Relationship of Intracellular Free Ca^(2+) Concentration and Calcium-activated Chloride Channels of Pulmonary Artery Smooth Muscle Cells in Rats under Hypoxic Conditions

To investigate the relationship between intracellular free Ca^2＋ concentration （[Ca^2＋ ]i ） and calcium-activated chloride （Clca） channels of pulmonary artery smooth muscle cells （PASMCs） in rats under acute and chronic hypoxic conditions, acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca^2＋ indicator Fura-2/AM was used to observe [Ca^2＋ ]i of rat PASMCs under normal and chronic hypoxic condition. The effect of Clca channels on PASMCs proliferation was assessed by MTT assay. The Clca channel blockers niflumic acid （NFA） and indaryloxyacetic acid （IAA-94） exerted inhibitory effects on acute hypoxia-evoked contractions in the pulmonary artery. Under chronic hypoxic condition, [Ca^2＋ ]i was increased. Under normoxic condition, [Ca^2＋ If was （123.634-18.98） nmol/ L, and in hypoxic condition, [Ca^2＋]i wag （281. 754-16.48） nmol/L （P〈0. 01）. Under normoxic condition, [Ca^2＋ ]i showed no significant change and no effect on Clca channels was observed （P〉 0. 05）. Chronic hypoxia increased [Ca^2＋ ]i which opened Clca channels. The NFA and IAA-94 blocked the channels and decreased [Ca^2＋ ]i from （281.75± 16.48） nmot/L to （117.66 ±15.36） nmol/L （P〈0.01）. MTT assay showed that under chronic hypoxic condition NFA and IAA-94 decreased the value of absorbency （A value） from 0. 459±0. 058 to 0. 224±0. 025 （P〈0. 01）. Hypoxia increased [Ca^2＋ ]i which opened Cl~ channels and had a positive-feedback in [Ca^2＋ ]i. This may play an important role in hypoxic pulmonary hypertension. Under chronic hypoxic condition, Clca channel may play a part in the regulation of proliferation of PASMCs.

Functional remodeling of Ca^(2+)-activated Cl^-channel in pacing induced canine failing heart

Objective To determine whether Ca2+ activated Cl- current(Icl(Ca)) contributes to the functional remodeling of the failing heart.Methods Whole cell patch-clamp recording technique was employed to record the Icl(Ca) in cardiac myocytes enzymatically isolatedfrom rapidly pacing induced canine failing hearts at room temperature and compared that of the normal hearts (Nor).Results Thecurrent density of DIDS(200M)sensitive Icl(Ca) induced by intracellular Ca2+ release trigged by L-type Ca2+ current(Ica,L)wassignificantly decreased in heart failare(HE)cells compared to Nor cells.At membrane voltage of 20mV,the Icl(Ca) density was 3.02±0.54 pA/pF in Nor(n=6)vs.1.31±0.25 pA/pF in HF(n=8)cells,(P＜0.01),while the averaged Ica,L density did not show differencebetween two groups.The time constant of current decay of Icl(Ca) was similar in both types of cells.On the other hand,in intra cellularCa2+ clamped mode,where the[Ca2+];was maintained at 100nmol/L,Icl(Ca) density be increased significantly in HF cells when themembrane voltage at+30mV or higher.Conclusions Our results suggest that Icl(Ca) density was decreased in pacing induced failingheart but the channel function be enhanced.Impaired Ca2+ handing in HF cells rather than reduced,Icl(Ca) channel function itself may havecaused this abnormality.The Icl(Ca) density reduction might contribute to the prolongation of action potential in failing heart.The Icl(Ca)channel function up-rugulation is likely to cause cardiac arrhythmia by inducing a delayed after depolarization,when Ca2+ overloadoccurred in diastolic failing heart cells.

Changes of Ca^2+ activated potassium channels and cellular proliferation in autogenous vein grafts

Objective: To investigate changes of Ca2+ activated potassium channels (KCa) in autogenous vein grafts. Methods: Contraction of venous ring was measured by means of perfusion in vitro. The intimal rabbits proliferation of vascular and proliferation of cultured smooth muscle cells(vascular smooth muscle cells, VSMCs)were observed by the means of computerised image analysis and MTT method respectively. Furthermore, whole cell mode of patch clamp was used to record KCa of VSMCs isolated from autogenous vein grafts. Results: One week after transplantation there were no significant differences of contraction and intimal relative thickness between autogenous vein grafts and control. Contraction and intimal relative thickness of autogenous vein graft were significantly increased 2 weeks after transplantation (P<0.05, n=8 vs control), and they was more enhanced 4 weeks after vein transplantation (P<0.01, n=8 vs control).TEA(blocker of Ca2+ activated potassium channels)increased MTT A490 nm value of VSMCs from femoral vein in a dose dependent manner(P<0.05, n=8). KCa current density was significantly attenuated in VSMCs from autogenous vein grafts (1-4) week after transplantation(P<0.05, n=5).Conclusion: KCa is inhibited in autogenous vein graft, which account for vasospasm and intimal proliferation.

Correlation of large conductance Ca2+ activated K+ channelα andβ subunit expression in uterine smooth muscle with the postpartum hemorrhage induced by uterine inertia

Objective:To study the correlation of large conductance Ca2+ activated K+ channel (BKCa)α andβ subunit expression in uterine smooth muscle with the postpartum hemorrhage induced by uterine inertia.Methods: The puerperae who underwent cesarean section and had postpartum hemorrhage induced by uterine inertia in Panzhihua Women and Children Health Hospital between March 2015 and May 2017 were selected as the hemorrhage group of the study, and the puerperae who underwent cesarean section and were without postpartum hemorrhage in Panzhihua Women and Children Health Hospital during the same period were selected as the control group. Proper amount of uterine muscle tissue was collected during the cesarean section to measure the expression of BKCaα andβ subunits and the levels of contraction-related proteins in uterine muscle as well as the contraction characteristic parameters of the uterine muscle.Results: The mRNA expression and protein expression of BKCaα andβ subunits in uterine muscle tissue of hemorrhage group were significantly higher than those of control group;the contraction amplitude, contraction frequency and contraction activity of uterine muscle tissue as well as the OTR, COX2, CX43 and HSP27 levels in uterine muscle tissue of hemorrhage group were significantly lower than those of control group;the BKCaα andβ subunit expression in uterine muscle tissue of hemorrhage group were negatively correlated with the contraction amplitude, contraction frequency and contraction activity as well as the OTR, COX2, CX43 and HSP27 levels.Conclusion: The high expression of BKCa in uterine smooth muscle can reduce the uterine muscle contractility and decrease the levels of contraction-related proteins, and it is closely related to the occurrence of postpartum hemorrhage induced by uterine inertia.

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.

Effects of calcium-activated chloride channels on proliferation of pulmonary artery smooth muscle cells in rats under chronic hypoxic condition

Objective：To investigate the effects of calcium-activated chloride （ClCa） channels on proliferation of pulmonary artery smooth muscle cells（PASMCs） in rats under chronic hypoxic condition. Methods：The cultured PASMCs were placed under normoxic and chronic hypoxic conditions：The cells were observed by light and electron microscope; The cell cycles were observed by flow-cytometry; Immunocytochemistry staining was used to detect the expressions of PCNA, c-fos and c-jun of PASMCs; Cytoplasmic free Ca^2＋ concentration （[Ca^2＋]i） in PASMCs was investigated by fluorescent quantitation using fluorospectrophotometer. Results：The PASMCs were contractile phenotype under normoxic conditions. Observation by transmission electron microscope： In kytoplasm of contractile phenotype cells, myofilament bundles were abundant and the content of cell organs such as Golgi＇s bodies were rare. The PASMCs were synthetic phenotype under chronic hypoxic condition. There were increased free ribosomes, dilated rough endoplasmic reticulums, highly developed Golgi complexes, decreased or disappeared thick filaments and dense body in kytoplasm of synthetic phenotype cells. After NFA and IAA-94, the situations were reversed The number of S ＋G2M PASMCs were significantly increased in chronic hypoxic condition; The NFA and IAA-94 were shown to significantly decrease them from （28.6±1.0）% to （16.0±1.6）% and the number of G0G1 PASMCs significantly increased from （71.4± 1.9）% to （83.9 ± 1.6）% （P〈 0.01）. In chronic hypoxic conditions, the expression of proliferating cell nucleus antigen was significantly increased; The NFA and IAA-94 were shown to significantly decrease it from （81 ± 6）% to （27 ± 7）%（P 〈 0.01）. The expression of c-fos and c-jun were significantly increased in＇chronic hypoxic conditions; The NFA and IAA-94 were shown to significantly decrease them from 0.15 ±0.02, 0.32 ± 0.05 to 0.05 ± 0.01, 0.12 ± 0.05, respectively （P〈 0.01）; Under chronic hypoxic conditions, [Ca^2＋]i was increased; The NFA and IAA-94 decreased it from （281.8±16,5）nmol/L to （117.7 ± 15.4）nmol/L（P 〈 0.01）. Conclusion：Hypoxia initiated the change of PASMCs from contractile to synthetic phenotype and increased proliferation of PASMCs. NFA and IAA-94 depressed cell proliferation by blocking ClCa channels in hypoxic condition. These may play an important role in proliferation of PASMCs under chronic hypoxic conditions.

PDGFRα^(+)细胞上P2Y1-SK3通路对功能性消化不良大鼠胃肠动力的调控机制

目的探究血小板衍生生长因子受体α^(+)(PDGFRα^(+))细胞上P2Y1-小电导Ca^(2+)激活K^(+)(SK3)通道对功能性消化不良(FD)大鼠胃肠动力的影响。方法将30只SD大鼠随机分为空白组、模型组、P2Y1受体抑制剂(MRS2500)组,每组10只。除空白组外,其余两组采用多因素干预法建立FD大鼠模型。造模成功后抑制剂组予以尾静脉注射P2Y1抑制剂MRS2500,其他组不采取干预措施。处理结束后进行行为学和胃肠动力学检测;用BL-420S生物信号系统采集并分析胃肠生物电信息;取胃窦组织评估病理变化;采用免疫印迹、实时荧光定量PCR技术检测各组大鼠胃窦PDGFRα、C-kit(卡介尔间质细胞特异性指标)、P2Y1和SK3的表达情况;采用免疫荧光法检测胃窦PDGFRα和C-kit、P2Y1、SK3的组织表达和共定位情况;用钙检测试剂盒检测胃窦组织中Ca^(2+)含量变化。结果FD模型建立后,大鼠活动度、体重增长速度和进食量都显著降低,胃肠动力减弱,胃窦内PDGFRα、C-kit、P2Y1和SK3表达水平降低。MRS2500干预后,P2Y1受体抑制剂组大鼠较模型组大鼠体重增长率和进食量升高,胃肠动力减弱情况改善,PDGFRα、P2Y1和SK3表达水平进一步降低,C-kit表达水平升高,Ca^(2+)含量降低。PDGFRα与C-kit在胃窦中不存在共表达,而PDGFRα与P2Y1、SK3共表达。结论长期的饮食和情绪失调会刺激肠神经系统释放抑制性神经递质,这一过程通过PDGFRα^(+)细胞上P2Y1受体引起SK3通道的Ca^(2+)敏感性降低,在多因素刺激诱导的FD模型大鼠胃肠动力障碍中起重要作用。

IgA肾病患者肾组织内电压依赖性Ca^(2+)通道和高通透性Ca^(2+)激活钾通道mRNA的表达

目的观察IgA肾病(IgAN)患者肾组织内电压依赖性Ca2+通道(VOCC)和高通透性Ca2+激活钾通道(BKCa)mRNA表达情况,以评价VOCC和BKCa通道mRNA表达与IgAN患者肾脏病理损害的相关性。方法提取对照组(n=11)和IgAN患者(n=25)肾组织总RNA,以RT-PCR方法检测VOCC和BKCa mRNA表达量,同时将IgAN患者肾活检组织的一部分进行病理诊断和病理损害评分,病理损害评分采用Katafuchi半定量法。结果与对照组比较,IgAN患者肾组织内VOCC和BKCa mRNA的表达均呈显著增高(P<0.05);IgAN患者肾组织内VOCC mRNA表达与病理损害总积分、肾小球系膜细胞增生程度均呈正相关(r=0.6962,P<0.01;r=0.4220,P<0.05),而与肾小球系膜基质增多和肾小球硬化程度无相关性(P>0.05);IgAN患者肾组织内BKCa mRNA表达与病理损害总积分呈正相关(r=0.5582,P<0.05),而与肾小球系膜细胞增生程度、肾小球系膜基质增多以及肾小球硬化程度均无相关性(均P>0.05)。结论IgAN患者肾组织内VOCC和BKCa mRNA表达异常,两种通道蛋白的mRNA表达异常与肾小球组织病理损害总积分呈一定程度正相关,提示IgAN患者肾组织内VOCC和BKCa的表达情况,可作为IgA肾病进展与否的指标。

Ca^(2+)经钙激活非选择性阳离子通道进入ECV304内皮细胞

目的 研究钙离子进入ECV30 4内皮细胞株的途径和血管紧张素Ⅱ (AⅡ )对钙内流的影响。方法 用膜片钳的细胞贴附式和全细胞方式记录ECV30 4内皮细胞的通道活动。结果 (1 )在记录单通道电流时电极液含 1 2 0mmol·L- 1 CaCl2 ,细胞浴液不含K+ 、Na+ 时 ,Ca2 + 经非选择性阳离子通道 (CAN)内流的电导为γ0 =(1 2 90± 2 1 1 ) pS(n =4)。1× 1 0 - 7mol·L- 1 AⅡ可显著增强通道电流幅度和延长通道开放时 ,其电导增大为γ1 =(2 2 1 8± 2 2 9)pS(n =4)。全细胞记录得到的结果与单通道的一致。 (2 )用全细胞方式记录到ECV30 4内皮细胞的电压依赖性钙通道电流 ,记录到该峰值电流为 (2 9 32± 3 56)pA(n =4) ,2 0 μmol·L- 1 nifedepine能抑制这个峰值电流 ,被抑制后的电流峰值为 (6 0 0± 3 94)pA(n =4)。 2 μmol·L- 1 BayK8644能显著激活通道活动。结论 Ca2 + 经CAN进入ECV30 4细胞 。

血管钠肽对大鼠肠系膜动脉平滑肌细胞Ca^(2+)激活K^+通道的作用

目的:研究血管钠肽(VNP)对大鼠肠系膜动脉血管平滑肌细胞(VSMCs)Ca2+激活K+通道(KCa)的作用及其机制。方法:采用全细胞膜片钳技术观察VNP对KCa的影响,以及HS-142-1、8-Br-cGMP和美蓝(MB)在这一过程中的作用。结果:①VNP(10-6mol/L)显著增强KCa(P<0.05,n=5)。②8-Br-CGMP(10-3mol/L)模拟VNP增强KCa的作用(P<0.05,n=6)。③HS-142-1(2×10-5mol/L)或MB(10-5mol/L)完全阻断VNP增加KCa电流密度的作用。结论:VNP通过作用于VSMCs的钠尿肽GC耦联受体,升高细胞内的cGMP水平,激活KCa。

下丘脑神经元中游离Ca^(2+)的电生理测定法

目的 采用膜片钳技术通过对SD乳鼠下丘脑神经元上Ca2 + 激活K+ 通道 (KCa)Ca2 + 敏感性的研究测定其细胞内游离Ca2 + 浓度 ([Ca2 + ]i)。方法 首先应用内面向外式研究不同 [Ca2 + ]i 时KCa通道的开放概率 (P0 ) ,作出量效曲线 ,再应用细胞贴附式求得生理状态下此通道的开放概率 ,从量效曲线上查出的细胞内游离Ca2 + 浓度。结果 试验表明SD乳鼠下丘脑神经元上KCa通道的P0 具有对 [Ca2 + ]i 的高度敏感性 ,求得下丘脑神经元内的 [Ca2 + ]i 为 0 1μmol/L。 结论 实验结果说明此电生理法测定 [Ca2 + ]i 具有较高的可靠性和准确性。

Ca^(2+)在链霉素对大鼠颈动脉窦压力感受器反射影响中的作用

在 2 3只隔离灌流颈动脉窦区的麻醉大鼠上 ,观察了链霉素 (streptomycin ,SM)对动脉压力感受器反射影响的离子机制。结果 :(1)用SM (2 0 0 μmol/L)隔离灌流大鼠颈动脉窦区时 ,压力感受器机能曲线向右上方移位 ,曲线最大斜率及反射性血压下降幅度均减小 (P <0 0 1) ,提示SM对压力感受器反射有抑制作用 ;(2 )预先灌流高Ca2 +溶液 (4mmol/L)后 ,可部分消除SM (2 0 0 μmol/L)对压力感受器反射的抑制作用 (P <0 0 1) ,使其压力感受器机能曲线向左下方移位 ,曲线的最大斜率由 0 2 7± 0 0 4增至 0 37± 0 0 2 (P <0 0 1) ,反射性血压下降幅度由4 32± 0 14kPa增至 6 18± 0 17kPa (P <0 0 1) ,而阈压和饱和压则分别从 10 2 9± 0 2 9和 2 7 2 6± 0 42kPa降至9 98± 0 33和 2 5 2 2± 0 38kPa (P <0 0 5 ) ;(3)用Ca2 +通道激动剂BayK 86 44 (5 0 0nmol/L)预处理 ,可完全消除SM(2 0 0 μmol/L)对压力感受器反射的抑制效应 ;(4 )预先给予Ca2 +激活性K+通道阻断剂 (charybdotoxin ,ChTX ,10 0nmol/L) ,对压力感受器反射无明显影响 ,加入SM后仍呈现抑制作用。以上结果表明 ,SM可能是通过抑制颈动脉窦压力感受器中机械敏感性通道的Ca2 +内流而发挥作用。

Effects of ethanol on the tonicity of corporal tissue and the intracellular Ca^2＋ concentration of human corporal smooth muscle cells

Heavy alcohol consumption is associated with an increased risk of erectile dysfunction （ED）; however, the acute effects of ethanol （EtOH） on penile tissue are not fully understood. We sought to investigate the effects of EtOH on corporal tissue tonicity, as well as the intracellular Ca^2＋ concentration （[Ca^2＋]i） and potassium channel activity of corporal smooth muscle. Strips of corpus cavernosum （CC） from rabbits were mounted in organ baths for isometric tension studies. Electrical field stimulation （EFS） was applied to strips precontracted with 10 μmol L^-1 phenylephrine as a control. EtOH was then added to the organ bath and incubated before EFS. The [Ca^2＋]i levels were monitored by the ratio of fura-2 fluorescence intensities using the fura-2 loading method. Single-channel and whole-cell currents were recorded by the conventional patch-clamp technique in short-term cultured smooth muscle cells from human CC tissue. The corpus cavernosal relaxant response of EFS was decreased in proportion to the concentration of EtOH. EtOH induced a sustained increase in [Ca^2＋]i in a dose-dependent manner, Extracellular application of EtOH significantly increased whole-cell K^＋ currents in a concentration-dependent manner （P 〈 0.05）. EtOH also increased the open probability in cell-attached patches; however, in inside-out patches, the application of EtOH to the intracellular aspect of the patches induced slight inhibition of Ca^2＋-activated potassium channel （KCa） activity. EtOH caused a dose-dependent increase in cavemosal tension by alterations to [Ca^2＋]i. Although EtOH did not affect KCa channels directly, it increased the channel activity by increasing [Ca^2＋]i. The increased corpus cavemosal tone caused by EtOH might be one of the mechanisms of ED after heavy drinking.

Tacrolimus Inhibits Vasoconstriction by Increasing Ca^(2+) Sparks in Rat Aorta

The present study attempted to test a novel hypothesis that Ca^2＋ sparks play an important role in arterial relaxation induced by tacrolimus. Recorded with confocal laser scanning microscopy, tacrolimus（10 μmol/L） increased the frequency of Ca^2＋ sparks, which could be reversed by ryanodine（10 μmol/L）. Electrophysiological experiments revealed that tacrolimus（10 μmol/L） increased the large-conductance Ca^2＋-activated K＋ currents（BKCa） in rat aortic vascular smooth muscle cells（AVSMCs）, which could be blocked by ryanodine（10 μmol/L）. Furthermore, tacrolimus（10 and 50 μmol/L） reduced the contractile force induced by norepinephrine（NE） or KCl in aortic vascular smooth muscle in a concentration-dependent manner, which could be also significantly attenuated by iberiotoxin（100 nmol/L） and ryanodine（10 μmol/L） respectively. In conclusion, tacrolimus could indirectly activate BKCa currents by increasing Ca^2＋ sparks released from ryanodine receptors, which inhibited the NE- or KCl-induced contraction in rat aorta.

TRPC1与BK-α的表达对大鼠糖尿病肾病的影响

目的 探究瞬时受体电位C1(transient receptor potential channel 1,TRPC1)蛋白和大电导钙离子激活钾通道α亚单位(large conductance Ca^(2+)-activated K^(+)channel α subunit,BK-α)蛋白对大鼠糖尿病肾病(diabetic kidney disease,DKD)的影响。方法 将SD大鼠随机分为对照组(n=15)和模型组(n=15)。利用高脂饲料和链脲佐菌素(streptozocin,STZ)构建DKD模型。采用血糖分析仪检测大鼠血糖变化;采用全自动生化分析仪检测大鼠肾功能水平;HE染色检测肾组织的病理变化以确定造模成功。实时荧光定量PCR(RT-qPCR)和蛋白免疫印迹分别检测肾组织TRPC1和BK-α的mRNA和蛋白表达水平;免疫组化检测TRPC1和BK-α的分布和表达情况。结果 模型组大鼠空腹血糖(fasting plasma glucose,FPG)、尿白蛋白排泄率(urinary albumin excretion rates,UAER)、血尿素氮(blood urea nitrogen,BUN)和肌酐(creatinine,Cr)均显著高于对照组(P <0.01);模型组大鼠肾小管内壁细胞出现膨胀现象,部分细胞脱离;可见肾小管发生病变或死亡;此外,在许多肾小管及肾间质区域发现有中性白细胞及其残骸;以上HE染色结果提示,DKD模型复制成功。TRPC1和BK-α在肾小球部位最为丰富,且模型组大鼠肾组织中TRPC1和BK-α的mRNA和蛋白水平都显著高于对照组(P <0.05)。结论 大鼠糖尿病肾病影响TRPC1和BK-α在肾组织中的分布和表达。

XIRP2和HCN4在心肌脂肪浸润致猝死者心脏传导系统表达研究

目的:研究心肌脂肪浸润致猝死的法医病理学特征及传导组织中含心肌动蛋白重复序列蛋白2(XIRP2)和超极化激活环核苷酸门控阳离子通道4(HCN4)蛋白含量表达变化情况,探讨XIRP2和HCN4蛋白作为心肌脂肪浸润致猝死的潜在分子标记物的可能性。方法:收集某司法鉴定中心2015~2022年死因为心肌脂肪浸润致猝死者8例为猝死组,选取死因为外伤致死者10例为对照组进行统计、归纳和分析。通过大体检查、HE染色和Masson三色染色观察组织病理学改变,免疫组织化学染色(IHC)观察XIRP2和HCN4蛋白表达情况。结果:组织病理学检查示心肌脂肪浸润主要侵犯右心室、窦房结、房室结;IHC结果示猝死组较对照组的XIRP2和HCN4蛋白在窦房结、房室结表达升高,在左右束支表达降低(P<0.05)。心肌脂肪浸润猝死者窦房结和房室结XIRP2和HCN4蛋白表达水平高于左右束支(P<0.05),表达趋势与对照组相反。心肌脂肪浸润猝死者HCN4、XIRP2蛋白表达在窦房结与房室结之间差异有统计学意义。传导系统XIRP2与HCN4蛋白表达相关(P<0.05)。结论:心肌脂肪浸润主要表现为右心室合并窦房结、房室结内大量脂肪浸润,可能通过影响传导系统离子通道蛋白表达致心律失常引起猝死,XIRP2和HCN4蛋白在传导系统的表达差异可为该类案件的法医学鉴定提供参考。

Molecular mechanisms of diabetic coronary dysfunction due to large conductance Ca2＋-activated K＋ channel impairment

Background Diabetes mellitus is associated with coronary dysfunction, contributing to a 2- to 4-fold increase in the risk of coronary heart diseases. The mechanisms by which diabetes induces vasculopathy involve endothelial-dependent and -independent vascular dysfunction in both type 1 and type 2 diabetes mellitus. The purpose of this study is to determine the role of vascular large conductance Ca2＋-activated K＋ （BK） channel activities in coronary dysfunction in streptozotocin-induced diabetic rats. Methods Using videomicroscopy, immunoblotting, fluorescent assay and patch clamp techniques, we investigated the coronary BK channel activities and BK channel-mediated coronary vasoreactivity in streptozotocin-induced diabetic rats. Results BK currents （defined as the iberiotoxin-sensitive K＋ component） contribute （65＋4）% of the total K＋currents in freshly isolated coronary smooth muscle cells and 〉50% of the contraction of the inner diameter of coronary arteries from normal rats. However, BK current density is remarkably reduced in coronary smooth muscle cells of streptozotocin-induced diabetic rats, leading to an increase in coronary artery tension. BK channel activity in response to free Ca2＋ iS impaired in diabetic rats. Moreover, cytoplasmic application of DHS-1 （a specific BK channel i~ subunit activator） robustly enhanced the open probability of BK channels in coronary smooth muscle cells of normal rats. In diabetic rats, the DHS-1 effect was diminished in the presence of 200 nmol/L Ca2＋ and was significantly attenuated in the presence of high free calcium concentration, i.e., 1 μmol/L Ca2＋. Immunoblotting experiments confirmed that there was a 2-fold decrease in BK-β1 protein expression in diabetic vessels, without alterinq the BK channel a-subunit expression.Although the cytosolic Ca2＋ concentration of coronary arterial smooth muscle cells was increased from （103±23） nmol/L （n=5） of control rats to （193±22） nmol/L （n=6, P 〈0.05） of STZ-induced diabetic rats, reduced BK-β1 expression made these channels less sensitive to intracellular Ca2＋, which in turn led to enhanced smooth muscle contraction. Conclusions Our results indicated that BK channels are the key determinant of coronary arterial tone. Impaired BK channel function in diabetes mellitus is associated with down-regulation of BK-β1 expression and reduction of the β1-mediated BK channel activation in diabetic vessels.

氯唑沙腙对抗HepG2肿瘤细胞的作用研究

目的研究氯唑沙腙(chlorzoxazone)对HepG2细胞存活和凋亡的影响。方法采用MTT法检测氯唑沙腙对体外培养的HepG2细胞存活率的影响,通过检测LDH释放观察氯唑沙腙对HepG2细胞的致坏死作用,通过TUNEL法评价细胞凋亡率,透射镜评价细胞的超微结构。结果氯唑沙腙在50～500μmol.L-1浓度范围内对HepG2细胞的存活率均有抑制作用,呈明显的剂量依赖效应关系。荧光显微镜和透射电镜下可见典型的肿瘤细胞凋亡改变。氯唑沙腙在100、200、300及500μmol.L-1浓度下作用48h均可诱导HepG2细胞凋亡,具有明显的剂量效应关系。氯唑沙腙100、200、300及500μmol.L-14种浓度分别作用于HepG2细胞24、48及72h,发现以上各种浓度在48h即可诱导细胞凋亡,作用时间越长,凋亡率越高,有明显的时间效应关系。结论氯唑沙腙能够抑制HepG2细胞存活和诱导细胞凋亡。

11,12-表氧化二十碳三烯酸对COPD大鼠气道平滑肌细胞钙激活性钾通道的作用

目的观察COPD大鼠气道平滑肌细胞(ASMCs)的钙激活性钾通道(KCa)的活性变化,以及11,12表-氧化二十碳三烯酸(11,12-EETs)对气道平滑肌细胞KCa的作用。方法40只雄性SD大鼠随机分为正常对照组和COPD组,每组20只。在相对密闭舱内吸入纸烟建立COPD动物模型,采用急性酶分离法分离大鼠ASMCs,应用膜片钳技术,分离出KCa电流,测定KCa开放概率(Po)、平均开放时间(To)和平均关闭时间(Tc)。比较COPD组和正常对照组KCa上述活性指标的变化,以及应用3μmol/L的11,12-EETs对COPD组ASMCs细胞进行灌流后KCa活性的变化。结果与正常对照组比较,COPD组ASMCs的KCa通道Po明显降低(0.084±0.028比0.198±0.029,P<0.01),To显著缩短[(0.732±0.058)m s比(1.648±0.152)m s,P<0.01],Tc显著延长[(12.259±2.612)m s比(6.753±1.237)m s,P<0.01]。而用11,12-EETs灌流ASMCs细胞后可明显激活KCa活性,Po增加(0.227±0.059比0.084±0.028,P<0.01),To延长[(2.068±0.064)m s比(0.732±0.058)m s,P<0.01],Tc缩短[(4.273±0.978)m s比(12.259±2.612)m s,P<0.01]。结论COPD大鼠ASMCs细胞KCa通道活性降低可能在COPD发病机制中起着一定作用,11,12-EETs可以直接激活COPD大鼠ASMCs细胞KCa活性,提高膜电位稳定性,从而松弛COPD大鼠气道平滑肌。

BKca下调RhoA/ROCK信号通路改善糖尿病大鼠阴茎勃起功能

目的:观察大电导钙激活钾通道(big conductance Ca2+-activated K+channel,BKca)对糖尿病勃起功能障碍(diabetes mellitus-induced erectile dysfunction,DMED)大鼠阴茎海绵体平滑肌Rho A/ROCK信号通路的影响。方法:实验大鼠分为空白对照组8只,DMED组8只,NS1619组(DMED治疗组)7只,NS1619组大鼠采用特异性BKca激活剂(NS1619)阴茎海绵体注入2周。观察各组勃起行为,并电刺激检测阴茎海绵体内压(intracavernous pressure,ICP)/平均动脉压(mean arterial pressure,MAP),取各组阴茎海绵体平滑肌检测肌张力,采用Western blot和定量PCR(real-time PCR)方法检测Rho A、ROCK1、ROCK2表达,反应Rho A/ROCK信号通路差异,同时检测MYPT-1表达反应肌球蛋白轻链磷酸酶(myosin light chain phosphatase,MLCP)磷酸化程度。结果:成功构建DMED大鼠模型,NS1619组大鼠与DMED组大鼠比较,勃起次数和ICP/MAP明显改善(P=0.025、0.024),阴茎海绵体平滑肌舒缩顺应性提高(P=0.031、0.024)并且Rho A、ROCK2以及肌球蛋白磷酸酶靶向结合亚基(myosin phosphatase target subunit,MYPT)-1蛋白和m RNA表达水平下调(P=0.029、0.003、0.002),但仍未达到正常对照组大鼠水平(P=0.018、0.040、0.003),ROCK1表达无明显差异(P=0.533)。结论:DMED大鼠因Rho A/ROCK信号通路上调和MLCP磷酸化增强导致阴茎海绵体平滑肌舒缩顺应性降低以致勃起功能障碍发生,激活BKca可以通过下调Rho A/ROCK信号通路和抑制MLCP磷酸化,改善勃起功能。