Lipid raft organization and function in brush borders of epithelial cells

Polarized epithelial cells of multicellular organisms confront the environment with a highly specialized apical cell membrane that differs in composition and function from that facing the internal milieu. In the case of absorptive cells, such as the small intestinal enterocyte and the kidney proximal tubule cell, the apical cell membrane is formed as a brush border, composed of regular, dense arrays of microvilli. Hydrolytic ectoenzymes make up the bulk of the microvillar membrane proteins, endowing the brush border with a huge digestive capacity. Several of the major enzymes are localized in lipid rafts, which, for the enterocyte in particular, are organized in a unique fashion. Glycolipids, rather than cholesterol, together with the divalent lectin galectin-4, define these rafts, which are stable and probably quite large. The architecture of these rafts supports a digestive/absorptive strategy for nutrient assimilation, but also serves as a portal for a large number of pathogens. Caveolae are well-known vehicles for internalization of lipid rafts, but in the enterocyte brush border, binding of cholera toxin is followed by uptake via a clathrin-dependent mechanism. Recently, 'anti-glycosyl' antibodies were shown to be deposited in the enterocyte brush border. When the antibodies were removed from the membrane, other carbohydrate-binding proteins, including cholera toxin, increased their binding to the brush border. Thus, anti-glycosyl antibodies may serve as guardians of glycolipid-based rafts, protecting them from lumenal pathogens and in this way be part of an ongoing 'cross-talk' between indigenous bacteria and the host.     

Molecular organization of the intestinal brush border

The brush border of enterocytes represents one of the more specialized apical poles of epithelial cells. It is formed by particularly well-developed apical plasma membrane microvilli, whose shape is ensured by a highly organized cytoskeleton. The molecular organization of the cytoskeleton is described. Whereas several cytoskeleton proteins are ubiquitous, villin is highly specific for intestinal cells and can be used as a differentiation marker of these cells. The major glycoproteins, in particular hydrolases, of the brush border membrane have been characterized. They have many common structural features, in particular their mode of integration into the membrane by their N-terminal hydrophobic sequences that also plays the role of the 'signal peptide' responsible for their co-translational insertions into the endoplasmic reticulum. Studies on the biosynthesis and intracellular pathway of aminopeptidase N strongly suggest that sorting of apical and basolateral glycoproteins could occur after their integration into the basolateral domain.     

Lipid asymmetry in rabbit small intestinal brush border membrane as probed by an intrinsic phospholipid exchange protein.

All classes of phospholipids present in brush border membrane are exchanged in a 1:1 ratio for egg phosphatidylcholine when brush border membrane vesicles from rabbit small intestine are incubated with small unilamellar vesicles of egg phosphatidylcholine. The exchange reaction exhibits biphasic kinetics similar to those of the hydrolysis of brush border membrane phospholipids by phospholipase A2 and sphingomyelinase C. In both reactions there is an initial fast phase followed by a markedly slower one. The phospholipid exchange appears to be catalyzed by intrinsic brush border membrane protein(s), while the digestion by phospholipases is mediated by externally added enzymes. From a comparison of the kinetics of phospholipid exchange and phospholipid hydrolysis, the following conclusions can be drawn: Both sets of experiments indicate the presence of two phospholipid pools differing in the rate of phospholipid exchange and hydrolysis. Except for sphingomyelin, the size of the two phospholipid pools derived from phospholipid exchange is in good agreement with that derived from phospholipid hydrolysis. This is the main finding of this work, and on the basis of this result the two lipid pools are tentatively assigned to phospholipid molecules located on the outer and inner layer of the brush border membrane. The slow rate of phospholipid exchange reflects the rate of transverse or flip-flop movement of phospholipids. The half-time of this motion is approximately 8 h for isoelectric (neutral) phospholipids such as phosphatidylethanolamine and approximately 80 h for negatively charged phosphatidylserine and phosphatidylinositol. Isoelectric phospholipids (phosphatidylcholine, phosphatidylethanolamine) are preferentially located on the inner (cytoplasmic) side (to about 70%) while the negatively charged phospholipids are more evenly distributed: 55-60% are located on the inner side.     

Uptake of cholesterol by small intestinal brush border membrane is protein-mediated

Absorption of cholesterol by small intestinal brush border membrane from either mixed micelles or small unilamellar vesicles is protein-mediated. It is a second-order reaction. The kinetic data are consistent with a mechanism involving collision-induced transfer of cholesterol. With micelles as the donor particle, there is net transfer of cholesterol while with small unilamellar vesicles as the donor, cholesterol is evenly distributed between the two lipid pools at equilibrium. The cholesterol absorption by brush border membrane from both mixed micelles and small unilamellar vesicles reveals saturation kinetics. Proteolytic treatment of brush border membrane with papain releases about 25% of the total membrane protein. As a result, the cholesterol uptake by brush border membrane changes from a second-order reaction to a first-order one. The reaction mechanism changes from collision-induced cholesterol uptake to a mechanism involving diffusion of monomeric cholesterol through the aqueous phase. The protein(s) released into the supernatant by papain treatment of brush border membrane exhibit(s) cholesterol exchange activity between two populations of small unilamellar vesicles. The supernate-protein(s) bind(s) the spin-labeled cholesterol analogue 3-doxyl-5 alpha-cholestane.     

Orientation and motion of spin-labels in rabbit small intestinal brush border vesicle membranes

The temperature dependence of the packing (order) and fluidity (microviscosity) of rabbit small, intestinal brush border vesicle membranes and of liposomes made from their extracted lipids has been investigated by using a variety of lipid spin probes. The lipids in the brush border membrane are present essentially as a bilayer. Compared to other mammalian membranes, the brush border membrane appears to be characterized by a relatively high packing order as well as microviscosity. At body temperature, the lipid molecules undergo rapid, anisotropic motion, which is essentially a fast rotation about an axis approximately perpendicular to the bilayer normal. Both the order (motional anisotropy) and the microviscosity increase with decreasing temperature and with increasing distance from the center of the bilayer. Qualitatively similar motional or fluidity gradients have been reported for other mammalian and bacterial membranes. The liposomes made from the extracted lipids have a somewhat lower packing order and a slightly higher fluidity than brush border vesicle membranes. The differences are, however, small indicating that the packing and the fluidity (microviscosity) of the membrane are primarily determined by the lipid composition. Membrane-associated proteins and cytoskeleton cannot play a dominant role in determining the order and fluidity of the lipid bilayer. Discontinuities are observed in the temperature dependence of various spectral parameters, the order parameter S, the rotational correlation time tau, and 2,2,6,6-tetramethylpiperidinyloxy partitioning. They are assigned to phase transitions and/or phase separations of the membrane lipids. These discontinuities occur at about 30, 20, and 13 degrees C for 5-doxyl-, 12-doxyl-, and 16-doxylstearic acid, respectively. The apparent transition temperature depends on the location of the spin probe along the bilayer normal, being higher the closer the probe is to the membrane surface. This indicates the possibility that chain melting is progressive and spreads with increasing temperature from the center of the membrane outward.     

Molecular characteristics of small intestinal and renal brush border thiamin transporters in rats

The molecular characteristics of thiamin (T) transport were studied in the small intestinal and renal brush border membrane vesicles of rats, using [³H]T at high specific activity. The effects of various chemical modifiers (amino acid blockers) on T uptake were examined and their specificity assessed. Treatment with the carboxylic specific blockers 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, (1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride and N-ethyl-5-phenylisoaxolium-3′-sulfonate (Woodward’s Reagent K) and with the sulfhydryl specific blocker p-chloromercuribenzene sulfonate inhibited T transport in both types of vesicles. Phenylglyoxal, but not ninhydrin, both reagents for arginine residues, and diethylpyrocarbonate, a reagent for histidine residues, specifically decreased T transport only in renal and small intestinal vesicles respectively. Similarly 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacted, but not N-acetylimidazole, both of which are reagents for tyrosine residues. However, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibition was aspecific. Acetylsalicylic acid, a reagent for lysine and serine residues, decreased T transport, but the lysine effect was aspecific. Acetylsalicylic acid serine blockage also eliminated T/H⁺ exchange in small intestinal vesicles. Taken together, these results suggest that for T transport carboxylic and sulfhydryl groups and serine residues are essential in both renal and small intestinal brush border membrane vesicles. In addition, arginine and histidine residues are also essential respectively for renal and small intestinal transporters. Serine was essential for the T/H⁺ antiport mechanism.     

Use of percollTM in the isolation and purification of rabbit small intestinal brush border membranes

(1) Intestinal absorption is altered under a variety of circumstances in health and disease and to determine a possible relationship between intestinal absorptive function and intestinal brush border membrane composition, we undertook the isolation and purification of rabbit jejunal and ileal brush borders, to allow further studies of their lipid composition under varied experimental conditions. (2) A modification of an established method (Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B.K., Cerda, J.J. and Crane, R.K. (1973) Biochim. Biophys. Acta 323, 98-112) utilized CaCl2 aggregation and sequential centrifugation followed by purification of the brush border pellet (P2) at 27,000 X g on a PercollTM (Pharmacia) self-forming gradient. The PercollTM was removed by ultracentrifugation for 30 min at 100 000 X g, utilizing a batch rotor in the Beckman airfugeTM. (3) Pure brush border membrane vesicles were obtained and characterized by specific marker analysis and electron microscopy. Comparative marker analyses performed on P2 and final PercollTM preparations from animals showed that the purification achieved was 8-11-fold greater when compared to the original homogenates. Verification of purity was also demonstrated by the absence of DNA and very low levels of Beta-gluconridase and (Na+ + K+)-ATPase in the PercollTM preparations. (4) Comparative lipid analyses of P2 and final PercollTM preparations showed that levels of total phospholipid and free fatty acids were several-fold higher in the PercollTM preparations on a per mg protein basis. (5) A comparison of the activity of enzyme markers and the levels of total free fatty acids in P2 pellets obtained after Cacl2 and MgCl2 aggregation showed that CaCl2 aggregation gave the more consistently reproducible results. (6) Although standard procedures of membrane preparations not involving density gradient separation provide membranes of reasonable purity for the estimation of lipid components, we consider the final purification step of density gradient separation using PercollTM is essential for determining small quantitative changes which might occur in the membrane lipid composition under experimental conditions were intestinal absorptive function is altered.     

The uptake of phosphatidylcholine by small intestinal brush border membrane is protein-mediated

Brush border membrane vesicles prepared from rabbit small intestine are essentially free of basolateral membranes and nuclear, mitochondrial, microsomal and cytosolic contaminants. The resulting brush border membrane is unstable due to intrinsic lipases and proteinases. The PC transfer between small unilamellar lipid vesicles or mixed lipid micelles as the donor and the brush border membrane vesicles as the acceptor is protein-mediated. After proteolytic treatment of brush border membrane with papain or proteinase K the PC transfer activity is lost and the kinetics of PC uptake are similar to those measured with erythrocytes under comparable conditions. Evidence is presented to show that the PC transfer activity resides in the apical membrane of the enterocyte and not in the basolateral part of the plasma membrane. Furthermore, the activity is localized on the external surface of the brush border membrane exposed to the aqueous medium with its active centre probably not in direct contact with the lipid bilayer of the membrane. Proteins released from brush border membrane by proteolytic treatment catalyze PC exchange between different populations of small unilamellar vesicles. Furthermore, these protein(s) bind(s) PC forming a PC-protein complex.     

Spontaneous cholesterol movement between lipid vesicles and monkey small intestinal brush border membrane

[14C]Cholesterol movement between egg phosphatidylcholine-cholesterol lipid vesicles and vesicles prepared from monkey small intestinal brush border membrane (BBMV) was studied in physiological buffer at 37 degrees C. The rate of cholesterol transfer from sonicated unilamellar vesicles (ULV) to BBMV follows apparently first-order kinetics. Intermembrane cholesterol movement was strikingly similar in both the directions. However, from BBMV to ULV, the transfer rate was three times faster than that of ULV to brush border membrane (BBM). Similarity in the rate constant was observed when cholesterol transfer was studied using either large multilamellar lipid vesicles or ULV as the donor and BBMV as the acceptor membrane. Rate constant was also the same when the acceptor membrane used was either intact BBMV or ULV prepared from BBM lipids. The rate of transfer of label was not affected even when the acceptor vesicle concentration was increased over fivefold, indicating the first-order nature of the reaction. Transfer of cholesterol from ULV to BBMV was accelerated by the presence of acetone, dimethyl sulfoxide (DMSO), deoxycholate, and papain. Partially purified nonspecific lipid-exchange protein increased the rate of cholesterol transfer by about threefold. Reduction in BBM cholesterol and phospholipid content was noted by DMSO, acetone, and deoxycholate, while papain caused a small depletion of membrane protein. Cholesterol transfer is temperature dependent with an activation energy of 31 kJ X mol-1, which is almost identical in the presence or absence of nonspecific lipid-exchange protein. The molecular mechanism of intermembrane cholesterol movement is discussed in view of the kinetic data obtained.     

Molecular characteristics of small intestinal and renal brush border thiamin transporters in rats.

The molecular characteristics of thiamin (T) transport were studied in the small intestinal and renal brush border membrane vesicles of rats, using [(3)H]T at high specific activity. The effects of various chemical modifiers (amino acid blockers) on T uptake were examined and their specificity assessed. Treatment with the carboxylic specific blockers 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, (1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride and N-ethyl-5-phenylisoaxolium-3'-sulfonate (Woodward's Reagent K) and with the sulfhydryl specific blocker p-chloromercuribenzene sulfonate inhibited T transport in both types of vesicles. Phenylglyoxal, but not ninhydrin, both reagents for arginine residues, and diethylpyrocarbonate, a reagent for histidine residues, specifically decreased T transport only in renal and small intestinal vesicles respectively. Similarly 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacted, but not N-acetylimidazole, both of which are reagents for tyrosine residues. However, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibition was aspecific. Acetylsalicylic acid, a reagent for lysine and serine residues, decreased T transport, but the lysine effect was aspecific. Acetylsalicylic acid serine blockage also eliminated T/H(+) exchange in small intestinal vesicles. Taken together, these results suggest that for T transport carboxylic and sulfhydryl groups and serine residues are essential in both renal and small intestinal brush border membrane vesicles. In addition, arginine and histidine residues are also essential respectively for renal and small intestinal transporters. Serine was essential for the T/H(+) antiport mechanism.     

Effect of glucose and insulin on small intestinal brush border enzymes in fasted rats

Fasting reduced small intestinal length. It also decreased mucosal weight, DNA and protein content, and concentrations of enterokinase, maltase, and sucrase in both duodenal and jejunal segments. In contrast, the concentrations of lactase and leucine aminopeptidase were not affected. Concomitantly, serum insulin levels dropped to one-fifth of the control levels while serum glucose concentrations showed a lesser degree of reduction. Glucose supplementation alone raised the serum insulin level, prevented the decrease in DNA content, and showed a protective effect on mucosal protein, mucosal weight, mucosal thickness, and villus height. Glucose also protected the sucrase and maltase concentrations; more significantly for maltase in the jejunal segment. Insulin alone, although it increased the serum insulin level to that found with glucose supplementation alone, had no protective effect on the loss in protein, DNA, and most enzymes except for maltase concentration in the jejunal segment. Addition of insulin to glucose did not modify the glucose effect on the contents of DNA, protein, and concentrations of sucrase and maltase. These results suggest that the glucose effect on the mucosa is not mediated by insulin. In addition, the retention of both maltase and sucrase activities through only glucose supplementation suggests the loss of maltase and sucrase in fasting is due to nutrient rather than specific substrate restriction.