共查询到13条相似文献,搜索用时 109 毫秒
1.
目的:定量分析生育男性获能后单个精子的[Ca^2 ]i基础水平以及10μM孕酮刺激后的[Ca^2 ]i动态改变。方法:生育健康男子10例,手淫法留取精液。精液液化后,应用上游技术分离活动良好的精子,之后将精子悬液置于3‘70C培养2h使之获能。获能后精子移人Petri培养皿,每皿加入终浓度为8.85μmoL/L的钙荧光探针Fluo-3/AM,370C避光孵育40min。 相似文献
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羟基磷灰石溶胶对W-256癌肉瘤细胞内钙离子浓度及细胞形态结构的影响 总被引:11,自引:0,他引:11
前言羟基磷灰石具有很好的生物相容性和生物活性,能与骨组织形成骨性结合,烧结成多孔或致密的块状及颗粒,可用于骨缺损的修复[1,2]。也可通过喷涂在金属上形成羟基磷灰石涂层,用于牙根种植以及人工关节的植入[3]。近期的研究发现,将羟基磷灰石制备成溶胶(H... 相似文献
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Marina G. Mozhayeva 《Pflügers Archiv : European journal of physiology》1996,433(1-2):180-187
Sustained Ca2+ elevation (”Ca2+ response”), caused by subsequent readdition of Ca2+ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca2+-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular
Ca2+ concentration ([Ca2+]i) was between about 50 and 110 nM, a massive Ca2+ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca2+]i of over 110 nM displayed small plateau-like Ca2+ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted,
the dependence of the Ca2+ response amplitude on resting [Ca2+]i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca2+ responses, the amplitudes of which were almost constant and in the same range as resting [Ca2+]i. Ni2+, known to partly block Ca2+ entry, caused no change in the half-decay time of [Ca2+]i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni2+] when added at the peak of the phasic component of the Ca2+ response. However, it lowered the sustained phase of the Ca2+ response by 42%. When applied at the start of the readdition of Ca2+, Ni2+ blocked the phasic component of the Ca2+ response, there being a threefold decrease in the initial rate of [Ca2+]i rise. In cells with a resting [Ca2+]i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca2+ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca2+ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP
3-) sensitive store by a submaximal concentration of agonist (in Ca2+-free medium) was followed, on readdition of Ca2+, by Ca2+ entry, which appeared to trigger IP
3-sensitive Ca2+ release (IICR) which, in turn, initiated Ca2+-sensitive Ca2+ release (CICR), thus resulting in a massive elevation of [Ca2+]i.
Received: 3 July 1996 / Received after revision and accepted: 9 September 1996 相似文献
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The aim of the present study was to examine the possible role of protein kinase C (PKC) in thrombin-induced Ca2+ signalling. As shown before, continuous superfusion of rat glioma cells with thrombin caused sustained [Ca2+]i oscillations through activation of cell surface receptors [Czubayko U, Reiser G (1995) Neuroreport 6: 1249]. These oscillations
were inhibited by protease nexin-1. Addition of PKC inhibitors, i. e. staurosporine (0.2–20 μM), bisindolylmaleimide (1 μM)
or chelerythrine (1 μM), irreversibly suppressed thrombin-induced [Ca2+]i oscillations. Thereafter application of 2,5-di(tert-butyl)-1,4-benzohydroquinone (t-BuBHQ, 20 μM) or thapsigargin (1 μM) (inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase) caused no [Ca2+]i response, indicating that intracellular Ca2+ stores were completely empty. We tested whether PKC affects the refilling of internal Ca2+ stores in thrombin-stimulated cells, by monitoring the amount of Ca2+ release caused by t-BuBHQ in the presence or absence of PKC inhibitors or activators. The amount of Ca2+ released by t-BuBHQ, which was normalized by comparison with the thrombin-induced Ca2+ response, was decreased by simultaneous incubation with staurosporine or chelerythrine, but enhanced with the PKC activator
oleoyl acetyl glycerol. Furthermore, the capacitative Ca2+ entry was reduced by inhibition or downregulation, and increased by activation, of PKC. Capacitative Ca2+ entry was induced in these experiments by depletion of Ca2+ stores by the addition of thapsigargin or t-BuBHQ. In contrast, the inhibition of PKC during thrombin-induced depletion of
intracellular stores did not influence the Ca2+ entry but nearly completely abolished the refilling of the internal stores. Thus we conclude that during thrombin receptor
stimulation activation of PKC is required to maintain the refilling of intracellular Ca2+ stores for sustained [Ca2+]i oscillations. Thus, the control by PKC of the capacitative Ca2+ entry is apparently different depending on whether it is induced by sarco/endoplasmic reticulum Ca2+-ATPase inhibition or by activation of the thrombin receptor.
Received: 5 July 1996 / Received after revision and accepted: 7 October 1996 相似文献
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A. Shmigol D. A. Eisner S. Wray 《Pflügers Archiv : European journal of physiology》1998,437(1):158-160
In myometrial smooth muscle cells the rate of decline of intracellular calcium ([Ca2+]i) is determined by Ca2+ extrusion from the cell and uptake into intracellular stores. The relative quantitative contribution of these processes however,
has not been established. We therefore examined the effect of the sarcolemmal Ca2+ pump inhibitor, carboxyeosin, on the rate of the [Ca2+]i transient decline in myocytes isolated from pregnant rat uterus. Indo-1 was used in conjunction with the whole-cell patch-clamp
technique to measure [Ca2+]i simultaneously with transmembrane calcium current (I
Ca). [Ca2+]i transients were elicited by repetitive membrane depolarization to simulate the natural pattern of uterine electrical activity.
The rate of [Ca2+]i removal was calculated from the falling phase of the [Ca2+]i transient. Pre-treatment of the cells with 2 μM carboxyeosin led to a marked decrease in the rate of [Ca2+]i transient decay, suggesting that the sarcolemmal Ca2+ pump is involved in the calcium extrusion process. Removal of the extracellular Na also decreased the rate of [Ca2+]i decay, indicating an important role for the Na+/Ca2+ exchange. When both the sarcolemmal Ca2+ pump and Na+/Ca2+ exchange were inhibited the cell failed to restore [Ca2+]i after the stimulation. Comparison of the rate constants of [Ca2+]i decay in control conditions and after carboxyeosin treatment shows that approximately 30% of [Ca2+]i decay is due to the sarcolemmal calcium pump activity. The remaining 70% can be attributed to the activity of Na+/Ca2+ exchanger and the intracellular calcium stores.
Received: 17 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998 相似文献
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Marina G. Mozhayeva Galina N. Mozhayeva 《Pflügers Archiv : European journal of physiology》1996,432(4):614-622
In single bovine aortic endothelial (BAE) cells pre-loaded with Fura-2, Ca2+ transients in a Ca2+-free medium have been revealed, which evidently reflects Ca2+ release from intracellular stores. In cells with different levels of resting basal cytoplasmic Ca2+ ([Ca2+]i) from about 50 to 110 nM, a biphasic dependence of the Ca2+ transients on resting [Ca2+]i was shown and spontaneous Ca2+ oscillations were observed. At a [Ca2+]i level over 110 nM, a pronounced rise in Ca2+ transients occurred and only single transients were observed. Ryanodine (10 μM) produced a transient [Ca2+]i elevation, suggesting the presence of ryanodine receptors in intracellular store membranes. The results imply that both inositol
1,4,5-trisphosphate-sensitive Ca2+ release (IICR) and Ca2+-sensitive Ca2+ release (CICR) take place in BAE cells. Only IICR seems to be sufficient for generating baseline Ca2+ oscillations in BAE cells, whereas the ATP-induced (5–100 μM) Ca2+ response involves the CICR set in motion by an oscillatory IICR of high frequency. The completion of both the spontaneous
and ATP-induced Ca2+ transients was associated with a [Ca2+]i decrease to a level below the initial resting [Ca2+]i (undershoot). Its depth biphasically depended on the resting [Ca2+]i from 50 to 110 nM, suggesting that the lack of a Ca2+ leak from inositol 1,4,5-trisphosphate-sensitive stores is responsible for the undershoot in this range. The Ca2+ leak is concluded to play a key role in the initiation and termination of regenerative IICR both in spontaneous oscillations
and in ATP-induced transients.
Received: 13 November 1995/Received after revision and accepted 27 March 1996 相似文献
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目的探讨红车轴草提取物(Trifoliumpratense Leguminosae extract,TLE)体外对小鼠淋巴细胞[Ca2+]i及腹腔巨噬细胞NO分泌和吞噬微球的影响。方法无菌条件下制备小鼠淋巴细胞悬液及小鼠腹腔巨噬细胞悬液;MTT法检测药物对细胞悬液的毒性情况;Fluo-4/AM染色结合流式细胞术分析TLE对小鼠淋巴细胞[Ca2+]i的影响;Griess反应系统检测TLE对脂多糖(LPS)刺激的小鼠腹腔巨噬细胞NO分泌的影响;1μm与2μm直径的荧光微球结合流式细胞术分析TLE对小鼠腹腔巨噬细胞吞噬作用的影响。结果终质量浓度为20、40mg/L的TLE对细胞的毒性小;TLE促进了淋巴细胞的Ca2+内流;TLE抑制了巨噬细胞的NO分泌与吞噬作用,与非TLE组比较P<0.01。结论对淋巴细胞[Ca2+]i及巨噬细胞的NO分泌和吞噬的作用可能是TLE调节小鼠免疫系统的途径。 相似文献
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Activation of large conductance Ca2+-activated K+ channels (BK channels) in intact clonal rat pituitary cells (GH4 cells) was investigated using the cell-attached patch-clamp configuration. This method prevents loss of intracellular factors which might influence channel activity. BK channels are generally considered to be inactive at the resting membrane potential in excitable cells. However, at the resting potential (0 mV pipette potential), 40% of the cell-attached patches displayed spontaneously active BK channels, which remained active even at 20 mV hyperpolarization. The peptide thyroliberin (TRH) elevates the cytosolic Ca2+ concentration ([ Ca2+]i) in GH cells by IP3-induced release of Ca2+ from intracellular stores. This rise in [Ca2+]i occurs concomitantly with membrane hyperpolarization. TRH stimulation caused activation of BK channels in nine out of 30 silent cell-attached patches, and caused enhanced channel activity in seven out of 29 cell-attached patches containing spontaneously active BK channels. The Ca2+ ionophore ionomycin activated silent BK channels in three out of 10 cell-attached patches, and increased the activity of spontaneously active BK channels in seven out of 16 cell-attached patches. The pipette potential was clamped to 0 mV in all these experiments. We conclude that the BK channels in GH4 cells may be active at the resting membrane potential and more negative membrane potentials. The channels may also be activated further by physiological elevations of [Ca2+]i in the same potential range. Our results point towards new possible physiological roles for the BK channels in GH4 cells. This is in agreement with the emerging picture of BK channels highly sensitive to [Ca2+]i in a wide variety of cell types. 相似文献
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目的探讨氟伐他汀(fluvastatin,Flu)对小牛血清(CS)干预的高血压大鼠胸主动脉血管平滑肌细胞增殖及血管重构的影响并初步探讨其可能的机制。方法CS、Flu和甲羟戊酸(mevalonic acid,Meva)处理血管平滑肌细胞4d,将细胞分为SHR组、SHR+20%CS组、SHR+20%CS+Flu组和SHR+20%CS+Flu+Meva组,采用MTT检测细胞的增殖能力,Transwell检测细胞的迁移能力,体外管样实验、天狼星红染色以及免疫荧光的方法分别检测细胞的管样结构的生成能力、胶原纤维以及F-actin的表达,Western印迹检测PMCA、SERCA和LTCC的表达。结果Flu能够抑制CS促进的VSMCs增殖效应、迁移能力,管样结构生成,胶原蛋白表达,以及骨架蛋白的聚合,并下调VSMCs中PMCA的表达,上调SERCA和LTCC的表达,而Meva则对Flu的作用进行逆转。结论氟伐他汀对CS诱导的VSMCs增殖及血管重构产生抑制,该过程可能是通过调节钙泵的表达实现的,而Meva代谢途径可能在其中发挥了重要的作用。 相似文献
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[Ca(2+)](i) elevation is a key event when O(2) sensitive cells, e.g. PC12 cells and pulmonary artery smooth muscle cells, face hypoxia. Ca(2+) entry pathways in mediating hypoxia-induced [Ca(2+)](i) elevation include: voltage-gated Ca(2+) channels (VGCCs), transient receptor potential (TRP) channel and Na(+)-Ca(2+) ex-changer (NCX). In the pulmonary artery, accumulated evidence strongly suggests that prostaglandins (PGs) are involved in pulmonary inflammation and cause vasoconstriction during hypoxia. In this study, we investigated the effect of arachidonic acid (AA), the upstream substrate for PGs, on hypoxia response in O(2) sensitive cells. Exogenous application of AA significantly inhibited hypoxia-induced [Ca(2+)](i) elevation. This effect was due to AA itself rather than its degenerative products. The pharmacological modulation of endogenous AA showed that the prevention of AA generation by blockage of cPLA2, diacylglycerol (DAG) lipase and fatty acid hydrolysis (FAAH), augments hypoxia-induced [Ca(2+)](i) elevation, whereas prevention of AA degeneration attenuates hypoxia-induced [Ca(2+)](i) elevation. Over-expression of COX2 enhances hypoxia-induced [Ca(2+)](i) elevation and this enhancement is reversed by exogenous AA. Our results suggest that AA inhibits hypoxia response. The dynamic alterations in cellular lipids might determine cell response to hypoxia. 相似文献
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J. Robbins R. Cloues D. A. Brown 《Pflügers Archiv : European journal of physiology》1992,420(3-4):347-353
Receptor-mediated formation of inositol 1,4,5-trisphosphate (IP3) can induce an outward Ca2+-activated K+ current [I
K(Ca)] some neural cells. We have investigated I
K(Ca) activated by intracellular injections of IP3 in whole-cell patch-clamped neuroblastoma×glioma hybrid cells. The current could only be recorded reliably using citrate as the anion in the pipette, but not using acetate, aspartate, chloride, fluoride, gluconate or methylsulphate. This could be attributed to buffering of intracellular Mg2+ by citrate. Theoretical calculations suggested free [Mg2+] of 1.0 and 0.07 mM respectively in the acetate- and citrate-based recording solutions. Further, IP3-activated I
K(Ca) could be recorded when the free Mg2+ level in the acetate, chloride or methylsulphate solutions was lowered to the range (0.05 mM) calculated for the citrate solution. Thus, raised [Mg2+] blocks I
K(Ca). This appeared to be due to inhibition of the response to released Ca2+, since high [Mg2+] also blocked the response to intracellular injections of Ca2+ ions. Mean Mg2+ levels in intact neuroblastoma×glioma hybrid cells measured by Mag-Indo-1/AM fluorescence were estimated to be less than 0.14 mM. We therefore conclude that IP3-induced I
K(Ca) is expressed under normal conditions, but may be subject to regulation by intracellular Mg2+. 相似文献