Lysophosphatidylcholine, a Component of Stromal Phospholipids, as a Candidate Vasoconstrictive Factor in Stroma-Free Hemoglobin

Abstract: Stroma-free hemoglobin (SFH) contains a trace amount of phospholipids, which has been implicated in the toxic reactions associated with SFH. We analyzed stromal phospholipids by high-performance liquid chromatography and found that SFH contained small quantities of lysophosphatidylcholine (LPC), which is known to be capable of producing a defect in endothe-lium-dependent arterial relaxation, in addition to major classes of constituent phospholipids in red cell membrane. LPC content was determined to be 1.65 nmol/ml (hemoglobin 8.1 g/dl). To evaluate the role of these stromal phospholipids in SFH-induced vasoconstriction, we next examined the effect of lipids on vascular tone in rabbit aortic strips. Preincubation with the crude lipid extract or the LPC purified from SFH significantly inhibited acetylcholine-induced relaxation in phenylephrine-precontracted tissues. The LPC-induced inhibition was reversed by incubation of the tissues in the absence of lipids, indicating the functional integrity of endothelium. From these results, we propose a possibility that LPC, a component of stromal phospholipids, is a candidate for vasoconstrictive factors present in SFH.     

二苯乙烯苷对LPC损伤血管内皮细胞的保护作用及其机制

目的: 观察在溶血性磷脂酰胆碱(LPC)诱导下,二苯乙烯苷(TSG)对血管内皮细胞的保护作用及非对称性二甲基精氨酸(ADMA)、NO和细胞凋亡的影响。方法: 体外培养人脐静脉血管内皮细胞(HUVECs),细胞经培养、传代,将第3、4代用于实验。随机将细胞分为对照组、LPC组和TSG+LPC组。10 mg/L LPC作用于HUVECs,孵育24 h诱导内皮细胞损伤,建立细胞损伤的模型;10.0、1.0、0.1 μmol/L TSG预先作用于HUVECs 1 h后,再给予10 mg/L LPC作用于HUVECs共同孵育24 h,建立TSG+LPC组。然后,分别检测细胞的存活率、ADMA、NO和凋亡率的变化。结果: LPC组与对照组比较,细胞培养上清液中ADMA含量显著增加,而NO含量明显降低,细胞的凋亡有所增加,细胞的存活率降低。与LPC损伤组比较,10.0、1.0、0.1 μmol/L TSG作用HUVECs 1 h后,再予10 mg/L LPC作用HUVECs 24 h后,细胞培养上清液中ADMA显著降低, NO含量显著升高,细胞凋亡率降低和细胞存活率显著升高。结论: 二苯乙烯苷能够通过抑制ADMA的表达,增加NO 的生成,并抑制细胞的凋亡,增加细胞的存活,从而对LPC损伤的血管内皮细胞发挥保护作用。     

Lysophosphatidylcholine causes cardiac arrhythmia

The precipitation of malignant cardiac arrhythmias after onset of ischemia is well-documented [7]. Although many biochemical and physiological changes were observed during ischemia, the exact cause for disturbances in cardiac rhythm after ischemia remains obscure. Recently, several investigators reported that lysophosphoglycerides were accumulated in the ischemic myocardium [3, 11, 12] of several mammalian species. These lysophosphoglycerides are thought to originate from hydrolytic deacylation of phospholipids [4]. Since lysophosphoglycerides are cytolytic, [13] the accumulation of these lipids in myocardium may be important in the genesis of arrhythmias associated with ischemia. Depression of action potential in isolated cardiac fibers by lysophosphatidylcholine (LPC) further suggests the arrhythmogenic nature of these lipids [4]. More recent work showed that LPC and acyl carnitine, another amphiphilic compound, had similar electrophysiological effects on canine Purkinje fibers [5] and the effect might be dependent on a critical micelle concentration [1]. In this communication, we provide direct evidence that perfusion of hamster hearts with a solution containing LPC, the major lysophosphoglyceride in mammalian hearts, causes cardiac arrhythmias as a function of the quantity present in the free form. Exclusively bound lysophosphatidylcholine, associated with serum proteins, is not arrhythmogenic, as has been noted by others [5]. The results indicate also that low concentrations of free lysophosphatidylcholine, simulating those found in ischemic hearts, are arrhythmogenic.     

Modulation of Ecto-5′-Nucleotidase by phospholipids in human umbilical vein endothelial cells (HUVEC)

Ecto-5′-nucleotidase, the major enzyme controlling extracellular adenosine production, can be activated by phospholipids, e.g. lysophosphatidylcholine (LPC). This study examined the structural requirements of phospholipids to evoke this enzyme activation and figured out two new activators of ecto-5′-nucleotidase: platelet activating factor (PAF) and sphingosylphosphorylcholine (SPC). Potential signal transduction pathways including an involvement of protein kinase C and PAF-receptor were evaluated on the model of human umbilical vein endothelial cells (HUVEC). Cells were pre-incubated with 10 μM of various phospholipids including lysophosphatidylcholine, β-arachidonyl-γ-palmityl-α-phosphatidylcholine, β, γ-dipalmityl-α-phosphatidyl-choline, β,γ-dipalmityl-α-phosphatidylethanolamine, β,γ-dipalmityl-α-phosphatidylserine, γ-acyl-β-lyso-α-phosphatidylethanolamine, β-acetyl-γ-O-hexadecyl-α-phosphatidylcholine (platelet activating factor), lysophosphatidylic acid, sphingosine-1-phosphate and sphingosylphosphorylcholine. In the cell supernatant the extracellular dephosphorylation rate of the fluorescent AMP-analogue 1, N⁶-etheno-5′AMP to 1, N⁶-etheno-adenosine was measured by HPLC. Out of these ten structurally related phospholipids only lysophosphatidylcholine, sphingosylphosphatidylcholine and platelet activating factor dose-dependently increased the activity of ecto-5′-nucleotidase. Pharmacological blocking experiments revealed that neither the activation of PAF-receptor nor of protein kinase C were important for mediating the activation of ecto-5′-nucleotidase. Thus, using information on the known molecular structures of tested phospholipids, a phosphatidylcholine residue in α-position and a short chain length fatty acid esterified in β-position seem essential for activation of ecto-5′-nucleotidase by glycerophospholipids. Since all tested phospholipids have similar fatty acid chain lengths and residues in α-position, they should act similarly on membrane fluidity. It is concluded that the observed effects are not based on changes in membrane fluidity by the added phospholipids, but rather involve a yet to be determined phospholipid-receptor.     

溶血卵磷脂对血管内皮细胞增殖及凋亡的影响

目的观测溶血卵磷脂(LPC)对血管内皮细胞增殖及凋亡的影响,从而探讨动脉硬化发生的机制。方法取体外培养的人脐静脉内皮细胞(HUVECs),在含不同浓度LPC(5、10、20mg/L)的培养基中分别培养6、12、24、48h,用四甲基偶氮唑蓝(MTT)比色法、流式细胞仪、荧光显微镜观测血管内皮细胞增殖及凋亡的变化。结果与正常对照组比较,LPC抑制内皮细胞增殖,促进细胞凋亡,且其作用呈时间-效应、浓度-效应依赖关系,但随着LPC浓度增加,细胞凋亡增加的同时细胞死亡比例也增加。结论LPC抑制血管内皮细胞增殖,促进细胞凋亡,可能是其致动脉粥样硬化机制之一。     

切应力和溶血磷脂酰胆碱对内皮细胞表面黏附分子表达的影响

在体内 ,内皮细胞的功能不仅受化学因子的调节 ,而且还受力学因素的影响。为探讨流体切应力和溶血磷脂酰胆碱 ( L ysophosphatidylcholine,L yso- PC)的双重作用对培养的人脐静脉内皮细胞 ( Hum an um bilical veinendothelial cells,HU VECs)表面黏附分子 ICAM- 1、VCAM- 1、E- selectin表达的影响 ,采用流式细胞仪技术检测了L yso- PC( 3 0 μg/m l)和流体切应力 ( 2 .2 3、4.2 0 dyne/cm2 )的协同作用下内皮细胞黏附分子表达的变化。结果显示 :在受剪切作用之前 ,用 L yso- PC孵育激活内皮细胞 ,或预先剪切后再用 L yso- PC孵育 ,内皮细胞的 ICAM- 1和VCAM- 1表达与两种刺激同时作用相比 ,显著增加 ( P<0 .0 5 ) ;切应力或 L yso- PC的单独作用 ,以及两种刺激同时存在对 HU VEC的 E- selectin表达无显著影响。而在受剪切作用之前 ,用 L yso- PC孵育激活内皮细胞 ,或预先剪切后再用 L yso- PC孵育 ,内皮细胞的 E- selectin表达与两种刺激同时作用相比 ,显著增加 ( P<0 .0 5 )。结论认为 :即使在不利于细胞黏附的力学环境中 ,流体切应力与 L yso- PC的协同作用 ,也可能是在炎症部位单核细胞对内皮细胞募集增加的重要原因之一     

Inhibitory effects of palmitoylcarnitine and lysophosphatidylcholine on the sodium current of cardiac ventricular cells

We investigated the effects of ischemia-related amphipathic compounds, palmitoylcarnitine (PamCar, 0.5–50 M) and lysophosphatidylcholine (lysoPtdCho, 5–50 M) on sodium current (I _Na) of guinea-pig ventricular myocytes. The cells were perfused with low-Na⁺ (60 mM) Tyrode's solution, and Ca²⁺ and K⁺ currents were blocked by external Co²⁺ (3 mM) and internal Cs⁺ (140 mM), respectively. I _Na was elicited by depolarizing voltage steps from a holding potential of –100mV at a temperature of 33 °C. PamCar (5 M) decreased the peak I _Na (attained at –20mV or –30mV) from 6.1±2.1 nA to 3.9±1.4 nA (n=11), or by 36.1% within 2 min, and shifted the curve of steady-state I _Na inactivation by 5.4 mV in the positive direction (from –76.3±4.6 mV, control to –70.9±4.0 mV, in PamCar, n=4). Partial restoration of the amplitude and the shift of the steady-state inactivation curve of I _Na was attained after washout of PamCar. In contrast, lysoPtdCho at concentrations over 10 M irreversibly depressed the I _Na within 0.5–3 min and the reduction of IinNa was followed by cell contracture or cell death (n=9). The survival time, defined as a period from the start of lysoPtdCho application to the time of the last successful recording of the I _Na (before evolution of sudden changes in the holding current), depended on the concentrations of lysoPtdCho. Both PamCar and lysoPtdCho retarded the time course of activation and inactivation of I _Na. These findings are compatible with the idea that PamCar and lysoPtdCho decrease the maximum Na⁺ conductance and alter the surface negative charge of the membrane, perhaps via amphiphilic intervention in the phospholipid bilayers. However, PamCar had an additional effect that indicates more direct but reversible incorporation of this agent with the Na⁺ channels or integral membrane proteins.     

Exogenous lysophosphatidylcholine increases non-selective cation current in guinea-pig ventricular myocytes

Whole cell, patch-clamp studies were performed to examine the effect of lysophosphatidylcholine (LPC) on the membrane current in guinea-pig ventricular myocytes. The addition of 10 μM LPC to the external solution induced a membrane current which had a reversal potential of 0 mV. When Na⁺, the main cation in the external solution, was replaced by either K⁺, N-methyl-D-glucamine (NMG) or 90 mM Ca²⁺, LPC induced a current with the reversal potential near 0 mV, indicating that the current passed through a Ca²⁺-permeable non-selective cation channel. The order of the cationic permeability calculated from the reversal potential of the current was Cs⁺ > K⁺ > NMG > Na⁺ > Ca²⁺. Cl⁻ did not pass through the LPC-induced channel. The LPC-induced current was not blocked by Gd³⁺ in the external solution, nor by the absence of Ca²⁺ in the pipette solution. In conclusion, LPC induces a Ca²⁺-permeable non-selective cation channel in guinea-pig ventricular myocytes. Received: 11 September 1995/Received after revision: 3 January 1996/Accepted: 12 February 1996     

溶血磷脂酰胆碱诱导内皮细胞表达血管内皮生长因子及丹酚酸B的抑制作用

研究溶血磷脂酰胆碱对内皮细胞中血管内皮生长因子表达的影响以及丹酚酸B的保护作用。在人脐静脉内皮细胞株ECV30 4培养基中加入溶血磷脂酰胆碱或溶血磷脂酰胆碱 +丹酚酸B ,用酶联免疫吸附试验检测各组内皮细胞培养上清液中血管内皮生长因子蛋白含量 ;用原位杂交检测血管内皮生长因子mRNA的表达。结果显示 ,培养的ECV30 4中未见血管内皮生长因子mRNA的表达 ,溶血磷脂酰胆碱刺激后可见血管内皮生长因子mRNA的高表达 ,加入丹酚酸B后阳性反应明显低于溶血磷脂酰胆碱组。酶联免疫吸附试验结果显示 ,溶血磷脂酰胆碱可使ECV30 4细胞条件培养基中血管内皮生长因子蛋白表达明显增加 ,丹酚酸B可明显降低其含量。以上结果提示 ,溶血磷脂酰胆碱能诱导ECV30 4表达高水平的血管内皮生长因子 ,丹酚酸B可明显降低其含量