Cytosolic calcium during contraction of isolated mammalian gastric muscle cells

During activation of visceral smooth muscle there is an increase in cytosolic-free calcium, but the source (intracellular calcium release or calcium influx), kinetics, and stoichiometry of this increase have not been determined. Here, the fluorescent indicator, quin2-acetoxymethyl ester, was used to measure directly cytosolic-free calcium during contraction of isolated stomach muscle cells induced by the two neuropeptides cholecystokinin-octapeptide and Met-enkephalin as well as acetylcholine. An increase in cytosolic-free calcium was seen that was (i) dependent on the concentration of contractile agonist, (ii) derived from intracellular sources (that is, not significantly affected by removal of ambient calcium or addition of a calcium channel blocker), and (iii) kinetically and stoichiometrically related to net calcium efflux and contraction. In contrast, the increase in cytosolic-free calcium induced by depolarizing concentrations of potassium was caused by influx of calcium through voltage-dependent calcium channels.     

拳参-413对大鼠离体胸主动脉环的舒张作用机制研究

[目的]研究拳参-413对大鼠离体胸主动脉血管的舒张作用机制。[方法]采用离体血管环灌流方法观察拳参-413在含Ca+或无Ca+的Krebs液孵育条件下对去甲肾上腺素(NA)引起的血管平滑肌收缩的影响,考察拳参-413舒张血管作用的时间依赖性,并观察拳参-413对浓度40和80 mmol/L的KCl引起的血管平滑肌收缩的影响。[结果]拳参-413能舒张NA引起的血管收缩,且呈浓度依赖性;拳参-413(100μmol/L)在30 min达到最大舒张效应;无Ca+组拳参-413抑制NA所致血管平滑肌收缩效应大于含Ca+组;拳参-413对浓度40和80 mmol/L的KCl引起的血管平滑肌收缩均有抑制作用,且两者量效曲线明显上移。[结论]拳参-413可舒张血管平滑肌,其作用机制可能与该药促进NO合成释放,开放钙激活的钾通道以及抑制血管平滑肌细胞外钙内流和内钙释放有关。     

影响獭兔离体肠道平滑肌收缩的因素

[目的]探讨影响獭兔离体肠道平滑肌收缩的因素。[方法]以獭兔十二指肠平滑肌为试验材料,以平滑肌收缩波的振幅和周期为评价指标,考察影响试验的各主要因素。[结果]在(36.5±0.5)℃台氏液中,獭兔离体肠管的存活时间和保质期最长,振幅最稳定,肾上腺素使离体肠管平滑肌收缩幅度减弱,乙酰胆碱、氢氧化钠及氯化钙均导致平滑肌收缩频率及幅度升高,收缩力增强。[结论]獭兔离体肠道平滑肌易受到温度、pH、其他化学因素及药物的影响。     

Regulation of calcium concentration in voltage-clamped smooth muscle cells

The regulation of intracellular calcium concentration in single smooth muscle cells was investigated by simultaneously monitoring electrical events at the surface membrane and calcium concentration in the cytosol. Cytosolic calcium concentration rose rapidly during an action potential or during a voltage-clamp pulse that elicited calcium current; a train of voltage-clamp pulses caused further increases in the calcium concentration up to a limit of approximately 1 microM. The decline of the calcium concentration back to resting levels occurred at rates that varied with the calcium concentration in an apparently saturable manner. Moreover, the rate of decline at any given calcium concentration was enhanced after a higher, more prolonged increase of calcium. The process responsible for this enhancement persisted for many seconds after the calcium concentration returned to resting levels. Thus, the magnitude and duration of a calcium transient appear to regulate the subsequent calcium removal.     

Vasoconstriction: a new activity for platelet-derived growth factor

Platelet-derived growth factor (PDGF) is a potent mitogen for vascular smooth muscle cells that has been implicated in the pathogenesis of atherosclerosis. The potential role of PDGF in the altered vasoreactivity of atherosclerotic vessels has been studied through an examination of its effects on contractility in the rat aorta. PDGF caused a concentration-dependent contraction of aortic strips and was significantly more potent on a molar basis than the classic vasoconstrictor peptide angiotensin II. Furthermore, PDGF increased the cytosolic free calcium concentration in cultured rat aortic smooth muscle cells. These observations suggest a new biological activity for PDGF that may contribute to the enhanced vasoreactivity of certain atherosclerotic vessels.     

Free calcium at rest during "catch" in single smooth muscle cells

Tension and intracellular free calcium concentration [( Ca2+]i) were measured simultaneously in single smooth muscle cells isolated from the anterior byssus retractor muscle (ABRM) of Mytilus edulis that were loaded with the fluorescent Ca2+ indicator fura-2. Electrical stimulation evoked a transient elevation of [Ca2+]i associated with a "catch" contraction. During the catch state, however, [Ca2+]i was effectively at its resting level and was unaffected by 5-hydroxytryptamine, which induced a rapid relaxation from catch. The results indicate that a maintained high [Ca2+]i is not required for the maintenance of catch tension in intact ABRM and that there was no significant change in [Ca2+]i upon abolition of catch.     

Calcium-dependent stress maintenance without myosin phosphorylation in skinned smooth muscle

Stress development depended on calcium-stimulated myosin phosphorylation in an arterial smooth muscle preparation in which the concentration of calcium was controlled. However, developed stress was maintained at a concentration of calcium that did not support phosphorylation. These results, in conjunction with other evidence, suggest that the interaction of two regulatory mechanisms with different calcium sensitivities regulate both stress and the rate and energetics of contraction.     

血根碱对兔离体肠平滑肌运动的舒张作用

【目的】探讨血根碱对兔离体肠平滑肌的舒张作用及机制。【方法】采用离体肠肌试验,应用BL-420E生物机能实验系统,比较血根碱对正常肠肌及工具药乙酰胆碱、酚妥拉明、氯化钡作用下兔离体肠平滑肌活动的影响,记录肠肌的收缩频率和振幅,并计算抑制率。【结果】血根碱可浓度依赖性的抑制兔小肠平滑肌自发性收缩的频率和振幅。0.4 mg•mL-1的血根碱对由乙酰胆碱、氯化钡引起的离体肠肌的兴奋性收缩有明显的拮抗作用,而对酚妥拉明阻断肾上腺素能受体后,仍能显著降低离体肠平滑肌收缩的振幅。【结论】血根碱对小肠平滑肌的舒张作用可能与乙酰胆碱竞争,阻断M受体、与酚妥拉明竞争,兴奋α受体及直接抑制肠肌兴奋性有关。     

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.     

Microfilaments in cellular and developmental processes

In our opinion, all of the phenomena that are inhibited by cytochalasin can be thought of as resulting from contractile activity of cellular organelles. Smooth muscle contraction, clot retraction, beat of heart cells, and shortening of the tadpole tail are all cases in which no argument of substance for alternative causes can be offered. The morphogenetic processes in epithelia, contractile ring function during cytokinesis, migration of cells on a substratum, and streaming in plant cells can be explained most simply on the basis of contractility being the causal event in each process. The many similarities between the latter cases and the former ones in which contraction is certain argue for that conclusion. For instance, platelets probably contract, possess a microfilament network, and behave like undulating membrane organelles. Migrating cells possess undulating membranes and contain a similar network. It is very likely, therefore, that their network is also contractile. In all of the cases that have been examined so far, microfilaments of some type are observed in the cells; furthermore, those filaments are at points where contractility could cause the respective phenomenon. The correlations from the cytochalasin experiments greatly strengthen the case; microfilaments are present in control and "recovered" cells and respective biological phenomena take place in such cells; microfilaments are absent or altered in treated cells and the phenomena do not occur. The evidence seems overwhelming that microfilaments are the contractile machinery of nonmuscle cells. The argument is further strengthened if we reconsider the list of processes insensitive to cytochalasin (Table 2). Microtubules and their sidearms, plasma membrane, or synthetic machinery of cells are presumed to be responsible for such processes, and colchicine, membrane-active drugs, or inhibitors of protein synthesis are effective at inhibiting the respective phenomena. These chemical agents would not necessarily be expected to affect contractile apparatuses over short periods of time, they either do not or only secondarily interfere with the processes sensitive to cytochalasin (Table 1). It is particularly noteworthy in this context that microtubules are classed as being insensitive to cytochalasin and so are not considered as members of the "contractile microfilament" family. The overall conclusion is that a broad spectrum of cellular and developmental processes are caused by contractile apparatuses that have at least the common feature of being sensitive to cytochalasin. Schroeder's important insight (3) has, then, led to the use of cytochalasin as a diagnostic tool for such contracile activity: the prediction is that sensitivity to the drug implies presence of some type of contractile microfilament system. Only further work will define the limits of confidence to be placed upon such diagnoses. The basis of contraction in microfilament systems is still hypothetical. Contraction of glycerol-extracted cells in response to adenosine triphosphate (53), extraction of actin-like or actomyosin-like proteins from cells other than muscle cells (54), and identification of activity resembling that of the actomyosin-adenosine triphosphatase system in a variety of nonmuscle tissues (40, 54) are consistent with the idea that portions of the complex, striated muscle contractile system may be present in more primitive contractile machinery. In the case of the egg cortex, calcium-activated contractions can be inhibited by cytochalasin. If, as seems likely, microfilaments are the agents activated by calcium, then it will be clear that they have the same calcium requirement as muscle. Biochemical analyses of primitive contractile systems are difficult to interpret. Ishikawa's important observation (31), that heavy meromyosin complexes with fine filaments oriented parallel to the surface of chondrocytes and perpendicular to the surface of intestinal epithelial cells, implies that both types of filaments are "actin-like" in this one respect. Yet, it is very likely that these actin-like filaments correspond respectively to the cytochalasin-insensitive sheath of glial and heart fibroblasts and the core filaments of oviduct microvilli. No evidence from our studies links contractility directly to these meromyosin-binding filaments. Apart from this problem, activity resembling that of the myosin-adenosine triphosphatase has been associated with the microtubule systems of sperm tails and cilia (55), but those organelles are insensitive to cytochalasin in structure and function. Clearly, a means must be found to distinguish between enzymatic activities associated with microfilament networks, microfilament bundles, microtubules, and the sheath filaments of migratory cells. Until such distinctions are possible, little of substance can be said about the molecular bases of primitive contractile systems. Three variables are important for the control of cellular processes dependent upon microfilaments: (i) which cells of a population shall manufacture and assemble the filaments; (ii) where filaments shall be assembled in cells; and (iii) when contractility shall occur. With respect to distribution among cells, the networks involved in cell locomotion are presumed to be present in all cells that have the potential to move in cell culture. In this respect, the networks can be regarded as a common cellular organelle in the sense that cytoplasmic microtubules are so regarded. In some developing systems, all cells of an epithelium possess microfilament bundles (7, 13), whereas, in others, only discrete subpopulations possess the bundles (5, 6). In these cases the filaments can be regarded as being differentiation products associated only with certain cell types. These considerations may be related to the fact that microfilament networks are associated with behavior of individual cells (such as migration, wound healing, and cytokinesis), whereas the bundles are present in cells that participate in coordinated changes in shape of cell populations. With respect to placement in cells, two alternatives are apparent, namely, localized or ubiquitous association with the plasma membrane. Microfilament bundles of epithelial cells are only found extending across the luminal and basal ends of cells. In this respect they contrast with desmosomal tonofilaments and with microtubules, each of which can curve in a variety of directions through the cell. The strict localization of microfilament bundles probably rests upon their association with special junctional complex insertion regions that are only located near the ends of cells. In the case of mitotically active cells, the orientation of the spindle apparatus may determine the site at which the contractile ring of microfilaments will form (4, 56); this raises the question of what sorts of cytoplasmic factors can influence the process of association between filament systems and plasma membranes. In contrast to such cases of localized distribution, contractile networks responsible for cell locomotion are probably found beneath all of the plasma membrane, just as the network of thrombosthenin may extend to all portions of the periphery of a blood platelet. This ubiquitous distribution probably accounts for the ability of a fibroblast or glial cell to establish an undulating membrane at any point on its edge, or of an axon to form lateral microspikes along its length. The third crucial aspect of control of these contractile apparatuses involves the choice of when contraction shall occur (and as a corollary the degree or strength of contraction that will occur). In the simplest situation, contraction would follow automatically upon assembly of the microfilament bundles or networks. In cleavage furrows of marine embryos (4), for instance, microfilaments are seen beneath the central cleavage furrow and at its ends, but not beyond, under the portion of plasma membrane that will subsequently become part of the furrow. This implies that the furrow forms very soon after the contractile filaments are assembled in the egg cortex. In other cases, microfilaments are apparently assembled but not in a state of (maximal?) contraction. Thus, networks are seen along the sides of migratory cells, although such regions are not then active as undulating membrane organelles. Similarly, microfilament bundles occur in all epithelial cells of the salivary gland (13), or pancreatic anlage (7), although only the ones at discrete points are thought to generate morphogenetic tissue movements. Likewise, bundles begin to appear as early as 12 hours after estrogen administration to oviduct, although visible tubular gland formation does not start until 24 to 30 hours. Finally, streaming in plant cells can wax and wane, depending upon external factors such as auxin (57). All of these cases imply a control mechanism other than mere assembly of the microfilament systems and even raise the possibility that within one cell some filaments may be contracting while others are not. In discussing this problem, it must be emphasized that different degrees of contraction or relaxation cannot as yet be recognized with the electron microscope. In fact, every one of the cases cited above could be explained by contraction following immediately upon some subtle sort of "assembly." Inclusive in the latter term are relations between individual filaments, relations of the filaments and their insertion points on plasma membrane, and quantitative alterations in filament systems. Furthermore, the critical role of calcium and high-energy compounds in muscle contraction suggest that equivalent factors may be part of primitive, cytochalasinsensitive systems. The finding that calcium-induced contraction in the cortex of eggs is sensitive to cytochalasin strengthens that supposition and emphasizes the importance of compartmentalization of cofactors as a means of controlling microfilaments in cells.     

Tetrodotoxin and manganese ions: effects on electrical activity and tension in taenia coli of guinea pig

Tetrodotoxin, at concentrations up to 5 x 10(-6) gram per milliliter, has no effect on the spontaneous discharge in the smooth muscle of taenia coli. However, the spontaneous discharge is abolished by Mn(++) at a concentration of 0.5 millimole per liter. The contraction induced by immersing the muscle in isotonic KCl solution is also suppressed in the presence of Mn(++). Because Mn(++) is a specific suppressor of the spike induced by Ca(++) and tetrodotoxin is an inhibitor of the spike induced by Na(+), we suggest that Ca(++) is a charge carrier in the production of spike potential in the smooth muscle and that the entry of intervening Ca(++) through the membrane acts as a trigger for the contraction of smooth muscle.     

刺糖对小鼠肠推进和离体小肠平滑肌运动的影响

通过观察刺糖对小鼠离体小肠和在体小肠平滑肌运动的影响,以中华墨汁的推进距离与小肠总长的比值计算小肠推进率;采用离体肠管平滑肌收缩实验,以小鼠小肠平滑肌的收缩幅度、收缩频率及运动指数为指标,观察在正常台氏液中刺糖对小鼠离体肠管的影响,同时观察胃肠通和阿托品对不同浓度刺糖的作用。结果表明,刺糖可增强小鼠小肠运动功能（P〈0.01）,但与胃肠通相比作用缓和（P〈0.01）。35%,70%刺糖溶液均可促进正常离体肠管的收缩功能（P〈0.01,P〈0.01）,能拮抗阿托品引起的舒张作用（P〈0.01,P〈0.01）和胃肠通引起的收缩运动（P〈0.01,P〈0.01）。体内外实验结果表明,刺糖对小鼠小肠运动在体内外均具有调节作用。     

Contraction of granulation tissue in vitro: similarity to smooth muscle

Strips of granulation tissue from three different experimental models contract in vitro when treated with substances that induce contraction of smooth muscle. Because the fibroblasts in such tissues have some ultrastructural features typical of smooth muscle, our findings indicate that fibroblasts are able to modulate toward a cell type that is morphologically and functionally close to smooth muscle.     

Calcium and sodium channels in spontaneously contracting vascular muscle cells

Electrophysiological recordings of inward currents from whole cells showed that vascular muscle cells have one type of sodium channel and two types of calcium channels. One of the calcium channels, the transient calcium channel, was activated by small depolarizations but then rapidly inactivated. It was equally permeable to calcium and barium and was blocked by cadmium, but not by tetrodotoxin. The other type, the sustained calcium channel, was activated by larger depolarizations, but inactivated very little; it was more permeable to barium than calcium. The sustained calcium channel was more sensitive to block by cadmium than the transient channel, but also was not blocked by tetrodotoxin. The sodium channel inactivated 15 times more rapidly than the transient calcium channel and at more negative voltages. This sodium channel, which is unusual because it is only blocked by a very high (60 microM) tetrodotoxin concentration but not by cadmium, is the first to be characterized in vascular muscle, and together with the two calcium channels, provides a basis for different patterns of excitation in vascular muscles.     

忍冬藤提取物对兔离体肠平滑肌的舒张作用及其机制

【目的】胃肠平滑肌过度收缩可引起腹痛腹泻等临床常见的疾病。目前,临床上对治疗胃肠平滑肌过度收缩的药物主要以西药为主,如钙离子通道阻断药硝苯地平和抗胆碱药阿托品等,硝苯地平长期使用可引起负性肌力和负性传导的现象,而阿托品由于其不良反应较大,在临床应用中受到一定的限制。因此,开发具有有效防治胃肠平滑肌痉挛、低毒、低残留的天然中草药意义重大。试验以兔离体小肠平滑肌为研究对象,利用丰富的忍冬藤资源,研究忍冬藤提取物对兔离体小肠平滑肌收缩的影响,并探讨其作用机制。【方法】采用兔离体小肠平滑肌试验,应用BL-420E生物机能实验系统,观察忍冬藤提取物对正常状态下兔离体小肠平滑肌自发性收缩的影响;进而使用工具药乙酰胆碱、组胺和氯化钡致兔小肠痉挛性收缩后,观察忍冬藤提取物对其痉挛性收缩的影响;为研究忍冬藤提取物抑制兔离体小肠平滑肌收缩的作用机制,应用IP_3受体阻断剂肝素(HP)、肌浆网ryanodine受体阻断剂钌红(RR)和一氧化氮合酶抑制剂左旋硝基精氨酸甲酯(L-NAME),探明忍冬藤提取物对兔离体小肠平滑肌作用的机制。【结果】忍冬藤提取物可浓度依赖性抑制兔离体小肠平滑肌自发性收缩,药物浓度在7.5 g·L~(-1)时可显著抑制兔离体小肠平滑肌收缩的频率(P0.05),药物浓度在5g·L~(-1)时可极显著抑制兔离体小肠平滑肌收缩的振幅(P0.05);工具药乙酰胆碱、组胺和氯化钡可显著诱导兔小肠平滑肌收缩的振幅,忍冬藤提取物可显著抑制由乙酰胆碱、组胺和氯化钡诱导的兔离体小肠平滑肌收缩的频率(P0.05),可极显著抑制兔离体小肠平滑肌收缩的振幅(P0.01)。IP_3受体阻断剂肝素能增强忍冬藤提取物舒张兔离体小肠平滑肌收缩的作用(P0.01),而肌浆网ryanodine受体阻断剂钌红对忍冬藤提取物舒张兔小肠平滑肌的作用无明显影响(P0.05)。左旋硝基精氨酸甲酯能够部分阻断忍冬藤提取物舒张兔离体小肠平滑肌收缩的作用(P0.01)。【结论】忍冬藤提取物可显著抑制兔离体小肠平滑肌收缩的频率和振幅,其机制可能与增加一氧化氮浓度,抑制IP_3受体介导的内钙释放有关,但对肌浆网ryanodine受体途径引起的内钙释放无关。     

Platelet-mediated cholesterol accumulation in cultured aortic smooth muscle cells

Cholesterol accumulates within smooth muscle cells and macrophages in atherosclerotic lesions, thereby contributing to the progressive enlargement of these lesions. The mechanism of this cellular accumulation of cholesterol is not known. The possibility that platelets may have a role in the cellular cholesterol accumulation that occurs during atherogenesis was investigated. Incubation of thrombin-activated washed rat platelets (or platelet-free supernatants prepared from thrombin-activated platelets) with cultured rat aortic smooth muscle cells induced cholesteryl ester lipid droplet accumulation within the smooth muscle cells. No cholesteryl ester lipid droplets accumulated when smooth muscle cells were incubated with unactivated platelets. Smooth muscle cell lipid droplet accumulation occurred in the absence of serum lipoproteins and was not inhibited by mevinolin, a drug that blocks cholesterol synthesis. These findings suggest that activated platelets may release cholesterol, which can be accumulated by cells and stored as lipid droplets.     

山羊创伤愈合时组织病理学和超微结构的观察

山羊创伤愈合过程组织病理学和超微结构的动态变化观察结果表明:在几天内伤口中出现许多肌-成纤维细胞,它具有成纤维细胞和平滑肌细胞的共同特征。肌-成纤维细胞的核细长,有深的缺刻,并有发育完善的高尔基体和粗面内质网以及在胞浆中有很多微丝团块等。所以,肌-成纤维细胞在创伤收缩中起重要作用。     

Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent

A 70 percent reduction in the rate of blood flow through the common carotid artery in rabbits caused a 21 percent decrease in the diameter of this artery within 2 weeks. The smooth muscle relaxant papaverine did not attenuate the response; therefore, such reductions in diameter probably reflect a structural modification of the arterial wall rather than sustained contraction of smooth muscle. This arterial response to reduced blood flow was abolished when the endothelium was removed from the vessels. It appears that the endothelium is essential for the compensatory arterial response to long-term changes in luminal blood flow rates.     

Highly cooperative opening of calcium channels by inositol 1,4,5-trisphosphate

The kinetics of calcium release by inositol 1,4,5-trisphosphate (IP3) in permeabilized rat basophilic leukemia cells were studied to obtain insight into the molecular mechanism of action of this intracellular messenger of the phosphoinositide cascade. Calcium release from intracellular storage sites was monitored with fura-2, a fluorescent indicator. The dependence of the rate of calcium release on the concentration of added IP3 in the 4 to 40 nM range showed that channel opening requires the binding of at least three molecules of IP3. Channel opening occurred in the absence of added adenosine triphosphate, indicating that IP3 acts directly on the channel or on a protein that gates it. The channels were opened by IP3 in less than 4 seconds. The highly cooperative opening of calcium channels by nanomolar concentrations of IP3 enables cells to detect and amplify very small changes in the concentration of this messenger in response to hormonal, sensory, and growth control stimuli.