Changes of calcium release in myocardial sarcoplasmic reticulum during heart failure

目的:探讨慢性心力衰竭时心肌细胞肌浆网内钙离子释放的变化.方法:采用冠状动脉左前降支结扎法制备大鼠慢性心衰模型,酶解法分离成年大鼠心室肌细胞.用全细胞模式膜片钳技术和共聚焦显微镜钙成像技术同步实时记录心肌细胞的L-型钙电流以及胞质内的钙瞬变.结果:心衰组的钙诱导钙瞬变幅度小于对照组,并且心衰组的咖啡因诱导钙瞬变幅度也低于对照组.结论:心力衰竭的发生与心肌细胞内钙离子释放变化有着十分密切的关系.     

四逆汤对过氧化氢所致心肌细胞L型钙电流异常的作用

目的探讨四逆汤对氧自由基所致心肌细胞L型钙电流异常的作用。方法用急性酶解法获得单个大鼠心室肌细胞,用标准的全细胞膜片钳技术记录钙通道电流,首先观察5mmol/LH2O2对心肌细胞ICa,L的改变,然后观察预先用四逆汤含药血清孵育30min后5mmol/LH2O2对心肌细胞ICa,L的影响。结果在H2O2作用下,大鼠心室肌细胞ICa,L电流密度增加,电流-电压曲线显著下移,稳态失活曲线明显左移。四逆汤含药血清对离子电流无直接影响,但可以减轻H2O2所引起的稳态失活曲线的异常改变。结论四逆汤可减轻H2O2对心肌细胞钙通道的损伤作用。     

三七总皂甙对慢性低氧大鼠右心室心肌细胞钙电流的影响

目的:探讨长期应用三七总皂甙对慢性常压低氧大鼠右心室心肌细胞钙电流的影响。方法:采用全细胞膜片钳方法分别记录并比较正常对照组、慢性低氧组、慢性低氧加三七总皂甙组大鼠单个右心室心肌细胞钙电流和电流-电压关系曲线。结果:正常对照组、慢性低氧组、慢性低氧加三七总皂甙组细胞钙电流峰值分别为(571.1±145.9)pA,(835.1±194.1)pA,(526.3±152.6)pA。缺氧组显著大于另两组(P＜0.05)。结论:应用三七总皂甙能阻滞慢性低氧引起的钙内流增高。     

阿托伐他汀通过调节钠-钙交换体改善心力衰竭大鼠心脏功能

目的:探讨阿托伐他汀对心力衰竭大鼠心脏功能及心肌细胞钠-钙交换体表达的影响。方法:结扎大鼠冠状动脉左前降支8周,制备心肌梗死后心力衰竭模型。随机分3组,即假手术组、心力衰竭组与阿托伐他汀治疗组,分别于结扎后第2天给予安慰剂或阿托伐他汀治疗。8周后测定大鼠心脏功能及钠-钙交换体蛋白表达水平。培养原代乳鼠心肌细胞,通过缺氧模型分析阿托伐他汀对细胞内钙水平的影响。结果:阿托伐他汀治疗组较心衰组BNP水平降低,左室舒张末内径缩小,左室短轴缩短率升高。阿托伐他汀治疗明显降低心力衰竭时钠-钙交换体蛋白表达量,抑制缺氧诱导细胞内钙水平增加。结论:阿托伐他汀治疗改善心功能,可能与影响心力衰竭心肌细胞钠-钙交换体的表达及功能有关。     

白细胞介素-2改变心肌细胞钙瞬态的频率依赖性

目的:观察白细胞介素-2(IL-2)对大鼠心肌细胞内钙处理能力的影响。方法:采用细胞内双波长钙荧光系统检测稳定状态及不同电刺激频率下单个心肌细胞钙瞬态的变化。结果:在稳定状态(0.2Hz)下, 2×10⁵U/LIL-2抑制心肌细胞钙瞬态的幅度和峰值, 使舒张末钙水平升高, 钙瞬态下降相时间常数延长。细胞外钙浓度为1.25mmol/L时, 心肌细胞舒张末钙水平、钙瞬态的幅度均随刺激频率(0.2-1.0Hz)的增高而增高。IL-2抑制了这种正性的频率依赖关系, 当将外钙浓度增加至2.5mmol/L时并不能改变其抑制作用。咖啡因诱导的内贮钙释放也呈现正性的频率依赖关系, 2×10⁵U/LIL-2抑制其频率依赖关系。在细胞外钙浓度为1.25mmol/L时, 两组间的钙瞬态机械恢复特性无差异, 当外钙为2.5mmol/L时, IL-2组的恢复率减慢。结论:IL-2通过抑制肌浆网内贮钙的释放而抑制了细胞内钙瞬态的正性频率依赖关系。     

缺氧预处理对自发性高血压大鼠心肌细胞内钙的影响

目的:探讨缺氧预处理对后继缺氧再给氧所致心肌细胞内钙超载的影响及蛋白激酶C(PKC)和百日咳毒素(PTX)敏感的G-蛋白在缺氧预处理的作用.方法:循环灌流法分离自发性高血压大鼠(SHR)肥大心肌细胞,用Fu ra-2AM测定钙浓度.结果:[Ca2+].为1.0 mmol/L时,预处理组缺氧再给氧后其心肌细胞内游离钙浓度[Ca2+]i为(194.0±24.8) nmol/L,未预处理组为(297.5 ±24.5) nmol/L;无外钙预处理组缺氧再给氧后其心肌细胞[Ca2+]i为(162.0 ±30.7) nmol/L,未预处理组为(236.5±28.3) nmol/L,提示缺氧预处理可减少缺氧再给氧所致心肌细胞内钙释放及外钙内流.在[Ca2+].为1.0 mmol/L组,于预处理前分别预先给予PKC拮抗剂Staurosporine(10 nmol/L)及Gi-蛋白失活剂百日咳毒素(PTX,200 ng /L),可见经后继缺氧再给氧后其心肌细胞[Ca2+]i分别为(264.8±19.3)及(25 8.0±27.7) nmol/L,高于缺氧预处理组但低于未预处理组.结论:SH R肥大心肌细胞,缺氧预处理可减轻后继缺氧再给氧所致心肌细胞内钙超载;PKC及PTX敏感的G- 蛋白可能部分参与缺氧预处理的保护作用.     

钙／钙调素依赖的蛋白激酶Ⅱ对心肌细胞钙循环的影响

在心衰、心肌肥厚和心肌缺血等情况下,心肌细胞钙离子循环常出现障碍,导致心脏电-机械活动异常,临床发现心律失常和心源性猝死常和钙循环异常有关.钙调素和钙/钙调素依赖的蛋白激酶Ⅱ(CaMKII)通过调节钙循环的各个环节来影响心肌细胞信号传导和兴奋-收缩偶联,在心衰或心肌肥厚等病理条件下细胞内表达升高.对其深入研究有利于了解这些病理情况下心律失常发生的机制.     

慢性心衰大鼠心肌细胞内雷尼丁受体及集钙蛋白的表达

目的:研究慢性心衰大鼠心肌细胞内雷尼丁受体(RyR2)及其调节蛋白集钙蛋白的表达,探讨心衰心肌细胞内钙容量降低的机制。方法:雄性SD大鼠被随机分为心衰组和假手术组,运用激光扫描共聚焦显微镜、透射电子显微镜、免疫印迹等方法研究2组大鼠心肌细胞肌浆网内钙容量、心肌细胞超微结构及心肌细胞内肌浆网钙释放通道RyR2和其调节蛋白集钙蛋白表达的变化。结果:慢性心衰大鼠心功能左心室舒张末期压力、左室压力最大上升速率均显著低于假手术组。假手术组大鼠心肌细胞的肌小节完整,肌丝排列整齐,线粒体结构正常;心衰组大鼠部分心肌细胞肌丝溶解,线粒体肿胀,嵴断裂。慢性心衰大鼠心肌细胞肌浆网内钙容量显著低于假手术组。慢性心衰大鼠心肌细胞内RyR2的表达量无明显变化,集钙蛋白表达明显下降。结论:慢性心衰大鼠心肌细胞内集钙蛋白表达下调,可能是导致心肌细胞肌浆网钙容量减少的原因之一。     

神经肽Y活化大鼠心肌细胞内Ca2+及钙调素依赖的钙调神经磷酸酶途径

目的 观察神经肽 Y (NPY)对心肌细胞内Ca2+及钙调素(CaM)依赖的钙调神经磷酸酶(CaN)信号传导途径的影响.方法 以NPY刺激心肌细胞.用环胞素A(CsA)特异性抑制CaN活性.应用Western印迹测细胞内CaN-α蛋白表达.采用组织化学法测CaN酶活性.用Fluo3-AM荧光标记法观察心肌细胞胞质及核内钙离子浓度变化.结果 加入100 nmol/L NPY刺激后,心肌细胞CaN酶活力较对照组明显增高(P＜0.05),10 nmol/L NPY组和CsA 组(100 nmol/L NPY + 5 μg/ml CsA) CaN酶活性与对照组相比,差异无统计学意义.100 nmol/L NPY组心肌细胞内CaN-α蛋白表达较对照组显著增高(P＜0.05). 加入100 nmol/L NPY在10 min内,心肌细胞胞浆及核内钙含量与对照组相比,差异无统计学意义.经100 nmol/L NPY刺激24 h后,心肌细胞胞浆及核内钙含量明显高于对照组(P均＜0.01).结论 NPY可活化心肌细胞内Ca2+及CaM-CaN途径.NPY刺激下产生的细胞内钙增加,可能是其活化CaN途径的始动环节.     

钙/钙调素依赖的蛋白激酶II对心肌细胞钙循环的影响

在心衰、心肌肥厚和心肌缺血等情况下 ,心肌细胞钙离子循环常出现障碍 ,导致心脏电 机械活动异常 ,临床发现心律失常和心源性猝死常和钙循环异常有关。钙调素和钙 /钙调素依赖的蛋白激酶II(CaMKII)通过调节钙循环的各个环节来影响心肌细胞信号传导和兴奋 收缩偶联 ,在心衰或心肌肥厚等病理条件下细胞内表达升高。对其深入研究有利于了解这些病理情况下心律失常发生的机制。     

Free radical-dependent Ca2+ signaling: role of Ca2+-induced Ca2+ release

Previously we have shown that Fe3+/ascorbate-induced Ca2+ release from scallop sarcoplasmic reticulum (SR) is due to Ca2+-channel gating by free radicals. This study is aimed at demonstrating that Ca2+-induced Ca2+ release (CICR) plays a role in this kind of Ca2+ release. Scallop SR vesicles were incubated with fluo-3 and exposed to Fe3+/ascorbate. Fluorimetric recordings showed massive Ca2+ release, with maximum rate and 50% release occurring at 30 min after exposure. Conversely, the use of the probe for reactive oxygen species dihydrorhodamine or the assay of malondialdehyde allowed oxyradical production to be traced for approximately 5 min only. Hence, although Ca2+ release started just after exposure to Fe3+/ascorbate, most release occurred after free radical exhaustion. Ruthenium red addition after Fe3+/ascorbate slowed down the Ca2+ release, whereas cyclic adenosine 5'-diphosphoribose addition accelerated it, indicating that the free radical-induced Ca2+ release from SR vesicles triggers a mechanism of CICR that dramatically increases the initial effect.     

Ca2+-dependent inactivation of Ca2+-induced Ca2+ release in bullfrog sympathetic neurons

We studied inactivation of Ca(2+)-induced Ca(2+) release (CICR) via ryanodine receptors (RyRs) in bullfrog sympathetic neurons. The rate of rise in [Ca(2+)](i) due to CICR evoked by a depolarizing pulse decreased markedly within 10-20 ms to a much slower rate despite persistent Ca(2+) entry and little depletion of Ca(2+) stores. The Ca(2+) entry elicited by the subsequent pulse within 50 ms, during which the [Ca(2+)](i) level remained unchanged, did not generate a distinct [Ca(2+)](i) rise. This mode of [Ca(2+)](i) rise was unaffected by a mitochondrial uncoupler, carbonyl cyanide p-trifluromethoxy-phenylhydrazone (FCCP, 1 microm). Paired pulses of varying interval and duration revealed that recovery from inactivation became distinct >or= 50 ms after depolarization and depended on [Ca(2+)](i). The inactivation was prevented by BAPTA (>or= 100 microm) but not by EGTA (相似文献   

Ca2+ signaling by T-type Ca2+ channels in neurons

Among the major families of voltage-gated Ca²⁺ channels, the low-voltage-activated channels formed by the Ca_v3 subunits, referred to as T-type Ca²⁺ channels, have recently gained increased interest in terms of the intracellular Ca²⁺ signals generated upon their activation. Here, we provide an overview of recent reports documenting that T-type Ca²⁺ channels act as an important Ca²⁺ source in a wide range of neuronal cell types. The work is focused on T-type Ca²⁺ channels in neurons, but refers to non-neuronal cells in cases where exemplary functions for Ca²⁺ entering through T-type Ca²⁺ channels have been described. Notably, Ca²⁺ influx through T-type Ca²⁺ channels is the predominant Ca²⁺ source in several neuronal cell types and carries out specific signaling roles. We also emphasize that Ca²⁺ signaling through T-type Ca²⁺ channels occurs often in select subcellular compartments, is mediated through strategically co-localized targets, and is exploited for unique physiological functions. Lucius Cueni and Marco Canepari contributed equally to this review.     

Voltage-activated ion channels and Ca2+-induced Ca2+ release shape Ca2+ signaling in Merkel cells

Ca²⁺ signaling and neurotransmission modulate touch-evoked responses in Merkel cell–neurite complexes. To identify mechanisms governing these processes, we analyzed voltage-activated ion channels and Ca²⁺ signaling in purified Merkel cells. Merkel cells in the intact skin were specifically labeled by antibodies against voltage-activated Ca²⁺ channels (Ca_V2.1) and voltage- and Ca²⁺-activated K⁺ (BK_Ca) channels. Voltage-clamp recordings revealed small Ca²⁺ currents, which produced Ca²⁺ transients that were amplified sevenfold by Ca²⁺-induced Ca²⁺ release. Merkel cells’ voltage-activated K⁺ currents were carried predominantly by BK_Ca channels with inactivating and non-inactivating components. Thus, Merkel cells, like hair cells, have functionally diverse BK_Ca channels. Finally, blocking K⁺ channels increased response magnitude and dramatically shortened Ca²⁺ transients evoked by mechanical stimulation. Together, these results demonstrate that Ca²⁺ signaling in Merkel cells is governed by the interplay of plasma membrane Ca²⁺ channels, store release and K⁺ channels, and they identify specific signaling mechanisms that may control touch sensitivity.     

Inositol trisphosphate enhances Ca2+ oscillations but not Ca2+-induced Ca2+ release from cardiac sarcoplasmic reticulum

The role of inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] in excitation-contraction coupling in cardiac muscle is still unclear, although many laboratories are beginning to assume a critical role for this putative second messenger. Earlier studies from this laboratory [Nosek et al. (1986) Am J Physiol 250:C807] found that Ins(1,4,5)P ₃ enhanced spontaneous Ca²⁺ release and the caffeine sensitivity of Ca²⁺ release from myocardial sarcoplasmic reticulum (SR) and proposed an increase in the Ca²⁺ sensitivity of the release as a possible mechanism. In order to clarify the phyisological relevance of these actions of Ins(1,4,5)P ₃ and specifically to test the effect of Ins(1,4,5)P ₃ on the Ca²⁺ sensitivity of Ca²⁺ release, we compared the effects of Ins(1,4,5)P ₃ on Ca²⁺ oscillations and on Ca²⁺-induced Ca²⁺ release (CICR) from the SR in saponin-skinned rat papillary muscle. We found that: (a) 30 M Ins(1,4,5)P ₃ enhanced the Ca²⁺ oscillations (measured by tension oscillations) from the rat cardiac SR, consistent with the previous report on guinea pig tissue; (b) both GTP and GTP[S] enhanced Ca²⁺ oscillations. The effect was not additive to that of Ins(1,4,5)P ₃ indicating that two different Ca²⁺-release pools do not exist in cardiac SR; (c) 30 M Ins(1,4,5)P ₃ had no effect on the Ca²⁺ sensitivity of CICR; (d) Ins(1,4,5)P ₃ (up to 30 M) had no effect on SR Ca²⁺ loading. The studies were performed in the presence of Cd²⁺ or 2,3-bisphosphoglycerate, agents that inhibit Ins(1,4,5)P ₃ hydrolysis. These results suggest that: (a) two different mechanisms underlie Ca²⁺ oscillations and CICR, Ins(1,4,5)P ₃ influencing Ca²⁺ oscillations but not CICR; (b) Ins(1,4,5)P ₃ does not increase the Ca²⁺ sensitivity of Ca²⁺ release from the SR; (c) cardiac muscle is different from smooth muscle where Ca²⁺ release from the SR is dependent upon GTP; (d) the physiological role of Ins(1,4,5)P ₃ in excitation-contraction coupling in cardiac muscle is minimal. In contrast, Ins(1,4,5)P ₃ may play a pathological role in cardiac arrhythmogenesis by enhancing spontaneous Ca²⁺ ocsillations.     

ATP suppresses activity of Ca2+ -activated K+ channels by Ca2+ chelation

Ca²⁺-activated maxi K⁺ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NP _o) was stimulated by 3 M [Ca²⁺]_c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na₂ATP. The following results suggest that suppression of NP _o by Na₂ATP is due to Ca²⁺ chelation and hence reduction of [Ca²⁺]_c and reduced Ca²⁺ activation of the channel. The effect was absent when Mg ATP was used instead of Na₂ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca²⁺]_c was buffered with 10 mM HDTA (apparent pK _Ca 5.58) instead of EGTA (pK _Ca 6.8). A Ca²⁺-sensitive electrode system indicated that 1 mM Na₂ATP reduced [Ca²⁺]_c in 1 mM Ca-EGTA from 3 M to 1.4 M. Na₂ATP, Na₂GTP, Li₄AMP-PNP and NaADP reduced measured [Ca²⁺]_c in parallel with their suppression of NP _o. After the Na₂ATP-induced reduction of [Ca²⁺]_c was re-adjusted by adding either CaCl₂ or MgCl₂, the effect of Na₂ATP on NP _o disappeared. In vivo, intracellular [Mg²⁺] exceeds free [ATP^4–], hence ATP modulation of maxi K⁺ channels due to Ca²⁺ chelation is without biological relevance.     

Ca2+ channels that activate Ca2+-dependent K+ currents in neostriatal neurons

It is demonstrated that not all voltage-gated calcium channel types expressed in neostriatal projection neurons (L, N, P, Q and R) contribute equally to the activation of calcium-dependent potassium currents. Previous work made clear that different calcium channel types contribute with a similar amount of current to whole-cell calcium current in neostriatal neurons. It has also been shown that spiny neurons possess both "big" and "small" types of calcium-dependent potassium currents and that activation of such currents relies on calcium entry through voltage-gated calcium channels. In the present work it was investigated whether all calcium channel types equally activate calcium-dependent potassium currents. Thus, the action of organic calcium channel antagonists was investigated on the calcium-activated outward current. Transient potassium currents were reduced by 4-aminopyridine and sodium currents were blocked by tetrodotoxin. It was found that neither 30 nM omega-Agatoxin-TK, a blocker of P-type channels, nor 200 nM calciseptine or 5 microM nitrendipine, blockers of L-type channels, were able to significantly reduce the outward current. In contrast, 400 nM omega-Agatoxin-TK, which at this concentration is able to block Q-type channels, and 1 microM omega-Conotoxin GVIA, a blocker of N-type channels, both reduced outward current by about 50%. These antagonists given together, or 500 nM omega-Conotoxin MVIIC, a blocker of N- and P/Q-type channels, reduced outward current by 70%. In addition, the N- and P/Q-type channel blockers preferentially reduce the afterhyperpolarization recorded intracellularly. The results show that calcium-dependent potassium channels in neostriatal neurons are preferentially activated by calcium entry through N- and Q-type channels in these conditions.     

Ca2+ channels that activate Ca2+-dependent K+ currents in neostriatal neurons

It is demonstrated that not all voltage-gated calcium channel types expressed in neostriatal projection neurons (L, N, P, Q and R) contribute equally to the activation of calcium-dependent potassium currents. Previous work made clear that different calcium channel types contribute with a similar amount of current to whole-cell calcium current in neostriatal neurons. It has also been shown that spiny neurons posses both “big” and “small” types of calcium-dependent potassium currents and that activation of such currents relies on calcium entry through voltage-gated calcium channels. In the present work it was investigated whether all calcium channel types equally activate calcium-dependent potassium currents. Thus, the action of organic calcium channel antagonists was investigated on the calcium-activated outward current. Transient potassium currents were reduced by 4-aminopyridine and sodium currents were blocked by tetrodotoxin. It was found that neither 30 nM ω-Agatoxin-TK, a blocker of P-type channels, nor 200 nM calciseptine or 5 μM nitrendipine, blockers of L-type channels, were able to significantly reduce the outward current. In contrast, 400 nM ω-Agatoxin-TK, which at this concentration is able to block Q-type channels, and 1 μM ω-Conotoxin GVIA, a blocker of N-type channels, both reduced outward current by about 50%. These antagonists given together, or 500 nM ω-Conotoxin MVIIC, a blocker of N- and P/Q-type channels, reduced outward current by 70%. In addition, the N- and P/Q-type channel blockers preferentially reduce the afterhyperpolarization recorded intracellularly.The results show that calcium-dependent potassium channels in neostriatal neurons are preferentially activated by calcium entry through N- and Q-type channels in these conditions.     

Ryanodine- and thapsigargin-insensitive Ca2+-induced Ca2+ release is primed by lowering external Ca2+ in rabbit autonomic neurons

Rises in cytosolic Ca2+ induced by a high K+ concentration (30 or 60 mM) (K+-induced Ca2+ transient) were recorded by fluorimetry of Ca2+ indicators in cultured rabbit otic ganglion cells. When external Ca2+ ([Ca2+]o) was reduced to a micromolar (10-40 microM) or nanomolar (<10 nM) level prior to high-K+ treatment, K+-induced Ca2+ transients of considerable amplitude (50% of control) were generated in most cells, although those initiated at normal [Ca2+]o were reduced markedly or abolished by reducing [Ca2+]o during exposure to a high K+ concentration. Lowering [Ca2+]o alone occasionally caused a transient rise in cytosolic Ca2+. K+-induced Ca2+ transients at micromolar [Ca2+]o were repeatedly generated and propagated inwardly at a speed slower than that at normal [Ca2+]o, while those at nanomolar [Ca2+]o occurred only once. K+-induced Ca2+ transients at micromolar [Ca2+]o were not blocked by ryanodine (10 microM), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 5 microM: at 20-22 degrees C but blocked at 31-34 degrees C) or thapsigargin (1-2 microM), but were blocked by Ni2+ (1 mM) or nicardipine (10 microM). Thus, there is a ryanodine-insensitive Ca2+-release mechanism in FCCP- and thapsigargin-insensitive Ca2+ stores in rabbit otic ganglion cells, which is primed by lowering [Ca2+]o and then activated by depolarization-induced Ca2+ entry. This Ca2+-induced Ca2+ release may operate when [Ca2+]o is decreased by intense neuronal activity.