A study of selenate efflux from human placental microvillus membrane vesicles

Selenate efflux from human placental brush border membrane vesicles was studied using an ion-exchange column assay. Selenate efflux was found to be mediated almost exclusively by a temperature dependent DIDS-sensitive pathway. Chromate markedly inhibited selenate efflux: in contrast medium selenate had no effect. It is concluded that selenate and sulphate share a common pathway for transport across the human placental microvillus membrane.Abbreviations DIDS 4-acetamido-4-isothiocyanostilbene-2-2-disulphonate     

Lipid peroxidation of rabbit small intestinal microvillus membrane vesicles by iron complexes

Fe(II)- and Fe(III)-induced lipid peroxidation of rabbit small intestinal microvillus membrane vesicles was studied. Ferrous ammonium sulphate, ferrous ascorbate at a molar ratio of 10:1, and ferric citrate, at molar ratios of 1:1 and 1:20, did not stimulate lipid peroxidation. Ferrous ascorbate, 1:1, induced low stimulation, while ferrous ascorbate, 1:20 gave higher stimulation of lipid peroxidation. These results show that in our experimental system, ascorbate is a promotor rather than an inhibitor of lipid peroxidation. Ferric nitrilotriacetate (at molar ratios of 1:2 and 1:10), at an iron concentration of 200 microM, was by far the most effective in inducing lipid peroxidation. Superoxide dismutase, mannitol and glutathione had no effect, while catalase, thiourea and vitamin E markedly decreased ferrous ascorbate 1:20-induced lipid peroxidation. Ferric nitrilotriacetate-induced lipid peroxidation was slightly reduced by catalase and mannitol, significantly reduced by superoxide dismutase, and completely inhibited by thiourea. Glutathione caused a 100% increase in the ferric nitrilotriacetate-induced lipid peroxidation. These results suggest that Fe(II) in the presence of trace amounts of Fe(III), or an oxidizing agent and Fe(III) in the presence of Fe(II) or a reducing agent, are potent stimulators of lipid peroxidation of microvillus membrane vesicles. Addition of deferoxamine completely inhibited both ferrous ascorbate, 1:20 and ferric nitrilotriacetate-induced lipid peroxidation, demonstrating the requirement for iron for its stimulation. Iron-induced peroxidation of microvillus membrane may have physiological significance because it could already be demonstrated at 2 microM iron concentration.     

Purification and Na+ uptake by human placental microvillus membrane vesicles prepared by three different methods

Three methods were used to prepare microvillus membrane vesicles from each of six human placentas. Two of these incorporated an agitation stage to preferentially remove microvilli and either Ca2+ (Method 1) or Mg2+ (Method 2) aggregation of non-microvillus membrane. The third method involved homogenisation of the tissue followed by Mg2+ aggregation of non-microvillus membrane (Method 3). Enrichment of alkaline phosphatase activity (27.6 +/- 1.9, 25.3 +/- 2.7) and ouabain binding (5.9 +/- 2.6, 5.3 +/- 2.2, respectively) was similar in vesicles prepared by Methods 1 and 2, respectively. Method 3 vesicles showed a significantly (P less than 0.01) lower alkaline phosphatase enrichment (18.1 +/- 1.2), but ouabain binding enrichment (6.3 +/- 1.3) was not different and vesicle protein recovery (mg/g placenta) was 5-fold greater. Na+ uptake in the presence of an outwardly directed proton gradient was significantly inhibited in all microvillus membrane vesicles by amiloride (0.5 mM). However, the amiloride sensitive component of Na+ uptake was 3-6-fold greater in Method 3 vesicles than in Method 1 and 2 vesicles, and showed overshoot above equilibrium in the former but not the latter. Further experiments using the pH sensitive dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein suggested that the proton gradient dissipated faster from Method 1 than from Method 3 vesicles. Thus methodological differences can have a marked effect on transport processes in microvillus membrane vesicles prepared from the human placenta.     

Intramembranous particles are clustered on microvillus membrane vesicles

Many intramembranous particles in pig jejunal microvillus membranes cluster during cell disruption and membrane vesiculation with the MgCl2 aggregation technique (Hauser, H., Howell, K., Dawson, R.M.C. and Bowyer, D.E. (1980) Biochim. Biophys. Acta 602, 567-577). Isolated brush borders and purified microvillus membrane vesicles were jet-frozen and examined by freeze-fracture electron microscopy. From 30 to 60% of purified vesicles exhibited no intramembranous particles on their fracture face and 22-39% exhibited clustered or aggregated intramembranous particles. Only 6-15% of the vesicles exhibited the random distribution of intramembranous particles that is characteristic of intact enterocytes. Aggregation was not reversed after dialysis to remove divalent cations. Prior freezing of tissue or vesicles (-70 degrees C) gave the same results as fresh unfrozen material. Heterogeneity of microvillus vesicles may occur among the vesicles generated from a single microvillus.     

Lipid composition of the isolated rat intestinal microvillus membrane

1. Rat intestinal microvillus plasma membranes were prepared from previously isolated brush borders and the lipid composition was analysed. 2. The molar ratio of cholesterol to phospholipid was greatest in the membranes and closely resembled that reported for myelin. 3. Unesterified cholesterol was the major neutral lipid. However, 30% of the neutral lipid fraction was accounted for by glycerides and fatty acid. 4. Five phospholipid components were identified and measured, including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine. Though phosphatidylethanolamine was the chief phospholipid, no plasmalogen was detected. 5. In contrast with other plasma membranes in the rat, the polar lipids of the microvillus membrane were rich in glycolipid. The cholesterol:polar lipid (phospholipid+glycolipid) ratio was about 1:3 for the microvillus membrane. Published data suggest that this ratio resembles that of the liver plasma membrane more closely than myelin or the erythrocyte membrane. 6. The fatty acid composition of membrane lipids was altered markedly by a single feeding of safflower oil. Membrane polar lipids did not contain significantly more saturated fatty acids than cellular polar lipids. Differences in the proportion of some fatty acids in membrane and cellular glycerides were noted. These differences may reflect the presence of specific membrane glycerides.     

Lipid regulation of cell membrane structure and function

Recent studies of structure-function relationships in biological membranes have revealed fundamental concepts concerning the regulation of cellular membrane function by membrane lipids. Considerable progress has been made in understanding the roles played by two membrane lipids: cholesterol and phosphatidyl-ethanolamine. Cholesterol has been shown to regulate ion pumps, which in some cases show an absolute dependence on cholesterol for activity. These studies suggest that an essential role that cholesterol plays in mammalian cell biology is to enable crucial membrane enzymes to provide function necessary for cell survival. Studies of phosphatidylethanolamine regulation of membrane protein activity and regulation of membrane morphology led to hypotheses concerning the roles for this particular lipid in biological membranes. New information on lipid-protein interactions and on the nature of the lipid head groups has permitted the development of mechanistic hypotheses for the regulation of membrane protein activity by phosphatidyl-ethanolamine. In addition, intermediates in the lamellar-nonlamellar phase transitions of membrane systems containing phosphatidylethanolamine, or other lipids with similar properties, have recently been implicated in facilitating membrane fusion. Finally, studies of transmembrane movement of lipids have provided new insight into the regulation of membrane lipid asymmetry and the biogenesis of cell membranes. These kinds of studies are harbingers of a new generation of progress in the field of cell membranes.     

Macromolecule entrapment in human placental microvillous membrane vesicles

An osmotic lysis technique was developed to induce transient permeability in human placental microvillous membrane vesicles. The degree of vesicle opening and resealing was quantitated using the fluorescent markers, 6-carboxyfluorescein and fluorescein dextran. Compared to freeze-thaw and sonication methods, hypotonic lysis was significantly more efficient, causing greater than 90% lysis with greater than 90% subsequent resealing under optimal conditions. The transient increase in vesicle permeability permitted the unrestricted entry of macromolecules with molecular masses up to 70,000 kDa. Passive transport of water, protons, and erythritol and carrier-mediated transport of L-valine and sodium-proton exchange were unaltered by the lysis/resealing procedure. Bovine tracheal vesicles were lysed to an extent similar to placental microvillous vesicles, but rabbit renal cortical brush border and basolateral membranes were lysed to a lesser extent (approximately 60%). These results show that hypotonic lysis is a suitable method for the loading and trapping of macromolecules in isolated membrane vesicles for studies of intracellular regulation of transport.     

Dicarboxylate transport in human placental brush-border membrane vesicles

Pathways for transport of dicarboxylic acid metabolites by human placental epithelia were investigated using apical membrane vesicles isolated by divalent cation precipitation. The presence of Na+/dicarboxylate cotransport was assessed directly by [14C]succinate tracer flux measurements and indirectly by fluorescence determinations of voltage sensitive dye responses. The imposition of an inwardly directed Na+ gradient stimulated vesicle uptake of succinate achieving levels approximately 5-fold greater than those observed at equilibrium. The increased succinate uptake was specific for Na+ as no stimulation was observed in the presence of Li+, K+ or choline+ gradients. In addition to concentrative accumulation of succinate, a direct coupling of Na+/succinate cotransport was suggested by the absence of a sizeable conductive pathway for succinate uptake and decreased succinate uptake levels associated with a more rapid decay of an imposed Na+ gradient. Na+ gradient-driven succinate uptake was not the result of parallel Na+/H+ and succinate/OH- exchange activities but was reduced by the Na+-coupled inhibitor harmaline. The voltage sensitivity of Na+ gradient-driven succinate uptake suggests Na+/succinate cotransport is electrogenic occurring with net transfer of positive charge. Substrate-specificity studies suggest the tricarboxylic acid cycle intermediates as candidates for transport by the Na+-coupled pathway. Decreasing pH increased the citrate-induced inhibition of succinate uptake suggesting divalent citrate as the preferred substrate for transport. Initial rate determinations of succinate uptake indicate succinate interacts with a single saturable site (Km 33 microM) with a maximal transport rate of 0.5 nmol/mg per min.     

Elucidating membrane structure and protein behavior using giant plasma membrane vesicles

The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts. These observations are made in giant plasma membrane vesicles (GPMVs), which can be isolated by chemical vesiculants from a variety of cell types and microscopically observed using basic reagents and equipment available in any cell biology laboratory. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. This protocol describes the methods to prepare and isolate the vesicles, equipment to observe them under temperature-controlled conditions and three examples of fluorescence analysis: (i) fluorescence spectroscopy with an environment-sensitive dye (laurdan); (ii) two-photon microscopy of the same dye; and (iii) quantitative confocal microscopy to determine component partitioning between raft and nonraft phases. GPMV preparation and isolation, including fluorescent labeling and observation, can be accomplished within 4 h.     

Calcium uptake by placental plasma membrane vesicles

The Placental plasma membrane vesicles are capable of accumulating up to 190 mM Ca²⁺. This is 24-fold higher than the external Ca²⁺ concentration.This process is dependent on ATP hydrolysis by the placental Ca²⁺-ATPase.The $\text{P}{}_{\mathrm{i}}\text{Ca}$ ratio is dependent on the external Ca²⁺ concentration, and reaches the value of 2 at 10 mM Ca²⁺.Phosphate (5 mM) can double Ca²⁺ uptake when measured in the presence of 5 mM Ca²⁺.Mg²⁺; increased Ca²⁺ uptake only at low Ca²⁺ concentrations, and had no significant effect at 5 mM Ca²⁺.     

Zinc uptake by human placental microvillous membrane vesicles

Zinc uptake by syncytiotrophoblast microvillous membrane vesicles (SMMV) from human placentas was characterized and the effects of maternal serum zinc levels at term and of gestational age on kinetic parameters were evaluated. Zinc uptake at pH 7.2 was rapid for the first 2 min, followed by a slower increase, approaching equilibrium after 30 min. Uptake was saturable at a zinc concentration of 30 micromol/L, higher than the upper range of the physiological serum zinc level. Kinetic analysis of uptake at 1 min in SMMV from term placenta showed similar Km values (mean: 6.9+/-0.6 micromol/L) for different levels of maternal serum zinc. However, Vmax was higher (p < 0.05) in SMMV from mothers with serum zinc lower than 7.6 micromol/L compared to those with higher serum zinc levels (35.8+/-1.6 and 26.6+/-1.6 nmol 65Zn/mg protein/min, respectively). Km values were similar in term (>37 wk of gestation) and preterm (20-25 wk of gestation) placentas, whereas Vmax was higher (p < 0.05) in the preterm (34.3+/-1.6 nmol Zn/mg protein/min) compared to term placentas from mothers with serum zinc levels above 7.6 micromol/L. These results suggest that whereas afffinity for zinc was not altered with gestational age or maternal serum zinc levels, zinc-uptake capacity in human placenta is influenced both by gestational age and by low levels of maternal serum zinc in order to ensure an adequate maternal-fetal zinc transfer.     

Calcium uptake by placental plasma membrane vesicles.

The placental plasma membrane vesicles are capable of accumulating up to 190 mM Ca2+. This is 24-fold higher than the external Ca2+ concentration. This process is dependent on ATP hydrolysis by the placental Ca2+-ATPase. The Pi/Ca ratio is dependent on the external Ca2+ concentration, and reaches the value of 2 at 10 mM Ca2+. Phosphate (5 mM) can double Ca2+ uptake when measured in the presence of 5 mM Ca2+. Mg2+ increased Ca2+ uptake only at low Ca2+ concentrations, and had no significant effect at 5 mM Ca2+.     

Differences in composition and fluidity of intestinal microvillus membrane vesicles prepared by different methods

Multiple methods have been developed to isolate the intestinal microvillus membrane and facilitate the study of its composition and function. Variations in membrane composition and fluidity may result from different preparative techniques. This study shows that the use of MgCl2 and/or KSCN in vesicle preparation alters phospholipid and protein composition of the membrane compared to CaCl2 precipitation. The use of MgCl2 in membrane preparation increased phosphatidylethanolamine and decreased phosphatidylinositol content. The use of KSCN in membrane preparation decreased the protein content. The structural changes seen with the use MgCl2 alone are accompanied by an increase in both static and dynamic membrane fluidity. These results suggest that different methods of membrane vesicle preparation affect membrane phospholipid and protein content as well as membrane fluidity.     

Preparation of microvillus membrane vesicles from the intestine of embryonic and young chicks

1. A procedure is described for the isolation of microvillus membranes from 19-day old embryonic, newly hatched, and 2-day old chicken intestine. 2. The magnesium concentration of epithelial cell homogenates is shown to be a crucial factor in obtaining membranes of equal purity from the three age groups. 3. Microvillus membranes are purified 20-25 fold over the original homogenate and form vesicles which are tightly sealed based on the Na+-dependent accumulation of glucose to levels four to five times equilibrium values. 4. These membrane preparations should prove useful in future studies concerning the embryonic and neonatal development of microvillus enzymes and nutrient transport systems in the chicken.     

Interaction of external H+ with the Na+-H+ exchanger in renal microvillus membrane vesicles

We examined the effects of external H+ on the kinetics of Na+-H+ exchange in microvillus membrane vesicles isolated from the rabbit renal cortex. The initial rate of Na+ influx into vesicles with internal pH 6.0 was optimal at external pH 8.5 and was progressively inhibited as external pH was reduced to 6.0. A plot of 1/V versus [H+]o was linear and yielded apparent KH = 35 nM (apparent pK 7.5). In vesicles with internal pH 6.0 studied at external pH 7.5 or 6.6, apparent KNa was 13 or 54 mM, Ki for inhibition of Na+ influx by external Li+ was 1.2 or 5.2 mM, Ki for inhibition by external NH4+ was 11 or 50 mM, and Ki for inhibition by external amiloride was 7 or 25 microM, respectively. These findings were consistent with competition between each cation and H+ at a site with apparent pK 7.3-7.5. Lastly, stimulation of 22Na efflux by external Na+ (i.e. Na+-Na+ exchange) was inhibited as external pH was reduced from 7.5 to 6.0, also consistent with competition between external H+ and external Na+. Thus, in contrast with internal H+, which interacts at both transport and activator sites, external H+ interacts with the renal microvillus membrane Na+-H+ exchanger at a single site, namely the external transport site, where H+, Na+, Li+, NH4+, and amiloride all compete for binding.     

The microvillus 110K cytoskeletal protein is an integral membrane protein

A protein of 110,000 MW connects actin filaments to the plasma membrane in microvilli of intestinal epithelial cells. In the present study four independent lines of evidence suggest that the 110K protein is directly bound to the lipid bilayer. The solubilization of the 110K protein requires detergents and removal of detergent after solubilization results in aggregation. The 110K protein partitions into the detergent phase in Triton X-114 solutions. It is selectively incorporated into liposomes. It is specifically labeled with the hydrophobic probe ¹⁴C-phenylisothiocyanate. In addition we present a purification scheme for the 110K protein in milligram amounts. This represents the simplest system of membrane to filament attachment, in which an integral membrane protein is also a cytoskeletal protein.