CD3 antigen-mediated calcium signals and protein kinase C activation are higher in CD45R0+ than in CD45RA+ human T lymphocyte subsets

T lymphocytes may be separated into subsets according to their expression of CD45 isoforms. The CD45R0⁺ T cell subset has been reported to proliferate in response to recall antigen and to mitogenic mAb to a much greater extent than the CD45RA⁺ subset. This difference could be due to more efficient coupling of the T cell antigen receptor complex to mitogenic signaling pathways. To investigate this possibility, CD3 antigen-induced calcium signals, diacylglycerol (DAG) production and protein kinase C (PKC) activation levels were compared in CD45RA⁺ and CD45R0⁺ human T lymphocyte subsets derived from peripheral blood. The mean CD3-induced rise in intracellular calcium was 80% greater in CD45R0⁺ than in CD45RA⁺ cells. Basal DAG levels in CD45R0⁺ cells were found to be, on average, 60% higher than in CD45RA⁺ cells (p = 0.002), but the CD3-induced production of DAG over background was not different in the two subsets (p = 0.4). Basal PKC activity, and CD3-induced PKC activation levels over background, were found to be 50% and 140% higher, respectively, in CD45R0⁺ cells than in CD45RA⁺ cells (p = 0.015 and 0.023). The CD45R0⁺ subset contained a higher proportion of cells expressing activation markers, such as CD25, CD71 and major histocompatibility complex class II, when compared to the CD45RA⁺ subset. Our results suggest that the elevated basal DAG levels observed in the CD45R0⁺ subset may reflect the recent activation of these cells. Both the higher basal DAG and CD3-induced elevation in intracellular calcium observed in the CD45R0⁺ cells may contribute to the greater PKC activation signals triggered by CD3 mAb in this subset. These findings elucidate the greater response of CD45R0⁺ T cells to mitogenic stimuli compared to CD45RA⁺ cells.     

A fast perfusion system for single cell physiology optimized for microscopes with water immersion objectives

A perfusion system was constructed which allows the fast application of different solutes underneath a water immersion objective. The perfusion system is mounted into the immersion objective by milling a slot into the frontal metal plate of the lens holder. It consists of a five-channel pipette fixed to the objective and solution reservoirs gated by computer controlled magnetic valves. Up to five different solutions can be applied to the specimen under study. The solution between objective and specimen is completely exchanged after 1–2 s as determined from fluorescence measurements. This arrangement is optimized for [Ca²⁺] measurements with a fluorescence measurement system in tissue slices, where upright microscopes are required. It offers the advantage of saving a micromanipulator for the perfusion pipette and facilitates a fast, reproducible and precise positioning of the perfusion system.     

Influence of caffeine,Ca2+, and Mg2+ on ryanodine depression of the tension transient in skinned myocardial fibers of the rabbit

Ryanodine, a blocker for Ca²⁺-release channels of the sarcoplasmic reticulum (SR Ca²⁺-release channels), induces depression of myocardial contraction in isolated intact muscle, which is consistent with depression of the caffeine-induced tension transient in skinned muscle fibers. In isolated SR, ryanodine binds to a specific receptor with high affinity, and this binding is enhanced by caffeine and increasing Ca²⁺ and decreased by increasing Mg²⁺. The aim of this study was to test the hypothesis that depression of myocardial contraction is mediated by changes in ryanodine-receptor binding properties. Accordingly, factors (caffeine, Ca²⁺, and Mg²⁺) affecting ryanodine-receptor binding properties in the isolated SR membrane were studied in skinned myocardial fibers from adult rabbits. The depression of the caffeine-induced tension transient by ryanodine (ryanodine depression) influenced by these three factors was measured. In a dose-dependent manner, increasing caffeine or Ca²⁺ concentrations enhanced the ryanodine depression. The concentrations for 50% ryanodine depression (IC₅₀) approximated 7mM for caffeine, and pCa 5.25 for Ca²⁺. When 1 M ryanodine and 25 mM caffeine were combined, ryanodine depression was independent of Ca²⁺ at low Ca²⁺ concentrations (20%–30% at pCa>8 and 7.5) and was a direct function of Ca²⁺ at higher concentrations (pCa 7.5–6.0 with IC₅₀ approx. pCa 6.75). In contrast, increasing Mg²⁺ reduced the ryanodine depression with IC₅₀ approximately equal to pMg 3.3. In conclusion, the caffeineor Ca²⁺-enhanced, and Mg²⁺-reduced ryanodine depression observed in this study is consistent with known ryanodinereceptor binding properties.     

骨组织工程用聚磷酸钙的结构和性能的研究

多孔聚磷酸钙（Calcium polyphosphate，CPP）生物陶瓷是一种新型的骨组织工程支架材料，国外有研究表明聚磷酸钙作为骨组织工程支架材料具有良好的生物相容性及可降解性。通过对原料煅烧过程的研究，探索了聚合度的计算方法，制备出不同聚合度的材料。通过对材料烧结温度的试验，制得了不同晶型的聚磷酸钙多孔材料。在Tris—HCl缓冲液中进行的体外降解实验表明，CPP多孔支架材料是可控降解的，不同聚合度和晶型的支架材料力学强度和降解性能不同。随着聚合度的增加，材料的力学强度增大，降解速率变小；非晶态的CPP17d就可以完全降解，7晶型的CPP25d可完全降解，β和α晶型的CPP降解缓慢，30d分别降解了大约12％和5％。因此聚磷酸钙是一种很有前途的骨组织工程支架材料，凭借其独特的无机聚合物结构及降解性能，有望实现可控速率的降解。     

Effect of hindlimb unloading on resting intracellular calcium in intrafusal fibers and ramp-and-hold stretches evoked responsiveness of soleus muscle spindles in conscious rats

The present study was to investigate and evaluate the dynamic changes of calcium homeostasis of soleus muscle spindle for the exploration of the potential mechanisms of muscle spindle degeneration induced by hindlimb unloading. We systematically observed the changes in immunoreactivity of calbindin D28K (CaBP-D28K), intracellular resting calcium in intrafusal fibers of soleus muscle spindle, and the responsiveness of muscle spindles to ramp-and-hold stretches after short- and long-term (3, 7, 14 d) hindlimb unloading. The immunoreactivity of CaBP-D28K started to decrease after 7-d hindlimb unloading, while its decrease was obviously different compared with the control group 14 d following the hindlimb unloading. The resting calcium concentration was increased significantly at 3 d, and reached the peak level 14 d after the hindlimb unloading. The responsiveness of muscle spindles, assessed by investigating Index3-L₂, Index3-L₃, and Index5, to ramp-and-hold stretches started to decrease during the period of 7-d hindlimb unloading. All Indexs, in particular Index3-L₃ and Index5, were significantly decreased at 14 d after the hindlimb unloading. The data suggest the disturbance of calcium homeostasis in intrafusal fibers during the exposure to hindlimb unloading might gradually influence the structure and function of muscle spindles.     

Ketamine inhibits LPS-induced calcium elevation and NF-kappa B activation in monocytes

Objective:To investigate whether ketamine could inhibit lipopolysaccharide (LPS)-induced intracellular calcium elevation and NF-kappa B activation in monocytes. Materials and methods:Isolated rat monocytes were challenged with 10 g/ml LPS with or without the presence of various concentrations of ketamine (10, 100, 1000 M). Intracellular calcium was monitored by laser confocal microscopy. NF-kappa B activity of the nuclear extracts of monocytes was analyzed by electrophoretic mobility shift assay (EMSA). Results:LPS provoked a significant calcium elevation and enhanced NF-kappa B activity in monocytes. Ketamine above concentration of 100 M inhibited endotoxin-induced intracellular calcium elevation and NF-kappa B activity. Ketamine itself had no effect on either of them. Conclusions:These findings suggest that ketamine could suppress NF-kappa B in monocytes exposed to endotoxin, and this anti-inflammatory effect might act through attenuating intracellular calcium elevation.Received 31 October 2003; returned for revision 18 December 2003; accepted by I. Ahnfelt-Rønne 26 Januaryy 2004     

What does TnCDANZ fluorescence reveal about the thin filament state?

Fluorescence of skinned psoas fibres reconstituted with the troponin C subunit labelled with the fluorescent probe dansylaziridine (TnC_DANZ) increases upon activation with Ca²⁺. This fluorescence enhancement is due to Ca²⁺ binding to the Ca²⁺-specific binding sites of TnC_DANZ and attachment of cross-bridges to the actin filament. We found that approximately 20% of the enhanced fluorescence signal derived from Ca²⁺ binding to TnC_DANZ and 80% from cross-bridge attachment during maximal activation. Furthermore we studied the effects of different cross-bridge states on TnC_DANZ fluorescence. Weakly bound, non-force-generating cross-bridge states (pCa 8, low ionic strength) and rigor cross-bridges revealed similar effects on the TnC_DANZ fluorescence. Strongly attached, force-generating states, however, increased fluorescence to the greatest extent. These results suggests a complex system of reciprocal couplings between TnC and different attached cross-bridge states. Cooling or increase of inorganic phosphate decreased isometric force but hardly decreased fluorescence, suggesting the accumulation of attached cross-bridge states with low tension output.     

心房颤动时心房肌细胞L型Ca2+通道与肌浆网间Ca2+信号转导的探讨

目的：探讨房颤时心房肌细胞膜上L型Ca2+通道与肌浆网之间的Ca2+信号转导。 方法： 杂种犬10条，随机分为正常对照组和单纯房颤组。房颤组用起搏器行右心房快速起搏（500±20）次/分，术后观测24周。正常对照组不植入起搏器。胶原酶Ⅱ型分离心房肌细胞，用激光共聚焦显微镜检测L型Ca2+ 通道对细胞内Ca2+浓度变化的影响；L型Ca2+通道与肌浆网三磷酸肌醇受体（IP3R）和兰尼碱受体（RyR）之间的Ca2+信号转导。 结果： （1）L型Ca2+通道与肌浆网IP3R之间的Ca2+信号转导：正常对照组、单纯房颤组的心房肌细胞在用mibefradil和丁卡因分别阻滞T型Ca2+通道和RyR后给予细胞膜激动剂时，细胞内Ca2+浓度均升高（分别为1.4000±0.0776和1.5169±0.4414），组间比较无显著差异（P＞0.05）；（2）L型Ca2+通道与肌浆网RyR之间的Ca2+信号转导：正常对照组的心房肌细胞在用mibefradil和肝素分别阻滞T型Ca2+通道和IP3R后给予细胞膜激动剂时，细胞内Ca2+浓度升高（1.5576±0.1989），单纯房颤组的细胞内Ca2+浓度也升高（1.5372±0.2952），两组间比较无显著差异（P＞0.05）。 结论： 房颤时L型Ca2+通道与RyR和IP3R之间可能存在信号转导，但其可能在房颤时的细胞内Ca2+超载及异常Ca2+信号转导方面不起重要作用。     

大鼠内毒素休克肝线粒体Ca^2＋,Mg^2＋含量变化与线粒体损伤

本文在大鼠内毒素休克模型上，观察了肝线粒体结构和功能与Ｃａ＾２＋，Ｍｇ＾２＋含量变化的关系。结果发现：休克动物肝组织和线粒体的Ｃａ＾２＋浓度升高，Ｍｇ＾２＋浓度下降与正常对照组相比有非常显著性差异；用图像分析仪定量分析显示线粒体结构明显受损，线粒体标志酶之一－琥珀酸脱氢酶（ＳＤＨ）活性明显下降，本文认为组织和线粒体内Ｍｇ＾２＋／Ｃａ＾２＋比值下降是休克细胞不可逆损伤重要因素之一。     

Ultrastructure,function and composition of smooth muscle

Filamentous myosin is present in both relaxed (myosin light chains unphosphorylated) and contracted (light chains phosphorylated) vascular smooth muscle. The organization of myosin and actin filaments and the insertion of the latter on cytoplasmic and plasma membrane bound dense bodies is consistent with a mini sarcomere-like organization and a sliding filament mechanism of contraction in smooth muscle. Mitochondria are high capacity, low affinity Ca stores in smooth muscle. They do not play a role in the regulation of cytoplasmic Ca²⁺ at physiological levels. The localization and Ca content of the junctional sarcoplasmatic reticulum (SR) is consistent with this organelle being the major intracellular source of activator Ca released by excitatory transmitters. Repeated contractions in the absence of extracellular Ca²⁺ (thought to represent recycling of intracellular activator Ca²⁺) can be demonstrated if the excitatory agent is not allowed to remain in contact with the smooth muscle throughout relaxation; the demonstration of “recycling” is facilitated if the efflux of cellular Ca²⁺ is blocked. The rise in total cytoplasmic calcium measured with electron probe analysis during a maintained (30 min) contracture in rabbit portal-anterior mesenteric vein smooth muscle (∼0.9 mmol/kg dry cytoplasm) is greater than the amount of Ca that could be bound to calmodulin.