Effects of lysophosphatidylcholine and arachidonic acid on the regulation of intracellular Ca2+ transport

Summary The role of lysophosphatidylcholine and arachidonic acid in signal transduction was investigated using subcellular organelles and permeabilized cells from liver. Both substances can be generated intracellularly by the action of phospholipase A₂ on phosphatidy1choline. Lysophosphatidylcholine as well as arachidonic acid raised the free Ca²⁺ concentration in the incubation media of permeabilized cells, isolated mitochondria and microsomes. The half maximally effective concentrations for Ca²⁺ release from mitochondria were 78 ± 1 mol/l for lysophosphatidylcholine and 80 ± 11 mol/l for arachidonic acid. Though isolated microsomes released Ca²⁺ in response to both agents, the combined presence of mitochondria and microsomes did not exhibit a synergism in Ca²⁺ release in response to arachidonic acid; the increase in the free Ca²⁺ concentration in response to lysophosphatidylcholine was even smaller than with mitochondria alone. It is concluded that the two reaction products of phospholipase A₂ can raise the cytoplasmic Ca²⁺ concentration and therefore may participate in cellular signal transduction. Send offprint requests to I. Rustenbeck at the above address     

氟伐他汀拮抗溶血磷脂酰胆碱致心律失常作用的机制研究

摘要：目的探讨降脂药氟伐他汀抑制外源性溶血磷脂酰胆碱(LPC)致心律失常作用的机理。方法20只雄性SD大鼠,随
机分为LPC组和氟伐他汀处理组,应用Langendorff装置行离体心脏灌注。LPC 5 μmol/L灌注5 min后冲洗30 min;氟伐
他汀组先灌注10 μmol/L 氟伐他汀30 min,然后灌注5 μmol/L LPC 5 min。细胞膜电流测定部分：采用全细胞膜片钳技
术,测定LPC对大鼠心室肌细胞膜电流的影响,并观察氟伐他汀的拮抗LPC的作用。结果LPC可引起离体心脏严重的心
律失常,以短阵室速和室颤常见,氟伐他汀可基本拮抗LPC的致心律失常作用;LPC可诱发心肌细胞的巨大非选择性阳离
子电流(INSC),这一作用可被氟伐他汀显著抑制;LPC诱发的INSC亦可被小分子G蛋白Rho的抑制剂和Rho激酶抑制剂所拮
抗。结论氟伐他汀通过抑制LPC诱发的非选择性阳离子电流来拮抗LPC的致心律失常作用,LPC的致心律失常作用与
小分子G蛋白Rho/Rho激酶信号途径有关。
     

串联质谱法在过氧化物酶体病筛查中的应用

目的：探讨串联质谱法检测极长链酰基肉碱（VLCAC）和溶血磷脂酰胆碱（LPC）在过氧化物酶体病筛查中的价值。方法：选取2017年1月至2021年3月以发育迟缓等神经系统异常就诊于上海市儿童医院,根据临床症状、磁共振成像和基因检测结果明确诊断为X-连锁肾上腺脑白质营养不良（X-ALD）患儿14例和脑肝肾综合征（ZS）患儿4例。另选取同年龄段体检儿童200名为健康对照组。使用含稳定同位素内标的溶剂萃取所有对象干血斑标本中的VLCAC和LPC,直接采用串联质谱法检测二十碳酰基肉碱（C20）、二十二碳酰基肉碱（C22）、二十四碳酰基肉碱（C24）、二十六碳酰基肉碱（C26）、二十碳溶血磷脂酰胆碱（C20:0-LPC）、二十二碳溶血磷脂酰胆碱（C22:0-LPC）、二十四碳溶血磷脂酰胆碱（C24:0-LPC）和二十六碳溶血磷脂酰胆碱（C26:0-LPC）水平,并计算C24/C20、C24/C22、C26/C20、C26/C22、C24:0-LPC/C20:0-LPC、C24:0-LPC/C22:0-LPC、C26:0-LPC/C20:0-LPC、C26:0-LPC/C22:0-LPC比值。采用Kruskal-Wallis H检验和Mann-WhitneyU检验比较各组间VLCAC和LPC各指标检测值及比值,采用偏最小二乘法和变量投影重要度权重评分分析各指标对判断疾病的贡献度。结果：除C24:0-LPC/C20:0-LPC外,所有指标和比值在各组间差异均有统计学意义（P<0.05或P<0.01）;X-ALD组与健康对照组、ZS组与健康对照组间,各指标有不同程度的差异,但X-ALD组与ZS组间差异无统计学意义（P>0.05）;偏最小二乘法分析显示X-ALD和ZS组与健康对照组能够完全分离,C26的变量投影重要度值最大。结论：串联质谱法检测VLCAC和LPC可作为过氧化物酶体病筛查的方法,其中C26或可作为诊断敏感指标。     

葛根素对溶血磷脂酰胆碱损伤血管内皮依赖性舒张功能的影响

目的 探讨葛根素（puerarin）对溶血磷脂酰胆碱（lysophosphatidylcholine，LPC）损伤家兔血管内皮依赖性舒张功能的影响。方法 采用离体血管环张力实验法检测乙酰胆碱诱导的内皮依赖性舒张反应来评价LPC对血管内皮功能的损害和葛根素的保护作用。结果 分别用2．5～10mg／LLPC孵育血管环30min，出现剂量依赖性抑制乙酰胆碱诱导的内皮依赖性舒张反应。用0．25～1g／L葛根素分别孵育血管环15min，再与5mg／LLPC共同孵育30min，明显改善LPC所致的血管舒张功能的损害。结论 葛根素对LPC所致的血管内皮依赖性舒张功能的损伤具有明显的保护作用。     

Different mechanisms of lysophosphatidylcholine-induced Ca mobilization in N2a mouse and SH-SY5Y human neuroblastoma cells

In mice, lysophosphatidylcholine (LPC) was found to be a physiological substrate of neuropathy target esterase, which is also bound by organophosphates that cause a delayed neuropathy in human and some animals. However, the mechanism responsible for causing the different symptoms in mice and humans that are exposed to neuropathic organophosphates still remains unknown. In the present study, we examined and compared the effect of exogenous LPC on intracellular Ca²⁺ overload in mouse N2a and human SH-SY5Y neuroblastoma cells. LPC caused an intracellular Ca²⁺ level ([Ca²⁺]_i) increase in both N2a and SH-SY5Y cells; moreover, the amplitude was higher in N2a cells than that in SH-SY5Y cells. Preincubation of the cells with verapamil, an L-type Ca²⁺ channel blocker, did not affect the LPC-induced Ca²⁺ increase in N2a cells, verapamil inhibited the response by 23% in SH-SY5Y cells. In Ca²⁺-free medium, LPC produced a significant [Ca²⁺]_i decrease in N2a cells, while it caused 64% of total [Ca²⁺]_i increase in SH-SY5Y cells. The results of a cell viability test suggest that N2a cells were more sensitive to LPC than were SH-SY5Y cells. These data suggested that the LPC-induced [Ca²⁺]_i increase was produced in each cell line through different mechanisms. In particular, the [Ca²⁺]_i increase occurred via entry through a permeabilized membrane in N2a cells, but through L-type Ca²⁺ channels as well as by Ca²⁺ release from intracellular Ca²⁺ stores in SH-SY5Y cells. Thus, the symptomatic differences of organophosphate-induced neurotoxicity between mice and humans are probably not related to the diverse amplitudes of intracellular Ca²⁺ overload produced by LPC. Moreover, the demyelination effect induced by LPC in mice may be a consequence of its detergent effect on membranes.     

HPLC-ELSD法测定大豆磷脂注射液中有关物质

目的:建立HPLC-ELSD法测定大豆磷脂注射液中有关物质溶血性磷脂酰胆碱的含量。方法:采用C18色谱柱(250mm×4.6mm,5μm),以甲醇-冰醋酸(500∶10,用三乙胺调pH至6.2)为流动相,柱温为室温,流速为1.0mL/min,进样量为10μL;蒸发光散射检测器参数:载气为空气,载气压力为250kPa,载气流量为2.0L/min,漂移管温度为60℃。结果:大豆磷脂注射液中主成分磷脂酰胆碱和溶血性磷脂酰胆碱能很好地分离检出,其线性浓度范围分别为20.4~801.6(r=0.9995)和10.652~532.6(r=0.9994)mg/L,溶血性磷脂酰胆碱的最低检测限为5.326ng。辅料对检测无干扰。结论:该方法准确、灵敏、快捷,适用于大豆磷脂注射液中有关物质的测定。     

银杏叶提取物对溶血卵磷脂胆碱致血管内皮细胞损伤的保护作用

应用传代培养的小牛主动脉内皮细胞(BAEC)为实验模型,观察银杏叶提取物(EGb)对溶血卵磷脂胆碱(LPC)损伤BAEC及其分泌功能的影响。结果发现培养BAEC与LPC(2.5μg·mL^-1)孵育24h后,出现细胞皱缩成团,大多细胞脱落、甚至细胞解体。细胞内漏出LDH增加,脂质过氧化物(MDA)含量增高,SOD活性降低,而PAI活性增高。LPC与不同浓度EGb共孵育24h,大部分细胞排列规则,形态基本正常,LDH明显减少,MDA含量下降,SOD活性增高及PAI活性降低。结果表明LPC可致培养血管内皮细胞损伤,而EGb可对抗LPC所造成的血管内皮细胞损伤,可能通过清除氧自由基、抗脂质过氧化作用和提高EC产生抗氧化能力。     

Betaine attenuates lysophosphatidylcholine-mediated adhesion molecules in aged rat aorta: Modulation of the nuclear factor-κB pathway

We previously reported that lysophosphatidylcholine (LPC) is a mediator of endothelial dysfunction in the expression of adhesion molecules (AMs) during aging. This study aimed at investigating the effects of betaine on LPC-related expression of AMs and the molecular modulation of nuclear factor-κB (NF-κB) activation in the aorta of aged rats and rat endothelial YPEN-1 cells. The experiment was performed on young (7 months) and old (21 months) rats; 2 groups of old rats were fed betaine (3 or 6 mg·kg^− 1·day^− 1 for 10 days). Betaine inhibited the expression of LPC-related AMs in the serum and tissue of aged rats, without affecting the elevated levels of serum LPC. Betaine also prevented the generation of reactive species, thereby maintaining the redox status via the enhancement of the thiol status during aging. Furthermore, betaine attenuated NF-κB activation via the dephosphorylation of IκB kinase (IKK) and mitogen-activated protein kinases (MAPKs) in aged aorta and LPC-treated YPEN-1 cells. Thus, betaine suppressed the LPC-related AM expression associated with NF-κB activation via the upregulation of IKK/MAPKs. Our findings provide insights into the prevention of vascular disorders and the development of interventions based on natural compounds, such as betaine.     

Chelation of copper reduces inhibition by oxidized lipoproteins of endothelium-dependent relaxation in porcine coronary arteries

Summary We examined the effect of dialyzing oxidized low-density lipoprotein (oLDL) against Krebs-Ringer solution, in the absence (yielding d-oLDL) or presence (yielding EDTA-oLDL) of ethylenediamine tetraacetic acid (EDTA), to investigate the mechanism that underlies the inhibition of endothelium-dependent relaxation (EDR) by o-LDL. Oxidation of LDL by exposure to Cu²⁺ resulted in the formation of a thiobarbituric acid-reacting substance (TBARS) and lipid hydroperoxide (LPO). At a concentration of 5mg/dl, d-oLDL markedly attenuated EDR in the porcine coronary artery. Analysis of doLDL by gel filtration revealed that TBARS was distributed in both the lipoprotein and the aqueous phases, whereas LPO was present only in the lipoprotein particles. Lysophosphatidylcholine (LPC), which has been suggested to be responsible for the impairment of EDR by oLDL, was present not only in the lipoprotein but also in the aqueous phase. However, EDR inhibitory activity was observed only in the oLDL particles, not in the aqueous phase. Almost all Cu²⁺ associated with the oLDL particles was removed by dialysis of oLDL against Krebs-Ringer solution containing EDTA. EDTA-oLDL or native LDL, at concentrations as high as 75mg/dl, exerted only a moderate inhibitory action on EDR, Both TBARS and LPO in EDTA-oLDL were distributed only in the lipoprotein particles, not in the aqueous phase. These results demonstrate that the impairment of EDR by oLDL is related both to LPO and to transition metal ions such as Cu²⁺ associated with the lipoprotein particles, not to the amount of the TBARS or negative charge, and that factors other than LPC may affect EDR.This work was partially supported by the Ministry of Health and Welfare of Japan (05670603).     

Inverse relations of serum phosphatidylcholines and lysophosphatidylcholines with vascular damage and heart rate in patients with atherosclerosis

Background and aims

The rapidly growing discipline of lipidomics allows the study of a wide spectrum of lipid species in body fluids and provides new insights into the pathogenesis of cardiovascular disease. We investigated serum phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) species in relation to arterial stiffness, hemodynamics, and endothelial dysfunction in symptomatic patients with atherosclerosis and in healthy controls.