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Arginine (Arg), as a basic amino acid, has been reported to be involved in regulation of gut motility. However, the evidence is limited and the underlying mechanism is not fully understood. Our study was conducted to investigate the effects of L-Arg on spontaneous contraction of the longitudinal muscle strip (LMS) of the rat colon and the relevant mechanisms. An organ bath system was used to detect the contractile force of the LMS. Whole-cell voltage-clamp techniques were applied to observe alterations in the currents of large conductance Ca2+-activated K+ (KCa) channels, voltage-dependent potassium (KV) channels, and L-type Ca2+ channels (LTCCs) in smooth muscle cells (SMCs) of the colon. We found that L-Arg within the physiological concentration had no effect on contraction of LMS, while 1 mM L-Arg significantly increased both the amplitude and frequency of LMS contractility. And the increase in force was mucosa-dependent, whereas changes in frequency as well as in amplitude were inhibited by atropine. In addition, L-Arg (1 mM) activated the LTTCs and inhibited both KCa channels and KV channels on SMCs. Thus, L-Arg above the physiological concentration exerted an excitatory effect on colonic LM contraction, and stimulation by L-Arg was mediated by ACh. In addition, LTCCs, KCa channels, and KV channels on SMCs were involved in the action of L-Arg. [ABSTRACT FROM AUTHOR]

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-β-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 (200μΜ) 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. [ABSTRACT FROM AUTHOR]

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. 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. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H 2 S), a novel gasotransmitter, is involved in the regulation of gut motility. Alterations in the balance of H 2 S play an important role in the pathogenesis of diabetes. This study was conducted to investigate the role of H 2 S in the colonic hypomotility of mice with streptozotocin (STZ)-induced diabetes. A single intraperitoneal injection of STZ was used to induce the type 1 diabetes model. Male C57BL/6 mice were randomized into a control group and an STZ-treated group. Immunohistochemistry, Western blotting, H 2 S generation, organ bath studies and whole-cell patch clamp techniques were carried out in single smooth muscle cells (SMCs) of the colon. We found that STZ-induced diabetic mice showed decreased stool output, impaired colonic contractility, and increased endogenous generation of H 2 S ( p < 0.05). H 2 S-producing enzymes were upregulated in the colon tissues of diabetic mice ( p < 0.05). The exogenous H 2 S donor sodium hydrosulfide (NaHS) elicited a biphasic action on colonic muscle contraction with excitation at lower concentrations and inhibition at higher concentrations. NaHS (0.1 mM) increased the currents of voltage-dependent calcium channels (VDCCs), while NaHS at 0.5 mM and 1.5 mM induced inhibition. Furthermore, NaHS reduced the currents of both voltage-dependent potassium (K V ) channels and large conductance calcium-activated potassium (BK) channels in a dose-dependent manner. These results show that spontaneous contraction of colonic muscle strips from diabetic mice induced by STZ was significantly decreased, which may underlie the constipation associated with diabetes mellitus (DM). H 2 S overproduction with subsequent suppression of muscle contraction via VDCCs on SMCs may contribute in part to the pathogenesis of colonic hypomotility in DM. SIGNIFICANCE STATEMENT: Hydrogen sulfide may exhibit a biphasic effect on colonic motility in mice by regulating the activities of voltage-dependent calcium channels and voltage-dependent and large conductance calcium activated potassium channels. H 2 S overproduction with subsequent suppression of muscle contraction via VDCCs may contribute to the pathogenesis of colonic hypomotility in diabetes mellitus., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

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.

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.

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 Ca²⁺-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.

Recent evidence shows that altered microRNA-126 (miR-126) expression is implicated in the progression of colorectal cancer (CRC). However, the precise roles and mechanisms of miR-126 in CRC remain unclear. The aim of this study was to investigate the roles of miR-126 in CRC cells and to elucidate miR-126-mediated mechanisms in CRC cells. First, miR-126 expression was analyzed using qRT-PCR in 4 human CRC cell lines (SW480, SW620, HT-29 and HCT-116). Furthermore, the biological properties of miR-126 in CRC cells in vitro were examined by applying Cell Counting Kit 8, cell cycle, cell apoptosis and transwell assays. The mechanisms and pathways of miR-126-mediated in CRC cells were detected by using qRT-PCR, western blotting and luciferase reporter assay. We found that miR-126 overexpression inhibited cell proliferation, migration and invasion, and induced cell arrest in the G0/G1 phase of CRC cells, suggesting that miR-126 functions as a tumor suppressor in CRC cells. Furthermore, we identified the CXC chemokine receptor 4 (CXCR4) as a target of miR-126, and showed that it was negatively regulated by miR-126. We demonstrated that miR-126-mediated tumor suppression might be partly dependent on AKT and ERK1/2 signaling pathways. In conclusion, our data revealed that miR-126 functions as a tumor suppressor in CRC cells by regulating CXCR4 expression via the AKT and ERK1/2 signaling pathways and might be a novel target for therapeutic strategies in CRC.