Action on phosphatidylcholine of the toxic phospholipase A2 from the venom of Bulgarian viper (Vipera ammodytes ammodytes)

The action of the neurotoxic complex and its components from the venom of the Bulgarian viper (Vipera ammodytes ammodytes) on phosphatidylcholine was studied. The nontoxic acidic component partially inhibited the phospholipase A₂ activity of the strongly toxic basic component. The basic component, separated from the acidic, was unstable and in the course of 12–14 days lost its enzymatic activity. The Michaelis constant, K_m = 1 × 10⁻³M was the same for the free phospholipase A₂ and the neurotoxic complex. Temperature optimum was 23–26°C and pH optimum was 9.9–10. A concentration of 1–3 × 10⁻³ M CaCl₂ was required for maximum enzyme activity. The influence of divalent cations on the initial velocity of the enzyme hydrolysis was studied. Although composed of a basic toxic phospholipase A₂ and a nontoxic acidic component the neurotoxic complex exhibited an insignificant enzyme activity.     

卵磷脂脂质体滴眼液治疗眼干燥症的研究

目的研究卵磷脂(PC)脂质体滴眼液治疗兔眼干燥症的疗效,并探讨其流变学性质。方法测定0.1%,0.2%和0.4%PC脂质体滴眼液的流变学性质,并通过建立兔眼干燥症模型,考察3种不同浓度的PC脂质体滴眼液的疗效。结果3种不同浓度的PC脂质体滴眼液均为非牛顿流体,且均可显著改善兔眼干燥症,PC在0.1%和0.2%浓度效果较好。结论PC脂质体滴眼液可有效治疗兔眼干燥症,且与其独特的流变学性质有关。     

Mastoparan-induced phosphatidylcholine hydrolysis by phospholipase D activation in human astrocytoma cells.

1. The effect of mastoparan on phosphatidylcholine hydrolysis was examined in 1321N1 human astrocytoma cells. Mastoparan (3-30 microM) caused an accumulation of diacylglycerol (DG) and phosphatidic acd (PA) accompanied by choline release in a concentration- and time-dependent manner. 2. In the presence of 2% n-butanol, mastoparan (3-100 microM) induced phosphatidylbutanol (PBut) accumulation in a concentration- and time-dependent manner, suggesting that mastoparan activates phospholipase D (PLD). Propranolol (30-300 microM), a phosphatidate phosphohydrolase inhibitor, inhibited DG accumulation induced by mastoparan, supporting this idea. 3. Depletion of extracellular free calcium ion did not alter the effect of mastoparan on PLD activity. 4. A protein kinase C (PKC) inhibitor, calphostin C (1 microM), did not inhibit mastoparan-induce PLD activation but the ability of mastoparan to stimulate phospholipase D activity was decreased in the PKC down regulated cells. 5. PLD activity stimulated by mastoparan was not prevented by pretreatment of the cells with pertussis toxin (PT) or C3 ADP-ribosyltransferase. Furthermore, guanine nucleotides did not affect PLD activity stimulation by mastoparan in membrane preparations. 6. Mastoparan stimulated PLD in several cell lines such as RBL-2H3, RBL-1, HL-60, P388, endothelial cells, as well as 1321N1 human astrocytoma cells. 7. These results suggest that mastoparan induces phosphatidylcholine (PC) hydrolysis by activation of PLD, not by activation of phosphatidylcholine-specific phospholipase C (PC-PLC); mastoparan-induced PLD activation is not mediated by G proteins.     

Influence of cationic amphiphilic drugs on the phosphatidylcholine hydrolysis by phospholipase A2

On chronic treatment certain amphiphilic drugs induce a generalized phospholipidosis. This drug side effect has been related to an inhibition of the lysosomal phospholipases due to the interaction of the drugs with phospholipids (PL). In the present experiments, the influence of the amphiphilic drugs ambroxol, imipramine, chloroquine and chlorphentermine on the hydrolysis of dipalmitoyl-phosphatidylcholine (DPPC) unilamellar liposomes by bee venom phospholipase A2 (PLase A2) was studied. Special emphasis was laid on the initial phase and temperature dependence. The activity of PLase A2 was measured continuously with a spectrophotometric assay using cresol red as indicator. In most cases a lag-phase of different duration was observed before the enzyme exhibited its full activity. The duration of the lag-phase and the rate of hydrolysis in the second phase are inversely related. The temperature dependence of the hydrolysis reveals a maximum of activity near the phase transition of the bilayer and a gradually decreasing activity at lower and higher temperatures, respectively. The analysis of the influence of amphiphilic drugs reveals three types of interaction. Imipramine and ambroxol shift the temperature activity profile towards lower temperatures without a substantial influence on the shape of the profile and on the maximal rate of hydrolysis. Chloroquine inhibits the enzyme activity without any temperature dependence. Chlorphentermine, the classical lipidosis inducing drug, exhibits a third type of interaction which seems to be a combination of the two former types.     

Effects of norepinephrine and cardiotrophin-1 on phospholipase D activity and incorporation of myristic acid into phosphatidylcholine in rat heart

The present study is part of a project on phospholipase D (PLD) in cardiac hypertrophy and analyzed effects on PLD activity of two growth stimuli, norepinephrine (NE) and cardiotrophin-1 (CT-1), in incubated rat heart. Phosphatidylcholine (PC) was labeled by (3)H-myristic acid. PLD produced (3)H-phosphatidylethanol ((3)H-PEth) from (3)H-PC in the presence of ethanol and maintained a basal formation of (3)H-PEth. Short-term and long-term exposure to NE for 2 or 13 h, respectively, enhanced the formation of (3)H-PEth, which was blocked by prazosin. Long-term pretreatment with NE or CT-1 increased the incorporation of (3)H-myristic acid into PC, which was blocked by atenolol. When the (3)H-PEth formation was expressed as a fraction of (3)H-PC, PLD activity seemingly was unchanged (NE) or markedly reduced (CT-1); the true effects, namely, stimulation by NE and nonresponsiveness towards CT-1, were unraveled by atenolol (NE) or when PLD activity was expressed as (3)H-PEth per ng protein. In conclusion, alpha-adrenoceptor activation increased PLD activity. Long-term treatment with NE (via beta-receptors) or CT-1 enhanced the (3)H-myristic acid incorporation into a PC compartment, that was not available for the alpha-receptor-mediated PLD activation. These results were discussed in regard to cellular mechanisms of cardiac hypertrophy and to the transphosphatidylation assay of PLD.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) primes human neutrophils for enhanced phosphatidylcholine breakdown by phospholipase D.

GM-CSF has previously been shown to increase human neutrophil phospholipase D (PLD) activity in response to fMLP. To further define the mechanism by which GM-CSF up-regulates PLD activity, we investigated the effect of GM-CSF pretreatment of neutrophils on phosphatidylcholine breakdown in response to a receptor-coupled stimulus N-formyl-methionyl-leucyl-phenylalanine (fMLP) and to a receptor-independent stimulus phorbol-myristate-acetate (PMA). Treatment of 1-0-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine-prelabeled human neutrophils with 200 pM GM-CSF for 1 hour at 37 degrees C led to a more rapid and increased accumulation (2-3 fold) of [3H]-alkyl-phosphatidic acid (or [3H]-alkyl-phosphatidylethanol when cells are stimulated in presence of 0.5% ethanol) in response to both fMLP or PMA. The data indicate GM-CSF up-regulates phosphatidylcholine hydrolysis by a PLD by interfering with the excitation-response coupling sequence at a site distal to the fMLP receptor.     

Effect of cholesterol on egg yolk phosphatidylcholine peroxidation in multilamellar liposomes.

Lipid peroxidation of aerated multilamellar liposomes composed of egg yolk phosphatidylcholine (EYPC) and cholesterol (CHOL) at molar ratios CHOL:EYPC = 0, 0.1, 0.2, 0.3, 0.4, 0.6 and 1.0 was studied during autooxidation and during Fe2+/H2O2-induced peroxidation by following the formation of conjugated diene and thiobarbituric acid reactive substances. The presence of cholesterol in the fluid lipid bilayers has an inhibiting effect on the EYPC peroxidation in the propagation phase of both the autooxidation and Fe(2+)-induced peroxidation free radical chain reactions. This inhibiting effect increases with the increase in CHOL:EYPC molar ratio. The inhibition of EYPC peroxidation by cholesterol probably originates a) from the increased lateral separation of polyunsaturated EYPC acyl chains caused by insertion of cholesterol between EYPC molecules, b) from the increased molecular packing of both the bilayer polar and hydrophobic regions due to the reduced bilayer hydration, and c) from the antioxidant properties of cholesterol.     

Optimization and characterization of a sphingomyelin/cholesterol liposome formulation of vinorelbine with promising antitumor activity

Vinorelbine (VRL) is a particularly lipophilic member of the vinca alkaloids which, as a class of drugs, exhibit improved cytotoxicity and therapeutic activity through increased duration of exposure. Here, we describe and optimize a sphingomyelin/cholesterol (SM/Chol) liposome formulation of VRL to maximize in vivo drug retention, plasma circulation time, and therapeutic activity. VRL was efficiently encapsulated (>90%) into 100 nm liposomes using an ionophore-mediated loading method. VRL retention in SM/Chol liposomes after intravenous injection in mice was dependent on drug-to-lipid ratio (D/L), with higher D/L ratios exhibiting increased drug retention (0.3 > 0.2 > 0.1, wt/wt) and improved pharmacokinetics. Cryo-electron microscopic examination of a high D/L ratio formulation indicated that the intravesicular regions of these liposomes were electron dense compared with empty liposomes. The optimized, high D/L ratio SM/Chol VRL formulation showed promising activity against subcutaneous B16 melanoma tumors compared with VRL or SM/Chol formulations of vincristine or vinblastine. Finally, the stability of the formulation was excellent (<5% drug leakage, >99% intact VRL, no changes in liposome size after 1 year at 2-8 degrees C). The optimized drug retention properties of the SM/Chol formulation of VRL, combined with its promising antitumor activity and pharmaceutical stability, make this formulation an excellent candidate for future clinical development.     

HPLC—ELSD法测定复合磷脂固醇脂质中磷脂酰胆碱的含量

目的:采用 HPLC-ELSD 法测定复合磷脂固醇脂质中磷脂酰胆碱(PC)的含量。方法:采用 Waters Nova-pak(?) silica60(?)径向柱(100 mm×8 mm,4 μm),流动相:己烷-异丙醇(3:4)为流动相 A,己烷-异丙醇-水(3:4:0.75)为流动相 B,梯度洗脱,流速1.5 mL·min~(-1);蒸发光散射检测,ELSD 漂移管温度:40℃,雾化气:氮气,载气压力:340 kPa。结果:在选定的色谱条件下,棕榈酸甘油三酯、磷脂酰乙醇胺、溶血磷脂酰乙醇胺、磷脂酰胆碱、鞘磷脂、溶血磷脂酰胆碱各成分之间可达到很好分离。磷脂酰胆碱进样量在21.2～127.2 μg范围内与峰面积的线性关系良好(r=0.9969),最低检测限为424 ng(S/N=3),3个浓度水平下磷脂酰胆碱的平均同收率(n=9)为100.6%。结论:方法灵敏,快速,准确,重复性好,可用于产品的质量控制。     

Phenylbutazone action on dimiristoyl phosphatidylcholine liposome phase transition and 8-anilino-1-naphthalene sulfonate binding.

Phenylbutazone (PhB), a powerful anti-inflammatory drug, is able to modify the phase transition of phospholipid bilayers without changing its calorimetric enthalpy (delta Hcal), as can be shown by differential scanning calorimetry (DSC) experiments. Under PhB interaction, dimiristoyl phosphatidylcholine (DMPC) multilamellar liposomes (MLV) undergo lateral phase separation as a result of immiscibility in the bilayer plane. On the other hand, the binding of the anionic fluorescent probe 8-anilino-1-naphthalene sulfonate (ANS) to the surface of DMPC liposomes is altered by PhB. Even though the quantum efficacy of the probe fluorescence emission remains unaffected, the negative cooperativity of the binding process disappears, with the intrinsic dissociation constant showing only a minor variation. From these results it is concluded that PhB would be most likely located close to the lipid:water interface.     

A structure based model for liposome disruption and the role of catalytic activity in myotoxic phospholipase A2s

Venom phospholipase A₂s (PLA₂s) display a wide spectrum of pharmacological activities and, based on the wealth of biochemical and structural data currently available for PLA₂s, mechanistic models can now be inferred to account for some of these activities. A structural model is presented for the role played by the distribution of surface electrostatic potential in the ability of myotoxic D49/K49 PLA₂s to disrupt multilamellar vesicles containing negatively charged natural and non-hydrolyzable phospholipids. Structural evidence is provided for the ability of K49 PLA₂s to bind phospholipid analogues and for the existence of catalytic activity in K49 PLA₂s. The importance of the existence of catalytic activity of D49 and K49 PLA₂s in myotoxicity is presented.     

Hydroxyethylated cationic cholesterol derivatives in liposome vectors promote gene expression in the lung

Three cationic cholesterol derivatives (CCDs), which differ in their types of amine and bear a hydroxyethyl group at the amine group, were synthesized and formulated into liposomes and nanoparticles as gene delivery vectors. In vitro transfection into A549 cells proved that liposomes formulated with CCDs and dioleoylphosphatidylethanolamine (DOPE) of 1/2 molar ratio were more effective than the corresponding nanoparticles with CCDs and Tween 80 at charge ratios (+/-) of 1/2, 3/1 and 5/1. Among the liposomal formulations, non-hydroxyethylated CCDs were more effective than hydroxyethylated ones in vitro. However, gene transfection in the lung through intratracheal injection showed opposite results to those in vitro, with liposomes containing hydroxyethylated CCDs being more potent than those containing non-hydroxyethylated CCDs. Transfection by liposomes with N,N-methyl hydroxyethyl aminopropane carbamoyl cholesterol iodide (MHAPC) showed the highest luciferase activity, resulting in 2- and 60-fold higher gene expression than jet-PEI and naked DNA, respectively. The distribution of MHAPC lipoplex after intratracheal injection was heterogeneous, and luciferase was expressed in epithelial cells lining the bronchi and bronchioles. All the lipoplexes led to higher TNF-alpha levels in the lung compared to the nanoplex and jet-PEI, but our findings suggested that modification of the cationic cholesterol with a hydroxyethyl group at the tertiary amine terminal, MHAPC, promoted gene expression in the lung without increasing the toxicity compared with other CCDs. This work firstly proved that liposomes containing hydroxyethylated CCDs could promote gene expression in the lung through intratracheal injection.     

In human monocytes interleukin-1 stimulates a phospholipase C active on phosphatidylcholine and inactive on phosphatidylinositol.

Interleukin-1 (IL-1) can initiate the synthesis of prostaglandins which in turn act as endogenous modulators of IL-1 production. The human monocyte/macrophage synthesizes various eicosanoids through the activation of the cellular phospholipase system. Cell stimulation results in the activation of phospholipase A2 (PLA2) whose major substrate is phosphatidylcholine (PC) and the release of the eicosanoid precursor arachidonic acid (AA) from PC. Another pathway is the stimulation of a phospholipase C (PLC) mainly active on phosphoinositides and the resulting formation of inositol phosphates (IPs) and diacylglycerol (DAG). Phospholipids other than phosphoinositides can also be hydrolysed by PLC to give rise to DAG. Studies have shown that IL-1 does not activate the IP pathway, but it primarily stimulates a PLC linked to phosphatidylethanolamine in cultured rat mesangial cells, and a PLC linked to PC in Jurkart cells. We have stimulated human monocytes with IL-1 and calcium ionophore A23187 and we have observed their effect on the phospholipase system. The results indicate that IL-1 does not activate the formation of IPs in cells labeled with [3H]myo-inositol. In contrast, in cells labeled with [3H]AA, IL-1 causes the formation of DAG associated with the hydrolysis of PC. Moreover, after stimulation with IL-1 there is no accumulation of free AA which would indicate that there has been no activation of PLA2, which occurs instead with A23187 stimulation. These data suggest that, in monocytes, IL-1 does not directly stimulate a PLA2 or a PLC active on phosphatidylinositol; instead it primarily stimulates a PLC active on PC.     

Action of phospholipase A2 on bilayers containing lysophosphatidylcholine analogs and the effect of inhibitors

The effects of several lysophospholipid analogs on the phase properties of codispersions with diacylphosphatidylcholine with or without fatty acids were examined. These ternary codispersions were readily hydrolyzed by phospholipase A2, and they underwent a rapid change in turbidity. Nonideal mixing or phase separation in the ternary codispersions is postulated to be responsible for their enhanced susceptibility to pig pancreatic phospholipase A2, as well as for their tendency to undergo spontaneous change in turbidity, presumably due to spontaneous fusion of the vesicles. Both of these processes were inhibited by a variety of structurally unrelated solutes like n-hexanol and mepacrine. These and other observations are interpreted to suggest that structural defects in bilayers of ternary codispersions are a common locus for the binding of phospholipase A2 and are responsible for the process underlying the change in turbidity. The experiments described here suggest that many of the common inhibitors of phospholipase A2 owe their effects to their ability to modify the quality of the substrate interface, rather than to a direct interaction with the enzyme.     

Protein kinase C-dependent coupling of alpha(2A/D)-adrenergic receptors to phospholipase D

To clarify the role of protein kinase C (PKC) in regulating the coupling pathway of alpha(2)-adrenergic receptors, we examined receptor activation of phospholipase D (PLD) in PC12 cells overexpressing alpha(2A/D) receptors, using [(3)H]phosphatidylbutanol formation as an index of PLD activity. In intact PC12/alpha(2A/D) cells, the ability of either epinephrine or the alpha(2)-receptor-selective agonist UK14304 to stimulate PLD was completely dependent on concomitant PKC activation. Pretreatment with the PKC activator phorbol dibutyrate revealed an agonist-stimulated PLD activity which was blocked by the alpha(2)-receptor-selective antagonist rauwolscine and by pertussis toxin treatment. Removal of extracellular calcium or tyrosine kinase inhibition by genistein pretreatment also eliminated the ability of epinephrine to stimulate PLD. These results indicate that alpha(2A/D)-adrenergic receptors couple via pertussis toxin-sensitive G proteins to PLD in a PKC-requiring and tyrosine kinase regulated manner.     

革兰阴性菌磷脂酶D致病机制的研究进展

磷脂酶D (PLD)是致病因子的一种,通过多种机制帮助病原菌感染和复制,其有望成为治疗病原菌感染的新靶标.本文综述几种革兰阴性菌PLD致病机制的研究进展.     

两性霉素B及其卵磷脂-胆固醇脂质体降解动力学研究

目的考察以卵磷脂、胆固醇为膜材制备的两性霉素B脂质体的稳定性及降解动力学。方法应用高效液相色谱法考察光和热对两性霉素B溶液及其脂质体中药物稳定性及降解动力学的影响,并拟合其降解动力学模型,求算降解半衰期。结果两性霉素B溶液及其脂质体光降解分别符合一级和零级动力学过程,降解半衰期分别为2.6和24.7d;热降解过程分别符合零级动力学和Higuchi方程;40℃下半衰期分别为27.8和1532.8d;60℃下半衰期分别为8.2和17.9d。结论脂质体可明显降低两性霉素B的光和热降解,延长药物降解半衰期,显著提高两性霉素B的稳定性。