M型受体在大鼠肾上腺嗜铬细胞内钙库释放和激素分泌中的作用

在单个大鼠肾上腺嗜铬细胞上,用显微荧光测量和碳纤电极记录方法,测量可激活毒蕈碱（ｍｕｓｃａｒｉｎｅ,Ｍ）受体的激动剂乙酰甲胆碱（ｍｅｔｈａｃｈｏｌｉｎｅ,ＭＣｈ）对胞内游离钙浓度「Ｃａ＾２＋」ｉ和儿茶酚胺激素分泌的影响。在细胞外液含２ｍｍｏｌ／Ｌ Ｃａ＾２＋时,用含钙或不含钙的ＭＣｈ（１ｍｍｏｌ／Ｌ）刺激细胞,均引起「Ｃａ＾２＋」ｉ的升高或钙振荡,并诱发激素的分泌。     

降钙素基因相关肽对缺氧时海马细胞内游离Ca^2＋的影响

本实验用Ｆｕｒａ－２荧光测定技术直接监测了缺氧时大鼠海马细胞内游离Ｃａ２＋浓度（［Ｃａ２＋］ｉ）的变化,并观察了降钙素基因相关肽（ＣＧＲＰ）对这种变化的影响。结果发现,缺氧可使海马细胞［Ｃａ２＋］ｉ显著增高;４或８ｎｍｏｌ／ＬＣＧＲＰ能明显地降低缺氧引起的［Ｃａ２＋］ｉ增高,但在无胞外Ｃａ２＋的情况下,ＣＧＲＰ的作用消失。结果表明,ＣＧＲＰ的降钙作用是通过抑制缺氧时胞外Ｃａ２＋的内流来实现的。     

SNP抑制5-HT诱导的胞内游离钙浓度升高和内钙释放

用Ｆｕｒａ － ２／ＡＭ 荧光测量技术研究了５ － 羟色胺（５－ ＨＴ） 诱导的大鼠尾动脉平滑肌细胞胞内钙升高和一氧化氮（ＮＯ） 的抑制效应。实验表明, 胞外０ｍ ｍｏｌ／ Ｌ Ｃａ２ ＋ 时胞内静息［Ｃａ２ ＋ ］ ｉ 为２０ ．２±８ ．６ｎｍｏｌ／Ｌ（ｎ ＝ ８） 。１０μｍｏｌ／Ｌ ５－ ＨＴ 可诱导出胞内钙库释放引起的瞬态［Ｃａ２ ＋］ｉ 升高,其峰值达２４５ ．７ ±７１．６ｎｍｏｌ／ Ｌ（ｎ ＝ ６） 。１０ － ７ ｍｏｌ／Ｌ 硝普钠（ＳＮＰ） 可抑制５－ ＨＴ 诱导的［Ｃａ２ ＋］ｉ 升高,其峰值浓度降为７５．１±３５ ．９ｎｍｏｌ／Ｌ（ｎ ＝ ５） 。当细胞浴液含２．５ｍ ｍｏｌ／Ｌ Ｃａ２ ＋ 时,静息［Ｃａ２ ＋］ｉ为１１２ ．８ ±１０ ．３ｎｍｏｌ／ Ｌ（ｎ ＝ ５） , 这时１０μｍｏｌ／ Ｌ ５ － ＨＴ 可诱导［Ｃａ２ ＋ ］ ｉ 的峰值为２５２ ．３ ±８０ ．６ｎｍｏｌ／Ｌ（ｎ ＝ ４） ,以及其后平台浓度为１４３ ．０ ±３７ ．６ｎｍｏｌ／Ｌ（ｎ ＝ ４） ,略大于［Ｃａ２ ＋］ｉ 为１１２．８ ±１０ ．３ｎｍｏｌ／Ｌ 的静息浓度,为外钙内流引起。１０ － ７ ｍｏｌ／Ｌ ＳＮＰ 也可抑制５－ ＨＴ 诱导［Ｃａ２ ＋ ］ｉ 平台相浓度。平台浓度由１４３ ±４７     

低频、低压交变电场对成骨细胞增殖的影响

低频、低压交变电场能够促进成骨细胞的增殖。成骨细胞受交变电场作用以后,运用ＭＴＴ方法和流式细胞术,检测细胞的增殖情况。结果表明：电场作用后的细胞,与对照组细胞相比,细胞数目增多,Ｓ期细胞百分比增高。运用荧光标记技术检测细胞膜流动性以及胞内游离Ｃａ２＋浓度（［Ｃａ２＋］ｉ）的变化,初步探索电场对细胞增殖影响的作用机制。     

药物诱导后的人大肠癌细胞的[Ca^2＋]i的变化

采用荧光分光光度计法检测维甲酸（ＲＡ）、１,２５（ＯＨ）２ＶＤ３及佛波酯（ＰＭＡ）诱导ＣＣＬ２２９细胞分化后［Ｃａ２＋］ｉ变化,并观察内质网（ＥＲ）特异的Ｃａ２＋－ＡＴＰａｓｅ抑制剂Ｔｈａｐｓｉｇａｒｇｉｎ（ＴＧ）、ＩＰ３受体抑制剂Ｈｅｐａｒｉｎ对ＲＡ诱导［Ｃａ２＋］ｉ变化的影响,从而探讨ＲＡ诱导［Ｃａ２＋］ｉ变化与ＥＲ的关系。结果显示：ＲＡ和１,２５（ＯＨ）２ＶＤ３在数秒内引起［Ｃａ２＋］ｉ显著升高。在ＥＧＴＡ和Ｖｅｒａｐａｍｉｌ预处理细胞条件下,ＴＧ不能抑制ＲＡ引起Ｃａ２＋从细胞内钙池中外流,ＲＡ作用后ＴＧ仍能升高［Ｃａ２＋］ｉ。另外,Ｈｅｐａｒｉｎ也不能完全抑制ＲＡ升高［Ｃａ２＋］ｉ。提示ＲＡ诱导大肠癌细胞升高［Ｃａ２＋］ｉ可能通过ＥＲ上ＩＰ３敏感性和非敏感性钙池,亦可能细胞内存在除ＥＲ外对ＲＡ敏感的钙池。     

γ—氨基丁酸抑制缺氧所致神经元钙超载

本文以体外分散培养的新生大鼠海马ＣＡ１区神经细胞为标本,分别采用激光扫描共聚集显微镜动态监测单个细胞［Ｃａ＾２＋］ｉ和膜片箝全细胞记录的电生理技术检测细胞的ＮＭＤＡ电流和电压依赖性Ｃａ＾２＋电流等方法,较为深入地研究了抑制性神经递质γ－氨基丁酸（ＧＡＢＡ）及ＧＡＢＡ－Ａ受体激动剂蝇蕈醇对急性缺氧时海马ＣＡ１神经元［Ｃａ＾２＋］ｉ升高过程的影响方式及其作用机制。结果表明：对照组细胞缺氧后比缺氧前［Ｃ     

羟墓自由基引起神经细胞［Ca~（2＋）］i增高的机制和Ebselen的抑制作用

用Ｆｕｒａ－２显微荧光测量技术研究了羟基自由基对单个皮层神经细胞内游离钙离子浓度［Ｃａ２＋］ｉ影响和硒化合物Ｅｂｅｌｅｎ对［Ｃａ２＋］ｉ的抑制作用。结果表明羟基自由基的作用首先引起胞内［Ｃａ２＋］ｉ以时间常数τ＝３８９５．４±５０７．２Ｓ速度缓慢增加,然后加入了以τ＝４２０．６±１２２．０Ｓ的外钙大量涌入。钙通道阻断剂、疏基还原剂、疏基还原制和自由基清除剂对羟基自由基损伤作用的影响提示外钙的大量涌入部分与通道的开放有关,疏基损伤在羟基自由基引起的［Ｃａ２＋］ｉ升高中起着重要的作用。具有类谷胱甘肽过氧化酶活性的小分子硒化合物Ｅｂｓｅｌｅｎ（１０－５ｍｏｌ／Ｌ和１０－６ｍｏｌ／Ｌ）抑制羟基自由基引起的［Ｃａ２＋］ｉ升高,推测它可以抑制钙库的释放或促进内钙的外排以及抑制外钙的流入。     

培养乳鼠心肌细胞自发性〔Ca~（2＋）〕_i瞬间变化的实验模型

使用Ｃａ￣（２＋）敏感的荧光指示剂Ｆｕｒａ－２／ＡＭ,对培养的心肌细胞测定细胞内Ｃａ￣（２＋）的瞬间变化。结果为,在一个心搏周期中,经过大约１８０ｍｓ的时间,［Ｃａ￣（２＋）］＿ｉ瞬间变化达到最大值,然后恢复到基线水平（最小值）,经历了２６０ｍｓ。测得平均［Ｃａ￣（２＋）］＿ｉ瞬间变化的最大值及最小值分别为３４６±３８ｎｍｏｌ／Ｌ和１０２±１８ｎｍｏｌ／Ｌ。血管紧张素Ⅱ１００ｎｍｏｌ／Ｌ引起［Ｃａ￣（２＋）］＿ｉ瞬间变化最大值明显增加,但对［Ｃａ￣（２＋）］＿ｉ瞬间变化的基线水平没有明显的影响。ｒｙａｎｏｄｉｎｅ３μｍｏｌ／Ｌ使［Ｃａ￣（２＋）］＿ｉ瞬间变化的幅度明显降低。ＫＣｌ５０ｍｍｏｌ／Ｌ使［Ｃａ￣（２＋）］＿ｉ瞬间变化完全停止,并明显增加［ｃａ￣（２＋）］＿ｉ的基线水平。结果提示,本实验模型可用来观察［Ｃａ￣（２＋）］＿ｉ的瞬间变化。     

腺苷对缺氧时海马神经元内游离Ca^2＋的影响

实验用Ｆｌｕｏ－３负载细胞,在激光扫描共聚焦显微镜下直接监测缺氧后分散培养的大鼠海马ＣＡ１区神经元内游离Ｃａ＾２＋浓度（［Ｃａ＾２＋］ｉ）的变化,观察腺苷对这种变化的影响并初步探讨其作用机制。结果发现,急性缺氧使海马神经元［Ｃａ＾２＋］ｉ显著升高;腺苷（１００μｍｏｌ／Ｌ）明显抑制缺氧引起的［Ｃａ＾２＋］ｉ增高,腺苷Ａ１受体拮抗剂ＣＰＴ以及Ｋ＾＋通道阻断剂４－ＡＰ和ＡＴＰ敏感性Ｋ＾＋通道阻断剂ｇｌ     

缺氧时大鼠红细胞变形性损伤的机制研究

本实验通过测定平原和模拟高原减压缺氧３０天大鼠红细胞滤过指数（ＩＦ）、红细胞内［ｐＨ］ｉ、［Ｋ＋］ｉ／［Ｎａ＋］ｉ比值、［Ｃａ２＋］ｉ、［Ｍｇ２＋］ｉ、平均红细胞体积（ＭＣＶ）及平均红细胞血红蛋白浓度（ＭＣＨＣ）,从而探讨缺氧条件下大鼠红细胞变形性损害的机制。结果发现：１．缺氧组大鼠红细胞［Ｃａ２＋］ｉ明显升高,且与ＩＦ呈显著正相关,但［Ｍｇ２＋］ｉ无明显差异;２．缺氧组［Ｋ＋］ｉ／［Ｎａ＋］ｉ值较平原组明显降低,且与ＩＦ呈显著负相关;３．缺氧组ＭＣＨＣ与平原组无明显差异,但ＭＣＶ显著升高;４．缺氧组红细胞内［ｐＨ］ｉ较平原组明显升高。提示：缺氧时红细胞［Ｃａ２＋］ｉ升高,［Ｋ＋］ｉ／［Ｎａ＋］ｉ值降低,ＭＣＶ增大以及红细胞［ｐＨ］ｉ值的改变在其变形性损伤中起重要作用。     

Coordination of hormone-induced calcium signals in isolated rat hepatocyte couplets: demonstration with confocal microscopy.

Excitable cells often display rapid coordination of hormone-induced intracellular calcium signals. Calcium elevations that begin in a single epithelial cell also may spread to adjacent cells, but coordination of hormone-induced signals among epithelial cells has not been described. We report the use of confocal microscopy to determine the inter- and intracellular distribution of cytosolic calcium in isolated rat hepatocyte couplets, an isolated epithelial cell system in which functional polarity is maintained. Both vasopressin and phenylephrine evoked sequential coordinated calcium signals in the couplets, even during cytosolic calcium oscillations. The coupling was abolished by closure of intercellular gap junction channels by treatment with octanol. These observations demonstrate that hormone-induced intracellular calcium signals are coordinated among hepatocytes and suggest that gap junction channels mediate this intercellular integration of tissue responsiveness.     

A membrane model for cytosolic calcium oscillations. A study using Xenopus oocytes.

Cytosolic calcium oscillations occur in a wide variety of cells and are involved in different cellular functions. We describe these calcium oscillations by a mathematical model based on the putative electrophysiological properties of the endoplasmic reticulum (ER) membrane. The salient features of our membrane model are calcium-dependent calcium channels and calcium pumps in the ER membrane, constant entry of calcium into the cytosol, calcium dependent removal from the cytosol, and buffering by cytoplasmic calcium binding proteins. Numerical integration of the model allows us to study the fluctuations in the cytosolic calcium concentration, the ER membrane potential, and the concentration of free calcium binding sites on a calcium binding protein. The model demonstrates the physiological features necessary for calcium oscillations and suggests that the level of calcium flux into the cytosol controls the frequency and amplitude of oscillations. The model also suggests that the level of buffering affects the frequency and amplitude of the oscillations. The model is supported by experiments indirectly measuring cytosolic calcium by calcium-induced chloride currents in Xenopus oocytes as well as cytosolic calcium oscillations observed in other preparations.     

Model for receptor-controlled cytosolic calcium oscillations and for external influences on the signal pathway.

The external stimulation of many cells by a hormone, for example, often leads to an oscillating cytosolic calcium concentration. This periodic behavior is now designated the cytosolic calcium oscillator. A theoretical model is presented that describes this behavior on the basis of inositol(1,4,5)trisphosphate-induced calcium oscillations. In contrast to other models only a single positive feedback loop is taken into account to obtain oscillations. The model includes important innovations compared to other approaches. It includes the contribution of extracellular calcium and its modification after the stimulation of the cell. Furthermore, the signal pathway that leads to cytosolic calcium oscillations is described in more detail than in other models. This enables investigations on the influence of additional parameters like external electromagnetic fields on the signal transduction pathway. The model and the calculations are based on the theory of nonlinear self-sustained oscillators.     

Complex calcium oscillations and the role of mitochondria and cytosolic proteins

Intracellular calcium oscillations, which are oscillatory changes of cytosolic calcium concentration in response to agonist stimulation, are experimentally well observed in various living cells. Simple calcium oscillations represent the most common pattern and many mathematical models have been published to describe this type of oscillation. On the other hand, relatively few theoretical studies have been proposed to give an explanation of complex intracellular calcium oscillations, such as bursting and chaos. In this paper, we develop a new possible mechanism for complex calcium oscillations based on the interplay between three calcium stores in the cell: the endoplasmic reticulum (ER), mitochondria and cytosolic proteins. The majority ( approximately 80%) of calcium released from the ER is first very quickly sequestered by mitochondria. Afterwards, a much slower release of calcium from the mitochondria serves as the calcium supply for the intermediate calcium exchanges between the ER and the cytosolic proteins causing bursting calcium oscillations. Depending on the permeability of the ER channels and on the kinetic properties of calcium binding to the cytosolic proteins, different patterns of complex calcium oscillations appear. With our model, we are able to explain simple calcium oscillations, bursting and chaos. Chaos is also observed for calcium oscillations in the bursting mode.     

Hormone-induced calcium oscillations in liver cells can be explained by a simple one pool model

Hormone-induced oscillations of the free intracellular calcium concentration are thought to be relevant for frequency encoding of hormone signals. In liver cells, such Ca2+ oscillations occur in response to stimulation by hormones acting via phosphoinositide breakdown. This observation may be explained by cooperative, positive feedback of Ca2+ on its own release from one inositol 1,4,5-trisphosphate-sensitive pool, obviating oscillations of inositol 1,4,5-trisphosphate. The kinetic rate laws of the associated model have a mathematical structure reminiscent of the Brusselator, a hypothetical chemical model involving a rather improbable trimolecular reaction step, thus giving a realistic biological interpretation to this hallmark of dissipative structures. We propose that calmodulin is involved in mediating this cooperativity and positive feedback, as suggested by the presented experiments. For one, hormone-induced calcium oscillations can be inhibited by the (nonphenothiazine) calmodulin antagonists calmidazolium or CGS 9343 B. Alternatively, in cells overstimulated by hormone, as characterized by a non-oscillatory elevated Ca2+ concentration, these antagonists could again restore sustained calcium oscillations. The experimental observations, including modulation of the oscillations by extracellular calcium, were in qualitative agreement with the predictions of our mathematical model.     

Stimulus-dependent control of inositol 1,4,5-trisphosphate-induced Ca(2+) oscillation frequency by the endoplasmic reticulum Ca(2+)-ATPase

In many cell types, receptor stimulation evokes cytosolic calcium oscillations with a frequency that depends on agonist dose. Previous studies demonstrated controversial effects of changing the activity of the endoplasmic reticulum Ca(2+)-ATPase upon the frequency of oscillations. By numerical simulations, we found that the model of De Young and Keizer (J. Keizer and G.W. De Young, 1994, J. Theor. Biol. 166: 431-442), unlike other models, can explain the observed discrepancies, assuming that the different experiments were performed at different stimulus levels. According to model predictions, partial inhibition of internal calcium pumps is expected to increase frequency at low stimulus strength and should have an opposite effect at strong stimuli. Similar results were obtained using an analytical estimation of oscillation period, based on calcium-dependent channel activation and inactivation. In experiments on HeLa cells, 4 nM thapsigargin increased the frequency of calcium oscillations induced by 1 and 2.5 microM histamine but had no effect on supramaximally stimulated cells. In HEp-2 cells, 2 nM thapsigargin slowed down the rapid, ATP-induced oscillations. Our results suggest that in the investigated cell types, the De Young-Keizer model based on inositol 1,4,5-trisphosphate-dependent calcium-induced calcium release can properly describe intracellular calcium oscillations.     

A model of the dynamics of intracellular calcium based on refined kinetics of inhibiting the inositol-1,4,5-trisphoshate-sensitive calcium channel

Several new models of intracellular calcium dynamics based on refined inositol-1,4,5-triphosphate-sensitive calcium channel kinetics were studied. The refined kinetic schemes take into account that a cytosolic calcium cannot inhibit inositol-1,4,5-triphosphate receptors when they are bound to inositol-1,4,5-triphosphate. The mathematical analysis of intracellular calcium dynamics based on one of these schemes allowed us to show how different types of Ca response to extracellular stimuli, such as excitability, oscillations, sustained elevation of Ca and frequency encoding can arise with a reasonably good fit to experimental data.     

Real-time control of neutrophil metabolism by very weak ultra-low frequency pulsed magnetic fields

In adherent and motile neutrophils NAD(P)H concentration, flavoprotein redox potential, and production of reactive oxygen species and nitric oxide, are all periodic and exhibit defined phase relationships to an underlying metabolic oscillation of approximately 20 s. Utilizing fluorescence microscopy, we have shown in real-time, on the single cell level, that the system is sensitive to externally applied periodically pulsed weak magnetic fields matched in frequency to the metabolic oscillation. Depending upon the phase relationship of the magnetic pulses to the metabolic oscillation, the magnetic pulses serve to either increase the amplitude of the NAD(P)H and flavoprotein oscillations, and the rate of production of reactive oxygen species and nitric oxide or, alternatively, collapse the metabolic oscillations and curtail production of reactive oxygen species and nitric oxide. Significantly, we demonstrate that the cells do not directly respond to the magnetic fields, but instead are sensitive to the electric fields which the pulsed magnetic fields induce. These weak electric fields likely tap into an endogenous signaling pathway involving calcium channels in the plasma membrane. We estimate that the threshold which induced electric fields must attain to influence cell metabolism is of the order of 10(-4) V/m.     

Effects of inositol 1,4,5-trisphosphate receptor-mediated intracellular stochastic calcium oscillations on activation of glycogen phosphorylase

In various cell types cytosolic calcium (Ca(2+)) is an important regulator. The possible role of Ca(2+) release from the inositol 1,4,5-trisphosphate (IP(3)) receptor channel in the regulation of the phosphorylation-dephosphorylation cycle process involved in glycogen degradation by glycogen phosphorylase have theoretically investigated by using the Li-Rinzel model for cytosolic Ca(2+) oscillations. For the case of deterministic cytosolic Ca(2+) oscillations, there exists an optimal frequency of cytosolic Ca(2+) oscillations at which the average fraction of active glycogen phosphorylase reaches a maximum value, and a mutation for the average fraction of active glycogen phosphorylase occurs at the higher bifurcation point of Ca(2+) oscillations. For the case of stochastic cytosolic Ca(2+) oscillations, the fraction of active phosphorylase is strongly affected by the number of IP(3) receptor channels and the level of IP(3) concentration. Small number of IP(3) receptor channels can potentiate the sensitivity of the activity of glycogen phosphorylase. The average frequency and amplitude of active phosphorylase stochastic oscillations are increased with the level of increasing IP(3) stimuli. The various distributions for the amplitude of active glycogen phosphorylase oscillations in parameters plane are discussed.     

Low frequency electromagnetic fields and the Belousov-Zhabotinsky reaction

Low frequency magnetic fields can influence biochemical reactions and consequently physiological rhythms and oscillations. To test this for a model reaction we used the chemical Belousov-Zhabotinsky (BZ) reaction, which is one of the simplest chemical oscillators. The oscillation frequency of the reaction was tracked optically by the absorption of blue light. Field treatment was carried out at room temperature in the middle of two Helmholtz coils. After starting the reaction, for 5 min the oscillations were monitored as control measurement, then during the next 10 min monitoring was with a magnetic field switched on, followed by a period of 5 min with the field switched off. A variety of exposure conditions have been tested: the frequency was varied between 5 and 1000 Hz, the field strength was varied up to 2.7 mT, different pulse shapes were used, the influence of the exposure temperature was tested, and the influence of the optimum exposure conditions (static magnetic field and the frequency of the dynamic field) as predicted by the ion parametric resonance (IPR) model has been measured. In conclusion, in no case any statistical significant influence of the magnetic treatment on the oscillation frequency of the BZ reaction could be detected (P > .05, t-test).