Cystic fibrosis: II. Altered microviscosity of erythrocyte membrane

Erythrocyte membrane fluidity alterations in cystic fibrosis are described. The relative flexibility of the membrane was studied using lipid spin label, i.e. methyl-5-doxylpalmitate (M5DP), and pyrene as a fluorescence probe. It was found that there was a decrease of membrane fluidity in the hydrophobic midzone of the membrane, probed by pyrene, as well as at the hydrophilic surface region, probed by M5DP.     

Effects of native and oxidized apolipoprotein A-I on lipid bilayer microviscosity of erythrocyte plasma membrane

Using a pyrene as a fluorescent probe, we investigated the influence of native and oxidized apolipoprotein A-I (apo A-I) and their complexes with tetrahydrocortisol (THC) on the microviscosity of the erythrocyte plasma membrane. The addition of THC to isolated membranes led to a 17% increase in the membrane microviscosity. In contrast, native apo A-I reduced the microviscosity (i.e., increased the fluidity) of the membranes by 15%. A more pronounced increase (by 25%) in the membrane fluidity was found in the presence of the complex of apo A-I with THC. Unlike native apo A-I, oxidized apo A-I and its complex with THC did not change the membrane viscosity. In view of the fact that apo A-I plays an important role in the binding of membrane cholesterol we suggest that the observed increase in the membrane fluidity under the influence of the native apo A-I is associated with the cholesterol efflux from plasma membrane. Oxidative modification of apo A-I likely disturbs the mechanisms of the cholesterol efflux and prevents the decrease in the membrane microviscosity.     

Regulation of insulin receptor activity of human erythrocyte membrane by prostaglandin E1

Incubation of human erythrocyte membrane with low concentration of prostaglandin E1 or prostacyclin increased the binding of 125I-labeled insulin to the membrane. The binding of the radioiodinated hormone was maximally stimulated at 3 nM prostaglandin E1 and the use of higher concentrations (above 8 nM) of the autacoid tended to reverse its own effect at lower concentrations. While prostaglandins A1, A2, B1, B2, D2, F1 alpha, F2 alpha or 6-keto-prostaglandin F1 alpha had no effect on the binding of insulin to the erythrocyte membrane, prostaglandin E2 at similar concentrations decreased the binding of the hormone. The effect of prostaglandin E1 on the increased binding of the insulin was found to be reversible and depended on the occupancy of the autacoid molecules on the membrane and showed positive cooperativity. Scatchard analysis of the binding of 125I-labeled insulin to the erythrocyte ghosts indicated that in the presence of the autacoid, the binding capacity of the insulin receptor increased 2-fold (from 207 to 424 fmol/mg protein) without any change in the ghosts affinity for the ligand (Kd 2.4 X 10(-9) versus 2.49 X 10(-9) M). As a consequence of increased binding of insulin to the erythrocyte membrane in the presence of prostaglandin E1 (3.0 nM), the optimal concentration of the peptide hormone for the maximal reduction of the membrane microviscosity decreased from approx. 1.6 to approx. 0.4 nM. Addition of prostaglandin E1 alone at the above concentration to the assay mixture had no effect on the membrane microviscosity.     

A method for measuring membrane microviscosity using pyrene excimer formation. Application to human erythrocyte ghosts.

In order to determine the microviscosity of human erythrocyte membrane suspensions, a method has been developed which is based on pyrene excimer formation. First, measurements of partitioning of pyrene into membranes, in conjunction with known values for the volume of the lipid compartment of erythrocyte ghosts are used to determine the concentration of pyrene in the membrane lipid. Secondly, reported measurements of the diffusion constants of aromatic hydrocarbons similar in structure to pyrene, are used to derive an empirical equation relating solvent viscosity and the diffusion constant of pyrene. Then, measurements of pyrene excimer formation in a series of solvents ranging up to several poise in viscosity are used to determine that the interaction diameter of the excimer formation reaction is 3 +/- 1 A. Finally all these data are brought together in order to conclude that the viscosity of the lipid in the human erythrocyte ghost is 8.0, 4.0 and 1.6 P at 10, 25 and 40 degrees C, respectively.     

Control of NADH ferricyanide reductase activity in the human erythrocyte by somatotrophin and insulin

Reduction of external ferricyanide by the human erythrocyte is significantly stimulated by insulin and somatotrophin at concentrations above physiological levels. Basal (in absence of hormones) and hormone-stimulated activities are attenuated in the presence of glycolytic inhibitors iodoacetate and vanadate indicating the requirement of glycolytic substrates for the reduction process and for the activation of cellular metabolism in response to the hormones. Sulfhydryl reagents like N-ethylmaleimide also attenuate the basal and hormone-stimulated activities and this effect was rationalized on the basis of action at SH sites which trigger responses to hormones. Stimulation of ferricyanide reduction by insulin and somatotrophin may be also the result of Na⁺/H⁺ antiport activation which may be prevented by amiloride. This suggests that Na⁺/H⁺ antiport is part of the membrane transduction system for insulin and somatotrophin in the human erythrocyte. These observations are a contribution to the study of plasma membrane oxidoreductase systems involved in physiological and metabolic functions of the cell.     

Calcium binding by the erythrocyte membrane

Calcium binding to isolated erythrocyte membranes was stimulated by ATP. This stimulatory effect of ATP required Mg²⁺.Ethacrynic acid and ruthenium red inhibited the stimulatory effect of ATP.About 80% of the bound Ca²⁺ was associated with the membrane protein.The strongly bound Ca²⁺ was confined to two high molecular weight membrane proteins.Increasing amounts of Ca²⁺ bound to the membrane inhibited Na⁺ binding in the presence of ATP.     

A new spin label method for the measurement of erythrocyte internal microviscosity

A new spin-label method for the measurement of the internal microviscosity of erythrocyte is presented. The spin label used is 2,2',5,5'-tetramethyl-3-maleimidopyrrolidinyl-N-oxyl (MAL-5) which penetrates inside the red blood cell and binds covalently on cytoplasmic glutathione. After washing off the external label, 98% of the electron paramagnetic signal is due to the labelled glutathione. This signal allows one to measure the rotational correlation time of the label. A calibration curve established with spin-labelled glutathione in sucrose solutions of increasing viscosity is used to convert the measured rotation times into viscosity units. This method avoids the use of unphysiological salts like potassium ferricyanide, and permits the study of red blood cells in various suspension media. In normal human subjects, the mean value of microviscosity is 4.45 +/- 0.16 mPa . s at 20 degrees C in isotonic saline (25 subjects) and 6 +/- 0.25 mPa . s in plasma. The variations of microviscosity as a function of the osmolarity of the medium are explained according to a theoretical model taking into account the variations of the red blood cell volume and the viscometric properties of haemoglobin.     

Polarization of plasma membrane microviscosity during endothelial cell migration

Cell movement is characterized by anterior-posterior polarization of multiple cell structures. We show here that the plasma membrane is polarized in moving endothelial cells (EC); in particular, plasma membrane microviscosity (PMM) is increased at the cell leading edge. Our studies indicate that cholesterol has an important role in generation of this microviscosity gradient. In vitro studies using synthetic lipid vesicles show that membrane microviscosity has a substantial and biphasic influence on actin dynamics; a small amount of cholesterol increases actin-mediated vesicle deformation, whereas a large amount completely inhibits deformation. Experiments in migrating ECs confirm the important role of PMM on actin dynamics. Angiogenic growth factor-stimulated cells exhibit substantially increased membrane microviscosity at the cell front but, unexpectedly, show decreased rates of actin polymerization. Our results suggest that increased PMM in lamellipodia may permit more productive actin filament and meshwork formation, resulting in enhanced rates of cell movement.     

Erythrocyte insulin receptor characteristics and erythrocyte membrane lipid composition in healthy men

The cell membrane plays an important role in the mechanism of insulin action. To test whether erythrocyte insulin receptor characteristics are related to the erythrocyte membrane lipid composition, 11 healthy volunteers were studied. The relationship between insulin binding to erythrocytes, the number of receptors per cell and the affinity of receptors to insulin on the one hand and total phospholipid fatty acid (FA) composition and cholesterol/phospholipid molar ratio in the erythrocyte membrane on the other hand were evaluated. 1. We found a significant negative correlation between specific insulin binding and the proportion of n-6 essential FA in erythrocyte membrane phospholipids, especially linoleic acid (r = -0.82, p less than 0.01) and arachidonic acid (r = -0.73, p less than 0.05). On the other hand, a significant positive correlation between insulin binding and the proportion of nonessential FA (r = +0.65, p less than 0.05) was seen. Number of receptors per cell and the affinity of receptors were not significantly related to phospholipid FA composition. 2. There was no significant correlation between insulin receptor characteristics and the cholesterol/phospholipid molar ratio in the erythrocyte membrane. The data presented support the hypothesis that the FA pattern of membrane total phospholipids may modify the properties of insulin receptors.     

Regulation of glycerol 3-phosphate oxidation in mitochondria by changes in membrane microviscosity

The inhibition of glycerol 3-phosphate oxidation by oleic acid correlates with changes in membrane microviscosity monitored by the steady-state fluorescence anisotropy of DPH. The dynamic measurements indicate that the changes of both the limiting anisotropy and rotational relaxation time occur in a concentration range where the enzyme activity is strongly inhibited.     

Biosynthesis of insulin secretory granule membrane proteins. Control by glucose.

The biosynthesis of a component SGM 110, specifically localized to the membrane of insulin secretory granules, was studied in rat insulinoma cells and in normal islets of Langerhans. Cells or islets were labelled with [35S]methionine or [3H]mannose and SGM 110 was immunoprecipitated by using a monoclonal antibody. Pulse-chase experiments demonstrated that the nascent polypeptide was cotranslationally glycosylated to form a 97,000 Da peptide which in turn was processed to the mature 110,000 Da form. A 50,000 Da form detected by immunoblotting with the same antibody was not conspicuously labelled even after a 20 h chase incubation, suggesting that it represented late processing of SGM 110 in lysosomes. With insulinoma cells, an increase in medium glucose concentration from 3 mM to 20 mM was without effect on the secretion of insulin or on the biosynthesis of (pro)insulin or SGM 110. In normal islets, however, 20 mM-glucose produced a 17-fold increase in (pro)insulin biosynthesis and a 13-fold increase in SGM 110 biosynthesis, compared with only a 2-fold increase in total protein synthesis, as judged by incorporation of [35S]methionine during a 1 h incubation. The effect of glucose on both (pro)insulin and SGM 110 biosynthesis was blocked by the addition of mannoheptulose, but not by the removal of extracellular calcium, both of which conditions inhibit insulin secretion. In contrast tolbutamide, an agent which stimulates insulin secretion, did not enhance the biosynthesis of (pro)insulin or SGM 110. It is concluded that at least one protein component of the insulin secretory granule membrane is synthesized co-ordinately with proinsulin and is subject to similar regulatory mechanisms. Factors which acutely control insulin secretion may also control granule biogenesis, although the two processes are not coupled in an obligatory fashion.     

Absence of covalent binding by insulin to erythrocyte and reticulocyte insulin receptors

We investigated whether insulin forms covalent bonds with its receptors on erythrocytes and reticulocytes, as it does in adipocytes (1). Of the [125I]-insulin specifically bound at 37 degrees C to human and rat erythrocytes and rat reticulocytes, only 1.5-2.3% was non-dissociable on extensive washing. When ghosts prepared from the washed cells were solubilized in Triton X-100, only 0.6-1.5% of the specifically bound radioactivity appeared in the void volume of a Sephadex G-50 column. Moreover in contrast to adipocytes, this high molecular weight radioactivity was not immunoprecipitable by antibodies to the insulin receptor and was dissociated during chromatography in sodium dodecyl sulphate. Thus we have been unable to demonstrate the formation of covalent bonds between insulin and its receptors on erythrocytes and reticulocytes. This finding is consistent with the hypothesis that covalent binding of insulin is a necessary receptor modification for insulin's metabolic effects.     

Calcium transport by pigeon erythrocyte membrane vesicles

Membrane vesicles from pigeon erythrocytes show a rapid, ATP-dependent accumulation of ⁴⁵Ca²⁺. Ca²⁺ accumulation ratios greater than or approximately equal to 10⁴ are readily attained. For ATP-dependent Ca²⁺ uptake, V is 1.5 mmol · 1^?1 · min^?1 at 27°C (approx. 0.9 nmol · mg^?1 protein · min^?1), [Ca²⁺]_{$\text{1}\text{2}$} is 0.18 μM, [ATP]_{$\text{1}\text{2}$} is 30–60 μM, the Ca²⁺ uptake rate depends on [Ca²⁺]² and the dependence of uptake rate on ATP concentration implies strong ATP-ATP cooperativity. The Arrhenius activation energy is 19.1 ± 1.4 kcal/mol and the pH optimum is approx. 6.9.     

Control of erythrocyte shape by calmodulin

Erythrocytes are deformable cells whose shapes can be altered by treatments with a variety of drugs. The forms the erythrocyte may assume vary continuously from the spiny "echinocytes" or crenated cells at one extreme to highly folded and dented "cupped" cells at the other extreme. Examination of 39 compounds for cup-forming activity revealed a remarkable correlation between their ability to form cupped cells and their inhibitory activity against the calcium regulatory protein, calmodulin. Calmodulin is known to interact with several erythrocyte proteins including spectrin, spectrin kinase, and the Ca++ ATPase calcium pump of the membrane. These proteins regulate the form of the cytoskeleton as well as intracellular calcium and ATP levels. It is proposed that calmodulin is required to maintain normal erythrocyte morphology and that in the presence of calmodulin inhibitors, the cell assumes a cupped shape.