Decrease In Microviscosity Research Articles

SPECIFIC AND NONSPECIFIC ALTERATIONS IN MEMBRANE MICROVISCOSITY INDUCED BY RETINOIDS IN EMBRYONAL CARCINOMA AND FIBROBLAST CELLS

Retinoids can induce structural changes in erythrocyte and lysosomal membranes.1,z These alterations may also effect lipid-protein interactions and the fluid state of the membrane. Membrane microviscosity can be monitored quantitatively by fluorescence polarization analysis3 with the aid of the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene (DPH). In this study this technique was used to determine the effect of retinoids on the membrane microviscosity of some eukaryotic cells. FIGURE 1 shows the effect of various concentrations of retinoic acid (RA) on membrane microviscosity of 3T6 cells. RA up to lO-7M had no influence on microviscosity; at higher concentrations microviscosity dropped rapidly and leveled off at 5.10'6M. A decrease in microviscosity (increase in fluidity) could be observed even after short treatments (2 hr) with RA. Similar results had been obtained previously with red blood cells.4

Changes in phospholipid methyltransferases and membrane microviscosity during induction of rat liver microsomal cytochrome P-450 by phenobarbital and 3-methylcholanthrene.

Rat liver microsomes contained two methyltransferases which converted phosphatidylethanolamine (PE) to phosphatidylcholine (PC). The first methyltransferase converted PE to phosphatidyl-N-methylethanolamine (PME) and the second methyltransferase converted PME to PC. Previous work has shown that increased PME synthesis decreases membrane microviscosity. Therefore, changes in the rat liver microsomal cytochrome P-450, phospholipid methyltransferases and membrane microviscosity after induction by phenobarbital and 3-methylcholanthrene were studied. Phenobarbital and 3-methylcholanthrene increased cytochrome P-450 levels 2- to 3-fold. At low SAM concentration, the proportion of PME among the total phospholipids formed increased significantly, and at a high SAM concentration, the proportion of PC among the total phospholipids formed decreased significantly in microsomes of treated rats. Treatment of rats with phenobarbital and 3-methylcholanthrene also decreased microviscosities of the microsomal membranes and liposomes which were prepared from phospholipids extracted from the microsomes. In synthetic liposomes containing PE, PME and PC, microviscosity decreased when the proportion of PME was increased or the proportion of PC was decreased. These results suggest that the membrane fluidity increases with phenobarbital and 3-methylcholanthrene treatment, and changes in phospholipid methyltransferases may contribute to the process of enzyme induction. During induction with phenobarbital, all three factors known to increase membrane fluidity (linoleic acid content, the formation of phosphatidyl-N-methylethanolamine, and decreases in the cholesterol/phospholipid ratio) contribute to the decrease in microviscosity. During induction with 3-methylcholanthrene, alterations in phospholipid methylation is possibly the primary cause of the decrease in membrane microviscosity.

Correlation of hyperthermic sensitivity and membrane microviscosity in E. coli K1060.

We have demonstrated a positive correlation between membrane microviscosity and the temperature required to kill E. coli. Batches of cells with differing unsaturated fatty acid (u.f.a.) compositions were prepared from the u.f.a.-requiring E. coli K12 mutant K1060. The membrane microviscosity of these cells is estimated from the extent of fluorescence polarization of the probe molecule 1,6-diphenyl-1,3-5,-hexatriene dissolved in the membrane. For the same growth temperature, cells grown in oleic acid (18:1) have a greater microviscosity and u.f.a. content than linolenic acid (18:3) grown cells. the rate of decrease in microviscosity with increasing temperature is correlated with the amount of u.f.a. present in the membrane. From survival curves determined at several hyperthermic exposures, one can interpolate the hyperthermic temperature required to kill 90 per cent of the cells in three hours. These equivalent kill temperatures are directly related to the cell microviscosity. These data support the hypothesis that cell membrane microviscosity plays a critical role in hyperthermic killing.

Protein-lipid interaction: Biophysical studies of (Ca 2+ + Mg 2+)-ATPase reconstituted systems

Differential scanning calorimetry, fluorescence spectroscopy and freeze-fracture electron microscopy have been applied to a study of the reconstituted Ca 2+-ATPase proteins from sarcoplasmic reticulum when they are incorporated into pure lipid/water systems. The results obtained with these techniques have been used to examine the effects of this intrinsic protein upon the surrounding lipid at temperatures above and below the main lipid solid-fluid phase transition temperature ( T c). 1. 1. Above this T c value, the freeze-fracture data show that the proteins are randomly distributed within the plane of the bilayer. The fluorescence data show that as the protein content in the bilayer increases, so does the ‘microviscosity’. 2. 2. Below T c the proteins occur in high protein to lipid patches, separate from the remaining crystalline lipid. The fluorescence data indicate that at these temperatures the presence of the protein causes a decrease in microviscosity, whilst the calorimetric data indicate a decrease in enthalpy of the main lipid transition.

Microviscosity of mucosal cellular membranes in toad urinary bladder: Relation to antidiuretic hormone action on water permeability

The microviscosity of cellular membranes (or membrane fluidity) was measured in suspensions of single mucosal cells isolated from the urinary bladder of the toad, Bufo marinus, by the technique of polarized fluorescence emission spectroscopy utilizing the hydrophobic fluorescent probe, perylene. At 23 degrees C, 5 mM dibutyryl cyclic 3',5'-AMP decreased the apparent microviscosity of the cell membranes from 3.31 to 3.07 P, a minimum decrease of 7.3% (P less than 0.001) with a physiological time course. Direct visualization of the cell suspension indicated that 98% of the cells were viable, as indicated by Trypan Blue dye exclusion. The fluorescent perylene could be seen only in plasma membranes, suggesting that the measured viscosity was that of plasma membrane with little contribution from the membranes of cellular organelles. Addition of antidiuretic hormone to intact hemibladders stained with perylene produced changes in fluorescence consistent with a similar 7% decrease in apparent microviscosity with a physiological time course. However, finite interpretation of the findings in intact tissue cannot be made because the location and the fluorescent lifetime of the probe could only be conducted on the isolated cells. Comparison with previously determined relationships between water permeability and microviscosity in artificial bilayers suggests that the 7% (a lower limit) decrease in microviscosity would produce only a 6.5% increase in water permeability.

Decreased microviscosity of membrane lipids in leukemic cells: Two possible mechanisms

Steady-state fluorescence polarization studies with the fluorescent lipid probe 1,6-diphenyl 1,3,5-hexatriene were done to determine the degree of microviscosity of cellular membrane lipids and serum lipoproteins in human normal donors and leukemic patients. The results show a marked decrease in microviscosity of cellular membrane lipids in both intact lymphocytes and isolated cellular plasma membranes obtained from leukemic patients in clinical relapse as compared to intact lymphocytes and isolated cellular plasma membranes obtained from normal donors and leukemic patients in complete clinical remission. Concomitant to these dynamic changes in cellular membrane lipids, the degree of microviscosity of lipids in the blood serum of leukemic patients in clinical relapse is markedly reduced as compared to serum obtained from normal donors and leukemic patients in complete clinical remission. Moreover, an in vitro incubation of leukemic lymphocytes with normal low density lipoproteins results in an increased microviscosity of cellular membrane lipids. In addition to the interrelation between cellular membrane lipids and serum lipoproteins, plasma membrane vesicles with a high degree of lipid microviscosity were isolated from the blood serum and pleural effusion of leukemic patients in clinical relapse. Such membrane vesicles could not be detected in normal serum. Therefore, we suggest that the two major mechanisms associated with the decreased microviscosity of membrane lipids in human leukemic cells are an abnormal exchange in lipids between the leukemic cell surface membrane and leukemic serum lipoproteins and an exfoliation of plasma membrane vesicles with a high degree of microviscosity from the cell surface of leukemic cells.

Increased fluidity of human platelet membranes during complement-mediated immune platelet injury.

Complement appears to be involved in the destruction of platelets in certain clinical disorders, such as quinidine purpura and post-transfusion purpura. In both disorders, the classical complement sequence is activated by antigen-antibody complexes. It has been suggested that the terminal components of the complement sequence insert into the hydrophobic core of cell surface membranes and that this process leads to cell lysis. Fluidity is a fundamental property of lipids within the membrane's hydrophobic core. To examine the interaction of complement with membranes, we investigated the effect of complement activation on the fluidity of human platelet membranes. Complement was fixed to platelets using a post-transfusion purpura antibody, and membrane lipid fluidity was assessed in terms of fluorescence anisotropy using two fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene and 9-(12-anthroyl) stearic acid. Microviscosity, expressed in poise, was derived from the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene.Post-transfusion purpura antibody plus complement made platelet membranes more fluid as evidenced by a 21% decrease in anisotropy and a 35% decrease in microviscosity of platelets at 37 degrees C, and this was associated with platelet lysis ((51)Cr release). Complement damage to platelets was accompanied by a 10-15% increase in DeltaE, the fusion activation energy for microviscosity, indicating that complement not only decreased membrane microviscosity but also made membrane lipids less ordered. These changes were consistent and rapid, with platelet lysis and the reduction in microviscosity being half-maximal by 6 min. They were prevented by inactivation of complement with heat or with EDTA, and they were not observed when C5-deficient plasma was used as the complement source. Qualitatively similar changes in platelet membrane fluidity were observed when complement was fixed to platelets by a quinidine-dependent anti-platelet antibody rather than by post-transfusion purpura antibody. Post-transfusion purpura antibody plus complement also decreased the microviscosity of isolated platelet membranes. Moreover, the lipids extracted from platelets lysed by complement had a 22% decrease in microviscosity (P < 0.01), with no associated changes in the amount of cholesterol relative to phospholipid or in the amounts of the various phospholipids. These studies demonstrate that lipids within the hydrophobic core of platelet membranes damaged by complement become more fluid, and this is associated with platelet lysis. These findings are consistent with the concept that the insertion of the terminal complement components into the platelet membrane bilayer perturbs lipid-lipid interactions within the membrane's hydrophobic core.

Changes in membrane microviscosity associated with phagocytosis: effects of colchicine.

The effects of phagocytosis on plasma membrane microviscosity were studied by fluorescence depolarization techniques. It was shown that lipophilic probes are accumulated in intracellular vesicles to a significant degree in fibroblasts and neutrophils. Microviscosity was thus determined from the behavior of probes in isolated membranes. Phagocytosis of oil emulsions or polystyrene beads by rabbit polymorphonuclear leukocytes induces a marked decrease in plasma membrane microviscosity that parallels the extent of phagocytosis. Liposomes made from extracts of membrane lipid show qualitatively the same changes, indicating that the alteration of microviscosity results at least in part from changes in lipid composition. The decrease in microviscosity is abolished when colchicine is present during phagocytosis. Addition of colchicine to membranes previously isolated from control or phagocytic cells has no effect on their microviscosity. The results suggest that phagocytosis is accompanied by a microtubule-dependent reorganization of membrane lipids.

Decrease in microviscosity of lymphocyte surface membrane associated with stimulation induced by concanavalin A.

CM factor (CVF) - treated normal mouse serum produced a profound inhibition of the antibvody responses, affecting IgG responses more than IgM, and being T-dependent more than T-independent, but no part of the response being unaffected...