Effect of calcium, insulin and growth hormone on membrane fluidity. A spin label study of rat adipocyte and human erythrocyte ghosts

ESR spectra were recorded from rat epididymal adipocyte ghosts labeled with the 5-nitroxide stearic acid spin probe, I(12,3). Polarity-corrected and approximate order parameters, that are sensitive to the flexibility of the incorporated label, were used to evaluate the membrane lipid fluidity. Addition of CaCl2 a 37 degrees C decreased the fluidity, as indicated by positive increases in the order parameters. The ordering effect of Ca2+ was concentration-dependent, reached saturation at approx. 3--4 mM, and was completely reversed by excess EGTA. Previous studies indicated that low- and high-affinity sites on adipocyte plasma membranes are able to bind 45Ca2+, and our results suggest that Ca2+-induced alterations in the lipid fluidity involve cation binding to low-affinity sites. The cellular movements of Ca2+ and, in particular, the binding of Ca2+ to the plasma membrane may play important roles in insulin's action on fat cell function. The possibility that insulin directly alters the membrane fluidity was tested by adding hormone to freshly-prepared I(12,3)-labeled adipocyte ghosts. Insulin, at concentrations (10(-6) M) that enhance glucose uptake into intact adipocytes, did not affect the fluidity of ghosts suspended in buffers with or without Ca2+. The fluidities of I(12,3)-labeled rat adipocyte ghosts or human erythrocyte ghosts were also unaffected by various forms of human growth hormone.     

A spin label study of E. coli membrane vesicles

The phase transition in E. coli membrane vesicles has been investigated by the spin labeling technique. N-oxyl-4′,4′-dimethyloxazolidine derivatives of stearic acid were incorporated into the vesicles. The results suggest that there are two phase transitions in these bacterial membrane vesicles (one at ≈20°C and the other at ≈30°C). These two phase transitions may be related to some of the functional properties of the membranes.     

A spin label study of erythrocyte membranes during simulation of freezing

Summary Human erythrocytes were labeled with stearic acid spin labels, and no change was detected in membrane fluidity under hyperosmotic stress, going from isotonicity to about 3000 mOsm. Intact erythrocytes labeled with an androstane spin label and submitted to simulation of freezing show the onset of irreversible structural breakdown occurring in a saline solution at 2,000 mOsm. Ghosts labeled with maleimide spin label (4-maleimide-2,2,6,6-tetramethylpiperidinooxyl) when submitted to solutions of increasing osmolalities (pH 7.4), exhibit protein conformational changes that are irreversible after a simulated freeze-thaw cycle. After sonication of maleimide spin-labeled ghosts, membrane buried sulfhydryl groups become exposed. Such preparations showed behavior similar to the unsonicated when in saline hyperosmolal medium (pH 7.4). Such results suggest the ionic strength of the medium as the determining factor of the detected conformational changes. Maleimide spin-labeled ghosts in 300 mOsm saline solution (pH 7.4) were treated with ascorbic acid (spin destruction of nitroxides), and the kinetic analysis indicates that 65% of the labeled sites are located at the external interface of the membrane or in hydrophilic channels. Deformation and rearrangements of membrane components in solutions of increasing osmolalities apparently are related to protein conformational changes, on the outside surface of erythrocyte membranes, with a significant amount being structurally dissociated of lipids.     

Interactions of the cholesterol side-chain with egg lecithin. A spin label study.

The effect in egg lecithin liposomes of cholesterol and cholesterol analogues with side-chains of reduced length on the order parameters of two steroid spin labels has been studied. Analogues with side-chains shorter than cholesterol by more than three carbon cause significantly less ordering than cholesterol. Liposomes containing a cholesterol analogue in which the side-chain is absent cause very little increase in the ordering of a new sterol spin label in which the nitroxide is incorporated into the side-chain. The results suggest that the sterol side-chain exerts a great influence on membrane rigidity within its immediate environment.     

Irradiation increases proteolysis in erythrocyte ghosts: A spin label study

Summary X- and gamma-irradiation of human erythrocyte membranes (250–1000 Gy) was found to decrease the ratio of weakly to strongly immobilized signal height of membrane-bound maleimide spin label (Mal-6). Subsequent incubation of spin-labeled membranes at ambient temperature (21 °C) induced a progressive increase in this ratio, faster for membranes irradiated with low doses which was hampered by protease inhibitors. These results demonstrate that ionizing radiation stimulates proteolysis of erythrocyte membrane proteins by membrane-associated proteases.     

A spin label study on human low density lipoprotein

Selective labeling with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimide of human serum LDL has been performed. The spin-labeled LDL exhibited an ESR spectrum containing signals of a strongly immobilized component only. The signals were completely reversible between 4 degrees C and 37 degrees C and fairly stable at each temperature. The spin-labeled LDL which was prepared by the usual method exhibited an ESR spectrum containing signals of both strongly immobilized and weakly immobilized components (5, 6). The latter was unstable above 25 degrees C and changed irreversibly. The strongly binding site showed higher affinity for the nitroxide radical than the weakly binding site, and two kinds of the strongly binding site were demonstrated kinetically. The rate of binding of the nitroxide radical to the two kinds of strongly binding site were estimated to be 4.7 x 10(4) M-1 . day-1 and 0.16 x 10(4) M-1 . day-1 at pH 7.4 and 4 degrees C, respectively. Both the strongly immobilized and weakly immobilized radicals were reduced with ascorbate at the same rate. It was also shown on gel filtration of the SDS-treated LDL derivatives that the strongly immobilized component was on the apoprotein B moiety, whereas either noncovalent binding to LDL or binding to some small molecular species other than protein was suggested for the weakly immobilized component.     

The decreased membrane fluidity of in vivo aged, human erythrocytes. A spin label study.

The decreased membrane fluidity of the in vivo aged, human erythrocytes is found, by monitoring the electron paramagnetic resonance (EPR) spectra of fatty acid spin labels incorporated into the membrane. In addition, the decreased cell sizes and the decreased cholesterol and phospholipids contents, without significant changes of the quantity of the membrane proteins, also the decrease of ATP and 2,3-diphosphoglycerate and the increase of ADP and AMP, in the aged cells, were observed. Further the functional impairments of the aged cells, i.e. the increased oxygen affinity and the decreased deformability, were shown. On the basis of these quantitative data, the alteration of the protein-lipid organization, due to decreased lipid/protein ratio, the modified protein-lipid interaction and/or the influences of the diminished ATP content, is suggested to contribute towards the decreased membrane fluidity of the in vivo aged erythrocytes.     

The decreased membrane fluidity of in vivo aged,human erythrocytes. A spin label study

The decreased membrane fluidity of the in vivo aged, human erythrocytes is found, by monitoring the electron paramagnetic resonance (EPR) spectra of fatty acid spin labels incorporated into the membrane.In addition, the decreased cell sizes and the decreased cholesterol and phospholipids contents, without significant changes of the quantity of the membrane proteins, also the decrease of ATP and 2,3-diphosphoglycerate and the increase of ADP and AMP, in the aged cells, were observed. Further the functional impairments of the aged cells, i.e. the increased oxygen affinity and the decreased deformability, were shown.On the basis of these quantitative data, the alteration of the protein-lipid organization, due to decreased lipid/protein ratio, the modified protein-lipid interaction and/or the influences of the diminished ATP content, is suggested to contribute towards the decreased membrane fluidity of the in vivo aged erythrocytes.     

Spin label studies on the human erythrocyte membrane. Two sites and two phases for fatty acid spin labels.

Human erythrocytes, untreated and glutaraldehyde-treated, were spin labeled with three kinds of fatty acid labels, and their electron spin resonance (ESR) spectra were studied in detail at various temperatures. 1. The better spectral resolution could be obtained by packing the erythrocytes in a hematocrit capillary tube, because of the preferential parallel orientation of the cylindrical axes of erythrocyte-disc to the centrifugal axis. 2. It was demonstrated by the incorporation and the release of the labels that the membrane possessed two kinds of the fatty acid "sites": the tightly and weakly binding "sites" at the approximate molar ratio of 1:1. The rough estimates of the binding constants were obtained, which reproducibly varied with the blood donors over a period of a year. 3. The temperature dependency of the ESR spectra revealed the presence of two distinct phases, perhaps the solid and fluid phases. With lowering of the temperature, the fluid phase became more solid but the solid phase unchanged. The pretreatment of the erythrocytes with glutaraldehyde increased the amount of the frozen phase, corresponding to the decrease of the membrane flexibility.     

A spin label study of human erythrocyte ghost membranes damaged by methyl phenyldiazenecarboxylate (azoester)

The mechanism of methyl phenyldiazenecarboxylate (azoester) damage to human erythrocyte membranes has been investigated by means of spin labels. Azoester treatment exposed protein binding sites for non-covalently bound stearic acid and androstane spin labels that were occult in the untreated membrane. Experiments with iodoacetamide and N-alkylmaleimide spin labels suggested that azoester destroyed membrane sulfhydryl groups. No change in the structural integrity of membrane lipid components could be detected.     

A spin label study of the urinary bladder luminal membrane

Spin label experiments have been carried out on the urinary bladder luminal membrane of the bovine transitional epithelium employing the 5-, 7-, 12-, and 16-doxyl substituted stearic acid methyl esters, and compared for reference to similarly labeled bovine erythrocytes. The bladder membranes are significantly different from the bovine red blood cell membranes and show a lower order and polarity near the membrane surface. This fact and the general similarity of results for the bladder and isolated plaque membranes suggests that the highly organized proteins of the bladder membrane may act as a coat on the lipid bilayer and, while intrinsic in nature, do not significantly perturb the hydrophobic core of the lipid bilayer.     

The exchangeability of human erythrocyte membrane cholesterol

A new method has been used to determine what fraction of human erythrocyte cholesterol is available for exchange with plasma unesterified cholesterol. Erythrocytes labeled with ³H-cholesterol by this exchange process were incubated with sonicated phosphatidylcholine vesicles, giving rise to a net movement of cholesterol out of the cells. The specific activity of cholesterol taken up by the vesicles depended on the length of time of incubation. Initially the specific activity in the vesicles was greater than that in the cells, but after approximately 10% of cell cholesterol had been removed, the specific activity of subsequently removed cholesterol was equal to that of the remaining erythrocyte cholesterol. We conclude from these data that (a) all of the cholesterol in the erythrocyte is exchangeable with plasma, and (b) approximately 10% of erythrocyte cholesterol is in a more rapidly exchangeable pool than the remainder.     

A spin label study of the membrane effect of various psychoactive drugs in human erythrocytes

The effect of psychoactive agents with different clinical actions: three sedative neuroleptics (trifluoperazine, alimemazine tartrate, chlorpromazine), an anticholinergic agent (trihexyphenidyl hydrochloride), two tricyclic antidepressants (imipramine, desipramine) and lithium carbonate on the rotational correlation frequency (V⁺) of the spin label 16NS has been comparatively investigated in whole human erythrocytes. V⁺ was about 40% increased by the three neuroleptics, the anticholinergic agent and the antidepressant molecules at 0.2mM. By contrast, lithium did not induce any significant change in V⁺ at the same concentrations. It can be suggested that the increase in “membrane fluidity”, observed with a wide variety of drugs, is a non specific effect, unrelated to the psychotropic action, that can be ascribed to the amphiphilic properties of the tested drugs.