Study Of The Effects Of Brain-derived Neurotrophic Factor And Hydrogen Sulfide On Colonic Motility

Part I The effects of Brain-derived neurotrophic factors and Substance P on colonic motilityObjective To investigate the possible effects of Brain-derived neurotrophic factors (BDNF) and Substance P (SP) on colonic motility.Methods Male Wistar rats were used to prepare colonic muscle strips, including longitudinal muscle (LM) and circular muscle (CM). Organ bath system was applied to record the spontaneous contractile activities of LM and CM before and after adding drugs. Whole-cell patch clamp techniques were used to record the currents of L-type calcium channel and large conductance Ca2+-activated potassium channel (BKCa).Results BDNF had no effect on contractile activities of both LM and CM. And BDNF had no effect on currents of both L-type calcium channel and BKCa neither. TrkB-antibody (1:1000) significantly inhibited the contraction of both LM and CM. Furthermore, Trk antagonist K252a also had inhibitory effect on the spontaneous contractile activities of both LM and CM. SP significantly increased the contraction of colonic smooth muscle through L-type calcium channel and BKCa. The interstitial cells of Cajal in the deep muscular plexus (ICC-DMP) network exhibits stimulus-induced synchronized rhythmicity, SP could induce quiet ICC-DMP into active ICC-DMP with spontaneous calcium oscillation. BDNF could enhance SP-induced contraction of CM but not LM.Conclusions BDNF had no direct effect on contractile activity of colonic smooth muscle, but can enhance SP-induced circular muscle contraction. SP can increase smooth muscle contraction through L-type calcium channel and BKCa.Part II The possible relationship between brain-derived neurotrophic factor and chronic stress-induced colonic hypermotilityObjective To investigate the possible involvement of brain-derived neurotrophic factors (BDNF) in stress-induced colonic hypermotility.Methods Male Wistar rats were exposed to daily 1-hour water avoidance stress (WAS) or sham WAS (SWAS) for 10 consecutive days. The levels of BDNF and substance P (SP) in serum and the presence of BDNF and SP in the colonic mucosa were determined using Enzyme Immunoassay Kits. Immunohistochemistry and Western blotting were performed to assess the expression of BDNF and its receptor, trkB. The contractions of muscle strips were studied in an organ bath system.Results Repeated WAS increased the fecal pellet expulsion and spontaneous contractile activities of the colonic muscle strips. Both BDNF and SP in the serum and colonic mucosa were elevated following WAS. Immunohistochemistry revealed the presence of BDNF and TrkB in the mucosa and myenteric plexus. BDNF and TrkB were both up-regulated in colon devoid of mucosa and submucosa from the stressed rats compared with the control. BDNF pretreatment caused an enhancement of the SP-induced contraction of the circular muscle (CM) strips. TrkB-Antibody significantly inhibited the contraction of the colonic muscle strips and attenuated the excitatory effects of SP on contractions of the CM strips. Repeated WAS increased the contractile activities of the CM strips induced by SP after BDNF pretreatment, and this effect was reversed by TrkB-Antibody.Conclusions The colonic hypermotility induced by repeated WAS may be associated with the increased expression of endogenous BDNF and TrkB. BDNF may have potential clinical therapeutic use in modulating gut motility.PartⅢ Effects of Hydrogen sulfide on colonic contraction of rats and ion channel mechanismsObjective To test the hypothesis that hydrogen sulfide (H2S) regulates the colonic motility by modulating both L-type voltage-dependent calcium channels and large conductance Ca2+-activated K+(BKCa) channels.Methods Immunohistochemistry was used on rat colonic samples to observe the localization of the H2S-producing enzymes cystathionine-β-synthase (CBS) and cystathionine-y-lyase (CSE). Organ bath system was applied to study the contractions of proximal colonic smooth muscle. The whole-cell patch-clamp technique was used to record L-type calcium currents (Ica,L) and BKCa currents in colonic smooth muscle cells (SMCs) isolated from rat colon.Results Immunohistochemistry revealed the presence of CBS and CSE in mucosa, smooth muscle cells and myenteric neurons. The H2S donor NaHS inhibited spontaneous contractions of the longitudinal muscle and circular muscle strips in a dose-dependent manner, and the inhibitory effects were not blocked by tetrodotoxin. NaHS inhibited the peak ICa,L in colonic SMCs at a membrane potential of 0 mV. The current-voltage (I-V) relationship of L-type calcium channels was modified by NaHS, and the peak of the I-V curve was shifted to the right. NaHS (200μM) evoked a significant rightward shift of the steady-state activation curve and inhibited the inactivation of L-type calcium channels. Furthermore, NaHS reversibly decreased the peak ICa,L in a dose-dependent manner. Likewise, BKCa channels were significantly inhibited by NaHS, and the addition of NaHS caused a time- and dose-dependent reduction in the BKCa current.Conclusions both CBS and CSE participated in the endogenous production of H2S synthesis. And the relaxant effect of H2S on colonic muscle strips may be associated with the direct inhibition of H2S on L-type calcium channels. The inhibitory effect of H2S on BKCa indicated an excitatory effect on contraction of SMCs.