A Novel Functional GABAergic Signal System In Regulating Intestinal Fluid Secretion And The Underlying Mechanisms

Part1GABAergic signal system is expressed in ileum epithelium and is involved in the regulation of fluid secretionObjectivey-aminobutyric acid (GABA for short) is one of the most important inhibitory neurotransmitter in mammalian CNS which mediates the fast inhibition of neurons. Physiologically, GABA is synthesized by glutamate decarboxylase (GAD) from glutamate in neurons which are called GABAergic neurons. And there are two isoforms of GAD:GAD65and67identified by the different molecular weight. GAD65is found to sit in the GABA-contained vesicles near the axon terminals, and the GABA is released to the synaptic cleft acting on the GABAA receptors in postsynaptic membrane mediating fast inhibition in adult brain. While GAD67is distributed randomly in the cytosol, and GABA synthesized by GAD67is thought to act on GABA receptors located in extrasynaptic membrane. As for the release mechanism of cytosolic GABA remains obscure. GABAA receptors are chloride ion channels. GABA or its analogous activates GABAA receptors whose opening leads to chloride influx with membrane hyperpolarization as a consequent. In immature neurons or some types of neurons, GABAA receptor hyperpolarized cell membrane to excite cells. GABA, GABA receptors and GAD are termed as GABAergic signal system. Besides CNS, GABAergic signal system is found in many peripheral tissues and organs like lungs, reproductive system, kidney as well as gastrointestinal tract. GABA and GABAA receptors are reported to play important roles in regulating pulmonary epithelial fluid and mucus secretion. The system is found to be upregulated in gastric and colon carcinoma, but whether it is there in normal gastrointestinal tract is still unknown despite its physiological functions. We hypothesized that GABAergic signal system is expressed in human and animal intestine, which might contributes to the fluid and mucus secretion when activated.MethodsRT-PCRRNAs were extracted from IEC-18cell line and cortex of healthy male Wistar rat by Trizol. RT-PCR KIT was used to reproduce cDNA(complementary DNA). And GABAA receptor subunits and GAD DNA were amplified using their primers. The DNA bands were got by running DNA agar gel.ImmunohistochemistryParaffin embedded slice staining:fresh tissues were soaked in4%PFA (Paraformal dehyde) for24hours before paraffin embedding. Slice the tissue with4-5μm thickness. Followed by deparaffination, hydration and antigen repair. Then block the slice with5%serum for1hour at room temperature. Incubate the slices at4℃overnight with primary antibody after the serum blocking. Rinse slices3times with PBS the next day,5minutes per rinse. Incubate with fluorescence labeled secondary antibodies for1hour at room temperature. Rinse slices3times and5minutes per time. Seal the slice with75%glycerol buffer and take pictures with fluorescence microscope.IEC-18cell staining:cells were planted on coverslips coated with10%Poly-L-Lysine. Fix cells with4%PFA for10minutes at room temperature, followed by PBS rinses for3times,5minutes per time. The rest procedures are the same as the paraffin embedded slice staining.Western Blot AnalysisFresh tissues and cells were homogenized, centrifuged at12000g for10min at4℃.Total proteins were fractionated on a5%to10%gradient SDS-PAGE (sodium d odecylsulfate polyacrylamide gel electrophoresis). They were transferred to0.45μm P olyvinylidene Fluoride (PVDF) membranes. Membranes were blocked in blocking bu ffer (5%non-fat dry milk, TTBS) for60min at room temperature, incubated with pri mary antibody at4℃overnight. After washing10minutes for three times, the PVDF membranes were incubated for1h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies followed by3times TTBS washing. Finally, immunoreactive proteins were detected by ECL (Electrochemiluminescence) plus.ElectrophysiologyIEC-18cells were incubated on PDL (Poly-D-Lysine) coated coverslips for24hours. Record cells using Axon700B amplifier. The cells were bathed in extracellular solution containing (in mmol/1):155NaCl,1.3CaC12,5.4KC1,25HEPES, and33glucose (pH7.4, osmolarity315mosmol/kgH2O) while the recording pipette was filled with intracellular solution containing (in mmol/1)155KC1,15KOH,10HEPES,2MgC12,1CaC12,10EGTA and2Tetraethylammonium (pH7.35, osmolarity:315mosmol/kgH2O).In vivo intestinal fluid secretion experimentHealthy male BALB/c mice (weight between25-30g) were used in this experiment. Animals were fast overnight while free drinking. Animals were anesthetized with2%pentobarbital sodium (45-50mg/kg intraperitoneal injection). Body temperature of mice was maintained between36and38℃during surgery using a heating pad. An abdominal incision (～1.5cm) was made to expose the small intestine, and a closed loop of empty ileum (20mm of length) proximal to the cecum was isolated by sutures. Ileum loops were injected with100μl normal saline (NS) alone or NS containing drugs. The abdominal incision was closed with sutures, and the animal was allowed to recover from anesthesia. Five hours later, sacrifice the mice, and ileum loops were isolated. The ileum loops were weighted after removal of mesentery and connective tissues. After the lumen fluid was released through a longitudinal incision, the weight and length of ileum loops were measured. The ratio of intestinal fluid weight to intestinal length was calculated.Statistical AnalysisIn the intestinal fluid secretion experiment, the relative values of intestinal fluid weight to intestinal length were used to analyze the difference between groups.All the values in these experiments were presented as mean±SEM. One way analysis of variance (ANOVA) and the student’s t-test was used to analyze. P<0.05was considered to be a significant difference.Results1, RT-PCR results showed GAD65/67and GABAA receptor subunits mRNA expression in intestinal epithelial cells. And GAD65/67,β2/β3and%subunits of GABAA receptor were found to be expressed in protein level using Western Blot technique.2, Immunohistochemistry staining located GAD65/67to be in cytosol of epithelial cells, while β2/β3and π subunits of GABAA receptor sit in the apical side of intestinal villi.3, GAD65/67and π subunits of GABAA receptor are also found in epithelial cells of human ileum.4, Whole cell patch clamp of IEC-18cells showed an inward current triggered by1mM GAB A when holding cell membrane potential at-60mV. Muscimol, the selective GABAA receptor agonist, works the same as GAB A on IEC-18cells.5, In the intestinal fluid secretion experiment, GAB A (100μm) and Muscimol (10μm) showed similar increasing fluid accumulation in intestine, while the increased fluid secretion can only be partly blocked by TTX1μm, Tetrodotoxin). Gabazine, the selective GABAA receptor blocker, decreased intestinal fluid accumulation caused by GABA.Conclusion1, GABAergic signal system is functionally expressed in mouse, rat and human intestinal epithelial cells.2, Exogenous GABAA receptor agonist and antagonist activate/deactivate GABAA receptor to regulate intestinal fluid secretion. Part Ⅱ The role of GABAergic signal system in diarrhea and the underlying mechanismsObjectiveDiarrhea happens commonly in patients with digestive diseases, and there are kinds of mechanisms involved in diarrhea. Viruses and bacteria toxins invade intestinal mucous leading to increased permeability of the intestinal epithelium, and disturb secretion and absorption of the epithelia, thus leading to diarrhea. Chloride secretion from the apical membrane to lumen is the driving force for other ions and water efflux. The malfunction of chloride channel activity is always the direct reason for diarrhea. For example, Cholera and Travelers’diarrhea need the involvement of CFTR and CaCC channels. Both of CFTR and CaCC are chloride channels whose activating leads to water, electrolytes and mucus accumulation in intestinal lumen. It’s important to discuss the roles that chloride channels. play in physiological and pathophysiological progress of intestine. Targeting chloride channels in intestinal epithelial membrane might be an alternative way to treat diarrhea.GABAA receptors are found to be functionally expressed in intestinal epithelial cells according to our previous results. Since GABAA receptors are chloride channels too, it’s necessary to explore the functional significance of GABAergic signal system. We aim to research the roles that GABAergic signal system plays in food allergen induced diarrhea and the underlying mechnisms.MethodsImmunohistochemistry procedure is the same as part1.Food allergen induced diarrhea animal model experiment protocolHealthy male adult BALB/c mice (body weight between20-25g) were divided into four groups randomly:control group, OVA (50mg, Ovalbumin) induced diarrhea group, OVA mixed with Gabazine (selective GABAA receptor antagonist), OVA mixed with Picrotoxin (nonselective GABAA receptor blocker). All the animal were injected with OVA (1mg) diluted in aluminum potassium sulfate adjuvant to sensitize animals. At the7th day after sensitization, mice were fasted for4h. Mice in different groups were given NS (control), OVA (50mg), OVA mixed with gabazine (1.2mg/kg), OVA mixed with picrotoxin (1.2mg/kg) via intragastric administration. The properties of mouse stool were observed1h after intragastric administration of NS and drugs. The occurrence of diarrhea in mice was determined by comparing the property of stools before and after OVA or drug administration. Mice were sacrificed by cervical dislocation after10times of intragastric drug administration every other day. The ileum (0.5cm from cecum,2-3cm length) was excised from each mouse and was prepared for immunohistochemistry assay or Western blot.Western Blot AnalysisTissue protein samples obtaining and testing procedure is the same as Part1.Cell protein analysis:IEC-18cells were rinsed with cold PBS3times after being treated with IL-13. Cell lysis was used to break cells followed by centrifuging at4℃. Protein is in the supernatant. Proteins were denatured at100℃for5minutes. Then they were allowed to go through the SDS-PAGE. Proteins were transferred to PVDF membrane. After blocking PVDF membrane with5%non-fat dry milk in TTBS for60min at room temperature, incubate membrane with primary antibody at4℃overnight. After washing10minutes for three times, the PVDF membranes were incubated for1h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies followed by3times TTBS washing. Finally, immunoreactive proteins were detected by ECL (Electrochemiluminescence) plus.Statistical AnalysisThe properties of mouse stool were observed1h after intragastric administration of NS and drugs. Animals with loose or watery stool were considered to be diarrhea. The diarrhea incidence rate of each intragastric administration (diarrhea animal number to the total number of each group) was taken as the statistical index.Results1, All the animals showed diarrhea symptom after the7th intragastric administration with OVA, while the NS group animals were of no diarrhea. Diarrhea incidence was significantly reduced when treating animals with OVA and GABAA receptor antagonists (Gabazine and Picrotoxin).2, OVA treatment increased expression of GAD67, π and β2/β3subunits of GABAA receptor in mouse ileum. The increased expression of β2/β3subunits was reversed by GABAA receptor antagonists, Gabazine and Picrotoxin, while GAD65/67and π subunit were still in higher expression. 3, Phosphorylated-AKT473and phosphorylated-AKT308reached the highest level at4and4.5minute after IL-13treatment on IEC-18cells. And GABAA receptor β2/β3subunits expression began to increase at4minute.Conclusion1, GABAergic signal system expression was significantly increased during the progress of allergic diarrhea, while GABAA receptors antagonists alleviated diarrhea symptoms and slowed down diarrhea incidence.2, PI3K-AKT-GABAA receptor signal pathway might account for the upregulation of GABAergic signal system.