败血症大鼠心肌核被膜和肌浆网ryanodine受体的变化

为观察败血症时心肌肌浆网（ＳＲ）和核被膜（ＮＥ）的ｒｙａｎｏｄｉｎｅ受体的变化,采用结扎及穿刺盲肠（ＣＬＰ）制作败血症动物模型,用密度梯度离心分离ＳＲ和ＮＥ,用放射配体结合法研究ｒｙａｎｏｄｉｎｅ受体的特征。结果表明,大鼠早期败血症（ＣＬＰ后９ｈ）时,ＳＲ的ｒｙａｎｏｄｉｎｅ受体的最大结合（Ｂｍａｘ）增加２３％,ＮＥ的ｒｙａｎｏｄｉｎｅ受体的Ｂｍａｘ则增加１倍,二者比值降低３９％（Ｐ＜００１）;在晚期败血症（ＣＬＰ后１８ｈ）时,ＳＲｒｙａｎｏｄｉｎｅ受体的Ｂｍａｘ降低了３８％,ＮＥ的ｒｙａｎｏｄｉｎｅ受体的Ｂｍａｘ增加１６倍,二者比值降低７６％;ＳＲ和ＮＥｒｙａｎｏｄｉｎｅ受体的离解常数无显著改变。败血症时,ＳＲｒｙａｎｏｄｉｎｅ受体早期上调,晚期下调,而ＮＥｒｙａｎｏｄｉｎｅ受体均上调,这些变化可能与休克时相有关。     

肾上腺髓质素对大鼠损伤性心肌肌浆网功能的改善

通过观察下述五个指标,评价肾上腺髓质素(adrenomedullin,Adm)对大鼠损伤性心肌肌浆网功能的改善程度左心室压力最大变化速率(±dp/dtmax)、肌浆网钙摄取和释放及钙泵活性.皮下注射异丙肾上腺素(isoproterenol,ISO,69μmol/kg体重)制备大鼠心肌损伤坏死模型.摘取心脏后用Adm灌流,观察左心室压力最大变化速率(±dp/dtmax);制备并提纯心肌肌浆网(sarcoplasmicreticulum,SR)膜,测定SRCa2+摄取和释放速率、SR钙泵活性和钙通道蛋白～3H-ryanodine受体的最大结合量.结果发现,5×10-5mol/LAdm灌流能使ISO损伤的大鼠心脏左室±dp/dtmax分别增加16.9%(2?135±281vs1?980±302)和29.2%(1?375±267vs1?064±355,均P<0.05);SRCa2+摄取和释放率分别增加23.0%(15.0±1.4vs12.2±1.2)和43.5%(6.6±1.0vs4.6±0.6,均P<0.01);SRCa2+-ATPase活性和～3H-ryanodine受体最大结合量(Bmax)分别增加24.2%(P<0.01)和42.2%(P<0.05).提示Adm对ISO诱导的大鼠心肌损伤具有保护作用,其机制可能与Adm增加SRCa2+-ATPase活性、增加～3H-ryanodine所致SRCa2+摄取和释放升高有关.外源性给予Adm对损伤心肌可能具有临床治疗作用.     

心肌肥厚中L-型钙通道与ryanodine受体分子间信号耦联的衰退

心脏压力负荷导致心肌肥厚的过程是心衰发生的关键环节。已有研究表明,控制心脏收缩的细胞钙致钙释放过程在心肌肥厚及心衰状态下发生缺损,但分子机制尚未阐明。我们以主动脉结扎手术建立压力负荷的大鼠心肌肥厚模型：实验组分为假手术组、代偿性肥厚组（CHT）和失代偿性肥厚组（DHT）,以松钳一共聚焦成像技术研究单个L-型钙通道（LCC）与ryanodine受体（RyR）间的钙信号耦联。我们发现DHT中LCC—RyR分子耦联潜伏期延长49％,耦联成功率降低47％,失败概率提高72％,证明DHT进入了一种“分子间衰退”状态。出人意料的是,心功能正常的CHT也发生分子间衰退,并与锚定肌质网与细胞膜的junctophilin蛋白表达下降有关,表明分子间耦联衰退在细胞功能变化显现之前已经潜性地发生。与此一致,细胞兴奋期钙释放同步性降低,但钙释放总量和细胞钙瞬变在CHT并无变化。这些结果提示,在一个我们称为“稳定余量”（stability margin）的范围内,分子间耦联衰退不会影响细胞兴奋收缩耦联能力,只有分子间耦联衰退超出稳定余量,心衰才会发生。潜性的分子间耦联衰退的发现对早期防治心衰有重要意义。     

蓝氏贾第虫核被膜缺口的电镜观寨

双滴虫类是迄今所知的现存最原始的真核生物类群。以蓝氏贾第虫作为双滴虫类的代表,对其细胞核进行了电镜观察。除了未见有核仁外,还发现其核被膜的横切面上存在有缺口。在缺口的边缘处,核内膜与校外膜是相互连接着的,表明并非切片时所造成的假象。核被膜缺口处常有一核纤层样的薄层分隔核质与细胞质。用高锰酸钾固定细胞以求只保存膜结构时,核被膜缺口仍然可见,上述的薄层即未见到。核被膜缺口的发现证实了李靖炎（１９７９）的核被膜起源假说所作出的推断。     

慢性缺氧大鼠心肌肌浆网功能及其钙泵基因表达的动态变化

将实验大鼠放置模拟５０００ｍ海拔高度低压舱内１、２和４周。结果表明：与对照组比较,１,２和４周组动物的心肌重量分别增加１５％,１８％和５７％;心肌ＳＲＣａ２＋摄取分别降低３３％,３８％和５３％;心肌ＳＲＣａ２＋ＡＴＰａｓｅ活性分别降低５４％,６０％和７４％;钙泵ｍＲＮＡ含量（基因表达分别降低１４％,４６％和６８％。这些结果提示,缺氧导致的ＳＲ钙泵功能降低可能是心肌功能受损的重要生化基础之一,而钙泵数目减少可能是钙泵功能降低的分子生物学机制。     

Ryanoid modification of the cardiac muscle ryanodine receptor channel results in relocation of the tetraethylammonium binding site

The interaction of ryanodine and derivatives of ryanodine with the high affinity binding site on the ryanodine receptor (RyR) channel brings about a characteristic modification of channel function. In all cases, channel open probability increases dramatically and single-channel current amplitude is reduced. The amplitude of the ryanoid-modified conductance state is determined by structural features of the ligand. An investigation of ion handling in the ryanodine-modified conductance state has established that reduced conductance results from changes in both the affinity of the channel for permeant ions and the relative permeability of ions within the channel (Lindsay, A.R.G., A. Tinker, and A.J. Williams. 1994. J. Gen. Physiol. 104:425-447). It has been proposed that these alterations result from a reorganization of channel structure induced by the binding of the ryanoid. The experiments reported here provide direct evidence for ryanoid-induced restructuring of RyR. TEA+ is a concentration- and voltage-dependent blocker of RyR in the absence of ryanoids. We have investigated block of K+ current by TEA+ in the unmodified open state and modified conductance states of RyR induced by 21-amino-9alpha-hydroxyryanodine, 21-azido-9alpha-hydroxyryanodine, ryanodol, and 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine. Analysis of the voltage dependence of block indicates that the interaction of ryanoids with RyR leads to an alteration in this parameter with an apparent relocation of the TEA+ blocking site within the voltage drop across the channel and an alteration in the affinity of the channel for the blocker. The degree of change of these parameters correlates broadly with the change in conductance of permeant cations induced by the ryanoids, indicating that modification of RyR channel structure by ryanoids is likely to underlie both phenomena.     

Block of the sheep cardiac sarcoplasmic reticulum Ca2+-release channel by tetra-alkyl ammonium cations

Summary The purified ryanodine receptor channel of the sheep cardiac muscle sarcoplasmic reticulum (SR) membrane functions as a calcium-activated cation-selective channel under voltage-clamp conditions following reconstitution into planar phospholipid bilayers. We have investigated the effects of the tetra-alkyl ammonium (TAA) cations, (C _n H_2n+1)₄N⁺ and the trimethyl ammonium cations, ethyltrimethyl ammonium and propyltrimethyl ammonium, on potassium conductance through the receptor channel. Small TAA cations (n = 1–3) and the trimethyl ammonium derivatives act as asymmetric, voltage-dependent blockers of potassium current. Quantitative analysis of the voltage dependence of block indicates that the conduction pathway of the sheep cardiac SR ryanodine receptor channel contains two distinct sites for the interaction of these small organic cations. Sites are located at approximately 50% for tetramethyl ammonium (TMA ⁺) and 90% for tetraethyl ammonium (TEA⁺) and tetrapropyl ammonium (TPrA⁺) of the voltage drop across the channel from the cytosolic face of the protein. The chemical substitution of an ethyl or propyl group for one of the methyl groups in TMA⁺ increases the voltage dependence of block to a level similar to that of TEA ⁺ and TPrA⁺. The zero-voltage dissociation constant (K _b(0)) falls with the increasing number of methyl and methylene groups for those blockers acting 90% of the way across the voltage drop. This is interpreted as suggesting a hydrophobic binding site at this point in the conduction pathway. The degree of block increases as the concentration of small TAA cations is raised. The concentration dependence of tetraethyl ammonium block indicates that the cation interacts with a single site within the conduction pathway with a K _m of 9.8±1.7 mm (mean±sd) at 40 mV. Larger TAA cations (n = 4–5) do not induce voltage-dependent block of potassium current of the form seen with the smaller TAA cations. These data support the contention that the sheep cardiac SR ryanodine receptor channel may be occupied by at most one ion at a time and suggest that a large proportion of the voltage drop falls over a relatively wide region of the conduction pathway.This work was supported by funds from the Medical Research Council and the British Heart Foundation. We would like to thank Richard Montgomery for his considerable help with the chemical synthesis. We are grateful to Drs. John Chambers, Nick Price and staff for showing us the intricacies of NMR spectroscopy.     

Thermodynamics of calmodulin binding to cardiac and skeletal muscle ryanodine receptor ion channels

The skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptor calcium release channels contain a single, conserved calmodulin (CaM) binding domain, yet are differentially regulated by CaM. Here, we report that high-affinity [(35)S]CaM binding to RyR1 is driven by favorable enthalpic and entropic contributions at Ca(2+) concentrations from <0.01 to 100 microM. At 0.15 microM Ca(2+), [(35)S]CaM bound to RyR2 with decreased affinity and binding enthalpy compared with RyR1. The rates of [(35)S]CaM dissociation from RyR1 increased as the temperature was raised, whereas at 0.15 microM Ca(2+) the rate from RyR2 was little affected. The results suggest major differences in the energetics of CaM binding to and dissociation from RyR1 and RyR2.     

The interaction of local anesthetics with the ryanodine receptor of the sarcoplasmic reticulum

The effects of various local anesthetics (LAs) on the skeletal muscle ryanodine receptor were tested. The LAs were divided into three categories according to their effects on the binding of ryanodine to the junctional sarcoplasmic reticulum membranes. Ryanodine binding was assayed in the presence of 0.2 m NaCl and 10 m CaCl₂. Tetracaine and dibucaine inhibit the binding with half-maximal inhibition (CI₅₀) of 0.12 and 0.25 mm, respectively, while inhibition by benzocaine and procaine occurs with CI₅₀ of about 10-fold higher. Lidocaine, its analogue QX-314, and prilocaine, on the other hand, stimulate the binding up to fourfold with half-maximal stimulation occurring with about 2 mm of the drugs. Lidocaine increases both the receptor affinity for ryanodine by about fivefold and the rate of ryanodine association with its binding site by about 10-fold.Tetracaine interacts with the ryanodine receptor in a non-competitive fashion with respect to ryanodine but it competes with lidocaine for its binding site, suggesting the existence of a single site for the inhibitory and stimulatory LA.     

Rapid activation of the cardiac ryanodine receptor by submillisecond calcium stimuli

The local control concept of excitation-contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca(2+) entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (<0.5 ms) Ca(2+) elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca(2+) signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca(2+) generated by flash photolysis of DM-nitrophen. Application of such Ca(2+) spikes (amplitude approximately 10-30 microM, duration approximately 0.1-0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope approximately 2.5) with spike amplitude. The time constants of RyR activation were 0.07-0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a power n approximately 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca(2+)-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca(2+) triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca(2+) ions in accordance with the tetrameric organization of the channel protein.     

The action of carboxyl modifying reagents on the ryanodine receptor/Ca2+ release channel of skeletal muscle sarcoplasmic reticulum

Summary

In this work we show that ryanodine binding to junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor (RyR) is inhibited in a time — and concentration-dependent fashion by prior treatment with the carboxyl reagent dicyclohexylcarbodiimide (DCCD). Exposure of the membrane-bound RyR to the water soluble carboxyl reagents 1-ethyl-3 (3-(dimethylamino) propyl carbodiimide (EDC) or N-ethyl-pheny-lisoxazolium-3 -sulfonate (WRK) only slightly affects their ryanodine binding capacity. The amphipathic reagent N-ethoxy cabonyl-2-ethoxy-1, 2-dihydroquinaline (EEDQ) inhibited ryanodine binding at relatively high concentrations. DCCD-modifica-tion of the SR decreased the binding affinities of the RyR for ryanodine and Ca²⁺ by about 3- and 18-fold, respectively.

The single channel activity of SR membranes modified with DCCD and then incorporated into planar lipid bilayers is very low (5–8%) in comparison to control membranes. Application of DCCD to either the myoplasmic (c/s) or luminal (trans) side of the reconstituted unmodified channels resulted in complete inhibition of their single channel activities. Similar results were obtained with the water soluble reagent WRK applied to the myoplasmic, but not to the luminal side. The DCCD-modified non-active channel is re-activated by addition of ryanodine in the presence of 250_üM Ca²⁺ and is stabilized in a sub-conductance state. With caffeine, ryanodine re-activated the channel in the presence of 100_üM of Ca^2+. The results suggest that a carboxyl residue(s) in the RyR is involved either in the binding of Ca²⁺, or in conformational changes that are produced by Ca²⁺ binding, and are required for the binding of ryanodine and the opening of the Ca²⁺ release channel.     

Spectroscopic determination of sarcoplasmic reticulum Ca2+ uptake and Ca2+ release

In this report we describe the application of spectroscopic methods to the study of Ca2+ release by isolated native sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle. To date, dual-wavelength spectroscopy of arsenazo III and antipyrylazo III difference absorbance have been the most common spectroscopic methods for the assay of SR Ca2+ transport. The utility of these methods is the ability to manipulate intraluminal Ca2+ loading of SR vesicles. These methods have also been useful for studying the effect of both agonists and antagonists upon SR Ca2+ release and Ca2+ uptake. In this study, we have developed the application of Calcium Green-2, a long-wavelength excitable fluorescent indicator, for the study of SR Ca2+ uptake and release. With this method we demonstrate how ryanodine receptor Ca2+ channel opening and closing is regulated in a complex manner by the relative distribution of Ca2+ between extraluminal and intraluminal Ca2+ compartments. Intraluminal Ca2+ is shown to be a key regulator of Ca2+ channel opening. However, these methods also reveal that the intraluminal Ca2+ threshold for Ca2+-induced Ca2+ release varies as a function of extraluminal Ca2+ concentration. The ability to study how the relative distribution of a finite pool of Ca2+ across the SR membrane influences Ca2+ uptake and Ca2+ release may be useful for understanding how the ryanodine receptor is regulated, in vivo.     

The role of the sarcoplasmic reticulum in various types of cardiomyocytes

The relative importance of the sarcoplasmic reticulum (SR) as a source of Ca²⁺ in the excitation-contraction coupling of mammalian myocytes was tested. Shortening and intracellular Ca²⁺ transients of electrically paced, isolated,adult rat myocytes were found to be absolutely dependent on the presence of a functional SR and were completely abolished by the SR Ca²⁺-ATPase inhibitors cyclopiazonic acid and thapsigargin or by the Ca²⁺-release channel opener ryanodine.Neonatal rat cardiomyocytes, on the other hand, elicited consistent intracellular Ca²⁺-transients even after complete functional inhibition of the SR. The transients, however, were markedly prolonged. Also isolatedadult guinea pig myocytes maintained the ability to shorten after a complete inhibition of the SR Ca²⁺-ATPase by either thapsigargin or cyclopiazonic acid. The twitches and the intracellular Ca²⁺-transients, however, were considerably longer after inhibition of the SR Ca²⁺-ATPase. Different results were obtained after preincubation of the cells with 10 M ryanodine to induce emptying of the SR Ca²⁺ pool. In this case, Ca²⁺ spikes and twitches were also markedly reduced in size, in addition to being prolonged. When a SR Ca²⁺-pump inhibitor was added to ryanodine-treated cells, the size of the Ca²⁺-transients and the capacity of the cells to shorten increased. Ryanodine leaves the activity of the Ca²⁺-pump of the SR intact and thus leads to an underestimation of the amount of excitatory Ca²⁺-flowing into the cell.The results show that, while the significance of the SR in regulating the Ca²⁺-transients and shortening of cardiomyocytes varies depending on the species and the stage of development, SR function is of paramount importance for the occurrence of rapid twitches.Abbreviations EGTA ethylene glycol-bis-(beta amino ethyl ether)N,N,N,N tetraacetic acid - MOPS morpholinopropane sulfonic acid - SR sarcoplasmic reticulum - BSA bovine serum albumin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid