage step from-80mV to+50mV. One minute after administration of NaHS (1mM), the outward currents increased significantly. The changes of outward currents was increased from45.7±37.6pA (control group, n=8) to619.2±47.9pA (NaHS group, n=17,P<0.001).3. Involvement of KATP channels in NaHS-induced increase of outward currentAfter pre-incubation with the KATP channels antagonist (Glib,10μM), the NaHS-induced increase of the outward current was significantly reduced. One minuite following NaHS (1mM) administration, the changes of outward current was reduced from673.8±51.8pA (DMSO+NaHS group, n=13) to73.7±46.1pA (Glib+NaHS group, n=8, P<0.001).4. Expression of KATP channels in the duodenal myenteric plexus neuronsKir6.2is a subunit of KATP channels and NeuN is neuron-specific nuclear protein. We use these two antibodies to mark KATP channels and neurons. Double labeling of Kir6.2and NeuN in isolated myenteric plexus cells and LMMP showed that KATP channels were expressed in the NeuN-possitive neurons of myenteric plexus.5. Involvement of protein s-sulfhydration in NaHS-induced increase of outward currentPretreatment with NEM (0.1mM), a wildly used sulfhydryl alkylation compound, significantly reduced the outward current induced by NaHS on the S neurons. One minute after NaHS (1mM) administration, the changes of outward current was reduced from619.2±47.9pA (vehicle+NaHS group, n=17) to137.7±63.3pA (NEM+NaHS group, n=7, P<0.001).Conclusion:Exogenous H2S hyperpolarized myenteric plexus S neurons by activating KATP channels via protein s-sulfhydration. |
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