Downregulation Of Excitatory Effect Of GABA On Contraction Of Circular Muscle Strips Of Colon In Rats Following Shigellosis

INTRODUCTIONGamma-aminobutyric acid(GABA)has been shown to be a neurotransmitter within the enteric nervous system(ENS).Immunohistochemical and in situ hybridization studies have revealed the presence of the three kinds of GABA receptors in the myenteric and submucosal neurons within ENS.As an important neurotransmitter in ENS,GABA exerts complex and variable effect on gastrointestinal motility depending on the specific GABA receptors involved,the part of the gut and the animal species studied.Dysentery is a common clinical symptom.It causes long-term disorder of the gastrointestinal tract, structural and functional changes of ENS,but the mechanism has not been clarified. Because of the important function of GABA in ENS on gastrointestinal motility,we hypothesized that GABA might be involved in the dysmotility of gut and plasticity of ENS following gastrointestinal inflammation.In order to testify this hypothesis,we made an animal model of acute shigellosis by administrating the rats orally with Shigella flexneri and monitored the motor response of the circular muscle strips of colon to GABA following the gut inflammation.The expression of GABA receptors was quantified by the method of Western blot.MATERIALS AND METHODSAnimal and Muscle Strips PreparationMale Wistar rats,weighing 280-320 g,was fed Shigella flexneri(1×10⁸ CFU/ml)in order to induce the dysentery.The rats were sacrificed at 10,14,21,32 and 45 after the bacteria administration.Immediately after a midline laparotomy,a segment of colon(5 cm from anus)was removed and the intraluminal content of the colon was rinsed out,and then the colon was opened along the mesenteric border and the resulting rectangular sheet was pinned flat(mucosa up).Muscle strips(10×4 mm)were cut parallel to the circular fibres.The mucosa layer on each strip was carefully scraped away.The muscle strip was suspended in tissue chamber containing 5 mL Krebs solution(37℃)and bubbled continuously with 95%O₂ and 5%CO₂.Isometric contraction of rat circular smooth muscle was monitored. HE stainingSegments of colon(5 cm from anus)was prepared from a control healthy rat,and the infected rats at 10 days,14 days,21 days,32 days and 45 days after the oral administration of Shigella flexneri.The colon of each animal was sectioned with paraffin slices set for a thickness of 4μm.Sections were collected on gelatin coated glass slides and stained for hematoxylin/eosin.Western blotExpression of GABA_AR_aland GABA_BR₁ protein was evaluated by Western blot analysis. Muscle strip of the colon was prepared as described above,and the mucosa layer on each strip was carefully scraped away.Then the tissues were homogenized,protein samples (50μg)were separated using SDS polacrylamide,gelelectrophoresis,transferred to nitrocellulose membranes and probed with a multiclonal antirat GABA_AR_alIgG(1:500, sc 7348;Santa Cruz Biotechnology)or GABA_BR₁ IgG(1:500,sc 7339;Santa Cruz Biotechnology).After autoradiography,immunoreactive bands were quantified.Data analysisThe value of muscle tension was presented as mean±SEM.Differences between groups were evaluated by One Way ANOVA on ranks followed by bunnett's test,P＜0.05 was considered to be significantly different. RESULTS(1)GABA(100μM-10 mM)excited the contraction of circular muscle strips of colon. Spontaneous contraction of the strips of the circular muscle was enhanced immediately after GABA administration and reached the greatest level at the first minute.Lower dose of GABA(10μM)did not influence the contraction of the muscle strips.(2)Muscimol(Mus)(100μM),an agonist of GABA_A receptor,increased the tension of muscle strips,but Bac(100μM),an agonist of GABA_B receptor,did not influence it.(3)Pretreatment of bicuculline(Bic)(10μM),an antagonist of GABA_A receptors, completely blocked the excitatory effect GABA on the contraction of the muscle strips. Pretreatment of TTX(10μM),the specific blocker of voltage-gated Na⁺ channels on the nerve fiber,completely abolished the excitatory effect of GABA(10μM-10 mM) on the circular muscle strips of colon.(4)The activity of myeloperoxidase(MPO)increased significantly 10-14 days following Shigella flexneri administration and returned to normal 21 days later.The excitatory effect of GABA on the colon motility was downregulated after gastrointestinal infection. At 10,14 and 21 days after the Shigella flexneri administration,the increase of mean tension of the muscle strips following GABA(1 mM)was 0.062±0.010 g,0.018±0.007g and 0.021±0.009 g,significantly lower than that of the control group(n=11,P＜0.05).At 32 and 45 days,GABA did not influence the colon contraction. (5)Ten days after the gastrointestinal infection,the expression of GABA_AR_alprotein significantly decreased(n=6,p＜0.05).It returned to normal 14 days later(n=6,P＞0.05).GABA_BR₁ protein was not detectable by the Western blot(data not shown).(6)The contractibility of the colonic muscle strips 32-45 days after gastrointestinal infection was comparable to that of control.CONCLUSION AND DISCUSSIONGABA_A receptors in ENS excite the circular muscle strips of colon in rats.This effect was depressed following acute shigellosis.This depression persisted after the disappearance of the colon inflammation.The long-term downregulation of the excitatory effect of GABA on colon motility following gut inflammation was not attributed to the change of amount of GABA_A receptors expressed on colon and the change of contractility of colonic muscle..