缺氧对培养的猪肺内皮细胞胞浆游离钙的影响

本文应用荧光探针Ｆｕｒａ－１／ＡＭ测定胞浆游离钙浓度（［Ｃａ＾２＋］ｉ）技术，观察培养的猪肺动脉内皮细胞［Ｃａ＾２＋］ｉ在缺氧时变化。实验发现：向细胞悬液中充氮气造成缺氧时，肺动脉内皮细胞［Ｃａ＾２＋］ｉ升高８１±２１％（Ｐ＜０．０５，ｎ＝８）。结果提示肺动脉内皮细胞钙信使系统可能参与缺氧所致血管反应。     

低氧对肺动脉内皮细胞分泌一氧化氮的影响

以内皮细胞合成了一氧化氮（ＥＤＮＯ）的代谢产物ＮＯ＾－２为指标，观察了低氧条件下猪肺动脉内皮细胞合成分泌ＥＤＮＯ及细胞内Ｃａ＾２＋浓度的变化，发现低氧时内皮细胞合成分泌的ＥＤＮＯ明显增加，但随着低氧时间延长，增加的幅度减小，内皮细胞的（Ｃａ２＋）ｉ显著增加。而低氧培养时间的长短对（Ｃａ＾２＋）ｉ没有明显的影响。结果表明，低氧条件下内皮细胞（Ｃａ＾２＋）ｉ的增加是ＥＤＮＯ合成分泌增加的重要原因之一。     

缺氧对肺动脉内皮细胞和平滑肌细胞5-羟色胺转载体基因表达的影响

目的：探讨无氧（Ｏ％Ｏ２＋９５％Ｎ２＋５％ＣＯ２）和低氧（２５～３％Ｏ２＋９２％Ｎ２＋５％ＣＯ２）对新生小牛肺动脉内皮细胞（ＰＡＥＣ）和肺动肺平滑肌细胞（ＰＡＳＭ）５－羟色胺转载体基因表达的影响。方法：应用细胞培养，核酸分子杂交技术。结果：无氧组和低氧组（１５ｈ、３ｈ、６ｈ）ＰＡＥＣ５－羟色胺转载体ｍＲＮＡ表达显著高于常氧组（Ｐ＜００５），而无氧和低氧１２ｈ至４８ｈ对ＰＡＥＣ５－羟色胺转载体ｍＲＮＡ表达无明显影响。无氧和低氧（３ｈ、６ｈ、１２ｈ、２４ｈ）组ＰＡＳＭ５－羟色胺ｍＲＮＡ表达均显著高于常氧组（Ｐ＜００１），结论：推测缺氧早期通过促进ＰＡＥＣ５－羟色胺转载体基因表达，加强ＰＡＥＣ对５－羟色胺的摄取和降解，随缺氧时间的延长，ＰＡＥＣ的此功能受损。缺氧ＰＡＳＭ５－羟色胺转载体基因表达的持续增加，则可能参与缺氧肺动脉高压的形成。     

H~＋－Ca~（2＋）交换在心肌细胞缺氧复氧过程中所致Ca~（2＋）超负荷的意

多数研究资料表明：在心肌细胞发生氧反常和ｐＨ反常后，Ｈ￣＋一Ｎａ￣＋、Ｎａ￣＋一Ｃａ￣（２＋交换加强是细胞内Ｃａ￣（２＋）超载的重要机制。我们的研究表明，造成细胞Ｃａ￣（２＋）超载的原因，除Ｈ￣＋一Ｎａ￣＋、Ｎａ￣＋一Ｃａ￣（２＋）交换外，尚有Ｈ￣＋一Ｃａ￣（２＋）交换参加．本实验证实，在细胞缺氧１０、２０、３０和４０ｍｉｎ时，经Ｈ￣＋一Ｃａ￣（２＋）交换进入细胞的Ｃａ２＋量占同一时点细胞摄Ｃａ￣（２＋）总量的比率分别为（％）：１０．１±０．５、１２４±０．７、１１．８±０．４和１１．２±０．５、平均值为（％）：１１．４±０．９．当缺氧细胞再复氧后，这一比率显著增加。各时点的比率分别为（％）：２３．７±０．６、２２．３±０．５、２２．１±０．７和２０．５±０．８、平均值为（％）：２２．２±１２。这一结果表明：在ｐＨ反常所致Ｃａ￣（２＋）超载过程中，Ｈ￣＋一Ｃａ￣（２＋）交换的作用不容忽视。     

维拉帕米和Mn~（2＋）对缺氧及复氧单心肌细胞游离Ca~（2＋）的影响

本文采用荧光探针Ｆｕｒａ－２结合计算机图像处理技术观察不同时间的缺氧及复氧单心肌细胞内游离Ｃａ￣（２＋）含量的变化以及Ｃａ￣（２＋）通道阻滞剂及Ｎａ￣＋－Ｃａ￣（２＋）交换抑制剂对其的响。结果显示随着缺氧和缺氧复氧时间的延长，细胞内Ｃａ￣（２＋）浓度逐渐增加。缺氧复氧时细胞内Ｃａ￣（２＋）增加幅度较单纯缺氧大。Ｍｎ￣（２＋）及维拉帕米均能降低缺氧时心肌细胞内Ｃａ￣（２＋）超负荷（Ｐ＜０．０５）。Ｍｎ￣（２＋）同时也能降低缺氧复氧时细胞内Ｃａ￣（２＋）含量（Ｐ＜０．０５），而维拉帕米降钙作用不明显（Ｐ＞０．０５）。本文提示缺氧和缺氧复氧时细胞内Ｃａ￣（２＋）超负荷的机制并非完全一致。     

镁对大鼠缺氧再给氧心肌细胞内游离钙的影响

目的和方法：用ＡＣＡＳ５７０粘附式细胞仪，以荧光素染色法观察镁（Ｍｇ２＋）对体外培养乳鼠心肌细胞内游离钙（Ｃａ２＋）的影响及对缺氧再给氧时细胞内Ｃａ２＋作用。结果：细胞外Ｍｇ２＋降至０３ｍｍｏｌ·Ｌ－１，细胞内Ｃａ２＋荧光强度上升速度加快，达到稳定所需时间延长，出现Ｃａ２＋振荡曲线。增加Ｍｇ２＋浓度可使细胞内Ｃａ２＋降低。Ｍｇ２＋还可以显著减少缺氧再给氧时细胞内Ｃａ２＋，Ｐ＜００１。结论：细胞外低Ｍｇ２＋可导致细胞内Ｃａ２＋增加，Ｍｇ２＋有维持正常心肌细胞内Ｃａ２＋稳定性及拮抗缺氧再给氧时细胞内Ｃａ２＋超载作用     

Ca^2＋预处理对离体大鼠心肌缺血再灌注的保护作用

本文旨在探讨Ｃａ＾２＋预处理对离体大鼠心肌缺血再灌（即停灌复灌）的保护作用，实验应用Ｌａｎｇｅｎｄｏｒｆｆ灌流模型，经房室瓣向左心室内插入一导管球囊，用多导记录仪测量心功能指标。Ｃａ＾２＋预处理方案为３次３０ｓ无Ｃａ＾２＋继１０ｍｉｎ复Ｃａ＾２＋灌流。缺血处理方案为３次５ｍｉｎ停灌继５ｍｉｎ再灌，持续缺血方案为３０ｍｉｎ停灌继２０ｍｉｎ再灌，实验结果显示，对照组３０ｍｉｎ停灌后，再灌２０ｍｉｎ时支     

缺氧对肺动脉内皮细胞和平滑肌细胞5—羟色胺转载体基因表达 …

目的：探讨无氧（０％Ｏ２＋９５％Ｎ２＋５％ＣＯ２）和低氧（２．５￣３％＋９％Ｎ２＋５％ＣＯ２）对新生小牛肺动脉内皮细胞（ＰＡＥＣ）和肺动脉平滑肌细胞（ＰＡＳＭ）５－羟色胺转载体基因表达的影响。方法：应用细胞培养，核到分子杂交技术。结果：无氧组和低氧组（１．５ｈ、３ｈ、６ｈ）ＰＡＥＣ５－羟色胺转载体ｍＲＮＡ表达显著同于常氧组（Ｐ〈０．０５），而无氧和低氧１２ｈ至４８ｈ对ＰＡＥＣ５－羟色胺转载体ｍＲＮ     

低强度He－Ne激光照射对缺氧／再给氧心肌细胞内钙的影响

本文以离体大鼠心肌细胞作为缺血／再灌注损伤的《氧反常》模型。为测定低强度Ｈｅ－Ｎｅ激光照射前、照射后心肌细胞内游离钙浓度（［Ｃａ￣（２＋）］ｉ）变化，我们应用了最新一代钙荧光探针（Ｆｌｕｏ－３），结果表明：（１）缺氧／再给氧组和激光照射前、后组心肌细胞［Ｃａ￣（２＋）］ｉ明显高于正常对照组。（２）缺氧／冉给氧织心肌细胞［Ｃａ￣（２＋）］ｉ明显高于激光照射前、后组，但是激光照射前与照射后心肌细胞［Ｃａ￣（２＋）］ｉ没有明显变化。同时我们分别观察了各组心肌细胞超微结构变化。我们的研究表明Ｈｅ－Ｎｅ激光照射对缺氧／再给氧损伤有预防和治疗作用。     

钙离子与牛磺酸两者跨心肌细胞膜内流的相互影响

目的和方法：取新生ＳＤ大鼠心室肌制备培养搏动心肌细胞，采用放射性同位素４５Ｃａ２＋及３Ｈ－牛磺酸示踪技术，观察钙离子与牛磺酸跨心肌细胞膜内流的相互影响。结果：（１）牛磺酸内流在低胞外Ｃａ２＋浓度（［Ｃａ２＋］ｏ，０．５ｍｍｏｌ／Ｌ）和高［Ｃａ２＋］ｏ（４０ｍｍｏｌ／Ｌ）均比正常［Ｃａ２＋］ｏ（１５ｍｍｏｌ／Ｌ）和高［Ｃａ２＋］ｏ（４０ｍｍｏｌ／Ｌ）时增加，尤以低［Ｃａ２＋］。时明显；异搏定（１０μｍｏｌ／Ｌ）能促进牛磺酸的内流；（２）１０～２０ｍｍｏｌ／Ｌ牛磺酸对不同［Ｃａ２＋］。（０５、１５和４０ｍｍｏｌ／Ｌ）下的Ｃａ２＋内流均有抑制作用，而１ｍｍｏｌ／Ｌ牛磺酸只抑制高［Ｃａ２＋］ｏ时Ｃａ２＋内流，对低［Ｃａ２＋］ｏ和正常［Ｃａ２＋］ｏ时Ｃａ２＋内流无明显作用；１～２０ｍｍｏｌ／Ｌβ－丙氨酸对Ｃａ２＋内流无明显影响。结论：胞外Ｃａ２＋浓度的变化可直接影响牛磺酸内流，而牛磺酸能抑制Ｃａ２＋的内流，两者相互影响，从而发挥牛碘酸调节细胞内Ｃａ２＋浓度的作用     

The vasopressin receptor of the blood-brain barrier in the rat hippocampus is linked to calcium signalling.

The signal transduction system of the vasopressin receptor in cerebral microvessels is not known but appears not to be adenylate cyclase/cyclic AMP. We determined the effect of arginine vasopressin (AVP) on the intracellular free calcium concentration [Ca2+]i in endothelial cells of isolated hippocampal microvessels of rats, using the fura-2 fluorescence technique. AVP administration caused a rapid and transient rise of cytosolic free calcium which was absent after extracellular calcium was removed, and could be blocked with the vasopressin V1 receptor antagonist, d(CH2)5 Tyr(Me)AVP. The vasopressin V2 receptor agonist, 1-deamino-8,D-AVP, on the contrary, failed to affect the intracellular free calcium level, and was unable to inhibit the AVP-induced rise of [Ca2+]i in the preparation. Our results, therefore, demonstrate the presence of a calcium-signalling, i.e. V1 vasopressin receptor at the blood-brain barrier in the hippocampus of the rat.     

卡托普利对缺氧/复氧乳鼠心室肌细胞游离钙的影响及其离子通道机制

目的：观察卡托普利晚期预处理对缺氧/复氧乳鼠心室肌细胞游离钙的影响及其离子通道机制。方法:建立培养乳鼠心肌细胞缺氧/复氧损伤模型。设正常对照组、缺氧/复氧组、缺氧预适应组和卡托普利组。经Flou-3/AM负载染色后，采用流式细胞分析技术，测定细胞内钙离子浓度(［Ca²⁺］i)；利用膜片钳技术，观察L-型钙通道和钠钙交换电流的变化。结果:（1）缺氧/复氧时，［Ca²⁺］i和Na⁺/Ca²⁺交换电流高于正常对照组（P<0.01），L-型钙电流（I_Ca-L）峰值下降，I-V曲线上移，半数失活电压(V_0.5)减小，ICa-L失活曲线左移。（2）晚期预处理和卡托普利使缺氧/复氧时［Ca²⁺］i低于缺氧/复氧组（P<0.01）；I_Ca-L增加，I-V曲线下移，V_0.5增大及稳态失活曲线右移；Na⁺/Ca²⁺ 交换电流减少；但［Ca²⁺］i和Na⁺/Ca²⁺交换电流高于对照组（P<0.05）。（3）卡托普利组与缺氧预适应组比较上述指标均无显著差异 。结论：心肌细胞缺氧/复氧，通过Na⁺/Ca²⁺交换电流的异常增加可引起［Ca²⁺］i的异常升高及其钙超载；卡托普利通过轻度增加Na⁺/Ca²⁺交换电流及其［Ca²⁺］i而触发晚期预处理，抑制后续缺氧/复氧引起的Na⁺/Ca²⁺交换电流及其［Ca²⁺］i的异常增加。     

Hyperoxia-induced reactive oxygen species formation in pulmonary capillary endothelial cells in situ

Lung capillary endothelial cells (ECs) are a critical target of oxygen toxicity and play a central role in the pathogenesis of hyperoxic lung injury. To determine mechanisms and time course of EC activation in normobaric hyperoxia, we measured endothelial concentration of reactive oxygen species (ROS) and cytosolic calcium ([Ca(2+)](i)) by in situ imaging of 2',7'-dichlorofluorescein (DCF) and fura 2 fluorescence, respectively, and translocation of the small GTPase Rac1 by immunofluorescence in isolated perfused rat lungs. Endothelial DCF fluorescence and [Ca(2+)](i) increased continuously yet reversibly during a 90-min interval of hyperoxic ventilation with 70% O(2), demonstrating progressive ROS generation and second messenger signaling. ROS formation increased exponentially with higher O(2) concentrations. ROS and [Ca(2+)](i) responses were blocked by the mitochondrial complex I inhibitor rotenone, whereas inhibitors of NAD(P)H oxidase and the intracellular Ca(2+) chelator BAPTA predominantly attenuated the late phase of the hyperoxia-induced DCF fluorescence increase after > 30 min. Rac1 translocation in lung capillary ECs was barely detectable at normoxia but was prominent after 60 min of hyperoxia and could be blocked by rotenone and BAPTA. We conclude that hyperoxia induces ROS formation in lung capillary ECs, which initially originates from the mitochondrial electron transport chain but subsequently involves activation of NAD(P)H oxidase by endothelial [Ca(2+)](i) signaling and Rac1 activation. Our findings demonstrate rapid activation of ECs by hyperoxia in situ and identify mechanisms that may be relevant in the initiation of hyperoxic lung injury.     

Recovery of intracellular pH in cortical brain slices following anoxia studied by nuclear magnetic resonance spectroscopy: role of lactate removal, extracellular sodium and sodium/hydrogen exchange.

[31P]- and [1H]nuclear magnetic resonances recorded in an interleaved fashion were used in order to quantify high-energy phosphates, intracellular pH and lactate in cortical brain slices of the guinea-pig superfused in a CO2/HCO3(-)-buffered medium during and after anoxic insults. The volume-averaged intracellular pH and energy status of the preparation following anoxia were determined. In the presence of external Na+, intracellular pH normalized in 3 min and was significantly more alkaline from 10 to 12 min of recovery, but lactate remained elevated for 12 min of reoxygenation following anoxia. The amount of lactate removed was only 40% of the quantity of acid extruded showing operation of H+ neutralizing transmembrane mechanisms other than transport of lactic acid. Amiloride (1 or 2 mM) did not prevent the recovery of intracellular pH, but it blocked the "overshoot" of the alkalinization at 10-12 min of recovery. In a medium containing 70 mM K+, 60 mM Na+ and 0.1 mM Ca2+, the recovery of pH, but not lactate washout, was significantly delayed. Removal of external Na+ caused severe energetic failure, decreases both in oxygen uptake and in N-acetyl aspartate concentration, indicating loss of viable tissue. In Na(+)-free superfusion, lactic acidosis caused a more severe drop in intracellular pH than in the presence of Na+. Complexing of extracellular Ca2+ in the Na(+)-free medium inhibited the acidification by 0.38 pH units during anoxia which is as much as the acidification caused by lactate accumulation in the absence of Na+. In Na(+)-free medium intracellular pH recovered, however, from an anoxic level to a normoxic value in 6 min. Metabolic damage of the slice preparation induced by anoxia in the absence of Na+ was as profound in the presence as in the absence of Ca2+ showing that accumulation of Ca2+ is not the only reason for the damage. It is concluded that recovery of intracellular pH from lactic-acidosis can occur independently of energetic recovery and involves acid extrusion mechanism(s) that is(are) dependent on external Na+ and sensitive to high K+.     

Effects of polyvalent cations and dihydropyridine calcium channel blockers on recovery of CNS white matter from anoxia

The effects of anoxic injury on the functional integrity of mammalian central white matter were studied electrophysiologically using the rat optic nerve model. Previous studies on this model have shown that extracellular Ca2+ is critical to the production of irreversible anoxic injury, and suggest that during anoxia Ca2+ crosses the membrane to enter the intracellular compartment. We attempted to elucidate the mechanism by which this damaging Ca2+ influx occurs. The inorganic Ca2+ channel blockers Mn2+ (1 mM), Co2+ (1 mM) or La3+ (0.1 mM) had no effect on recovery of the area under the compound action potential after a standard 60 min period of anoxia; only Mg2+ (10 mM) significantly improved recovery (54.9 +/- 8.9% vs. 28.7 +/- 10.1%, P less than 0.005). Treatment with organic Ca2+ channel blockers of the dihydropyridine class, nifedipine (1-10 microM) or nimodipine (1-40 microM), also had no effect on recovery from anoxia. We conclude that Ca2+ influx during anoxia does not occur via conventional Ca2+ channels sensitive to polyvalent cations or dihydropyridines.     

多胺对大鼠缺氧-复氧心肌细胞内钙的影响

目的：观察外源性低浓度多胺对大鼠缺氧-复氧心肌细胞钙超载的影响。 方法: 酶解分离大鼠心室肌细胞，用正常氧合Tyrode液灌流8 min, 换为缺氧液灌流32 min, 再转换为正常氧合Tyrode液灌流8 min,复制心肌缺氧-复氧模型。分别在缺氧前给予精胺，缺氧-复氧后给予精胺、精脒、腐胺。应用激光扫描共聚焦显微镜（LSCM）连续观察细胞内钙荧光强度的动态变化。 结果: 精胺（1 mol/L）对正常静息状态下大鼠心室肌［Ca2+］i无影响。缺氧前给予精胺，可取消复氧引起的心肌［Ca2+］i增高；缺氧-复氧后给予精胺、精脒、腐胺对缺氧-复氧引起［Ca2+］i升高也有不同程度的降低作用，其中以精胺的作用最强。复氧后给予精胺降低缺氧-复氧［Ca2+］i 升高的作用小于缺氧前给予。 结论: 缺氧前给予精胺可拮抗缺氧-复氧心肌细胞钙超载发生；复氧后给予精胺可使缺氧-复氧心肌细胞钙超载减轻，但其作用力度不如缺氧前给药。多胺拮抗缺氧-复氧心肌细胞钙超载的作用以精胺＞精脒＞腐胺的顺序递减。     

Anoxia-evoked intracellular pH and Ca2+ concentration changes in cultured postnatal rat hippocampal neurons.

The ratiometric indicators 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein and Fura-2 were employed to examine, respectively, intracellular pH (pHi) and calcium ([Ca2+]i) changes evoked by anoxia in cultured postnatal rat hippocampal neurons at 37 degrees C. Under both HCO3-/CO2- and HEPES-buffered conditions, 3-, 5- or 10-min anoxia induced a triphasic change in pHi consisting of an initial fall in pHi, a subsequent rise in pHi in the continued absence of O2 and, finally, a further rise in pHi upon the return to normoxia, which recovered towards preanoxic steady-state pHi values if the duration of the anoxic insult was < or = 5 min. In parallel experiments performed on sister cultures, anoxia of 3, 5 or 10 min duration evoked rises in [Ca2+]i which, in all cases, commenced after the start of the fall in pHi, reached a peak at or just following the return to normoxia and then declined towards preanoxic resting levels. Removal of external Ca2+ markedly attenuated increases in [Ca2+]i, but failed to affect the pHi changes evoked by 5 min anoxia. The latency from the start of anoxia to the start of the increase in pHi observed during anoxia was increased by perfusion with media containing either 2 mM Na+, 20 mM glucose or 1 microM tetrodotoxin. Because each of these manoeuvres is known to delay the onset and/or attenuate the magnitude of anoxic depolarization, the results suggest that the rise in pHi observed during anoxia may be consequent upon membrane depolarization. This possibility was also suggested by the findings that Zn2+ and Cd2+, known blockers of voltage-dependent proton conductances, reduced the magnitude of the rise in pHi observed during anoxia. Under HCO3-/CO2-free conditions, reduction of external Na+ by substitution with N-methyl-D-glucamine (but not Li+) attenuated the magnitude of the postanoxic alkalinization, suggesting that increased Na+/H+ exchange activity contributes to the postanoxic rise in pHi. In support, rates of pHi recovery from internal acid loads imposed following anoxia were increased compared to control values established prior to anoxia in the same neurons. In contrast, rates of pHi recovery from acid loads imposed during anoxia were reduced, suggesting the possibility that Na+/H+ exchange is inhibited during anoxia. We conclude that the steady-state pHi response of cultured rat hippocampal neurons to transient anoxia is independent of changes in [Ca2+]i and is characterized by three phases which are determined, at least in part, by alterations in Na+/H- exchange activity and, possibly, by a proton conductance which is activated during membrane depolarization.     

Activation of neurotransmitter release in hippocampal nerve terminals during recovery from intracellular acidification.

Intracellular pH may be an important variable regulating neurotransmitter release. A number of pathological conditions, such as anoxia and ischemia, are known to influence intracellular pH, causing acidification of brain cells and excitotoxicity. We examined the effect of acidification on quantal glutamate release. Although acidification caused only modest changes in release, recovery from acidification was associated with a very large (60-fold) increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons. This was accompanied by a block of evoked EPSCs and a rise in intracellular free Ca2+ ([Ca2+]i). The rise in mEPSC frequency required extracellular Ca2+, but influx did not occur through voltage-operated channels. Because acidic pH is known to activate the Na+/H+ antiporter, we hypothesized that a resulting Na+ load could drive Ca2+ influx through the Na+/Ca2+ exchanger during recovery from acidification. This hypothesis is supported by three observations. First, intracellular Na+ rises during acidification. Second, the elevation in [Ca2+]i and mEPSC frequency during recovery from acidification is prevented by the Na+/H+ antiporter blocker EIPA applied during the acidification step. Third, the rise in free Ca2+ and mEPSC frequency is blocked by the Na+/Ca2+ exchanger blocker dimethylbenzamil. We thus propose that during recovery from intracellular acidification a massive activation of neurotransmitter release occurs because the successive activation of the Na+/H+ and Na+/Ca2+ exchangers in nerve terminals leads to an elevation of intracellular calcium. Our results suggest that changes in intracellular pH and especially recovery from acidification have extensive consequences for the release process in nerve terminals. Excessive release of glutamate through the proposed mechanism could be implicated in excitotoxic insults after anoxic or ischemic episodes.     

Calcium-imaging with Fura-2 in isolated cerebral microvessels

Cerebral microvessels were isolated from rat hippocampus with a modified, mild collagenase digestion and loaded with the calcium fluoroprobe, Fura-2. Using a fluorescence microscope with quartz optics and an image analyser, it was possible to measure the intracellular concentration of free calcium ions [Ca2+]i in single microvessels for the first time. A resting level of (90 +/- 28) nmol/l (+/- SD, n = 24) was calculated which immediately rose after ionomycin application. The temporal resolution for [Ca2+]i of our set-up was 0.7 s. By image processing, sequences of digitized fluorescence images of single microvessels were colour-coded in terms of [Ca2+]i with a spatial resolution up to 1.5 microns (pixel size). Both the temporal and spatial resolution make our system suitable for investigation of calcium-mobilizing receptors of the blood-brain barrier.     

A transient acidification linked to intracellular Ca2+ in anti-mu-stimulated human B-lymphocytes.

Mitogen-induced cellular proliferation is in many cell types preceded by rapid changes in intracellular pH and free Ca2+ concentration. We studied the patterns of pH and Ca2+ changes in normal resting human B-lymphocytes after exposure to anti-mu antibodies and the monoclonal antibody IF5, reactive with the CD20 antigen, both able to activate resting B-lymphocytes to enter the G1 phase of the cell cycle. Monitoring intracellular pH with the pH-sensitive, fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein, we demonstrated that poly- and monoclonal anti-mu antibodies induced a rapid (maximum change within 2 min) intracellular acidification of 0.06 pH units followed by a slower (10-15 min) alkalinization towards, or slightly above, the resting pH of 6.88. The acidification response was amiloride-resistant, whereas the return to baseline was sensitive. Intracellular free Ca2+ was measured by using the fluorescent Ca2+ dye, indo-I. Exposure of cells to anti-mu resulted in a rapid increase (maximum change within 2 min) in cytoplasmic Ca2+ of 340 nM and a slower decline in fluorescence back to baseline of about 180 nM. In contrast to anti-mu, IF5 caused no change in cytoplasmic Ca2+ and pH. However, the Ca2+ ionophore ionomycin at low concentrations mimicked the Ca2+ response as well as the pH response to anti-mu. In Ca2(+)-free solutions the intracellular Ca2+ stores are usually rapidly depleted and, indeed, the Ca2+ and pH responses to anti-mu were reduced after 5 min and almost abolished after 35 min under such conditions.(ABSTRACT TRUNCATED AT 250 WORDS)