Neuronal Nitric Oxide Synthase Regulates Cerebellar Parallel Fiber Slow EPSC in Purkinje Neurons by Modulating STIM1-Gated TRPC3-Containing Channels.

Responding to burst stimulation of parallel fibers (PFs), cerebellar Purkinje neurons (PNs) generate a convolved synaptic response displaying a fast excitatory postsynaptic current (EPSCFast) followed by a slow EPSC (EPSCSlow). The latter is companied with a rise of intracellular Ca2+ and critical for motor coordination. The genesis of EPSCSlow in PNs results from activation of metabotropic type 1 glutamate receptor (mGluR1), oligomerization of stromal interaction molecule 1 (STIM1) on the membrane of endoplasmic reticulum (ER) and opening of transient receptor potential canonical 3 (TRPC3) channels on the plasma membrane. Neuronal nitric oxide synthase (nNOS) is abundantly expressed in PFs and granule neurons (GNs), catalyzing the production of nitric oxide (NO) hence regulating PF-PN synaptic function. We recently found that nNOS/NO regulates the morphological development of PNs through mGluR1-regulated Ca2+-dependent mechanism. This study investigated the role of nNOS/NO in regulating EPSCSlow. Electrophysiological analyses showed that EPSCSlow in cerebellar slices of nNOS knockout (nNOS-/-) mice was significantly larger than that in wildtype (WT) mice. Activation of mGluR1 in cultured PNs from nNOS-/- mice evoked larger TRPC3-channel mediated currents and intracellular Ca2+ rise than that in PNs from WT mice. In addition, nNOS inhibitor and NO-donor increased and decreased, respectively, the TRPC3-current and Ca2+ rise in PNs. Moreover, the NO-donor effectively decreased TRPC3 currents in HEK293 cells expressing WT STIM1, but not cells expressing a STIM1 with cysteine mutants. These novel findings indicate that nNOS/NO inhibits TRPC3-containig channel mediated cation influx during EPSCSlow, at least in part, by S-nitrosylation of STIM1.

Advances in acupuncture regulation on the autonomic nervous system from 2013 to 2022: A bibliometric analysis via citespace.

OBJECTIVE: To understand research advances and frontiers of acupuncture regulation on the autonomic nervous system (ANS) over the past decade through a bibliometric analysis. METHODS: Publications related to acupuncture regulation on the ANS were retrieved from the Web of Science Core Collection (WoSCC) database. CiteSpace software was used to analyze the datasets and generate knowledge maps. RESULTS: A total of 445 relevant publications published between 2013 and 2022 were included in this bibliometric analysis. The number of annual publications fluctuated from 2013 to 2016 but increased gradually from 2016 to 2022. China produced the highest number of publications, while the USA established the most extensive cooperation relationships. China Academy of Chinese Medical Science was the most productive institution. Chen Jiande D.Z. was the most prolific author and Rong Peijing holds the most extensive cooperation network. Han Jisheng was the most co-cited author. Relevant research involved mechanism exploration and clinical efficacy research, and "anti-inflammatory effect" was the most active research topic, especially cholinergic anti-inflammatory mechanisms. The most cited references mainly focused on inflammation. Gastrointestinal and cardiovascular disorders were the most active medical conditions studied in this field. CONCLUSIONS: Research related to acupuncture regulation on the ANS mainly focused on anti-inflammation, and regulating gastrointestinal and cardiovascular function over the past decade. However, the mechanisms of the autonomic effects of acupuncture need further investigation. High-quality clinical studies are required to determine the optimal parameters of acupuncture for clinical application.

Electroacupuncture Promotes Functional Recovery after Facial Nerve Injury in Rats by Regulating Autophagy via GDNF and PI3K/mTOR Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To explore the mechanism of electroacupuncture (EA) in promoting recovery of the facial function with the involvement of autophagy, glial cell line-derived neurotrophic factor (GDNF), and phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway.@*METHODS@#Seventy-two male Sprague-Dawley rats were randomly allocated into the control, sham-operated, facial nerve injury (FNI), EA, EA+3-methyladenine (3-MA), and EA+GDNF antagonist groups using a random number table, with 12 rats in each group. An FNI rat model was established with facial nerve crushing method. EA intervention was conducted at Dicang (ST 4), Jiache (ST 6), Yifeng (SJ 17), and Hegu (LI 4) acupoints for 2 weeks. The Simone's 10-Point Scale was utilized to monitor the recovery of facial function. The histopathological evaluation of facial nerves was performed using hematoxylin-eosin (HE) staining. The levels of Beclin-1, light chain 3 (LC3), and P62 were detected by immunohistochemistry (IHC), immunofluorescence, and reverse transcription-polymerase chain reaction, respectively. Additionally, IHC was also used to detect the levels of GDNF, Rai, PI3K, and mTOR.@*RESULTS@#The facial functional scores were significantly increased in the EA group than the FNI group (P<0.05 or P<0.01). HE staining showed nerve axons and myelin sheaths, which were destroyed immediately after the injury, were recovered with EA treatment. The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats (P<0.01); however, EA treatment reversed these abnormal changes (P<0.01). Meanwhile, EA stimulation significantly increased the levels of GDNF, Rai, PI3K, and mTOR (P<0.01). After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist, the repair effect of EA on facial function was attenuated (P<0.05 or P<0.01).@*CONCLUSIONS@#EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI. EA may exert this neuroreparative effect through mediating the release of GDNF, activating the PI3K/mTOR signaling pathway, and further regulating the autophagy of facial nerves.

L-alanyl-L-glutamine modified perfusate improves human lung cell functions and extend porcine ex vivo lung perfusion.

BACKGROUND: The clinical application of normothermic ex vivo lung perfusion (EVLP) has increased donor lung utilization for transplantation through functional assessment. To develop it as a platform for donor lung repair, reconditioning and regeneration, the perfusate should be modified to support the lung during extended EVLP. METHODS: Human lung epithelial cells and pulmonary microvascular endothelial cells were cultured, and the effects of Steen solution (commonly used EVLP perfusate) on basic cellular function were tested. Steen solution was modified based on screening tests in cell culture, and further tested with an EVLP cell culture model, on apoptosis, GSH, HSP70, and IL-8 expression. Finally, a modified formula was tested on porcine EVLP. Physiological parameters of lung function, histology of lung tissue, and amino acid concentrations in EVLP perfusate were measured. RESULTS: Steen solution reduced cell confluence, induced apoptosis, and inhibited cell migration, compared to regular cell culture media. Adding L-alanyl-L-glutamine to Steen solution improved cell migration and decreased apoptosis. It also reduced cold preservation and warm perfusion-induced apoptosis, enhanced GSH and HSP70 production, and inhibited IL-8 expression on an EVLP cell culture model. L-alanyl-L-glutamine modified Steen solution supported porcine lungs on EVLP with significantly improved lung function, well-preserved histological structure, and significantly higher levels of multiple amino acids in EVLP perfusate. CONCLUSIONS: Adding L-alanyl-L-glutamine to perfusate may provide additional energy support, antioxidant, and cytoprotective effects to lung tissue. The pipeline developed herein, with cell culture, cell EVLP, and porcine EVLP models, can be used to further optimize perfusates to improve EVLP outcomes.

Effect of electroacupuncture on enteric neuronal autophagy in functional constipation mice / 中国针灸

OBJECTIVES@#To explore the effect mechanism of electroacupuncture (EA) on functional constipation (FC) at the combined lower he-sea and front-mu points of large intestine based on enteric neuronal autophagy.@*METHODS@#A total of 40 SPF Kunming mice were randomly divided into 5 groups (n = 8), i.e. a control group, a model group, an acupuncture group, a 3-methyl adenine (3-MA) group, and a 3-MA + acupuncture group. Except the control group, the FC model was established by gavage with compound diphenoxylate suspension for 14 days in the other 4 groups. After successful modeling, the mice of the acupuncture group and the 3-MA + acupuncture group received EA at bilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37), stimulated for 30 min with disperse-dense wave, 2 Hz/15 Hz of frequency, 1 mA of intensity. EA was delivered once daily. One course of treatment was composed of 5 days and 2 courses were needed, with an interval of 2 days. An intraperitoneal injection of 3-MA (15 mg/kg) was administered 30 min before EA in the mice of the 3-MA group and the 3-MA + acupuncture group, once daily. Before and after intervention, the time of the first black stool defecation and defecation behaviors in 6 h were observed in each group. After intervention, in every group, the small intestine propulsion rate was calculated, the colon tissue morphology was observed using HE staining, the ultrastructure of enteric neuronal autophagy was observed under transmission electron microscope, and the expressions of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1 and neuronal nuclear antigen protein (NeuN) in neurons of colonic muscularis were determined by immunohistochemistry.@*RESULTS@#Before intervention, when compared with those in the control group, the time of the first black stool defecation was prolonged (P<0.01, P<0.05), and numbers (P<0.01), wet weight (P<0.01, P<0.05) and water content (P<0.05, P<0.01) of stool in 6 h were reduced in the model, acupuncture, 3-MA and 3-MA + acupuncture groups. After intervention, compared with those in the control group, the time of the first black stool defecation was longer (P<0.05), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were decreased in the model group. The time of the first black stool defecation was shortened (P<0.01), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were increased in the acupuncture group when compared with those in the model group. The time of the first black stool defecation was extended (P<0.01), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were declined in the 3-MA + acupuncture group in comparison with those in the acupuncture group. All layers of colon tissue were normal and intact in each group. When compared with the control group, the small intestine propulsion rate and the average optical density (OD) values of LC3, Beclin-1 and NeuN in neurons of colonic muscularis were decreased (P<0.01), and autophagosomes were dropped in the model group. In the acupuncture group, the small intestine propulsion rate and the average OD values of NeuN, LC3 and Beclin-1 in neurons of colonic muscularis increased (P<0.01,P<0.05), and autophagosomes were elevated when compared with those in the model group. The small intestine propulsion rate and the average OD values of NeuN, LC3 and Beclin-1 in neurons of colonic muscularis were dropped (P<0.05,P<0.01) in the 3-MA + acupuncture group in comparison with those in the acupuncture group.@*CONCLUSIONS@#Electroacupuncture may promote enteric neuronal autophagy and increase the number of neurons so that the intestinal motility can be improved and constipation symptoms can be relieved in FC mice.

XB130 Plays an Essential Role in Folliculogenesis Through Mediating Interactions Between Microfilament and Microtubule Systems in Thyrocytes.

Background: XB130 (actin filament-associated protein 1-like 2, AFAP1L2) is a thyroid-abundant adaptor/scaffold protein. Xb130-/- mice exhibit transient growth retardation postnatally due to congenital hypothyroidism with diminished thyroglobulin iodination and release at both embryonic and early postnatal stages due to disorganized thyroid apical membrane structure and function. We hypothesized that XB130 is crucial for polarity and folliculogenesis by mediating proper cytoskeletal structure and function in thyrocytes. Methods: Primary thyrocytes isolated from thyroid glands of Xb130-/- mice and their wild-type littermates at postnatal week 2 were cultured in 10% Matrigel for different time periods. Folliculogenesis was studied with immunofluorescence staining, followed by confocal microscopy. Cells were also transfected to express human XB130 fused Green Fluorescent Protein (XB130-GFP) or Green Fluorescent Protein (GFP) only before morphological analysis. Cytoskeletal structures from embryo and postnatal thyroid glands were also studied. Results: In three-dimensional cultures of thyrocytes, XB130, aligned with actin filaments, participated in defining the site of apical membrane formation and coalescence to form a thyroid follicle lumen. Xb130-/- thyrocytes displayed delayed folliculogenesis, reduced recruitment of a microtubule (MT)-associated proteins, and disorganized acetylated tubulin under the apical membrane, resulting in delayed folliculogenesis with reduced efficiency in formation of the thyroid follicle lumen. Conclusions: XB130 critically regulates thyrocyte polarization by functioning as a link between the actin filament cortex and MT network at the apical membrane of thyrocytes. Defects of adaptor scaffold proteins may affect cellular polarity and cytoskeletal structure and function and result in disorders of epithelial function, such as congenital hypothyroidism.

Pathogenesis of Multinodular Goiter in Elderly XB130-Deficient Mice: Alteration of Thyroperoxidase Affinity with Iodide and Hydrogen Peroxide.

Background: Multinodular goiter (MNG) is the most common disorder of the thyroid gland. Aging and genetic mutations that impair thyroid hormone (TH) production have been implicated in the development of MNG. XB130 is an adaptor/scaffold protein predominantly expressed in the thyroid gland. XB130 deficiency leads to transient postnatal growth retardation in mice due to congenital hypothyroidism. We studied the formation of MNG and possible mechanisms in elderly mice. Methods: Thyroid glands of male and female Xb130-knockout (Xb130-/-), heterozygous (Xb130+/-), and wild-type (Xb130+/+) mice at the ages of 12-20 months were harvested for visual examination, histopathological, and immunohistological analyses. Blood and thyroid samples were collected after feeding elderly mice with a low iodine diet for 125I uptake and perchlorate discharge assay. The activity of thyroperoxidase (Tpo) was examined by spectrophotometric evaluation of iodide oxidation. Results: While moderate MNG was seen in Xb130+/+ and Xb130+/- mice, severe MNG, characterized by multiple nodules intermixed with dilated colloid-rich macrofollicles, was found only in Xb130-/- mice at 18 months. Thyrocyte cytoskeletal structure and cell adhesion molecules were disorganized, and TH production was significantly reduced. Reduced iodide organification was seen in elderly Xb130+/+ mice and further enhanced in Xb130-/- mice. In Xb130+/+ mice, Tpo shows high affinity with hydrogen peroxide (H2O2) throughout aging, but reduced affinity with iodide in an age-dependent manner. By contrast, in elderly Xb130-/- mice, the affinity of Tpo for iodide remained high, but the affinity of Tpo for H2O2 was reduced. Conclusions: The pathophysiological features in the thyroid glands of aged Xb130-/- mice closely resemble the features of MNG in humans. Moderate MNG in elderly mice was dramatically aggravated by XB130 deficiency. Reduced affinity of Tpo for H2O2 may contribute to MNG development via oxidative stress. This could be specific to XB130 deficiency but also could be a common mechanism in MNG. Its clinical relevance should be further investigated.

XB130 Deficiency Causes Congenital Hypothyroidism in Mice due to Disorganized Apical Membrane Structure and Function of Thyrocytes.

Background: Congenital hypothyroidism is often caused by genetic mutations that impair thyroid hormone (TH) production, resulting in growth and development defects. XB130 (actin filament associated protein 1 like 2) is an adaptor/scaffold protein that plays important roles in cell proliferation, migration, intracellular signal transduction, and tumorigenesis. It is highly expressed in thyrocytes, however, its function in the thyroid remains largely unexplored. Methods:Xb130-/- mice and their littermates were studied. Postnatal growth and growth hormone levels were measured, and responses to low or high-iodine diet, and levothyroxine treatment were examined. TH and thyrotropin in the serum and TH in the thyroid glands were quantified. Structure and function of thyrocytes in embryos and postnatal life were studied with histology, immunohistochemistry, immunofluorescence staining, Western blotting, and quantitative reverse transcription polymerase chain reaction. Results:Xb130-/- mice exhibited transient growth retardation postnatally, due to congenital hypothyroidism with reduced TH synthesis and secretion, which could be rescued by exogenous thyroxine supplementation. The thyroid glands of Xb130-/- mice displayed diminished thyroglobulin iodination and release at both embryonic and early postnatal stages. XB130 was found mainly on the apical membrane of thyroid follicles. Thyroid glands of embryonic and postnatal Xb130-/- mice exhibited disorganized apical membrane structure, delayed folliculogenesis, and abnormal formation of thyroid follicle lumina. Conclusion: XB130 critically regulates folliculogenesis by maintaining apical membrane structure and function of thyrocytes, and its deficiency leads to congenital hypothyroidism.

Nitric oxide displays a biphasic effect on calcium dynamics in microglia.

Calcium is a critical secondary messenger in microglia. In response to inflammation, microglia mobilize intracellular calcium and increase the expression of inducible nitric oxide synthase (iNOS), which produces nitric oxide (NO). This study set to explore whether NO regulates intracellular calcium dynamics through transient receptor potential (TRP) channels in primary wildtype (WT) and iNOS knockout (iNOS-/-) microglia, and the BV2 microglial cell line using calcium imaging and voltage-clamp recordings. Our results demonstrated that application of the NO-donor SNAP induced a biphasic calcium response in naïve murine microglia. Specifically, phase I was characterized by a rapid decline in calcium influx that was attenuated by pretreatment of the store operated calcium channel (SOCC) inhibitor 2APB, while phase II presented as a slow calcium influx that was abolished by pretreatment with the TRP vanilloid type 2 (TRPV2) channel inhibitor tranilast. Importantly, in the presence of a protein kinase G (PKG) inhibitor, the SNAP-mediated calcium decline in phase I persisted while the calcium influx in phase II was abolished. Application of thapsigargin to activate SOCCs caused a calcium influx through a nonselective cation conductance in BV2 microglia, which was abruptly attenuated by SNAP. Importantly, iNOS-/- microglia displayed a significantly larger calcium influx though SOCCs while expressing less stromal interaction molecule 1, Orai1, and TRP canonical type 1 and 3 mRNA, when compared to WT microglia. Together, these results demonstrate that NO signaling restricts calcium influx through SOCCs independent of PKG signaling and increases calcium influx through TRPV2 channels in a PKG-dependent mechanism in microglia.

Nitric Oxide Critically Regulates Purkinje Neuron Dendritic Development Through a Metabotropic Glutamate Receptor Type 1-Mediated Mechanism.

Nitric oxide (NO), specifically derived from neuronal nitric oxide synthase (nNOS), is a well-established regulator of synaptic transmission in Purkinje neurons (PNs), governing fundamental processes such as motor learning and coordination. Previous phenotypic analyses showed similar cerebellar structures between neuronal nitric oxide null (nNOS-/-) and wild-type (WT) adult male mice, despite prominent ataxic behavior within nNOS-/- mice. However, a study has yet to characterize PN molecular structure and their excitatory inputs during development in nNOS-/- mice. This study is the first to explore morphological abnormalities within the cerebellum of nNOS-/- mice, using immunohistochemistry and immunoblotting. This study sought to examine PN dendritic morphology and the expression of metabotropic glutamate receptor type 1 (mGluR1), vesicular glutamate transporter type 1 and 2 (vGluT1 and vGluT2), stromal interaction molecule 1 (STIM1), and calpain-1 within PNs of WT and nNOS-/- mice at postnatal day 7 (PD7), 2 weeks (2W), and 7 weeks (7W) of age. Results showed a decrease in PN dendritic branching at PD7 in nNOS-/- cerebella, while aberrant dendritic spine formation was noted in adult ages. Total protein expression of mGluR1 was decreased in nNOS-/- cerebella across development, while vGluT2, STIM1, and calpain-1 were significantly increased. Ex vivo treatment of WT slices with NOS inhibitor L-NAME increased calpain-1 expression, whereas treating nNOS-/- cerebellar slices with NO donor NOC-18 decreased calpain-1. Moreover, mGluR1 agonist DHPG increased calpain-1 in WT, but not in nNOS-/- slices. Together, these results indicate a novel role for nNOS/NO signaling in PN development, particularly by regulating an mGluR1-initiated calcium signaling mechanism.

Nitric oxide signaling inhibits microglia proliferation by activation of protein kinase-G.

Microglia population is primarily determined by a finely-regulated proliferation process during early development of the central nervous system (CNS). Nitric oxide (NO) is known to inhibit proliferation in numerous cell types. However, how NO signaling regulates microglia proliferation remains elusive. Using wildtype (WT) and inducible nitric oxide synthase knockout (iNOS-/-) mice, this study investigated the role and underlying mechanisms of iNOS/NO signaling in microglia proliferation. Here we reported that iNOS-/- mice displayed significantly more BrdU-labeled proliferating microglia in the cortex than that in WT mice at postnatal day 10. Compared to microglia isolated from WT mouse cortex, significantly more iNOS-/- microglia displayed the specific cell-cycle markers Ki67 and phospho-histone H3 (pH3) in their nuclei. In addition, treating WT microglia with the NOS inhibitor LNAME drastically increased the percentage of cells expressing Ki67 and pH3, whereas treating iNOS-/- microglia with NOC18, a slow-release NO-donor, significantly decreased the percentage of microglia expressing the two cell-cycle markers. Moreover, inhibition of protein kinase-G (PKG) in WT microglia increased the proportion of microglia expressing Ki67 and pH3, whereas activation of PKG signaling using 8Br-cGMP in iNOS-/- microglia significantly decreased the fraction of microglia displaying Ki67 and pH3. Interestingly, in the presence of a PKG inhibitor, NOC18 increased the quantity of iNOS-/- microglia expressing Ki67 and pH3. Together, these results indicate that basal activity of iNOS/NO signaling impedes microglial cell-cycle progression and attenuates proliferation through activation of the cGMP-PKG pathway. However, NO increases microglia cell-cycle progression in the absence of cGMP-PKG signaling.

Neurexin-1α regulates neurite growth of rat hippocampal neurons.

The growth of neurites underlies the axonal pathfinding and synaptic formation during neuronal development and regeneration. Neurite growth is regulated by specific interactions between growth cone receptors and their ligands that function as molecular cues existing in microenvironments. Neurexins (NRXNs) are concentrated on growth cones and they may function to constrain axonal branches of invertebrate neurons. The present study explored the role of NRXN-1α in regulating neurite growth of mammalian neurons. Results showed that transfecting an effective NRXN-1α siRNA to cultured rat hippocampal neurons significantly increased neurite length. Adding NRXN-1α ligands including neuroligin (NLGN) peptide and/or α-latrotoxin (α-LTX) to the culture media largely decreased neurite growth of naïve neurons in a Ca2+-dependent manner, but had no effect on neurite growth of neurons transfected with NRXN-1α siRNA. Our results suggest that NRXN-1α regulates neurite development of mammalian neurons.

Nitric oxide upregulates microglia phagocytosis and increases transient receptor potential vanilloid type 2 channel expression on the plasma membrane.

Microglia phagocytosis is critical for central nervous system development, and dysregulation of phagocytosis may contribute to a variety of neurological disorders. During initial stages of phagocytosis, microglia display increased nitric oxide (NO) production via inducible nitric oxide synthase (iNOS) activity and amplified calcium entry through transient receptor potential vanilloid type 2 (TRPV2) channels. The present study investigated the regulatory role of iNOS/NO signaling in microglial phagocytosis and TRPV2 channel activation using phagocytosis assay, calcium imaging, patch clamp electrophysiology, immunocytochemistry, and immunoblot assays. Results showed that primary microglia from iNOS-knockout (iNOS-/- ) mice exhibited substantial deficits in phagocytic capacity and TRPV2 channel activity relative to wild-type (WT) controls. Specifically, iNOS-/- microglia displayed a lower level of TRPV2 protein localized on the plasma membrane (PM) without any significant change in the mRNA levels of Fc-gamma receptors and TRPV2. In addition, iNOS-/- microglia, unlike their WT controls, failed to elicit a calcium influx in response to application of the TRPV2-agonist 2-aminoethoxydiphenyl borate (2APB). Importantly, the phagocytic capacity and the PM expression and activity of TRPV2 in iNOS-/- microglia were largely corrected by pretreatment with NO-donors. Accordingly, the 2APB-evoked calcium influx and the PM expression of TRPV2 in WT microglia were significantly decreased by selective inhibition of iNOS, protein kinase-G (PKG), or phosphoinositide-3-kinase (PI3K), respectively. Together, results from this study indicated that iNOS/NO signaling upregulates microglial phagocytosis and increases TRPV2 trafficking to the PM via PKG/PI3K dependent pathway(s).

XB130 deficiency enhances carcinogen-induced skin tumorigenesis.

XB130 is an adaptor protein that functions as a mediator of multiple tyrosine kinases important for regulating cell proliferation, survival, migration and invasion. Formerly predicted as an oncogene, alterations of its expression are documented in various human cancers. However, the exact role of XB130 in tumorigenesis is unknown. To address its function in skin tumorigenesis, a two-stage dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA) study was performed on XB130 knockout (KO), heterozygous (HZ) and wild-type (WT) littermate mice. DMBA/TPA-treated XB130 KO and HZ males developed a significantly higher number of epidermal tumors that were notably larger in size than did WT mice. Interestingly, DMBA/TPA-treated female mice did not show any difference in tumor multiplicity regardless of the genotypes. The skin tumor lesions of XB130 KO males were more progressed with an increased frequency of keratoacanthoma. Deficiency of XB130 dramatically increased epidermal tumor cell proliferation. The responses to DMBA and TPA stimuli were also individually investigated to elucidate the mechanistic role of XB130 at different stages of tumorigenesis. DMBA-treated male XB130 KO mice showed compensatory p53-mediated stress response. TPA-treated XB130 KO males demonstrated more skin ulceration with more severe edema, enhanced cell proliferation, accumulation of infiltrating neutrophils and increased production of pro-inflammatory cytokine genes compared with WT mice. Enhanced activities of nuclear factor-kappa B pathway, increased protein expression of metalloproteinase-9 and ERK1/2 phosphorylation were found in these KO mice. These findings demonstrate that XB130 acts as a tumor suppressor in carcinogen-induced skin tumorigenesis that may be mediated through inhibiting inflammation.

Autocrine GABA signaling distinctively regulates phenotypic activation of mouse pulmonary macrophages.

In response to micro-environmental cues such as microbial infections or T-helper 1 and 2 (TH1 and TH2) cytokines, macrophages (Mϕs) develop into M1- or M2-like phenotypes. Phenotypic polarization/activation of Mϕs are also essentially regulated by autocrine signals. Type-A γ-aminobutyric acid receptor (GABAAR)-mediated autocrine signaling is critical for phenotypic differentiation and transformation of various cell types. The present study explored whether GABAAR signaling regulates lung Mϕ (LMϕ) phenotypic activation under M1/TH1 and M2/TH2 environments. Results showed that GABAAR subunits were expressed by primary LMϕ of mice and the mouse Mϕ cell line RAW264.7. The expression levels of GABAAR subunits in mouse LMϕs and RAW264.7 cells decreased or increased concurrently with classical (M1) or alternative (M2) activation, respectively. Moreover, activation or blockade of GABAARs distinctively influenced the phenotypic characteristics of Mϕ. These results suggested that microenvironments leading to LMϕ phenotypic polarization concurrently modulates autocrine GABA signaling and its role in Mϕ activation.

Paracrine GABA and insulin regulate pancreatic alpha cell proliferation in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: This study aimed to elucidate the mechanism of increased proliferation of alpha cells in recent-onset type 1 diabetes. Pancreatic beta cells express GAD and produce γ-aminobutyric acid (GABA), which inhibits alpha cell secretion of glucagon. We explored the roles of GABA in alpha cell proliferation in conditions corresponding to type 1 diabetes in a mouse model and in vitro. METHODS: Type 1 diabetes was induced by injecting the mice with streptozotocin (STZ). Some of the STZ-injected mice were treated with GABA (10 mg/kg daily) for 12 days. Isolated pancreatic islets were treated with STZ or STZ together with GABA for 2 days. The effects of GABA treatment on STZ-induced alpha cell proliferation in vivo and in vitro were assessed. The effect of muscimol, a GABA receptor agonist, on αTC1-6 cell proliferation was also examined. RESULTS: STZ injection substantially decreased levels of GAD, GABA and insulin in pancreatic beta cells 12 h after injection; this was followed by an upsurge of phosphorylated mechanistic target of rapamycin (p-mTOR) in the alpha cells at day 1, and a significant increase in alpha cell mass at day 3. Treating STZ-injected mice with GABA largely restored the immunodetectable levels of insulin and GAD in the beta cells and significantly decreased the number of aldehyde dehydrogenase 1 family, member A3 (ALDH1a3)-positive cells, alpha cell mass and hyperglucagonaemia. STZ treatment also increased alpha cell proliferation in isolated islets, which was reversed by co-treatment with GABA. Muscimol, together with insulin, significantly lowered the level of cytosolic Ca2+ and p-mTOR, and decreased the proliferation rate of αTC1-6 cells. CONCLUSIONS/INTERPRETATION: GABA signalling critically controls the alpha cell population in pancreatic islets. Low intraislet GABA may contribute to alpha cell hyperplasia in early type 1 diabetes.

Protective roles of hepatic GABA signaling in acute liver injury of rats.

γ-Aminobutyric acid (GABA) is produced by various cells through the catalytic activity of glutamic acid decarboxylase (GAD). Activation of type-A GABA receptor (GABAAR) inhibits stem cell proliferation but protects differentiated cells from injures. The present study investigated hepatic GABA signaling system and the role of this system in liver physiology and pathophysiology. RT-PCR and immunoblot assays identified GAD and GABAAR subunits in rat livers and in HepG2 and Clone 9 hepatocytes. Patch-clamp recording detected GABA-induced currents in Clone 9 hepatocytes and depolarization in WITT cholangiocytes. The function of hepatic GABA signaling system in rats was examined using models of d-galactosamine (GalN)-induced acute hepatocytic injury in vivo and in vitro. The expression of GAD increased whereas GABAAR subunits decreased in the liver of GalN-treated rats. Remarkably, treating rats with GABA or the GABAAR agonist muscimol, but not the GABABR agonist baclofen, protected hepatocytes against GalN toxicity and improved liver function. In addition, muscimol treatment decreased the formation of pseudobile ductules and the enlargement of hepatocytic canaliculi in GalN-treated rats. Our results revealed that a complex GABA signaling system exists in the rat liver. Activation of this intrahepatic GABAergic system protected the liver against toxic injury.NEW & NOTEWORTHY Auto- and paracrine GABAergic signaling systems exist in the rat hepatocytes and cholangiocytes. Activation of GABA signaling protects liver function from d-galactosamine injury by reducing toxic impairment of hepatocytes and by decreasing cholangiocyte proliferation.

Interaction of acetylcholinesterase with neurexin-1ß regulates glutamatergic synaptic stability in hippocampal neurons.

BACKGROUND: Excess expression of acetylcholinesterase (AChE) in the cortex and hippocampus causes a decrease in the number of glutamatergic synapses and alters the expression of neurexin and neuroligin, trans-synaptic proteins that control synaptic stability. The molecular sequence and three-dimensional structure of AChE are homologous to the corresponding aspects of the ectodomain of neuroligin. This study investigated whether excess AChE interacts physically with neurexin to destabilize glutamatergic synapses. RESULTS: The results showed that AChE clusters colocalized with neurexin assemblies in the neurites of hippocampal neurons and that AChE co-immunoprecipitated with neurexin from the lysate of these neurons. Moreover, when expressed in human embryonic kidney 293 cells, N-glycosylated AChE co-immunoprecipitated with non-O-glycosylated neurexin-1ß, with N-glycosylation of the AChE being required for this co-precipitation to occur. Increasing extracellular AChE decreased the association of neurexin with neuroligin and inhibited neuroligin-induced synaptogenesis. The number and activity of excitatory synapses in cultured hippocampal neurons were reduced by extracellular catalytically inactive AChE. CONCLUSIONS: Excessive glycosylated AChE could competitively disrupt a subset of the neurexin-neuroligin junctions consequently impairing the integrity of glutamatergic synapses. This might serve a molecular mechanism of excessive AChE induced neurodegeneration.

An autocrine γ-aminobutyric acid signaling system exists in pancreatic ß-cell progenitors of fetal and postnatal mice.

Gamma-aminobutyric acid (GABA) is produced and secreted by adult pancreatic ß-cells, which also express GABA receptors mediating autocrine signaling and regulating ß-cell proliferation. However, whether the autocrine GABA signaling involves in ß-cell progenitor development or maturation remains uncertain. By means of immunohistochemistry we analyzed the expression profiles of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) and the α1-subunit of type-A GABA receptor (GABAARα1) in the pancreas of mice at embryonic day 15.5 (E15.5), E18.5, postnatal day 1 (P1) and P7. Our data showed that at E15.5 the pancreatic and duodenum homeobox-1 (Pdx1) was expressed in the majority of cells in the developing pancreata. Notably, insulin immunoreactivity was identified in a subpopulation of pancreatic cells with a high level of Pdx1 expression. About 80% of the high-level Pdx-1 expressing cells in the pancreas expressed GAD and GABAARα1 at all pancreatic developmental stages. In contrast, only about 30% of the high-level Pdx-1 expressing cells in the E15.5 pancreas expressed insulin; i.e., a large number of GAD/GABAARα1-expressing cells did not express insulin at this early developmental stage. The expression level of GAD and GABAARα1 increased steadily, and progressively more GAD/GABAARα1-expressing cells expressed insulin in the course of pancreatic development. These results suggest that 1) GABA signaling proteins appear in ß-cell progenitors prior to insulin expression; and 2) the increased expression of GABA signaling proteins may be involved in ß-cell progenitor maturation.

Isoflurane regulates atypical type-A γ-aminobutyric acid receptors in alveolar type II epithelial cells.

BACKGROUND: Volatile anesthetics act primarily through upregulating the activity of γ-aminobutyric acid type A (GABAA) receptors. They also exhibit antiinflammatory actions in the lung. Rodent alveolar type II (ATII) epithelial cells express GABAA receptors and the inflammatory factor cyclooxygenase-2 (COX-2). The goal of this study was to determine whether human ATII cells also express GABAA receptors and whether volatile anesthetics upregulate GABAA receptor activity, thereby reducing the expression of COX-2 in ATII cells. METHODS: The expression of GABAA receptor subunits and COX-2 in ATII cells of human lung tissue and in the human ATII cell line A549 was studied with immunostaining and immunoblot analyses. Patch clamp recordings were used to study the functional and pharmacological properties of GABAA receptors in cultured A549 cells. RESULTS: ATII cells in human lungs and cultured A549 cells expressed GABAA receptor subunits and COX-2. GABA induced currents in A549 cells, with half-maximal effective concentration of 2.5 µM. Isoflurane (0.1-250 µM) enhanced the GABA currents, which were partially inhibited by bicuculline. Treating A549 cells with muscimol or with isoflurane (250 µM) reduced the expression of COX-2, an effect that was attenuated by cotreatment with bicuculline. CONCLUSIONS: GABAA receptors expressed by human ATII cells differ pharmacologically from those in neurons, exhibiting a higher affinity for GABA and lower sensitivity to bicuculline. Clinically relevant concentrations of isoflurane increased the activity of GABAA receptors and reduced the expression of COX-2 in ATII cells. These findings reveal a novel mechanism that could contribute to the antiinflammatory effect of isoflurane in the human lung.