Different distributions of interstitial cells of Cajal and platelet-derived growth factor receptor-α positive cells in colonic smooth muscle cell/interstitial cell of Cajal/plateletderived growth factor receptor-α positive cell syncytium in mice

AIM To investigate the distribution and function of interstitialcells of Cajal(ICCs) and platelet-derived growth factor receptor-α positive(PDGFRα+) cells in the proximal and distal colon.METHODS The comparison of colonic transit in the proximal and distal ends was performed by colonic migrating motor complexes(CMMCs). The tension of the colonic smooth muscle was examined by smooth muscle spontaneous contractile experiments with both ends of the smooth muscle strip tied with a silk thread. Intracellular recordings were used to assess electrical field stimulation(EFS)-induced inhibitory junction potentials(IJP) on the colonic smooth muscle. Western blot analysis was used to examine the expression levels of ICCs and PDGFRα in the colonic smooth muscle.RESULTS Treatment with NG-nitro-L-arginine methyl ester hydrochloride(L-NAME) significantly increased the CMMC frequency and spontaneous contractions, especially in the proximal colon, while treatment with MRS2500 increased only distal CMMC activity and smooth muscle contractions. Both CMMCs and spontaneous contractions were markedly inhibited by NPPB, especially in the proximal colon. Accordingly, CyPPA sharply inhibited the distal contraction of both CMMCs and spontaneous contractions. Additionally, the amplitude of stimulationinduced nitric oxide(NO)/ICC-dependent slow IJPs(sIJPs) by intracellular recordings from the smooth muscles in the proximal colon was larger than that in the distal colon, while the amplitude of electric field stimulationinduced purinergic/PDGFRα-dependent fast IJPs(fIJPs) in the distal colon was larger than that in the proximal colon. Consistently, protein expression levels of c-Kit and anoctamin-1(ANO1) in the proximal colon were much higher, while protein expression levels of PDGFRα and small conductance calcium-activated potassium channel 3(SK3) in the distal colon were much higher.CONCLUSION The ICCs are mainly distributed in the proximal colon and there are more PDGFRα+ cells are in the distal colon, which generates a pressure gradient between the two ends of the colon to propel the feces to the anus.

Hydrogen sulfide-induced enhancement of gastric fundus smooth muscle tone is mediated by voltagedependent potassium and calcium channels in mice

AIM:To investigate the effect of hydrogen sulfide(H2S)on smooth muscle motility in the gastric fundus.METHODS:The expression of cystathionineβ-synthase(CBS)and cystathionineγ-lyase(CSE)in cultured smooth muscle cells from the gastric fundus was examined by the immunocytochemistry technique.The tension of the gastric fundus smooth muscle was recorded by an isometric force transducer under the condition of isometric contraction with each end of the smooth muscle strip tied with a silk thread.Intracellular recording was used to identify whether hydrogen sulfide affects the resting membrane potential of the gastric fundus in vitro.Cells were freshly separated from the gastric fundus of mice using a variety of enzyme digestion methods and whole-cell patch-clamp technique was used to find the effects of hydrogen sulfide on voltage-dependent potassium channel and calcium channel.Calcium imaging with fura-3AM loading was used to investigate the mechanism by which hydrogen sulfide regulates gastric fundus motility in cultured smooth muscle cells.RESULTS:We found that both CBS and CSE were expressed in the cul tured smooth muscle cel ls from the gastric fundus and that H2S increased the smooth muscle tension of the gastric fundus in mice at low concentrations.In addition,nicardipine and aminooxyacetic acid(AOAA),a CBS inhibitor,reduced the tension,whereas Nω-nitro-L-arginine methyl ester,a nonspecific nitric oxide synthase,increased the tension.The AOAA-induced relaxation was significantly recovered by H2S,and the Na HS-induced increase in tonic contraction was blocked by 5 mmol/L4-aminopyridine and 1μmol/L nicardipine.Na HS significantly depolarized the membrane potential and inhibited the voltage-dependent potassium currents.Moreover,Na HS increased L-type Ca2+currents and caused an elevation in intracellular calcium([Ca2+]i).CONCLUSION:These findings suggest that H2S may be an excitatory modulator in the gastric fundus in mice.The excitatory effect is mediated by voltagedependent potassium and L-type calcium channels.

Gastric nNOS reduction accompanied by natriuretic peptides signaling pathway upregulation in diabetic mice

AIM:To investigate the relationship between neuronal nitric oxide synthase(nNOS)expression and the natriuretic peptide signaling pathway in the gastric fundus of streptozotocin(STZ)-induced diabetic mice.METHODS:Diabetic mice were induced by injection of STZ solution.Immunofluorescence labeling of HuC/D,nNOS and natriuretic peptide receptor-A,B,C(NPRs)in the gastric fundus(GF)was used to observe nNOS expression and whether NPRs exist on enteric neurons.The expression levels of nNOS and NPRs in the diabetic GF were examined by western blotting.An isometric force transducer recorded the electric field stimulation(EFS)-induced relaxation and contraction in the diabetic GF.An intracellular recording method assessed EFSinduced inhibitory junction potentials(IJP)on the GF.GF smooth muscles acquired from normal mice were incubated with different concentrations of the NPRs agonist C-type natriuretic peptide(CNP)for 24 h,after which their nNOS expressions were detected by western blotting.RESULTS:Eight weeks after injection,43 diabetic mice were obtained from mouse models injected with STZ.Immunofluorescence indicated that the number of NOS neurons was significantly decreased and that nNOS expression was significantly downregulated in the diabetic GF.The results of physiological and electrophysiological assays showed that the EFS-induced relaxation that mainly caused by NO was significantly reduced,while the contraction was enhanced in the diabetic GF.EFSinduced IJP showed that L-NAME sensitive IJP in the diabetic GF was significantly reduced compared with control mice.However,both NPR-A and NPR-B were detected on enteric neurons,and their expression levels were upregulated in the diabetic GF.The nNOS expression level was downregulated dose-dependently in GF smooth muscle tissues exposed to CNP.CONCLUSION:These findings suggested that upregulation of the NPs signaling pathway may be involved in GF neuropathy caused by diabetes by decreasing nNOS expression.