Françoise Giraud

The effects of membrane lipid order and cholesterol on the internal and external cationic sites of the Na+−K+ pump in erythrocytes

cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the Na+-K+ pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the Na+-K+ pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the Na+-K+ pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the Na+-K+ pump. This difference in sensitivity to membrane lipids order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments.

Sphingosine Contributes to Glucocorticoid-Induced Apoptosis of Thymocytes Independently of the Mitochondrial Pathway

During the selection process in the thymus, most thymocytes are eliminated by apoptosis through signaling via TCR or glucocorticoids. The involvement of ceramide (Cer) and sphingosine (SP), important apoptotic mediators, remains poorly defined in glucocorticoid-induced apoptosis. We report that, in mouse thymocytes, apoptosis triggered by 10−6 M dexamethasone (DX) was preceded by a caspase-dependent Cer and SP generation, together with activation of acidic and neutral ceramidases. Apoptosis was drastically reduced by blocking either sphingolipid production (by acid sphingomyelinase inhibitor) or SP production (by ceramidase inhibitors), but not by inhibition of de novo Cer synthesis. Thus, SP generated through acid sphingomyelinase and ceramidase activity would contribute to the apoptotic effect of DX. Consistent with this hypothesis, SP addition or inhibition of SP kinase induced thymocyte apoptosis. DX induced a proteasome-dependent loss of mitochondrial membrane potential (Δψm) and caspase-8, -3, and -9 processing. Apoptosis was abolished by inhibition of Δψm loss or caspase-8 or -3, but not caspase-9. Δψm loss was independent of SP production and caspase-8, -3, and -9 activation. However, inhibition of SP production reduced caspase-8 and -3, but not caspase-9 processing. Proteasome inhibition impaired activation of the three caspases, whereas inhibition of Δψm loss solely blocked caspase-9 activation. These data indicate that DX-induced apoptosis is mediated in part by SP, which contributes, together with proteasome activity, to caspase-8-3 processing independently of mitochondria, and in part by the proteasome/mitochondria pathway, although independently of caspase-9 activation.

Alterations in human erythrocyte shape and the state of spectrin and phospholipid phosphorylation induced by cholesterol depletion.

Cholesterol depletion of erythrocytes, obtained after incubation with phosphatidylcholine vesicles, induces in most of the experiments: (1) a discocytestomatocyte transformation as observed by scanning electron microscopy; (2) a specific decrease in spectrin phosphorylation of intact erythrocytes; (3) an increase in lipid phosphorylation. It is concluded that the effect of cholesterol on erythrocyte shape is probably mediated through its action on the activity o of membrane-bound enzymes, proteases or kinases.

The involvement of the Ca-dependent K channel and of the KCl co-transport in sickle cell dehydration during cyclic deoxygenation

We have investigated the mechanisms involved in sickle cell dehydration upon continuous or cyclic deoxygenation: the Ca(2+)-activated K+ channel and the KCl co-transport system. Short-term continuous deoxygenation (1 h) of sickle cells in a Ca(2+)-containing medium promoted a stimulation of the efflux of K+ and cell dehydration. This latter was reduced by the replacement of Ca2+ in the medium by EGTA, but not by addition of [(dihydro-indenyl) oxy] alkanoic acid (DIOA), an inhibitor of the KCl co-transport. During cycles of deoxygenation-reoxygenation, cell dehydration was partly prevented by EGTA and significantly reduced by DIOA only in the presence of Ca2+. The present data support the view that sickle cell dehydration during deoxygenation arises from the stimulation of the Ca(2+)-dependent K+ permeability leading to water loss, whereas during reoxygenation periods, subsequent activation of the KCl co-transport also contributes to cell dehydration.

Involvement of Sodium in Early Phosphatidylserine Exposure and Phospholipid Scrambling Induced by P2X7 Purinoceptor Activation in Thymocytes

Extracellular ATP (ATPec), a possible effector in thymocyte selection, induces thymocyte death via purinoceptor activation. We show that ATPec induced cell death by apoptosis, rather than lysis, and early phosphatidylserine (PS) exposure and phospholipid scrambling in a limited thymocyte population (35–40%). PS externalization resulted from the activation of the cationic channel P2X7 (formerly P2Z) receptor and was triggered in all thymocyte subsets although to different proportions in each one. Phospholipid movement was dependent on ATPec-induced Ca2+ and/or Na+ influx. At physiological external Na+ concentration, without external Ca2+, PS was exposed in all ATPec-responsive cells. In contrast, without external Na+, physiological external Ca2+ concentration promoted a submaximal response. Altogether these data show that Na+ influx plays a major role in the rapid PS exposure induced by P2X7 receptor activation in thymocytes.

α-Adrenergically mediated changes in membrane lipid fluidity and Ca2+ binding in isolated rat liver plasma membranes

Abstract Noradrenaline (0.1–5 μM, in the presence of 5 μM propranolol to block β-receptors), ATP (100 μM) and angiotensin II (0.1 μM), which are thought to increase cytosolic Ca 2+ concentration by mobilizing Ca 2+ from internal stores, increased the lipid fluidity as measured by diphenylhexatriene fluorescence polarization in plasma membranes isolated from rat liver. The effect of noradrenaline was dose-dependent and blocked by the α-antagonists phenoxybenzamine (50 μM) and phentolamine (1 μM). The response to a maximal dose of noradrenaline (5 μM) and that to ATP (100 μM) were not cumulative, suggesting that both agents use a common mechanism to alter the membrane lipid fluidity. In contrast, the addition of noradrenaline (5 μM) along with the foreign amphiphile Na + -oleate (1–30 μM) resulted in an increase in membrane lipid fluidity which was equivalent to the sum of individual responses to the two agents. In the absence of Mg 2+ , reducing free Ca 2+ concentration by adding EGTA increased membrane lipid fluidity and abolished the effect of noradrenaline, suggesting that Ca 2+ is involved in the mechanism by which the hormone exerts its effect on plasma membranes. Noradrenaline (5 μM) and angiotensin II (0.1 μM) also promoted a small release of 45 Ca 2+ (16 pmol/mg membrane proteins) from prelabelled plasma membranes. The effect of noradrenaline was suppressed by the α-antagonist phentolamine (5 μM). It is proposed that noradrenaline, via α-adrenergic receptors and other Ca 2+ -mobilizing hormones, increases membrane lipid fluidity by displacing a small pool of Ca 2+ bound to phospholipids, removing thus the mechanical constraints brought about by this ion.

Cholesterol depletion affects the Ca2+ influx but not the Ca2+ pump in human erythrocytes

Control and cholesterol-depleted human erythrocytes were loaded with permeant Ca2+ chelators (Benz2-AM or Quin2-AM) in order to increase their exchangeable Ca2+ pool and to measure both Ca2+ fluxes and [Ca]i (free cytoplasmic calcium concentration). The fluxes were independent of the concentration and of the nature of the intracellular chelator. The ATP content was not decreased by more than 50% under our experimental conditions. Cholesterol depletion (up to 28%) induced a decrease in both Ca2+ fluxes and [Ca]i which was proportional to the extent of the depletion. It is shown that cholesterol depletion primarily altered the properties of the system responsible for Ca2+ entry causing a diminution of the [Ca]i. This, in turn, induced a diminution of the activity of the Ca2+ pump without affecting the properties of this pump.

Extracellular ATP Induces Phosphatidylserine Externalization Earlier than Nuclear Apoptotic Events in Thymocytes

Abstract: The present study shows for the first time that ATPec induces a very early PS exposure in thymocytes and that this process can be induced in the absence of Ca2+ influx and caspase activation.

The measurement in vivo of the rate of unesterified cholesterol exchange between rat plasma and erythrocytes

In order to investigate the rate of unesterified cholesterol exchange between plasma and erythrocytes in vivo, cholesterol labelling in rats was achieved in one of the following ways: intravenous injection of cholesterol-labelled erythrocytes, subcutaneous injection of labelled acetate, feeding of labelled cholesterol. The specific activity of the unesterified cholesterol was measured at intervals up to 24 h and a kinetic analysis of the data was performed. It assumes that both the cholesterol in the erythrocytes and the unesterified cholesterol in the plasma were homogeneous pools. The rate constants obtained for the movements of unesterified cholesterol from erythrocytes to plasma and from plasma to erythrocytes were not significantly different in the three labelling conditions (mean values: 0.26 and 1.5 h-1, respectively).

Formation of protein polymers in erythrocyte ghosts incubated with sonicated lipid vesicles. Effects on spectrin extractibility, permeability of ghosts to vesicles, intramembrane particle distribution and bleb formation.

Abstract The incubation of human erythrocyte white ghosts with phosphatidylcholine (PC) vesicles or cholesterol/phosphatidylcholine (C/PC) vesicles under hypotonic or isotonic conditions generated membrane protein cross-links. The latter appeared in the form of a high molecular weight polymer after SDS-polyacrylamide gel electrophoresis. The polymer started to develop within a few minutes of incubation, arising largely from spectrin, and required 24 h or more for completion. It occurred regardless of cholesterol depletion undergone by the ghosts in the presence of PC vesicles. It was not reversed upon further incubation in a hypotonic, vesicle-free medium. When initial incubation was carried out under hypotonic conditions, a number of other alterations were recorded: (i) spectrin extractibility was abolished; (ii) ghosts became gradually impermeable to vesicles within a few hours, a process referred to as slow ‘resealing’ and generating an irreversible sequestration of the vesicles; (iii) intramembrane particles aggregated and blebs free of intramembrane particles pinched off inward or outward. When initial incubation was conducted under isotonic conditions, the following was observed: (i) spectrin was unextractible, as could be expected; (ii) vesicles did not enter the ghosts, a fact indicating an immediate and complete impermeabilization of ghosts to vesicles, a process referred to as fast ‘resealing’; (iii) intramembrane particle aggregation and blebs free of intramembrane particles were also present. When initial incubation was performed under isotonic conditions, but in the absence of vesicles, the polymer failed to be associated with spectrin inextractibility. These data support the view that lipid vesicles generate a high molecular weight polymer-associated, slow resealing of erythrocyte ghosts that differs, at least in part, from the polymer-free, fast resealing induced by a vesicle-free isotonic medium. Resistance to β-mercaptoethanol of the polymer makes unlikely the sole participation of disulfide bonds. Absence of added Ca 2+ in the medium is inconsistent with the transglutaminase-catalyzed formation of amide linkages. When ghosts were separated from the vesicles by a cellophane membrane upon hypotonic incubation, spectrin remained extractible and no polymer developed. Sonication of the vesicles under nitrogen and in the presence of butylated hydroxytoluene did not prevent the formation of the polymer.