消逝波显微镜观察“一吻即逝”的胰岛素出胞作用

目的用消逝波显微镜观察胰岛β细胞瘤来源的Ins-1细胞的胰岛素出胞作用,旨在为"一吻即逝"胰岛素分泌方式提供新的证据。方法用含囊泡相关膜蛋白2和增强型绿色荧光蛋白融合蛋白基因的质粒标记Ins-1细胞的胰岛素分泌囊泡,用含高葡萄糖和高氯化钾的溶液刺激Ins-1细胞,用基于物镜的消逝波显微镜观察胰岛素的出胞作用。结果 Ins-1细胞经高葡萄糖高氯化钾溶液刺激后,消逝波显微镜可观察到两种现象:①荧光基团(亮点)在细胞膜出现并一直或长期停留在细胞膜;②荧光基团在细胞膜出现,停留较短时间后又逐渐消失(离开细胞膜)。前者应属于传统的膜融合分泌方式,即胰岛素分泌囊泡与细胞膜融合,其内容物被释放,囊泡膜成为细胞膜的一部分。后者应属于"一吻即逝"分泌方式,即胰岛素分泌囊泡与细胞膜接触并部分融合,释放部分内容物后,囊泡离开细胞膜,囊泡膜不成为细胞膜的一部分。结论该项研究结果支持胰岛素的分泌方式,除了传统的膜融合方式外,还存在"一吻即逝"的分泌方式。     

神经垂体多肽激素分泌颗粒释放形式和转运途径的形态学研究

目的探讨神经垂体多肽激素分泌颗粒的释放形式和在细胞外正常转运途径的结构基础。方法对大鼠神经垂体进行光镜、透射电镜和扫描电镜观察。结果神经垂体主要由无髓神经纤维、垂体细胞及富含毛细血管的结缔组织组成。毛细血管内皮为有孔型(50nm)，外有基膜与血管周隙分隔。完整的大型膜包分泌颗粒(100～300nm)不仅大量存在于无髓神经纤维内，而且也见于血管周隙内。垂体囊由单层扁平上皮细胞和上皮下结缔组织构成，囊上皮细胞间存在许多不规则的囊上皮孔(2～5μm)，上皮孔附近经常可见分泌颗粒。结论这些结构特征提示，神经垂体多肽激素或分泌颗粒的释放形式，存在一种连同颗粒被膜的整体释放；释放入血管周隙内的多肽激素或分泌颗粒，更易经组织间隙、囊上皮孔进入脑脊液发挥旁分泌作用，而非直接通过毛细血管壁进入血液。     

大鼠松果体囊上皮表面的扫描电镜观察

目的 探讨松果体激素释放到蛛网膜下隙脑脊液途径的结构基础.方法 对1.5月龄和12月龄大鼠松果体浅部,进行松果体囊表面的扫描电镜观察.结果 在大鼠松果体囊表面发现存在筛状囊上皮孔和喇叭口状囊上皮孔.筛状囊上皮孔主要存在于1.5月龄大鼠的松果体囊上皮,是由许多密集排列的囊上皮细胞周边部细胞膜凹陷形成的圆形孔洞,筛孔直径200～500 nm;喇叭口状囊上皮孔见于1.5月龄和12月龄大鼠的松果体囊表面,由相邻上皮细胞向松果体内部凹陷,形成圆形或椭圆形开口,孔径1～4 μm.在两组动物的松果体囊表面,可见到直径800～1 000 nm的球形分泌颗粒样结构.结论 松果体分泌颗粒的释放形式,可能存在连同颗粒被膜的整体释放;松果体激素或分泌颗粒,可能会通过松果体囊上皮孔,直接进入蛛网膜下隙的脑脊液.     

胎儿垂体前叶激素释放的超微结构研究

本文通过5例水囊引产、不同胎龄胎儿垂体前叶超微结构研究,从形态上证明前叶激素的释放有多种方式:1.胞吐和错位胞吐;2.以胞质小块释放入血窦:有突起的细胞将突起伸入血管间隙,末端与细胞脱离形成含分泌颗粒的胞质小块,经内皮窗孔进入血窦;3、越膜渗出:分泌颗粒内容物跨越颗粒膜和细胞膜渗出细胞;4.有脑颗粒的排出;5.血窦内皮的吞噬和转运作用。[关     

胞外囊泡在肿瘤发生发展中的研究进展

<正>胞外囊泡(extracellular vesicles,EVs)是释放到细胞外的膜性小囊泡,是传递细胞间信号的一种新方式,在生理或病理情况下发挥重要调节作用。几乎所有类型的细胞均可以产生并释放EVs,尤其是肿瘤细胞~([1-2])。越来越多的证据表明,在肿瘤的发生和发展过程中,肿瘤来源的胞外囊泡能够通过传递囊泡中的内容物来改变受体细胞的生物学功能,比如导致免疫抑制,诱导血管生成,甚至促进肿瘤转移~([3-4])。     

胰组织结构提示多肽胰岛素的正常转运经淋巴而非门脉途径

目的　探讨胰岛多肽激素释放形式和细胞外正常转运途径的结构基础。方法　对大鼠和人胰尾部组织进行了光镜 (LM )和透射电镜 (TEM)观察。结果　胰组织主要由腺泡构成的腺小叶和分隔腺小叶的结缔组织所组成。胰腺的导管、血管和淋巴管 ,有髓和无髓神经纤维 ,均穿行于胰结缔组织内。在胰岛周围的结缔组织间隙内也可见到完整的膜包分泌颗粒 (2 0 0～ 5 0 0nm)。胰腺的毛细血管为窗孔 (5 0nm)型。结论　胰组织结构特点提示 :胰岛多肽激素的释放形式 ,可能是连同颗粒膜的整体释放而非传统认为的胞吐分泌 ;释放入胰组织液中的多态胰岛素或分泌颗粒 ,更易进入淋巴而非门脉血液     

大鼠神经垂体催产素能神经元内神经分泌颗粒的免疫电镜观察

目的　观察大鼠神经垂体催产素（ＯＴ）能神经元轴突及终末内颗粒囊泡形态学及免疫细胞化学特点。方法　应用电镜包埋后免疫细胞化学方法,对大鼠神经垂体进行ＯＴ免疫染色。结果　在ＯＴ能神经元轴突终末内,可见ＯＴ免疫反应性的致密核芯大颗粒囊泡和无ＯＴ免疫反应性的清亮小泡存在。部分囊泡与轴膜靠近,并可见颗粒囊泡与轴膜融合形成的胞吐现象;在轴突非终末部分,也可见ＯＴ免疫反应性的颗粒囊泡,散在分布轴浆内,还可见无ＯＴ免疫反应性的颗粒囊泡,呈散在或聚集分布,两种囊泡均可位于微管之间。结论　位于轴突和轴突终末内的囊泡,在形态学和ＯＴ免疫染色方面有明显差异,推测它们在功能上也有不同。     

神经垂体分泌物释放入脑脊液主要途径结构基础的电镜研究

目的探讨神经垂体分泌物直接释放入脑脊液途径的结构基础。方法透射电镜和扫描电镜观察大鼠神经垂体。结果神经垂体无髓神经纤维终止于毛细血管或垂体细胞周围,垂体细胞突起与神经纤维膨体及终末之间形成细胞间池。有孔型毛细血管内皮外有基膜与血管周隙分隔,血管周隙内可见到完整的膜包分泌颗粒(100~300 nm)。神经垂体表面的囊上皮孔(2~5μm)附近经常可见分泌颗粒。结论细胞间池-血管周隙-囊上皮孔,构成了神经垂体分泌物释放入脑脊液主渠道的结构基础。     

免疫组化电镜示垂体LH细胞

应用免疫组化电镜显示垂体黄体生成素(LH)细胞的形态特徵、超微结构以及反应阳性颗粒的分布状况等,并按此将LH细胞分为四型。一、二型细胞内大小分泌粒均含有LH;三型细胞长(?)的150 nm及200 nm颗粒显免疫反应阳性,且部分胞膜和微绒毛顶部界膜内外附着致密细粒;四型细胞出现反应阴性颗粒,仅在rER膜囊外显有直径30～40 nm黑色细粒。LH细胞的分泌可能以外排、分子渗透和局部分泌三种方式排出LH。     

Exosome一种新型的潜在的抗肿瘤疫苗

胞外体是真核细胞释放的, 含有多种细胞膜分子以及相关蛋白的小囊泡.系由细胞内的多泡体(multivesicular bodies, MVBs)膜与细胞膜融合后释放到细胞外环境中囊泡状结构, 被称之为胞外体(exosome, EXO).     

Elementary properties of spontaneous fusion of peptidergic vesicles: fusion pore gating

The release of hormones and neurotransmitters by regulated exocytosis requires the delivery of secretory vesicles to the plasma membrane, where they dock and become primed for fusion with the plasma membrane. Upon stimulation a fusion pore is formed through which cargo molecules diffuse out of the vesicle lumen into the extracellular space. After the cargo release the fusion pore either closes (kiss-and-run, transient exocytosis), fluctuates between an open and a closed state (for short times, fusion pore flickering, or for rather longer periods, 'pulsing pore') or expands irreversibly (full fusion exocytosis). In almost all secretory cells spontaneous secretion of vesicle cargo can be detected in the absence of stimulation. Spontaneous and stimulated exocytosis were thought to exhibit similar properties at elementary level, differing only in the probability of occurrence. However, recent studies indicate that spontaneous exocytosis differs from the stimulated one in many respects, therefore opening questions about the physiological role of spontaneous exocytosis. In this report we address the elementary properties of spontaneous and stimulated peptidergic vesicle discharge which appears to be modulated by fusion pore conductance (diameter) and fusion pore gating.     

Neurotransmitter release through the exocytotic fusion pore

The exocytotic fusion pore transiently connects the secretory granule and plasma membrane during secretory vesicle fusion. Recent evidence on the properties of the fusion pore measured electrophysiologically suggest that the fusion pore is a lipid structure formed by a tension-driven fusion mechanism. We propose that a macromolecular scaffold of proteins directs and regulates the fusion process by drawing a characteristic plasma membrane dimple into close proximity with the secretory granule. Although the identity of the proteins making up the scaffold are largely unknown, studies on the regulation of exocytosis implicate the GTP binding protein Rab3 and unknown Ca²⁺ binding proteins as part of the scaffold, which appears to behave as a coincidence detector. Simultaneous measurement of fusion and the release of secretory products, along with some unusual properties of secretory granule matrices, have led to the unexpected conclusion that the release of secretory products from the matrix may also be regulated.     

Activation of GPCRs modulates quantal size in chromaffin cells through G(betagamma) and PKC

Exocytosis proceeds by either full fusion or 'kiss-and-run' between vesicle and plasma membrane. Switching between these two modes permits the cell to regulate the kinetics and amount of secretion. Here we show that ATP receptor activation reduces secretion downstream from cytosolic Ca2+ elevation in rat adrenal chromaffin cells. This reduction is mediated by activation of a pertussis toxin-sensitive G(i/o) protein, leading to activation of G(betagamma) subunits, which promote the 'kiss-and-run' mode by reducing the total open time of the fusion pore during a vesicle fusion event. Furthermore, parallel activation of the muscarinic acetylcholine receptor removes the inhibitory effects of ATP on secretion. This is mediated by a G(q) pathway through protein kinase C activation. The inhibitory effects of ATP and its reversal by protein kinase C activation are also shared by opioids and somatostatin. Thus, a variety of G protein pathways exist to modulate Ca2+-evoked secretion at specific steps in fusion pore formation.     

Presynaptic vesicles, exocytosis, membrane fusion and basic physical forces

The theoretical hypothesis is presented trying to explain the vesicle release from presynaptic nerve ending and membrane fusion. This theoretical concept implies only essential physical forces such as electrostatic force and surface tension force. Transmembrane resting potential of approximately -70 to -80 mV means that the intracellular fluid is electronegative in comparison with extracellular one. In this concept it is supposed that the inner and outer lipid layer of the membrane also have different electrostatic charges. Presynaptic vesicles are made from cell membrane by endocytic process through which the vesicle loses the contact with cell membrane. Also, during the endocytic process, the inner lipid layer of the cell membrane becomes the outer lipid layer of presynaptic vesicle and vice versa. During the resting phase, equally charged lipid layers of presynaptic vesicle and cell membrane repel each other, but during the action potential, differently charged lipid layers strongly attract each other, bringing the presynaptic vesicle and cell membrane in close contact. Immediately thereafter, the surface tension forces open the pore and fuse both membranes trying to minimize the area of the contact between water fluids (extra and intracellular fluid) and lipid fluids (lipid membrane bilayer). Since only fundamental physical forces are involved in this process, it could be very fast, effective and almost inexhaustible. Similar mechanisms could be responsible for all exocytic processes and all membrane fusion processes in the cells.     

Role of Rab GTPases in membrane traffic and cell physiology

Intracellular membrane traffic defines a complex network of pathways that connects many of the membrane-bound organelles of eukaryotic cells. Although each pathway is governed by its own set of factors, they all contain Rab GTPases that serve as master regulators. In this review, we discuss how Rabs can regulate virtually all steps of membrane traffic from the formation of the transport vesicle at the donor membrane to its fusion at the target membrane. Some of the many regulatory functions performed by Rabs include interacting with diverse effector proteins that select cargo, promoting vesicle movement, and verifying the correct site of fusion. We describe cascade mechanisms that may define directionality in traffic and ensure that different Rabs do not overlap in the pathways that they regulate. Throughout this review we highlight how Rab dysfunction leads to a variety of disease states ranging from infectious diseases to cancer.     

Secretion without membrane fusion: porocytosis

We have recently proposed a mechanism to describe secretion, a fundamental process in all cells. That hypothesis, called porocytosis, embodies all available data and encompasses both forms of secretion, i.e., vesicular and constitutive. The current accepted view of exocytotic secretion involves the physical fusion of vesicle and plasma membranes; however, that hypothesized mechanism does not fit all available physiological data. Energetics of apposed lipid bilayers do not favor unfacilitated fusion. We consider that calcium ions (e.g., 10(-4) to 10(-3) M calcium in microdomains when elevated for 1 ms or less), whose mobility is restricted in space and time, establish salt bridges among adjacent lipid molecules. This establishes transient pores that span both the vesicle and plasma membrane lipid bilayers; the diameter of this transient pore would be approximately 1 nm (the diameter of a single lipid molecule). The lifetime of the transient pore is completely dependent on the duration of sufficient calcium ion levels. This places the porocytosis hypothesis for secretion squarely in the realm of the physical and physical chemical interactions of calcium and phospholipids and places mass action as the driving force for release of secretory material. The porocytosis hypothesis that we propose satisfies all of the observations and provides a framework to integrate our combined knowledge of vesicular and constitutive secretion.     

Hydrogen ions control synaptic vesicle ion channel activity in Torpedo electromotor neurones

During exocytosis the synaptic vesicle fuses with the surface membrane and undergoes a pH jump. When the synaptic vesicle is inside the presynaptic nerve terminal its internal pH is about 5.5 and after fusion, the inside of the vesicle comes in contact with the extracellular medium with a pH of about 7.25. We examined the effect of such pH jump on the opening of the non-specific ion channel in the synaptic vesicle membrane, in the context of the post-fusion hypothesis of transmitter release control. The vesicles were isolated from Torpedo ocellata electromotor neurones. The pH dependence of the opening of the non-specific ion channel was examined using the fused vesicle-attached configuration of the patch clamp technique. The rate of opening depends on both pH and voltage. Increasing the pH from 5.5 to 7.25 activated dramatically the non-specific ion channel of the vesicle membrane. The single channel conductance did not change significantly with the alteration in the pH, and neither did the mean channel open time. These results support the hypothesis that during partial fusion of the vesicle with the surface membrane, ion channels in the vesicle membrane open, admit ions and thus help in the ion exchange process mechanism, leading to the release of the transmitter from the intravesicular ion exchange matrix. This process may have also a pathophysiological significance in conditions of altered pH.     

Vacuolar ATPase in phago(lyso)some biology

Many eukaryotic cells ingest extracellular particles in a process termed phagocytosis which entails the generation of a new intracellular compartment, the phagosome. Phagosomes change their composition over time and this maturation process culminates in their fusion with acidic, hydrolase-rich lysosomes. During the maturation process, degradation and, when applicable, killing of the cargo may ensue. Many of the events that are pathologically relevant depend on strong acidification of phagosomes by the ‘vacuolar’ ATPase (V-ATPase). This protein complex acidifies the lumen of some intracellular compartments at the expense of ATP hydrolysis. We discuss here the roles and importance of V-ATPase in intracellular trafficking, its distribution, inhibition and activities, its role in the defense against microorganisms and the counteractivities of pathogens.     

Tethering Membrane Fusion: Common and Different Players in Myoblasts and at the Synapse

Membrane fusion is essential for the communication of membrane-defined compartments, development of multicellular organisms and tissue homeostasis. Although membrane fusion has been studied extensively, still little is known about the molecular mechanisms. Especially the intercellular fusion of cells during development and tissue homeostasis is poorly understood. Somatic muscle formation in Drosophila depends on the intercellular fusion of myoblasts. In this process, myoblasts recognize each other and adhere, thereby triggering a protein machinery that leads to electron-dense plaques, vesicles and F-actin formation at apposing membranes. Two models of how local membrane stress is achieved to induce the merging of the myoblast membranes have been proposed: the electron-dense vesicles transport and release a fusogen and F-actin bends the plasma membrane. In this review, we highlight cell-adhesion molecules and intracellular proteins known to be involved in myoblast fusion. The cell-adhesion proteins also mediate the recognition and adhesion of other cell types, such as neurons that communicate with each other via special intercellular junctions, termed chemical synapses. At these synapses, neurotransmitters are released through the intracellular fusion of synaptic vesicles with the plasma membrane. As the targeting of electron-dense vesicles in myoblasts shares some similarities with the targeting of synaptic vesicle fusion, we compare molecules required for synaptic vesicle fusion to recently identified molecules involved in myoblast fusion.     

Phospholipases and fatty acid signalling in exocytosis

Vesicle fusion is a ubiquitous biological process involved in general membrane trafficking and a variety of specialized events, for example release of neurotransmitters and hormones, sperm acrosome exocytosis, plasma membrane repair and neurite outgrowth. Many vesicle fusion events have long been known to be activated by phospholipases and products of their activity, such as polyunsaturated arachidonic acid. Polyunsaturated fatty acids (PUFAs) have been proposed to have a number of multiple effectors, including ion channels and the cytoskeleton, but the precise mechanism of PUFA action is still unclear. It was recently reported that omega-3 and omega-6 PUFAs can act on syntaxin, a plasma membrane protein directly involved in vesicle fusion. In this review, we will discuss the role of this new mode of PUFA action in exocytosis.