Use of chlorotetracycline fluorescence to demonstrate Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum of skinned cardiac cells.

It has been proposed that the trans-sarcolemmal influx of Ca2+ occurring during the plateau of the mammalian cardiac action potentials is insufficient in itself to activate the myofilaments, but can trigger a release of Ca2+ from the sarcoplasmic reticulum (SR) which is sufficient for activation. The demonstration of this Ca2+-induced release of Ca2+ relied entirely on experiments in which the tension developed by the myofilaments was used as a sensor of the changes of myoplasmic free Ca2+ concentration ([free Ca2+]) in segments of single cardiac cells from which the sarcolemma had been removed by microdissection (skinned cardiac cells). The small size of these preparations has previously prevented the use of more direct methods for the detection of myoplasmic Ca2+ movements. The present study is a direct demonstration of Ca2+-induced release of Ca2+ from the SR of skinned cardiac cells treated with chlorotetracycline (CTC), a fluorescent chelate probe which enables changes in the amount of Ca2+ bound to a variety of biological membranes or micelles to be monitored. The fluorescence increases when more Ca2+ is bound.     

Ca2+-dependent and Ca2+-independent phagocytosis in human neutrophils

The phagocytic function of neutrophils is a crucial element in host defence against invading microorganisms. Two main specific receptor-mediated mechanisms operate in the phagocyte plasma membrane, one recognizing the C3b/bi fragment of complement and the other the Fc domain of immunoglobulin G (ref. 1). There is evidence that phagocytosis mediated by these receptors differs in the number and nature of the intracellular signals generated. However, the mechanisms by which receptor binding is transduced into a signal that generates the formation of the phagocyte pseudopod is not known, although extensive biochemical evidence has allowed the postulate that calcium ion gradients in the peripheral cytoplasm, by interacting with calcium-sensitive contractile proteins, initiate the process of engulfment. Using the high-affinity fluorescent calcium indicator quin2 both to measure and to buffer intracellular calcium ([Ca2+]i), we show here that in human neutrophils two mechanisms of phagocytosis coexist: a [Ca2+]i-dependent and modulated phagocytosis, triggered by activation of the Fc receptor, and a [Ca2+]i-independent mechanism triggered by the activation of the C3b/bl receptors.     

Alpha-haemolysin of uropathogenic E. coli induces Ca2+ oscillations in renal epithelial cells

Pyelonephritis is one of the most common febrile diseases in children. If not treated appropriately, it causes irreversible renal damage and accounts for a large proportion of end stage renal failures. Renal scarring can occur in the absence of inflammatory cells, indicating that bacteria may have a direct signalling effect on renal cells. Intracellular calcium ([Ca2+]i) oscillations can protect cells from the cytotoxic effects of prolonged increases in intracellular calcium. However, no pathophysiologically relevant protein that induces such oscillations has been identified. Here we show that infection by uropathogenic Escherichia coli induces a constant, low-frequency oscillatory [Ca2+]i response in target primary rat renal epithelial cells induced by the secreted RTX (repeats-in-toxin) toxin alpha-haemolysin. The response depends on calcium influx through L-type calcium channels as well as from internal stores gated by inositol triphosphate. Internal calcium oscillations induced by alpha-haemolysin in a renal epithelial cell line stimulated production of cytokines interleukin (IL)-6 and IL-8. Our findings indicate a novel role for alpha-haemolysin in pyelonephritis: as an inducer of an oscillating second messenger response in target cells, which fine-tunes gene expression during the inflammatory response.     

低功率激光通过细胞内Ca^2＋对细胞功能的调控作用

综述了Ｈｅ－Ｎｅ激光照射使细胞溶质内［Ｃａ＾２＋］ｉ增加，产生钙信号，从而调控细胞功能，引起细胞内一系列生理和生物化学反应。     

Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel

High-conductance voltage- and Ca(2+)-activated K(+) channels function in many physiological processes that link cell membrane voltage and intracellular Ca(2+) concentration, including neuronal electrical activity, skeletal and smooth muscle contraction, and hair cell tuning. Like other voltage-dependent K(+) channels, Ca(2+)-activated K(+) channels open when the cell membrane depolarizes, but in contrast to other voltage-dependent K(+) channels, they also open when intracellular Ca(2+) concentrations rise. Channel opening by Ca(2+) is made possible by a structure called the gating ring, which is located in the cytoplasm. Recent structural studies have defined the Ca(2+)-free, closed, conformation of the gating ring, but the Ca(2+)-bound, open, conformation is not yet known. Here we present the Ca(2+)-bound conformation of the gating ring. This structure shows how one layer of the gating ring, in response to the binding of Ca(2+), opens like the petals of a flower. The degree to which it opens explains how Ca(2+) binding can open the transmembrane pore. These findings present a molecular basis for Ca(2+) activation of K(+) channels and suggest new possibilities for targeting the gating ring to treat conditions such as asthma and hypertension.     

Ca2+-activated ATPase and ATP-dependent calmodulin-stimulated Ca2+ transport in islet cell plasma membrane

Calcium is known to play an essential part in the regulation of insulin secretion in the pancreatic beta cell. Calcium influx/efflux studies indicate that glucose promotes an accumulation of calcium by the beta cell. However, interpretation of such data is particularly difficult due to the complex compartmentalization of calcium within the cell. Although indirect evidence using chlorotetracycline suggests that control of calcium homeostasis at the plasma membrane may be central to insulin secretion, the mechanism by which secretagogues influence the handling of calcium remains unknown. Despite its continuous diffusive entry, intracellular calcium is maintained in the submicromolar range by energy-dependent mechanisms. One such process which has been well characterized in erythrocytes is a plasma membrane calcium extrusion pump whose enzymatic basis is a high affinity (Ca+2 + Mg+2)ATPase. A similar mechanism regulated by insulin has recently been identified in adipocyte plasma membranes. We report here the presence of a high affinity (Ca+2 + Mg+2)ATPase and ATP-dependent calmodulin-stimulated calcium transport system in rat pancreatic islet cell plasma membranes.     

Ca2+ signalling between single L-type Ca2+ channels and ryanodine receptors in heart cells

Ca2+-induced Ca2+ release is a general mechanism that most cells use to amplify Ca2+ signals. In heart cells, this mechanism is operated between voltage-gated L-type Ca2+ channels (LCCs) in the plasma membrane and Ca2+ release channels, commonly known as ryanodine receptors, in the sarcoplasmic reticulum. The Ca2+ influx through LCCs traverses a cleft of roughly 12 nm formed by the cell surface and the sarcoplasmic reticulum membrane, and activates adjacent ryanodine receptors to release Ca2+ in the form of Ca2+ sparks. Here we determine the kinetics, fidelity and stoichiometry of coupling between LCCs and ryanodine receptors. We show that the local Ca2+ signal produced by a single opening of an LCC, named a 'Ca2+ sparklet', can trigger about 4-6 ryanodine receptors to generate a Ca2+ spark. The coupling between LCCs and ryanodine receptors is stochastic, as judged by the exponential distribution of the coupling latency. The fraction of sparklets that successfully triggers a spark is less than unity and declines in a use-dependent manner. This optical analysis of single-channel communication affords a powerful means for elucidating Ca2+-signalling mechanisms at the molecular level.     

地西泮对大鼠脑突触体内Ca2+水平和Ca2+, Mg2+-ATP酶活性的作用

用Quin 2法测得大鼠脑突触体内静息游离Ca2+浓度([Ca2+]i)为85±13μmol/g protein.地西泮0.1,1和10 mmol/L对静息突触体[Ca2+]i无明显影响,但能以剂量依赖方式增高65 mmol/LKCl所致突触体[Ca2+]i升高,从105±23μmol/g protein分别达到185±28,267±49和291±48μmol/gprotein.地西泮1和10 mmol/L分别使突触体Ca2+,Mg2+-ATP酶活性降低28.7%和42.5%;使Mg2+-ATP酶活性分别降低12.6%和32.8%,提示地西泮可能是通过抑制钙调素,进而抑制Ca2+,Mg2+-ATP酶活性,使突触体[Ca2+]1升高,促进神经末梢释放递质.     

Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels

Acute modulation of P/Q-type (alpha1A) calcium channels by neuronal activity-dependent changes in intracellular Ca2+ concentration may contribute to short-term synaptic plasticity, potentially enriching the neurocomputational capabilities of the brain. An unconventional mechanism for such channel modulation has been proposed in which calmodulin (CaM) may exert two opposing effects on individual channels, initially promoting ('facilitation') and then inhibiting ('inactivation') channel opening. Here we report that such dual regulation arises from surprising Ca2+-transduction capabilities of CaM. First, although facilitation and inactivation are two competing processes, both require Ca2+-CaM binding to a single 'IQ-like' domain on the carboxy tail of alpha1A; a previously identified 'CBD' CaM-binding site has no detectable role. Second, expression of a CaM mutant with impairment of all four of its Ca2+-binding sites (CaM1234) eliminates both forms of modulation. This result confirms that CaM is the Ca2+ sensor for channel regulation, and indicates that CaM may associate with the channel even before local Ca2+ concentration rises. Finally, the bifunctional capability of CaM arises from bifurcation of Ca2+ signalling by the lobes of CaM: Ca2+ binding to the amino-terminal lobe selectively initiates channel inactivation, whereas Ca2+ sensing by the carboxy-terminal lobe induces facilitation. Such lobe-specific detection provides a compact means to decode local Ca2+ signals in two ways, and to separately initiate distinct actions on a single molecular complex.     

Intracellular Ca indicator Quin-2 inhibits Ca2+ inflow via Na/Ca exchange in squid axon

Until recently, intracellular free calcium has been amenable to measurement and investigation only in cells large enough to permit either microinjection of a suitable Ca sensor such as a aequorin or arsenazo III or insertion of a Ca-sensitive microelectrode. This constraint on cell size was removed by the development of the fluorescent Ca2+ -sensitive dye Quin-2 and its acetoxymethyl ester, which can be introduced into a wide range of cell types. A major requirement of any intracellular Ca2+ indicator is that it should not disturb intracellular Ca2+ homeostasis and Quin-2 is generally considered to be satisfactory in this respect. We now report that injection of Quin-2 into squid (Loligo forbesi) axons can almost completely abolish one component of Ca2+ entry--intracellular Na+ (Nai)-dependent Ca2+ inflow, which occurs via Na/Ca exchange. Mixtures of Ca and Quin-2 that buffer an ionized Ca2+ at close to physiological concentrations also block Nai-dependent Ca2+ influx but these same mixtures fail to block the extracellular Na+ (Na0)-dependent extrusion of Ca2+, showing that Quin-2 acts specifically on Ca2+ inflow.     

cGMP-dependent protein kinase enhances Ca2+ current and potentiates the serotonin-induced Ca2+ current increase in snail neurones

Protein phosphorylation catalysed by cyclic AMP-dependent, Ca2+/calmodulin-dependent and Ca2+/diacylglycerol-dependent protein kinases is important both in the modulation of synaptic transmission and in the regulation of neuronal membrane permeability (for reviews see refs 5-7). However, there has previously been no evidence for the involvement of cyclic GMP-dependent protein kinase (cGMP-PK) in the regulation of neuronal function. Serotonin induces an increase of Ca2+ current in a group of identified ventral neurones of the snail Helix aspersa. This effect is probably mediated by cGMP because it is mimicked by the intracellular injection of cGMP or the application of zaprinast, an inhibitor of cGMP-dependent phosphodiesterase. We have now found that the effect of either serotonin or zaprinast on the Ca2+ current is potentiated by the intracellular injection of cGMP-PK. Moreover, the intracellular injection of activated cGMP-PK (cGMP-PK + 1 microM cGMP) greatly enhances the Ca2+ current of the identified ventral neurones seen in the absence of serotonin. These results indicate that cGMP-PK has a physiological role in the control of the membrane permeability of these neurones.     

Rapid photochemical inactivation of Ca2+-antagonists shows that Ca2+ entry directly activates contraction in frog heart

'Calcium-antagonists' are a group of pharmacological agents which are potent vasodilators and are clinically used for the treatment of angina. They are thought to block Ca2+ channels in vascular smooth muscle and myocardium but other sites of action have been proposed. These agents bind tightly to heart muscle and suppress action potential and contraction. Nifedipine and nisoldipine (BAY K 5552) are Ca2+ antagonists which have o-nitrobenzyl groups and are photolabile. We have found that short pulses of UV light rapidly inactivate these drugs in ventricular muscle. This observation allowed us to study the effect of Ca2+ antagonists on action potential, Ca2+ current and tension in conditions in which diffusion of those drugs from their site of action was not rate limiting. Our studies, described here, suggest that the primary mechanism of action of Ca2+ antagonists is the blockade of the Ca2+ channel and support the idea that extracellular space is the immediate source of contractile Ca2+ in the frog heart.     

钙离子对学习记忆的影响

Ca^2 作为神经信号传递的重要信使，参与学习记忆的神经机制。突触前和突触后细胞内钙离子在长时程突触的可塑性中发挥重要的信息传递作用。研究表明衰老性记忆障碍与中枢神经系统的Ca^2 稳态调节失衡有关。神经细胞内游离钙水平[Ca^2 ]i受多种机制的调节，主要包括Ca^2 的跨膜转运、细胞内钙池摄取与释放Ca^2 等过程；最近的研究表明神经胶质细胞也参与其调节。     

不同Ca~(2+)浓度对斑节对虾光感受器细胞内储存的Ca~(2+)的影响

应用草酸-焦锑酸盐结合的沉淀技术研究了在不同Ca^2 浓度条件下斑节对虾光感受器细胞胞内储存的Ca^2 的变化，电镜观察表明：在高钙溶液培育后，细胞内的多囊体、色素颗粒、板膜体中都存在大量的焦锑酸钙沉淀的黑色颗粒，线粒体中则无；在生理溶液培育后，线粒体中出现沉淀，而其他Ca^2 储存器中焦锑酸钙沉淀的黑色颗粒则大量减少，低钙溶液培育后焦锑酸钙沉淀的黑色颗粒情况与生理溶液条件下的相似，研究证实光感受器细胞内储存的Ca^2 受到胞外Ca^2 浓度的影响。     

龙柚果实总钙、可溶性Ca2+含量及Ca2+-ATPase活性的变化

研究以柚类品种龙柚为材料,对发育过程中果皮、果肉总钙和可溶性Ca~(2 )含量及Ca~(2 )-ATPase活性进行了测定.结果表明:龙柚花前至花期子房总钙含量相对较低,花后有明显上升;龙柚果皮总钙和可溶性Ca~(2 )均在果实增大期保持持续增长的趋势,此时的果肉总钙呈明显下降趋势,而对应可溶性Ca~(2 )的变化幅度较大;龙柚Ca~(2 )-ATPase活性在花前较低,花期开始上升;而果皮和果肉的可溶性Ca~(2 )含量和Ca~(2 )-ATPase活性在增大期内均有一明显增长的过程或相对较高的水平.     

离子色谱法测定饮用水中钠、镁、钙

本文研究了离子色谱法测定饮用水及源水中钠、镁、钙离子的方法.实验表明,该方法灵敏度高,重现性好,操作简便,分析快速.在本方法实验条件下,钠、镁、钙方法检出限,分别为0.013mg/L;0.013mg/L;0.025mg/L.相对标准偏差分布在0.07-0.76%,加标回收率分布在99-102%.     

甲基对硫磷诱发大鼠肝细胞内钙浓度升高的初步机制研究

应用激光扫描共聚焦显微镜观察甲基对硫磷(M ETHYLPARATH ION,MP)对正常大鼠肝细胞株(BRL)内游离钙浓度([CA2 ]I)的影响。实验结果表明,在正常生理盐溶液(N-PSS)中,MP能引起细胞内的钙升高,并呈浓度依赖性;在无钙生理盐溶液(0 CA2 -PSS)中,400 PMOL/L MP不显著改变细胞内的钙浓度。用不同的非选择性阳离子通道阻断剂(300ΜMOL/L GD3 ,2 MMOL/L SR2 ,2 MMOL/L N I2 )作用于由MP引起的钙升高时,发现均能有效地抑制钙升高,其中N I2 的抑制效果最为明显。同时MP引起的钙升高的时程远远大于ATP的作用。以上结果显示,MP对BRL细胞胞内的CA2 具有升高作用,此作用可能是MP激活非选择性阳离子通道,并最终导致细胞膜上的其它钙离子通道开放。