Effect of taurine on intracellular free calcium concentration in cultured neonatal rat heart cells

在培养的单个ＳＤ乳鼠心肌细胞，观察了牛磺酸对ＫＣｌ，去甲肾上腺素（ＮＥ）和毒毛花苷Ｇ引起的胞浆游离Ｃａ２＋浓度（［Ｃａ２＋］ｉ）变化的影响．当细胞外ＣａＣｌ２浓度为１．３ｍｍｏｌ·Ｌ－１时，牛磺酸１０，２０ｍｍｏｌ·Ｌ－１不影响心肌细胞静息［Ｃａ２＋］ｉ；但能浓度依赖性地抑制３５ｍｍｏｌ·Ｌ－１ＫＣｌ和１μｍｏｌ·Ｌ－１毒毛花苷Ｇ升高［Ｃａ２＋］ｉ的作用．１０μｍｏｌ·Ｌ－１ＮＥ在含Ｃａ２＋的缓冲液中能引起双相的［Ｃａ２＋］ｉ变化，即快速升高相和持续升高相．牛磺酸２０ｍｍｏｌ·Ｌ－１能抑制ＮＥ引起的［Ｃａ２＋］ｉ持续升高，而对快速升高相无显著影响．在无Ｃａ２＋的缓冲液中，牛磺酸不影响ＮＥ升高［Ｃａ２＋］ｉ的作用．结果提示牛磺酸可能通过减少心肌细胞电压依赖性Ｃａ２＋内流和Ｎａ＋／Ｃａ２＋交换而抑制ＫＣｌ，ＮＥ和毒毛花苷Ｇ引起的［Ｃａ２＋］ｉ升高．     

Effects of dexamethasone on intracellular Ca 2 in its sensitive cells from neonatal mouse hippocampus and cultured cortical neurogliocytes

目的：研究地塞米松（Ｄｅｘ）对神经元和胶质细胞内钙浓度（［Ｃａ２＋］ｉ）的影响．方法：Ｆｕｒａ２ＡＭ负载小鼠海马细胞（ＮＭＨＣ）和培养的胶质细胞（ＣＣＮ）．单细胞内［Ｃａ２＋］ｉ由ＡＲＣＭＭＩＣ检测系统测定．结果：Ｄｅｘ使多数ＮＭＨＣ［Ｃａ２＋］ｉ浓度依赖地迅速升高,９６个ＮＭＨＣ中仅１０％出现［Ｃａ２＋］ｉ降低．［Ｃａ２＋］ｉ升高被无镁细胞外液阻滞、被氯化镧逆转,但不受氯化锂影响．无钙Ｈａｎｋｓ液悬浮、米非司酮（Ｍｉｆ）或河毒素均可阻断Ｄｅｘ４０－９０μｍｏｌ·Ｌ－１的升［Ｃａ２＋］ｉ效应,而Ｄｅｘ２００μｍｏｌ·Ｌ－１的效应仍被保持．４０个ＣＣＮ中５０％对Ｄｅｘ产生浓度依赖的［Ｃａ２＋］ｉ升高,并被无钙或无镁的细胞外液和Ｍｉｆ预处理抑制．结论：Ｄｅｘ快速改变海马神经元和胶质细胞内［Ｃａ２＋］ｉ．［Ｃａ２＋］ｉ的这种改变是由Ｍｇ２＋和受体相关的外钙内流及高浓度Ｄｅｘ诱发的内钙释放介导的．     

钙拮抗剂TMB-8对培养牛大脑中动脉内皮细胞[Ca2+]i和一氧化氮释放的影响

目的旨在观察ＴＭＢ８对血管内皮细胞［Ｃａ２＋］ｉ水平和ＮＯ释放的影响，探讨扩张脑血管的机制。用ＡＲＣＭＭＩＣ阳离子测定系统，测量单个细胞内游离钙浓度（［Ｃａ２＋］ｉ），用血红蛋白法测量一氧化氮（ＮＯ）的释放。结果表明，在细胞外钙浓度为１３ｍｍｏｌ·Ｌ－１时，ＴＭＢ８１２５及２５０μｍｏｌ·Ｌ－１对静息［Ｃａ２＋］ｉ和甲基血红蛋白ΔＥ无明显影响，而５０及１００μｍｏｌ·Ｌ－１时可升高静息［Ｃａ２＋］ｉ和甲基血红蛋白ΔＥ。表明ＴＭＢ８５０及１００μｍｏｌ·Ｌ－１升高脑血管内皮［Ｃａ２＋］ｉ，激活ＮＯ合酶，促进ＮＯ合成和释放，这可能是其扩张脑血管的重要机制之一。     

钙拮抗剂TMB-8抑制BHQ,NE和KCl引起的培养乳牛基底动脉单个平滑肌细胞内游离钙的升高

用ＡＲＣＭＭＩＣ阳离子测定系统，测量单个细胞内游离钙浓度（［Ｃａ２＋］ｉ），研究８（Ｎ，Ｎ二乙胺）ｎ辛基３，４，５三甲氧基苯甲酸酯（ＴＭＢ８）对培养乳牛基底动脉平滑肌［Ｃａ２＋］ｉ的作用。在细胞外钙浓度为１３ｍｍｏｌ·Ｌ－１时，ＴＭＢ８（３０μｍｏｌ·Ｌ－１）可明显抑制ＢＨＱ，ＮＥ及ＫＣｌ引起［Ｃａ２＋］ｉ的升高。在细胞外钙为零＋ＥＧＴＡ０１ｍｍｏｌ·Ｌ－１时，ＴＭＢ８（１０，３０及１００μｍｏｌ·Ｌ－１）可浓度依赖性地降低静息［Ｃａ２＋］ｉ，ＴＭＢ８（３０μｍｏｌ·Ｌ－１）可几乎完全阻断ＢＨＱ及ＮＥ引起［Ｃａ２＋］ｉ的增加。研究表明ＴＭＢ８降低培养乳牛基底动脉平滑肌［Ｃａ２＋］ｉ的机制，主要是抑制肌浆网Ｃａ２＋的释放，或增加肌浆网对Ｃａ２＋的摄入，并由此间接地抑制细胞外钙的内流。     

小檗碱对培养的新生大鼠心肌细胞内游离钙含量的影响

采用Ｃａ２＋指示剂Ｆｕｒａ－２作为细胞内钙离子的荧光探针，利用ＡＲ－ＣＭ－ＭＩＣ阳离子测定系统。检测了培养新生大鼠心肌细胞内游离钙的浓度，并观察了小檗碱对去甲肾上腺素，Ｈ２Ｏ２，高Ｃａ２＋及高Ｋ＋引起细胞内钙离子浓度（［Ｃａ２＋］ｉ）变化的影响。小檗碱对心肌细胞静息［Ｃａ２＋］ｉ无明显影响，能浓度依赖地抑制去甲肾上腺素和Ｈ２Ｏ２引起的［Ｃａ２＋］ｉ的升高。小檗碱５０μｍｏｌ·Ｌ－１能抑制高Ｋ＋引起的［Ｃａ２＋］ｉ的升高，而小剂量（１－１０μｍｏｌ·Ｌ－１）则无作用。对搏动细胞，小檗碱能抑制其［Ｃａ２＋］ｉ瞬间变化的最大值，对最小值则无作用。     

8-(N,N-Diethylamino)-n-octyl-3,4,5-trimethoxybenzoate reduced (Ca~(2 ))_i elevation induced by histamine,serotonin,and glutamate in cultured calf basilar artery smooth muscle cells

目的：研究８（Ｎ，Ｎ二乙胺）ｎ辛基３，４，５三甲氧基苯甲酸酯对培养乳牛基底动脉平滑肌［Ｃａ２＋］ｉ的作用．方法：采用ＡＲＣＭＭＩＣ阳离子测定系统，测量细胞内游离钙浓度（［Ｃａ２＋］ｉ）．结果：在细胞外钙浓度为１３ｍｍｏｌ·Ｌ－１时，ＴＭＢ８３０μｍｏｌ·Ｌ－１可明显抑制组胺，５羟色胺和谷氨酸引起的［Ｃａ２＋］ｉ的升高．在外钙为零＋依他酸０１ｍｍｏｌ·Ｌ－１时，ＴＭＢ８３０μｍｏｌ·Ｌ－１可明显降低静息［Ｃａ２＋］ｉ，ＴＭＢ８３０μｍｏｌ·Ｌ－１可几乎完全阻断组胺和５羟色胺增加［Ｃａ２＋］ｉ的作用．结论：ＴＭＢ８降低培养乳牛基底动脉平滑肌静息［Ｃａ２＋］ｉ，抑制Ｈｉｓ，５ＨＴ和Ｇｌｕ引起的［Ｃａ２＋］ｉ的增加．     

小檗碱对培养大鼠神经细胞内游离Ca^2＋的影响

以Ｆｕｒａ２／ＡＭ为细胞内钙离子的荧光指示剂，用ＡＲＣＭＭＩＣ阳离子测定系统，直接测定了体外培养的新生大鼠神经细胞内游离钙（［Ｃａ２＋］ｉ）值，并观察了小檗碱（Ｂｅｒ）的影响。结果表明，Ｂｅｒ对神经细胞静息［Ｃａ２＋］ｉ无明显影响，Ｂｅｒ１～１００μｍｏｌ·Ｌ－１能剂量依赖地抑制去甲肾上腺素和Ｈ２Ｏ２引起的［Ｃａ２＋］ｉ升高，其ＩＣ５０分别为３９．９和１７．９μｍｏｌ·Ｌ－１。高剂量Ｂｅｒ（１０～１００μｍｏｌ·Ｌ－１）能抑制高Ｋ＋引起的［Ｃａ２＋］ｉ升高。姐果提示，Ｂｅｒ对去甲肾上腺素，高Ｋ＋及Ｈ２Ｏ２引起的［Ｃａ２＋］ｉ升高的抑制作用可能是其抗脑缺血作用机制之一。     

小檗碱对神经递质引起的新生大鼠脑细胞内游离钙升高的影响

以Ｆｕｒａ－２／ＡＭ为荧光指示剂，利用ＡＲ－ＣＭ－ＭＩＣ阳离子系统测定小檗碱（Ｂｅｒ）对新生大鼠脑细胞静息Ｃａ２＋和神经递质引起的脑细胞内游离钙浓度（［Ｃａ２＋］ｉ）变化的影响．Ｂｅｒ１，１０，３０μｍｏｌ·Ｌ－１对脑静息［Ｃａ２＋］ｉ无明显影响．Ｂｅｒ１～１００μｍｏｌ·Ｌ－１剂量依赖的抑制谷氨酸引起的［Ｃａ２＋］ｉ升高．Ｂｅｒ１０μｍｏｌ·Ｌ－１能降低Ｈａｎｋｓ液有Ｃａ２＋和无Ｃａ２＋时去甲肾上腺素引起的［Ｃａ２＋］ｉ升高，且能降低５－羟色胺引起的［Ｃａ２＋］ｉ升高（在细胞外液有Ｃａ２＋时）．结果提示Ｂｅｒ可降低谷氨酸，去甲肾上腺素和５－羟色胺引起的［Ｃａ２＋］ｉ升高，这可能是其抗脑缺血机理之一．     

丙泊酚抑制去甲肾上腺素诱导大鼠肺动脉平滑肌细胞内游离钙浓度升高作用的机理

用荧光分光光度法及同位素放射免疫分析法检测丙泊酚（３０－３００μｍｏｌ·Ｌ－１）影响大鼠肺动脉平滑肌细胞（ＰＡＳＭＣ）内游离钙离子浓度（［Ｃａ２＋］ｉ）与肌醇－１,４,５－三磷酸（ＩＰ３）合成作用,以探讨丙泊酚舒张肺动脉平滑肌的作用机理．结果表明,与丙泊酚共同培养７２ｈ,对ＰＡＳＭＣ［Ｃａ２＋］ｉ基础水平无明显影响,但可浓度依赖性抑制去甲肾上腺素（ＮＥ３μｍｏｌ·Ｌ－１）引起的［Ｃａ２＋］ｉ升高作用;当细胞外液无钙或存在钙通道阻滞剂维拉帕米（３０μｍｏｌ·Ｌ－１）时,丙泊酚抑制ＮＥ升高［Ｃａ２＋］ｉ作用被增强;丙泊酚还可浓度依赖性抑制ＮＥ促进ＩＰ３合成作用．结果提示丙泊酚舒张血管平滑肌作用与抑制ＩＰ３介导的细胞内钙释放密切相关．     

槲皮素对异丙肾上腺素所致大鼠心肌肥厚的影响

目的　研究槲皮素（ Ｑｕｅ） 对异丙肾上腺素（ Ｉｓｏ） 所致大鼠心肌肥厚的抑制作用及作用机制。方法　 Ｉｓｏ ００２ ｍｇ·ｋｇ－ １ ,每日两次,连续ｓｃ ６ ｗｋ ,形成大鼠心肌肥厚模型,分别测定心脏各重量参数、心肌过氧化脂质（ Ｌ Ｐ Ｏ） 含量、超氧化物歧化酶（ Ｓ Ｏ Ｄ） 活性及心肌 Ｃａ２ ＋ 和主动脉 Ｃａ２ ＋ 含量,培养乳鼠心肌细胞,应用 Ｆｕｒａ ２／ Ａ Ｍ 钙荧光指示剂技术测定心肌细胞内游离钙浓度。结果　 Ｉｓｏ 连续ｓｃ ６ ｗｋ 后,心肌和左心室重量明显增加,心肌 Ｌ Ｐ Ｏ 含量显著增加, Ｓ Ｏ Ｄ 活性下降,心肌 Ｃａ２ ＋ 和主动脉 Ｃａ２ ＋ 含量明显增加,给 Ｑｕｅ ７５ ｍｇ·ｋｇ－ １ ,１５０ ｍｇ·ｋｇ － １ 和维拉帕米１０ ｍｇ·ｋｇ － １ 后均能明显减轻心肌肥厚,降低 Ｌ Ｐ Ｏ 含量,增加 Ｓ Ｏ Ｄ 活性,降低心肌 Ｃａ２ ＋和主动脉 Ｃａ２ ＋ 的含量,应用 Ｆｕｒａ ２／ Ａ Ｍ 钙荧光指示剂技术发现 Ｉｓｏ 和 Ｈ２ Ｏ２ 能引起培养乳鼠心肌细胞内游离钙浓度明显升高。槲皮素对心肌细胞静息钙无明显影响,能抑制 Ｉｓｏ和 Ｈ２ Ｏ２ 致培养乳鼠心肌细胞内游离钙浓度的升高。结论　 Ｑｕｅ 能抑制 Ｉｓｏ 所引起的心肌肥厚, 该作用与清     

CD40-CD40配体相互作用对培养的人单核细胞二酰基甘油-蛋白激酶C信号通路及胞内Ca^2＋的影响

目的：探讨CD40-CD40配体(CD40L)相互作用是否能激活人外周血单核细胞(PBMC)内二酰基甘油(DAG)-蛋白激酶C(PKC)信号通路．方法：细胞内DAG含量采用放射酶标记、薄层层析和放射自显影方法检测．细胞PKC活性及胞内游离钙分别采用[γ-^32P]ATP磷酸转移法和Fluo-3荧光负载流式细胞术检测．结果：CD40L以剂量依赖方式刺激人外周血单核细胞合成DAG,并具有双时限性变化,第一峰值在20s,第二峰值在10min时出现．然后DAG水平缓慢下降,至少持续20-30min．单核细胞蛋白激酶,C总活性受CD40L刺激后明显增加,峰值在12min,持续20min以上．并且这种作用主要是胞浆PKC活性向胞膜PKC活性转位所致．CD40L,能刺激胞内游离Ca^2 出现短暂的快速升高,继之为持续阶段．移去细胞外Ca^2 ,胞内快速阶段无影响,而持续阶段明显受到抑制．抗CD40抗体能显著抑制CD40L引起的胞内DAG-PKC信号通路激活及[Ca^2 ]i的动态变化．结论：CD40-CD40L相互作用能激活人外周血单核细胞[Ca^2 ]i动态变化及二酰基甘油-蛋白激酶C信号通路．     

Inhibitory action of dilazep on histamine-stimulated cytosolic Ca2+ increase in cultured human endothelial cells.

Using a fluorescent Ca(2+)-sensitive dye, fura-2, and photometric fluorescence microscopy, we measured changes in cytosolic Ca2+ concentration ([Ca2+]i) in cultured human endothelial cells and studied the effect of dilazep on [Ca2+]i elevation induced by histamine. Histamine (1 microM) caused a rapid transient peak in the average [Ca2+]i of a group of cells (approximately 10(2) cells), followed by a decrease to a sustained elevation. Dilazep as well as diltiazem (1.0 to 100 microM) concentration-dependently inhibited the latter sustained elevation, which was eliminated by removal of extracellular Ca2+, while the initial transient response was not changed by dilazep at concentrations up to 100 microM. The IC50 values of dilazep and diltiazem were 16 and 58 microM, respectively. The patterns of the [Ca2+]i elevation responses to histamine were variable among individual cells. Some single cells showed a transient peak and a sustained elevation as observed in a group of cells. Some single cells caused a repetitive spikelike elevation of [Ca2+]i. Dilazep lowered the sustained elevation to the resting level and in some single cells, changed the sustained elevation to the spikelike elevation. The frequency of the spikelike [Ca2+]i elevation was also decreased by dilazep. Decrease in extracellular [Ca2+] showed the same pattern of inhibitory actions as dilazep did. These results indicate that dilazep inhibits the extracellular Ca2+ influx in endothelial cells.     

Bradykinin induces elevations of cytosolic calcium through mobilisation of intracellular and extracellular pools in bovine aortic endothelial cells.

1. In the presence of 1.8 mM extracellular calcium, bradykinin (0.3 nM-100 nM) induced a biphasic elevation of intracellular calcium ([Ca2+]i) in bovine aortic endothelial cells, consisting of an initial, large transient component followed by a lower sustained component. 2. When endothelial cells were bathed in nominally calcium-free solution containing 0.5 mM EGTA, bradykinin induced only a transient elevation of [Ca2+]i: the magnitude of this was significantly smaller than that obtained in the presence of extracellular calcium and the sustained phase was abolished. In the continued presence of bradykinin, re-addition of extracellular calcium to achieve a level of around 1.8 mM resulted in the induction of a biphasic elevation of [Ca2+]i consisting of a large initial component followed by a lower sustained component. 3. In the presence of 1.8 mM extracellular calcium, caffeine (5 mM) induced a small elevation of [Ca2+]i. When endothelial cells were bathed in nominally calcium-free solution containing 0.5 mM EGTA, the caffeine-induced elevation of [Ca2+]i was almost completely abolished. 4. In the presence of 1.8 mM extracellular calcium, treatment of endothelial cells with the calcium influx blocker, nickel chloride (4 mM), had no effect on resting [Ca2+]i or on the magnitude of the bradykinin-induced initial transient elevation of [Ca2+]i but abolished the sustained component. 5. In the presence of 1 mM extracellular calcium, treatment with the calcium chelator EGTA (2 mM; 1 min) had no effect on resting [Ca2+]i but the magnitude of the bradykinin-induced initial transient elevation of [Ca2+]i was significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of extracellular and intracellular sources of Ca2+ in sarafotoxin S6b-induced contraction of strips of the rat aorta.

1. The effect of sarafotoxin S6b (sarafotoxin), a vasoconstrictor peptide, on cytosolic Ca2+ concentration ([Ca2+]i) and force in rat aortic strips loaded with fura-2 was determined by front-surface fluorometry. The objective was to elucidate the role of extracellular and intracellular Ca2+ in the mechanism of action of this peptide. 2. In the presence of extracellular 1.25 mM Ca2+, sarafotoxin induced a biphasic response consisting of an initial rapid increase in [Ca2+]i followed by a secondary sustained increase. Tension developed slowly but was sustained during the application of sarafotoxin. Diltiazem (10 nM-0.1 mM) partially inhibited both the increases in [Ca2+]i and tension. 3. In the presence of extracellular Ca2+, the force developed in relation to the increase in [Ca2+]i ([Ca2+]i-force relationship) observed with sarafotoxin was much greater than that observed upon K+ depolarization. In the presence of diltiazem the sarafotoxin-induced [Ca2+]i-force relationship was shifted even further to the left. 4. In the absence of extracellular Ca2+, sarafotoxin induced a transient increase in [Ca2+]i and a sustained contraction. Extending the incubation time in Ca(2+)-free physiological solution, resulted in smaller responses. However, after 60 min in Ca(2+)-free solution, sarafotoxin induced a sustained contraction but no change in [Ca2+]i. This residual contraction was inhibited by H-7, which is known to inhibit protein kinase C. 5. After treatment with caffeine to reduce intracellular stored Ca2+, sarafotoxin could still elicit increases in [Ca2+]i and in tension, showing that the caffeine-sensitive intracellular Ca2+ store partially overlaps with the sarafotoxin-sensitive store.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of endothelium-dependent relaxation induced by substance P in the coronary artery of the pig.

1. Using front-surface fluorometry of fura-2-loaded porcine coronary arterial strips with the endothelium intact, we investigated the mechanisms of vasorelaxation induced by substance P (SP). Fura-2 fluorescence signals which indicated the cytosolic Ca2+-concentration ([Ca2+]i), were observed to arise exclusively from teh smooth muscle cells in these strips. 2. During the contractions induced by U46619 (100 nM), a thromboxane A2 analogue, an SP-induced endothelium-dependent, biphasic vasorelaxation was observed, which consisted of an initial rapid relaxation phase followed by a sustained phase, with a transient decrease in [Ca2+]i. Pretreatment with indomethacin (Ind) had no effect on the SP-induced relaxation; however, pretreatment with NG-nitro-L-arginine (L-NOARG) partially, but significantly inhibited the decrease in both the [Ca2+]i and tension abolished. Thus, part of the relaxation was considered to be mediated by L-NOARG-sensitive relaxing factor (endothelium-derived relaxing factor: EDRF). 3. During the 40 mM K+-depolarization-induced contraction which may eliminate the effects of endothelium-derived hyperpolarizing factor (EDRF), the vasorelaxation reduced by SP was completely inhibited by L-NOARG. 4. During the vasorelaxation induced SP, the [Ca2+]i-tension relationships shifted to the right of the contractions induced by either U46619 or high K+-depolarization. 5. Using front-surface fluorometry of fura-2 loaded porcine aortic valvular strips, we examined the effects of SP on [Ca2+]i in endothelial cells in situ. SP induced a rapid increase in [Ca2+]i of endothelial cells in situ followed by a small sustained phase in normal PSS (5.9 mM K+). The increase in extracellular K+ had no apparent effect on the SP-induced [Ca2+]i elevation of endothelial cells.     

The mechanism of the decrease in cytosolic Ca2+ concentrations induced by angiotensin II in the high K(+)-depolarized rabbit femoral artery

1. Using front-surface fluorometry of fura-2-loaded strips, and measuring the transmembrane 45Ca2+ fluxes of ring preparations of the rabbit femoral artery, the mechanism underlying a sustained decrease in the cytosolic Ca2+ concentration ([Ca2+]i) induced by angiotensin II (AT-II) was investigated. 2. The application of AT-II during steady-state 118 mM K(+)-induced contractions caused a sustained decrease in [Ca2+]i following a rapid and transient increase in [Ca2+]i, while the tension was transiently enhanced. 3. When the intracellular Ca2+ stores were depleted by thapsigargin, the initial rapid and transient increase in [Ca2+]i was abolished, however, neither the sustained decrease in [Ca2+]i nor the enhancement of tension were affected. 4. Depolarization with 118 mM K+ physiological salt solution containing 1.25 mM Ba2+ induced a sustained increase in both the cytosolic Ba2+ concentration ([Ba2+]i) level and tension. However, the application of 10(-6) M AT-II during sustained Ba(2+)-contractions was found to have no effect on [Ba2+]i, but it did enhance tension. 5. After thapsigargin treatment, AT-II neither decreased nor increased the enhanced Ca2+ efflux rate induced by 118 mM K(+)-depolarization, whereas AT-II did increase the enhanced 45Ca2+ influx and the 45Ca2+ net uptake induced by 118 mM K(+)-depolarization. 6. Pretreatment with calphostin-C, partially, but significantly inhibited the decrease in [Ca2+]i induced by AT-II. 7. These findings therefore suggest that AT-II stimulates Ca2+ sequestration into the thapsigargin-insensitive Ca2+ stores, and thus induces a decrease in [Ca2+]i in the high external K(+)-stimulated rabbit femoral artery.     

Effect of probucol on intracellular pH and proliferation of human vascular endothelial cells.

We investigated the effect of probucol on the intracellular pH ([pH]i) and proliferation of human umbilical vein endothelial cells (HUVEC), as well as their production of prostacyclin (PGI2). The addition of probucol produced a biphasic shift in [pH]i, with a brief initial acidification followed by a rapid alkaline shift. After pretreatment with EGTA, the initial decrease in [pH]i was abolished, and the subsequent increase was inhibited. After pretreatment with amiloride, only the increase of [pH]i was abolished. These results suggest that the probucol-induced increase of [pH]i was mainly dependent on Na+/H+ exchange and partly on extracellular Ca2+. In contrast, the addition of LDL produced a decrease of [pH]i. Under Ca2+-free condition, [pH]i was further decreased by LDL. In cells pretreated with amiloride, however, [pH]i was not further decreased by LDL. It was found that probucol promoted cell proliferation, and LDL inhibited cell proliferation. Addition of probucol also enhanced prostacyclin generation by HUVEC. This enhancement of PGI2 generation resulted from increased release of Ca2+ from the storage sites, due not only to increased production of inositol 1,4,5-triphosphate (IP3) but also to the increase of [pH]i. These findings may help to explain the antiatherosclerotic action of probucol.     

心肌肥厚时κ阿片受体介导的信号通路受损(英文)

目的:分离正常及慢性缺氧性有心肥厚的心室肌细胞,观察细胞内[Ca~(2 )]_i及细胞内pH_i对心肌к-阿片受体激动后的反应。方法:以fura-2和BCECF分别为[Ca~(2 )]_i和pH_i的指示剂,用光谱荧光法测定电刺激引起的细胞内[Ca~(2 )]_i瞬变及pH_i。结果:к阿片受体选择性激动剂U50,488H可降低电刺激引起的[Ca~(2 )]_i瞬变和增加pH_i,该作用是由蛋白激酶C(PKC)所介导。在肥厚的心室肌细胞,U50,488H的上述作用显著减弱。与此相对应,PKC的激动剂PMA引起的[Ca~(2 )]_i瞬变降低和pH_i的增加作用在肥厚的心室肌细胞亦消失。用NH_4Cl法观察Na~ -H~ 交换器的功能显示其在肥厚的心室肌细胞无明显改变。结论:心肌肥厚时к-阿片受体介导的信号通路受损,受损部位发生在PKC与效应器之间。     

N-desethylamiodarone modulates intracellular calcium concentration in endothelial cells

The main in-vivo metabolite of amiodarone, N-desethylamiodarone (DEAM), possesses clinically relevant class-II antiarrhythmic and vasodilator activities. Vasodilation by DEAM is endothelium dependent and involves a sustained and biphasic increase in cytosolic free Ca2+ concentration ([Ca2+]i). The aims of this study were to explore the mechanisms mediating the DEAM-induced increase in [Ca2+]i in endothelial cells and to determine whether this increase in [Ca2+]i was associated with altered cell proliferation. Cultured bovine aortic endothelial cells were loaded with the Ca2+-sensitive fluorescent dye Fura-2/AM, and [Ca2+]i measured spectrofluorimetrically. DEAM increased [Ca2+]i concentration dependently (EC50 approximately 6 microM) both in the presence and absence of extracellular Ca2+. In the presence of extracellular Ca2+, the response of [Ca2+]i to DEAM (10 microM) consisted of an initial rise to a plateau followed by a second increase to micromolar levels. The initial plateau was reduced by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (200 nM) and by the antioxidant ascorbic acid (100 microM). The initial rate of rise in [Ca2+]i was decreased by blocking mitochondrial Ca2+ release with cyclosporine A (1 microM). Under Ca2+-free conditions, the response of [Ca2+]i to DEAM (10 microM) was also biphasic, consisting of an initial transient peak and a second slow increase. When extracellular Ca2+ was restored, [Ca2+]i rose to micromolar concentrations. The initial peak was abolished by thapsigargin, but not altered by ascorbic acid or cyclosporine A. Both the second [Ca2+]i increase and that due to restoring extracellular Ca2+ were reduced by ascorbic acid but not affected by thapsigargin or cyclosporine A. The DEAM-induced generation of free radicals and sustained increase in [Ca2+]i might alter cell proliferation and endothelial cell proliferation was indeed concentration-dependently inhibited by DEAM (IC50 approximately 2.5 microM). In conclusion, the DEAM-induced [Ca2+]i increase in endothelial cells is due to Ca2+ influx from the extracellular space and to Ca2+ release from endoplasmic reticulum and mitochondria and involves enhanced generation of free radicals.