ASIC2 Synergizes with TRPV1 in the Mechano-Electrical Transduction of Arterial Baroreceptors / 神经科学通报·英文版

Mechanosensitive ion channels (MSCs) are key molecules in the mechano-electrical transduction of arterial baroreceptors. Among them, acid-sensing ion channel 2 (ASIC2) and transient receptor potential vanilloid subfamily member 1 (TRPV1) have been studied extensively and documented to play important roles. In this study, experiments using aortic arch–aortic nerve preparations isolated from rats revealed that both ASIC2 and TRPV1 are functionally necessary, as blocking either abrogated nearly all pressure-dependent neural discharge. However, whether ASIC2 and TRPV1 work in coordination remained unclear. So we carried out cell-attached patch-clamp recordings in HEK293T cells co-expressing ASIC2 and TRPV1 and found that inhibition of ASIC2 completely blocked stretch-activated currents while inhibition of TRPV1 only partially blocked these currents. Immunofluorescence staining of aortic arch–aortic adventitia from rats showed that ASIC2 and TRPV1 are co-localized in the aortic nerve endings, and co-immunoprecipitation assays confirmed that the two proteins form a compact complex in HEK293T cells and in baroreceptors. Moreover, protein modeling analysis, exogenous co-immunoprecipitation assays, and biotin pull-down assays indicated that ASIC2 and TRPV1 interact directly. In summary, our research suggests that ASIC2 and TRPV1 form a compact complex and function synergistically in the mechano-electrical transduction of arterial baroreceptors. The model of synergism between MSCs may have important biological significance beyond ASIC2 and TRPV1.

ASIC2 Synergizes with TRPV1 in the Mechano-Electrical Transduction of Arterial Baroreceptors / 神经科学通报·英文版

Mechanosensitive ion channels (MSCs) are key molecules in the mechano-electrical transduction of arterial baroreceptors. Among them, acid-sensing ion channel 2 (ASIC2) and transient receptor potential vanilloid subfamily member 1 (TRPV1) have been studied extensively and documented to play important roles. In this study, experiments using aortic arch-aortic nerve preparations isolated from rats revealed that both ASIC2 and TRPV1 are functionally necessary, as blocking either abrogated nearly all pressure-dependent neural discharge. However, whether ASIC2 and TRPV1 work in coordination remained unclear. So we carried out cell-attached patch-clamp recordings in HEK293T cells co-expressing ASIC2 and TRPV1 and found that inhibition of ASIC2 completely blocked stretch-activated currents while inhibition of TRPV1 only partially blocked these currents. Immunofluorescence staining of aortic arch-aortic adventitia from rats showed that ASIC2 and TRPV1 are co-localized in the aortic nerve endings, and co-immunoprecipitation assays confirmed that the two proteins form a compact complex in HEK293T cells and in baroreceptors. Moreover, protein modeling analysis, exogenous co-immunoprecipitation assays, and biotin pull-down assays indicated that ASIC2 and TRPV1 interact directly. In summary, our research suggests that ASIC2 and TRPV1 form a compact complex and function synergistically in the mechano-electrical transduction of arterial baroreceptors. The model of synergism between MSCs may have important biological significance beyond ASIC2 and TRPV1.

Expression of acid-sensing ion channel 1a in the central nervous system of mice with panic-like behavior / 中华行为医学与脑科学杂志

Objective To explore the expression of acid-sensing ion channel 1a ( ASIC1a) in cen-tral nervous system of mice with panic like behavior. Methods 20 male C57BL/6 mice were randomly di-vided into two groups according to their weight( 10 mice in each group):the group experienced rat exposure test of panic-like behavior model ( RET group ) and the control group ( Ctr group ) . A panic-like behavior model was established by rat exposure stimuli. Ten minutes defensive and avoidance behaviors of mice were recorded with a horizontal video camera. The anxiety level of mice was evaluated by elevated plus maze ( EPM) test.Western blot was used to detect the ASIC 1a expression in different brain areas of prefrontal cor-tex,hippocampus and periaqueductal gray (PAG). Results Compared with Ctr group,mice in RET group spent significantly more time in freezing ((5.83±1.92)s) than that of Ctr group ((1.00±0.45)s) (P<0.01),had significantly higher frequency of risk assessment behavior (5.33±0.49) than that of Ctr group (0.60±0.40) (P<0.01),spent significantly less time to contact the wire mesh ((17.83±4.38)s) than that of Ctr group((50.00±6.90)s) (P<0.01),and significantly more time of staying in the protected area((431.00±33.13)s) than that of Ctr group((264.40±40.43)s) (P<0.01).At the same time,RET group showed sig-nificantly lower time percent ((8.28±1.12)％) than Ctr group ((16.81±2.13)％) in opened arm (P<0.05) and significantly higher time percent ((80.08±4.26)％) than Ctr group ((60.91±5.27)％) in the closed arm (P<0.05).Western blot suggested that the expression level of ASIC 1a in the prefrontal cortex (1.32± 0.05) and hippocampus (2.56±0.30) significantly increased than that of Ctr group((0.98±0.07),(1.56± 0.16)( P<0.05),while significantly decreased in the PAG (0.83± 0.02) than that of Ctr group(1.26±0.05) ( P<0.05) . Conclusion Rat exposure stimuli can induce panic-like behavior among mice,which increases the expression of ASIC 1a in the prefrontal cortex and hippocampus,but decreases the level of ASIC 1a in the PAG.

The expressions of acid sensing ion channel 1a and activator protein-1 in hepatocellular carcinoma and their relationships with clinical features / 中华肝胆外科杂志

Objective To investigate the expressions of acid sensing ion channel 1a(ASIC1a) and activator protein-1 (AP-1) in hepatocellular carcinoma,and to analyze the relationship between expressions and clinical features.Methods At the transcription level,expression spectroscopy chips and bioinformatics were used to analyze the changes of signal pathways before and after ASICla interference in hepatocellular carcinoma cells.63 cases of HCC and 42 cases of tumor adjacent tissue samples were chosen from the Third People's Hospital of Wuxi,between January 2010 and December 2014.Immunohistochemical staining was utilized to detect the expressions of ASIC1 a,c-Jun,c-Fos.The relationships among the three were analyzed by the nonparametric Spearman rank correlation coefficient.Results After ASIC1a inhibition,the expression of AP-1 (c-Jun and c-Fox) decreased significantly.The expressions of c-Jun and c-Fox were greatly decreased in interference group compared with control group.The positive rates of ASIC1a,c-Jun and c-Fos in HCC tissues were significantly higher than that of the adjacent tissues,68.3％ vs.19.0％,55.6％ vs.11.9％,47.6％ vs.11.7％ (P ＜ 0.05).Correlation analysis showed that the expression of ASIC1a was positively correlated with c-Jun and c-Fos expression (r =0.404,0.309,P ＜ 0.05).The expressions of ASIC 1 a,c-Jun and c-Fos were not related to age,tumor diameter and gender (P ＞ 0.05),which were related to the clinical stage,AFP and lymph node metastasis of the tumor (P ＜ 0.05).Conclusion ASIC1a may.affect the development of hepatocellular carcinoma through the downstream gene AP-1.

Role of acid-sensing ion channel 1a in pathogenesis of autoimmune diseases / 中国药理学通报

Acid-sensing ion channels ( ASICs) are proton-gated channels expressed widely in the central nervous systems and pe-ripheral tissues, among which ASIC1a is a core part and plays an important role in many physiological and pathological proces-ses.As a key receptor for extracellular protons , ASIC1a is in-volved in a variety of pathophysiological processes involving tis-sue acidosis, such as pain, inflammation, seizures and multiple sclerosis.Autoimmune disease is a chronic inflammatory dis-ease , and the excessive activation of T , B cells leads to multiple tissue and organ damage when the body responds to autoantigen immune response . In recent years , studies have found that ASIC1a plays an important role in the development of various au-toimmune diseases.In this paper, the biological characteristics of ASIC1a are briefly reviewed , and the research progress of ASIC1a in the development and progression of autoimmune dis-eases is discussed .

Relation between esophageal visceral hyperesthesia and expression of acid-sensing ion channel 1 in rats with reflux esophagitis / 中华消化杂志

Objective To explore the role of the visceral afferent nerve hyperesthesia and acid-sensing ion channel 1 (ASIC1) in rats with reflux esophagitis (RE).Methods Sixty male Sprague-Dawley rats were selected and animal model was established.Rats were divided into control group (n=20) and RE group (n=40).The esophageal mocosa biopsy were routinely performed in two groups.The esophageal specific DRG neurons were identified by 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate tracing method and the whole-cell patch clamp assay was performed.The expression of ASIC1 in esophageal mucosa and thoracic spine cord three to five segments at protein level and mRNA level were detected by Western blotting and quantitative real time-polymerase chain reaction (qPCR).Two independent samples t test was performed for statistical analysis.Results The body weight of RE group was significantly lower than that of control group ((179.41±-16.38) g vs (290.75 ±-22.20) g),and the difference was statistically significant (t=17.090,P＜ 0.01).Esophageal basal cell hyperplasia,papillary elongation,vascular dialation and congestion,inflammatory cells infiltration were found in RE group rats.The results of whole-cell patchclamp showed depolarization of the resting potential of esophageal-specific DRG neurons of RE group was more significant than that of control group (-(46.20 ± 1.92) mV vs-(51.60 ± 1.52) mV),and the difference was statistically significant (t=4.930,P＜0.01).The threshold current of RE group was much lower than that of control group ((18.00±13.04) pAvs (80.00±12.25) pA),and the difference was statistically significant (t=7.750,P＜0.01).When stimulated with two to three times the threshold current,the frequency of action potential of RE group significantly increased (5.80 ±1.48 vs 3.00 ±1.58,10.60±2.30 vs 5.20±1.92),and the differences were statistically significant (t=2.890 and 4.030,both P＜0.01).The results of Western blotting indicated that the expression of ASIC1 in esophageal mucosa of RE group was significatly lower than that of control group (0.614±0.120 vs 0.976±0.283),and the difference was statistically significant (t =2.885,P＜ 0.05),while there was no statistically significant difference in the expression of ASIC1 in DRG between RE group and control group (0.804 ± 0.182 vs 1.032±0.316;t=1.528,P＞0.05).The results of qPCR showed that the expression of ASIC1 mRNA in esophageal mucosa of RE group was lower than that of control group (0.694 ± 0.118 vs 1.036 ±0.137),and the difference was statistically significant (t=4.642,P＜0.01).However there was no statistically significant difference in ASIC1 at mRNA level between RE group and control group (1.002± 0.074 vs 0.985±0.120;t=0.294,P＞0.05).Conclusion The sensitivity of esophageal visceral afferent nerve of rats in RE group increases and ASIC1 may negatively regulate the formation of esophageal visceral hypersensitivity.

Relation between esophageal visceral hyperesthesia and expression of acid-sensing ion channel 1 in rats with reflux esophagitis / 中华消化杂志

Objective To explore the role of the visceral afferent nerve hyperesthesia and acid-sensing ion channel 1 (ASIC1) in rats with reflux esophagitis (RE).Methods Sixty male Sprague-Dawley rats were selected and animal model was established.Rats were divided into control group (n=20) and RE group (n=40).The esophageal mocosa biopsy were routinely performed in two groups.The esophageal specific DRG neurons were identified by 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate tracing method and the whole-cell patch clamp assay was performed.The expression of ASIC1 in esophageal mucosa and thoracic spine cord three to five segments at protein level and mRNA level were detected by Western blotting and quantitative real time-polymerase chain reaction (qPCR).Two independent samples t test was performed for statistical analysis.Results The body weight of RE group was significantly lower than that of control group ((179.41±-16.38) g vs (290.75 ±-22.20) g),and the difference was statistically significant (t=17.090,P＜ 0.01).Esophageal basal cell hyperplasia,papillary elongation,vascular dialation and congestion,inflammatory cells infiltration were found in RE group rats.The results of whole-cell patchclamp showed depolarization of the resting potential of esophageal-specific DRG neurons of RE group was more significant than that of control group (-(46.20 ± 1.92) mV vs-(51.60 ± 1.52) mV),and the difference was statistically significant (t=4.930,P＜0.01).The threshold current of RE group was much lower than that of control group ((18.00±13.04) pAvs (80.00±12.25) pA),and the difference was statistically significant (t=7.750,P＜0.01).When stimulated with two to three times the threshold current,the frequency of action potential of RE group significantly increased (5.80 ±1.48 vs 3.00 ±1.58,10.60±2.30 vs 5.20±1.92),and the differences were statistically significant (t=2.890 and 4.030,both P＜0.01).The results of Western blotting indicated that the expression of ASIC1 in esophageal mucosa of RE group was significatly lower than that of control group (0.614±0.120 vs 0.976±0.283),and the difference was statistically significant (t =2.885,P＜ 0.05),while there was no statistically significant difference in the expression of ASIC1 in DRG between RE group and control group (0.804 ± 0.182 vs 1.032±0.316;t=1.528,P＞0.05).The results of qPCR showed that the expression of ASIC1 mRNA in esophageal mucosa of RE group was lower than that of control group (0.694 ± 0.118 vs 1.036 ±0.137),and the difference was statistically significant (t=4.642,P＜0.01).However there was no statistically significant difference in ASIC1 at mRNA level between RE group and control group (1.002± 0.074 vs 0.985±0.120;t=0.294,P＞0.05).Conclusion The sensitivity of esophageal visceral afferent nerve of rats in RE group increases and ASIC1 may negatively regulate the formation of esophageal visceral hypersensitivity.

Effects of Deep Electroacupuncture on Cartilage in Knee Osteoarthritis Rabbits / 中国康复理论与实践

Objective To observe the effects of deep electroacupuncture on carlilage tissue in knee osteoarthritis (KOA) rabbits. Meth-ods 40 New Zealand rabbits were randomly divided into normal group (A, n=10) and model group (n=30). The model group was modeled KOA with Hulth-Telhag way, and identified with X-ray. Then they were divided into no-treated group (B, n=10), deep electroacupuncture group (C, n=10) and routine electroacupuncture group (D, n=10) randomly. The groups C and D accepted electroacupuncture since 6 weeks after modeling, for 4 weeks. They were measured with pH of joint fluid, observed structure and pathology of cartilage under transmission electron microscope, detected apoptosis index, and determined the expression of acid-sensing ion channel 1 (ASIC1), p38 mitogen-activated protein kinases (p38MAPK) and p53 with Western blotting, and distribution of ASIC1 with immunohistochemistry in cartilage tissue. Re-sults The pHs of joint fluid from high to low were ranged as the groups A=C>D>B (P<0.01). The cartilage structure was more complete in the groups A, C and D than in the group B. The apoptosis rates from less to more were ranged as the groups A=C

Effect of ASIC1 a on hepatic fibrosis under high glucose / 中国药理学通报

Aim To investigate the effect of ASIC1 a ( acid-sensing ion channel 1 a ) on the pathological change of diabetes complication liver fibrosis and the proliferation and activation of hepatic stellate cell ( HSC-T6 ) stimulated by PDGF-BB under hyperglyce-mia. Methods Diabetes rats model was established by streptozotocin ( STZ) , and liver fibrosis rats model was induced by carbon tetrachloride ( CCl4 ) . Then, the liver extent of damage and the expression of ASIC1 a were observed in the diabetic rats, liver fibrosis rats and diabetes complication liver fibrosis rats. In vitro, after pretreated with amiloride, HSC-T6 was treated with high glucose for 24 h and then stimulated with PDGF-BB for another 24 h. The proliferation and acti-vation of HSC-T6 were observed, and the expression of ASIC1a, α-SMA and collagen Ⅰ were detected by Western blot. Results Compared with the control group, rats from diabetic group induced by STZ, liver fibrosis group induced by CCl4 , and the diabetes com-plication liver fibrosis rats co-induced by STZ and CCl4 were all observed with liver damage at different levels, and tissue injury of complication group was most seri-ous. However, the expression of ASIC1a in the three model groups was significantly increased compared to the control group. ASIC1a level was most obvious in the diabetes complication liver fibrosis rats. Amiloride pretreatment significantly decreased ASIC1 a expression and inhibited PDGF-BB mediated proliferation and the expression ofα-SMA and collagenⅠin HSC-T6 under high glucose environment. Conclusion High ambient glucose aggravates HSC activation and hepatic fibrosis, and this may be related with the increasing expression of ASIC1a.

The fate of medications evaluated for ischemic stroke pharmacotherapy over the period 1995-2015

Stroke is a brain damage caused by a loss of blood supply to a portion of the brain, which requires prompt and effective treatment. The current pharmacotherapy for ischemic stroke primarily relies on thrombolysis using recombinant tissue plasminogen activators (rt-PAs) to breakdown blood clots. Neuroprotective agents that inhibit excitatory neurotransmitters are also used to treat ischemic stroke but have failed to translate into clinical benefits. This poses a major challenge in biomedical research to understand what causes the progressive brain cell death after stroke and how to develop an effective pharmacotherapy for stroke. This brief review analyzes the fate of about 430 potentially useful stroke medications over the period 1995-2015 and describes in detail those that successfully reached the market. Hopefully, the information from this analysis will shed light on how future stroke research can improve stroke drug discovery.

Effect of APETx2 on the expression of ASIC3 in dorsal root ganglia in rat AGML model / 实用医学杂志

Objective To study the effect of APETx2 on the expression of ASIC3 APETx2 in a rat model of acute gastric mucosal lesion(AGML). Methods Twenty-four Wistar rats were randomly assigned to three groups in equal number : normal control group, water immersion restraint stress (WIRS) group, APETx2 treatment group. AGML was induced by WIRS for 6 hours, and APETx2 (25 μg/kg) was injected intraperitoneally before the onset of stress. Intragastric pH and gastric histopathological changes were measured and the expression of ASIC3 mRNA in DRG neurons projecting to rat stomach was examined by real-time PCR. Immunohistochemistry was performed to detect the localization of ASIC3. Results Compared with the normal control group, the WIRS group showed obvious gastric injury with lower values of intragastric pH and extensive expression of ASIC3 in the DRG neurons (P < 0.05). The treatment with APETx2 before the onset of WIRS significantly alleviated the gastric mucosal injury, decreased gastric acidity and reduced ASIC3 expression in DRG neurons (P < 0.05). Conclusions ASIC3 expression in DRG neurons projecting to rat stomach is strongly associated with gastric mucosal lesion and acidosis in the WIRS model. APETx2 can improve gastric acidosis and prevent the occurrence of these lesions.

Role of acid-sensing ion channel 1 a in matrix turnover and MAPK expression of articular chondrocytes of rats / 中国药理学通报

Aim TostudytheroleofASIC1aonthe matrix turnover and MAPK expression of the rat articu-lar chondrocytes with extracellular acidosis. Methods ArticularchondrocyteswereisolatedfromSprague-Dawley rats, and their phenotype was determined by toluidine blue and immunocytochemical staining. The GAG content of cell culture supernatant was deter-mined by dimethyl-methylene blue spectrophotometric assay, while Hyp content by chloramine T assay. ELISA assay was used to measure MMP-2 , TIMP-2 content. Furthermore, the ERK1/2, p38 MAPK phos-phorylation protein expression levels were tested by Westernblotassay.Results ASIC1acontributedto the effect of GAG, Hyp and TIMP-2 levels reduction induced by extracellular acidification, while the effect of MMP-2 was weaker. Moreover, ASIC1a could in-crease the ERK1/2 , p38 MAPK phosphorylation pro-teinexpressionlevels.Conclusion ASIC1acould regulate rat articular chondrocytes matrix turnover via ERK1/2 and p38 MAPK signaling pathway, and there-by inhibit the rat articular cartilage damage induced by acidosis.

Co-localization of metabotropic glutamate receptor 2 with ASIC3 or TRPV1 in the dorsal root / 神经解剖学杂志

Metabotropie glutamate receptor (mGluR) 2/3 plays an important role on the nociceptive transmission from periphery to spinal cord.The previous studies demonstrated that mGluR2 can contribute to mechanical hypersensitivity and thermal hypersensitivity in rat.Therefore,in the present study,by using the immunofluorescenee histochemical technique,we try to explore that whether mGluR2 is colocalized with acid-sensing ion channel 3 (ASIC3),a muhi-modulator of mechanosensation,or transient receptor potential/vanilloid receptor subtype-1 (TRPV1),which responses for thermosensation in dorsal root ganglion (DRG).Morphological observations showed that mGluR2-immunoreactivity was mainly distributed in cellular plasma of neurons in DRG.The counting number results indicated that 35.84% of DRG neurons were mGluR2-immunoreactive (ir).On the other hand,82.61% of mGluR2-ir cells were the small-diameter neurons (diameter:＜30 μm),5.79% of which were the medium-diameter neurons (diameter:30-50μm) and 11.59% of which was the large-diameter neurons (diameter:＞50 tun).Furthermore,42.45% and 79.78% of mGiuR2-ir cells was individually co-localized with ASIC3-or TRPVI-ir in small-diameter neurons in the double-labeled immunofluorescence sections.The present results suggest that mGhiR2 mainly exists in small neurons of the DRG,which are regarded as nociceptors consisting of AS-and C-fibers.While mGluR2 is highly co-localized with ASIC3 and TRPV1,implying their potential relationship in DRG may be involved in mechanical hypersensitivity and thermal hypersensitivity.

El plomo inhibe la corriente activada por protones (ASIC) en las neuronas de los ganglios dorsales / Lead inhibits proton gated currents (ASIC) in dorsal root ganglion neurons

Antecedentes. Los canales iónicos ASIC (del inglés Acid Sensing Ion Channel) son canales iónicos activados por reducciones transitorias en el pH extracelular. Pese a no conocerse con exactitud su mecanismo, la activación ocurre por medio de la unión de protones al dominio extracelular del canal y es modulada por iones calcio y zinc. Objetivo. El hecho de que los cationes divalentes modifiquen el funcionamiento del canal nos llevó a preguntar si el plomo, otro catión divalente, sería capaz de alterar el funcionamiento de los ASIC. Métodos y resultados. Mediante el uso de la técnica de fijación de voltaje en configuración de célula completa en las neuronas de los ganglios de la raíz dorsal de la rata, encontramos que el plomo inhibe la corriente ASIC en forma dependiente de la concentración. Conclusiones. Estos resultados contribuyen a definir los mecanismos de activación de los canales ASIC y a explicar algunos de los mecanismos tóxicos del plomo en el organismo.

BACKGROUND: Acid sensing ion channels (ASIC) are ionic channels activated by transient pH reductions in the ext raceilularenvi ronment. Although the activation mechanism is not fully elucidated, it is clear that the channel is activated by proton binding to its extraceilular domain, a process that is modulated by calcium and zinc. OBJECTIVE: The fact that divalent cations are able to modify ASIC operation, lead us to consider if lead, anotherdivalent cation and widely distributed neurotoxicant, is also capable to affect ASIC function. METHODS: For this purpose, we recordedASiC currents in rat dorsal root ganglion neurons using the whole cell patch-clamp technique. RESULTS: The results indicated that lead inhibits ASIC currents in a concentration -dependent fashion. CONCLUSIONS: These results contribute to the understanding of the activation mechanism of ASIC and to explain some of the toxic mechanisms of lead in the organism.

Biological Characteristics of Acid Sensing Ion Channels (ASICs) and Their Modulations / 生物化学与生物物理进展

ASICs are H+-gated novel cation ion channels, which belong to the epithelial sodium channels (NaC/DEG) superfamily. As recent studies focus, ASICs are expected to be pharmacological targets on protecting the neuron from ischemia and damage, improving the ability of memory and study, curing epilepsy and analgesia. It is not until the most recentness that the subunits of ASICs have been cloned. Now, researchers have paid more attention to the distribution, expression, function and modulation of ASICs in the organism.

Research progress on the acid-sensing ion channel 1a / 中国药理学通报

Acid-sensing ion channels (ASICs), which are activated by extracelluar H+, are H+-gated cation channels. At present, seven subunits of ASICs family have been identified. Numerous studies revealed that ASIC1a plays important roles in synaptic transmission and plasticity,spatial learning and memory, nociception, as well as cerebral ischemia under physiological and pathological conditions. This review concerns the latest research about ASIC1a to promote the understanding of its physiological and pathological functions.