Abstract Background Brain‐derived neurotrophic factor ( BDNF ) is a neurotrophin present in the intestine where it participates in survival and growth of enteric neurons, augmentation of enteric circuits, and stimulation of intestinal peristalsis and propulsion. Previous studies largely focused on the role of neural and mucosal BDNF . The expression and release of BDNF from intestinal smooth muscle and the interaction with enteric neuropeptides has not been studied in gut. Methods The expression and secretion of BDNF from smooth muscle cultured from the rabbit intestinal longitudinal muscle layer in response to substance P ( SP ) and pituitary adenylate cyclase‐activating peptide ( PACAP ) was measured by western blot and enzyme‐linked immunosorbent assay. BDNF mRNA was measured by reverse‐transcription polymerase chain reaction. Key Results The expression of BNDF protein and mRNA was greater in smooth muscle cells ( SMC s) from the longitudinal muscle than from circular muscle layer. PACAP and SP increased the expression of BDNF protein and mRNA in cultured longitudinal SMC s. PACAP and SP also stimulated the secretion of BDNF from cultured longitudinal SMC s. Chelation of intracellular calcium with BAPTA (1,2‐bis‐(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid) prevented SP ‐induced increase in BDNF mRNA and protein expression and SP ‐induced secretion of BDNF. Conclusions & Inferences Neuropeptides known to be present in enteric neurons innervating the longitudinal layer increase the expression of BDNF mRNA and protein in SMC s and stimulate the release of BDNF . Considering the ability of BDNF to enhance smooth muscle contraction, this autocrine loop may partially explain the characteristic hypercontractility of longitudinal muscle in inflammatory bowel disease.
Brain-derived neurotrophic factor (BDNF) has been postulated to participate in inflammation-induced visceral hypersensitivity by modulating the sensitivity of visceral afferents through the activation of intracellular signalling pathways such as the extracellular signal-regulated kinase (ERK) pathway. In the current study, we assessed the expression levels of BDNF and phospho-ERK in lumbosacral dorsal root ganglia (DRG) and spinal cord before and during tri-nitrobenzene sulfonic acid (TNBS)-induced colitis in rats with real-time PCR, ELISA, western blot and immunohistochemical techniques. BDNF mRNA and protein levels were increased in L1 and S1 but not L6 DRG when compared with control (L1: two- to five-fold increases, P < 0.05; S1: two- to three-fold increases, P < 0.05); however, BDNF protein but not mRNA level was increased in L1 and S1 spinal cord when compared with control. In parallel, TNBS colitis significantly induced phospho-ERK1/2 expression in L1 (four- to five-fold, P < 0.05) and S1 (two- to three-fold, P < 0.05) but not in L6 spinal cord levels. Immunohistochemistry results showed that the increase in phospho-ERK1/2 expression occurred at the region of the superficial dorsal horn and grey commisure of the spinal cord. In contrast, there was no change in phospho-ERK5 in any level of the spinal cord examined during colitis. The regional and time-specific changes in the levels of BDNF mRNA, protein and phospho-ERK with colitis may be a result of increased transcription of BDNF in DRG and anterograde transport of BDNF from DRG to spinal cord where it activates intracellular signalling molecules such as ERK1/2.
Hydrogen sulfide (H 2 S) plays an important role in the regulation of gastrointestinal motility and secretion. H 2 S is synthesized mainly from L‐cysteine via cystathionine‐γ‐lyase (CSE) and cystathionine‐β‐synthase (CBS). We showed that smooth muscle cells of stomach and colon of rabbit, mouse and human express CSE, but not CBS. Activation of CSE causes inhibition of Rho kinase activity and muscle contraction and the inhibition was blocked by CSE siRNA and by a selective CSE inhibitor, propargylglycine (PPG). Aim To identify the mechanism of activation of CSE in gastrointestinal smooth muscle cells. Methods Activation of CSE was analyzed by the measurement of H 2 S in response to L‐cysteine and NO donor sodium nitroprusside (SNP) in gastric and colonic muscle of rabbit and human. Filter paper disks dipped in zinc acetate were exposed to H 2 S trapped in N 2 ‐perfused glass vials and the amount of reduced zinc acetate in the presence of H 2 S was measured spectrophotometrically. Results The basal levels of H 2 S in gastric and colon muscle from rabbit and human were ~20‐25 nM/mg. L‐Cysteine (10 mM) increased H 2 S generation up to 50 nM/mg; the effect of L‐cysteine was inhibited by PPG. L‐cysteine‐induced H 2 S generation was augmented in the presence of SNP and cGMP analog, 8‐bromo cGMP to 125 µM/g and 120 nM/mg, respectively. Augmentation of CSE activity in response to SNP and 8‐br‐cGMP was blocked by the cGMP‐dependent protein kinase (PKG) inhibitor, Rp‐cGMPS, suggesting phosphorylation‐dependent activation. Conclusion We have identified a mechanism of stimulation of CSE activity in gastric and colonic muscle involving cGMP‐dependent activation of PKG.
H 2 S, synthesized endogenously from L‐cysteine via cystathionine‐γ‐lyase (CSE) and cystathionine‐β‐synthase (CBS), play a role in the regulation of gastrointestinal (GI) motility and secretion. Neither the expression of enzymes nor the mechanism of action of H 2 S in GI smooth muscle is fully known. Methods: Expression of CSE and CBS was analyzed by RT‐PCR and western blot. The effect of H 2 S on carbachol (CCh)‐induced Rho kinase activity and sustained contraction, and nitric oxide‐induced PDE5 activity, cGMP formation and muscle relaxation was examined using L‐cysteine (activator of CSE) or NaHS (H 2 S donor). Results: CSE, but not CBS was expressed in colonic smooth muscle cells of mouse, rabbit and human. CCh‐induced contraction in muscle strips and cells, and increased Rho kinase activity were inhibited by L‐cysteine and NaHS in a concentration‐dependent manner. Inhibition of Rho kinase activity and muscle contraction by L‐cysteine (but not NaHS) was blocked by CSE siRNA and CSE inhibitor, propargylglycine, respectively. L‐cysteine and NaHS also inhibited PDE5 activity, and augmented cGMP formation and muscle relaxation leading to increased (35%) colonic pellet propulsion. Conclusion: Activation of H 2 S‐producing CSE causes muscle relaxation via a dual mechanism involving inhibition of Rho kinase and PDE5 activity leading to downregulation of RhoA/Rho kinase pathway and upregulation of cGMP/PKG pathway. Grant Funding Source : DK‐28300
Deficiency of dystrophin, a cytoskeletal protein localized in the inner face of the plasma membrane in skeletal, cardiac and smooth muscle, results in Duchenne Muscular Dystrophy (DMD). Through its interactions with extracellular matrix and plasma membrane proteins, dystrophin plays a role in contraction and signal transduction. In DMD, gastrointestinal disorders such as gastric dilation and intestinal pseudo‐obstruction resulting from altered motility have been reported. The role of dystrophin in the regulation of contractile protein expression and smooth muscle function, per se, is not known. Studies have suggested that inflammation contributes to the pathophysiology of DMD. Exogenous H 2 S had been shown to exert beneficial cardiovascular and gastrointestinal functions, probably via exerting anti‐inflammatory actions. Aim To test the hypothesis that a lack of dystrophin causes a decrease in contractile protein expression and smooth muscle function and that treatment with H 2 S restores the effects of dystrophin deficiency. Methods The role of dystrophin was examined using mice deficient in dystrophin alone ( mdx ) and mice deficient in dystrophin plus telomerase RNA ( mdx/mTR ), which exhibit increased disease severity. The effect of an orally‐active, slow releasing H 2 S agent (SG1002) was tested in mdx/mTR mice (40 mg/kg body weight in chow/every 3 days starting from 3 weeks to 9 months). Contraction in response to acetylcholine (ACh) was measured in gastric muscle strips isolated from mdx , mdx/mTR and SG1002‐treated mdx/mTR mice. Age‐matched control mice were used for each group. Contraction was also measured in muscle cells isolated from the stomach of control and mdx mice by scanning micrometry and expressed as the percent decrease in muscle cell length. Expression of contractile proteins such as smoothelin, caldesmon, calponin and tropomyosin was measured by qRT‐PCR and western blot. Results Acetylcholine‐induced contraction was reduced in muscle strips from mdx/mTR mice (19 ± 5 mN/100 mg tissue) compared to age‐matched 9‐month old control mice (34 ± 7 mN/100 mg tissue). Treatment of mdx/mTR mice with SG1002 restored contraction to above normal levels (56 ± 8 mN/100 mg tissue). Contraction was also reduced in mdx mice (22 ± 8 mN/100 mg tissue) compared to age‐matched 3‐month old control mice (40 ± 5 mN/100 mg tissue). Acetylcholine‐induced contraction was also decreased in gastric muscle cells from mdx mice (25 ± 3% decrease in cell length) compared to control mice (41 ± 5% decrease in cell length). Expression of contractile proteins was decreased in gastric smooth muscle from mdx and mdx/mTR mice compared to age‐matched controls. Treatment of mdx/mTR mice with SG1002 restored expression of contractile proteins to levels similar to control. Conclusion The results support our hypothesis that dystrophin deficiency affects contractile protein expression leading to decreased muscle contraction. Furthermore, treatment with H 2 S restores gastric smooth muscle function and contractile protein expression suggesting therapeutic potential of H 2 S in the treatment of motility disorders in DMD. Support or Funding Information Supported by DK15564, DK28300, DK34153, and HL133167. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
