Effects of exendin-4 on colonic motility in rats and its underlying mechanism.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists modulate gastrointestinal motility; however, the effects of GLP-1R agonists on colonic motility are still controversial, and the molecular mechanism is unclear. Exendin-4 shares 53% homology with GLP-1 and is a full agonist of GLP-1R. In this study, our aims were to explore the role and mechanism of exendin-4 in isolated rat colonic tissues and cells. METHODS: An organ bath system was used to examine the spontaneous contractions of smooth muscle strips. The whole-cell patch-clamp technique was used to investigate the currents of L-type voltage-dependent calcium channels and large conductance Ca2+ -activated K+ (BKCa ) channels in smooth muscle cells. KEY RESULTS: Exendin-4 decreased both the amplitude and frequency of spontaneous contractions of smooth muscle strips in a concentration-dependent manner. The inhibitory effect was completely blocked by exendin-4(9-39), a GLP-1R antagonist. Moreover, this effect was partially abolished by tetrodotoxin (TTX), a blocker of neuronal voltage-dependent Na+ channels, Nω-Nitro-l-arginine (L-NNA), a nitric oxide synthase (NOS) inhibitor, apamin, an inhibitor of small-conductance Ca2+ -activated K+ (SK) channels. Whole-cell patch-clamp recordings revealed that exendin-4 inhibited the peak current of L-type calcium channels in colonic smooth muscle cells, but did not change the shape of the current-voltage (I-V) curves. The steady-state activation and steady-state inactivation of L-type calcium channels were not affected. Likewise, BKCa currents were significantly inhibited by exendin-4. CONCLUSIONS: Exendin-4 indirectly inhibits colonic muscle activity via a nitrergic and a purinergic neural pathway through NO and ATP release and inhibits L-type voltage-dependent calcium channels and BKCa channels in smooth muscle cells.

Diallyl trisulfide regulates rat colonic smooth muscle contractions by inhibiting L-type calcium channel currents.

Diallyl trisulfide (DATS) is an active organosulfide component of allicin and has several beneficial effects, including antimicrobial, antioxidant, cardioprotective and anticancer effects. Few studies have shown the modulatory effect of DATS on L-type calcium channels in rat colonic smooth muscle cells and colonic motility. To investigate the modulatory effect of DATS on L-type calcium channels in rat colonic smooth muscle and colonic contraction, L-type calcium channel currents were recorded, and colonic contractility in longitudinal and circular smooth muscle strips was measured. DATS attenuated L-type calcium channel currents without affecting steady-state activation or inactivation kinetics and inhibited the spontaneous contractions of both longitudinal and circular smooth muscle strips dose-dependently. In conclusion, DATS has an inhibitory effect on the contractions of colonic muscle strips that is related to its regulation of L-type calcium channels.

Fusobacterium nucleatum promotes the progression of colorectal cancer by interacting with E-cadherin.

Increasing evidence suggests that Fusobacterium nucleatum is involved in colorectal carcinogenesis. Previous studies have explored whether F. nucleatum may trigger colonic epithelial-mesenchymal transition. The results of the present study demonstrated that F. nucleatum enhances the proliferation and invasion of NCM460 cells compared with that of normal control and DH5α cells. Furthermore, F. nucleatum significantly increased the phosphorylation of p65 (a subunit of nuclear factor-κB), as well as the expression of interleukin (IL)-6, IL-1ß and matrix metalloproteinase (MMP)-13. Additionally, F. nucleatum infection did not affect the expression levels of epithelial (E-)cadherin and ß-catenin. E-cadherin knockdown in NCM460 cells did not induce the activation of inflammatory responses in response to F. nucleatum infection, whereas it increased inflammation in response to ß-catenin silencing. F. nucleatum infection could not increase the proportion of cells at S phase when E-cadherin was silenced. Nevertheless, F. nucleatum infection enhanced the proportion of NCM460 cells at S phase when transfected with small interfering RNAs to knock down ß-catenin expression. In conclusion, the results of the present study demonstrated that F. nucleatum infection interacted with E-cadherin instead of ß-catenin, which in turn enhances the malignant phenotype of colorectal cancer cells.

Mechanism of sodium hydrosulfide modulation of L-type calcium channels in rat colonic smooth muscle cells.

Hydrogen sulfide (H2S) can exert different effects on the gastrointestinal tract by modulating ion channels. Previously, we found that H2S donor sodium hydrosulfide (NaHS) regulates colonic motility through L-type calcium channels, but the molecular mechanism remains unknown. The present study was designed to investigate possible mechanisms underlying the modulation of L-type calcium channels by NaHS in rat colonic smooth muscle cells. L-type calcium currents in colonic smooth muscle cells were recorded using the whole-cell patch-clamp technique. Spontaneous contractions of mid-colonic smooth muscle strips were measured in an organ bath system and a biological signal acquisition system. NaHS evoked a significant rightward shift in the steady-state activation curve of L-type calcium channels, changed the shape of the current-voltage (I-V) curve, and decreased the peak current density at 0mV, although it significantly increased with higher stimulatory voltage. The sulfhydryl-modifying reagent DL-dithiothreitol (DTT) enhanced the effects of NaHS on L-type calcium channels, while diamide (DM) and reduced L-glutathione (GSH) alleviated the effects of NaHS. Additionally, NaHS inhibited the spontaneous high-amplitude contractions of both longitudinal and circular smooth muscle strips in a dose-dependent manner. The inhibitory effects were reversible. DTT and GSH enhanced the effects of NaHS, while DM attenuated the effects of NaHS. In conclusion, NaHS modulates L-type calcium channels in rat colonic smooth muscle cells and regulates the contractile activity of colonic smooth muscle, potentially by modifying the free sulfhydryl groups of L-type calcium channels.

The role of substance P in the maintenance of colonic hypermotility induced by repeated stress in rats.

BACKGROUND: The mechanism underlying chronic stress-induced gastrointestinal (GI) dysmotility has not been fully elucidated and GI hormones have been indicated playing a role in mediating stress-induced changes in GI motor function. AIMS: Our objective was to study the possible role of substance P (SP) in the colonic hypermotility induced by repeated water avoidance stress (WAS) which mimics irritable bowel syndrome. METHODS: Male Wistar rats were submitted to WAS or sham WAS (SWAS) (1h/day) for up to 10 consecutive days. Enzyme Immunoassay Kit was used to detect the serum level of SP. The expression of neurokinin-1 receptor (NK1R) was investigated by Immunohistochemistry and Western blotting. The spontaneous contraction of muscle strip was studied in an organ bath system. L-type calcium channel currents (ICa,L) of smooth muscle cells (SMCs) were recorded by whole-cell patch-clamp technique. RESULTS: Fecal pellet expulsion and spontaneous contraction of proximal colon in rats were increased after repeated WAS. The serum level of SP was elevated following WAS. Immunohistochemistry proved the expression of NK1R in mucosa, muscularis and myenteric plexus. Western blotting demonstrated stress-induced up-regulation of NK1R in colon devoid of mucosa and submucosa. Repeated WAS increased the contractile activities of longitudinal muscle and circular muscle strips induced by SP and this effect was reversed by a selective NK1R antagonist. The ICa,L of SMCs in the WAS rats were drastically increased compared to controls after addition of SP. CONCLUSIONS: Increased serum SP level and up-regulated NK1R in colon may contribute to stress-induced colonic hypermotility. And L-type calcium channels play a potentially important role in the process of WAS-induced dysmotility.

[Effect of substance P on the potassium and calcium currents of colonic smooth muscle cells].

OBJECTIVE: To investigate the effect of substance P(SP) on the spontaneous contractile activity of smooth muscle cells,the large-conductance calcium-activated potassium channel currents (IBKCa) and the L-type calcium channel currents (ICaL) in rat smooth muscle cells of the proximal colon. METHODS: A total of 24 healthy male Wista rats were used in this test. The change of smooth muscle strips spontaneous contraction of rat proximal colon after adding SP was recorded by a physiological signal stystem (RM6240). The IBKCa and ICaL were measured via the whole cell patch-clamp technique. RESULTS: The longitudinal muscle contraction was obviously increased concentration-dependently after adding different concentrations of SP (10(-7)-10(-6) mol/L), so as the circular muscle while adding SP(10(-8)-10(-6) mol/L) (all P<0.05). Compared with the control group, IBKCa was decreased after adding SP(10(-6) mol/L). Under the stimulating voltage of 60 mV, the IBKCa current density was (11.71±1.65) pA/pF, which was significantly lower compared with the control group (14.42±2.89) pA/pF (P<0.05). The ICaL) was apparently increased. Under the stimulating voltage of 0 mV, the ICaL) currents density was (-5.04±0.67) pA/pF, compared with the control group (-4.25±0.46) pA/pF, which was significantly increased (P<0.01). CONCLUSIONS: SP can promote the spontaneous contractile activity of colon smooth muscle of rats in vitro.And SP decrease IBKCa representatively while apparently increase ICaL). That is probably one of the mechanism SP regulate the gastrointestinal motility.

[Modulatory role and mechanism of BDNF and its receptor TrkB in colonic motility disorder in chronic stress rat].

OBJECTIVE: To investigate the role and mechanism of BDNF and its receptor TrkB in the disorder of colonic motility in chronic stress rats. METHODS: A total of 20 male Wistar rats were randomly divided into two groups using to completely random method after weighed, with 10 rats in each group. Chronic water avoidance stress model was established. Then the fecal pellets of water avoidance stress (WAS) group and sham water avoidance stress (SWAS) group were recorded. Enzyme linked immunosorbent assay (ELISA), real time PCR, Western blot and immunohistochemistry were used to detect the expression of BDNF and TrkB in serum and colon muscle. The amplitudes of contractions of circular smooth muscle strips of each group were recorded after the treatment of TTX, BDNF and K252a. RESULTS: The number of fecal pellets had obviously increased in WAS group comparing with SWAS group (P < 0.05). The level of serum BDNF in WAS group was higher than that of SWAS group ((158.30 ± 9.82) vs (84.68 ± 7.80) pg/ml). And the expression of TrkB in the colon muscle in WAS group was higher than that in SWAS group (0.44 ± 0.03 vs 0.30 ± 0.02, P < 0.05). There was no significant difference between the two groups in expression of BDNF mRNA in colon muscle (P > 0.05). TrkB was mainly expressed in the cell nucleus of muscular layer neurons, and the expression of TrkB had obviously increased in WAS rats. The amplitudes of contractions of circular smooth muscle in WAS rats had significantly increased compared with SWAS rats ((0.35 ± 0.02) vs (0.22 ± 0.03) g, P < 0.05). After adding TTX to block the function of enteric nervous, the difference was remaining ((0.89 ± 0.07) vs (0.53 ± 0.06) g, P < 0.05). BDNF was added to the bath and the R value at different time was recorded. The difference had statistically significant at 6 min and 12 min (both P < 0.05). BDNF could induced the contraction peak of the circular smooth muscle. The contraction peak induced by BDNF was delayed and reduced when K252a was added to the bath 30 min before adding BDNF. CONCLUSION: BDNF plays a modulatory role in the disorder of colonic motility in chronic stress rat by acting on its receptor TrkB.

[Effects of hydrogen sulfide on colonic contraction of rats and ion channel mechanisms].

OBJECTIVE: To explore the effects of hydrogen sulfide (H2S) on colonic contraction of rats at low concentration and ion channel mechanisms. METHODS: Organ bath recordings were used to examine the contraction of colonic smooth muscle strips. The whole-cell patch-clamp technique was used to record the currents of L-type calcium and large conductance Ca²âº-activated K⁺ (BKCa) channels in smooth muscle cells isolated from 30 male Wistar rats. RESULTS: The H2S donor NaHS ((1-12) × 10⁻5 mol/L) increased the spontaneous contractions of longitudinal and circular muscle strips in a dose-dependent manner (all P<0.05) and the effect could not be blocked by tetrodotoxin. NaHS (6 × 10⁻5 and 12 × 10⁻5 mol/L) reversibly increased the L-type calcium current (ICa,L) in a dose-dependent manner. And the peak of ICa,L at 0 mV increased from (-3.16 ± 0.47) to (-3.33 ± 0.54) and (-3.65 ± 0.66) pA/pF respectively (n=6, both P<0.05). Current-voltage (I-V) curve had no shift after NaHS treatment and H2S donor caused no change in the curves of steady-state activation. Likewise BKCa channel was significantly inhibited by NaHS (6 × 10⁻5 and 12 × 10⁻5 mol/L) in a dose-dependent manner. And IBKCa at 60 mV decreased from (16.68 ± 1.23) to (15.26 ± 2.67) and (13.80 ± 3.04) pA/pF respectively(both P<0.05). CONCLUSIONS: H2S at low concentrations increases the spontaneous contraction of rat colonic smooth muscle in a dose-dependent manner. Such an effect may be due to a direct activation of L-type calcium channel and an inhibition of BKCa channel in smooth muscle cells.

Brain-Derived Neurotrophic Factor Contributes to Colonic Hypermotility in a Chronic Stress Rat Model.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) has prokinetic effects on gut motility and is increased in the colonic mucosa of irritable bowel syndrome. AIMS: We aimed to investigate the possible involvement of BDNF in stress-induced colonic hypermotility. METHODS: Male Wistar rats were exposed to daily 1-h water avoidance stress (WAS) or sham WAS for 10 consecutive days. The presence of BDNF and substance P (SP) in the colonic mucosa was 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 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.

Hydrogen sulfide regulates the colonic motility by inhibiting both L-type calcium channels and BKCa channels in smooth muscle cells of rat colon.

OBJECTIVE: To examine 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 performed on rat colonic samples to investigate the localization of the H2S-producing enzymes cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE). The contractions of proximal colonic smooth muscle were studied in an organ bath system. The whole-cell patch-clamp technique was used to record both L-type calcium currents (ICa,L) and BKCa currents in colonic smooth muscle cells (SMCs) isolated from male Wistar rats. 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 (200 µΜ) 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. CONCLUSION: The relaxant effect of H2S on colonic muscle strips may be associated with the direct inhibition of H2S on L-type calcium channels. H2S may be involved in the regulation of calcium homeostasis in colonic SMCs of rat colon.