Supramolecular DNA-based catalysis in organic solvents.

The distinct folding accompanied by its polymorphic character renders DNA G-quadruplexes promising biomolecular building blocks to construct novel DNA-based and supramolecular assemblies. However, the highly polar nature of DNA limits the use of G-quadruplexes to water as a solvent. In addition, the archetypical G-quadruplex fold needs to be stabilized by metal-cations, which is usually a potassium ion. Here, we show that a noncovalent PEGylation process enabled by electrostatic interactions allows the first metal-free G-quadruplexes in organic solvents. Strikingly, incorporation of an iron-containing porphyrin renders the self-assembled metal-free G-quadruplex catalytically active in organic solvents. Hence, these "supraG4zymes" enable DNA-based catalysis in organic media. The results will allow the broad utilization of DNA G-quadruplexes in nonaqueous environments.

Shape morphing of hydrogels by harnessing enzyme enabled mechanoresponse.

Hydrogels have been designed to react to many different stimuli which find broad applications in tissue engineering and soft robotics. However, polymer networks bearing mechano-responsiveness, especially those displaying on-demand self-stiffening and self-softening behavior, are rarely reported. Here, we design a mechano-controlled biocatalytic system at the molecular level that is incorporated into hydrogels to regulate their mechanical properties at the material scale. The biocatalytic system consists of the protease thrombin and its inhibitor, hirudin, which are genetically engineered and covalently coupled to the hydrogel networks. The catalytic activity of thrombin is reversibly switched on by stretching of the hydrogels, which disrupts the noncovalent inhibitory interaction between both entities. Under cyclic tensile-loading, hydrogels exhibit self-stiffening or self-softening properties when substrates are present that can self-assemble to form new networks after being activated by thrombin or when cleavable peptide crosslinkers are constitutional components of the original network, respectively. Additionally, we demonstrate the programming of bilayer hydrogels to exhibit tailored shape-morphing behavior under mechanical stimulation. Our developed system provides proof of concept for mechanically controlled reversible biocatalytic processes, showcasing their potential for regulating hydrogels and proposing a biomacromolecular strategy for mechano-regulated soft functional materials.

Recombinant supercharged polypeptides for safe and efficient heparin neutralization.

Heparin is a widely used anticoagulant agent in the clinic. After application, its anticoagulant effect must be reversed to prevent potential side effects. Protamine sulfate (PS) is the only clinically licensed antidote that has been used for this purpose in the last 80 years, which, however, provokes severe adverse effects, such as systemic hypotension and even death. Herein, we demonstrate the potential of supercharged polypeptides as a promising alternative for protamine sulfate. A series of supercharged polypeptides with multiple positive charges was recombinantly produced, and the heparin-neutralizing performance of the polypeptides was evaluated in comparison with PS. It was found that increasing the number of charges significantly enhanced the ability to neutralize heparin and resist the screening effect induced by salt. In particular, the polypeptide bearing 72 charges (K72) exhibited an excellent heparin-neutralizing behavior that was comparable to that of PS. Further in vivo studies revealed that the heparin-triggered bleeding was almost completely alleviated by K72 while a negligible toxic effect was observed. Therefore, such recombinant supercharged polypeptides might replace protamine sulfate as heparin-reversal agents.

Polymer Mechanochemistry in Microbubbles.

Polymer mechanochemistry is a promising technology to convert mechanical energy into chemical functionality by breaking covalent and supramolecular bonds site-selectively. Yet, the mechanochemical reaction rates of covalent bonds in typically used ultrasonication setups lead to reasonable conversions only after comparably long sonication times. This can be accelerated by either increasing the reactivity of the mechanoresponsive moiety or by modifying the encompassing polymer topology. Here, a microbubble system with a tailored polymer shell consisting of an N2 gas core and a mechanoresponsive disulfide-containing polymer network is presented. It is found that the mechanochemical activation of the disulfides is greatly accelerated using these microbubbles compared to commensurate solid core particles or capsules filled with liquid. Aided by computational simulations, it is found that low shell thickness, low shell stiffness and crosslink density, and a size-dependent eigenfrequency close to the used ultrasound frequency maximize the mechanochemical yield over the course of the sonication process.

Programming DNA Circuits for Controlled Immunostimulation through CpG Oligodeoxynucleotide Delivery.

Herein, we present a DNA circuit programmed for the delivery of CpG oligodeoxynucleotides (CpG ODNs) with the pharmacological immunostimulation function. The circuit employs a complementary DNA (cDNA) strand to deactivate the biological function of CpG ODNs via hybridization, while T7 exonuclease mediates the activation by hydrolyzing the cDNA and releasing the CpG ODN as an active moiety. We investigated the influence of several factors on the kinetic profile and temporal behavior of the circuit. These include the design of the cDNA strand, the concentration of the DNA duplex, and the concentration of T7 exonuclease. The DNA circuit's in vitro activation resulted in toll-like receptor 9 stimulation in the HEK-engineered cell line, as well as tumor necrosis factor-alpha release by J774A.1 macrophages. By programming the DNA circuit to control the release of the CpG ODN, we achieved an altered pharmacological profile with acute and potent immunostimulation, in comparison to a system without controlled CpG ODN release, which exhibited a slow and delayed response. Our findings demonstrate the potential of DNA circuits in controlling the pharmacological activity of DNA strands for controlled drug delivery.

Optically Controlled Construction of Three-Dimensional Protein Arrays.

Protein crystallization is an important tool for structural biology and nanostructure preparation. Here, we report on kinetic pathway-dependent protein crystals that are controlled by light. Photo-responsive crystallites are obtained by complexing the model proteins with cationic azobenzene dyes. The crystalline state is readily switched to a dispersed phase under ultraviolet light and restored by subsequent visible-light illumination. The switching can be reversibly repeated for multiple cycles without noticeable structure deterioration. Importantly, the photo-treatment not only significantly increases the crystallinity, but creates crystallites at conditions where no ordered lattices are observed upon directly mixing the components. Further control over the azobenzene isomerization kinetics produces protein single crystals of up to ≈50 µm. This approach offers an intriguing method to fabricate metamaterials and study optically controlled crystallization.

Fracture Detection in Bio-Glues with Fluorescent-Protein-Based Optical Force Probes.

Glues are being used to bond, seal, and repair in industry and biomedicine. The improvement of gluing performance is hence important for the development of new glues with better and balanced property spaces, which in turn necessitates a mechanistic understanding of their mechanical failure. Optical force probes (OFPs) allow the observation of mechanical material damage in polymers from the macro- down to the microscale, yet have never been employed in glues. Here, the development of a series of ratiometric OFPs based on fluorescent-protein-dye and protein-protein conjugates and their incorporation into genetically engineered bio-glues is reported. The OFPs are designed to efficiently modulate Förster resonance energy transfer upon force application thereby reporting on force-induced molecular alterations independent of concentration and fluorescence intensity both spectrally and through their fluorescence lifetime. By fluorescence spectroscopy in solution and in the solid state and by fluorescence lifetime imaging microscopy, stress concentrations are visualized and adhesive and cohesive failure in the fracture zone is differentiated.

Piezoelectric Metal-Organic Frameworks Mediated Mechanoredox Borylation and Arylation Reactions by Ball Milling.

Herein, metal-organic frameworks UiO-66 and UiO-66-NH2 were synthesized, and their piezoelectric properties were characterized by piezoresponse force microscopy. Under mechanical agitation, the highly polarized UiO-66 and UiO-66-NH2 can act as electron donors to reduce aryl diazonium salts, forming the aryl radical species, which further react with bis(pinacolato)diborane or heteroarenes to generate the desired products with broad substrate scope. For all substrates, it was found that the reactions mediated by UiO-66-NH2 , which exhibited better piezoelectric property, were more efficient than those promoted by UiO-66. Moreover, it was shown that the convenient regeneration of spent catalysts can be reused for promoting mechanoredox reactions without loss of activities by taking advantage of the unmatched repairability of metal-organic frameworks. Mechanistic studies revealed that a radical pathway was involved in these transformations.

[Dual regulation of Qizhiweitong particles on gastric motility in the model rats with gastroparesis].

This paper was aimed to study the effects of Qizhiweitong particles (QZWT) on gastric motility in gastroparesis model rats, and to provide a theoretical and experimental basis for its clinical treatment. Rat gastroparesis model was established by bilateral injection of 6-hydroxydopamine into the substantia nigra in male Sprague-Dawley (SD) rats. The model rats received single gastric feeding of 1, 10, 30, 100, 200, 450, or 675 mg/kg QZWT or continuous administration of 675 mg/kg QZWT per day for 7 days. The gastric motility was measured by gastric emptying study and in vivo digital X-ray imaging system. The in vivo and ex vivo gastric longitudinal muscle contraction was recorded by PowerLab biological signal acquisition system. Gastric myoelectric signals were recorded by wireless implantable telemetry system. Protein expression levels of proinflammatory proteases in the myometrium were determined by Western blot. The results showed that the single administration of QZWT dose-dependently inhibited the contractile activity of isolated gastric strips from normal rats. The single administration of QZWT inhibited the in vivo contraction of gastric smooth muscle and gastric myoelectric signal in the control and model rats. The gastric emptying rate, in vivo and ex vivo gastric motility and gastric myoelectric signal in the model rats were significantly decreased compared with those in the control rats; While the continuous administration of QZWT markedly improved all the above indices of gastric motility function. The single administration of QZWT inhibited isolated gastric muscle strip contraction, and neither atropine nor nitric oxide synthase inhibitor pretreatments affected QZWT's inhibitory effects. The continuous administration of QZWT down-regulated the increased protein expression levels of nitric oxide synthase and cyclooxygenase 2 in the model group. These results suggest that, in clinical treatment, the single administration of QZWT may induce an analgesic effect by rapidly inhibiting gastric motility, while this effect is not related to acetylcholine or nitric oxide pathways. Long-term treatment with QZWT may ameliorate gastric motility through enhancing myoelectric activities, gastric smooth muscle contraction and gastric emptying, and this effect may partly be related to its anti-inflammatory effect.

Mechanochemical Divergent Syntheses of Oxindoles and α-Arylacylamides via Controllable Construction of C-C and C-N Bonds by Copper and Piezoelectric Materials.

Herein, we present the divergent syntheses of α-arylacylamides and oxindoles via mechanoredox chemistry by using easily accessible α-bromo N-sulfonyl amides as starting materials. Our system consists of a catalytic amount of CuII precatalyst and piezoelectric materials. The highly polarized BaTiO3 or PbTiO3 under mechanical agitation can act as an electron donor to realize the recycling of CuII and CuI . Control experiments and density functional theory calculations have been performed to support the proposed mechanistic rationale for the cascade reactions.

Impaired nitrergic relaxation in pyloric sphincter of the 6-OHDA Parkinson's disease rat.

Patients with Parkinson's disease (PD) often suffer from delayed gastric emptying, but the underlying mechanism remains unclear. We have shown previously that a PD rat model comprising bilateral substantia nigra destruction by 6-hydroxydopamine (6-OHDA rats) exhibits gastroparesis with alteration of neural nitric oxide synthase (nNOS) and acetylcholine in gastric corpus. However, changes in pyloric motility in the 6-OHDA rats have not been characterized. Solid gastric emptying test, immunofluorescence, Western blot, and in vitro pyloric motility recordings were used to assess pyloric motor function in the 6-OHDA rats. The 6-OHDA-treated rats displayed delayed solid gastric emptying and a lower basal pyloric motility index. In the 6-OHDA rats, high K+-induced transient contractions were weaker in pyloric sphincters. Electric field stimulation (EFS)-induced pyloric sphincter relaxation was lower in the 6-OHDA rats. NG-nitro-l-arginine methyl ester (l-NAME), a nonselective inhibitor of NOS, markedly inhibited the EFS-induced relaxation in both control and 6-OHDA rats. Pretreatment of tetrodotoxin abolished the effect of EFS on the pyloric motility. In addition, nNOS-positive neurons were extensively distributed in the pyloric myenteric plexus, whereas the number of nNOS-immunoreactive neurons and the protein expression of nNOS were significantly decreased in the pyloric muscularis of 6-OHDA rats. However, sodium nitroprusside-induced pyloric relaxations were similar between the control and 6-OHDA rats. These results indicate that the pyloric sphincters of 6-OHDA rats exhibit both weakened contraction and relaxation. The latter may be due to reduced nNOS in the pyloric myenteric plexus. The dysfunction of the pyloric sphincter might be involved in the delayed gastric emptying.NEW & NOTEWORTHY Reduced nitrergic neurons in pyloric myenteric plexus potently contributed to the attenuated relaxation in 6-hydroxydopamine (6-OHDA) rats, subsequently affecting gastric emptying. SNP could well improve the relaxation of pylori in 6-OHDA rats. The present study provides new insight into the diagnosis and treatment of delayed gastric emptying in patients with PD.

Activation of dopamine D2 receptor promotes pepsinogen secretion by suppressing somatostatin release from the mouse gastric mucosa.

In vivo administration of dopamine (DA) receptor (DR)-related drugs modulate gastric pepsinogen secretion. However, DRs on gastric pepsinogen-secreting chief cells and DA D2 receptor (D2R) on somatostatin-secreting D cells were subsequently acquired. In this study, we aimed to further investigate the local effect of DA on gastric pepsinogen secretion through DRs expressed on chief cells or potential D2Rs expressed on D cells. To elucidate the modulation of DRs in gastric pepsinogen secretion, immunofluorescence staining, ex vivo incubation of gastric mucosa isolated from normal and D2R-/- mice were conducted, accompanied by measurements of pepsinogen or somatostatin levels using biochemical assays or enzyme-linked immunosorbent assays. D1R, D2R, and D5R-immunoreactivity (IR) were observed on chief cells in mouse gastric mucosa. D2R-IR was widely distributed on D cells from the corpus to the antrum. Ex vivo incubation results showed that DA and the D1-like receptor agonist SKF38393 increased pepsinogen secretion, which was blocked by the D1-like receptor antagonist SCH23390. However, D2-like receptor agonist quinpirole also significantly increased pepsinogen secretion, and D2-like receptor antagonist sulpiride blocked the promotion of DA. Besides, D2-like receptors exerted an inhibitory effect on somatostatin secretion, in contrast to their effect on pepsinogen secretion. Furthermore, D2R-/- mice showed much lower basal pepsinogen secretion but significantly increased somatostatin release and an increased number of D cells in gastric mucosa. Only SKF38393, not quinpirole, increased pepsinogen secretion in D2R-/- mice. DA promotes gastric pepsinogen secretion directly through D1-like receptors on chief cells and indirectly through D2R-mediated suppression of somatostatin release.

Light manipulation for fabrication of hydrogels and their biological applications.

The development of biocompatible materials with desired functions is essential for tissue engineering and biomedical applications. Hydrogels prepared from these materials represent an important class of soft matter for mimicking extracellular environments. In particular, dynamic hydrogels with responsiveness to environments are quite appealing because they can match the dynamics of biological processes. Among the external stimuli that can trigger responsive hydrogels, light is considered as a clean stimulus with high spatiotemporal resolution, complete bioorthogonality, and fine tunability regarding its wavelength and intensity. Therefore, photoresponsiveness has been broadly encoded in hydrogels for biological applications. Moreover, light can be used to initiate gelation during the fabrication of biocompatible hydrogels. Here, we present a critical review of light manipulation tools for the fabrication of hydrogels and for the regulation of physicochemical properties and functions of photoresponsive hydrogels. The materials, photo-initiated chemical reactions, and new prospects for light-induced gelation are introduced in the former part, while mechanisms to render hydrogels photoresponsive and their biological applications are discussed in the latter part. Subsequently, the challenges and potential research directions in this area are discussed, followed by a brief conclusion. STATEMENT OF SIGNIFICANCE: Hydrogels play a vital role in the field of biomaterials owing to their water retention ability and biocompatibility. However, static hydrogels cannot meet the dynamic requirements of the biomedical field. As a stimulus with high spatiotemporal resolution, light is an ideal tool for both the fabrication and operation of hydrogels. In this review, light-induced hydrogelation and photoresponsive hydrogels are discussed in detail, and new prospects and emerging biological applications are described. To inspire more research studies in this promising area, the challenges and possible solutions are also presented.

Activation of α7nAChR Protects Against Gastric Inflammation and Dysmotility in Parkinson's Disease Rats.

The cholinergic anti-inflammatory pathway (CAIP) has been proposed to regulate gastrointestinal inflammation via acetylcholine released from the vagus nerve activating α7 nicotinic receptor (α7nAChR) on macrophages. Parkinson's disease (PD) patients and PD rats with substantia nigra (SN) lesions exhibit gastroparesis and a decayed vagal pathway. To investigate whether activating α7nAChR could ameliorate inflammation and gastric dysmotility in PD rats, ELISA, western blot analysis, and real-time PCR were used to detect gastric inflammation. In vitro and in vivo gastric motility was investigated. Proinflammatory mediator levels and macrophage numbers were increased in the gastric muscularis of PD rats. α7nAChR was located on the gastric muscular macrophages of PD rats. The α7nAChR agonists PNU-282987 and GTS-21 decreased nuclear factor κB (NF-κB) activation and monocyte chemotactic protein-1 mRNA expression in the ex vivo gastric muscularis of PD rats, and these effects were abolished by an α7nAChR antagonist. After treatment with PNU-282987 in vivo, the PD rats showed decreased NF-κB activation, inflammatory mediator production, and contractile protein expression and improved gastric motility. The present study reveals that α7nAChR is involved in the development of gastroparesis in PD rats and provides novel insight for the treatment of gastric dysmotility in PD patients.

Identification and a phased pH control strategy of diosgenin bio-synthesized by an endogenous Bacillus licheniformis Syt1 derived from Dioscorea zingiberensis C. H. Wright.

Diosgenin is widely used as one precursor of steroidal drugs in pharmaceutical industry. Currently, there is no choice but to traditionally extract diosgenin from Dioscorea zingiberensis C. H. Wright (DZW) or other plants. In this work, an environmentally friendly approach, in which diosgenin can be bio-synthesized by the endophytic bacterium Bacillus licheniformis Syt1 isolated from DZW, is proposed. Diosgenin produced by the strain was identified by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and Fourier transform infrared spectroscopy (FTIR). The thermal gravimetric analysis (TGA) showed that the melting point of the diosgenin product was 204 °C. The optical rotation measurement exhibited that the optical rotation was α20589 = - 126.1° ± 1.5° (chloroform, c = 1%): negative sign means that the product is left-handed, which is very important to further produce steroid hormone drugs. Cholesterol may be the intermediate product in the diosgenin biosynthesis pathway. In the batch fermentation process to produce diosgenin using the strain, pH values played an important role. A phased pH control strategy from 5.5 to 7.5 was proved to be more effective to improve production yield than any single pH control, which could get the highest diosgenin yield of 85 ± 8.6 mg L-1. The proposed method may replace phyto-chemistry extraction to produce diosgenin in the industry in the future.Key points• An endophytic Bacillus licheniformis Syt1 derived from host can produce diosgenin.• A dynamic pH industrial control strategy is better than any single pH control.• Proposed diosgenin-produced method hopefully replaces phyto-chemistry extraction.

Reduced acetylcholine and elevated muscarinic receptor 2 in duodenal mucosa contribute to the impairment of mucus secretion in 6-hydroxydopamine-induced Parkinson's disease rats.

Patients with Parkinson's disease (PD) have a higher incidence rate of duodenal ulcers. The mucus barrier provides the first line of defense for duodenal mucosal protection. However, it is unknown whether duodenal mucus secretion is affected in PD. In the present study, we used the rats microinjected 6-hydroxydopamine (6-OHDA) into the bilateral substantia nigra to investigate duodenal mucus secretion and potential therapeutic targets in duodenal ulcer in PD. Alcian blue-periodic acid-Schiff, transmission electron microscopy, immunofluorescence, duodenal mucosal incubation, and enzyme-linked immunosorbent assays were used. The 6-OHDA rats exhibited mucin accumulation and retention in duodenal goblet cells. Mucin granules were unable to fuse with the apical membranes of goblet cells, and the exocytosis ratio of goblet cells was significantly reduced. Moreover, decreased acetylcholine and increased muscarinic receptor 2 (M2R) levels were detected in the duodenal mucosa of 6-OHDA rats. Bilateral vagotomy rats were also characterized by defective duodenal mucus secretion and decreased acetylcholine with increased M2R levels in the duodenal mucosa. Application of the cholinomimetic drug carbachol or blocking M2R with methoctramine significantly promoted mucus secretion by goblet cells and increased MUC2 content in duodenal mucosa-incubated solutions from 6-OHDA and vagotomy rats. We conclude that the reduced acetylcholine and increased M2R contribute to the impaired duodenal mucus secretion of 6-OHDA rats. The study provides new insights into the mechanism of duodenal mucus secretion and potential therapeutic targets for the treatment of duodenal ulcers in PD patients.

Photochemical control of bacterial gene expression based on trans encoded genetic switches.

Controlling gene expression by light with fine spatiotemporal resolution not only allows understanding and manipulating fundamental biological processes but also fuels the development of novel therapeutic strategies. In complement to exploiting optogenetic tools, photochemical strategies mostly rely on the incorporation of photo-responsive small molecules into the corresponding biomacromolecular scaffolds. Therefore, generally large synthetic effort is required and the switching of gene expression in both directions within a single system remains a challenge. Here, we report a trans encoded ribo-switch, which consists of an engineered tRNA mimicking structure (TMS), under control of small photo-switchable signalling molecules. The signalling molecules consist of two amino glycoside molecules that are connected via an azobenzene unit. The light responsiveness of our system originates from the photo-switchable noncovalent interactions between the signalling molecule and the TMS switch, leading to the demonstration of photochemically controlled expression of two different genes. We believe that this modular design will provide a powerful platform for controlling the expression of other functional proteins with high spatiotemporal resolution employing light as a stimulus.

Enhanced Contractive Tension and Upregulated Muscarinic Receptor 2/3 in Colorectum Contribute to Constipation in 6-Hydroxydopamine-Induced Parkinson's Disease Rats.

Constipation and defecatory dysfunctions are frequent symptoms in patients with Parkinson's disease (PD). The pathology of Lewy bodies in colonic and rectal cholinergic neurons suggests that cholinergic pathways are involved in colorectal dysmotility in PD. However, the underlying mechanism is unclear. The aim of the present study is to examine the effect of central dopaminergic denervation in rats, induced by injection 6-hydroxydopamine into the bilateral substania nigra (6-OHDA rats), on colorectal contractive activity, content of acetylcholine (ACh), vasoactive intestinal peptide (VIP) and expression of neural nitric oxide synthase (nNOS) and muscarinic receptor (MR). Strain gauge force transducers combined with electrical field stimulation (EFS), gut transit time, immunohistochemistry, ELISA, western blot and ultraperformance liquid chromatography tandem mass spectrometry were used in this study. The 6-OHDA rats exhibited outlet obstruction constipation characterized by prolonged transit time, enhanced contractive tension and fecal retention in colorectum. Pretreatment with tetrodotoxin significantly increased the colorectal motility. EFS-induced cholinergic contractions were diminished in the colorectum. Bethanechol chloride promoted colorectal motility in a dose-dependent manner, and much stronger reactivity of bethanechol chloride was observed in 6-OHDA rats. The ACh, VIP and protein expression of nNOS was decreased, but M2R and M3R were notably upregulated in colorectal muscularis externa. Moreover, the number of cholinergic neurons was reduced in sacral parasympathetic nucleus (SPN) of 6-OHDA rats. In conclusion, central nigrostriatal dopaminergic denervation is associated with decreased cholinergic neurons in SPN, decreased ACh, VIP content, and nNOS expression and upregulated M2R and M3R in colorectum, resulting in colorectal dysmotility, which contributes to outlet obstruction constipation. The study provides new insights into the mechanism of constipation and potential therapeutic targets for constipation in PD patients.

Na+-K+-2Cl- symporter contributes to γ-aminobutyric acid-evoked excitation in rat enteric neurons.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the adult central nervous system (CNS), however, it causes excitation in the immature CNS neurons. The shift from GABA-induced depolarization to hyperpolarization in postnatal brain is primarily due to progressive decrease in the expression of the Na+-K+-2Cl- symporter 1 (NKCC1) and increased expression of the K+-Cl- cotransporter 2 (KCC2). Unlike CNS neurons, both immature and mature neurons in the enteric nervous system (ENS) are depolarized by GABA. Molecular mechanisms by which GABA excites ENS neurons are unclear. It is understood, however, that the excitatory action depends on elevated intraneuronal Cl-. We aimed to test a hypothesis that high intracellular Cl- in ENS neurons is maintained by activity of the NKCCs. We found that NKCC2 immunoreactivity (IR) was expressed in the ENS of the rat colon on postnatal day 1 (P1). The expression level of NKCC2 continuously increased and reached a steady high level on P14 and maintained at that level in adulthood. NKCC1 IR appeared in ENS on P14 and maintained through adulthood. KCC2 IR was not detectable in the ENS in any of the developmental stages. Both NKCC1 IR and NKCC2 IR were co-expressed with GABAA receptors in ENS neurons. Exogenous GABA (1 mmol/L) caused membrane depolarization in the ENS neurons. The reversal potential of GABA-induced depolarization was about -16 mV. Blockade of NKCC by bumetanide (50 µmol/L) or furosemide (300 µmol/L) suppressed the depolarizing responses to GABA. Bumetanide (50 µmol/L) shifted the reversal potential of GABA-induced depolarization in the hyperpolarizing direction. Neither the KCC blocker DIOA (20 µmol/L) nor the Cl-/HCO3- exchanger inhibitor DIDS (200 µmol/L) suppressed GABA-evoked depolarization. The results suggest that ENS neurons continuously express NKCC2 since P1 and NKCC1 since P14, which contribute to the accumulation of Cl- in ENS neurons and GABA-evoked depolarization in neonate and adult ENS neurons. These results provide the first direct evidence for the contribution of both NKCC2 and NKCC1 to the GABAA-mediated depolarization.