Resection of rabbit ileum: effect on brush border membrane enzyme markers and lipids

Alterations in transport function have been described 6 weeks after surgical resection of 50% of the distal small intestine. Previous studies demonstrated a modest increase in the jejunal uptake of medium chain length fatty acids following resection, while the uptake of many other lipids (cholesterol, bile acids, fatty alcohols, short and long chain length fatty acids) appears to be unaffected. Marked changes in the kinetic constants for the carrier-mediated uptake of four sugars and leucine were observed following resection, but the changes in transport were not associated with changes in the mucosal surface area. This study was undertaken to examine the possible adaptive mechanisms that occur with ileal resection in the rabbit. A 29% increase in the wet weight of jejunal mucosal scrapings and a 53% increase in jejunal brush border membrane (BBM) protein was observed following resection. The jejunal BBM sucrase (S) was unchanged following ileal resection, but alkaline phosphatase (AP) total activities were increased in the resected rabbits. This resulted in a 45% increase in the ratio of AP/S with resection. The lipid composition (total free fatty acids, total bile acids, total cholesterol, total phospholipids, individual phospholipids, and the ratio of total phospholipids/total cholesterol) of BBM was similar in control and resected rabbits. This suggests that quantitative rather than qualitative changes in the membrane composition may be responsible for the transport changes observed in resected animals.     

Taurocholate uptake by membrane vesicles prepared from ileal brush borders

Taurocholate uptake by vesicles prepared from brush borders obtained from the small intestines of guinea pigs was studied. Vesicles obtained from the brush borders of ileums demonstrated an enhanced initial uptake in those incubations where a sodium ion gradient (extravesicular sodium concentration greater than intravesicular) was present at the outset. With the dissipation of this sodium gradient the intravesicular concentration of taurocholate declined. This overshoot phenomenon was absent in parallel incubations of vesicles made from jejunal tissue. When the sodium chloride was replaced by isosmotic amounts of mannitol no overshoot was observed in incubations of ileal vesicles until subsequent addition of sodium chloride to these incubations. These observations are in accord with the idea that those subcellular structural elements operating in the ileal bile salt transport system are associated with the brush border membranes of the ileal mucosal cells.     

Histochemistry of small intestinal dysplasia in familial polyposis coli

Biopsies of duodenal and ileal mucosa from patients with familial polyposis coli were studied. Areas of atypia were identified in the duodenum of six patients and in the ileum of three patients. Grade I atypia was characterized by crowding and elongation of cells and nuclei, a slight reduction in the number of goblet cells and the presence of a brush border; grade II atypia was further characterized by pseudo- or pluristratification of cells, a marked reduction in the number of goblet cells and the absence of a brush border. In areas of atypia, columnar cells often contained PAS-positive apical granules, which were diastase-resistant and unstained by alcian blue at any pH; the brush border, even where recognizable in haematoxylin-eosin and PAS-stained sections, was unreactive histochemically for alkaline phosphatase. Goblet cells were few in areas of atypia, but those present were regularly stained by PAS and alcian blue pH 2.6. Apical granules, similar in their histochemical characteristics to those observed in columnar cells in areas of atypia, were also found in otherwise normal mucosal areas, even in some patients with no overt areas of atypia in the biopsies studied. These granules have been interpreted as an abnormality, possibly preceding the onset of atypia. Hyperplasia of goblet cells, secreting mucins with the same staining pattern as in normal intestine, was found in some patients, either adjacent to areas of atypia or independent of them. Intervening columnar cells had a normal morphology, alkaline phosphatase-reactive brush borders and no sign of mucus secretion. This goblet cell hyperplasia has been interpreted as a reactive, nonspecific alteration of the mucosa.     

Role of the intestinal brush border in the absorption of cholesterol in rats

1. Short-term incubation of the everted intestinal sacs of rats in media containing cholesterol oleate or cholesterol plus oleic acid resulted in rapid hydrolysis, but no synthesis, of the sterol ester. 2. On separation of the brush border from the rest of the mucosal cell, almost all of the hydrolytic activity and appreciable amounts of the synthetic activity of the whole cell were found to be present in the brush-border fraction. 3. The isolated brush-border fraction contained considerable amounts of cholesterol, which was always present in the unesterified state; the rest of the cell contained about an equal amount of unesterified cholesterol, but, in addition, small but definite amounts of the esterified sterol were also found in this fraction. 4. On feeding rats with [4-(14)C]cholesterol, which was diluted with 3mg. of cholesterol, it was found that the brush border very rapidly took up the fed sterol without changing its net content of cholesterol. No traces of radioactive cholesterol ester could ever be detected in the isolated brush border after feeding with (14)C-labelled esterified or unesterified cholesterol. 5. The appearance of the labelled sterol was quite rapid in the rest of the cell also, where small proportions were found in the esterified state. 6. Therefore the sequence of events in the absorption of cholesterol appears to be: the dietary cholesterol esters are hydrolysed by the cholesterol ester hydrolase of pancreas or of the mucosal brush border or both, after which the brush border rapidly absorbs the de-esterified sterol and transfers it into the mucosal cell, by a mechanism of displacement, where it is slowly re-esterified for transport through the lymph.     

Effect of external abdominal irradiation on intestinal morphology and brush border membrane enzyme and lipid composition

Previous studies have shown that external abdominal irradiation is associated with alterations in intestinal morphology and function. The activity of the jejunal brush border membrane (BBM) enzyme markers sucrase (S) and alkaline phosphate (AP) were not altered by 600 rad irradiation in the rat. In contrast, ileal BBM, AP, and AP/S were increased 3, 7/8, and 28 days postirradiation. The total lipid composition of the jejunal BBM was lower than in control animals only at 3 days postirradiation; this was due to a decrease in the total free fatty acid content. In addition to a lower total free fatty acid content, the ileal BBM contained an increased amount of total phospholipid (PL) which resulted in an increased phospholipid/cholesterol ratio at 3 days following irradiation. Variations in the BBM phospholipid composition occurred in both jejunum and ileum. In the jejunal BBM, the phospholipid composition changes did not alter the choline or amine phospholipid content; therefore, the choline/amine phospholipid ratio was unaffected by irradiation at 600 rad. In the ileal BBM, the phosphatidyl ethanolamine was increased at 3, 7/8, 14, and 28 days following irradiation. The choline/amine phospholipid ratio was not altered in the ileal BBM due to concomitant increases in lecithin content. Jejunal villus height, villus surface area, and the number of cells per villus were decreased at 3 days postirradiation, but increased by day 7/8 and 14 postirradiation to levels much higher than observed in control jejunal villi. The mucosal surface area was decreased at 3 and 7/8 days following irradiation but returned to control values by Day 14. Jejunal microvillus morphology was unaffected by irradiation. Few significant changes were observed in ileal villus morphology following irradiation at 600 rad. Ileal villus height, villus surface area, and mucosal surface area did not change, but the number of cells per villus initially decreased at 3 days and then increased beyond control values at 7/8 and 14 days postirradiation. Ileal microvillus height was significantly decreased only at 7 days postirradiation, while the number of microvilli per micron was increased only at 3 days postirradiation. This study suggests that changes in intestinal morphology and brush border composition may contribute to the altered passive permeation toward lipids which has been reported following abdominal radiation.     

Effect of covalent modification on the binding of cholera toxin B subunit to ileal brush border surfaces.

A competitive binding assay has been developed to determine how modifications to the B subunit of cholera toxin affect the binding affinity of the subunit for an ileal brush border membrane surface. The Ricinus communis120 agglutinin (RCA120) specifically binds to terminal beta-D-galactosyl residues such as those found in oligosaccharide side chains of glycoproteins and ganglioside GM1. Conditions were designed to produce binding competition between the B subunit of cholera toxin and the RCA120 agglutinin. Displacement of RCA120 from brush border surfaces was proportional to the concentration of B subunit added. This assay was used to study the effect of modification of B subunit on competitive binding affinity for the ileal brush border surface. The B subunit of cholera toxin was modified by coupling an average of five sulfhydryl groups to each B subunit molecule and by reaction of the SH-modified B subunit with liposomes containing a surface maleimide group attached to phosphatidylethanolamine. SH-modified B subunit was approximately 200-fold more effective than native B subunit in displacing lectin from brush border surfaces in the competitive binding assay. The enhanced binding activity was retained on covalent attachment of the modified B subunit to the liposome surface. We conclude that the B subunit of cholera toxin may be a useful targeting agent for directing liposomes to cell surfaces that contain a ganglioside GM1 ligand.     

Mucosal surface ferricyanide reductase activity in mouse duodenum

Mouse duodenum possesses mucosal surface ferricyanide reductase activity. The reducing activity, determined in vitro by measuring ferrocyanide production from ferricyanide, was found to be greater in duodenal fragments when compared with ileal fragments. Experiments with right-side out tied-off duodenal sacs show that reduction occurs mainly on the mucosal side and indicates that the reducing activity is associated with the brush border membrane. Experiments using mice with increased levels of iron absorption (hypoxic, iron-deficient) showed corresponding increases in reducing activity. The increase was present in duodenal but not ileal fragments. Inhibitor studies showed no effect of several compounds which inhibit other, more characterized, transplasma membrane reductases. In particular, doxorubicin (10 m) and quinacrine (1 mm) were without effect on duodenal mucosal transplasma membrane reducing activity. Depolarization of the membrane potential with high medium K ⁺ inhibited reducing activity. N-ethyl malemide (1 mm) was a potent inhibitor, but iodoacetate was found to be less inhibitory. Comparision with inhibitory effects on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) demonstrated that the effect of N-ethyl malemide on reducing activity was not secondary to GAPDH. Collectively these results indicate that mouse duodenum possesses mucosal surface transplasma membrane ferricyanide reductase activity and that the activity is correlated with the process of intestinal iron absorption. Furthermore, the reducing activity appears to be distinct from other reported transplasma membrane reductases.     

Intestinal lactase (beta-galactosidase) and other glycosidase activities in suckling and adult tammar wallabies (Macropus eugenii)

The activities of various glycosidases in homogenates of the small intestinal mucosa of two adult and 18 suckling tammar wallabies (M. eugenii) aged from 6 to 50 weeks were investigated. Lactase (beta-D-galactosidase), beta-N-acetylglucosaminidase, alpha-L-fucosidase and neuraminidase activities were high during the first 34 weeks post partum and then declined to very low levels. Maltase, isomaltase, sucrase and trehalase activities were very low or absent during the first 34 weeks, and then increased. The lactase activity was unusual in being greater in the distal than the middle or proximal thirds of the intestine, and in its low pH optimum (pH 4.6), inhibition by p-chloromercuribenzene sulfonate but not by Tris, and lack of cellobiase activity. These properties are those of a lysosomal acid beta-galactosidase rather than of a brush border neutral lactase. The maltase activity had the characteristics of a lysosomal acid alpha-glucosidase early in lactation and of a brush border neutral maltase in adult animals. The significance of these findings is discussed in relation to changes in dietary carbohydrates during weaning and to the mode of digestion of milk carbohydrates by the pouch young.     

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.     

Effect of massive proximal small bowel resection on intestinal brush border membrane proteins in the dog.

Dog enterocyte brush border proteins have been studied after a 75% proximal resection of the small bowel. This study was carried on microvillar membrane preparations purified from ileal mucosa sampled before and after regeneration on neighbouring intestinal segments, each animal acting as its own control. After six weeks of regeneration a statistically significant decrease of the following enzyme specific activities was observed: lactase, cellobiase, maltase, sucrase, palatinase, dextranase, trehalase, alkaline phosphatase, aminopeptidase and gamma-glutamyl transferase. Analysis of brush border proteins by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate have shown after regeneration a decreased rate for the proteins with a molecular weight higher than 100,000 daltons. Modifications of electrophoretic patterns seem to be related to the specific activity decreases observed for brush border enzymes after regeneration, since the molecular weight of these enzymes were found between 116,000 and 285,000 daltons, after gel filtration.     

3-Hydroxy-3-methylglutaryl-CoA reductase, mevalonate-5-pyrophosphate decarboxylase and acyl-CoA: cholesterol acyl-transferase from mucosa scrapings and isolated enterocytes from chick duodenum, jejunum and ileum

3-Hydroxy-3-methylglutaryl-CoA reductase, mevalonate-5-pyrophosphate decarboxylase and acyl-CoA: cholesterol acyltransferase activities were assayed in mucosal scrapings and isolated enterocytes from chick duodenum, jejunum and ileum. Maximal reductase and decarboxylase specific activities were found in ileum and jejunum, while ileum exhibited the minimal acyltransferase specific activity. The isolated epithelial cells showed levels of reductase and acyltransferase specific activities higher than those found in mucosa scrapings, probably due to the contact of these microsomal proteins with proteolytic enzymes during homogenization of the mucosa. However, no protecting effect of the trypsin inhibitor (2mg/ml) could be observed on reductase activity in mucosa scrapings. The cytosolic location of decarboxylase may account for the similar levels of specific activities found in mucosa scrapings and isolated enterocytes.     

Effect of weaning on the activities of glycogen synthase and phosphorylase in rat liver

The effects of weaning on the level of glycogen and the activities of glycogen synthase and phosphorylase were determined in rat liver. Glycogen levels in rat liver increased at the start of the weaning period and reached a plateau on postnatal day 20. The active form of glycogen synthase increased until postnatal day 19 and then declined. Total glycogen synthase (active + inactive) remained high during the suckling period and declined to a new low level during the weaning period. The activity ratio (active/total) increased from day 16 to days 18-22 and then decreased to the same level as found during the suckling period. At the onset of weaning the active form of phosphorylase decreased, whereas total phosphorylase initially increased and then decreased after postnatal day 20. Both forms of phosphorylase increased again at the end of the weaning period. The activity ratio decreased at the start of weaning and remained low throughout the rest of the weaning period. The effects of premature weaning were similar to those observed in normally weaned animals, but the changes occurred sooner and were more pronounced.     

Effect of pentagastrin, secretin and cholecystokinin on growth and differentiation in organ cultured rabbit small intestine

The effect of pentagastrin, secretin and cholecystokinin on biochemical parameters of mucosal growth and differentiation was studied in organ cultured rabbit jejunum and ileum. Pentagastrin at 0.05-5.0 microgram/ml did not affect DNA content of the biopsy, but led to a significant decrease of sucrase and alkaline phosphatase activity in the ileum. Secretin prompted a significant decrease of DNA and protein in the ileum at a level of 10(-7) and 10(-5) M, but had no effect in the jejunum. Of the brush border enzymes, sucrase and alkaline phosphatase were suppressed in both parts of the intestine both with respect to specific activity and total biopsy content. Cholecystokinin, like pentagastrin, did not influence DNA or protein content, but reduced sucrase, maltase and alkaline phosphatase activity. HMG-CoA reductase, the key enzyme of cholesterol synthesis, was not significantly affected by any of the three hormones tested. When brush border enzymes or DNA from desquamated cells were measured in the post-culture medium, no consistent effect of any gastrointestinal hormone was apparent. The present study demonstrates a direct "antitrophic" effect of secretin in cultured mucosa. Pentagastrin and cholecystokinin did not influence mucosal DNA content in vitro but apparently inhibited villus cell differentiation.     

Subcellular localization of enterokinase in human small intestine

Enterokinase (enteropeptidase, EC 3.4.4.8) was found to be purified to the same extent as sucrase and alkaline phophatase when human intestinal brush border membrane was isolated. It is concluded that, in man as in other mammals, enterokinase activity occurs in close association with the brush border membrane.However, a second localization was also found. A fraction of the mucosal homogenate containing only small amounts of brush border but large amounts of endoplasmic reticulum, basolateral membranes and mitochondria (Fraction P₁) contained a disproportionately high amount of enterokinase. The enzyme in this particulate fraction occurred in a not fully active form.     

Spectrum of effects of dietary long-chain fatty acids on rat intestinal glucose and lipid uptake

Isocaloric modification in the ratio of dietary polyunsaturated-to-saturated fatty acids influences intestinal uptake of actively and passively transported nutrients. This study was undertaken to determine which dietary fatty acid was responsible for these alterations in absorption. Adult female rats were fed isocaloric semisynthetic diets high in palmitic and stearic acids (SFA), oleic acid (OA), linoleic acid (LA), or linolenic acid (LNA). An in vitro technique was used to measure the uptake of varying concentrations of glucose as well as a series of fatty acids and cholesterol. Jejunal uptake of 40 mM glucose was highest in rats fed SFA and lowest in those fed LA; ileal glucose uptake was similar in OA, LA, and LNA, but was lowest in SFA. Jejunal uptake of medium-chain fatty acids (8:0-12:0) was higher in OA than in other diet groups; ileal uptake of medium-chain fatty acids was unaffected by diet. Jejunal and ileal uptake of 18:2 was higher in LNA than in SFA or OA; the uptake of the other long-chain saturated or unsaturated fatty acids was unchanged by diet. The ileal but not the jejunal uptake of cholesterol was increased in LA as compared with SFA or OA, and reduced in LNA as compared with LA. These transport changes were not explained by differences in the animals' food consumption, body weight gain, intestinal mass, or mucosal surface area. We postulate that these diet-induced transport alterations may be mediated via changes in brush border membrane phospholipid fatty acyl composition. Thus, intestinal transport of nutrients may be varied by isocaloric changes in the dietary content of individual fatty acids.     

Labelling of intestinal brush border membrane proteins in vivo using diazotised [125I]iodosulfanilic acid

Membrane proteins of the intestinal brush border were labelled in vivo by intraluminal injection of diazotised [¹²⁵I]iodosulfanilic acid, a highly polar molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of brush border membranes labelled in this manner showed 20 protein bands, 11 of which contained significant radioactivity. The most heavily labelled proteins had molecular weights greater than 150 000, indicating that they were the most exposed to the intestinal lumen. Little radioactivity was detected in proteins with molecular weights of less than 94 000. The majority of these smaller proteins were likely to have been brush border core proteins. The evidence that diazotised [¹²⁵I]iodosulfanilic acid bound primarily to brush border membrane proteins when administered in this way, was: (a) the specific activity of brush border proteins was up to 3-fold greater than that of total cell particulate proteins (pelleted at 27 000 × $\text{g}$ from mucosal homogenates); (b) principal peaks in the gel radioactivity profile of total cell particulate proteins corresponded to the most heavily labelled proteins of the isolated brush border membrane; and (c) brush border core proteins showed minimal radioactivity in vivo, but considerably higher radioactivity when brush border membranes were labelled in vitro. A small amount of label was absorbed across the intestinal mucosa. However, secondary labelling of brush border proteins by this absorbed label was minimal, since the specific activity of brush border proteins in jejunum adjacent to the labelled loop was only 0.22% of the level for those proteins in the labelled segment. Since this technique did not affect the cellular morphology, enzyme activity or biochemical integrity of the membrane, it should prove useful as a means of accurately studying in vivo turnover rates of brush border membrane proteins.     

Inhibitory effects of somatostatin on growth and differentiation in cultured intestinal mucosa

The effect of somatostatin on mucosal DNA, protein and brush border enzymes was studied in organ cultured rabbit jejunum and ileum. Compared to control cultures, somatostatin reduced the biopsy DNA and protein content in parallel in the jejunum, but was ineffective in the ileum. This was probably due to a direct growth inhibition, since DNA and brush border enzyme activity from desquamated cells in the postculture medium were unaffected. In addition, a direct inhibition of jejunal villous cell differentiation by somatostatin was reflected in a significant decrease of sucrase, maltase and alkaline phosphatase activity. In the ileum, only the specific activity of alkaline phosphatase was reduced. The key enzyme of cholesterol synthesis, HMG-CoA-reductase, was measured as an intracellular enzyme control and was not influenced by the hormone. The high somatostatin concentrations necessary to achieve the effects are not an artefact of hormone degradation during culture, as shown by radioimmunoassay, and suggest a local or "paracrine" rather than systemic, inhibitory action of somatostatin on intestinal growth and differentiation.     

Determinants of regional sucrase-isomaltase expression in adult rat small intestine

Sucrase-isomaltase (SI) expression along the longitudinal and vertical axis of the small intestine was studied by sequentially isolating enterocytes from villus to crypt of rat proximal jejunum and distal ileum. Gradients of sucrase activity were observed with greatest activity occurring in jejunal and villus regions. Along the villus-to-crypt axis, gradients of SI mRNA abundance corresponded with activity. However, along the longitudinal axis no differences in SI mRNA levels were observed, thus not accounting for the observed 3-5-fold difference in SI activities between jejunum and ileum. Comparison of SI immunoprecipitates from jejunal and ileal mucosal scrapings showed significant differences in gel mobilities of the more mature forms, which did not appear to affect SI functional activities. When relative rates of de novo SI protein synthesis were compared, [35S]methionine incorporation into all SI forms was observed to be 3-5-fold greater in jejunum than in ileum at all time points. Because these results suggested differences in regional translational regulation, subcellular distribution of SI mRNA in jejunal and ileal epithelial cells was compared. A greater proportion of jejunal SI mRNA was found to be associated with membrane-bound polyribosomes. We conclude 1) sucrase expression along the villus-to-crypt axis correlates with SI mRNA abundance, 2) post-translational processing of SI differ in ileum and jejunum, but appear not to determine SI expression, and 3) differences in translational processing in distal ileum and proximal jejunum may determine sucrase activity along the longitudinal axis of rat small intestine.     

The effect of jejunectomy on alkaline phosphatase activity in the ileum

The activity of alkaline phosphatase activity in the small intestinal wall was studied in dogs after jejunectomy. The observations were made 3, 6, 8 or 9 and 12 weeks after operation. The studies aimed to obtain some information about the functional adaptation processes of the remaining intestinal segments. The enzyme activity in homogenates of duodenal and ileal mucosa was determined. Parallel interferometric measurements in the brush border and on the surface of the absorptive cells were performed. The results obtained indicate that after temporary reduction (especially 6 weeks postoperatively) a gradual rise of the alkaline phosphatase activity both in homogenates and in the brush border of the intestinal remnants took place. Several times repeated biopsies confirmed the ability of the intestinal segments (duodenum and distal ileum) significant increase in enzyme activity over the normal (control) level was observed.