Bioscience Reports

We have investigated the intramembranal ion traffic in apoptotic 3T6 cells in culture. Apoptosis was induced by various treatments, such as serum deprivation, high density growth and hydrogen peroxide at subnecrotic doses. Cell death was assessed by nucleosomal DNA fragmentation, single cell electrophoresis, immunofluorescence and histological staining. To study the modifications of membrane structure and function, we adopted a well established biophysical strategy based on the measurement of the electrical conductivity of cell suspensions, as a function of the frequency of the electrical field applied to the sample. A comparison between the conductivity of normal and apoptotic cell suspensions shows that programmed cell death causes a decrease of membrane conductivity which indicates a diminished intramembranal ion traffic. Our results strongly suggest that one of the early events in the triggering of apoptosis is represented by an overall reduction of plasma membrane function. Finally, our results are in agreement with the idea that the nucleus is not the sole target of the apoptotic process.

Mitochondria are known to participate in the initiation of programmed cell death (PCD) in animals and in plants. The role of chloroplasts in PCD is still unknown. We describe a new system to study PCD in plants; namely, leaf epidermal peels. The peel represents a monolayer consisting of cells of two types: phototrophic (guard cells) and chemotrophic (epidermal cells). The peels from pea (Pisum sativum L.) leaves were treated by cyanide as an inducer of PCD. We found an apoptosis-enhancing effect of illumination on chloroplast-containing guard cells, but not on chloroplastless epidermal cells. Antioxidants and anaerobiosis prevented the CN−-induced apoptosis of cells of both types in the dark and in the light. On the other hand, methyl viologen and menadione known as ROS-generating reagents as well as the Hill reaction electron acceptors (BQ, DAD, TMPD, or DPIP) that are not oxidized spontaneously by O2 were shown to prevent the CN−-induced nucleus destruction in guard cells. Apoptosis of epidermal cells was potentiated by these reagents, and they had no influence on the CN− effect. The light-dependent activation of CN−-induced apoptosis of guard cells was suppressed by DCMU, stigmatellin or DNP-INT, by a protein kinase inhibitor staurosporine as well as by cysteine and serine protease inhibitors. The above data suggest that apoptosis of guard cells is initiated upon a combined action of two factors, i.e., ROS and reduced plastoquinone of the photosynthetic electron transfer chain. As to reduction of ubiquinone in the mitochondrial respiratory chain, it seems to be antiapoptotic for the guard cell.

Nghiên cứu này là một phần trong nỗ lực hiểu vai trò của các hoạt động tế bào cụ thể trong quá trình tái hấp thu xương. Các thí nghiệm đã được thực hiện trong đó những tác nhân dược lý đã được sử dụng để ức chế từng chế độ hoạt động của tế bào hủy xương, chẳng hạn như sự di động và sự tiết. Các tác động của những điều trị này đối với quá trình tái hấp thu xương được đánh giá bằng phương pháp hiển vi điện tử quét định lượng. Các hợp chất bao gồm colchicine, được sử dụng để ức chế sự di động của tế bào hủy xương; ion molybdate được sử dụng để ức chế có chọn lọc hoạt động xúc tác của phosphatase acid tiết ra, và omeprazole được sử dụng để ức chế sự tiết ion hydro. Tất cả các hợp chất đều ức chế quá trình tái hấp thu xương của tế bào hủy xương, nhưng ảnh hưởng riêng lẻ đến các chế độ hoạt động xác định. Những phát hiện này gợi ý rằng mỗi chế độ hoạt động của tế bào hủy xương là thiết yếu cho quá trình tái hấp thu xương, và sự ức chế theo kiểu cụ thể có thể cung cấp một phương tiện để kiểm soát hoạt động quá mức của tế bào hủy xương trong bệnh lý.

Estrogens have been shown to have many positive effects on the function of arterial wall, and recent evidence suggest that 17β-estradiol has a direct action in reducing the accumulation of cholesteryl ester in macrophages. The mechanisms underlying the effects of 17β-estradiol on foam cell formation, however are poorly understood. The aim of this study is to investigate the role of 17β-estradiol in the regulation of the cholesteryl ester cycle and cholesterol efflux in human macrophages. In addition, the influence of 17β-estradiol on apolipoprotein E (apoE) and lipoprotein lipase (LDL) secretion by the cells was also tested. Human Monocyte Derived Macrophages (HMDM), matured in the presence or the absence of 17β-estradiol, were loaded with [3H]-cholesteryl ester-labeled-acetyl LDL (low density lipoprotein) and the efflux of radioactivity into the medium was measured. The effect of 17β-estradiol on cellular activities of acyl coenzyme A: cholesterol acyl transferase (ACAT), and both neutral and acid cholesteryl ester hydrolase (CEH) and the secretion of apoE and LDL into the medium, were also studied. The results indicate that 17β-estradiol induces an increase in the amount of labeled cholesterol released from the cells and, the data obtained from the measurements of ACAT and CEH activities showed that, in estrogen-treated HMDM, the cholesteryl ester cycle favors the hydrolysis of lipoprotein cholesterol by CEH in comparison with its acylation by ACAT. In particular, for the first time a strong enhancement of neutral and acid CEH in human macrophages by 17β-estradiol, was demonstrated. ApoE and LDL secretion increased during the maturation of monocytes to macrophages, and was not modified by 17β-estradiol. In contrast, loading the cells with cholesterol by incubation in the presence of acetylated or oxidized LDL produced an increase in the levels of apoE secreted by both estrogen-treated and control macrophages. The activity of LPL found in the cell medium, on the other hand, in lipid loaded cells tended to be increased only in estrogen treated macrophages, suggesting that the effects of estrogen on unloaded macrophages are different from those produced on lipid-loaded macrophages. On the whole, the present findings suggest that one of the mechanisms by which 17β-estradiol acts to reduce cholesterol accumulation in macrophages is by increasing reverse cholesterol transport through the enhancement of the cholesteryl ester cycle, so that the generation of intracellular unesterified cholesterol for excretion from the cells is favored.

Using high density and low density lipoproteins (HDL and LDL) labeled with fluorescent analogues of phosphatidylcholine or sphingomyelin it was found that low amounts (10−12 M) of prostaglandins E1 and F2α induced different structural rearrangements of the lipoprotein surface, whereas prostaglandins E2 and F1α had no effect. The effects of prostaglandin E1 on HDL were largely paralled by those of this prostaglandin on synthetic recombinants prepared from pure apolipoprotein A1, phospholipids and cholesterol and were demonstrated to be caused by prostaglandin-apolipoprotein interaction. The interaction resembled that of a ligand with a specific receptor protein because it was specific, reversible, concentration and temperature dependent and saturable. However the retaining capacity of HDL or LDL for prostaglandin E1 as determined by equilibrium dialysis was very low and a single prostaglandin E1 molecule was able to induce structural changes in large numbers of discrete lipoprotein particles. To explain this remarkable fact a non-equilibrium model of ligand-receptor interaction is proposed. According to that model in open systems characterized by weak ligand-receptor binding, high diffusion rate of the ligand and long relaxation times which exceed the interval between two successive receptor occupations, the ligand-induced changes will accumulate, resulting in transformation of the system into a new state which may be far away from equilibrium. It is emphasized that the low mobility of lipids constituting the environment of the receptor protein plays a critcal role in this type of signal amplification. It was further demonstrated that the PGE1-induced changes of the lipoprotein surface resulted in an enhancement of LDL-to-HDL transfer of cholesterol esters and phosphatidylcholine especially in the presence of serum lipid transfer proteins. The acceleration of the interlipoprotein transfer caused by prostaglandin E1 in turn increases the rate of cholesterol esterification in serum. It is suggested that in such a way prostaglandin E1 may influence the homeostasis of cholesterol.

Phospholipid packing has been suggested as a relevant variable in the control of membrane fusion events. To test this possibility in a model system, a comparison was made of the fusability of erythrocytes with a normal asymmetric transbilayer distribution of plasma membrane phospholipids (tightly packed exterior lipids) and erythrocytes with a symmetric transbilayer distribution of phospholipids (more loosely packed exterior lipids), using polyethylene glycol as fusogen. Not only were lipid-symmetric cells more readily fused, but fusions of mixtures of lipid-symmetric and lipid-asymmetric cells indicated that both fusing partners must have a symmetric distribution for fusion to be enhanced. Lipid-symmetric cells may fuse more readily because loose packing of the exterior lipids enhances hydrophobic interactions between cells. Alternatively, enhanced membrane fluidity may facilitate intramembranous particle clustering, previously implicated as a potentiator of fusion. Finally, exposure of phosphatidylserine on the surface of lipid-symmetric erythrocytes may be responsible for their enhanced fusion.