Identification and Function of Acid-sensing Ion Channels in RAW 264.7 Macrophage Cells

Activation of acid-sensing ion channels （ASICs） plays an important role in neuroinflammation. Macrophage recruitment to the sites of inflammation is an essential step in host defense. ASIC1 and ASIC3 have been reported to mediate the endocytosis and maturation of bone marrow derived macrophages. However, the expression and inflammation-related functions of ASICs in RAW 264.7 cells, another common macrophage, are still elusive. In the present study, we first demonstrated the presence of ASIC 1, ASIC2a and ASIC3 in RAW 264.7 macrophage cell line by using reverse transcriptase polymerase chain reaction （RT-PCR）, Western blotting and immunofluorescence experiments. The non-specific ASICs inhibitor amiloride and specific homomeric ASICla blocker PcTxl reduced the production of iNOS and COX-2 by LPS-induced activating RAW 264.7 cells. Furthermore, not only amiloride but also PcTxl inhibited the migration and LPS-induced apoptosis of RAW 264.7 cells. Taken together, our findings suggest that ASICs promote the inflammatory response and apoptosis of RAW 264.7 cells, and ASICs may serve as a potential novel target for immunological disease therapy.

Involvement of Acid-sensing Ion Channel 1a in Functions of Cultured Human Retinal Pigment Epithelial Cells

In the retina, pH fluctuations may play an important role in adapting retinal responses to different light intensities and are involved in the fine tuning of visual perception. Acidosis occurs in the subretinal space （SRS） under pathological conditions such as age-related macular degeneration （AMD）. Although it is well known that many transporters in the retinal pigment epithelium （RPE） cells can maintain pH homeostasis efficiently, other receptors in RPE may also be involved in sensing acidosis, such as acid-sensing ion channels （ASICs）. In this study, we investigated whether ASICla was ex- pressed in the RPE cells and whether it was involved in the function of these cells. Real-time RT-PCR and Western blotting were used to analyze the ASICla expression in ARPE-19 cells during oxidative stress induced by hydrogen peroxide （H202）. Furthermore, inhibition or over-expression of ASICla in RPE cells was obtained using inhibitors （amiloride and PCTxl） or by the transfection of cDNA encod- ing hASICla. Cell viability was determined by using the MTT assay. The real-time RT-PCR and West- ern blotting results showed that both the mRNA and protein of ASICla were expressed in RPE cells. In- hibition of ASICs by amiloride in normal RPE cells resulted in cell death, indicating that ASICs play an important physiological role in RPE cells. Furthermore, over-expression of ASICla in RPE cells pro- longed cell survival under oxidative stress induced by H2O2. In conclusion, ASICla is functionally expressed in RPE cells and may play an important role in the physiological function of RPE cells by pro-tecting them from oxidative stress.

Ischemic postconditioning protects against ischemic brain injury by up-regulation of acid-sensing ion channel 2a

Ischemic postconditioning renders brain tissue tolerant to brain ischemia,thereby alleviating ischemic brain injury.However,the exact mechanism of action is still unclear.In this study,a rat model of global brain ischemia was subjected to ischemic postconditioning treatment using the vessel occlusion method.After 2 hours of ischemia,the bilateral common carotid arteries were blocked immediately for 10 seconds and then perfused for 10 seconds.This procedure was repeated six times.Ischemic postconditioning was found to mitigate hippocampal CA1 neuronal damage in rats with brain ischemia,and up-regulate acid-sensing ion channel 2a expression at the m RNA and protein level.These findings suggest that ischemic postconditioning up-regulates acid-sensing ion channel 2a expression in the rat hippocampus after global brain ischemia,which promotes neuronal tolerance to ischemic brain injury.

Does closure of acid-sensing ion channels reduce ischemia/reperfusion injury in the rat brain?

Acidosis is a common characteristic of brain damage. Because studies have shown that permeable Ca2＋-acid-sensing ion channels can mediate the toxic effects of calcium ions, they have become new targets against pain and various intracranial diseases. However, the mechanism associated with expression of these channels remains unclear. This study sought to observe the expression characteristics of permeable Ca2＋-acid-sensing ion channels during different reperfusion inflows in rats after cerebral ischemia. The rat models were randomly divided into three groups： adaptive ischemia/reperfusion group, one-time ischemia/reperfusion group, and severe cerebral ischemic injury group. Western blot assays and immunofluorescence staining results exhibited that when compared with the one-time ischemia/reperfusion group, acid-sensing ion channel 3 and Bcl-x/I expression decreased in the adaptive ischemia/reperfusion group. Calmodulin expression was lowest in the adaptive ischemia/reperfusion group. Following adaptive reperfusion, common carotid artery flow was close to normal, and the pH value improved. Results verified that adaptive reperfusion following cerebral ischemia can suppress acid-sensing ion channel 3 expression, significantly reduce Ca2＋ influx, inhibit calcium overload, and diminish Ca2＋ toxicity. The effects of adaptive ischemia/reperfusion on suppressing cell apoptosis and relieving brain damage were better than that of one-time ischemia/reperfusion.

Effect of Activation of the Ca2+-Permeable Acid-Sensing Ion Channel 1a on Acid-Induced Vascular Endothelial Cell Injury of Henoch-Schönlein Purpura Children

Acidosis in local environment plays a critical role in cell injury. One key mediator of acidosis-induced cell injury is the acid-sensing ion channels (ASICs), particularly ASIC1a. Herein, we investigated the role of ASIC1a in acid-induced vascular endothelial cell injury of Henoch-Schonlein purpura (HSP) children. Acid-induced ASIC1a, Calpain and Calcineurin expression in vascular endothelial cells pretreated with IgA1 isolated from HSP were detected by real time quantitative polymerase chain reaction and western blot methods, respectively. Cell cytotoxicity was measured by interleukin-8 and nitric oxide production with ELISA. The results showed acid-induced ASIC1a, Calpain and Calcineurin expression in cells increased, especially at PH6.5. The cytotoxicity of vascular endothelial cells was increased by extracellular acidosis. Moreover non-specific or specific blockers of ASIC1a, Amiloride and PcTX-1 could remarkably decrease these parameters. These findings show that increased [Ca2+]i, mediated via ASIC1a, might contribute to acid-induced vascular endothelial cell injury of HSP.

Locus Coeruleus Acid-Sensing Ion Channels Modulate Sleep–Wakefulness and State Transition from NREM to REM Sleep in the Rat

The locus coeruleus(LC) is one of the essential chemoregulatory and sleep–wake(S–W) modulating centers in the brain. LC neurons remain highly active during wakefulness, and some implicitly become silent during rapid eye movement(REM) sleep. LC neurons are also involved in CO_2-dependent modulation of the respiratory drive. Acid-sensing ion channels(ASICs) are highly expressed in some brainstem chemosensory breathing regulatory areas, but their localization and functions in the LC remain unknown. Mild hypercapnia increases the amount of non-REM(NREM) sleep and the number of REM sleep episodes, but whether ASICs in the LC modulate S–W is unclear. Here, we investigated the presence of ASICs in the LC and their role in S–W modulation and the state transition from NREM to REM sleep. Male Wistar rats were surgically prepared for chronic polysomnographic recordings and drug microinjections into the LC. The presence of ASIC-2 and ASIC-3 in the LC was immunohistochemically characterized.Microinjections of amiloride(an ASIC blocker) and APETx2(a blocker of ASIC-2 and-3) into the LC significantly decreased wakefulness and REM sleep, but significantly increased NREM sleep. Mild hypercapnia increased the amount of NREM and the number of REM episodes. However, APETx2 microinjection inhibited this increase in REM frequency. These results suggest that the ASICs of LC neurons modulate S–W, indicating that ASICs could play an important role in vigilance-state transition. A mild increase in CO_2 level during NREM sleep sensed by ASICs could be one of the determinants of state transition from NREM to REM sleep.

触脑脊液神经元的研究进展

触脑脊液神经元（cerebrospinal fluid-contacting neurons,CSF-cNs）是一种分布于脑室、中央管、脑室周器及脑实质等处与脑脊液接触的特殊神经元。根据分布位置不同可将CSF-cNs分为室管膜上、室管膜下和远位CSF-cNs三类,不同部位的CSF-cNs分泌不同的神经递质。以往研究CSF-cNs多采用脑室注射辣根过氧化物酶标记的霍乱毒素B亚单位（cholera toxin subunit B labeled with horseradish peroxidase,CB-HRP）进行逆行追踪.

Lumbar radiculopathy and its neurobiological basis

Lumbar radiculopathy, a group of diseases in which the dorsal root ganglia(DRG) or dorsal roots are adversely affected by herniated discs or spinal stenosis, are clinically characterized by spontaneous and evoked types of pain. The pain is underpinned by various distinct pathophysiological mechanisms in the peripheral and central nervous systems. However, the diagnosis of lumbar radiculopathy is still unsatisfactory, because the association of the pain with the neurobiological basis of radiculopathy is largely unknown. Several animal models used to explore the underlying neurobiological basis of lumbar radiculopathy could be classified as mechanical, chemical, or both based on the component of injury. Mechanical injury elevates the intraneural pressure, reduces blood flow, and eventually establishes ischemia in the dorsal root and the DRG. Ischemia may induce ischemic pain and cause nerve damage or death, and the subsequent nerve damage or death may induce neuropathic pain. Chemical injury predominately induces inflammation surrounding the dorsal roots or DRG and consequent inflammatory mediators cause inflammatory pain. Furthermore, DRG neurons sensitized by inflammatory mediators are hypersensitive to innocuous mechanical force(stretch or compression) and responsible for mechanical allodynia in radiculopathy. As well, central sensitization in the spinal cord may play an important role in pain generation in lumbar radiculopathy. Increasing knowledge of pain-generating mechanisms and their translation into clinical symptoms and signs might allow for dissecting the mechanisms that operate in each patient. With precise clinical phenotypic characterization of lumbar radiculopathy and its connection to a specific underlying mechanism, we should be able to design optimal treatments for individuals. This review discusses the present knowledge of lumbar radiculopathy and proposes a novel mechanism-based classification.

Molecular mechanism of inflammatory pain

Chronic inflammatory pain resulting from arthritis, nerve injury and tumor growth is a serious public health issue. One of the major challenges in chronic inflammatory pain research is to develop new pharmacologic treatments with long-term efficacy and few side effects. The mediators released from inflamed sites induce complex changes in peripheral and central processing by directly acting on transducer receptors located on primary sensory neurons to transmit pain signals or indirectly modulating pain signals by activating receptors coupled with G-proteins and second messengers. High local proton concentration(acidosis) is thought to be a decisive factor in inflammatory pain and other mediators such as prostaglandin, bradykinin, and serotonin enhance proton-induced pain. Proton-sensing ion channels [transient receptor potential V1(TRPV1) and the acid-sensing ion channel(ASIC) family] are major receptors for direct excitation of nociceptive sensory neurons in response to acidosis or inflammation.G-protein-coupled receptors activated by prostaglandin, bradykinin, serotonin, and proton modulate functions of TRPV1, ASICs or other ion channels, thus leading to inflammation- or acidosis-linked hyperalgesia. Although detailed mechanisms remain unsolved, clearly different types of pain or hyperalgesia could be due to complex interactions between a distinct subset of inflammatory mediator receptors expressed in a subset of nociceptors. This review describes new directions for the development of novel therapeutic treatments in pain.

ASIC2 Synergizes with TRPV1 in the Mechano-Electrical Transduction of Arterial Baroreceptors

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 TRPV 1 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 synergisti-cally in the mechano-electrical transduction of arterial baroreceptors.The model of synergism between MSCs may have important biological significance beyond ASIC2 and TRPV 1.