Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A2

Summary The artificial insertion of increasing amounts of unsaturated fatty acids into human erythrocyte membranes modulated ATPase activities in a biphasic manner, depending on the number and position of double bonds, their configuration, and the chain length. Uncharged long-chain fatty acid derivatives with double bonds and short-chain fatty acids were ineffective. Stearic acid stimulated Na⁺K⁺-ATPase only. Anionic and non-ionic detergents and -lysophosphatidylcholine failed to stimulate ATPase activities at low, and inhibited them at high concentrations.Mg²⁺-ATPase activity was maximally enhanced by a factor of 2 in the presence of monoenoic fatty acids; half-maximal stimulation was achieved at a molar ratio ofcis(trans)-configurated C18 acids/membrane phopholipid of 0.16 (0.26).Na⁺K⁺-ATPase activity was maximally augmented by 20% in the presence of monoenoic C18 fatty acids at 37°C. Half-maximal effects were attained at a molar ratio oleic (elaidic) acid/phospholipid of 0.032 (0.075). Concentrations of free fatty acids which inhibited ATPase activities at 37°C were most stimulatory at reduced temperatures. AT 10°C, oleic acid increased Na⁺K⁺-ATPase activity fivefold (molar ratio 0.22).Unsaturated fatty acids simulated the effect of calmodulin on Ca²⁺-ATPase of native erythrocyte membranes (i.e., increase ofV _max from 1.6 to 5 mol PO ₄ ^3– ·phospholipid^–1·hr^–1, decrease of K _Ca from 6 m to 1.4–1.8 m). Stearic acid decreasedK _Ca (2 m) only, probably due to an increase of negative surface charges.A stimulation of Mg²⁺-ATPase, Na⁺K⁺-ATPase, and Ca²⁺-ATPase could be achieved by incubation of the membranes with phospholipase A₂.An electrostatic segregation of free fatty acids by ATPases with ensuing alterations of surface charge densities and disordering of the hydrophobic environment of the enzymes provides an explanation of the results.     

Modulation of endothelium function by fatty acids

Molecular and Cellular Biochemistry - The endothelium acts as the barrier that prevents circulating lipids such as lipoproteins and fatty acids into the arterial wall; it also regulates normal...     

Modulation of T-cell signalling by non-esterified fatty acids

Polyunsaturated fatty acids (PUFAs) have been shown to be immunosuppressive. In particular, they can decrease important T-cell functions that may have a profound impact on the acquired immune response. Several mechanisms may explain the immunosuppressive properties of PUFAs. Here we review the mechanisms by which they interfere with T-cell activation. PUFAs affect lipid rafts composition and function that play an essential role in T-cell signalling. The possible physiological and pathological significances of this immunomodulation by PUFAs are discussed. Further mechanistic studies and randomized controlled clinical trials are needed to assess more accurately their effects in healthy and pathological states.     

Membrane potential and human erythrocyte shape.

Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The process is fast and reversible at room temperature, so it seems to involve shifts in weak inter- or intramolecular bonds. This shape change has been reported to depend on changes in membrane potential, but control experiments excluding roles for other simultaneously varying cell properties (cell pH, cell water, and cell chloride concentration) were not reported. The present study examined the effect of independent variation of membrane potential on red cell shape. Red cells were equilibrated in a set of solutions with graduated chloride concentrations, producing in them a wide range of membrane potentials at normal cell pH and cell water. By using assays that were rapid and accurate, cell pH, cell water, cell chloride, and membrane potential were measured in each sample. Cells remained discoid over the entire range of membrane potentials examined (-45 to +45 mV). It was concluded that membrane potential has no independent effect on red cell shape and does not mediate the membrane curvature changes known to occur in red cells equilibrated at altered pH.     

Cytoplasmic pH and human erythrocyte shape.

Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The mechanism of this transformation is unknown. The preceding companion study (Gedde and Huestis) demonstrated that these shape changes are not mediated by changes in membrane potential, as has been reported. The aim of this study was to identify the physiological properties that mediate this shape change. Red cells were placed in a wide range of physiological states by manipulation of buffer pH, chloride concentration, and osmolality. Morphology and four potential predictor properties (cell pH, membrane potential, cell water, and cell chloride concentration) were assayed. Analysis of the data set by stratification and nonlinear multivariate modeling showed that change in neither cell water nor cell chloride altered the morphology of normal pH cells. In contrast, change in cell pH caused shape change in normal-range membrane potential and cell water cells. The results show that change in cytoplasmic pH is both necessary and sufficient for the shape changes of human erythrocytes equilibrated in altered pH environments.     

Modulation of 1,25-dihydroxyvitamin D3 receptor by phospholipids and fatty acids

The structural relationship between several lipids and their respective capacities to inhibit the specific binding of [3H]-1,25 (OH)2 vitamin D3 to chick intestinal cytosol preparations was investigated. The lipids investigated were: synthetic 3-sn-phosphatidylcholine and 3-sn-phosphatidic acid, egg yolk 3-sn-phosphatidylcholine and its corresponding phosphatidic acid, and free unsaturated fatty acids and their esters. The results indicate that at least three structural elements in the phospholipid molecule appear to be important; these are: 1) the structure of the fatty acid, 2) the anionic properties of the phospholipid phosphate group, and 3) the glycerol phosphate portion of the molecule. Our data also demonstrate that the position (1 or 2) and the amount (single vs. double) of unsaturated fatty acids in the phospholipid molecule do not play a major role in the receptor-1,25 (OH)2 vitamin D3 interaction. Furthermore, under equilibrium conditions, kinetic and Scatchard analysis suggest that phospholipids or free fatty acids may bind at a site different from the 1,25 (OH)2 vitamin D3 binding site, and therefore inhibit the hormone binding via a noncompetitive conformational change in the receptor molecule. A model for this phospholipid/free fatty acid binding site is proposed.     

Effects of fatty acids on activity and calmodulin binding of Ca-ATPase of human erythrocyte membranes

Activation and inhibition of Ca²⁺-ATPase of calmodulin-depleted human erythrocyte membranes by oleic acid and a variety of other fatty acids have been measured. Low concentrations of oleic acid stimulate the enzyme activity, both in the presence and in the absence of calmodulin. Concomitantly, the affinity of the membrane bound enzyme to calmodulin progressively decreases due to competitive interactions of calmodulin and oleic acid with the enzyme. Removal of oleic acid from the membrane by serum albumin extinguishes the activating effect of oleic acid and restores the ability of the enzyme to bind calmodulin with high affinity. High concentrations of oleic acid induce an almost complete and irreversible loss of enzyme activity which cannot be abolished by removal of oleic acid. Despite a complete loss of enzyme activity, binding of calmodulin to membranes is approximately normal after removal of oleic acid. Activities of (Na⁺ + K⁺)-ATPase, Mg²⁺-ATPase and acetylcholine esterase, as well as the total protein content, show no gross changes upon treatment of membranes with increasing amounts of oleic acid, which seems to exclude that membrane solubilisation by oleic acid causes an inactivation of the enzyme.     

Modulation of native T-type calcium channels by omega-3 fatty acids

Low voltage-activated, rapidly inactivating T-type Ca(2+) channels are found in a variety of cells where they regulate electrical activity and Ca(2+) entry. In whole-cell patch clamp recordings from bovine adrenal zona fasciculata cells, cis-polyunsaturated omega-3 fatty acids including docosahexaenoic acid (DHA), eicosapentaenoic acid, and alpha-linolenic acid inhibited T-type Ca(2+) current (I(T-Ca)) with IC(50)s of 2.4, 6.1, and 14.4microM, respectively. Inhibition of I(T-Ca) by DHA was partially use-dependent. In the absence of stimulation, DHA (5microM) inhibited I(T-Ca) by 59.7+/-8.1% (n=5). When voltage steps to -10mV were applied at 12s intervals, block increased to 80.5+/-7.2%. Inhibition of I(T-Ca) by DHA was accompanied by a shift of -11.7mV in the voltage dependence of steady-state inactivation, and a smaller -3.3mV shift in the voltage dependence of activation. omega-3 fatty acids also selectively altered the gating kinetics of T-type Ca(2+) channels. DHA accelerated T channel recovery from inactivation by approximately 3-fold, but did not affect the kinetics of T channel activation or deactivation. Arachidonic acid, an omega-6 polyunsaturated fatty acid, also inhibited T-type Ca(2+) current at micromolar concentrations, while the trans polyunsaturated fatty acid linolelaidic acid was ineffective. These results identify cis polyunsaturated fatty acids as relatively potent, new T-type Ca(2+) channel antagonists. omega-3 fatty acids are essential dietary components that have been shown to possess remarkable neuroprotective and cardioprotective properties that are likely mediated through suppression of electrical activity and associated Ca(2+) entry. Inhibition of T-type Ca(2+) channels in neurons and cardiac myocytes could contribute significantly to their protective actions.     

Modulation of the reactivity of the thiol of human serum albumin and its sulfenic derivative by fatty acids

The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted sixfold faster than FA-free HSA at pH 7.4 with the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and twofold faster with hydrogen peroxide and peroxynitrite. The apparent pK(a) of HSA-SH decreased from 7.9±0.1 to 7.4±0.1. Exposure to H(2)O(2) (2mM, 5min, 37°C) yielded 0.29±0.04mol of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ～threefold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pK(a) and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal.     

Modulation of adenylate cyclase activity of fibroblasts by free fatty acids and phospholipids

The effect of certain lipids on adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from fibroblasts in culture has been investigated. The unsaturated fatty acids, as well as lysolecithin, were found to act as potent inhibitors of fibroblast adenylate cyclase activity. Increasing the degree of unsaturation increases the extent of inhibition noted at a given fatty acid concentration. The inhibitory effect of the unsaturated fatty acids or lysolecithin is not selective for a specific function of the adenylate cyclase system since basal, and hormone- or fluoride-stimulated cyclase activities are inhibited to the same extent. The fatty acid-inactivated state of fibroblast adenylate cyclase is not readily reversed for enzyme activity is not restored when arachidonate-treated membranes are washed with Tris buffer containing 10 mm EDTA, 0.15 mm albumin, or 0.15 m KCl. Previous studies have shown that the adenylate cyclase system from Moloney sarcoma virus-transformed NRK (MNRK) cells is not stimulated by the addition of GTP or hormones. Of interest is the present finding that the addition of unsaturated fatty acids, or lysolecithin, over a narrow concentration range (0.1 – 0.2 mm) leads to partial restoration of GTP activation of MNRK cyclase activity. Hormonal responsiveness to l-epinephrine or prostaglandin E₁ is not restored to the MNRK enzyme with fatty acid or lysolecithin treatment.     

Modulation of nicotinic acetylcholine receptor conformational state by free fatty acids and steroids

Steroids and free fatty acids (FFA) are noncompetitive antagonists of the nicotinic acetylcholine receptor (AChR). Their site of action is purportedly located at the lipid-AChR interface, but their exact mechanism of action is still unknown. Here we studied the effect of structurally different FFA and steroids on the conformational equilibrium of the AChR in Torpedo californica receptor-rich membranes. We took advantage of the higher affinity of the fluorescent AChR open channel blocker, crystal violet, for the desensitized state than for the resting state. Increasing concentrations of steroids and FFA decreased the K(D) of crystal violet in the absence of agonist; however, only cis-unsaturated FFA caused an increase in K(D) in the presence of agonist. This latter effect was also observed with treatments that caused the opposite effects on membrane polarity, such as phospholipase A(2) treatment or temperature increase (decreasing or increasing membrane polarity, respectively). Quenching by spin-labeled fatty acids of pyrene-labeled AChR reconstituted into model membranes, with the label located at the gammaM4 transmembrane segment, disclosed the occurrence of conformational changes induced by steroids and cis-unsaturated FFA. The present work is a step forward in understanding the mechanism of action of this type of molecules, suggesting that the direct contact between exogenous lipids and the AChR transmembrane segments removes the AChR from its resting state and that membrane polarity modulates the AChR activation equilibrium by an independent mechanism.     

Spectrin phosphorylation and shape change of human erythrocyte ghosts

Human erthrocyte membranes in isotonic medium change shape from crenated spheres to biconcave disks and cup-forms when incubated at 37 degrees C in the presence of MgATP (M. P. Sheetz and S. J. Singer, 1977, J. Cell Biol. 73:638-646). The postulated relationship between spectrin phosphorylation and shape change (W. Birchmeier and S. J. Singer, 1977, J. Cell Biol. 73:647-659) is examined in this report. Salt extraction of white ghosts reduced spectrin phosphorylation during shape changes by 85-95%. Salt extraction did not alter crenation, rate of MgATP-dependent shape change, or the fraction (greater than 80%) ultimately converted to disks and cup-forms after 1 h. Spectrin was partially dephosphorylated in intact cells by subjection to metabolic depletion in vitro. Membranes from depleted cells exhibited normal shape-change behavior. Shape-change behavior was influenced by the hemolysis buffer and temperature and by the time required for membrane preparation. Tris and phosphate ghosts lost the capacity to change shape after standing for 1-2 h at 0 degrees C. Hemolysis in HEPES or N- tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid yielded ghosts that were converted rapidly to disks in the absence of ATP and did not undergo further conversion to cup-forms. These effects could not be attributed to differential dephsphorylation of spectrin, because dephosphorylation during ghost preparation and incubation was negligible. These results suggest that spectrin phosphorylation is not required for MgATP-dependent shape change. It is proposed that other biochemical events induce membrane curvature changes and that the role of spectrin is passive.     

N-3 fatty acids in the Mediterranean diet

Fats in the diet of countries in the Mediterranean basin are typically represented by olive oil, but the high consumptions of vegetables and to some extent also of fish result in appreciable intakes of n-3 fatty acids. In fact, various plant foods are relatively rich in the 18 carbon n-3 fatty acid, alpha linolenic acid, ALA, while the generally moderate consumption of fish, except for certain communities living close to the sea, contributes to the intake of the long-chain n-3. Although the amounts of fats in ALA-containing plant foods are low, the relatively high concentrations of this fatty acid and the large size of the portions consumed allow to reach appreciable doses of ALA, an n-3 fatty acid that has been shown to exert favourable effects on various relevant factors in cardiovascular protection. In addition, consumption of relatively small amounts of certain typical dry fruit components of the diet such as walnuts, provides a sizable supply of ALA that is also rather efficiently converted to the ALA derivative eicosapentaenoic acid (EPA). Additional rather typical wild food components of the diet in certain countries, i.e. snails and frogs, are also appreciable sources of ALA. It appears thus that the consumption of typical Mediterranean foods provides relevant intakes of n-3 fatty acids, especially ALA, that appears to be efficiently absorbed and also transformed at least to the long-chain derivative EPA.     

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.     

Fatty acid patterns of dog erythrocyte membranes after feeding of a fish-oil based DHA-rich supplement with a base diet low in n-3 fatty acids versus a diet containing added n-3 fatty acids

Background

Numerous signaling pathways function in the brain ventricular system, including the most important - GABAergic, glutaminergic and dopaminergic signaling. Purinergic signalization system - comprising nucleotide receptors, nucleotidases, ATP and adenosine and their degradation products - are also present in the brain. However, the precise role of nucleotide signalling pathway in the ventricular system has been not elucidated so far. The aim of our research was the identification of all three elements of purinergic signaling pathway in the porcine brain ventricular system.

Results

Besides nucleotide receptors on the ependymocytes surface, we studied purines and pyrimidines in the CSF, including mechanisms of nucleotide signaling in the swine model (Sus scrofa domestica). The results indicate presence of G proteins coupled P2Y receptors on ependymocytes and also P2X receptors engaged in fast signal transmission. Additionally we found in CSF nucleotides and adenosine in the concentration sufficient to P receptors activation. These extracellular nucleotides are metabolised by adenylate kinase and nucleotidases from at least two families: NTPDases and NPPases. A low activity of these nucleotide metabolising enzymes maintains nucleotides concentration in ventricular system in micromolar range. ATP is degraded into adenosine and inosine.

Conclusions

Our results confirm the thesis about cross-talking between brain and ventricular system functioning in physiological as well as pathological conditions. The close interaction of brain and ventricular system may elicit changes in qualitative and quantitative composition of purines and pyrimidines in CSF. These changes can be dependent on the physiological state of brain, including pathological processes in CNS.