IL—2对神经元NMDAR1m RNA表达和细胞内钙浓度的影响

Ｎ－甲基－Ｄ－天门冬氨酸（ＮＭＤＡ）受体和细胞内游离钙浓度（［Ｃａ２＋］ｉ）的变化与神经元的兴奋性关系密切。本文报道以大鼠大脑皮层神经元为研究对象，通过Ｎｏｒｔｈｅｒｎ印迹杂交并以Ｆｕｒａ－２作为Ｃａ２＋荧光指示剂，观察了外源性白细胞介素－２（ＩＬ－２）对ＮＭＤＡ受体１型亚单位（ＮＭＤＡＲ１）ｍＲＮＡ表达量及［Ｃａ２＋］ｉ的影响。结果显示：不同浓度的ＩＬ－２（１～２００Ｕ／ｍｌ）作用于原代培养的胚胎大鼠大脑皮层神经元６小时后其ＮＭＤＡＲ１ｍＲＮＡ表达量明显增加，并与ＩＬ－２浓度呈正相关关系。当其浓度为２５～２００Ｕ／ｍｌ时，ＮＭＤＡＲ１ｍＲＮＡ表达量增加４４．３％～２１９．７％（Ｐ＜０．０５）。ＩＬ－２（１～２００Ｕ／ｍｌ）与新生大鼠大脑皮层神经元共同孵育５～１０分钟后，［Ｃａ２＋］ｉ增加２７．１％～９４．２％（Ｐ＜０．０５）。钙通道阻断剂ｎｉｆｅｄｉｐｉｎｅ可完全阻断ＩＬ－２引起的［Ｃａ２＋］ｉ升高，而ＮＭＤＡ受体拮抗剂ＭＫ－８０１无此作用。本文结果提示：外源性ＩＬ－２具有兴奋性神经调质样作用，可能参与或介导了神经兴奋毒性作用和惊厥性疾病的发生与发展过程。     

蛋白激酶C抑制剂对去甲肾上腺素促进大鼠巨噬细胞I_a抗原表达的影响

我室以前的工作证明，在去甲肾上腺素（ＮＥ）促进大鼠巨噬细胞（ＭΦ）Ｉａ抗原表达的效应中，细胞内的第二信使物质三磷酸肌醇（ＩＰ－３）和Ｃａ￣２＋起着必不可少的作用。然而，另一第二信使物质甘油二酯（ＤＧ）的作用如何，尚待阐明。为此，我们用４α－佛　波醇－１２，１３－二癸酸盐（４α－ｐｈｏｒｂｏｌ－１２，１３－ｄｉｄｅｃａｎｏａｔｅ，４α－ＰＤＤ）抑制ＭΦ内的蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ，ＰＫＣ）以阻断ＤＧ的作用，结果使ＮＥ促进ＭΦＩａ抗原表达的效应显著减弱（Ｐ＜０．０１，Ｐ＜０．０１），说明在ＮＥ促进ＭΦＩａ抗原表达的效应中，ＩＰ－３、Ｃａ￣２＋和ＤＧ都起重要的作用。     

NMDA—Ca^2＋—NO路径与中枢神经系统

ＮＭＤＡ－Ｃａ２＋－ＮＯ路径是指存在于神经元内或神经元间由谷氨酸或Ｎ－甲基－Ｄ－天冬氨酸激活ＮＭＤＡ受体始发的胞内Ｃａ２＋增高、生成增加等一系列生理或病理变化途径，该路径存在于中枢神经系统，维持神经元的正常兴奋性，参与海马长时程增强的产生、维持及突触可塑性。在某些神经性疾病中起一定作用     

氯胺酮对缺氧小鼠存活时间的影响

氯胺酮对缺氧小鼠存活时间的影响湖北襄樊市中心医院麻醉科（襄樊４４１０２１）李明强，龚正声现已证明中枢神经系统内的兴奋性氨基酸递质和Ｎ－甲基－Ｄ－门冬氨酸盐（ＮＭＤＡ）受体在脑缺氧损伤中起重要作用，而ＮＭＤＡ受体拮抗剂可显著减轻脑的缺氧性损伤。氯胺酮（...     

粘附分子对CD3介导的胸腺细胞[Ca^2＋]i应答的影响

ＬＦＡ－１和ＩＣＡＭ－１广泛表达于各胸腺细胞亚群，但ＩＣＡＭ－１在ＰＮＡ￣＋细胞的表达下调。本文报道：用抗ＬＦＡ－１／ＩＣＡＭ－１和抗ＣＤ３单抗，分析了粘附分子ＬＦＡ－１／ＩＣＡＭ－１对抗ＣＤ３诱导的胸腺细胞［Ｃａ￣（２＋）］ｉ应答的影响。结果显示，可溶性抗ＬＦＡ－１／ＩＣＡＭ－１可抑制ＣｏｎＡ刺激的胸腺细胞增殖，且以抗ＬＦＡ－１抗体的作用更为显著，在ＣｏｎＡ或抗ＣＤ３诱导的胸腺细胞［Ｃａ￣（２＋）］ｉ应答中，抗ＬＦＡ－１单抗可明显抑制［Ｃａ￣（２＋）ｉ升高。但如果用二抗交联ＣＤ３和ＬＦＡ－１，胸腺细胞［Ｃａ￣（２＋）ｉ则显著高于单独交联ＣＤ３时的水平（Ｐ＜０．０１），而ＣＤ３与ＩＣＡＭ－ｌ交联却无此效应，此外，仅交联ＬＦＡ－１或ＩＣＡＭ－１也无诱导［Ｃａ￣（２＋）］ｉ应答的作用。提示在ＬＦＡ－ｌ与ＩＣＡＭ－１介导的胸腺细胞与胸腺基质细胞相互作用中，ＬＦＡ－１可为ＴＣＲ／ＣＤ３途径介导的胸腺细胞活化提供复合刺激信号。     

氯胺酮对脑缺血诱导Ca^2＋／CaM—PKⅡ活性抑制作用的影响

本文观察了ＮＭＤＡ受体非竞争性拮抗剂氯胺酮对沙土鼠脑缺血和再灌注时大脑皮质可溶性和颗粒性Ｃａ￣（２＋）／ＣａＭ－ＰＫⅡ活性变化的影响。实验结果如下：（１）随着脑缺血时间的增加，两部分酶活性均逐渐降低，而可溶性酶活性比颗粒性酶活性下降得更快。（２）脑缺血前腹腔注射氯胺酮可部分地拮抗脑缺血诱导该酶活性的下降，提示脑缺血时引起该酶活性的抑制作用是部分通过ＮＭＤＡ受体介导的，可看作脑缺血ＮＭＤＡ受体后兴奋毒事件。（３）随着脑缺血再灌注时间的延长两部分酶活性均可逐渐得到恢复，而氯胺酮不能加速再灌注过程中该酮活性恢复的速度。     

MTEC 1分泌的趋化因子引起特定亚群胸腺细胞的定向迁移

分析胸腺髓质上皮样细胞系ＭＴＥＣ１分泌的化学趋化因子对胸腺细胞亚群的趋化作用。方法以抗体加补体杀伤结合免疫磁珠及ｐａｎｎｉｎｇ法，将小鼠胸腺细胞分离纯化，获得ＣＤ４＋ＣＤ８＋（ＤＰ），ＣＤ４－ＣＤ８－（ＤＮ），ＣＤ４＋ＣＤ８－（ＣＤ４ＳＰ）及ＣＤ４－ＣＤ８＋（ＣＤ８ＳＰ）四亚群细胞，用Ｂｏｙｄｅｎ小室分析ＭＴＥＣ１┐ＳＮ对四群胸腺细胞的趋化作用。结果ＭＴＥＣ１┐ＳＮ对ＤＰ及ＣＤ４ＳＰ胸腺细胞有趋化活性（ＣＩ＝６．６±１．０及６．１±１．８）；对ＣＤ８ＳＰ细胞有中度趋化活性（ＣＩ＝３．２±１．０）；对ＤＮ趋化活性微弱（ＣＩ＝１．３±０．６）。化学趋化因子ＭＣＰ┐１纯品对ＣＤ４ＳＰ胸腺细胞显示强趋化活性（ＣＩ＝５．６），对ＤＮ胸腺细胞则无可测出趋化活性。结论ＭＴＥＣ１分泌的化学趋化因子对ＤＰ，ＣＤ４ＳＰ及ＣＤ８ＳＰ胸腺细胞有显著趋化作用，对ＤＮ胸腺细胞几乎无趋化作用。提示此类化学趋化因子有趋使胸腺发育中后期阶段的细胞向胸腺髓质区迁移和定位的作用。     

蛋白激酶C抑制剂对去甲肾上腺素促进大鼠巨噬细胞Ia抗原…

我室以前的工作证明，在去甲肾上腺素（ＮＥ）促进大鼠巨噬细胞（Ｍφ）Ｉａ抗原表达的效尖中，细胞内的第二信使物质三磷酸肌醇（ＩＰ２）和Ｃａ＾２＋起着必不可少的作用。然而，另一第二信使物质甘油二酯（ＤＧ）的作用如何，尚待阐明。为此，我们用４－α佛波醇－１２，１３－二癸酸盐（４α－ｐｈｏｒｂｏｌ－１２，１３－ｄｉｄｅｃａｎｏａｔｅ，４α－ＰＤＤ）抑制Ｍφ内的蛋白激酶Ｃ（ｐｒｏｔｅｉｎ ｋｉｎａｓｅ Ｃ，ｐ     

"缺血"对大鼠大脑皮层神经元NMDA受体通道的影响

目的：研究“缺血”大鼠大脑大皮层神经元Ｎ－甲基－Ｄ－天冬氨酸（ＮＭＤＡ）受体通道的单通道变化特征。方法：细胞贴附式膜片钳技术。结果：在“缺血”状态下，ＮＭＤＡ受体通道３５ｐＳ和１００ｐＳ电导水平的开放概率分别由对照组的０．０７９±０．００６和０．０６７±０．００４增加到０．３０８±０．１５５和０．４８８±０．１２６（Ｐ＜０．０１），３５ｐＳ通道开放时间常数τ２由对照值（４．１７±０．３８）ｍｓ增加到（８．５４±２．０５）ｍｓ（Ｐ＜０．０１），关闭时间由（７５．５０±１４．１０）ｍｓ缩短到（１１．８０±４．３０）ｍｓ（Ｐ＜０．０１）。结论：“缺血”可显著开放大鼠大脑皮层神经元ＮＭＤＡ受体通道，使钙内流增加，对胞内钙超载起重要作用，此可能是脑缺血细胞损伤的机制之一。     

牛磺酸对缺血大鼠心肌线粒体Ca2+-Mg2+-ATP酶活性与MDA含量影响

目的和方法：用Ｗｉｓｔａｒ大鼠皮下注射异丙肾上腺素（ＩＳＰ,５ｍｇ／ｋｇ）诱导心肌缺血模型。观测心肌线粒体（Ｍｉｔ）中丙二醛（ＭＤＡ）含量、Ｃａ２＋－Ｍｇ２＋－ＡＴＰ酶和Ｃａ２＋－ＡＴＰ酶活性及牛磺酸（Ｔａｕ）的影响。结果：缺血组大鼠心肌Ｍｉｔ中ＭＤＡ升高８７８３％、Ｃａ２＋－Ｍｇ２＋－ＡＴＰ酶和Ｃａ２＋－ＡＴＰ酶活性分别降低３７５６％和５０２０％（Ｐ＜０．０１）。Ｃａ２＋－Ｍｇ２＋－ＡＴＰ酶活性与ＭＤＡ含量也呈显著负相关（ｒ＝－０．８７,Ｐ＜０．０１）。Ｃａ２＋－ＡＴＰ酶活性与ＭＤＡ含量也呈显著负相关（ｒ＝－０７９,Ｐ＜０．０１）。在注射异丙肾上腺素（ＩＳＰ）前３０ｍｉｎ腹腔注射Ｔａｕ（２００ｍｇ／ｋｇ）则Ｍｉｔ中ＭＤＡ含量、Ｃａ２＋－Ｍｇ２＋－ＡＴＰ酶和Ｃａ２＋－ＡＴＰ酶活性均未见显著异常改变。结论：Ｔａｕ可能通过抑制ＭＤＡ的生成实现其保护Ｃａ２＋－ＡＴＰ酶和Ｃａ２＋－Ｍｇ２＋－ＡＴＰ酶活性的作用。     

Relationship between global cytosolic Ca2+ concentration and Ca2+-activated K+ current in rabbit cerebral arterial myocyte.

In smooth muscle cells, the sarcoplasmic reticulum (SR) has been identified as the primary storage site for intracellular Ca2+. The peripheral SR is in close proximity with plasma membrane to make a narrow subsarcolemmal space. In this study, we investigated the regulation of subsarcolemmal [Ca2+] ([Ca2+]sl) and global cytosolic [Ca2+] ([Ca2+]c) of rabbit arterial smooth muscle using whole cell patch clamp technique and microspectrofluorimetry. The Ca2+-activated K+ current (IK(Ca)) and the ratio of fura-2 fluorescence (R340/380) were considered to reflect the [Ca2+]sl and [Ca2+]c, respectively. At a holding potential of 0 mV, extracellular application of 10 mM caffeine, a well known Ca2+-releasing agent, induced transient increase of IK(Ca) and R340/380 (IK(Ca)-transient and R340/380-transient, respectively). The increase and decay of IK(Ca) transient was faster than R340/380-transient. By repetitive application of caffeine, when the refilling state of SR was supposed to be lower than the control condition, IK(Ca)-transient and R340/380 transient were suppressed to different levels; e.g. the second application 20 sec after the first could induce smaller IK(Ca) transient than R340/380-transient. Dissociation of IK(Ca)-transient and R340/380-transient was removed by sufficient (>3 min) washout of caffeine. Recovery from the dissociation was also dependent upon the membrane potential; faster recovery was observed at negative (-40 mV) holding potential than at depolarized (0 mV) condition. Dissociation of IK(Ca) from [Ca2+]c was also partially prevented by perfusion with Na+-free (replaced by NMDG+) extracellular solution. These results suggest that, 1) there is prominent spatial inhomogeneity of [Ca2+] in cerebral arterial myocyte, 2) [Ca2+]Sl is preferentially affected by the interference from nearby plasmalemmal Ca2+ regulation mechanism which is partly dependent upon extracellular Na+.     

Asphyxia and diuretic-induced changes in the Ca2+ concentration of endolymph

Using Ca2+ -selective microelectrodes based on the neutral carrier, ETH-1001 with polyvinyl chloride (PVC), we have measured changes in the free Ca2+ concentration of guinea pig cochlear endolymph ([Ca](e)) after transient asphyxia or intravenous administration of diuretics. Under the control conditions, the endocochlear potential (EP) was +80 mV, and the [Ca](e) was in the range 1.4 x 10(-7)-2.4 x 10(-6) M (n = 16). Transient asphyxia (1-1.5 min) produced an increase in the [Ca](e) with a fall in the EP, whereas the cessation of the asphyxia led to a quick recovery of both [Ca](e) and EP to their control levels. Intravenous administration of furosemide (60 mg/kg) or bumetanide (30 mg/kg) also caused an increase in the [Ca](e) with a fall in the EP, followed by a gradual recovery of both [Ca](e) and EP. From these results, we obtained a significant correlation between EP and p[Ca](e) (= -log[Ca](e)), and conclude that (1) the [Ca](e) is extremely low, around 10(-6) M or less, under normal conditions and (2) the [Ca](e) is directly correlated with EP under physiological conditions.     

The regulation of cytosolic Ca2+ concentration in a mammalian cell

The assumptions governing the regulation of free Ca2+ in a mammalian cell are presented. There is a possibility that mitochondria, microsomes and plasma membrane play a major role, by regulation of the very low concentration of free Ca2+ in cytosol, as the signal to biochemical activities. The possibility of the loss of very high-affinity Ca2(+)-binding and the number of its sites by preparation has not been taken into consideration in previous hypotheses.     

Pharmacological and electrographic properties of epileptiform activity induced by elevated K2+ and lowered Ca2+ and Mg2+ concentration in rat hippocampal slices

We studied some of the physiological and pharmacological properties of an in vitro model of epileptic seizures induced by elevation of [K⁺]₀ (to 8 mM and 10 mM) in combination with lowering of [Mg²⁺]₀ (to 1.4 mM and 1.6 mM) and [Ca²⁺]₀ (to 0.7 mM and 1 mM) in rat hippocampal slices. These concentrations correspond to the ionic constitution of the extracellular microenvironment during seizures in vivo. The resulting activity was rather variable in appearance. In area CA3 recurrent discharges were observed which resulted in seizure-like events with either clonic-like or tonic-cloniclike ictaform events in area CA1. With ion-sensitive electrodes, we measured the field potential and the changes in extracellular ion concentrations which accompany this activity. The recurrent discharges in area CA3 were accompanied by small fluctuations in [K⁺]₀ and [Ca²⁺]₀. The grouped clonic-like discharges in area CA1 were associated with moderate increases in [K⁺]₀ and small decreases in [Ca²⁺]₀ in the order of 2 mM and 0.2 mM, respectively. Large, negative field-potential shifts and increases in [K⁺]₀ to 13 mM, as well as decreases in [Ca²⁺]₀ by up to 0.4 mM, accompanied the tonic phase of ictaform events. The ictaform events were not blocked by D-2-aminophosphonovalerate (2-APV) but were sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) alone and in combination with 2-APV and ketamine. In order to determine the pharmacological characteristics of the ictaform events we bath-applied most clinically employed anticonvulsants (carbamazepine, phenytoin, valproate, phenobarbital, ethosuximide, trimethadione) and some experimental anticonvulsants (losigamone, vinpocetine, and apovincaminic acid). Carbamazepine, phenytoin, valproate, and phenobarbital were effective at clinically relevant doses. The data suggest that the high-K⁺ model of epileptiform activity is a good model of focal convulsant activity.     

Measurement of intracellular Ca2+ concentration using Indo-1 during simultaneous flash photolysis to release Ca2+ from DM-nitrophen

We have constructed a modular instrument to measure intracellular [Ca²⁺] ([Ca²⁺]_i) in single isolated cells while simultaneously imposing step changes in [Ca²⁺]_i using caged Ca²⁺. By combining the outputs of a xenon arc lamp with a frequency-tripled (Nd: YAG) laser, the instrument can operate with low maintained illumination to measure [Ca²⁺]_i using a ratiometric Ca²⁺-sensitive fluorophore and also activate the release of Ca²⁺ from a caged-Ca²⁺ compound with a high energy pulse of ultraviolet light. This instrument is simple to assemble, introduces little electrical noise, provides a wide range of illumination power, produces only moderate photobleaching of the Ca²⁺ indicator and can be readily adapted to diverse cellular preparations. We demonstrate the use of this system to measure step changes in [Ca²⁺]_i in adult rat ventricular myocytes and a human embryonic kidney cell line (293 cells) in culture.     

Modal gating transitions in cardiac ryanodine receptors during increases of Ca2+ concentration produced by photolysis of caged Ca2+

Channel adaptation is a basic property of the sarcoplasmic reticulum Ca²⁺-release channels/ryanodine receptors (RyRs). It allows channel activity to decay during sustained increases in the concentration of activating Ca²⁺. Despite the potential physiological importance of this self-confining process, its molecular mechanism is not well understood. To define the mechanism of adaptation we studied the dynamics of cardiac Ca²⁺-release channel (RyR) gating using the planar lipid bilayer technique in combination with photolysis of caged Ca²⁺ (DM-nitrophen). Channels activated by rapid and sustained increases in Ca²⁺ concentration (from 0.1 to 0.5 μmol/l) displayed three distinct gating modes, manifested as current records with frequent and long openings (H-mode), with rare and short openings (L-mode), and with no openings (I-mode). H-mode channel activity occurred primarily at early times while L- and I-modes predominated at late times after the rapid Ca²⁺ concentration increase. The decrease in probability of H-mode, mirrored by an increase in the probability of the I-mode, proceeded with a time constant similar to that observed for spontaneous decay in channel activity (i.e., adaptation) in ensemble average records. These results indicate that RyR adaptation transpires by a shift of channel gating from a high open probability mode to low open probability and inactivated modes of the channel. Received: 1 March 1999 / Received after revision: 16 April 1999 / Accepted: 19 April 1999     

The effect of extracellular Ca2+ concentration on the negative staircase of Ca2+ transient in field-stimulated rat ventricular cells

We performed experiments using the Ca²⁺ indicator dye, fura-2 to investigate the effect of extracellular Ca²⁺ concentration ([Ca²⁺]_o) on sarcoplasmic reticulum (SR) Ca²⁺ release and loading in single rat ventricular cells. In normal Tyrode solution (1.8 mM [Ca²⁺]_o) repetitive stimulation (0.5 Hz) resulted in a gradual decrease in calcium transients (the negative staircase phenomenon) without being accompanied by a gradual decrease in diastolic intracellular Ca²⁺ concentration. The rate of the slow decline in calcium transient was faster in lower [Ca²⁺]_o. However, the peak of the first calcium transient was relatively invariant over a wide range of [Ca²⁺]_o (0.5–5 mM). The size of the calcium transient elicited by field stimulation was proportional to that induced by 10 mM caffeine, applied following the field stimulation. These results suggest that the size of calcium transients depends mainly on the Ca²⁺ content of the SR. The quiescent period favoured the replenishment of the SR and this effect was promoted further by increasing the driving force for Ca²⁺ entry across the sarcolemma during this period. We conclude that in low [Ca²⁺]_o, short stimulation interval may limit Ca²⁺ influx across the sarcolemma during the quiescent period to cause a gradual reduction in calcium content of the SR and thus the calcium transient.     

Na+ and K+ concentration of rat parotid saliva

The effects of carbachol and auriculo-temporal stimulation on the Na⁺ and K⁺ concentrations of rat parotid saliva have been compared. The main duct perfused in situ, does not transport Na⁺ or K⁺ and is water impermeable. The Na⁺ concentration of secretion evoked by either stimulus is flow dependent, increasing with increasing flow rate and plateauing at near plasma Na⁺ levels. At low flow rate the carbachol evoked secretion has a higher Na⁺ concentration. This is not due to the release of catecholamines since neither sympathectomy nor adrenoceptor block altered the nature of the secretion. The K⁺ concentration, whilst flow dependent, decreasing with increasing flow rate, was the same for both stimuli.     

Activation and inactivation kinetics of a Ca2+-activated Cl- current: photolytic Ca2+ concentration and voltage jump experiments

The activation kinetics of the endogenous Ca²⁺-activated Cl⁻ current (I _Cl,Ca) from Xenopus oocytes was investigated in excised “giant” membrane patches with voltage and Ca²⁺ concentration jumps performed by the photolytic cleavage of the chelator DM-nitrophen. Currents generated by photolytic Ca²⁺ concentration jumps begin with a lag phase followed by an exponential rising phase. Both phases show little voltage dependence but are Ca²⁺-dependent. The lag phase decreases from about 10 ms after a small Ca²⁺ concentration jump (0.1 μM) to less than 1 ms after a saturating concentration jump (55 μM). The rate constant of the rising phase is half-maximal at about 5 μM. At saturating Ca²⁺ concentrations, the rate constant is 400 to 500 s⁻¹. The Ca²⁺ dependence of the stationary current can be described by the Hill equation with n=2.3 and K _0.5=0.5 μM. The amplitude of the stationary current decreases after the excision of the membrane patch with t _1/2≈5 min (run-down). The activation kinetics of the current elicited by a Ca²⁺ concentration jump is not affected by the run-down phenomenon. At low Ca²⁺ concentration (0.3 μM), voltage jumps induce a slowly activating current with voltage-independent time-course. Activation is preceded by an initial transient of about 1-ms duration. At saturating Ca²⁺ levels (1 mM), the initial transient decays to a stationary current. The transient can be explained by a voltage-dependent inactivation process. The experimental data reported here can be described by a linear five-state reaction model with two sequential voltage-dependent Ca²⁺-binding steps, followed by a voltage-independent rate-limiting transition to the open and a voltage-dependent transition to a closed, inactivated state.