Calcium potentiates the peroxidation of erythrocyte membrane lipids

A clonal cell line from rat osteosarcoma was found to possess parathyroid hormone and isoproterenol sensitive adenylate cyclase. This study examines the relationship between the two hormones and triphosphoguanine nucleotide with respect to enzyme activation. Concentration-dependence curves, analyzed by computer-aided curve fitting, revealed: (1) in the presence of 5 microM GTP there were two apparent affinities for parathyroid hormone (Km 9 and 89 nM) and isoproterenol (Km 72 and 340 nM; (2) and two affinities for guanosine-5' (beta, gamma-imido)triphosphate (Km 0.25 and 1.3 microM); (3) hormones and guanine nucleotides reciprocally shifted each other's concentration dependence curve to the high affinity sites; (4) parathyroid hormone and isoproterenol interacting with high affinity sites competed for the same adenylate cyclase; (5) parathyroid hormone and isoproterenol, acting on low affinity sites had additive effects and also stimulated adenylate cyclase in the absence of added guanine nucleotides. The findings are consistent with (i) competition of parathyroid hormone and isoproterenol for the activation of the high (hormone) affinity complex containing: receptors, nucleotide subunit, triphosphoguanine nucleotide, catalytic unit (ii) the apparent presence of receptor-nucleotide sub-unit GDP-catalytic unit complexes with low hormone affinity which are stimulated by parathyroid hormone and isoproterenol separately.     

Role of the nonspecific modification of erythrocyte membrane lipids during hibernation in the suslik Citellus parryi

Studies have been made on the content of cholesterol, phospholipids, fatty acid composition, the intensity of lipid peroxidation, the activity of Na+, K+-ATPase, as well as on the peroxide hemolysis in the erythrocytes in prehibernating and hibernating ground squirrels. Changes in partial content of cholesterol and in fatty acid composition of membranes are presumably due to the excessive lipid peroxidation during hibernation resulting from the decrease in the activity of antioxidative enzymes, which also accounts for the increase in peroxide hemolysis of erythrocytes.     

Role of the interaction between puerarin and the erythrocyte membrane in puerarin-induced hemolysis

Adverse drug reactions (ADR), especially intravenous hemolysis, have largely limited the application of puerarin injections in clinics. This study investigated the underlying mechanisms of puerarin-induced hemolysis. Our results show that puerarin induced concentration-dependent and time-dependent hemolysis when human erythrocytes were incubated in saline solution with more than 2 mM puerarin for over 2 h. However, incubation in PBS or addition of 1 mM of lidocaine to the saline solution completely abolished the hemolysis. Providing materials that could start ATP synthesis did not reverse the hemolysis, and puerarin did not affect Na⁺–K⁺–ATPase activity. In addition, puerarin (0.1–2 mM) did not cause calcium influx or exhibited pro-oxidant activity in erythrocytes. Puerarin exhibited different influences on the membrane microviscosity of erythrocytes in saline and PBS. Moreover, 1 mM lidocaine inhibited 8 mM puerarin-induced reduction of membrane microviscosity in saline solution. SDS–PAGE analysis of membrane proteins revealed that 2 mM puerarin treatment induced the appearance of several new protein bands but attenuated the expression of protein bands 2.1, 3, 4.1, 4.2 and 5. These results suggest that high concentrations of puerarin-induced hemolysis were associated with the changes of membrane lipids and of the composition of erythrocytes membrane proteins but not with ATP depletion, pro-oxidation and calcium influx. These changes could be related to the intercalation of amphiphilic puerarin at high concentration into the erythrocyte membrane in certain media, resulting in membrane disorganization and, eventually, cytolysis. Hence, in clinics, determining the optimal dose of puerarin is critical to avoid overdosing and ADR.     

Radiation-induced lipid peroxidation and the fluidity of erythrocyte membrane lipids

The effect of radiation-induced peroxidation on the fluidity of the phospholipids of the erythrocyte membrane was studied using both erythrocyte ghosts and liposomes formed from the polar lipids of erythrocytes. In liposomes, the oxidation of the phospholipids increased with radiation dose, but there was no change in the fluidity of the lipids as measured by spin-label motion. Under the same conditions of irradiation, no oxidation of phospholipid was detected in erythrocyte ghosts, although changes occurred in the motion of spin labels intercalated with the membrane. These changes were attributed to radiation-induced alterations in the membrane proteins. It is concluded that alterations in motion of spin labels, observed with intact membranes after irradiation, are most likely the result of changes in the structure of membrane proteins rather than the lipids.     

Effect of vitamin D3 on free-radical oxidation of lipids in low density lipoproteins in vitamin D deficiency

The lipid free radical oxidation in low density lipoproteins was investigated on D-deficiency model in vivo. The processes of lipid free radical oxidative activation in low density lipoproteins at D-deficiency occurred. The chemiluminescence kinetic parameters: the maximum intensity of the first flash and inclination angle tangent of an ascending branch of the second flash grew at D-deficiency in comparison with control group (p < 0.02 and p < 0.05, respectively). At the same time, the vitamin D3 introduction to the experimental animals diet failed result statistically reliable inclination angle tangent of an ascending branch of the second flash was reduced (p < 0.02). Increase of the products reacting with thiobarbituric acid content in low density lipoproteins in D-deficiency conditions (p < 0.001) was found. Vitamin D3 introduction to the diet reduced quantity of products reacting with thiobarbituric acid in low density lipoproteins (p < 0.01). However, their level remained higher than for the control animals (p < 0.01) as established.     

Gramicidin-induced enhancement of transbilayer reorientation of lipids in the erythrocyte membrane

Incorporation of the channel-forming antibiotic gramicidin into the membrane of human erythrocytes highly (up to 30-fold) enhances rates of reorientation (flip) of lysophosphatidylcholine and palmitoylcarnitine to the inner membrane layer after their primary incorporation into the outer layer. Despite the high increase of flip rates by gramicidin, the asymmetric orientation of the inner membrane layer phospholipids phosphatidylethanolamine and phosphatidylserine is stable as demonstrated by the lack of accessibility of these lipids toward cleavage by exogenous phospholipase A2. On the other hand, gramicidin enhances the rate of cleavage of outer membrane layer phosphatidylcholine by phospholipase A2, which indicates changes in the packing of phosphatidylcholine following gramicidin binding. The increase of flip becomes detectable when about 10(5) copies of gramicidin per cell have been bound (gramicidin to membrane phospholipid ratio of 1:2000). This is a 1000-fold higher concentration than that required for an increase of K+ permeability mediated by the gramicidin channel. Acceleration of flip is thus not simply correlated with channel formation. The enhancement of flip is markedly dependent on structural details of gramicidin. Formylation of its four tryptophan residues abolishes the effect. Even at high concentrations of formylated gramicidin at which the extents of binding of native and of formylated gramicidin to the membrane are comparable, no flip acceleration is produced. Enhancement of flip by gramicidin occurs after a temperature-dependent lag phase. At 37 degrees C, flip rates begin to increase within a few minutes and at 25 degrees C, only after 3 h. This lag phase is most likely not due to limitations by the rate of binding of gramicidin to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of vitamin D3 receptor in the synergistic differentiation of WEHI-3B leukemia cells by vitamin D3 and retinoic acid.

WEHI-3B D- cells differentiate in response to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) but not to all-trans-retinoic acid (RA) or other inducing agents. Combinations of RA with 1,25-(OH)2D3 interact to produce synergistic differentiation of WEHI-3B D- cells. To determine factors involved in the synergistic interaction, expression of the 1,25-(OH)2D3 receptor (VDR) and retinoid receptors, RARalpha and RXRalpha, was measured. No VDR was detected in untreated WEHI-3B D- cells; however, RA and 1,25-(OH)2D3 when used as single agents caused a slight induction of the VDR and in combination produced a marked increase in the VDR. In contrast, no changes in RARalpha and RXRalpha were initiated by these compounds. An RAR-selective agonist combined with 1,25-(OH)2D3 produced synergistic differentiation of WEHI-3B D- cells, whereas an RXR-selective agonist did not. To gain information on the role of the VDR in the synergistic interaction, the VDR gene was transferred into WEHI-3B D+ cells, in which no VDR was detected and no synergism was produced. Expression of the VDR conferred differentiation responsiveness to 1,25-(OH)2D3 in WEHI-3B D+ cells. These findings suggest that (a) induction of VDR expression is a key component in the synergistic differentiation induced by 1,25-(OH)2D3 and RA and (b) RAR and not RXR must be activated for enhanced induction of the VDR and for the synergistic differentiation produced by RA and 1, 25-(OH)2D3.     

Role of lipids in theNeurospora crassa membrane

Summary The effect of doubling the saturated fatty acid content on the electrophysiology ofNeurospora crassa membranes was studied. Intracellular membrane input resistance (R _m ) and potential (E _m ) were measured for wild-type (w/t) andcel ^– (Tween 40) organisms as a function of temperature. Over the 0 to 40°C temperature range studied, meanE _m values of bothw/t andcel ^– (Tw 40) organisms increased from –160 to –210 mV. This difference is greater than that expected from Nernst potential considerations, indicating an active component ofE _m . This active component is insensitive to a doubling of the saturated fatty acid content.R _m exhibits a temperature dependence and hysteresis. Averaged data indicate an increase inR _m with decreased temperature. The slope of the temperature dependence varies among individual hyphae. Above 17.5°Ccel ^– (Tw 40) hyphae averaged greater than 70% higher values ofR _m thanw/t. Below 17.5°Cw/t R _m data divided into low and high temperature dependence groups, whilecel ^– data exhibited a low temperature dependence. The results are discussed in relation to gel-liquid crystal phase transitions, membrane fluidity, and the contribution of fatty acid structure to membrane electrical properties.     

Alterations in erythrocyte membrane fluidity by phenylhydrazine-induced peroxidation of lipids

The addition of phenylhydrazine (.05 – 0.5 mM) to hemoglobin-free human erythrocyte membranes results in the peroxidation of endogenous phospholipids as measured by the thiobarbaturic acid reaction. The incorporation of 1,6-diphenyl-1,3-5-hexatriene into these membranes revealed a decrease in bulk lipid fluidity. Additionally, the fluorescence intensity of 1-anilino-8-naphthalene sulfonate was decreased and red-shifted after phenylhydrazine treatment of the membranes. The results obtained are consistent with the view that changes in the physical state of plasma membranes subsequent to the peroxidation of membrane lipids may be a determinant of the mechanical properties of drug-treated as well as aging cells.     

Effect of electrical brain stimulation on erythrocyte membrane lipids

Biliary obstruction was induced in rats by intermittent stimulation of the lateral hypothalamus for periods of 2 and 6 hours. Cholesterol and phospholipid concentrations of circulating red blood cells were found to be decreased, whereas in the plasma these lipids were elevated. The mechanism of membrane lipid depletion associated with early biliary obstruction is currently unknown. It is possible, however, that cells other than erythrocytes undergo similar changes. In previous experiments plasma membrane degeneration of endothelial cells was observed in rats subjected to hypothalamic stimulation for 6 hours. Thus, central nervous system stimulation may have a deleterious effect on cell membrane structure and function and be mediated through biliary obstruction.     

Polyunsaturated fatty acids in plasma and erythrocyte membrane lipids of diabetic children

While insulin is a potent activator of essential fatty acid metabolism, portal hypoinsulinemia is common in Type 1 diabetes. Fatty acids were determined by high-resolution capillary gas-liquid chromatography in plasma and erythrocyte membrane lipids in diabetic children (n = 40) and in age-matched healthy controls (n = 40). In plasma phospholipids, values of linoleic acid (23.00 [2.35] vs. 18.13 [2.54], % by wt, median [range from the first to the third quartile], P<0.000l) and alpha-linolenic acid (0.12 [0.06] vs. 0.07 [0.07], P<0.05) were significantly higher in diabetic children than in controls. In contrast, values of arachidonic acid (10.73 [2.34] vs. 11.53 [2.50], P<0.05) and docosahexaenoic acid (2.23 [0.63] vs. 2.77 [0.98], P<0.01) were significantly lower in diabetic children than in controls. Reduced availability of long-chain polyunsaturates in diabetic children suggests that an enhanced dietary supply of long-chain polyunsaturates may be beneficial.     

Modification of the erythrocyte membrane lipids in rats under conditions of alimentary stress

Erythrocyte phospholipids and lipid fatty acid composition was investigated at different stages of exposure to high-caloric diet in male Wistar rats. The revealed modification of erythrocyte lipids shows specific features of membrane response to stress factors of alimentary nature, in particular, cell protection compensatory mechanism initiation on the 90th day and their depletion by the 180th day of high-caloric diet.     

Role of membrane lipids in bacterial division-site selection

Heterogeneous distribution of specific phospholipids along the bacterial membrane results in the formation of domains enriched in anionic phospholipids at the cell poles and cell center, which appear to participate in the binding of amphitropic proteins responsible for selection and recognition of the division site. It was discovered that functioning of the Min system, which protects the cell poles from aberrant positioning of the Z-ring, is controlled by direct interaction of its MinD component with membrane phospholipids. There is also an accumulation of evidence that the mid-cell domain, formed in the cell at a defined step of the cell cycle, provides the optimal phospholipid composition first for initiation of DNA replication and then for Z-ring positioning.     

Interaction between plant polyphenols and the erythrocyte membrane

The purpose of these studies was to determine the effect of polyphenols contained in extracts from apple, strawberry and blackcurrant on the properties of the erythrocyte membrane, treated as a model of the biological membrane. To this end, the effect of the substances used on hemolysis, osmotic resistance and shape of erythrocytes, and on packing order in the hydrophilic region of the erythrocyte membrane was studied. The investigation was performed with spectrophotometric and fluorimetric methods, and using the optical microscope. The hemolytic studies have shown that the extracts do not induce hemolysis at the concentrations used. The results obtained from the spectrophotometric measurements of osmotic resistance of erythrocytes showed that the polyphenols contained in the extracts cause an increase in the resistance, rendering them less prone to hemolysis in hypotonic solutions of sodium chloride. The fluorimetric studies indicate that the used substances cause a decrease of packing order in the hydrophilic area of membrane lipids. The observations of erythrocyte shapes in a biological optical microscope have shown that, as a result of the substances’ action, the erythrocytes become mostly echinocytes, which means that the polyphenols of the extracts localize in the outer lipid monolayer of the erythrocyte membrane. The results obtained indicate that, in the concentration range used, the plant extracts are incorporated into the hydrophilic area of the membrane, modifying its properties.     

Up-regulation of the intestinal 1,25-dihydroxyvitamin D receptor during hypervitaminosis D: a comparison between vitamin D2 and vitamin D3

Concentrations of intestinal 1,25-dihydroxyvitamin D receptor were measured in rats receiving pharmacological amounts (25,000 IU/rat daily for 6 days) of either vitamin D2 or vitamin D3. The data showed that both hypervitaminosis D2 and hypervitaminosis D3 resulted in significant up-regulation of intestinal 1,25-dihydroxyvitamin D receptor (fmol/mg protein) relative to controls (409 +/- 24, vitamin D2-treated; 525 +/- 41, vitamin D3-treated; and 249 +/- 19, control). The 1,25-dihydroxyvitamin D receptor enhancement also was accompanied by elevated plasma 25-hydroxyvitamin D and hypercalcemia. These data suggest that increased target-tissue 1,25-dihydroxyvitamin D receptor may play a role in enhancing target-tissue responsiveness and, thus, have a significant role in mediating the toxic effects of hypervitaminosis D.     

Chromatographic separation of vitamin D3 sulfate and vitamin D3.

This paper describes a simple chromatographic technique on Sephadex LH20 for the separation of vitamin D3 sulfate from free vitamin D3 and its metabolites. This technique has been used in the study of vitamin D3 sulfate metabolism in rats. Seven hours after injection of vitamin D3 sulfate (35S or 35S and 3H) only the peak of vitamin D sulfoconjugate was found in chromatographic elution of serum extracts.     

Structural changes of erythrocyte membrane revealed by 3D confocal optical profilometer

We examined hematological changes influenced by the experimental hypervitaminosis A. The 3D confocal optical profilometer was applied for assessment of the erythrocytes' membrane structural changes influenced by an overdose of vitamin A. The blood smears were evaluated in terms of alterations of geometrical and optical parameters of erythrocytes for two groups of animals: oil base and retinol palmitate (n = 9 animals for each group). The results demonstrate that an overdose of retinol palmitate causes changes in the torus curvature and pallor of discocytes, their surface area and volume. The observed structural malformations of the shape of red blood cells become visible at the earlier preclinical stage of changes in animal state and behavior. With this in mind, the results of the study open a new area of research in the certain dysfunction diagnosis of red blood cells and have a great potential in the further development of new curative protocols.     

Alterations in erythrocyte membrane lipids in abetalipoproteinemia: phospholipid and fatty acyl composition

Scanning electron microscopic observation revealed that there were wide variations including typical acanthocytes in morphology of erythrocytes from a patient with abetalipoproteinemia. The erythrocyte membrane phospholipids and cholesterol contents from a patient was higher by 25% compared to an age-matched control subject. Analysis of phospholipid composition of red blood cells showed an increase of sphingomyelin (25.1----30.1%) with a concomitant decrease of lecithin (27.5----21.0%). Thus, the sphingomyelin/lecithin ratio was increased dramatically (0.91----1.43). As for fatty acyl chain composition of main phospholipids, an increased percentage of palmitic acid and docosahexaenoic acid and a decreased proportion of arachidonic acid and lignoceric acid were observed for sphingomyelin. There was an increment of palmitic acid which was accompanied with a decrease of linoleic acid in lecithin. On the other hand, no significant difference was shown in the fatty acid composition of phosphatidylethanolamine and phosphatidylserine plus phosphatidylinositol between a patient and control.