Isoproterenol-induced intramembrane particle aggregation and water flux in toad epidermis

Stimulation of toad skin with isproterenol resulted in a dramatic increase in water flow, and in the appearance of aggregates of intramembrane particles in the apical membrane of granular cells of the replacement layer, just beneath the stratum corneum. This membrane structural modification appears to be a general prerequisite for the change in water permeability of vasopressin-sensitive epithelia.     

Cross-linking of erythrocyte membrane proteins by periodate and intramembrane particle distribution.

Treatment of isolated human erythrocyte membranes at pH 7.4 with 0.1-0.5 mM-sodium periodate specifically cross-linked some of the spectrin polypeptides. Treatment with 2 mM-periodate resulted in complete cross-linking of spectrin and partial cross-linking of other polypeptides. The latter treatment also caused aggregation of the intramembrane particles made visible by freeze-fracturing. When membranes that had been treated with 2 mM-periodate were depleted of spectrin by treatment with 0.1 mM-EDTA, extensive aggregation of the intramembrane particles occurred.     

The ATP depletion process of human erythrocytes studied by freeze fracturing

By the freeze-fracture method it is shown that metabolic depletion of erythrocytes affects three levels of cell organization: the microstructural (erythrocyte form), microstructural (micro-relief of erythrocyte surface) and ultrastructural (ultrastructural state of erythrocyte plasma membranes) ones. As it is established, the size of spikes on the echinocyte surface and that of membrane vesicles budding from a cell coincide with each other. The structural modification of the membrane precedes the stage of erythrocyte crenation. The following model of vesicle budding process is suggested: reduction of ATP level and dephosphorylation of actin-spectrin network--structural modification of the protein and lipid membrane phases with the formation of regions disconnected from the spectrin framework--protrusion of these anomalous regions in the form of spikes--budding of spikes as spherical vesicles.     

Correlation between changes in the membrane organization and susceptibility to phospholipase C attack induced by ATP depletion of rat erythrocytes

About 20 and 43% of the total membrane phospholipids are hydrolized in fresh rat erythrocytes by treatment with phospholipase C (Bacillus cereus), or both sphingomyelinase and phospholipase C, respectively, without causing cell lysis. Treatment of ATP-depleted cells with phospholipase C alone results in 50% hydrolysis and extensive lysis. Depletion of ATP causes a marked increase in the aggregation of intramembranous particles accompanied by a similar increase in the smooth area between the particle clusters as revealed by the freeze-etch technique. Such changes are not induced by extensive phospholipid hydrolysis in absence of cell lysis in fresh cells.Based on these and additional data, it is suggested that the membrane phospholipid organization can be divided into 3 types: phospholipids exposed to phospholipase C; phospholipids protected against phospholipase C by presence of sphingomyelin; phospholipids which can be exposed following alteration of the proteinlipid interactions. Such alterations which might be induced by a variety of means, including ATP depletion, might result in clustering of intramembranous particles and increase of the free lipid bilayer phase of the membrane.     

Effects of intramembrane particle aggregation on erythrocyte membrane fluidity: an electron spin resonance study in normal and in dystrophic subjects

Mobilization and aggregation of intramembrane particles (IMPs) are physiological events observed in various cells. In erythrocyte membranes, aggregation of IMPs can be induced by the exposure of partially desprectrinized erythrocyte membranes to acidic pH. We investigated the association between IMPs aggregation, protein mobility, and membrane fluidity in erythrocyte membranes of healthy controls and Duchenne muscular dystrophy (DMD) patients by using electron spin resonance and specific spin labels for membrane proteins and lipids. In erythrocyte membranes of control subjects, the partial spectrin removal induced a decreased segmental motion of protein spin label indicating an increase of protein-protein interactions. Stearic acid spin labels 5- and 16-(N-oxyl-4,4'-dimethyloxazolidine) showed that the treatment induces an increase of membrane fluidity. In DMD patients, both treated and untreated erythrocyte membranes showed changes of membrane fluidity when compared to those of the controls. Our results suggest that defects in the interactions between skeletal proteins and/or between membrane and skeleton components may contribute to the alterations of erythrocyte membranes in DMD.     

Stimulation by local anesthetics of the metabolism of acidic phospholipids in the rat pineal gland

The efficacy of five local anesthetics in causing stimulation of phospholipid metabolism in rat pineal gland $\text{in vitro}$ paralleled their anesthetic potency and decreased in the order: dibucaine, tetracaine, cocaine, procaine, lidocaine. When stimulation occurred, the patterns of labeling resembled that produced by propranolol, a β-adrenergic receptor blocking agent with local anesthetic activity. Isotope incorporation into phosphatidylglycerol and CDP-diglyceride was markedly enhanced and increases of labeling of phosphatidic acid and phosphatidylinositol were also seen. At concentrations of 1–10 mM, propranolol and local anesthetics inhibited labeling of phosphatidylcholine and phosphatidylethanolamine by more than 90% and incorporation of ³²P_i into other phospholipids to a smaller extent.     

Lectin-mediated agglutination of erythrocyte ghost membranes following depletion of membrane protein and intramembrane particles

Profound digestion of unsealed human erythrocyte ghosts with high concentrations of Pronase results in a near complete loss of intramembrane particles while trypsin digestion is less effective. The small vesicles formed by proteolysis are agglutinable by soybean agglutinin (SBA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA), but not concanavalin A (ConA). Densitometer tracings of Pronase-treated vesicles analyzed on SDS-polyacrylamide gels demonstrated no detectable protein or glycoprotein migrating slower than the marking dye. The vesicles showed a loss of 90% Lowry positive material (the remainder may be non-protein chromogens), near depletion of sialyl residues, no significant change in lipid composition, and equal amounts of phospholipid phosphorus compared to an equal volume of ghosts. The lipid material extracted from Pronase-derived vesicles or intact ghosts inhibited hemagglutination with SBA and WGA but not ConA. SBA but not ConA was found to specifically bind to Pronase-derived vesicles while both lectins bound to native ghosts. These observations suggest that neither the integrity of the intramembrane particles nor the presence of membrane glycoprotein appears essential for SBA-, WGA-, and PHA-mediated agglutination. Furthermore, it appears that native membrane glycolipids (and perhaps glycopeptides) can bind SBA, WGA and PHA. The membrane glycolipids may play a larger role than heretofore realized in lectin-mediated agglutination of cells.     

Adenine nucleotide metabolism and nucleoside transport in human erythrocytes under ATP depletion conditions

The adenine nucleotides of human red cells were labeled by incubation of the cells with [3H]adenosine. Then, the cells were incubated in Tris-saline with various supplements that cause the loss of cellular ATP, and the degradation products were quantitated as a function of time of incubation at 37 degrees C. Incubation of the cells with 2.5 or 5 mM iodoacetate, iodoacetamide or 1 mM HCHO in combination with 5 mM KF and 50 mM deoxyglucose, 50 mM D-glucose or 10 mM inosine was most efficient in depleting the cells of ATP (100% in 0.5-1 h) without causing cell lysis. In iodoacetate- and iodoacetamide-treated cells practically all catabolism of ATP occurred via ADP----AMP----IMP----inosine----hypoxanthine with hypoxanthine accumulating in the medium. In HCHO-treated cells and in cells incubated in Tris-saline or in Tris-saline with deoxyglucose with and without KF, a substantial proportion of ATP (up to 50%) was catabolized via ADP----AMP----adenosine----inosine----hypoxanthine. Under all conditions, AMP deamination and IMP and AMP hydrolysis were rate-limiting reactions. IMP degradation was more rapid in iodoacetamide- and HCHO-treated than in iodoacetate-treated red cells. It was also more rapid in fresh than in outdated red cells, and it was inhibited by Pi. Treatment with iodoacetamide and HCHO under ATP-depletion conditions resulted in a 60-80% inhibition of uridine transport by the cells. Treatment with iodoacetate or deoxyglucose plus KF had only minor effects on nucleoside transport; thus, cells treated in this manner might be useful for studying the transport of adenosine and deoxyadenosine under conditions were their phosphorylation is prevented.     

Induction of sperm head abnormalities in mice by three tranquilizers

Toxic effects of diazepam (DZ), chlordiazepoxide (CDZ) and nitrazepam (NZ) on the spermatozoa of mice have been studied at the end of 1, 3, 4, 6, 8 and 12 weeks after 15 days repeated treatment with a daily oral dose of 0.5 mg. Different types of abnormalities involving both shape and size of the sperm head were noticed. Qualitatively NZ produced the maximum number of abnormal types. The incidences of abnormal sperm heads were significantly high in all the test weeks in the NZ series, at weeks 1 and 6 after DZ treatment, and at weeks 3, 4 and 6 after CDZ treatment. All three drugs produced maximum effects at week 6. The varying effects induced by the three benzodiazepines seem to be due to their differential pharmacokinetics.     

Effects of ATP depletion on the mechanism of hexose transport in intact human erythrocytes

Depletion of ATP is known to inhibit glucose transport in human erythrocytes, but the kinetic mechanism of this effect is controversial. Selective ATP depletion of human erythrocytes by 10 micrograms/ml A23187 in the presence of extracellular calcium inhibited 3-O-methylglucose influx noncompetitively and efflux competitively. ATP depletion also decreased the ability of either equilibrated 3-O-methylglucose or extracellular maltose to inhibit cytochalasin B binding in intact cells, whereas neither total high-affinity cytochalasin B binding nor its Kd was affected. Under the one-site model of hexose transport these data indicate that ATP depletion decreases both the affinity of the inward-facing glucose carrier for substrate and its ability to reorient outwardly in intact cells.     

Interaction of local anesthetics with the transport system of glucose in human erythrocytes.

Local anesthetics inhibit the exhange transport of glucose in human erythroytes. All compounds tested showed a competitive inhibition except lidocaine and baycaine causing a non-competitive one. Moreover the transport system can bind two inhibitor molecules to one transport site as described for tetracaine and oxybuprocaine.     

Concanavalin A-induced increase in the membrane fluidity of chicken erythrocytes.

To determine the fluidity of the membrane lipid phase, chicken erythrocytes were labeled with a stearic acid derivative spin label. When chicken erythrocytes were treated with concanavalin A (Con A), ESR spectra showed a change in the peaks of the labels in membrane lipids, indicating an increase of membrane fluidity. The degree of the increase in fluidity of the membrane lipid phase depended on the valency of the lectin used. Tetravalent Con A induced an increase of membrane fluidity at a concentration as low as 30 micrograms/ml, while a monovalent derivative of Con A did not affect membrane fluidity. This increase in membrane fluidity was observed within 10 min after the addition of Con A. If bound Con A was removed with methyl alpha-D-mannoside later than 60 min after its addition, a complete return of the fluidity to the normal level could not be observed. However, no change was found in the composition of phospholipids or in the fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine of chicken erythrocytes after the addition of Con A, indicating that this increase in membrane fluidity is not caused by a change of lipid composition. The clustering of membrane receptors of chicken erythrocytes for Con A was demonstrated when the two-dimensional distribution of ferritin-conjugated Con A on the membranes was assayed by transmission electron microscopy. Furthermore, it was shown that major receptors for Con A of chicken erythrocytes were transmembrane glycoproteins having apparent molecular weights of 100K, 45, and 33K.     

Chemical co-treatments and intramembrane particle patching in the poly(ethylene glycol)-induced fusion of turkey and human erythrocytes

Several chemical co-treatments were used to lower the threshold concentrations of poly(ethylene glycol) (PEG) required to induce fusion between turkey erythrocytes and between human erythrocytes. Concanavalin A was used in conjunction with 25% (w/w) PEG to induce turkey erythrocyte fusion. The fusion percentage increased with increasing concentrations of concanavalin A and the duration of concanavalin A treatment. In samples with high percentages of fusion, numerous hemispherical intramembrane particle-free zones (bubbles) in the plasma membrane were revealed by freeze-fracture electron microscopy. However, concanavalin A treatment did not facilitate fusion between human erythrocytes even at 35% PEG, although slight intramembrane particle patching was observed under this condition. Spermidine (0.05% w/v), trichloroacetic acid (100 mM) and ethanol (4% v/v) were found to promote fusion of human erythrocytes in 25% PEG. In all of these cases, intramembrane particle patching was observed by freeze-fracture electron microscopy in the presence of PEG. When applied alone, only ethanol caused a slight intramembrane particle patching. Neither dimethylsulfoxide (2% v/v), lysophosphatidylcholine (lysoPC, 0.15 mM), nor polylysine (mol. wt. 1000-4000, 0.05% w/v) promoted fusion of human erythrocyte in 25% PEG. None of these chemical treatments, alone, or in combination with PEG, caused intramembrane particle patching. We conclude that the positive effect of chemical treatments on PEG-induced cell fusion is closely related to the formation of intramembrane particle-free zones on the plasma membrane.     

Induction of aggregation and fusion of cholesterol-containing membrane vesicles by an anti-cholesterol monoclonal antibody

A monoclonal IgM antibody that reacts with cholesterol was able to aggregate small and large unilamellar lipid vesicles. Vesicles aggregated by the antibody could be dispersed by trypsin digestion. Inclusion of unsaturated phosphatidylethanolamine in the vesicle formulation lowered the relative amount of cholesterol necessary for aggregation, and prevented disaggregation by trypsin treatment. Fluorimetric assays indicated that membrane mixing occurred in aggregates resistant to trypsinization, but the vesicles did not mix or leak their aqueous contents. Analysis of the kinetics of lipid-mixing showed an increase in the aggregation and fusion rate constants with increasing antibody concentrations, indicating that the antibody reaction promotes both processes. An apparent inactivation process whose rate increased with antibody dose has been considered.We conclude that the simultaneous binding of antibodies to more than one vesicle at densities that allow the contact of membrane surfaces, induces first aggregation followed by hemifusion, and with excess of antibody also results in inactivation of the latter process.