Natural phosphorus-ferromanganese ore-based composites for the simultaneous remediation of arsenic- and lead-co-contaminated groundwater: synergistic effectiveness, kinetics, and mechanisms.

Natural phosphorus-ferromanganese ore (NPO-NFMO) based composites by mechanical ball milling method, applying for the simultaneous remediation of arsenic (As) and lead (Pb) co-contaminated groundwater. Kinetic behavior adopted pseudo-second-order adsorption mechanism attaining equilibrium in 120 min over a wide pH range (2.0-6.0). NPO-NFMO realized higher adsorption capacity for As(III) (6.8 mg g-1) and Pb(II) (26.5 mg g-1) than those of single NPO (1.7 and 7.8 mg g-1) and NFMO (2.9 and 5.1 mg g-1), indicating that synergistic effects of NPO and NFMO considerably enhanced the adsorption capacity in mixed adsorption system. Fresh and used NPO-NFMO were characterized, and indicated that NPO-NFMO formed stable minerals of PbAs2O6 and PbFe2(AsO4)2(OH)2. The underlying adsorption mechanism indicated that As(III) and Pb(II) removal was involved with multiple mechanisms, including electrostatic adsorption, oxidation, complexation, and coprecipitation. The effects of key reaction parameters including mass ratios of NPO and NFMO, initial metal ion concentration, dosage, solution pH, and co-existing anions in groundwater were systematically investigated. The novel designed NPO-NFMO-based composites can be deemed as a promising amendment for simultaneous immobilization of As(III) and Pb(II) in co-contaminated soil and groundwater.

Optimized concurrent hearing and genetic screening in Beijing, China: A cross-sectional study.

Concurrent screening has been proven to provide a comprehensive approach for management of congenital deafness and prevention of ototoxicity. The SLC26A4 gene is associated with late-onset hearing loss and is of great clinical concern. For much earlier detection of newborns with deafness-causing mutations in the SLC26A4 gene, the Beijing Municipal Government launched a chip for optimized genetic screening of 15 variants of 4 genes causing deafness based on a chip to screen for 9 variants of 4 genes, and 6 variants of the SLC26A4 gene have now been added. To ascertain the advantage of a screening chip including 15 variants of 4 genes, the trends in concurrent hearing and genetic screening were analyzed in 2019 and 2020. Subjects were 76,460 newborns who underwent concurrent hearing and genetic screening at 24 maternal and child care centers in Beijing from January 2019 to December 2020. Hearing screening was conducted using transiently evoked otoacoustic emissions (TEOAEs), distortion product otoacoustic emissions (DPOAE), or the automated auditory brainstem response (AABR). Dried blood spots were collected for genetic testing and 15 variants of 4 genes, namely GJB2, SLC26A4, mtDNA 12S rRNA, and GJB3, were screened for using a DNA microarray platform. The initial referral rate for hearing screening decreased from 3.60% (1,502/41,690) in 2019 to 3.23% (1,124/34,770) in 2020, and the total referral rate for hearing screening dropped form 0.57% (236/41,690) in 2019 to 0.54% (187/34,770) in 2020, indicating the reduced false positive rate of newborn hearing screening and policies to prevent hearing loss conducted by the Beijing Municipal Government have had a significant effect. Positivity according to genetic screening was similar in 2019 (4.970%, 2,072/41,690) and 2020 (4.863%,1,691/34,770), and the most frequent mutant alleles were c.235 del C in the GJB2 gene, followed by c.919-2 A > G in the SLC26A4 gene, and c.299 del AT in the GJB2 gene. In this cohort study, 71.43% (5/7) of newborns with 2 variants of the SLC26A4 gene were screened for newly added mutations, and 28.57% (2/7) of newborns with 2 variants of the SLC26A4 gene passed hearing screening, suggesting that a screening chip including 15 variants of 4 genes was superior at early detection of hearing loss, and especially in early identification of newborns with deafness-causing mutations in the SLC26A4 gene. These findings have clinical significance.

Protein Kinase CK2 Modulates the Calcium Sensitivity of Type 3 Small-conductance Calcium-activated Potassium Channels in Colonic Platelet-derived Growth Factor Receptor Alpha-positive Cells From Streptozotocin-induced Diabetic Mice.

Background/Aims: The gastrointestinal symptom of diabetes mellitus, chronic constipation, seriously affects patients' life. Whereas, the mechanism of chronic constipation is still ambiguous, resulting in a lack of effective therapies for this symptom. As a part of the smooth muscle cells, interstitial cells of Cajal, and platelet-derived growth factor receptor alpha-positive (PDGFRα+) cells syncytium (SIP syncytium), PDGFRα+ cells play an important role in regulating colonic motility. According to our previous study, in PDGFRα+ cells in colons of diabetic mice, the function of the P2Y1 purinergic receptor/type 3 small-conductance calcium-activated potassium (SK3) channel signaling pathway is strengthened, which may lead to colonic dysmotility. The purpose of this study is to investigate the changes in SK3 channel properties of PDGFRα+ cells in diabetic mice. Methods: Whole-cell patch clamp, Western blotting, superoxide dismutase activity measurement, and malondialdehyde measurement were main methods in the present study. Results: The present study revealed that when dialysed with low calcium ion (Ca2+) solution, the SK3 current density was significantly decreased in PDGFRα+ cells from diabetic mice. However, the SK3 current density in PDGFRα+ cells was enhanced from diabetic mice when dialysed with high Ca2+ solution. Moreover, hydrogen peroxide-treatment mimicked this phenomenon in SK3 transgenic HEK293 cells. The subunit of SK3 channels, protein kinase CK2, was up-regulated in colonic muscle layers and hydrogen peroxide-treated HEK293 cells. Additionally, protein phosphatase 2A, the subunit of SK3 channels, was not changed in streptozotocin-treated mouse colons or hydrogen peroxide-treated HEK293 cells. Conclusion: The diabetic oxidative stress-induced upregulation of CK2 contributed to modulating SK3 channel sensitivity to Ca2+ in colonic PDGFRα+ cells, which may result in colonic dysmotility in diabetic mice.

Corticotropin-releasing factor receptor agonists decrease interstitial cells of Cajal in murine colon.

BACKGROUND: Peripheral corticotropin-releasing factor (CRF) has been reported to affect gastrointestinal motility through corticotropin-releasing factor receptor located in enteric nervous system (ENS), but less is known about of the relationship between peripheral CRF and interstitial cells of Cajal (ICC). METHODS: Mice were intraperitoneally injected with CRF receptor agonists to determine their effects on colonic ICC. Chronic heterotypic stress (CHeS) was applied to mice to determine endogenous CRF-CRF receptor signaling on colonic ICC. RESULTS: We found that stressin1, a selective CRF receptor 1 (CRF1 ) agonist, significantly increased the expression of CRF1 but had no effect on the expression of CRF2 in the smooth muscles of murine colon. The protein expression of c-Kit, Anoctamin-1 (ANO1), and stem cell factor (SCF) in the colonic smooth muscles was significantly decreased in stressin1-treated mice. Accordingly, 2-(4-Chloro-2-methylphenoxy)-N'-(2-methoxybenzylidene) acetohydrazide (Ani 9), a selective ANO1 blocker, had a less significant inhibitory effect on CMMC in stressin1-treated mice compared to the saline-treated ones. Similarly, we also found that ICC and ANO1 were reduced in the colonic smooth muscles of mice by treatment with sauvagine (ip), a CRF2 agonist. However, different with stressin1, sauvagine decreased the expression of CRF2 besides increasing CRF1 expression in the colonic smooth muscles. Similar results of CRF1 and c-Kit expressions were also obtained from the colon of CHeS-treated mice. CONCLUSION: All these results suggest that CRF may be involved in the abnormality of colonic motility through peripheral CRF1 to decrease the number and function of ICC, which provides a potential target for treating stress-induced gastrointestinal motility disorder.

Propulsive colonic contractions are mediated by inhibition-driven poststimulus responses that originate in interstitial cells of Cajal.

The peristaltic reflex is a fundamental behavior of the gastrointestinal (GI) tract in which mucosal stimulation activates propulsive contractions. The reflex occurs by stimulation of intrinsic primary afferent neurons with cell bodies in the myenteric plexus and projections to the lamina propria, distribution of information by interneurons, and activation of muscle motor neurons. The current concept is that excitatory cholinergic motor neurons are activated proximal to and inhibitory neurons are activated distal to the stimulus site. We found that atropine reduced, but did not block, colonic migrating motor complexes (CMMCs) in mouse, monkey, and human colons, suggesting a mechanism other than one activated by cholinergic neurons is involved in the generation/propagation of CMMCs. CMMCs were activated after a period of nerve stimulation in colons of each species, suggesting that the propulsive contractions of CMMCs may be due to the poststimulus excitation that follows inhibitory neural responses. Blocking nitrergic neurotransmission inhibited poststimulus excitation in muscle strips and blocked CMMCs in intact colons. Our data demonstrate that poststimulus excitation is due to increased Ca2+ transients in colonic interstitial cells of Cajal (ICC) following cessation of nitrergic, cyclic guanosine monophosphate (cGMP)-dependent inhibitory responses. The increase in Ca2+ transients after nitrergic responses activates a Ca2+-activated Cl− conductance, encoded by Ano1, in ICC. Antagonists of ANO1 channels inhibit poststimulus depolarizations in colonic muscles and CMMCs in intact colons. The poststimulus excitatory responses in ICC are linked to cGMP-inhibited cyclic adenosine monophosphate (cAMP) phosphodiesterase 3a and cAMP-dependent effects. These data suggest alternative mechanisms for generation and propagation of CMMCs in the colon.

Involvement of PAR2 in platelet-derived growth factor receptor-α-positive cell proliferation in the colon of diabetic mice.

Our previous study indicated that streptozotocin (STZ)-induced diabetes leads to colonic platelet-derived growth factor receptor-α-positive (PDGFRα+ ) cell proliferation accompanied by slow colonic transit in mice; however, the mechanism of this effect is unclear. The present study used western blotting, immunohistochemistry, and quantitative PCR to investigate whether proteinase-activated receptor 2 (PAR2) mediates PDGFRα+ cell proliferation. Our results showed that PDGFRα, PAR2, and Ki-67 coexpression was increased in the diabetic colonic muscle layer. PDGFRα and PAR2 mRNA and protein expression levels were also markedly enhanced in the diabetic colonic muscle layer. Mice treated with 2-furoyl-LIGRLO-amide (2-F-L-a), a PAR2 agonist, exhibited significant colon elongation and increased smooth muscle weight. In the 2-F-L-a-treated mice, PDGFRα, PAR2, and Ki-67 coexpression was increased and PDGFRα and PAR2 mRNA and protein expression was significantly enhanced in the colonic smooth muscle layer. 2-F-L-a also increased proliferation and PDGFRα expression in NIH/3T3 cells cultured in high glucose, while LY294002, a PI3K antagonist, decreased cell proliferation and PDGFRα expression. PI3K and Akt protein and mRNA expression and p-Akt protein expression in diabetic and 2-F-L-a-treated mice were markedly reduced in colonic smooth muscle. 2-F-L-a also reduced PI3K, Akt, and p-Akt protein expression in NIH/3T3 cells, while the PI3K antagonist LY294002 increased this expression. The results indicate that PAR2 is involved in the proliferation of PDGFRα+ cells through the PI3K/Akt signaling pathway in the colon of STZ-induced diabetic mice, which may contribute to the slow transit and constipation that are associated with diabetes.

Optimizing the composition of LiFSI-based electrolytes by a method combining simplex with normalization.

The optimizing method of electrolyte formulation is always vital for the development of high-performance lithium-ion batteries. Traditional optimization methods are mainly aimed at the optimization of the electrolyte composition type, and less attention is paid to the optimization of the composition proportion in a certain electrolyte formulation. In this paper, in order to balance the relationship between aluminum (Al) foil corrosion inhibition and battery electrochemical performance, the electrolyte system LiFSI0.6-LiBOB0.4-EC/DEC/EMC (1 : 1 : 1, by volume) was optimized by combining the simplex method, normalization and electrochemical testing. A lithium iron phosphate (LiFePO4) cathode with the optimized electrolyte of LiFSI0.53-LiBOB0.35-EC/DEC/EMC (1.3 : 1.5 : 1.5) delivers a high capacity (143.1 mA h g-1 at 0.5C) and remarkable cycle life (94.9% retention after 100 cycles) at 45 °C. The outstanding performance is attributed to the composition of the cathode electrolyte interphase (CEI) containing the solid and dense LiF, AlF3, B2O3 and Li2CO3. This provides a new method and idea for future electrolyte formulation optimization.

[Role of platelet-derived growth factor receptor α positive cells in purinergic inhibitory nerve-smooth muscle transmission].

Under physiological conditions, the motility of smooth muscle in digestive tract is mainly regulated by enteric nervous system (ENS). However, how neural signal is transmitted to smooth muscle is not fully understood. Autonomic nerve endings in the smooth muscle layer form large number of varicosities which contain neurotransmitters. It was considered that nerve pulses arriving at the varicosities may cause the release of neurotransmitters, which may diffuse to the smooth muscle cells to induce contractile or relaxant responses. Over the past decade, a new understanding of the neurotransmission between ENS and smooth muscle has emerged, which emphasizes the role of a functional syncytium consisting of the interstitial cells of Cajal (ICC), the platelet-derived growth factor receptor α positive (PDGFRα+) cells and the smooth muscle cells. Within the syncytium, purine neurotransmitters bind to P2Y1 receptors on PDGFRα+ cells, activating small-conductance calcium activated potassium channel (SK3) to hyperpolarize PDGFRα+ cells, and thus hyperpolarize smooth muscle cells through gap junction, resulting in relaxation of smooth muscle. In this paper, we review the research progress in the field of inhibitory purinergic neurotransmission in the gastrointestinal tract.

Luteolin suppresses colonic smooth muscle motility via inhibiting L-type calcium channel currents in mice.

As a naturally occurring flavone, luteolin has received much attention due to its antioxidant, anti-inflammatory and anticancer functions. In the present study, we investigated the effect of luteolin on colonic motility and its mechanism using isometric muscle recording and the whole-cell patch-clamp technique in mice. Luteolin dose-dependently inhibited colonic smooth muscles motility and CMMC significantly. BayK8644, an L-type Ca2+ channel agonist, significantly attenuated the luteolin-induced inhibition. Moreover, the calcium currents recorded in colonic smooth muscle cells were dramatically inhibited by luteolin. However, no significant changes were found in the luteolin-induced inhibitory effect in the presence of TEA, a nonselective K+ channel blocker, glibenclamide, an ATP-dependent K+ channel blocker, and apamin, a small-conductance Ca2+-activated K+ channel blocker. Additionally, luteolin did not affect potassium currents. Furthermore, TTX, a Na+ channel blocker, L-NAME, an inhibitor of nitric oxide (NO) synthase, ODQ, an inhibitor of NO-sensitive guanylyl cyclase, and Ani9, a specific ANO1 channels blocker, had no effect on the luteolin-induced suppression. These results suggest that luteolin inhibited colonic smooth muscle motility by inhibiting L-type calcium channels in mice but not through potassium channels, the enteric nervous system (ENS), NO signaling pathways or ANO1 channels of interstitial cells of Cajal (ICCs).

Colonic Dysmotility in Murine Partial Colonic Obstruction Due to Functional Changes in Interstitial Cells.

BACKGROUND/AIMS: Interstitial cells play important roles in gastrointestinal (GI) neuro-smooth muscle transmission. The underlying mechanisms of colonic dysmotility have not been well illustrated. We established a partial colon obstruction (PCO) mouse model to investigate the changes of interstitial cells and the correlation with colonic motility. METHODS: Western blot technique was employed to observe the protein expressions of Kit, platelet-derived growth factor receptor-α (Pdgfra), Ca2+-activated Cl- (Ano1) channels, and small conductance Ca2+- activated K+ (SK) channels. Colonic migrating motor complexes (CMMCs) and isometric force measurements were employed in control mice and PCO mice. RESULTS: PCO mice showed distended abdomen and feces excretion was significantly reduced. Anatomically, the colon above the obstructive silicone ring was obviously dilated. Kit and Ano1 proteins in the colonic smooth muscle layer of the PCO colons were significantly decreased, while the expression of Pdgfra and SK3 proteins were significantly increased. The effects of a nitric oxide synthase inhibitor (L-NAME) and an Ano1 channel inhibitor (NPPB) on CMMC and colonic spontaneous contractions were decreased in the proximal and distal colons of PCO mice. The SK agonist, CyPPA and antagonist, apamin in PCO mice showed more effect to the CMMCs and colonic smooth muscle contractions. CONCLUSION: Colonic transit disorder may be due to the downregulation of the Kit and Ano1 channels and the upregulation of SK3 channels in platelet-derived growth factor receptor-α positive (PDGFRα+) cells. The imbalance between interstitial cells of Cajal-Ano1 and PDGFRαSK3 distribution might be a potential reason for the colonic dysmotility.

Concurrent Hearing and Genetic Screening of 180,469 Neonates with Follow-up in Beijing, China.

Concurrent hearing and genetic screening of newborns is expected to play important roles not only in early detection and diagnosis of congenital deafness, which triggers intervention, but also in predicting late-onset and progressive hearing loss and identifying individuals who are at risk of drug-induced HL. Concurrent hearing and genetic screening in the whole newborn population in Beijing was launched in January 2012. This study included 180,469 infants born in Beijing between April 2013 and March 2014, with last follow-up on February 24, 2018. Hearing screening was performed using transiently evoked otoacoustic emission (TEOAE) and automated auditory brainstem response (AABR). For genetic testing, dried blood spots were collected and nine variants in four genes, GJB2, SLC26A4, mtDNA 12S rRNA, and GJB3, were screened using a DNA microarray platform. Of the 180,469 infants, 1,915 (1.061%) were referred bilaterally or unilaterally for hearing screening; 8,136 (4.508%) were positive for genetic screening (heterozygote, homozygote, or compound heterozygote and mtDNA homoplasmy or heteroplasmy), among whom 7,896 (4.375%) passed hearing screening. Forty (0.022%) infants carried two variants in GJB2 or SLC26A4 (homozygote or compound heterozygote) and 10 of those infants passed newborn hearing screening. In total, 409 (0.227%) infants carried the mtDNA 12S rRNA variant (m.1555A>G or m.1494C>T), and 405 of them passed newborn hearing screening. In this cohort study, 25% of infants with pathogenic combinations of GJB2 or SLC26A4 variants and 99% of infants with an m.1555A>G or m.1494C>T variant passed routine newborn hearing screening, indicating that concurrent screening provides a more comprehensive approach for management of congenital deafness and prevention of ototoxicity.

Colonic Transit Disorder Mediated by Downregulation of Interstitial Cells of Cajal/Anoctamin-1 in Dextran Sodium Sulfate-induced Colitis Mice.

BACKGROUND/AIMS: Interstitial cells of Cajal (ICC) and their special calcium-activated chloride channel, anoctamin-1 (ANO1) play pivotal roles in regulating colonic transit. This study is designed to investigate the role of ICC and the ANO1 channel in colonic transit disorder in dextran sodium sulfate (DSS)-treated colitis mice. METHODS: Colonic transit experiment, colonic migrating motor complexes (CMMCs), smooth muscle spontaneous contractile experiments, intracellular electrical recordings, western blotting analysis, and quantitative polymerase chain reaction were applied in this study. RESULTS: The mRNA and protein expressions of c-KIT and ANO1 channels were significantly decreased in the colons of DSS-colitis mice. The colonic artificial fecal-pellet transit experiment in vitro was significantly delayed in DSS-colitis mice. The CMMCs and smooth muscle spontaneous contractions were significantly decreased by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), an ANO1 channel blocker, and NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of nitric oxide synthase activity, in DSS-colitis mice compared with that of control mice. Intracellular electrical recordings showed that the amplitude of NPPB-induced hyperpolarization was more positive in DSS-colitis mice. The electric field stimulation-elicited nitric-dependent slow inhibitory junctional potentials were also more positive in DSS-colitis mice than those of control mice. CONCLUSION: The results suggest that colonic transit disorder is mediated via downregulation of the nitric oxide/ICC/ANO1 signalling pathway in DSS-colitis mice.

Diabetes-induced damage of gastric nitric oxide neurons mediated by P2X7R in diabetic mice.

It is generally considered that enteric neuropathy is one of the causative factors in diabetic gastroparesis. Our previous study demonstrated that there is a loss of NOS neurons in diabetic mice. However, the underlying mechanism remains unclear. The present study was designed to clarify the relationship between neuronal P2X7R and NOS neuron damage. The effect of P2X7R on diabetes-induced gastric NOS neurons damage and its mechanism were investigated by using quantitative RT-PCR，immunofluorescence, western blot, isometric force recording, intracellular calcium ([Ca2+]i) measurement and whole-cell patch clamp techniques. The immunohistochemistry and western blot results showed that nNOS expression was significantly down-regulated in diabetic mice, meanwhile, electric field stimulation-induced NOS sensitive relaxation was significantly suppressed. Myenteric neurons expressed P2X7R and pannexin1, and the mRNA and protein level of P2X7R and pannexin1 were up-regulated in diabetic mice. BzATP, a P2X7R activator, evoked [Ca2+]i increase in Hek293 cells with heterologous expression of P2X7R (Hek293-P2X7R cells) and the same dose of ATP-induced [Ca2+]i was more obvious in Hek293-P2X7R cells than in Hek293 cells. Application of BzATP activated an inward current of Hek293-P2X7R in a dose dependent manner. Hek293-P2X7R but not untransfected Hek293 cells could take up of YO-PRO-1. In addition, the uptake of YO-PRO-1 by Hek293-P2X7R was blocked by oxATP, a P2X7 antagonist and CBX, a pannexin1 inhibitor. The results suggest that the P2X7R of enteric neurons may be involved in diabetes-induced NOS neuron damage via combining with pannexin-1 to form transmembrane pores which induce macromolecular substances and calcium into the cells.

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/platelet-derived growth factor receptor-α positive cell syncytium in mice.

AIM: To investigate the distribution and function of interstitial cells 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 stimulation-induced 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 stimulation-induced 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.

The functional role of protease-activated receptors on contractile responses by activation of Ca2+ sensitization pathways in simian colonic muscles.

It has been known that activation of protease-activated receptors (PARs) affects gastrointestinal motility. In this study, we tested the effects of PAR agonists on electrical and contractile responses and Ca2+ sensitization pathways in simian colonic muscles. The Simian colonic muscle was initially hyperpolarized by PAR agonists. After the transient hyperpolarization, simian colonic muscle repolarized to the control resting membrane potential (RMP) without a delayed depolarization. Apamin significantly reduced the initial hyperpolarization, suggesting that activation of small conductance Ca2+-activated K+ (SK) channels is involved in the initial hyperpolarization. In contractile experiments, PAR agonists caused an initial relaxation followed by an increase in contractions. These delayed contractile responses were not matched with the electrical responses that showed no after depolarization of the RMP. To investigate the possible involvement of Rho-associated protein kinase 2 (ROCK) pathways in the PAR effects, muscle strips were treated with ROCK inhibitors, which significantly reduced the PAR agonist-induced contractions. Furthermore, PAR agonists increased MYPT1 phosphorylation, and ROCK inhibitors completely blocked MYPT1 phosphorylation. PAR agonists alone had no effect on CPI-17 phosphorylation. In the presence of apamin, PAR agonists significantly increased CPI-17 phosphorylation, which was blocked by protein kinase C (PKC) inhibitors suggesting that Ca2+ influx is increased by apamin and is activating PKC. In conclusion, these studies show that PAR activators induce biphasic responses in simian colonic muscles. The initial inhibitory responses by PAR agonists are mainly mediated by activation of SK channels and delayed contractile responses are mainly mediated by the CPI-17 and ROCK Ca2+ sensitization pathways in simian colonic muscles. NEW & NOTEWORTHY In the present study, we found that the contractile responses of simian colonic muscles to protease-activated receptor (PAR) agonists are different from the previously reported contractile responses of murine colonic muscles. Ca2+ sensitization pathways mediate the contractile responses of simian colonic muscles to PAR agonists without affecting the membrane potential. These findings emphasize novel mechanisms of PAR agonist-induced contractions possibly related to colonic dysmotility in inflammatory bowel disease.

Molecular and functional characterization of inwardly rectifying K+ currents in murine proximal colon.

KEY POINTS: Interstitial cells of Cajal (ICC) from murine colonic muscles express genes encoding inwardly rectifying K+ channels. Transcripts of Kcnj2 (Kir2.1), Kcnj4 (Kir2.3), Kcnj14 (Kir2.4), Kcnj5 (Kir3.4), Kcnj8 (Kir 6.1) and Kcnj11 (Kir6.2) were found in colonic ICC. A conductance with properties consistent with Kir2 channels was observed in ICC but not in smooth muscle cells (SMC). Despite expression of gene transcripts, G-protein gated K+ channel (Kir3) and KATP (Kir6) currents were not resolved in ICC. KATP is a conductance prominent in SMC. Kir2 antagonist caused depolarization of freshly dispersed ICC and colonic smooth muscles, suggesting that this conductance is active under resting conditions in colonic muscles. The conclusion of the present study is that ICC express the Ba2+ -sensitive, inwardly rectifying K+ conductance in colonic muscles. This conductance is most probably a result of heterotetramers of Kir2 gene products, with this regulating resting potentials and the excitability of colonic muscles. ABSTRACT: Membrane potentials of gastrointestinal muscles are important because voltage-dependent Ca2+ channels in smooth muscle cells (SMC) provide the Ca2+ that triggers contraction. Regulation of membrane potential is complicated because SMC are electrically coupled to interstitial cells of Cajal (ICC) and PDGFRα+ cells. Activation of conductances in any of these cells affects the excitability of the syncytium. We explored the role of inward rectifier K+ conductances in colonic ICC that might contribute to regulation of membrane potential. ICC expressed Kcnj2 (Kir2.1), Kcnj4 (Kir2.3), Kcnj14 (Kir2.4), Kcnj5 (Kir3.4), Kcnj8 (Kir 6.1) and Kcnj11 (Kir6.2). Voltage clamp experiments showed activation of inward current when extracellular K+ ([K+ ]o ) was increased. The current was inwardly rectifying and inhibited by Ba2+ (10 µm) and ML-133 (10 µm). A similar current was not available in SMC. The current activated in ICC by elevated [K+ ]o was not affected by Tertiapin-Q. Gßγ, when dialysed into cells, failed to activate a unique, Tertiapin-Q-sensitive conductance. Freshly dispersed ICC showed no evidence of functional KATP . Pinacidil failed to activate current and the inward current activated by elevated [K+ ]o was insensitive to glibenclamide. Under current clamp, ML-133 caused the depolarization of isolated ICC and also that of cells impaled with microelectrodes in intact muscle strips. These findings show that ICC, when isolated freshly from colonic muscles, expressed a Ba2+ -sensitive, inwardly rectifying K+ conductance. This conductance is most probably a result of the expression of multiple Kir2 family paralogues, and the inwardly rectifying conductance contributes to the regulation of resting potentials and excitability of colonic muscles.

Colonic PDGFRα Overexpression Accompanied Forkhead Transcription Factor FOXO3 Up-Regulation in STZ-Induced Diabetic Mice.

BACKGROUND: Colonic transit disorder-induced constipation is a major complication in diabetic patients. PDGFRα+ (platelet-derived growth factor receptor α-positive) cells play critical roles in the inhibitory regulation of colonic motility, and FOXO3 (forkhead transcription factor 3) has a broad range of biological functions. The present study was designed to investigate the relationship between FOXO3 and PDGFRα+ cell proliferation in streptozotocin (STZ)-induced diabetic mice. METHODS: The major experimental techniques used in this paper are immunohistochemistry, quantitative RT-RCR and Western blotting for the evaluation of specific protein expression; ChIP assay for identifying the interaction between FOXO3 protein and the PDGFRα promotor; and lentiviral transfection for the overexpression of short hairpin RNAs (shRNAs) to down-regulate FOXO3. RESULTS: In proximal colonic smooth muscle tissue of STZ-induced diabetic mice, there was a significant increase in PDGFRα and Ki67 immunoreactivity. PDGFRα mRNA and protein expression levels were both significantly increased in colonic smooth muscle tissue, but PDGFRß expression was unchanged. Meanwhile, the expression of PDGF ligands, including both PDGFα and PDGFß, was significantly increased in diabetic colonic smooth muscle tissue. In whole cell and nuclear extracts, the expression of FOXO3 protein was also significantly increased; however, the expression of P-FOXO3 (phosphorylated FOXO3) protein was significantly decreased. When NIH cells were incubated with 50 mmol/L glucose for 12 h, 24 h and 48 h, the expression of PDGFRα significantly increased, and in whole cell and nuclear extracts, the expression of FOXO3 protein was significantly increased. However, the expression of P-FOXO3 protein was significantly decreased. FOXO3 could bind to a site on the PDGFRα promoter, and the basal expression of PDGFRα was significantly reduced when endogenous FOXO3 expression was knocked down with FOXO3 short hairpin RNA (shRNA) in NIH cells. The expression of phosphorylated Akt was significantly down-regulated in diabetic colonic muscle tissue. CONCLUSIONS: These results suggest that diabetes-induced colonic PDGFRα+ cell proliferation is mediated by FOXO3 up-regulation. FOXO3 up-regulation may be induced by inhibiting the PI3K/Akt signaling pathway in STZ-induced diabetic mice. PDGFRα+ cell proliferation could be a new target for clinical therapy of diabetes-induced colonic transit disorder.

H2 S-induced gastric fundus smooth muscle tension potentiation is mediated by the phosphoinositide 3-kinase/Akt/endothelial nitric oxide synthase pathway.

NEW FINDINGS: What is the central question of this study? The present study investigated the relationship between H2 S and NO in regulation of gastric fundus tension. What is the main finding and its importance? Endogenous or exogenous H2 S and NO have opposite effects on fundus tension, and H2 S-induced gastric fundus tension enhancements are mediated by inhibition of NO generation through the phosphoinositide 3-kinase/Akt pathway. These results are very important in exploring the mechanism of physiological accommodation and accommodation disorder. Hydrogen sulphide (H2 S) is considered a new gasotransmitter, along with NO and CO. It was recently confirmed that H2 S and NO play important roles in the regulation of gastrointestinal smooth muscle tension. The present study was designed to elucidate the interactions between H2 S and NO with respect to the regulation of gastric fundus smooth muscle tension using Western blotting, physiological and electrochemical techniques. Real-time H2 S and NO generation was detected in gastric smooth muscle tissue. NaHS, an H2 S donor, enhanced fundus smooth muscle tension, whereas SNP, an NO donor, decreased fundus smooth muscle tension in a dose-dependent manner. NaHS-induced increases in fundus smooth muscle tension were suppressed by l-NAME, an NO synthase inhibitor. Aminooxyacetic acid (AOAA), a cystathionine ß-synthase inhibitor, exerted inhibitory effects on fundus smooth muscle tension; these effects were also suppressed by l-NAME. Real-time NO generation was significantly potentiated by AOAA. Endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 and Akt phosphorylation at serine 308 and threonine 473 were significantly inhibited by NaHS. LY294002, a phosphoinositide 3-kinase inhibitor, blocked these NaHS-mediated effects. However, eNOS phosphorylation at serine 1177 and Akt phosphorylation at serine 308 and threonine 473 were significantly potentiated by AOAA. Cystathionine ß-synthase siRNA interference significantly increased eNOS phosphorylation at serine 1177 and Akt phosphorylation at serine 308 and threonine 473. Cystathionine ß-synthase siRNA interference also increased total eNOS protein expression levels but did not significantly change total Akt kinase protein expression levels. These results suggest that H2 S-induced enhancement of gastric fundus tension is mediated by inhibition of NO generation through the phosphoinositide 3-kinase/Akt pathway.

Hydrogen sulfide-induced enhancement of gastric fundus smooth muscle tone is mediated by voltage-dependent 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 cystathionine ß-synthase (CBS) and cystathionine γ-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 cultured smooth muscle cells 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 NaHS-induced increase in tonic contraction was blocked by 5 mmol/L 4-aminopyridine and 1 µmol/L nicardipine. NaHS significantly depolarized the membrane potential and inhibited the voltage-dependent potassium currents. Moreover, NaHS increased L-type Ca(2+) currents and caused an elevation in intracellular calcium ([Ca(2+)]i). CONCLUSION: These findings suggest that H2S may be an excitatory modulator in the gastric fundus in mice. The excitatory effect is mediated by voltage-dependent potassium and L-type calcium channels.

Protease-activated receptors modulate excitability of murine colonic smooth muscles by differential effects on interstitial cells.

Protease-activated receptors (PARs) are G protein-coupled receptors activated by proteolytic cleavage at their amino termini by serine proteases. PAR activation contributes to the inflammatory response in the gastrointestinal (GI) tract and alters GI motility, but little is known about the specific cells within the tunica muscularis that express PARs and the mechanisms leading to contractile responses. Using real time PCR, we found PARs to be expressed in smooth muscle cells (SMCs), interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor α positive (PDGFRα(+)) cells. The latter cell-type showed dominant expression of F2r (encodes PAR1) and F2rl1 (encodes PAR2). Contractile and intracellular electrical activities were measured to characterize the integrated responses to PAR activation in whole muscles. Cells were isolated and ICC and PDGFRα(+) cells were identified by constitutive expression of fluorescent reporters. Thrombin (PAR1 agonist) and trypsin (PAR2 agonist) caused biphasic responses in colonic muscles: transient hyperpolarization and relaxation followed by repolarization and excitation. The inhibitory phase was blocked by apamin, revealing a distinct excitatory component. Patch clamp studies showed that the inhibitory response was mediated by activation of small conductance calcium-activated K(+) channels in PDGFRα(+) cells, and the excitatory response was mediated by activation of a Cl(-) conductance in ICC. SMCs contributed little to PAR responses in colonic muscles. In summary, PARs regulate the excitability of colonic muscles; different conductances are activated in each cell type of the SMC-ICC-PDGFRα(+) cell (SIP) syncytium. Motor responses to PAR agonists are integrated responses of the SIP syncytium.