AMPA对视网膜L型水平细胞所接收的红、绿敏视锥信号的不同调制作用

在离体灌流的鲫鱼视网膜,应用细胞内记录技术,研究了谷氨酸受体激动剂——AMPA对L型水平细胞(LHC)的作用.AMPA压抑LHC由红敏视锥驱动的反应,却增强由绿敏视锥驱动的反应.此效应可为AMPA受体特异的拮抗剂(GYKI53655)所逆转,表明仅有AMPA受体参与其中.印防己毒素(PTX)或双氢红藻氨酸(DHK)存在时,依然可以观察到这种效应,提示自LHC向视锥的负反馈和视锥谷氨酸转运体在此效应中不起显著作用.AMPA的不同调制作用提示,LHC上可能存在不同特性的AMPA受体亚型介导红敏和绿敏视锥的信号传递.     

学习和记忆相关基因:Calsyntenins家族

Calsyntenins（Cstn）是一个独特的将胞外蛋白水解活性与胞内Ca^2＋信号转导相连在一起的家族,属于钙结合蛋白,与钙离子结合,参与信号转导和细胞交流。它包括3个成员,分别为calsyntenin-1、calsyntenin-2和calsyntenin-3,皆为突触后膜蛋白,主要在脑的神经元中表达,但表达模式各自不同,而且其蛋白也表现出高度的结构多样性。Calsyntenin-1位于中枢神经系统（CNS）突触后膜,是一个突触后膜蛋白水解的蛋白质。有一个结合钙的胞质酸性结构域,是一个通过胞外蛋白水解来调节突触后钙的动力调节子。Calsyntenin-1调节突触后膜下或胞内Ca^2＋储存库中的Ca^2＋瞬变,从而参与长时程增强（LTP）和长时程抑制（LTD）,与学习和记忆功能紧密相关。尤其是最近研究发现,β淀粉样蛋白前体（APP）和calsyntenins共同作用增加了β淀粉样蛋白（Aβ）的分泌,从而造成神经系统紊乱,促进阿尔采末病（AD）的发生,这对于AD发病机制的揭示和开发新一代治疗AD的药物具有重要的意义。     

INTERACTION OF CONE SIGNALS ON L-TYPE HORIZONTAL CELL IN CARP RETINA: EXPERIMENTS AND MODELING

本文应用胞内记录和动态模型分析方法, 研究了离体鲫鱼视网膜视锥驱动的亮度型水平细胞 (LHC) 上不同视锥信号的相互作用。 实验表明, 绿背景光的作用可以提高LHC的红光反应, 这种增强作用与绿敏视锥的活动程度密切相关。 模型分析表明, 绿背景光的作用使谷氨酸介导的前馈性通路和GABA介导的反馈性通路活动同时得以增强。 水平细胞对光反应的增强效应不能为外源性GABA所消除, 而其程度为前馈性通路和反馈性通路活动增加的相对程度所决定。     

钙依赖性突触的可塑性

突触前和突触后细胞内钙离子（[Ca^2 ]i)在短时程和长时程突触的可塑性中,发挥着重要的住处传递作用。兴奋后残留[Ca^2 ]i,可以激发短时程突触增强。突触前[Ca^2 ]i可以影响被抑制的突触前膜囊泡的更新,并准确编码突前和突触后信息,产生截然相反的长时程突触修（ＬＴＰ或ＬＴＤ）。     

钙-钙调蛋白依赖性蛋白激酶II向突触转运的动物模型和在体实验方法

钙 钙调蛋白依赖性蛋白激酶II(calcium /calmodulindependentproteinkinaseII ,CaMKII)在兴奋性突触长时程增强 (longtermpotentiation ,LTP)和其他形式的突触可塑性等生理现象中起重要作用。用观察神经元胞体内某种分子参与突触后致密物 (postsynapticdensities ,PSD)的组成可以判断此分子是否参与LTP等突触可塑性过程。体外实验发现 ,用绿色荧光蛋白 (greenfluorescentprotein ,GFP)标记的CaMKII分子可在神经元受到谷氨酸或者直接电刺激后形成突触后膜密度簇 (PDSclusters)。最近 ,纽约州大学学者MichelleR .Gleason等进一…     

STIM／SOCE通路参与细胞功能调控的研究进展

钙库操作性钙离子通道（store-operated calcium entry,SOCE）是介导胞外Ca^2＋进入细胞内的重要通道之一,其核心蛋白由位于内质网上的基质相互作用分子（stromalinteractionmolecule,STIM）和位于细胞膜上的Orai蛋白构成。目前研究发现,STIM蛋白存在STIM1和STIM2两种亚型,其主要功能略有不同。当内质网内钙库中Ca^2＋消耗之后,STIM蛋白通过其特殊的结构能够感受内质网内钙库中Ca^2＋浓度的变化,发生快速的转位和聚合化等激活反应,与质膜上的Orai蛋白偶联。实现SOCE通路的功能开放,引起Ca^2＋内流。当钙库中Ca^2＋得到补充之后,STIM蛋白与Orai蛋白缓慢解离即失活,通路关闭。目前对STIM蛋白结构的研究提示,通过其激活和失活机制不仅能够参与调节SOCE通路的开放与关闭,也参与对细胞内重要的细胞增殖、分化等功能活动调控。STIM蛋白可能成为治疗多种疾病的潜在的新靶点。     

突触后细胞电活动促进突触发育的条件

突触在发育过程中体现出极大的可塑性,突触的简单形式：神经肌肉接点（Ｎｅｕｒｏｍｕｓｃｕｌａｒｊｕｎｃｔｉｏｎ,ＮＭＪ）在发育中的自发电活动的规率性变化常用来代征其突触塑造发育的程度．利用膜片钳技术对爪蟾胚胎ＮＭＪ突触后肌肉细胞进行全细胞电流钳制,并施予高频阈上电刺激可诱发ＮＭＪ处自发电活动的增强。深入的研究表明这种增强来自反复的去极化过程而不是单纯的膜电位升高;这种增强只有在施予电刺激的数目,频率,幅度到达一定程度才能诱发;Ｃｄ２＋胞外灌流实验提示Ｃａ２＋在肌细胞内的激增与这种增强有关。     

兴奋-收缩偶联中DHPR与RyR的相互作用

兴奋-收缩偶联（E—C coupling）依赖纽胞膜二氢吡啶受体（DHPR）／L型电压门控Ca^2＋通道和肌浆网兰诺定受体（RyR）／Ca^2＋释放通道的相互作用。在骨骼肌细胞中,DHPR与RyRl在结构上二机械偶联,不依赖细胞外Ca^2＋即可激活RyRl;在心肌细胞中,去极化激活DHPR,细胞外Ca^2＋内流,内流的Ca^2＋通过钙诱导钙释放（CICR）机制激活RyR2。最近的研究表明,DHPR与RyR之间的信号转导通常是双向的。DHPR与RyR机械和化学的双向偶联机制调节这两种Ca^2＋通道的效率、精确度和活性。     

成年小鼠前脑NMDA受体参与神经元的动作电位发放

谷氨酸是中枢神经系统主要的快速兴奋性递质。AMPA受体和海人藻酸受体主要参与突触传递,而NMDA受体主要参与突触可塑性。基因操作的方法增强NMDA受体的功能,可以增强动物在正常生理状态下的学习能力,及在组织损伤情况下的反应敏感性。NMDA受体参与生理功能的主要机制是长时程增强（long—term potentiation,LTP）。我们的研究表明,NMDA受体不仅参与刺激前扣带皮层的第五层细胞或刺激白质诱导的突触反应,而且参与在胞体施加去极化跃阶电流诱导的动作电位的发放。钙一钙调蛋白敏感的腺苷酸环化酶1（adenylyl cyclase 1,AC1）和cAMP信号通路可能介导了这些反应。由于扣带皮层神经元在伤害性刺激和痛中发挥重要作用,我们的结果为前脑NMDA受体参与突触传递和动作电位发放,以及与前脑相关的行为,如感受伤害性刺激和痛,提供了一个新的机制。     

CaMKⅡ介导的谷氨酸受体磷酸化(英文)

钙/钙调蛋白依赖的蛋白激酶Ⅱ(Ca2+/calmodulin-dependent protein kinase Ⅱ,CaMKⅡ)在脑内兴奋性突触部位丰富表达。通过催化谷氨酸受体和众多突触蛋白磷酸化,CaMKⅡ调节磷酸化蛋白在基础或细胞兴奋时的转运、分布和功能。谷氨酸NMDA受体是CaMKⅡ的直接底物,有证据表明CaMKⅡ直接与NMDA受体胞内C末端相互结合,催化一特定丝氨酸(S1303)的磷酸化。CaMKⅡ也加强谷氨酸AMPA受体的磷酸化,通过磷酸化AMPA受体C末端特定的丝氨酸(S831),CaMKⅡ增强AMPA受体的功能。此外,CaMKⅡ可与代谢型谷氨酸受体mGluR1亚型的胞内C末端结合,促进一特定苏氨酸(T871)的磷酸化,从而促进受体兴奋后脱敏。CaMKⅡ在正常状态下与mGluR5受体结合以储存于突触内,刺激mGluR5受体时,CaMKⅡ与mGluR5受体分离,转运至NMDA受体,以介导mGluR5信号对NMDA受体的增强作用。总之,CaMKⅡ与谷氨酸受体相互作用,改变受体磷酸化水平,参与受体的数量和功能以及突触传导活动的调节。     

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.     

Ca(2+)-induced Ca(2+) release in sensory neurons: low gain amplification confers intrinsic stability

Ca(2+)-induced Ca(2+) release (CICR) is a ubiquitous mechanism by which Ca(2+) release from the endoplasmic reticulum amplifies the trigger Ca(2+) entry and generates propagating Ca(2+) waves. To elucidate the mechanisms that control this positive feedback, we investigated the spatial and temporal kinetics and measured the gain function of CICR in small sensory neurons from mammalian dorsal root ganglions (DRGs). We found that subsurface Ca(2+) release units (CRUs) are under tight local control by Ca(2+) entry, whereas medullar CRUs as a "common pool" system are recruited by inwardly propagating CICR. Active CRUs often displayed repetitive Ca(2+) sparks, conferring the ability to encode a "memory" of neuronal activity well beyond the duration of an action potential. Store Ca(2+) reserve was able to support all CRUs each to fire approximately 15 sparks, excluding use-dependent inactivation or store depletion as the major CICR termination mechanism. Importantly, CICR in DRG neurons operated in a low gain, linear regime (gain = 0.54), which conferred intrinsic stability to CICR. Combined with high Ca(2+) current density (-156 pA/pF at -10 mV), such a low gain CICR system generated large intracellular Ca(2+) transients without jeopardizing the stability. These findings provide the first demonstration that CICR operating in a low gain regime can be harnessed to provide a robust and graded amplification of Ca(2+) signal in the absence of counteracting inhibitory mechanism.     

Ca(2+)-induced Ca2+ release amplifies the Ca2+ response elicited by inositol trisphosphate in macrophages.

We have studied the rise in intracellular calcium concentration ([Ca2+]i) elicited in macrophages stimulated by platelet-activating factor (PAF) by using fura-2 measurements in individual cells. The [Ca2+]i increase begins with a massive and rapid release of Ca2+ from intracellular stores. We have examined the mechanism of this Ca2+ release, which has been generally assumed to be triggered by inositol trisphosphate (IP3). First, we confirmed that IP3 plays an important role in the initiation of the PAF-induced [Ca2+]i rise. The arguments are 1) an increase in IP3 concentration is observed after PAF stimulation; 2) injection of IP3 mimics the response to PAF; and 3) after introduction of heparin in the cell with a patch-clamp electrode, the PAF response is abolished. Second, we investigated the possibility of an involvement of Ca(2+)-induced Ca2+ release (CICR) in the development of the Ca2+ response. Ionomycin was found to elicit a massive Ca2+ response that was inhibited by ruthenium red or octanol and potentiated by caffeine. The PAF response was also inhibited by ruthenium red or octanol and potentiated by caffeine, suggesting that CICR plays a physiological role in these cells. Because our results indicate that in this preparation IP3 production is not sensitive to [Ca2+]i, CICR appears as a primary mechanism of positive feedback in the Ca2+ response. Taken together, the results suggest that the response to PAF involves an IP3-induced [Ca2+]i rise followed by CICR.     

Functional coupling of Ca(2+) channels to ryanodine receptors at presynaptic terminals. Amplification of exocytosis and plasticity

Ca(2+)-induced Ca(2+) release (CICR) enhances a variety of cellular Ca(2+) signaling and functions. How CICR affects impulse-evoked transmitter release is unknown. At frog motor nerve terminals, repetitive Ca(2+) entries slowly prime and subsequently activate the mechanism of CICR via ryanodine receptors and asynchronous exocytosis of transmitters. Further Ca(2+) entry inactivates the CICR mechanism and the absence of Ca(2+) entry for >1 min results in its slow depriming. We now report here that the activation of this unique CICR markedly enhances impulse-evoked exocytosis of transmitter. The conditioning nerve stimulation (10-20 Hz, 2-10 min) that primes the CICR mechanism produced the marked enhancement of the amplitude and quantal content of end-plate potentials (EPPs) that decayed double exponentially with time constants of 1.85 and 10 min. The enhancement was blocked by inhibitors of ryanodine receptors and was accompanied by a slight prolongation of the peak times of EPP and the end-plate currents estimated from deconvolution of EPP. The conditioning nerve stimulation also enhanced single impulse- and tetanus-induced rises in intracellular Ca(2+) in the terminals with little change in time course. There was no change in the rate of growth of the amplitudes of EPPs in a short train after the conditioning stimulation. On the other hand, the augmentation and potentiation of EPP were enhanced, and then decreased in parallel with changes in intraterminal Ca(2+) during repetition of tetani. The results suggest that ryanodine receptors exist close to voltage-gated Ca(2+) channels in the presynaptic terminals and amplify the impulse-evoked exocytosis and its plasticity via CICR after Ca(2+)-dependent priming.     

The variety of cytosolic calcium responses and possible roles of PLC and PKC

A model of ligand-induced intracellular calcium (Ca2+) responses incorporating phospholipase C (PLC) and protein kinase C (PKC) is developed for the purpose of understanding the mechanisms underlying the observed temporal patterns of intracellular calcium (Ca(i)2+) under sustained agonist stimulation. Some studies have suggested that inhibition of ligand receptors and PLC by PKC could generate sinusoidal Ca2+ oscillations, while PKC-independent Ca2+-induced Ca2+ release (CICR) via IP(3)-gated Ca2+ channels on the endoplasmic reticulum (ER) is believed to be responsible for baseline spiking. However, some evidence also indicates that baseline spiking can be observed under high-PKC activity, or under low-PKC activity with low agonist stimulus, as well. Insight into the basis of these observations regarding the role of PKC in Ca(i)2+ response patterns can be gained by developing and analyzing a mathematical model of Ca(i)2+ responses. We do this herein and find that (1) interaction of CICR and the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump is enough to generate both types of Ca(i)2+ oscillations, (2) there exist four possible Ca(i)2+ response patterns under sustained agonist stimulus: a sub-threshold response (SR), baseline spiking, sinusoidal oscillations (SO) and transient with plateau, and (3) the IP(3) concentration, which is controlled by the strength of the interaction between PKC and PLC, can be used to predict the Ca(i)2+ response patterns. From this analysis we conclude that the different patterns of Ca(i)2+ oscillations can be understood as a generic consequence of the interactions between CICR via the IP(3)-gated Ca(2+) channels in response to changes in the level of IP(3), and re-uptake into the ER/SR via the SERCA pump. PKC, in conjunction with PLC, can act as a switch between different Ca(i)2+ response patterns by modulating the cytosolic IP(3) level, which determines the Ca(i)2+ patterns.     

Activation of Ca(2+)-dependent currents in cultured rat dorsal root ganglion neurones by a sperm factor and cyclic ADP-ribose.

The effects of intracellular application of two novel Ca2+ releasing agents have been studied in cultured rat dorsal root ganglion (DRG) neurones by monitoring Ca(2+)-dependent currents as a physiological index of raised free cytosolic Ca2+ ([Ca2+]i). A protein based sperm factor (SF) extracted from mammalian sperm, has been found to trigger Ca2+ oscillations and to sensitize unfertilized mammalian eggs to calcium induced calcium release (CICR). In this study intracellular application of SF activated Ca(2+)-dependent currents in approximately two-thirds of DRG neurones. The SF induced activity was abolished by heat treatment, attenuated by increasing the intracellular Ca2+ buffering capacity of the cells and persisted when extracellular Ca2+ was replaced by Ba2+. In addition, activity could be triggered or potentiated by loading the cells with Ca2+ by activating a series of voltage-gated Ca2+ currents. Ca(2+)-activated inward current activity was also generated by intracellular application of cyclic ADP-ribose (cADPR), a metabolite of NAD+, which causes Ca2+ release in sea urchin eggs. This activity could also be enhanced by loading the cells with Ca2+. The cADPR induced activity, but not the SF induced activity, was abolished by depleting the caffeine sensitive Ca2+ store. Ruthenium red markedly attenuated SF induced activity but had little action on cADPR induced activity or caffeine induced activity. Our results indicate that both SF and cADPR release intracellular Ca2+ pools in DRG neurones and that they appear to act on subtly distinct stores or distinct intracellular Ca2+ release mechanisms, possibly by modulating CICR.     

Direction of action potential propagation influences calcium increases in distal dendrites of the cricket giant interneurons

To understand the relationship between the propagation direction of action potentials and dendritic Ca(2+) elevation, simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) and intradendritic membrane potential were performed in the wind-sensitive giant interneurons of the cricket. The dendritic Ca(2+) transients induced by synaptically-evoked action potentials had larger amplitudes than those induced by backpropagating spikes evoked by antidromic stimulation. The amplitude of the [Ca(2+)](i) changes induced by antidromic stimuli combined with subthreshold synaptic stimulation was not different from that of the Ca(2+) increases evoked by the backpropagating spikes alone. This result means that the synaptically activated Ca(2+) release from intracellular stores does not contribute to enhancement of Ca(2+) elevation induced by backpropagating spikes. On the other hand, the synaptically evoked action potentials were also increased at distal dendrites in which the Ca(2+) elevation was enhanced. When the dendritic and axonal spikes were simultaneously recorded, the delay between dendritic spike and ascending axonal spike depended upon which side of the cercal nerves was stimulated. Further, dual intracellular recording at different dendritic branches illustrated that the dendritic spike at the branch arborizing on the stimulated side preceded the spike recorded at the other side of the dendrite. These results suggest that the spike-initiation site shifts depending on the location of the activated postsynaptic site. It is proposed that the difference of spike propagation manner could change the action potential waveform at the distal dendrite, and could produce synaptic activity-dependent Ca(2+) dynamics in the giant interneurons.     

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic beta-cells

The effect of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition on the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was studied in primary insulin-releasing pancreatic beta-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca(2+)-deficient medium the individual primary beta-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca(2+)](i) followed by an explosive transient elevation. The [Ca(2+)](i) transients were preferentially observed at low intracellular concentrations of the Ca(2+) indicator fura-2 and were unaffected by pre-treatment with 100 microM ryanodine. Whereas 20mM caffeine had no effect on basal [Ca(2+)](i) or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca(2+)](i) transients as well as inositol 1,4,5-trisphosphate-mediated [Ca(2+)](i) transients in response to carbachol. In striking contrast to the primary beta-cells, caffeine readily mobilized intracellular Ca(2+) in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca(2+) from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca(2+)-induced Ca(2+) release (CICR). In primary pancreatic beta-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal beta-cells.     

Ca(2+)-induced Ca(2+) release via inositol 1,4,5-trisphosphate receptors is amplified by protein kinase A and triggers exocytosis in pancreatic beta-cells

Hormones, such as glucagon and glucagon-like peptide-1, potently amplify nutrient stimulated insulin secretion by raising cAMP. We have studied how cAMP affects Ca(2+)-induced Ca(2+) release (CICR) in pancreatic beta-cells from mice and rats and the role of CICR in secretion. CICR was observed as pronounced Ca(2+) spikes on top of glucose- or depolarization-dependent rise of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)). cAMP-elevating agents strongly promoted CICR. This effect involved sensitization of the receptors underlying CICR, because many cells exhibited the characteristic Ca(2+) spiking at low or even in the absence of depolarization-dependent elevation of [Ca(2+)](i). The cAMP effect was mimicked by a specific activator of protein kinase A in cells unresponsive to activators of cAMP-regulated guanine nucleotide exchange factor. Ryanodine pretreatment, which abolishes CICR mediated by ryanodine receptors, did not prevent CICR. Moreover, a high concentration of caffeine, known to activate ryanodine receptors independently of Ca(2+), failed to mobilize intracellular Ca(2+). On the contrary, a high caffeine concentration abolished CICR by interfering with inositol 1,4,5-trisphosphate receptors (IP(3)Rs). Therefore, the cell-permeable IP(3)R antagonist 2-aminoethoxydiphenyl borate blocked the cAMP-promoted CICR. Individual CICR events in pancreatic beta-cells were followed by [Ca(2+)](i) spikes in neighboring human erythroleukemia cells, used to report secretory events in the beta-cells. The results indicate that protein kinase A-mediated promotion of CICR via IP(3)Rs is part of the mechanism by which cAMP amplifies insulin release.