Effects of Mitochondrial ATP-sensitive K^+ Channel on Protein Kinase C Pathway and Airway Smooth Muscle Cell Proliferation in Asthma

The effects of ATP-sensitive mitochondrial K + channel(mitoK ATP) on mitochondrial membrane potential(Δψm),cell proliferation and protein kinase C alpha(PKCα) expression in airway smooth muscle cells(ASMCs) were investigated.Thirty-six Sprague-Dawley(SD) rats were immunized with saline(controls) or ovalbumin(OVA) with alum(asthma models).ASMCs were cultured from the lung of control and asthma rats.ASMCs were treated with diazoxide(the potent activator of mitoK ATP) or 5-hydroxydencanote(5-HD,the inhibitor of mitoK ATP).Rhodamine-123(R-123) was used to detect Δψm.The expression of PKCα protein was examined by using Western blotting,while PKCα mRNA expression was detected by using real-time PCR.The proliferation of ASMCs was measured by MTT assay and cell cycle analysis.In diazoxide-treated normal ASMCs,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and percentage of cells in S phase were markedly increased as compared with untreated controls.The ratio of G 0 /G 1 cells was decreased(P<0.05) in diazoxide-treated ASMCs from normal rats.However,there were no significant differences between the ASMCs from healthy rats treated with 5-HD and the normal control group.In untreated and diazoxide-treated ASMCs of asthmatic rats,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and the percentage of cells in S phase were increased in comparison to the normal control group.Furthermore,in comparison to ASMCs from asthmatic rats,these values were considerably increased in asthmatic group treated with diazoxide(P<0.05).After exposure to 5-HD for 24 h,these values were decreased as compared with asthma control group(P<0.05).In ASMCs of asthma,the signal transduction pathway of PKCα may be involved in cell proliferation,which is induced by the opening of mitoK ATP and the depolarization of Δψm.

Altered expression of stromal interaction molecule(STIM)-calcium release-activated calcium channel protein(ORAI) and inositol1,4,5-trisphosphate receptors(IP_3Rs)in cancer:will they become a new battlefield for oncotherapy?

The stromal interaction molecule(STIM)-calcium release-activated calcium channel protein(ORAI) and inositol1,4,5-trisphosphate receptors(IP_3Rs) play pivotal roles in the modulation of Ca^(2+)-regulated pathways from gene transcription to cell apoptosis by driving calcium-dependent signaling processes.Increasing evidence has implicated the dysregulation of STIM-ORAI and IP_3Rs in tumorigenesis and tumor progression.By controlling the activities,structure,and/or expression levels of these Ca^(2+)-transporting proteins,malignant cancer cells can hijack them to drive essential biological functions for tumor development.However,the molecular mechanisms underlying the participation of STIM-ORAI and IP_3Rs in the biological behavior of cancer remain elusive.In this review,we summarize recent advances regarding STIM-ORAI and IP_3Rs and discuss how they promote cell proliferation,apoptosis evasion,and cell migration through temporal and spatial rearrangements in certain types of malignant cells.An understanding of the essential roles of STIM-ORAI and IP_3Rs may provide new pharmacologic targets that achieve a better therapeutic effect by inhibiting their actions in key intracellular signaling pathways.

Antibody Therapies Targeting Complex Membrane Proteins

In analyses of protein families that may serve as drug targets,membrane-associated G-protein-coupled receptors(GPCRs)dominate,followed by ion channels,transporters,and—to a lesser extent—membrane-bound enzymes.However,various challenges put such membrane proteins among key groups of underutilized opportunities for the application of therapeutic antibodies.Antibodies hold the promise of exquisite specificity,as they are able to target even specific conformations of a particular membrane protein,as well as adaptability through engineering into various antibody formats.However,the ease of raising and isolating specific,effective antibodies targeting membrane proteins depends on many factors.In particular,the generation of specific antibodies is easier when targeting larger,simpler,extracellular domains with greater uniqueness of amino acid sequence.The rareness of such ideal conditions is illustrated by the limited number of approved biologics for targeting GPCRs and other complex membrane proteins.Challenges in developing antibodies to complex membrane proteins such as GPCRs,ion channels,transporters,and membrane-bound enzymes can be addressed by the design of the antigen,antibody-generation strategies,lead optimization technologies,and antibody modalities.A better understanding of the membrane proteins being targeted would facilitate mechanism-based drug discovery.This review describes the advantages and challenges of targeting complex membrane proteins with antibodies and discusses the preparation of membrane protein antigens and antibody generation,illustrated by select examples of success.

The Effects of Protein Kinase C (PKC) on the Tension of Normal and Passively Sensitized Human Airway Smooth Muscle and the Activity of Voltage-dependent Delayed Rectifier Potassium Channel (Kv)

The effects of protein kinase C （PKC） on the tension and the activity of voltage-dependent delayed rectifier potassium channel （K,,） were examined in normal and passively sensitized human airway smooth muscle （HASM）, by measuring tones and whole-cell patch clamp techniques, and the Kv activities and membrane potential （Em） were also detected. The results showed that phorbol 12-myristate 13-acetate （PMA）, a PKC activator, caused a concentration-dependent constriction in normal HASM rings. The constriction of the passively sensitized muscle in asthma serum group was significantly higher than that of the normal group （P〈0.05）, and the constrictions of both groups were completely abolished by PKC inhibitor Ro31-8220 and calcium channel inhibitor nifedipine. Kv activities of HASM cells were significantly inhibited by PMA, and the Em became more positive, as compared with the DMSO （a PMA menstruum）-treated group （P〈0.01）. This effect could be blocked by Ro31-8220 （P〈0.01 ）. It was concluded that activation of PKC could increase the tones of HASM, which might be related to the reduced Kv activity. In passively sensitized HASM rings, this effect was more notable.

Curcumin up-regulates B K channel by inhibition of protein degradation and activation of an ERK1/2 signaling pathway

Aim Large conductance Ca2^＋ -activated potassium channel （BK） , expressed in the distal nephron, me- diates potassium secretion. Loss-of-function of renal BK channel is closely related with aldosteronism resulting from renal potassium retention and hyperkalemia. Natural products affecting BK functions are still scarce, especially ac- tivators. Here, the pharmacological characterization of curcumin, one of the compounds isolated from the herb Cur- cuma longa. , on B K channels have been investigated. Methods B K currents were recorded by whole-cell patch- clamp, mRNA expressions of BK were measured by quantitative real-time PCR. The surface and total protein ex- pressions of B K were assessed by surface biotinylation and Western blot. Functional study was performed on aortic rings. Results Curcumin potently increased B K currents in transfected HEK293 cells as well as the current densi- ty in A7r5 cells （ endogenous expressed BK （ α ＋ β1） channels） with ECs0 - （6.76 ± 2.24） μmol · L^-1 and （7.19 ± 0.07） μmol · L^-1, respectively. Curcumin up-regulated B K protein abundance without affecting its mR- NA expression in A7r5 cells. Surface expression and half-life of B K channels were increased by curcumin in HEK293 cells, which were abolished by MG-132, a proteasome inhibitor. Simultaneously, ERK 1/2 phosphoryla- tion was also increased by curcumin. U0126, an inhibitor of ERK, attenuate the curcumin-induced up-regulation of BK protein level. Curcumin-induced relaxation in isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. Conclusion Curcumin increased BK currents and protein abundance by inhibiting proteasomal degradation and activating ERK signaling pathway. These findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and mechanisms.

Identification of Three Interactions to Determine the Conformation Change and to Maintain the Function of Kir2.1 Channel Protein

We find that a conserved mutation residue Glu to residue Asp （E303D）, which both have the same polar and charged properties, makes Kit2.1 protein lose its function. To understand the mechanism, we identify three interactions which control the conformation change and maintain the function of the Kit2.1 protein by combining homology modeling and molecular dynamics with targeted molecular dynamics. We find that the E303D mutation weakens these interactions and results in the loss of the related function. Our data indicate that not only the amino residues but also the interactions determine the function of proteins.

Effect of G_(αq/11) Protein and ATP-sensitive Potassium Channels on Ischemic Preconditioning in Rat Hearts

Objectives To investigate the effect of Gαq/11 signaling pathway and ATP-sensitive potassium channel ( KATP channel ) on ischemic preconditioning (IPC) protection in rat hearts. Methods Two series of experiments were performed in Wistar rat hearts. In the first series of experiment, ischemic preconditioning was induced by left anterior descending occlusion (three, 5 min episodes separated by 5 min of reperfusion), ischemia-reperfusion injury was induced by 30 min coronary artery occlusion followed by 90 min reperfusion. Hemodynamics, infarct size and scores of ventricular arrhythmias were measured. The expression of Gαq/11 protein in the heart was measured by Western blot analysis in the second series. Results Ischemic preconditioning rats showed decreased infarct size and scores of ventricular arrhythmia vs non-IP control rats. The effect of IPC was significantly attenuated by glibenclamide (1 mg/kg, ip), a nonselective KATP channel inhibitor. IPC caused a significant increase in the expression of Gαq/11 protein. Conclusions Activations of Gαq/11 signal pathway and KATP channel played significant roles in the classical cardioprotection of ischemic precon-ditioning rat heart and might be an important mechanism of signal transduction pathway during the ischemic preconditioning.

Involvement of Calcium dependent Protein Kinases in ABA regulation of Stomatal Movement

Patch clamp techniques were employed to investigate if calcium dependent protein kinases (CDPKs) be involved in the signal transduction pathways of stomatal movement regulation by the phytohormone abscisic acid (ABA) in Vicia faba. Stomatal opening was completely inhibited by external application of 1 μmol/L ABA, and such ABA inhibition was significantly reversed by the addition of CDPK inhibitor trifluoperazine (TFP). The inward whole cell K + currents were inhibited by 60% in the presence of 1 μmol/L intracellular ABA, and this inhibition was completely abolished by the addition of CDPK competitive substrate histone Ⅲ S. The results suggest that CDPKs may be involved in the signal transduction cascades of ABA regulated stomatal movements.

机械应力调控血管平滑肌细胞的凋亡

背景:随着生物力学的发展,其在心血管疾病中的研究也日益广泛,通过研究血管的力学特性可以有效地揭示动脉粥样硬化、再狭窄等心血管疾病的发病机制,开发心血管疾病治疗的新思路和新方法。目的:综述机械应力刺激对血管平滑肌细胞凋亡的研究现状,寻找临床治疗潜在靶分子和信号通路,以期改善临床动脉粥样硬化及再狭窄等心血管疾病的临床治疗效果。方法:检索1992年1月至2023年5月在中国知网、PubMed及ScienceDirect数据库收录的文献。中文检索词为“血管平滑肌细胞,机械应力,剪切力,牵张力,凋亡”;英文检索词为“vascular smooth muscle cell,mechanical stress,shear stress,stretch stress,apoptosis”,最终纳入63篇文献进行综述分析。结果与结论:①血管平滑肌细胞的生理性和病理性凋亡都是为了适应血管力学变化而发生的适应性重构。不同部位的平滑肌细胞承受不同的力学刺激,其发病机制也不同。②低剪切力、生理剪切力和高剪切力可通过直接与平滑肌细胞表面分子、受体及蛋白等作用调控凋亡信号分子和抑制增殖实现对血管平滑肌细胞凋亡的调控,该部分未对促进增殖的研究进行综述。③低牵张力、生理牵张力和高牵张力可导致平滑肌细胞凋亡,但仍存在争议。平滑肌表面存在多种力学感受分子(如整合素及受体络氨酸激酶等)可以将力学信号转导为细胞内的化学信号(如Hippo通路),启动平滑肌细胞的凋亡信号,调控平滑肌细胞的凋亡。④总之,不同力学刺激通过多种信号分子,启动多个信号通路共同作用,调控平滑肌细胞的凋亡,例如剪切力主要通过刺激分泌前列腺素、转化生长因子等影响Fas/FasL、Akt通路;而牵张力主要通过Yes相关蛋白等调控YAP通路和Notch通路等。这些分子在不同的作用时间,或不同作用强度下可能发挥相反的双向作用,比如,小鼠血管平滑肌细胞受到10%生理牵张1 h时,其增殖增加;然而,人的血管平滑肌细胞牵张12 h后可以减少其增殖。临床可以通过寻找其关键作用时间点干扰力学转导关键分子,达到预防和治疗临床心血管疾病的目的。

骨植入材料表面微结构对MC3T3-E1成骨细胞成骨分化的影响

背景:骨植入材料表面微纳结构仿生设计可模拟人体天然骨组织中细胞外环境的结构,从而获得较为理想的骨整合功能,但骨植入材料表面微纳结构调控细胞功能及促进成骨的机制还有待进一研究。目的:分析钛片材料微结构表面对MC3T3-E1成骨细胞成骨分化的影响。方法:(1)在5 V或20 V恒定电压下,采用酸蚀和阳极氧化技术在钛片表面制备不同管径的纳米管阵列,分别记为R5组、R20组。检测纯钛片(未经酸蚀和阳极氧化处理)、R5组、R20组的表面形貌、粗糙度与亲水性。(2)将对数生长期的MC3T3-E1成骨细胞分别接种于纯钛片、R5组、R20组钛片表面,成骨诱导培养24 h后,采用RT-PCR检测与免疫荧光染色分析细胞机械敏感通道蛋白1的表达;加入含或不含机械敏感通道蛋白1激活剂Yada1的成骨诱导基,培养7 d后进行碱性磷酸酶染色,培养14 d后进行茜素红染色。结果与结论:(1)扫描电镜下可见纯钛片表面光滑,R5组、R20组钛片表面可见分布相对均匀且有序的纳米管阵列,平均直径分别约30 nm和100 nm,原子力显微镜进一步验证了扫描电镜观察结果。R5组钛片表面粗糙度大于纯钛片(P <0.05),水接触角小于纯钛片(P <0.05);R20组钛片表面粗糙度大于R5组(P<0.05),水接触角小于R5组(P<0.05)。(2)RT-PCR检测与免疫荧光染色显示,R5组细胞机械敏感通道蛋白1的表达高于纯钛片组(P <0.05),R20组细胞机械敏感通道蛋白1的表达高于R5组(P <0.05)。在成骨诱导条件下,与未加入Yada1情况相比,纯钛片组加入Yada1后的细胞碱性磷酸酶活性、钙化结节沉积均无明显变化,R5组、R20组加入Yada1后的细胞碱性磷酸酶活性、钙化结节沉积均显著增加(P <0.05);在加入或未加入Yada1情况下,R5组细胞碱性磷酸酶活性与钙化结节沉积均多于纯钛片组(P <0.05),R20组细胞碱性磷酸酶活性与钙化结节沉积均多于R5组(P <0.05)。(3)结果表明,钛片表面微结构可通过激活机械敏感通道蛋白1促进成骨细胞MC3T3-E1的成骨分化。

C-reactive protein aggravates myocardial ischemia/reperfusion injury through activation of extracellular-signal-regulated kinase 1/2

Background Ischemia/reperfusion injury （IRI） is an inflammatory response that occurs when tissue is reperfused following a prolonged period of ischemia. Several studies have indicated that C-reactive protein （CRP） might play an important role in inducing IRI. However, the effects of CRP on myocardial IRI and the underlying mechanisms have not been fully elucidated. This study aimed to investigate the association between CRP and myocardial IRI and the underlying mechanisms. Methods We simulated ischemia/reperfusion using oxygen-glucose deprivation/ reoxygenation （OGD/R） in neonatal Sprague-Dawley rat cardiomyocytes; reperfusion injury was induced by three hours of hypoxia with glucose and serum deprivation followed by one hour of reperfusion. Cell viability was tested with MTS assays, and cardiomyocyte damage was evaluated by lactate dehydrogenase （LDH） leakage. Mitochondrial membrane potential was measured using tetramethylrhodamine ethyl ester （TMRE） and mitochondrial permeability transition pore （mPTP） opening was measured using calcein/AM; both TMRE and caocein/AM were visualized with laser scanning confocal microscopy. In addition, we studied the signaling pathways underlying CRP-mediated ischemia/reperfusion injury via Western blot analysis. Results Compared with the simple OGD/R group, after intervention with 10 pg/mL CRP, cell viability decreased markedly （82.36 % ± 6.18% vs. 64.84% ± 4.06%, P = 0.0007）, and the LDH leakage significantly increased （145.3 U/L ± 16.06 U/L vs. 208.2 U/L ± 19.23 U/L, P = 0.0122）. CRP also activated mPTP opening and reduced mitochondrial membrane potential during myocardial ischemia/reperfusion. Pretreatment with 1 pM atorvastatin （Ator） before CRP intervention protected cardiomyocytes from IRI. Mitochondrial KATP channel opener diazoxide and mPTP inhibitor cyclosporin A also offset the effects of CRP in this process. The level of phosphorylated extracellular-signal-regulated kinase （ERK） 1/2 was significantly higher after pre-treatment with CRP compared with the OGD/R group （170.4% ± 3.00% v.v. 93.53% ± 1.94%, P 〈 0.0001）. Western blot analysis revealed that Akt expression was markedly activated （184.2% ± 6.96% vs. 122.7% ± 5.30%, P = 0.0003） and ERK 1/2 phosphorylation significantly reduced after co-treatment with Ator and CRP compared with the level after CRP pretreatment alone. Conclusions Our results suggested that CRP directly aggravates myocardial IRI in myocardial cells and that this effect is primarily mediated by inhibiting mitochondrial ATP- sensitive potassium （mitoKATp） channels and promoting mPTP opening. Ator counteracts these effects and can reduce CRP-induced IRI. One of the mechanisms of CRP-induced IRI may be related to the sustained activation of the ERK signaling pathway.

Animal secretory endolysosome channel discovery

Secretory pore-forming proteins(PFPs) have been identified in organisms from all kingdoms of life. Our studies with the toad species Bombina maxima found an interaction network among aerolysin family PFPs(af-PFPs) and trefoil factors(TFFs). As a toad af-PFP, Bm ALP1 can be reversibly regulated between active and inactive forms, with its paralog Bm ALP3 acting as a negative regulator. Bm ALP1 interacts with Bm TFF3 to form a cellular active complex called βγ-CAT. This PFP complex is characterized by acting on endocytic pathways and forming pores on endolysosomes, including stimulating cell macropinocytosis. In addition, cell exocytosis can be induced and/or modulated in the presence of βγ-CAT. Depending on cell contexts and surroundings, these effects can facilitate the toad in material uptake and vesicular transport, while maintaining mucosal barrier function as well as immune defense. Based on experimental evidence,we hereby propose a secretory endolysosome channel(SELC) pathway conducted by a secreted PFP in cell endocytic and exocytic systems, with βγ-CAT being the first example of a SELC protein. With essential roles in cell interactions and environmental adaptations, the proposed SELC protein pathway should be conserved in other living organisms.

Migration-associated secretion of melanoma inhibitory activity at the cell rear is supported by KCa3.1 potassium channels

Malignant melanoma, characterized by invasive local growth and early formation of metastases, is the most aggressive type of skin cancer. Melanoma inhibitory activity （MIA）, secreted by malignant melanoma cells, interacts with the cell adhesion receptors, integrins a4131 and 05131, facilitating cell detachment and promoting formation of me- tastases. In the present study, we demonstrate that MIA secretion is confined to the rear end of migrating cells, while in non-migrating cells MIA accumulates in the actin cortex. MIA protein takes a conventional secretory pathway including coat protein complex I （COPI）- and coat protein complex II （COPII）-dependent protein transport to the cell periphery, where its final release depends on intracellular Ca2＋ ions. Interestingly, the Ca2＋-activated K＋-channel, subfamily N, member 4 （KCa3.1）, known to be active at the rear end of migrating cells, was found to support MIA secretion. Secretion was diminished by the specific KCa3.1 channel inhibitor TRAM-34 and by expression of dominant- negative mutants of the channel. In summary, we have elucidated the migration-associated transport of MIA protein to the cell rear and also disclosed a new mechanism by which KCa3.1 potassium channels promote cell migration.

Identification of TRPM7 channels in human intestinal interstitial cells of Cajal

AIM： To investigate the characteristics of slow electrical waves and the presence of transient receptor potential melastatin-type 7 （TRPM7） in the human gastrointestinal （GI） tract. METHODS： Conventional microelectrode techniques were used to record intracellular electrical responses from human GI smooth muscle tissue. Immunohistochemistry was used to identify TRPM7 channels in interstitial cells of Cajat （ICCs）. RESULTS： The human GI tract generated slow electrical waves and had ICCs which functioned as pacemak er cells. Flufenamic acid, a nonselective cation channel blocker, and 2-APB （2-aminoethoxydiphenyl borate） and La3＋, TRPM7 channel blockers, inhibited the slowwaves. Also, TRPM7 channels were expressed in ICCs in human tissue. CONCLUSION： These results suggest that the human GI tract generates slow waves and that TRPM7 channels expressed in the ICCs may be involved in the gen- eration of the slow waves.

Role of TRP channels and HSPs in thermal stress response in the aphid parasitoid Aphelinus asychis(Hymenoptera:Aphelinidae)

Aphelinus asychis is an important aphid endoparasitoid.Under field and greenhouse conditions,high temperature is one of the factors limiting the application of A asychis for biological pest control.To explore the potential role of transient receptor potential(TRP)channels and heat shock proteins(HSPs)in this process,we identified 11 genes encoding TRP channels and nine genes encoding HSPs.Three proteins(AasyTRPA5,AasyPyrexia,AasyPainless)that belong to transient receptor potential ankyrin(TRPA)subfamily and nine HSPs are involved in the response to high temperature.We also investigated the survival of A asychis and the response of the identified TRP channels and HSPs to high temperature.The results showed that the maximum temperature that allowed A.asychis survival was approximately 41°C;females had higher survival rates than that of the males at 40 and 41°C.Short-term heat-shock resulted in increased expression of Amsysbsp in males,and Aasyhsp40,Aasyhsp68,Aasyhsp70-4,Aasyhsp70-5 and Aasyhsp90 were upregulated and then down regulated,whereas Aasyhsp70-3was upregulated at41°C.Moreover,Aasyhsp40and Aasyhsp90showed higherexpression levels in females,while Aasyshsp and Aasyhsp70-3 presented opposite expression patterns.At temperature above 35°C,expression of AasyPyrexia in females was significant higher than that in males,whereas AasyPainless mnd AasyTRPA5 presented higher expression in males at 40 and 41°C,respectively.Altogether,these results indicate that protect!on against thermal stress in A.asychis is coordinated by TRP channels and HSPs.These findings provide a basis for understanding the potential mechanism of A asychis in response to high temperatures.

Synthesis and Characterization of A Small Molecule CFTR Chloride Channel Inhibitor

A thiazolidinone CFTR inhibitor(CFTR_ inh-172 ) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of 1H NMR and functionally in a CFTR-expressing cell line FRT/hCFTR/EYFP-H148Q by both fluorescent and electrophysiological methods. A large amount(100 g) of high-quality small molecule thiazolidinone CFTR chloride channel inhibitor,CFTR_ inh-172 ,can be produced with this simple three-step synthetic procedure. The structure of the final product 2-thioxo-3-(3-trifluromethylphenyl)-5-[4-carboxyphenyl- methylene]-4-thiazolidinone was confirmed by 1H NMR. The overall yield was 58% with a purity over 99% as analyzed by HPLC. The synthesized CFTR_ inh-172 specifically inhibited CFTR chloride channel function in a cell-based fluorescence assay( K _d≈1.5 μmol/L) and in a Ussing chamber-based short-circuit current assay( K _d≈0.2 μmol/L),indicating better quality than that of the commercial combinatorial compound. The synthesized inhibitor is nontoxic to cultured cells at a high concentration and to mouse at a high dose. The synthetic procedure developed here can be used to produce a large amount of the high-quality CFTR_ inh-172 suitable for antidiarrheal studies and for creation of cystic fibrosis models in large animals. The procedure can be used to synthesize radiolabled CFTR_ inh-172 for in vivo pharmacokinetics studies.

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.

Glabridin Relaxes Vascular Smooth Muscles by Activating BK_(Ca) Channels and Inhibiting Phosphodiesterase in Human Saphenous Vein

The aim of the current study was to investigate the pharmacological activity of glabridinon the isolated human saphenous vein (SV) and explore the underlying mechanisms. Samples of patients' SVs were removed during bypass surgery, and 4-mm lengths of the vessels were placedin Krebs solution at ±4℃ and hung in an isolated organ bath to assess their contraction/relaxationresponses. The contraction/relaxation responses were recorded to observe if the cyclic guanosinemonophosphate (cGMP)/protein kinase G (PKG) pathway mediates the relaxant effect of glabridinafter treatment with blockers like ODQ (a guanylate cyclase inhibitor), KT5823 (a PKG inhibitor),isobutylmethylxanthine [IBMX, a phosphodiesterase (PDE) inhibitor], and cantharidin [Cant,a myosin light-chain phosphatase (MLCP) inhibitor]. Moreover, nitric oxide (NO), cGMP, andPKG levels in SV tissues were determined by ELISA after incubation with glabridin, N(o)-nitro-L-arginine methyl ester (L-Name, a NO synthetase inhibitor), phenylephrine (PE), ODQ, IBMX,and KT5823. The results showed that glabridin relaxed the vascular smooth muscle of humanSV pretreated with PE in a dose-dependent manner, which was independent of the endothelium.The vasorelaxant effect of glabridin was only inhibited by iberiotoxin (IbTX), Cant, and KT5823.Glabridin increased cGMP and PKG levels in SV homogenates, whereas it did not alter the NOlevel. The enhancing efects of cGMP and PKG levels by glabridin were abolished by ODQ andKT5823. In conclusion, glabridin has a vasorelaxant effect, which is associated with the activationof BKc. channels and inhibition of PDE.

Roles of transient receptor potential channel 6 in glucose-induced cardiomyocyte injury

BACKGROUND Diabetic cardiomyopathy(DCM) is a serious complication of end-stage diabetes that presents symptoms such as cardiac hypertrophy and heart failure. The transient receptor potential channel 6(TRPC6) protein is a very important selective calcium channel that is closely related to the development of various cardiomyopathies.AIM To explore whether TRPC6 affects cardiomyocyte apoptosis and proliferation inhibition in DCM.METHODS We compared cardiac function and myocardial pathological changes in wild-type mice and mice injected with streptozotocin(STZ), in addition to comparing the expression of TRPC6 and P-calmodulin-dependent protein kinase Ⅱ(P-CaMKⅡ) in them. At the same time, we treated H9C2 cardiomyocytes with high glucose and then evaluated the effects of addition of SAR, a TRPC6 inhibitor, and KN-93, a CaMKⅡ inhibitor, to such H9C2 cells in a high-glucose environment.RESULTS We found that STZ-treated mice had DCM, decreased cardiac function, necrotic cardiomyocytes, and limited proliferation. Western blot and immunofluorescence were used to detect the expression levels of various appropriate proteins in the myocardial tissue of mice and H9C2 cells. Compared to those in the control group, the expression levels of the apoptosis-related proteins cleaved caspase 3 and Bax were significantly higher in the experimental group, while the expression of the proliferation-related proteins proliferating cell nuclear antigen(PCNA) and CyclinD1 was significantly lower. In vivo and in vitro, the expression of TRPC6 and P-CaMKⅡ increased in a high-glucose environment. However, addition of inhibitors to H9C2 cells in a high-glucose environment resulted in alleviation of both apoptosis and proliferation inhibition.CONCLUSION The inhibition of apoptosis and proliferation of cardiomyocytes in a high-glucose environment may be closely related to activation of the TRPC6/P-CaMKⅡ pathway.

Effect of genistein on voltage-gated potassium channels in guinea pig proximal colon smooth muscle cells

AIM： To investigate the action of genistein （GST）, a broad spectrum tyrosine kinase inhibitor, on voltagegated potassium channels in guinea pig proximal colon smooth muscle cells. METHODS： Smooth muscle cells in guinea pig proximal colon were enzymatically isolated. Nystatin-perforated whole cell patch clamp technique was used to record potassium currents including fast transient outward current （Ikto） and delayed rectifier current （Ikdr）, tWO of which were isolated pharmacologically with 10 mmol/L tetraethylammonium or 5 mmol/L 4-aminopyridine. Contamination of calcium-dependent potassium currents was minimized with no caldum and 0.2 mmol/L CdCl2 in an external solution. RESULTS： GST （10-100 μmol/L） reversibly and dosedependently reduced the peak amplitude of Ikto with an IC50 value of 22.0±6.9 μmol/L. To a lesser extent, Ikdr, was also inhibited in both peak current and sustained current. GST could not totally block the outward potassium current as a fraction of the outWard potassium current, which was insensitive to GST. GST had no effect on the steady-state activation （n =6） and inactivation kinetics （n =6） of Ikto. Sodium orthovanadate （1 retool/L）, a potent inhibitor of tyrosine phosphatase, significantly inhibited GST-induced inhibition （P〈 0.05）. CONCLUSION： GST can dose-dependently and reversibly block voltage-gated potassium channels in guinea pig proximal colon smooth muscle cells.