p-nitrophenyl phosphate hydrolysis and calcium ion transport in fragmented sarcoplasmic reticulum

The calcium ion pump of fragmented sarcoplasmic reticulum can be coupled to hydrolysis of p-nitrophenyl phosphate, in the absence of added adenosine triphosphate. Comparison of the activities obtained with the two substrates suggests an analogous mechanism of transport. Independent of the substrate, a 2 : 1 ratio between calcium ion transport and substrate hydrolysis is displayed by the system, and an identical amount of work is required for ion transport against a given gradient. A phosphate ester appears necessary for substrate utilization in the pump mechanism, whereas the structure of the substrate determines the rates of activity and the affinity of the system for calcium ion.     

PML regulates apoptosis at endoplasmic reticulum by modulating calcium release

The promyelocytic leukemia (PML) tumor suppressor is a pleiotropic modulator of apoptosis. However, the molecular basis for such a diverse proapoptotic role is currently unknown. We show that extranuclear Pml was specifically enriched at the endoplasmic reticulum (ER) and at the mitochondria-associated membranes, signaling domains involved in ER-to-mitochondria calcium ion (Ca(2+)) transport and in induction of apoptosis. We found Pml in complexes of large molecular size with the inositol 1,4,5-trisphosphate receptor (IP(3)R), protein kinase Akt, and protein phosphatase 2a (PP2a). Pml was essential for Akt- and PP2a-dependent modulation of IP(3)R phosphorylation and in turn for IP(3)R-mediated Ca(2+) release from ER. Our findings provide a mechanistic explanation for the pleiotropic role of Pml in apoptosis and identify a pharmacological target for the modulation of Ca(2+) signals.     

Calcium uptake by isolated sarcoplasmic reticulum treated with dithiothreitol

Isolated vesicles of the sarcoplasmic reticulum are known to take up calcium when provided with magnesium adenosine triphosphate as an energy source. Preparations of high activity are obtained by keeping the vesicles in 5 millimolar dithiothreitol (a reagent that reduces disulfide groups), and these preparations retain activity for a week or longer. The highly active preparations lend themselves to a spectrophotometric method for following calcium uptake, and continuous uptake kinetics are readily obtained. Calcium uptake appears to follow Michaelis-Menten kinetics (K(m) = 8x10(-6); V(mas), = 7x10(-7) mole per second per milligram of protein). These preparations are also useful for studying the effects of inhibitors of uptake, such as quinine. When extrapolated to the intact muscle, the results from these isolated vesicles should give a better estimate than has been available of the actual rates of calcium uptake and of the physiological effect of inhibitors of uptake.     

Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes

In skeletal muscle, intramembrane charge movement initiates the processes that lead to the release of calcium from the sarcoplasmic reticulum. In cardiac muscle, in contrast, the similarity of the voltage dependence of developed tension and intracellular calcium transients to that of calcium current suggests that the calcium current may gate the release of calcium. Nevertheless, a mechanism similar to that of skeletal muscle continues to be postulated for cardiac muscle. By using rapid exchange (20 to 50 milliseconds) of the extracellular solutions in rat ventricular myocytes in which the intracellular calcium transients or cell shortening were measured, it has now been shown that the influx of calcium through the calcium channel is a mandatory link in the processes that couple membrane depolarization to the release of calcium. Thus, intramembrane charge movement does not contribute to the release of calcium in heart muscle.     

Strontium accumulation by sarcoplasmic reticulum and mitochondria in vascular smooth muscle

Electron-opaque deposits of strontium were observed in the sarcoplasmic reticulum and in mitochondria of spontaneously contracting vascular smooth muscles that had been incubated in a strontium-containing solution prior to fixation. The deposits were present in those elements of the sarcoplasmic reticulum that are in close contact with the surface membrane and also in more centrally located portions. In vascular smooth muscle that does not contract spontaneously, similar deposits of strontium were only seen if the muscle was depolarized during or glycerinated before exposure to the strontium-containing solution. Strontium was also deposited in the sarcoplasmic reticulum of the endothelium. It is suggested that translocation of calcium from the sarcoplasmic reticulum that is in close contact with the surface membrane, and now shown to accumulate divalent cations, is responsible for the action potential-triggered contractions of rabbit and guinea pig mesenteric veins. Strontium may also be a suitable marker for identifying sites that accumulate calcium in other types of cells in which translocation of calcium plays a major regulatory function.     

Depolarization without calcium can release gamma-aminobutyric acid from a retinal neuron

Calcium influx is often an essential intermediate step for the release of neurotransmitter. However, some retinal neurons appear to release transmitter by a mechanism that does not require calcium influx. It was uncertain whether depolarization released calcium from an intracellular store or released transmitter by a mechanism that does not require calcium. The possibility that voltage, and not calcium, can regulate the release of transmitter was studied with pairs of solitary retinal neurons. Horizontal and bipolar cells were isolated from fish retinas and juxtaposed in culture. Communication between them was studied with electrophysiological methods. A horizontal cell released its neurotransmitter, gamma-aminobutyric acid, when depolarized during conditions that buffered the internal calcium concentration and prohibited calcium entry. The speed and amount of material released were sufficient for a contribution to synaptic transmission.     

Voltage-independent calcium release in heart muscle

The Ca2+ that activates contraction in heart muscle is regulated as in skeletal muscle by processes that depend on voltage and intracellular Ca2+ and involve a positive feedback system. How the initial electrical signal is amplified in heart muscle has remained controversial, however. Analogous protein structures from skeletal muscle and heart muscle have been identified physiologically and sequenced; these include the Ca2+ channel of the sarcolemma and the Ca2+ release channel of the sarcoplasmic reticulum. Although the parallels found in cardiac and skeletal muscles have provoked valuable experiments in both tissues, separation of the effects of voltage and intracellular Ca2+ on sarcoplasmic reticulum Ca2+ release in heart muscle has been imperfect. With the use of caged Ca2+ and flash photolysis in voltage-clamped heart myocytes, effects of membrane potential in heart muscle cells on Ca2+ release from intracellular stores have been studied. Unlike the response in skeletal muscle, voltage across the sarcolemma of heart muscle does not affect the release of Ca2+ from the sarcoplasmic reticulum, suggesting that other regulatory processes are needed to control Ca2(+)-induced Ca2+ release.     

Regenerative calcium release within muscle cells

Free calcium appears to trigger the release of stored calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibers immersed in solutions with a low concentration of magnesium ion.     

亚硒酸钠缓释剂的制备

以β-环状糊精为载体,以缓释效果为指标,选用饱和溶液法制备亚硒酸钠缓释剂．采用正交试验对缓释剂的制备条件进行优化,筛选出最佳制备条件,并用透析法检测其体外释放效果．试验结果表明,亚硒酸钠缓释剂释放速度明显慢于亚硒酸钠水溶液,制备亚硒酸钠β-CD缓释剂的最佳工艺条件为：温度20℃,搅拌时间3h,亚硒酸钠与β-CD投料体积比为1：3．     

交换性阳离子对磷灰石中磷和钙释放的影响

研究了以Ｌｉ~＋、Ｎａ~＋、Ｋ~＋、ＮＨ~＋_４等一价阳离子取代磷灰石表面交换性二价阳离子对磷灰石中磷和钙释放的影响，并就可能涉及的机理进行了探讨．     

骨骼肌肌浆网在收缩潜伏期内的超微结构形态变化

采用双向红外线探测器－计算机控制的电刺激与超低温快速冷冻固定同步技术对被电刺激后的骨骼肌作快速冷冻固定,采用透射电镜对电刺激后０．８ｍｓ,５．６ｍｓ,８．４ｍｓ及完全舒张时的蟾蜍腓肠肌超微结构变化进行了对比研究。骨骼肌组织在电刺激后０．８ｍｓ,５．６ｍｓ及８．４ｍｓ时发现肌浆网的前端,膜产生两个圆孔,而在骨骼肌处于全舒张时,肌浆网膜并无此形态结构。骨骼肌兴奋－收缩偶联发生时,发现肌浆网近Ｔ－管端的膜产生两个圆孔,而骨骼肌处于全舒张时,肌浆网膜也无此形态结构;骨骼肌收缩时出现的肌浆网膜小孔是否与肌浆网内某种物质（引起钙离子释放的某种未知物质？钙流？）在肌组织收缩时的转运或释放有关？     

Sarcoplasmic reticulum of striated muscle: localization of potential calcium binding sites

Fish branchial muscle stained at a low pH with thorium dioxide shows localization of the stain over the sacroplasmic reticulum. Binding of the positively charged thorium micelles with dissociated acid groups of polyanions in this region suggests a possible mechanism for the storage and release of divalent cations such as calcium.     

Deuterium oxide: inhibition of calcium release in muscle

Calcium release, measured as luminescence of the protein aequorin, was measured simultaneously with membrane potential and isometric tension in single muscle fibers of the barnacle (Balanus nubilus). Deuterium oxide inhibited calcium release and isometric tension but did not affect membrane potential, a result consistent with the postulate that deuterium oxide inhibits the coupling between excitation and contraction.     

Mechanism of transmitter release: voltage hypothesis and calcium hypothesis

The calcium hypothesis of synaptic transmission has been challenged by experimental results using the crayfish neuromuscular junction that suggest that presynaptic depolarization can trigger transmitter release directly without calcium influx. Results from electrophysiological experiments using the same preparation do not support this voltage hypothesis, but are consistent with the calcium hypothesis. Voltage may modulate, but not elicit, transmitter release.     

Calcium as a coagonist of inositol 1,4,5-trisphosphate-induced calcium release

Inositol 1,4,5-trisphosphate (IP3)-induced calcium release from intracellular stores is a regulator of cytosolic-free calcium levels. The subsecond kinetics and regulation of IP3-induced calcium-45 release from synaptosome-derived microsomal vesicles were resolved by rapid superfusion. Extravesicular calcium acted as a coagonist, potentiating the transient IP3-induced release of calcium-45. Thus, rapid elevation of cytosolic calcium levels may trigger IP3-induced calcium release in vivo. Extravesicular calcium also produced a more slowly developing, reversible inhibition of IP3-induced calcium-45 release. Sequential positive and negative feedback regulation by calcium of IP3-induced calcium release may contribute to transients and oscillations of cytosolic-free calcium in vivo.     

一种三角帆蚌肌浆网Ca2+-ATP酶基因cDNA的全长克隆与表达分析

采用RACE技术,从三角帆蚌(Hyriopsis cumingii)鳃组织中成功克隆得到一种肌浆网Ca²⁺-ATP酶(sarco/endoplasmic reticulum calcium ATPase,SERCA)基因的全长cDNA序列,共3 326 bp,包含201-bp 5’-UTR区域、3 060-bp编码框(ORF)和65-bp 3’-UTR。ORF共编码1 019个氨基酸,预测无信号肽。该基因氨基酸序列呈现出典型的Ca²⁺-ATP酶特征,由Cation_ATPase_N、E1-E2_ATPase、Hydrolase、Cation_ATPase_C四种类型结构域组成,其内含SERCAs的常见结构组成包括磷酸化区域、异硫氰酸荧光素位点、FSBA结合位点、受磷蛋白结合区以及毒胡萝卜素位点。分析显示,该基因序列高度保守且与海洋软体动物具有最高同源性。荧光定量PCR检测,该基因在三角帆蚌外套膜、斧足、鳃、肝胰腺、性腺等5个组织中均有表达,且在鳃、外套膜、肝胰腺组织中表达较高。不同Ca²⁺浓度处理试验的结果表明,随水体中Ca²⁺浓度逐渐升高,该基因在外套膜中的表达水平呈先下降后上升趋势,并在Ca²⁺浓度为60 mg·L^-1时达到最低值,80 mg·L^-1时达到最高值。同时在60 mg·L^-1 Ca²⁺浓度条件下,外套膜中SERCA基因的表达量随时间推移先上升,并于48 h时达到最高,而后逐渐下降。上述结果为进一步深入研究SERCA基因的功能及其调控机理奠定了基础。     

A G protein directly regulates mammalian cardiac calcium channels

A possible direct effect of guanine nucleotide binding (G) proteins on calcium channels was examined in membrane patches excised from guinea pig cardiac myocytes and bovine cardiac sarcolemmal vesicles incorporated into planar lipid bilayers. The guanosine triphosphate analog, GTP gamma S, prolonged the survival of excised calcium channels independently of the presence of adenosine 3',5'-monophosphate (cAMP), adenosine triphosphate, cAMP-activated protein kinase, and the protein kinase C activator tetradecanoyl phorbol acetate. A specific G protein, activated Gs, or its alpha subunit, purified from the plasma membranes of human erythrocytes, prolonged the survival of excised channels and stimulated the activity of incorporated channels. Thus, in addition to regulating calcium channels indirectly through activation of cytoplasmic kinases, G proteins can regulate calcium channels directly. Since they also directly regulate a subset of potassium channels, G proteins are now known to directly gate two classes of membrane ion channels.     

Intracellular calcium release mediated by sphingosine derivatives generated in cells

Soluble and hydrophobic lipid breakdown products have a variety of important signaling roles in cells. Here sphingoid bases derived in cells from sphingolipid breakdown are shown to have a potent and direct effect in mediating calcium release from intracellular stores. Sphingosine must be enzymically converted within the cell to a product believed to be sphingosine-1-phosphate, which thereafter effects calcium release from a pool including the inositol 1,4,5-trisphosphate-sensitive calcium pool. The sensitivity, molecular specificity, and reversibility of the effect on calcium movements closely parallel sphingoid base-mediated inhibition of protein kinase C. Generation of sphingoid bases in cells may activate a dual signaling pathway involving regulation of calcium and protein kinase C, comparable perhaps to the phosphatidylinositol and calcium signaling pathway.     

Sodium and potassium effects on skeletal muscle microsomal adenosine triphosphatase and calcium uptake

The relationship between the (Na(+) and K(+))-activated adenosine triphosphatase enzyme system implicated in sodium-transport by cell membranes and the calcium-activated adenosine triphosphatase, which is generally associated with calcium uptake, was examined in microsomes from skeletal muscle. Whereas sodium and potassium did not modify the relatively low adenosine triphosphatase activity seen in the absence of calcium, a pattern similar to that of the sodium-transport enzyme system was seen afer the addition of CaCl(2). The calcium-activated adenosine triphosphatase was stimulated equally by sodium or potassium alone, but both the rate and extent of calcium uptake were enhanced more by potassium than by sodium at concentrations below 0.12 mole per liter. In the absence of either of these ions addition of calcium failed to activate adenosine triphosphatase although significant amounts of calcium were taken up by the microsomes.     

海藻酸钙微胶囊包埋IgY及其释放特性研究

卵黄免疫球蛋白(IgY)是一种高活性的口服抗体,与人体健康密切相关。为最大限度保留IgY的生物活性,提高其稳定性,以海藻酸钠为主要壁材,使用锐孔-凝固浴法包埋IgY制备凝胶珠。以包埋率和载药量为主要指标,利用响应面法探究IgY-海藻酸钙凝胶珠的最佳制备工艺,并利用体外胃肠道消化模型评估凝胶珠释放效果。结果显示:当海藻酸钠为28.30 g·L^-1,氯化钙为10.80 g·L^-1,芯壁比(质量比)为0.46∶1时,新鲜制备得到的IgY-海藻酸钙凝胶珠成型效果较好,包埋率为(53.30±1.55)%,载药量为(10.90±0.26)%。通过宏观形态观察,凝胶珠颗粒具有较好的圆整性和均一性,粒径约为(2.10±0.05) mm,硬度为(4 396.87±331.62) g。干燥后的凝胶珠在模拟胃液中2 h后溶胀度为0.94±0.39,IgY累积释放率约在(11.10±3.20)%,在模拟肠液中2 h后,溶胀度持续增加至4.81±0.32,IgY累积释放率达(97.10±2.40)%以上。因此,IgY-海藻酸钙凝胶珠在胃液中具有缓慢释放芯材的作用,IgY在肠液中能基本全部释放。