| ObjectiveHydrogen sulfide (H2S) is a prominent gaseous constituent of the gastrointestinal (GI) tract with known cytotoxic properties. H2S is endogenously generated from L-cysteine (L-Cys) by the enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-Lyase (CSE). Endogenous concentrations of H2S are reported to range between 0.2-3.4 mmol/L in the GI tract of mice and humans. H2S stimulates intestinal epithelial K+ channels, but its effect on the nutrient absorption is not known. We have known that the absorption of nutrients is not only related to transport carrier,but also related to the electrochemical gradient on either side of the cell membrane.We sought to determine if H2S signaling involves the nutrient absorption in intestine.The aim of this study is to observe whether L-cysteine/H2S signaling pathways are expressed in jejunum epithelial, especially involved in nutrient absorption on the epithelial cells;Discuss physiological significance of L-cysteine/H2S signal system in jejunum epithelial.MethodsTissue preparation and short-circuit current measurement. Rats were sacrificed by dislocated of cervical spine.Jejunum were cut into somepieces about 1.5 cm.These pieces were cut along the longitudinal axis and washed with cold Krebs solution. The serosa and muscularis propria were stipped away to obtain a mucosa-submucosa preparation from jejunum.The mucosa-submucosa preparation was fixed in Ussing chamber bathed with a volume of 5 ml on each side of the preparation at 37℃ and gassed with 95% O2 and 5% CO2(exposed area of 0.5cm2).The tissue was short-circuited by a computer-controlled voltage-clamp device with correction for solution resistance (VCC MC4; Physiologic Instruments, San Diego, Calif).The tissue was continuously voltage-clamped to zero potential by the application of a current pulse. The baseline value of the electrical parameters was determined as the mean over the 3 min immediately prior to drug administration. The tissues were allowed to equilibrate to these conditions for approximately 30 min to stabilize the Isc in the presence of tetrodotoxin (TTX,10umol/L)prior to the addition of drugs.Hydrogen sulfide synthesis.The principal pathways for H2S synthesis in mammals involve conversion of L-cysteine to pyruvate, ammonium, and H2S via either of 2-pyroxidal-5’-phosphatedependent enzymes:cystathionine-y-lyase (CSE) and cystationine-β-synthase (CBS) and methylene blue formation following zinc acetate capture of H2S.Rat jejunum(mucosa and submucosa,0.1g)was homogenized in ice-cold 50mmol/L potassium phosphate buffer (PH=6.8) with a Polytron homogenizer.Homogenate(0.5ml)was preincubated at 37℃ with or without inbibitor for 5 minutes in the outside slot of a 20-ml reaction vial.A piece of filter paper(0.5×1.5cm)soaked with zinc acetate (1%;0.5ml)was put into the internal slot of the vial. The vial was flused with a slow stream of nitrogen gas for 20 seconds before L-cysteine(10mmol/L final concentration)and pyridoxal 5’-phosphate (2 mmol/L final concentration)were added. Then the vial was capped with an airtight serum cap and bathed at 37℃。After 90 minutes, trichloroacetic acid (TCA; 50%; 0.5 mL)was added into the outside slot to stop the reaction.In the next 60 minutes,evolved H2S was captured by the Zn acetate solution as Zn sulfide.Then the filter paper and Zn acetate solution was removed into a tube where N, N-dimethyl-p-phenylenediamine sulfate (DMPD,20 mmol/L; 0.5mL) in 7.2 mol/L HCl and FeCl3 (30 mmol/L; 0.4mL) in 1.2 mol/L HCl were added into.Twenty minutes later,absorbance at 670nm was measured with a micropate reader(Spectra Max 190,Molecular Devices,USA).A standard curve was generated with known concentrations of NaHS.Oral Glucose Tolerance Test.Thirty male C57 mice of 6-8weeks were randomly devided into 5 groups,6 mi ce per group.After a 9-hours fast (11:00 pm to 8:00 am), blood glucose levels were determined in 2 ul of blood from the tail with a handheld glucose mete r (ACCU-CHEK performa,Roche Diagnostics GmbH,Sandhofer Str.116.D-68305 Mannheim,Germany).The C57 mice were given an oral glucose bolus of lg/kg in 0.2 ml of water.Soon afterwards,mice of each group were intraperitoneal inje cted with physiological saline(0.2ml);sodium hydrogen sulfide(NaHS,5mg/kg);L-c ysteine(100mg/kg); aminoxyacetic acid (AOA,50mg/kg) and D, L-propargylglyc ine (PAG,100mg/kg);L-cysteine (100mg/kg),AOA (50mg/kg) and PAG(100mg/kg). After 30 minutes,about 40ul of blood from hepatic portal vein and 2ul of bloo d from tail were collected under pentobarbital sodium.Blood glucose levels of t hese samples were determined.Western Blot.Jejunum,ileum,liver(about O.lg, the serosa and muscularis propria were stipped away in jejunum and ileum) of Wistar rats and C57 mouse were homogenized in lml iced-cold lysis buffer(Beyotime).Then the homogenate was centrifuged(12,000 rpm) at 4℃ for 20 minutes and super-natants collected.The lysate was separated by 10% SDS-PAGE gels.After electrophoresis, the separated proteins were transferred onto the PVDF membrane and blocked with TBST (5% nonfat dried milk in TBS containing 0.2%Tween-20) for 2h at room temperature. Subsequently, the membranes were incubated with the appropriate primary antibodies overnight at 4℃ (CBS:sc-67154,rabbit polyclonal IgG, Santa Cruz Biotechnology;final dilution 1:500;CSE:abl31052,rabbit polyclonal antibody to cystathionase,Abcam;final dilution 1:500).Following three washes with TBST,the membrane was incubated with secondary antibody(ZB2301,goat anti rabbit IgG horseradish peroxidase-conjugated antibodies,ZSGB biology;final dilution 1:20000)for 1 hour followed by three washes.The target protein were detected with chemiluminescence method.(ECL amersham hyperfilm,Beyotime,China)Immunohistochemical experiments.The tissue was fixed with paraformaldehyde and embedded in paraffin. Sections(thick of 6 um) were cut and mounted on glass slides.Then the sections were baked at 65℃ for 1 hour and immersed in dimethyl benzene,100% alcohol,90%alcohol,80%alcohol and 70%alcohol in turn in order to dewaxing and rehydrating.After washing three times in PBS, the tissue antigen were exposed in antigen repaired liquid(0.482g sodium citrate and 0.075g citric acid in 200ml water) with microwave oven(high fire for 3 minutes and low fire for 30 minutes). Then the sections were washed for three times after cooling naturally. Hydrogen peroxide (3%)was added to the section and incubated in the dark for 10 minutes.After washing,sections were incubated with the respective primary antibodies(final dilution 1:50) overnight at 4℃.As negative control,sections were incubated with a solution that did not contain the primary antibodies.Washed for three times, Section were incubated with solution of type A(100ul; GTVision III anti rat/rabbit universal immuohistochemical kit)for 30 minutes at room temperature.Operating fluid of DAB was added to the tissue.Chromogenic time was controlled in the case of visible under inverted microscope.Tissue was double stained with wood grain for 5 minutes.Statistics.The data are presented as means±tandard error of the mean(SEM),n is the number of tissues examined.For the comparition of two groups,both one way analysis of variance,paired and unpaired two-tailed Student’s t-tests were applied as appropriate.P<0.05 was considered to be statistically significant.Results1,Western Blot results showed that rat or mouse jejunum epithelium tissue possesses both CBS and CSE.2, Immunohistochemical results showed that CBS was localized in absorptive cells near the tips of the villi and crypt cells of the jejunum, whereas CSE was primarily expressed in crypt cells of the jejunum.3, Hydrogen sulfide synthesis results showed that tissue homogenates efficiently convert L-Cys to H2S.4,Through Ussing Chamber,we found that exogenous H2S,sodium hydrogen sulfide(NaHS) or L-Cys causes a concentration-dependent decrease of short-circuit current (Isc) across epithelium of rat jejunum, which is blocked by glibenclamide, a K+ channel blocker. The serosal administration of L-Cys enhances either the L-alanine or glucose-evoked Isc. L-Cys enhancement was inhibited by the CBS inhibitor, aminoxyacetic acid (AOA), but not CSE inhibitor, D,L-propargylglycine(PAG) Exogenous H2S donor GYY4137, but not NaHS also markedly augmented the L-alanine-evoked response.5,Furthermore,we found an aberration in H2S signaling, either silenced or enhanced by pharmacologic or genetic methods, causes an abnormal 30-min postprandial blood glucose or weight change.Conclusion1,CBS,CSE are functionally expressed in mouse,rat jejunal epithelial cells. 2, The L-Cys/H2S pathway enhance jejunal nutrient transport. |
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