Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells.

A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and border preparations from the newborn are enriched in known marker enzymes. Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveals no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure.     

gamma-Glutamyltranspeptidase activity in newborn rat kidney brush border.

gamma-Glutamyltranspeptidase, known to be localized in the proximal tubule cell brush border in the rat, is a membrane-bound enzyme which transfers the gamma-glutamyl moiety of glutathione or its analogue gamma-glutamyl-p-nitroanilide to an amino acid or dipeptide acceptor. Brush borders were isolated from the kidneys of newborn and adult Sprague-Dawley rats and assayed for gamma-glutamyltranspeptidase activity. There is an increase in specific activity in the brush border with maturation. Newborn and adult brush border preparations exhibit similar pH optima, substrate affinities, apparent Km values, patterns of heat inactivation, inhibition by glutathione, and migration on polyacrylamide gels. Polyacrylamide gel electrophoresis of a deoxycholate extract of brush border proteins and subsequent reaction with substrate within the gel reveal the presence of two bands, suggesting the presence of two forms of gamma-glutamyltranspeptidase in the rat kidney brush border.     

Cytoskeletal protein and mRNA accumulation during brush border formation in adult chicken enterocytes

We have explored the development of the brush border in adult chicken enterocytes by analyzing the cytoskeletal protein and mRNA levels as enterocytes arise from crypt stem cells and differentiate as they move toward the villus. At the base of the crypt, a small population of cells contain a rudimentary terminal web and a few short microvilli with long rootlets. These microvilli appear to arise from bundles of actin filaments which nucleate on the plasma membrane. The microvilli apparently elongate via the addition of membrane supplied by vesicles that fuse with the microvillus and extend the membrane around the actin core. Actin, villin, myosin, tropomyosin and spectrin, but not myosin I (previously called 110 kD; see Mooseker and Coleman, J. Cell Biol. 108, 2395-2400, 1989) are already concentrated in the luminal cytoplasm of crypt cells, as seen by immunofluorescence. Using quantitative densitometry of cDNA-hybridized RNA blots from cells isolated from crypts, villus middle (mid), or villus tip (tip), we found a 2- to 3-fold increase in villin, calmodulin and tropomyosin steady-state mRNA levels; an increase parallel to morphological brush border development. Actin, spectrin and myosin mRNA levels did not change significantly. ELISA of total crypt, mid and tip cell lysates show that there are no significant changes in actin, myosin, spectrin, tropomyosin, myosin I, villin or alpha-actinin protein levels as the brush border develops. The G-/F-actin ratio also did not change with brush border assembly. We conclude that, although the brush border is not fully assembled in immature enterocytes, the major cytoskeletal proteins are present in their full concentration and already localized within the apical cytoplasm. Therefore brush border formation may involve reorganization of a pool of existing cytoskeletal proteins mediated by the expression or regulation of an unidentified key protein(s).     

Villin as a structural marker to study the assembly of the brush border

Summary Brush borders which are localized at the apical face of enterocytes, are composed of thousands of stiff microvilli containing bundles of microfilaments made of actin. Their assembly occurs during terminal differentiation of the enterocytes when these cells migrate along the villus of the intestinal mucosa. The cell line HT 29 derived from a human colonic adenocarcinoma whose differentiation can be induced, can also be used as a model to study in culture the assembly of the intestinal brush border.Villin is one of the actin binding proteins found in microvilli which compose brush borders. Villin is expressed in the adult and in the embryo before the appearance of the brush border. Villin can be used as a tissue-specific marker for normal diffentiated and undifferentiated cells derived from gastrointestinal tractus in the adult as well as in the embryo. Since villin is a good marker for intestinal cells and plays a structural role in the assembly of the brush border we have analysed its expression and its localization in HT 29 cells. In HT 29 cells, as in the tissue, villin is synthesized at low levels before the appearance of the brush border. The high rate of synthesis and the recruitement of villin at the apical pole of the cells can be correlated with the existence of a well developed brush border.     

Studies on the brush border membrane of the mouse duodenum

Summary Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.     

Studies on the brush border membrane of the mouse duodenum. I. Membrane isolation and analysis of protein components.

Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.     

The aging gut

A spectrum of changes occurs in the function of the gastrointestinal tract throughout the life of the animal. With aging, there is a decline towards newborn levels of the villus surface area and brush border membrane markers, but the absorption of some nutrients continues to increase (such as glucose and vitamin), whereas the absorption of some nutrients falls (such as cholesterol) and fatty acids). Clearly there is a distinction between age-related alterations in intestinal form and function, and some of the enzyme- or carrier-mediated changes are substrate specific. With aging, the structure and brush border membrane composition of the intestine tends to regress towards newborn levels. It remains to be clarified whether these changes are a benefit to the animal, whether they represent a degeneration of normal function, and whether these changes are the cause or the effect of various age-related alterations in metabolic and physiological function.     

Lipid composition of plasma membranes of jejunum enterocytes in cattle of different age

The lipid composition of plasma membranes (a brush margin and basolateral membranes) of enterocytes from the jejunum of cattle: adult animals (aged 1.5-3) and newborns (3-5 days) both healthy and suffering from diarrhea has been studied. Substantial differences are established in the content of phospholipids, cholesterol and fatty acids in the brush margin and basolateral membranes as well as in the both membrane fractions of the adult, newborn and sick animals.     

Subcellular localization of (Na+ + K+)-activated ATPase in the brush border membrane of the mucosal cell of the rat small intestine.

The localization of (Na+ + K+)-activated ATPase was investigated in isolated brush borders of rat small intestinal mucosa. The purity of the fractions has been checked by morphological and enzymatic criteria. The brush borders were found to contain a significant quantity of (Na+ + K+)-activated ATPase. Separation of isolated brush borders into their substructures suggests that (Na+ + K+)-activated ATPase is localized deeper within the brush border region than invertase. These findings are discussed in relation to active monosaccharide transport in the intestine.     

Characterization of the interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn calves

Enterotoxigenic Escherichia coli (ETEC) induces severe diarrhea in newborn calves through the elaboration of heat-stable enterotoxin (STa). We investigated the distribution and characteristics of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from anterior jejunum, posterior jejunum, ileum and colon of newborn calves. We found that density of the receptors and their affinity to STa were higher on enterocytes and BBMVs that were derived from the ileum than enterocytes and BBMVs prepared from other segments of the calf intestine. This study suggests that, in newborn calves, the ileum is the major part of the intestinal tract that is affected in the course of secretory diarrhea caused by STa-producing ETEC strains.     

Environmental cues and symbiont microbe‐associated molecular patterns function in concert to drive the daily remodelling of the crypt‐cell brush border of the Euprymna scolopes light organ

Recent research has shown that the microbiota affects the biology of associated host epithelial tissues, including their circadian rhythms, although few data are available on how such influences shape the microarchitecture of the brush border. The squid‐vibrio system exhibits two modifications of the brush border that supports the symbionts: effacement and repolarization. Together these occur on a daily rhythm in adult animals, at the dawn expulsion of symbionts into the environment, and symbiont colonization of the juvenile host induces an increase in microvillar density. Here we sought to define how these processes are related and the roles of both symbiont colonization and environmental cues. Ultrastructural analyses showed that the juvenile‐organ brush borders also efface concomitantly with daily dawn‐cued expulsion of symbionts. Manipulation of the environmental light cue and juvenile symbiotic state demonstrated that this behaviour requires the light cue, but not colonization. In contrast, symbionts were required for the observed increase in microvillar density that accompanies post dawn brush‐border repolarization; this increase was induced solely by host exposure to phosphorylated lipid A of symbiont cells. These data demonstrate that a partnering of environmental and symbiont cues shapes the brush border and that microbe‐associated molecular patterns play a role in the regulation of brush‐border microarchitecture.     

Comparison of brush border membrane glycoproteins and glycoenzymes in the proximal and distal rat small intestine

Brush border membranes isolated from the proximal and distal portions of the rat small intestine were examined to see whether qualitative differences exist in their glycoprotein constituents. After SDS-polyacrylamide gel electrophoresis distinct differences were observed, indicating that the protein and glycoprotein profiles of the distal intestine are less complex. A competitive radioassay of lectin receptors revealed that there are significantly more wheat germ agglutinin and succinylated wheat germ agglutinin receptors present on brush border membranes from proximal intestine as compared to distal intestine. However, binding of Ricinus communis agglutinin I to brush border membranes of distal intestine was 2-times higher than that of proximal intestine. These segmental differences were also reflected in the binding patterns of individual brush border membrane hydrolases to wheat germ agglutinin and R. communis agglutinin I. Carbohydrate analysis demonstrated that the overall sugar content of brush border membranes is higher in distal intestine, with more galactose and sialic acid residues. No difference was found in the content of N-acetylglucosamine between the two segments. When brush border membranes from both segments were used as acceptors for galactosyltransferase, those from proximal intestine were better acceptors. Neuraminidase treatment significantly enhanced galactose oxidase/sodium borotritide labeling of brush border membranes from distal intestine and altered the electrophoretic mobility of dipeptidyl aminopeptidase IV and aminopeptidase N. No significant changes in labeling or enzyme electrophoretic mobility were noted in brush border membranes from proximal intestine after neuraminidase treatment. These studies indicate that the glycoproteins from brush border membranes of proximal and distal intestine are qualitatively different and that the glycoproteins from distal intestine may have more completed oligosaccharide side chains.     

Interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn kids

The elaboration of heat stable enterotoxin (STa) is an important step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC), which causes severe diarrhea in newborn animals. In this study, the distribution of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from the anterior jejunum, posterior jejunum, ileum and colon of newborn kids was investigated. The density of STa-receptors on enterocytes and BBMVs was higher in the posterior jejunum than that in other segments of the kids' intestines. Additionally, the affinity of the posterior jejunum STa-receptors was higher than the affinity of receptors present on the epithelium of other intestinal segments. Our findings suggest that the posterior jejunum is a major target for STa within the intestinal tract of newborn kids.     

Identification and characterization of two huge protein components of the brush border cytoskeleton: evidence for a cellular isoform of titin

Two extremely high molecular weight proteins were found to be components of the intestinal epithelial cell brush border cytoskeleton. The largest brush border protein, designated T-protein, migrated on SDS gels as a doublet of polypeptides with molecular weights similar to muscle titin T I and T II. The other large brush border protein, designated N-protein, was found to have a polypeptide molecular weight similar to muscle nebulin. In Western analysis, a polyclonal antibody raised against brush border T-protein reacted specifically with T-protein in isolated brush borders and cross-reacted with titin in pectoralis and cardiac muscle samples. T-protein was distinguished from the muscle titins by an anti-cardiac titin mAb. A polyclonal antibody raised against N-protein was specific for N-protein in brush borders and cross-reacted with nothing in pectoralis muscle. Immunolocalization in cryosections of intestinal epithelia and SDS-PAGE analysis of fractionated brush borders revealed that both T-protein and N-protein are concentrated distinctly in the brush border terminal web region subjacent to the microvilli, but absent from the microvilli. EM of rotary-replicated T-protein samples revealed many of the molecules to be long (912 +/- 40 nm) and fibrous with a globular head on one end. In some of the molecules, the head domain appeared to be extended in a fibrous conformation yielding T-protein up to 1,700-nm long. The brush border N-protein was found as long polymers with a repeating structural unit of approximately 450 nm. Our findings indicate that brush border T-protein is a cellular isoform of titin and suggest that both T-protein and N-protein play structural roles in the brush border terminal web.     

Enterocytic differentiation and glucose utilization in the human colon tumor cell line Caco-2: modulation by forskolin

The human colon cancer line Caco-2 exhibits after confluency a concomitant increase of glycogen accumulation and an enterocytic differentiation. The purpose of this work was to investigate whether forskolin (FK), an activator of adenylate cyclase, would induce a permanent glycogenolysis and, if so, whether it would result in modifications of the differentiation pattern of the cells. FK activates adenylate cyclase in Caco-2 cells with an ED50 of 7 X 10(-6)M. Three different treatment protocols with FK (10(-5)M) were applied: 1) the cells were treated during all the time in culture (20 days); 2) the treatment was started after confluency; 3) the treatment was interrupted after confluency. The presence of FK results in a permanent stimulation of cAMP accumulation (10 to 20 fold the basal values) and in a permanently reduced glycogen content (30 or 50% of the control values). The rates of glucose consumption are increased three and five fold in protocols 1 and 3 respectively. These metabolic changes are associated with morphological changes (tightening of the intercellular spaces and shortening of the brush border microvilli) and with a dual inhibition of the activities of brush border hydrolases: a) an inhibition of the post-confluent increase of activity of sucrase, aminopeptidase N and alkaline phosphatase in the brush border enriched fraction; b) an inhibition of the post-confluent increase of activity of sucrase in the cell homogenate. A comparison of the results obtained in each protocol shows that the morphological modifications and the decrease of the enzyme activities in the brush border fraction are regularly associated with an increased cAMP accumulation, whereas the inhibition of the differentiation of sucrase is a direct consequence of the increase in glucose consumption and decrease in glycogen stores.     

Stimulation of lactase synthesis induced by starvation in the jejunum of adult rat

The effects of actinomycin D and of cycloheximide administration have been investigated on the enzyme activities of the jejunal brush border membrane in adult rats after a 48-hour period of starvation. The modifications in the protein and enzyme patterns of the brush border membrane and the incorporation of radiolabelled amino acid in the protein band corresponding to lactase have been studied in the nourished and in the starved animal. The results show that actinomycin D administration did not modify the stimulation of lactase activity caused by starvation whereas cycloheximide completely inhibited this process. The stimulation of lactase activity, in the starved animal, is related to a quantitative increase of the corresponding protein band and with enhanced incorporation of L-[3H]valine in this protein band after separation of brush border proteins by gel electrophoresis. It is concluded that the stimulation of lactase activity observed during starvation is the consequence of de novo synthesis of lactase molecules and that this process is regulated at a translational level. A general hypothesis is proposed in order to clear up partly the mechanism involved in the stimulation of lactase activity by food deprivation in the adult rat.     

Activity of calcium/phospholipid dependent protein kinase during rat kidney development

The activity of the calcium sensitive phospholipid dependent protein kinase C (PKC) was studied in cytosol and in the proximal tubular luminal membrane of rats during growth. Cytosolic activity was elevated at 14 and 21 days of age and fell to adult levels by day 60. Luminal brush border membrane activity on the other hand was low on day 14 but reached adult levels by day 21. Changes in brush border membrane PKC activity may have important consequences for the development of electrolyte transport in proximal tubular cells.     

Ca++-calmodulin-dependent phosphorylation of myosin, and its role in brush border contraction in vitro

We have reinvestigated the effects of Ca++ and ATP on brush borders isolated from intestinal epithelial cells. At 37 degrees C, Ca++ (1 microM) and ATP cause a dramatic contraction of brush border terminal webs, not a retraction of microvilli as previously reported (M. S. Mooseker, 1976, J. Cell Biol. 71:417-433). Terminal web contraction, which occurs over the course of 1-5 min at 37 degrees C, actively constricts brush borders at the level of their zonula adherens. Contraction requires ATP, is stimulated by Ca++ (1 microM), and occurs in both membrane-intact and demembranated brush borders. Ca++ - dependent-solation of microvillus cores requires a concentration of Ca++ slightly greater (10 microM) than that required for contraction. Under conditions in which brush borders contract, many proteins in the isolated brush borders become phosphorylated. However, the phosphorylation of only one of the brush border proteins, the 20,000 dalton (20-kdalton) light chain of brush border myosin (BBMLC20), is stimulated by Ca++. At 37 degrees C, BBMLC20 phosphorylation correlates directly with brush border contraction. Furthermore, both BBMLC20 phosphorylation and brush border contraction are inhibited by trifluoperazine, an anti-psychotic phenothiazine that inhibits calmodulin activity. These results indicate that Ca++ regulates brush border contractility in vitro by stimulating cytoskeleton-associated, Ca++- and calmodulin-dependent brush border myosin light chain kinase.     

Role of development in the evolution of the scapula of the giant sthenurine kangaroos (Macropodidae: Sthenurinae)

Extinct giant sthenurine kangaroos possessed scapulae morphologically distinct from those of all other extant and extinct adult macropodids, but qualitatively resembling those of newborn macropodids. The similarity between adult sthenurine and neonatal macropodid scapulae suggests that a developmental process, such as heterochrony, might have been behind the evolution of the unique sthenurine scapular morphology. By incorporating adult and ontogenetic data, this study examines the evolution and development of the sthenurine scapula. This study quantitatively upholds the previous qualitative morphological observations of macropodid scapulae and finds that ontogenetic and evolutionary morphological changes are correlated in macropodids. The similarity of scapula morphology in sthenurines and newborn macropodids, the correlation between ontogenetic and evolutionary morphological change, and information from other sources (i.e., sthenurine evolutionary history) suggests that pedomorphic shifts in morphology, most likely due to neotenic processes, occurred within the development of the scapula of giant sthenurines.