ADENOSINE TRIPHOSPHATE-LINKED CONCENTRATION OF CALCIUM IONS IN A PARTICULATE FRACTION OF RABBIT MUSCLE

ATPase and ATP-dependent calcium ion concentration was studied with a membrane fraction isolated from homogenized rabbit skeletal muscle by differential centrifugation. Electron micrographs of the fraction indicate that it consists mainly of resealed tubules and vesicles of the endoplasmic reticulum. The up-to-1400-fold concentration of calcium in this fraction might be explained by proposing the existence of an energy-requiring system for the transport of calcium ions into the tubules or vesicles.     

SEPARATION OF ACETYLCHOLINE AND CATECHOLAMINE CONTAINING SYNAPTIC VESICLES FROM BRAIN CORTEX

Abstract— Synaptic vesicles were prepared from guinea-pig cerebral cortex on a continuous D₂O-H₂O(1:1)-sucrose gradient and purified in the presence of 1 m m -EGTA by chromatography on columns of glass beads of controlled pore size. As markers, endogenous ACh, NA, dopamine and DβH were measured.
Two distinct populations of synaptic vesicles were recognized between the layers of 0.2–0.3 m - and 0.3–0.5 m -sucrose, which differed from each other both in electron microscopic appearance and transmitter content. The less dense vesicles had a much higher ACh content than the more dense vesicles which were composed mainly of somewhat larger particles with high NA and dopamine content. DβH was found to be present in substantial amounts in guinea-pig cortex and was located in the synaptic vesicle fractions having high CA content.
After glass bead chromatography the vesicle preparations were morphologically homogeneous, practically free from other subcellular elements and were contaminated with each other by not more than 10%
The yields were 0.2 and 0.1 mg protein g cortex⁻¹ tissue for 'cholinergic' and 'adrenergic' vesicle preparations, respectively.     

安定对兔心乳头肌动作电位和收缩的作用

本文观察了安定、戊脉安和PK11195对兔心乳头肌动作电位和收缩的作用,并比较了安定对兔与豚鼠心肌收缩作用的差异。安定(0.7×10~(-6)-22.4×10~(-6)mol/L)使兔乙乳头肌动作电位时程(APD)及钙依赖动作电位(Ca-AP)时程缩短,并使Ca-AP的最大上升速率(V_(max))和幅值减小;安定还抑制兔与豚鼠心室乳头肌的收缩,相比之下对豚鼠的作用更明显。在戊脉安(2×10~(-6)mol/L)作用下,兔心乳头肌Ga-AP逐渐消失。提高溶液中Ca~(2 )的浓度可拮抗安定和戊脉安的作用。PK11195(1×10~(-6)mol/L)可使安定对兔心乳头肌APD作用的剂量-反应曲线平行右移,且拮抗安定对Ca-AP的作用。预先加入PK11195(3×10~(-6)mol/L),则戊脉安不能取消Ca-AP。上述结果提示。(1)兔心乳头肌存在的苯二氮(艹卓)结合位点具有外周型苯二氮(艹卓)受体(BZR)的性质;(2)这种受体的激活可导致钙内流减少;(3)BZR的密度及反应性可能存在种属差异。     

THE SARCOTUBULAR SYSTEM OF FROG SKELETAL MUSCLE : A Morphological and Biochemical Study

In the frog skeletal muscle cell a well defined and highly organized system of tubular elements is located in the sarcoplasm between the myofibrils. The sarcoplasmic component is called the sarcotubular system. By means of differential centrifugation it has been possible to isolate from the frog muscle homogenate a fraction composed of small vesicles, tubules, and particles. This fraction is without cytochrome oxidase activity, which is localized in the mitochondrial membranes. This indicates that the structural components of this fraction do not derive from the mitochondrial fragmentation, but probably from the sarcotubular system. This fraction, called sarcotubular fraction, has a Mg⁺⁺-stimulated ATPase activity which differs from that of muscle mitochondria in that it is 3 to 4 times higher on the protein basis as compared with the mitochondrial ATPase, and is inhibited by Ca⁺⁺ and by deoxycholate like the Kielley and Meyerhof ATPase. We therefore conclude that the "granules" of the Kielley and Meyerhof ATPase, which were shown to have a relaxing effect, are fragments of the sarcotubular system. The isolated sarcotubular fraction has a high RNA content and demonstrable activity in incorporating labeled amino acids, even in the absence of added supernatant.     

乌桕桕脂中甾醇和其它成分分离鉴定

通过柱层析、薄层层析等化学方法,从乌桕桕脂中分离得到甾醇及脂肪酸酯类化合物。经气相色谱和气-质联用分析,鉴定出下列化合物:β-谷甾醇、菜油甾醇、Δ~7-豆甾烯醇、肉豆蔻酸甲酯、肉豆蔻酸乙酯、棕榈酸甲酯、硬酯酸甲酯和硬酯酸乙酯。     

THE ELECTRICAL IMPEDANCE OF MUSCLE DURING THE ACTION OF NARCOTICS AND OTHER AGENTS

1. The effect of certain inorganic cations upon the electrical impedance of the sartorius muscle of the frog was investigated. While Na, K, and Mg have little effect upon the resistance of muscle, Ba and Ca cause it to fall. The use of physiologically "unbalanced" salt solution does not in itself seem to affect muscle impedance. 2. The time course of the effect upon muscle impedance of the penetration of substances into the intercellular spaces was studied by treating the muscle with sugar solutions. Half of the effect is over in three-quarters of a minute when the sugar solution is permitted to circulate past both sides of the muscle. This sets an upper limit for the time necessary for inorganic cations and organic narcotics to reach the cell surfaces. The action of inorganic cations and organic narcotics upon muscle is slow compared to the time necessary for them to reach the scene of action. The penetration of the sugar solutions into the intercellular spaces of muscle was found to follow the well known diffusion law, the amount diffusing in being proportional to the square root of the time. Average values of 77.7 per cent for ρ, the volume concentration of fibers; 231 ohms specific resistance for r ₂, the resistance of the interior of the fibers; and 71.0° for θ, the phase angle of the impedance locus, were obtained for the muscle in Ringer''s solution. How these values change when the muscle is placed in various concentrations of sugar was also studied. 3. The action of a number of organic narcotics upon muscle was studied. All decrease 1000 cycle resistance if the concentration is sufficiently high. A detailed analysis of the action of the narcotic, iso-amyl carbamate, was made, and it was noted that low concentrations increase resistance while higher concentrations decrease it. By investigating the effect of narcotics upon muscle impedance over a wide frequency range, it was found that during narcosis the resistance of the fiber membranes first increases and then decreases, and, if the drug is present in sufficiently great concentration, membrane resistance may completely disappear. Membrane capacity is only very slightly affected.     

脑啡肽在豚鼠、兔回肠环肌收缩中的作用

豚鼠、兔回肠的未分离环肌,在电场刺激下,可发生缓慢的节律性收缩。此收缩可被纳洛酮完全消除,或被阿托品部分减弱。与此相反,亮脑啡肽和乙酰胆碱司使节律性收缩明显增强。此外,单独用乙酰胆碱(4.3—5.7μg/ml)或亮脑啡(1.4—2.8μg/ml)作用于分离环肌时,均未能使其发生任何收缩反应。但在乙酰胆碱4.3μg/ml作用的基础上,仅用1.4μg/ml的亮脑啡肽,或再用电场刺激时,则均能使其发生明显的收缩。上述结果提示,环肌的收缩可能需要脑啡肽和乙酰胆碱共同作用。本实验的结果还表明,脑啡肽对环肌收缩的作用与对纵肌相反,它使前者兴奋,使后者抑制。     

MITOCHONDRIA IN CARDIAC MUSCLE CELLS OF THE CANARY AND SOME OTHER BIRDS

The fine structure of mitochondria from the ventricular myocardium of canaries, sparrows, zebra finches, quail, and geese has been studied. The first three of these birds have very fast heart rates, the quail being intermediate, and the goose has a relatively slow rate. The canary heart has a unique form of mitochondrion containing large, parallel arrays of zigzag or angled cristae. Other cristae, continuous with the zigzag ones and also occupying large parts of the mitochondrial volume, are named retiform because of the hexagonal network which they form, sometimes in a single plane and sometimes three dimensional. These two types of cristae appear to be interconnectible. It is possible that there is a direct functional significance in these peculiar forms, but, in any case, the relative constancy of dimensions in these arrays is probably related to specific properties of the molecules of which the cristal membrane is composed. It is also demonstrated that this membrane is composed in part of approximately 30-A particles which are believed to be protein molecules. This unusual mitochondrial morphology is not seen either in the other fast bird hearts or in the slower ones, so that there is neither a simple correlation with heart rate nor probably with the separate parts of the cardiac cycle. Although none of the other four hearts shows more than an occasional angled crista, there does seem to be a rather gross correlation between heart rate and mitochondrial size and complexity of crista structure, but no correlation with presence or absence of zigzag forms. The cristae of quail heart mitochondria are disposed in unusually large close-packed whorls.     

MORPHOLOGY OF ISOLATED RABBIT HEART MUSCLE MITOCHONDRIA AND THE OXIDATION OF EXTRAMITOCHONDRIAL REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE

The morphology of rabbit heart muscle mitochondria isolated in several media has been compared by electron microscopy. The internal structure of isolated mitochondria differs from that of in situ mitochondria, with the type and degree of alteration depending on the isolation medium. Examination of the isolated mitochondria after incubation revealed that additional morphological changes occurred during incubation, but these changes were less pronounced when the incubation was conducted in a complete medium containing substrate. The isolated mitochondria have been shown to be capable of catalyzing a slow aerobic oxidation of extramitochondrial reduced diphosphopyridine nucleotide. The rate of DPNH oxidation observed is sufficient to account for the ability of the mitochondria to oxidize lactate in the presence of catalytic amounts of DPNH. The suspensions used were essentially free of mitochondrial fragments, which are known to oxidize DPNH. Possible relationships of these findings to metabolism in situ are discussed. The results indicate the desirability of correlating biochemical activities with the morphology of isolated mitochondria.     

RELEASE OF GLUTAMATE AND OTHER AMINO ACIDS FROM ARTHROPOD NERVE–MUSCLE PREPARATIONS

—A study has been made of the release of glutamate and other amino acids from perfused leg preparations from Carcinus and Schistocerca, at rest and during stimulation of motor nerves to the leg muscles. Stimulation caused an increase in release af many amino acids. No evidence was obtained to link glutamate release from such preparations with synaptic function. If release of transmitter glutamate is to be demonstrated in arthropods, techniques must be devised to avoid contamination with large quantities of non-transmitter glutamate.     

ON THE DEVELOPMENT OF SYMPATHETIC NERVE TRUNK VESICLES DURING AXONAL TRANSPORT: DENSITY GRADIENT ANALYSIS OF DOPAMINE β-HYDROXYLASE IN BOVINE SPLENIC NERVE

—Dopamine β-hydroxylase was used as a marker enzyme for sympathetic nerve vesicles which were studied by density gradient technique in bovine splenic nerves. The enzyme analyses were complicated by the occurrence of inhibitors which had to be carefully neutralized with copper. The inhibitor was mainly found in the soluble fraction and no evidence for the occurrence of endogenous inhibitors in the nerve vesicles was obtained. A great variation in density of the dopamine β-hydroxylase containing particles was observed. This was probably mainly due to the variation in vesicle maturation since dopamine β-hydroxylase was distributed more towards the lighter gradient fractions in the proximal nerve segment preparations compared with intrasplenic nerve segment preparations. Noradrenaline/protein and noradrenaline/dopaminc β-hydroxylase ratios were found to be increased about 1·7-fold in the vesicle fraction isolated from the proximal nerve segments to those from the intrasplenic segments. A further increase of the noradrenaline/dopamine β-hydroxylase ratio was observed in a fraction with the same density isolated from the spleen. On the basis of these findings the noradrenaline/protein ratio was calculated to be about 500-600 nmol/mg in the nerve terminal vesicles.     

家兔腓肠肌肌构筑及肌纤维型的实验研究

根据家兔（Ｏｒｙｃｔｏｌａｇｕｓｃｕｎｉｃｕｌｕｓｄｏｍｅｓｔｉｃａ）腓肠肌的肌构筑和神经供应特征,将该肌外侧头分为内侧、中间、外侧３个亚体,内侧头无肌亚体。用家兔８例１６侧腓肠肌,按上述３个亚体及内侧头分别取材,做冰冻横切片,肌动球朊ＡＴＰ酶染色,将肌纤维分为ＳＯ型、ＦＯＧ型和ＦＧ型。检测各亚体的肌纤维型构成比率,并以图象分析仪测量３型肌纤维的直径和横切面积。结果发现：ＳＯ型纤维在内侧头占１６．７％,在外侧头由内侧亚体的１１．３％向外侧亚体的２０．８％依次递增;ＦＯＧ型纤维由内侧亚体的２３．２％向外侧亚体的３０．９％递增。ＦＧ型纤维则从内侧亚体的６５．５％向外侧亚体的４８．３％逐渐递减。３型肌纤维成点缀式镶嵌型分布。各亚体中ＳＯ型纤维最细（５７．４—５８．２μｍ）,ＦＯＧ型纤维稍粗（６０．１—６１．１μｍ）,ＦＧ型纤维最粗（６３．６—６４．５μｍ）。     

SEPARATION OF HELA CELLS BY COLLOIDAL SILICA DENSITY GRADIENT CENTRIFUGATION : I. Separation and Partial Synchrony of Mitotic Cells

Using a colloidal silica density gradient, HeLa cells in mitosis were found to have a density of 1.040–1.046 g/cc, lighter than the remaining interphase cells. The mitotic cells could be harvested and cultured after centrifugation, showing growth synchrony by measurement of a peak in mitotic index 21 hr after establishing the culture. By using Colcemid or vinblastine sulfate, HeLa cells were arrested in metaphase and centrifuged on the colloidal silica density gradient. The blocked metaphase cells were lighter in density than the interphase cells but somewhat more dense than untreated cells selected by the density gradient centrifugation. Near-equilibrium conditions were established during the centrifugation of cells so that cell density measurements could be made, and the gradient medium employed was not measurably toxic to those cells tested.     

兔输卵管峡部平滑肌阵发性活动与卵巢激素的关系

本文记录了不同卵巢激素状态下兔离体输卵管峡部平滑肌收缩活动及肌电变化。成年动情兔和排卵兔输卵管峡部活动强,呈现阵发性收缩或放电;幼兔及切除卵巢成年兔峡部活动弱,无典型的阵发收缩,若给这种兔补充雌激素阵发性收缩又可出现。结果表明峡部平滑肌阵发性活动与卵巢激素有密切关系,雌激素在维持阵发性活动中起着相当重要的作用。     

兔肺内小动脉平滑肌细胞培养

肺内小动脉用低浓度胰蛋白酶从肺动脉灌洗后,分离并剪碎,反复用培养基清洗。将沉淀的组织块行肺内小动脉平滑肌细胞培养。培养的细胞经光镜、透射电镜及免疫组化验证为典型的平滑肌细胞。此法简单、方便,可推广应用于各种小血管平滑肌细胞培养,为阻力血管、容量血管疾病的研究提供了理想的实验材料。     

RU486对兔输卵管平滑肌的收缩效应

应用肌肉机械－电换能器和Ｇｉｌｓｏｎ生理记录仪,观察ＲＵ４８６对假孕４ｄ兔离体输卵管平滑肌的收缩效应。结果显示：（１）ＲＵ４８６可直接作用输卵管平滑肌,使其收缩频率增加,而未明显改变收缩张力及振幅,与在体肌内注射ＲＵ４８６观察到的结果相似;（２）ＲＵ４８６部分抑制ｃａ~（２＋）诱发的平滑肌收缩活动,它还与Ｖｅｒａｐａｍｉｌ诱发的抑制效应有协同作用,与ＮＥ诱发的收缩张力有拮抗作用,而对Ｆｏｒｓｋｏｌｉｎ诱发的效应未产生任何影响。以上结果表明,ＲＵ４８６对输卵管平滑肌的作用似乎是改变细胞内游离Ｃａ（２＋）的结果,可能干扰Ｃａ（２＋）的流入、或／和内质网Ｃａ（２＋）释放以及Ｃａ（２＋）－Ｉｐ３信息传递机制。