肾上腺髓质素对豚鼠心室肌细胞L-型钙通道的作用及其信号转导机制

目的: 研究肾上腺髓质素( adrenomedullin, ADM)抑制豚鼠心室肌细胞L-型钙通道的信号转导机制。方法: 应用全细胞膜片钳技术,记录应用ADM(1-100 nmol·L^-1)前后L-型钙电流(I_Ca,L),以及分别记录应用ADM特异性受体拮抗剂ADM_22-52(100 nmol·L^-1)+ADM(100 nmol·L^-1)、蛋白激酶A (PKA) 特异性拮抗剂H-89(10 μmol·L^-1) + ADM(100 nmol·L^-1)、蛋白激酶C (PKC) 特异性拮抗剂PKC_19-36(10 μmol·L^-1)+ADM(100 nmol·L^-1)、PKC特异性激动剂PMA(1 μmol·L^-1)前后I_Ca,L。结果: ADM(1-100 nmol·L^-1)浓度依赖性地抑制豚鼠心室肌细胞I_Ca,L,并可被ADM_22-52(100 nmol·L^-1)完全阻断;H-89(10 μmol·L^-1)对ADM抑制I_Ca,L的作用无影响。PKC_19-36(10 μmol·L^-1)可完全阻断ADM 对I_Ca,L的抑制效应,且PMA(1 μmol·L^-1)可模拟ADM 对I_Ca,L的抑制效应。结论: ADM作用于特异性ADM受体可浓度依赖性地抑制豚鼠心室肌细胞I_Ca,L,此作用有可能与PKC激活相关。     

金属硫蛋白对羟自由基损伤的大鼠肝细胞核核苷三磷酸酶的保护作用

目的:研究金属硫蛋白(MTs)是否对羟自由基(hydroxylradical,·OH^-)损伤的大鼠肝细胞核核苷三磷酸酶(NTPase)具有保护作用。方法:以羟自由基发生系统Fe³⁺/H₂O₂单独或与MTs共同孵育大鼠离体肝细胞核,检测分别用ATP和GTP作底物时大鼠肝细胞核NTPase活性。结果:不同浓度Fe³⁺/H₂O₂(μmol·L^-1/μmol·L^-1:0.1/0.5、0.5/2.5、1/5、5/25)孵育肝细胞核,浓度依赖地增强核NTPase活性,与对照组差异显著(P＜0.01)。用不同浓度的MT(10^-9-10^-4mol·L^-1)与Fe²⁺/H₂O₂(1μmol·L^-1/5μmol·L^-1)共孵育,浓度依赖地拮抗Fe³⁺/H₂O₂诱导的效应(P＜0.01)。用MT单独孵育肝细胞核对NTPase的活性没有影响(P＞0.05)。结论:Fe³⁺/H₂O₂系统产生的·OH对核NTPase活性具有强烈的抑制效应,MT浓度依赖地拮抗·OH导致的NTPase活性降低。     

钙调神经磷酸酶在血管紧张素Ⅱ刺激的大鼠心肌细胞肥大中的作用及其活性调节

目的:观察钙调神经磷酸酶(CaN)在血管紧张素Ⅱ(AngⅡ)刺激的大鼠心肌细胞肥大中的作用及其活性调节。方法:建立AngⅡ诱导的大鼠心肌细胞肥大模型,观察CaN抑制剂对AngⅡ刺激的心肌细胞 [³H]-亮氨酸掺入的影响,以及各种因素对心肌细胞CaN酶活性的影响。结果:10、 100、 1000 nmol·L^-1的AngⅡ作用12 h分别使心肌细胞的CaN活性增加了13%、 57%(P＜0.05)、 228%(P＜0.01)。AngⅡ(10 nmol·L^-1)刺激心肌细胞2 h内,CaN活性与对照组无明显差异(P＜0.05);AngⅡ刺激心肌细胞12 h以上,CaN活性才明显增高(P＜0.05)。Losartan(50 μmol·L^-1)、H₇(50 μmol·L^-1)及Fura-2/AM(4 μmol·L^-1)可明显抑制AngⅡ刺激的心肌细胞CaN活性;而PD98059(50 μmol·L^-1)对AngⅡ刺激的心肌细胞CaN活性无明显影响。AngⅡ(10^-7mol/L)刺激的大鼠心肌细胞 [³H]-亮氨酸掺入明显高于对照组(P＜0.01),而CaN特异性抑制剂-环孢素A(0.5~5 μg/mL)可以明显抑制AngⅡ刺激的心肌细胞 [³H]-亮氨酸掺入。结论:依赖Ca²⁺/CaM活化的CaN可能在AngⅡ刺激的心肌细胞肥大中起重要作用;CaN的活化可能有赖于胞内Ca²⁺水平的持续升高,另外,CaN的活性还可能受到蛋白激酶C等信号分子的磷酸化调节。     

甲基莲心碱对耐长春新碱人胃癌细胞多药耐药性的逆转

目的:研究甲基莲心碱(Nef)逆转耐长春新碱人胃癌细胞多药耐药性(MDR)的作用及机制。方法:采用噻唑蓝(MTT)比色法检测长春新碱(VCR)的细胞毒性;PI染色流式细胞计数测定VCR诱导细胞凋亡;间接免疫荧光流式细胞术检测细胞P-gp和MRP的表达。结果:Nef(5、10μmol·L^-1)对人胃癌细胞(SGC7901)和耐长春新碱人胃癌细胞(SGC7901/VCR)无显著毒性作用,VCR对敏感株SGC7901的IC₅₀为0.06mg·L^-1,而对MDR细胞株SGC7901/VCR的IC₅₀为2.32mg·L^-1,SGC7901/VCR较SGC7901对VCR耐药39倍,Nef(2.5、5、10μmol·L^-1)能使VCR对SGC7901/VCR细胞的IC₅₀从2.32mg·L^-1依次下降至0.34、0.12、0.05mg·L^-1,逆转倍数分别为6.8、18.1、43.8。Nef(2.5、5、10μmol·L^-1)能降低SGC7901/VCR细胞对VCR的凋亡抗性,其作用强于维拉帕米(VRP)。SGC7901/VCR细胞较SGC7901细胞高表达P-gp、MRP,Nef(10μmol·L^-1)处理24h后,SGC7901/VCR细胞P-gp、MRP的表达明显低下。结论:Nef具有逆转耐长春新碱人胃癌细胞的MDR作用,其作用机理与下调P-pg和MRP表达有关。     

1,6-二磷酸果糖对阿霉素引起大鼠心肌细胞内游离钙和肌浆网Ca2+-ATP酶活性改变的影响

目的:探讨1,6-二磷酸果糖(FDP)对阿霉素(ADR)所引起的大鼠心肌细胞内游离钙及心肌肌浆网钙-ATP酶(SRCa²⁺-ATPase)活性变化的影响。方法: 给大鼠腹腔注射ADR(2.5 mg·kg^-1,隔日1次,共6次),给ADR处理的大鼠腹腔注射不同剂量的FDP(隔日1次,共21次)进行干预。采用双抗体双夹心ELISA法定量测定血清肌钙蛋白I(CTnI);采用抗肌酸激酶同工酶(CK-MB)单克隆抗体包被小球测定血清CK-MB;用荧光分光光密度计测定心肌细胞内游离钙(MyoCa²⁺)浓度;用无机磷酸根法测定心肌肌浆网钙-ATP酶(SRCa²⁺-ATPase)活性。结果:FDP(300、600、1 200 mg·kg^-1)干预ADR处理的大鼠后,可显著降低血清CtnI、CK-MB及心肌细胞内MyoCa²⁺值,显著增高SRCa²⁺-ATPase活性(P＜0.01)。结论: FDP能通过降低心肌细胞内游离钙和对SRCa²⁺-ATPase活性的改变,起到减轻ADR对心肌的毒性作用。     

5-羟色胺对心肌细胞增殖及蛋白激酶C活性的影响

目的：观察5-羟色胺（5-HT）对新生大鼠心肌细胞增殖及蛋白激酶C（PKC）活性的影响，探讨5-HT在心肌肥大发病中的作用。方法：培养乳鼠心肌细胞,应用BCA法测定细胞总蛋白；流式细胞仪测定DNA含量；同位素底物掺入检测PKC活性。结果：5-HT在1 μmol·L^-1、10 μmol·L^-1和100 μmol·L^-1时均显著促进心肌细胞蛋白质和DNA合成，以10 μmol·L^-1剂量时作用最强。5-HT在1 μmol·L^-1和10 mol·L^-1剂量时可加强心肌细胞PKC活性，并促使PKC从胞浆移位到细胞膜，5-HT₂受体拮抗剂mianserin可逆转此作用。结论：5-HT可促进新生大鼠心肌细胞总蛋白和DNA合成，并通过心肌细胞5-HT₂受体激活PKC活性，促使PKC从胞浆移位到细胞膜，提示5-HT可能参与心肌肥大的发生发展过程.     

索拉非尼抑制人肝星状细胞胶原合成

目的: 研究索拉非尼(sorafenib)对人肝星状细胞胶原合成的影响。方法: 应用人肝星状细胞株LX-2进行体外研究,采用 -脯氨酸掺入法测定胶原的合成,采用免疫细胞化学法检测I型胶原蛋白表达,采用real-time PCR法测定I型胶原α1 mRNA表达。结果: 免疫细胞化学研究显示血小板源性生长因子(PDGF)刺激可引起LX-2细胞胶原合成增加,10.0 μmol·L^-1索拉非尼作用于LX-2细胞 24 h能明显抑制I型胶原蛋白的合成。无论有无PDGF的刺激,索拉非尼均呈剂量与时间依赖性地抑制LX-2细胞胶原合成(P<0.01);在10.0 μmol·L^-1浓度下,索拉非尼作用于LX-2细胞 12 h、24 h和48 h对胶原合成的抑制率为22.69%、37.52%和71.74%。索拉非尼剂量依赖性地抑制PDGF诱导的I型胶原α1 mRNA表达上调;在2.5 μmol·L^-1、5.0 μmol·L^-1和10.0 μmol·L^-1 索拉非尼作用下,I型胶原α1 mRNA表达较PDGF刺激组分别下调58.66%、 67.06%和81.64%。结论: 索拉非尼在体外能抑制人肝星状细胞胶原的合成,抑制I型胶原的表达,有可能成为一种新型的治疗肝纤维化药物。     

白三烯D4在促进培养的人气管 平滑肌细胞增殖中的作用

目的和方法:研究白三烯D₄(LTD₄)是否剌激培养的人气管平滑肌细胞(ASMC)增殖。将分离的人ASMC进行传代培养,在培养基中加入各种浓度的LTD₄,计数细胞并测定 [³H]-胸腺嘧啶核苷([³H]-TdR)掺入量和三磷酸肌醇(IP₃)累积量。结果: LTD₄在一定范围内(0.1 nmoL·L^-1~10 nmoL·L^-1)以浓度依赖的方式增加人ASMC(P＜0.01)。LTD₄也增加[³H]-TdR的掺入量和IP₃累积量(P＜0.01)。磷脂酶C抑制剂新霉素(1 μmol·L^-1)阻止IP₃累积量的增加(P＜0.01)。结论:LTD₄剌激培养的人ASMC增殖并且可能在哮喘的气道重塑中起了作用。     

过氧化物酶体增殖物激活受体α在实验性大鼠脂肪肝中的表达

目的:研究过氧化物酶体增殖物激活受体α(PPARα)表达异常在脂肪肝发生中的作用。方法:采用小剂量CCl₄后肢皮下注射, 并高脂饮食复制大鼠脂肪肝动物模型, 检测肝脏甘油三酯(TG)、总胆固醇(TC)和游离脂肪酸(FFA)含量及血清谷丙转氨酶(ALT)、肿瘤坏死因子-α(TNF-α)和FFA含量, 并做病理切片, 测定肝脂变面积。提取肝脏总RNA, 运用半定量RT-PCR方法对肝脏PPARαmRNA的表达情况进行分析。结果:脂肪肝模型组大鼠肝脏TG、TC、FFA含量分别为(1.88±0.20)mmol·L^-1、(11.03±1.12)mmol·L^-1和(1260.38±151.27)μmol·L^-1, 正常对照组则为(0.53±0.10)mmol·L^-1、(1.25±0.25)mmol·L^-1和(334.30±27.09)μmol·L^-1(P＜0.01)。血清ALT、TNF-α和FFA含量亦明显高于对照组。肝脏PPARα的灰度比值:脂肪肝模型组0.41±0.28, 正常对照组1.41±0.29(P＜0.01)。结论:肝细胞中毒性脂肪肝时, 肝脏PPARα表达减少, 使肝中脂质的利用和脂肪酸的氧化均发生障碍, 导致肝脂蓄积。     

测定细胞培养上清液中亚硝酸盐的荧光分光光度法

目的:建立检测细胞培养上清液中亚硝酸盐的方法。方法:以2, 3-二氨基萘与亚硝酸盐反应生成荧光产物2, 3-二氨基萘三唑或1--萘三唑, 用荧光分光光度法测定。结果:2, 3-二氨基萘浓度为0.63mmol·L^-1, 反应液和终止反应后pH值各为2.00和12.10, 20℃水浴15min是较适测定条件。本法的检出限为61.34nmol·L^-1, 日内和日间变异系数分别小于3%和5%, 加入NaNO₂0.36、1.45、2.54nmol的回收率各为99.12%±4.64%、103.28%±2.68%、105.14%±4.36%。测定大鼠腹腔巨噬细胞、大鼠胎鼠大脑半球神经细胞培养上清液亚硝酸盐含量分别为(109.95±4.57)μmol·L^-1和(6.52±1.57)μmol·L^-1, 后者的95%分布范围为3.44-9.60μmol·L^-1。核磁共振氢谱显示标准品化学位移δ7.33027ppm和细胞培养上清液化学位移δ7.33023ppm。结论:本法特异、敏感、快速、简便, 对一氧化氮的研究有一定价值。     

红景天苷促进培养乳鼠心肌细胞肌质网钙离子的释放

目的: 观察红景天苷对乳鼠心肌细胞胞浆Ca²⁺浓度的影响并分析其可能的作用机制。方法: 应用荧光指示剂Fluo-3/AM负载培养大鼠乳鼠的心肌细胞,用激光共聚焦显微镜动态观察胞内游离钙荧光信号强度的变化,检测不同浓度红景天苷对培养心肌细胞胞内游离钙离子浓度([Ca²⁺]_i)的影响。结果: 红景天苷浓度为15 mg/L、30 mg/L和60 mg/L时,细胞内的平均[Ca²⁺]_i升高,峰值分别为574.08±4.65、591.86±3.64和618.66±4.27(均P<0.01);有剂量依赖性而无时间依赖性。用维拉帕米阻断细胞膜外钙内流时,红景天苷同样引起细胞内[Ca²⁺]_i升高,峰值由357.74±3.13、387.17±2.37和391.43±1.34分别上升到480.86±3.98、496.70±3.08和522.18±3.19(均P<0.01)。结论: 红景天苷能升高乳鼠心肌细胞中[Ca²⁺]_i,其机制可能与其促进肌浆网钙离子释放有关。     

钙离子在半边旗提取物6F的细胞毒及诱导HL-60细胞DNA片段化中的作用

目的:研究蕨类植物半边旗提取物6F对HL-60细胞内游离钙浓度([Ca²⁺]_i)及Bcl-2蛋白表达的影响, Ca²⁺与6F细胞毒作用及诱导细胞DNA片段化的关系。方法:用荧光探针Fura-2/AM标记细胞内游离Ca²⁺, 在荧光分光光度计上测[Ca²⁺]_i;流式细胞仪检测Bcl-2的表达;噻唑蓝(MTT)法测定细胞成活率;二苯胺法测DNA片段化形成率。结果:6F作用后, HL-60细胞内[Ca²⁺]_i显著升高, 呈明显的时间剂量效应关系;6F降低Bcl-2的表达;于培养基中加2mmol/LCa²⁺、或加1mmol/LEDTA络合细胞外钙, 或加4μmol/L钙通道A₂₃₁₈₇升高细胞内钙浓度, 均可增强6F的细胞毒作用, 但均不影响6F诱导细胞DNA片段化程度。加入250μmol/LZn²⁺可显著降低6F所致DNA片段化率, 并可增强6F的细胞毒作用。结论:化合物6F可显著升高HL-60细胞[Ca²⁺]_i, 推测与6F降低Bcl-2的表达有关;6F诱导HL-60细胞DNA片段化可能是通过Ca²⁺-非依赖性DNA酶的作用所致。     

NCA增加心肌收缩力的作用研究

目的: 硝酰基 (HNO)作为一氧化氮 (NO) 的单电子还原产物,对活体心脏发挥正性肌力作用。我们对于这些效果是否由于乙酸1-亚硝基环已酯(NCA,HNO供体) 对肌原纤维的直接作用进行了研究。方法: 将大鼠右心室的完整的梳状肌连接在张力换能器与刺激电极之间,肌小节长度设定在2.2-2.3 μm之间,K-H液表面灌流后 (pH = 7.4,室温),Fura-2经玻璃微电极负载进行离子透入法检测[Ca²⁺]_i,同时测定心肌收缩张力的变化。稳态条件下对最大钙离子活化张力(F_max)及达到50%活性需要[Ca²⁺]_i(Ca₅₀)进行测定。Western blotting方法检测原肌球蛋白表达的变化。结果: 收缩力在NCA作用下呈剂量依赖性增加(20-100 μmol/L)。不同频率作用下(0.5-3.0 Hz,NCA 20 μmol/L,Ca²⁺ 0.5 mmol/L)收缩力的增加 (P< 0.01) 和[Ca²⁺]_i瞬变 (P>0.05) 没有受到明显的影响。舒张期作用力及[Ca²⁺]_i不受NCA的影响。与对照组相比,稳态活化过程中NCA (20 μmol/L) 能增加最大钙离子活力F_max [(124.0±15.0) mN/mm² vs (90.0± 4.2)mN/mm²,P< 0.05],降低Ca₅₀[ (0.39±0.01) μmol/L vs (0.57±0.03) μmol/L,P< 0.01],但不影响希尔系数 (3.94 ± 0.18 vs 4.92 ± 0.84, P>0.05)。同对照组相比,NCA处理去肌膜心肌收缩力明显增加 (P< 0.05)。非还原条件下Western blotting可见肌原纤维出现交联。巯基还原剂二硫苏糖醇 (DTT,5.0 mmol/L) 能够阻止并逆转NCA的活动,进一步证实氧化还原反应依赖HNO 的效应。结论: NCA提供的HNO是心脏钙离子增敏剂,心肌调节蛋白质巯基翻译后修饰可能是其靶点作用所在。     

白细胞介素-2对缺氧/复氧心肌细胞[Ca2+]i的作用

目的:观察白细胞介素-2(IL-2)对心肌细胞在缺氧/复氧过程中电刺激诱导的[Ca²⁺]i的作用。方法:采用酶解分离成年大鼠心室肌细胞化学缺氧模型, 以Fura-2/AM为钙探针, 用细胞内双波长钙荧光系统检测心肌[Ca²⁺]i的变化。结果:①缺氧/复氧过程中, 缺氧5min时, 心肌[Ca²⁺]i幅度降低、舒张末期[Ca²⁺]i升高, [Ca²⁺]i达峰时间(TTP)延长, 恢复时间(RT)延长。复氧10min后, 心肌[Ca²⁺]i幅度、舒张末期[Ca²⁺]i、TTP及RT逐渐回复, 但不能完全恢复到对照水平;②在缺氧期间加入IL-2(2×10⁵U/L), 复氧期间[Ca²⁺]i各参数回复减慢;③用κ-阿片受体拮抗剂nor-BNI(10^-8mol/L)预处理后, 缺氧+IL-2对复氧时[Ca²⁺]i作用的影响被减弱, 而δ-阿片受体拮抗剂纳曲吲哚(10^-6mol/L)预处理则无此作用。结论:缺氧时同时存在IL-2, 可加剧复氧时心肌[Ca²⁺]i的变化, 其机制可能是IL-2通过心肌κ-阿片受体而发挥作用。     

Agonist-induced intracellular Ca2+ transients in HT29 cells

In the present study we have investigated the mechanism of intracellular Ca²⁺ activity ([Ca²⁺]_i) changes in HT₂₉ cells induced by adenosine triphosphate (ATP), carbachol (CCH), and neurotensin (NT). [Ca²⁺]_i was measured with the fluorescent Ca²⁺ indicator fura-2 at the single-cell level or in small cell plaques with high time resolution (1–40Hz). ATP and CCH induced not only a dose-dependent [Ca²⁺]_i peak response, but also changes of the plateau phase. The [Ca²⁺]_i plateau was inversely dependent on the ATP concentration, whereas the CCH-induced [Ca²⁺]_i plateau increased at higher CCH concentrations. NT showed (from 10^–10 to 10^–7 mol/l) in most cases only a [Ca²⁺]_i spike lasting 2–3 min. The [Ca²⁺]_i plateau induced by ATP (10^–6 mol/l) and CCH (10^–5 mol/l) was abolished by reducing the Ca²⁺ activity in the bath from 10^–3 to 10^–4 mol/l (n=7). In Ca²⁺-free bathing solution the [Ca²⁺]_i peak value for all three agonists was not altered. Using fura-2 quenching by Mn²⁺ as an indicator of Ca²⁺ influx the [Ca²⁺]_i peak was always reached before Mn²⁺ influx started. Every agonist showed this delayed stimulation of the Ca²⁺ influx with a lag time of 23±1.5 s (n=15) indicating a similar mechanism in each case. Verapamil (10^–6–10^–4 mol/l) blocked dose dependently both phases (peak and plateau) of the CCH-induced [Ca²⁺]_i increase. Short pre-incubation with verapamil augmented the effect on the [Ca²⁺]_i peak, whereas no further influence on the plateau was observed. Ni²⁺ (10^–3 mol/l) reduced the plateau value by 70%.     

Effects of motilin on intracellular free calcium in cultured smooth muscle cells from the antrum of neonatal rats

Aim: The aim of this study was to determine the effects of motilin on [Ca²⁺]_i regulation and its underlying molecular mechanism in cultured antral smooth muscle cells (ASMCs). Methods: Antral cells were isolated and cultured from neonatal rats, and then the [Ca²⁺]_i in these cells was evaluated by calcium fluorescent probe Fluo-3/AM on a laser scanning confocal microscope. Results: We show that motilin dose-dependently increased [Ca²⁺]_i concentration in cultured ASMCs. Pre-incubation of cells with either the calcium antagonist verapamil (10⁻⁵ mol L⁻¹) or the calcium chelator Egtazic (EGTA, 0.1 mmol L⁻¹) significantly suppressed motilin (10⁻⁶ mol L⁻¹) induced [Ca²⁺]_i increase as indicated by fluorescent intensity. Interestingly, after mixing with the non-selective intracellular calcium release blocker TMB-8 (10⁻⁵ mol L⁻¹), guanosine triphosphate regulatory protein antagonist NEM (10⁻⁵ mol L⁻¹), phospholipase C (PLC) inhibitor compound 48/80 (1.2 μg mL⁻¹) and ryanodine at high concentration (10⁻⁵ mol L⁻¹), the motilin-induced [Ca²⁺]_i increase was only partially blocked. The protein kinase C inhibitor d -sphingosine (10⁻⁶ mol L⁻¹), however, did not show any inhibitory effect on motilin-induced [Ca²⁺]_i elevation. Conclusions: Our study suggests that motilin-stimulated [Ca²⁺]_i elevation in ASMCs is probably due to sustained extracellular Ca²⁺ influx and Ca²⁺ release from Ca²⁺ stores via inositol tris-phosphate receptors and ryanodine receptors. Specifically, motilin-induced [Ca²⁺]_i release is accompanied with guanosine triphosphate-binding protein-coupled receptor–PLC–inositol tris-phosphate signalling cascades.     

Ca2+ release in cultured rat epididymal cells during hypoosmotic swelling

Microfluorimetric studies were carried out to investigate the effects of hypoosmotic swelling on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in single rat epididymal cells. In Ca²⁺-free solution containing 50 mol/l ethylenebis(oxonitrilo)tetraacetate (EGTA) hypoosmotic swelling (–160 mosmol/l) induced a transient rise in [Ca²⁺]_i which was either monophasic, biphasic or oscillatory. The [Ca²⁺]_i responses to repeated hypoosmotic stimulations followed a decremental pattern. However, if 2.5 mmol/l Ca²⁺ was admitted during the recovery period between successive stimulations, the second and the third [Ca²⁺]_i responses were slightly greater than the first. Increasing the change in osmolarity from –14±1.0 to –154±1.5 mosmol/l increased the rise in [Ca²⁺]_i but reduced the [Ca²⁺]_i response to subsequent ionomycin stimulation (4 mol/l). The swelling- and the ionomycin-induced rises in [Ca²⁺]_i followed a reciprocal pattern. It was suggested that intracellular Ca²⁺ release in response to cell swelling in the epididymal epithelium might play a role in cell volume regulation and the control of epididymal fluid osmolarity.     

Intracellular Ca2+ transients in HT29 cells induced by hypotonic cell swelling

Cell swelling induced by hypotonic solution led to an osmolality-dependent increase in intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells. At moderate reductions in osmolality from 290 to 240 or 225 mosmol/l in most cases only a small monophasic increase of [Ca²⁺]_i to a stable plateau of 10–20 nmol/l above resting [Ca²⁺]_i was observed. Lower osmolalities resulted in a triphasic increase of [Ca²⁺]_i to a peak value. In a first phase after the volume change, lasting 20–40 s, [Ca²⁺]_i increased slowly by about 30 nmol/l. Thereafter [Ca²⁺]_i increased more rapidly within 20–30 s to a peak value. This peak was 189±45 nmol/l (190 mosmol/l, n=9) and 243±41 nmol/l (160 mosmol/l, n=20) above resting [Ca²⁺]_i. The peak was then followed by a decline of [Ca²⁺]_i over the next 2–3 min to a stable plateau value of 28±6 (n=6) and 32±11 nmol/l (n=11) above resting [Ca²⁺]_i at 190 and 160 mosmol/l, respectively. The plateau lasted as long as the hypotonic solution was present. Under Ca²⁺-free bath conditions the peak value for the cell-swelling-induced [Ca²⁺]_i transient was reached significantly later (60–100 s, compared to 40–60 s under control conditions). The peak values under Ca²⁺-free conditions were not significantly lower. This indicates that the [Ca²⁺]_i peak was mostly of intracellular origin. No [Ca²⁺]_i plateau phase was observed under Ca²⁺-free bath conditions. With the use of the fura-2-Mn ²⁺ quenching technique an increased Ca²⁺ influx induced by hypotonic cell swelling was shown (160 mosmol/l; n=4). This influx started immediately after or simultaneously with the cell swelling and preceded the [Ca²⁺]_i peak for more than 50 s.This study was supported by DFG grant Gr 480/10.     

Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential

We studied the role of the membrane potential in the control of the intracellular free calcium concentration ([Ca²⁺]_i) and release of the two autacoids endothelium-derived relaxing factor (EDRF = nitric oxide) and prostaglandin I₂ in endothelial cells. ATP (3 mol/l) and bradykinin (1 nmol/l) evoked rapid increases (sixfold) in [Ca²⁺]_i in cultured endothelial cells. [Ca²⁺]_i remained elevated over several minutes. When the cells were depolarized, either by K⁺ (70–90 mmol/l) or by preincubation with the blocker of K⁺ channels tetraethylammonium (3 mmol/l), the initial peak of [Ca²⁺]_i remained unaffected but [Ca²⁺]_i returned significantly faster to resting levels, indicating a reduction in Ca²⁺ influx. In native, freshly isolated endothelial cells, K⁺ abolished increases in [Ca²⁺]_i induced by acetylcholine (3 mol/l). Release of EDRF in response to bradykinin (cultured cells) and acetylcholine (native cells) was inhibited by K⁺ (by 70%), whereas release of prostaglandin I₂ was not significantly reduced. Preincubation of cultured endothelial cells with the receptor-independent stimulus thimerosal (5 mol/l, 40 min) evoked a long-lasting release of EDRF and small elevations of [Ca²⁺]_i (twofold) after washout of the drug. Depolarization with K⁺ decreased thimerosal-induced EDRF release and [Ca²⁺]_i in a reversible manner. In patch-clamped endothelial cells, bradykinin (1 nmol/l) induced transient hyperpolarizations that were significantly prolonged by BRL 34915 (1 mol/l), an activator of K⁺ channels. BRL 34915 also elicited increases in [Ca²⁺]_i, particularly in thimerosal-stimulated endothelial cells. These effects were abolished by K⁺. We conclude that the initial rise in [Ca²⁺]_i in response to receptor-binding agonists, caused by mobilization of Ca²⁺ from intracellular stores, activates K⁺ channels, thereby inducing hyperpolarization. This hyperpolarization provides the driving force for transmembrane Ca²⁺ influx into endothelial cells and is thus an important signal for synthesis and release of EDRF.     

钾通道对大鼠肺动脉平滑肌细胞[Ca2+]i的调节

目的:探讨在常氧、低氧条件下钾通道对大鼠肺动脉平滑肌细胞(PASMCs)[Ca²⁺]_i的调节。方法:采用钙荧光探针(Fura-2/AM)负载培养的大鼠PASMCs,观察常氧、低氧培养后3种钾通道抑制剂(4AP,TEA、Glib)对PASMCs[Ca²⁺]_i的调节,同时用四唑盐(MTT)比色法比较4AP、TEA、Glib对大鼠PASMCs增殖的影响。结果:(1)常氧状态下,PASMCs[Ca²⁺]_i为(156.91±8.60)nmol/L,低氧时为(294.01±16.81)nmol/L(P＜0.01)。(2)常氧状态下,4AP可引起PASMCs[Ca²⁺]_i升高,达(280.52±23.21)nmol/L(P＜0.01),而TEA、Glib无此作用。(3)低氧时,4AP和TEA都可引起PASMCs[Ca²⁺]_i的升高,分别为(422.41±24.28)nmol/L、(380.84±11.02)nmol/L(P<0.01),Glib无作用。(4)MTT比色法中,常氧和低氧状态下4AP均引起吸光度(A)值升高,分别是0.582±0.062,0.873±0.043(P＜0.01)。TEA仅在低氧时A值升高(0.729±0.041,P＜0.05),而Glib无论常氧还是低氧均无影响。结论:无论常氧还是低氧条件下,电压依赖性钾通道(K_V)对PASMCs[Ca²⁺]_i及其增殖起主要作用。钙激活的钾通道(K_Ca)在常氧条件下对[Ca²⁺]_i不起调节作用,而在低氧下使[Ca²⁺]_i降低,反应性地调节PASMCs增殖。ATP敏感性钾通道(K_ATP)无论在常氧还是低氧情况下对[Ca²⁺]_i的调节不起作用。