Brush Border Enzyme Activities Research Articles

Regulation of human Caco-2 intestinal epithelial brush border enzyme activity by cyclic nucleotides.

Regulation of intestinal epithelial differentiation is critical to normal function, malignant transformation, and healing. However, the intracellular regulation of intestinal epithelial differentiation is incompletely understood. We studied the effects of intracellular cyclic AMP and cyclic GMP on brush border enzyme activity in the human Caco-2 intestinal epithelial cell using pharmacologic agonists and antagonists of cAMP and cGMP mediated pathways as probes. The stable cyclic nucleotide analogs dibutyryl cAMP and dibutyryl cGMP selectively decreased Caco-2 dipeptidyl dipeptidase specific activity while increasing alkaline phosphatase. The inhibitors of adenylate and guanylate cyclase KT5720 and KT5823 each exerted the opposite effects. Combinations of dibutyryl cAMP and dibutyryl cGMP demonstrated synergistic effects on each brush border enzyme but KT5720 and KT5823 were less than additive. Thus, cAMP and CGMP may regulate human intestinal epithelial differentiation by interacting pathways.

Effect of chronic ethanol consumption on the activities of residual small bowel brush-border enzymes after proximal jejunum resection in the rat.

Ethanol consumption has a toxic effect on the epithelium of the small bowel, but enterocyte maturity is very difficult to measure under these circumstances. However, when ethanol intake is combined with enterectomy, enterocyte immaturity is greater, permitting an easier separation of these two effects. In a group of rats (13 male Wistar rats weighing approximately 220 g) fed a liquid diet containing 35% ethanol for 4 weeks after resection of the proximal jejunum, the residual small intestine brush border maltase, sucrase, and lactase activities were similar to those of a pair-fed control group (13 animals). However, alkaline phosphatase activity was decreased in the mucosa and in the enterocyte brush border, probably because of the lower activity of this enzyme in the jejunum-ileum remnant of the alcoholic group.

Dietary fat effects on brush border membrane composition and enzyme activities in rat intestine.

The effect of dietary fats on the chemical composition and enzyme activities has been studied in intestinal brush border membranes (BBM) or rats. Animals were given commercial rat pellet diet (RP) or semisynthetic diet rich in either saturated [coconut oil (CCO))] or polyunsaturated [n-6, corn oil (CO) or n-3, fish oil (FO)] fat at the 10% level for 5 weeks. The membrane cholesterol/phospholipid ratio was augmented in CO- or RP-fed rats. There was an increase in level of saturated fatty acids in BBM from CCO- or FO-fed animals. n-3 polyunsaturated fatty acid content was raised in FO-fed rats, while the proportion of linoleic acid and arachidonic acid was enhanced in animals given a CO diet. Membrane fluidity was in the order of CCO < RP = CO < FO. The membrane hexose content was high (p < 0.05) in the CCO group. Hexosamines were elevated (p < 0.05) in CCO- or FO-fed rat brush borders. Membrane fucose was unaltered, while sialic acid content was elevated in CO- (p < 0.05) and FO- (p < 0.01) fed vs. CCO-fed rats. Lectin binding to brush borders corroborated these findings. The activities of alkaline phosphatase, sucrase and lactase were augmented (p < 0.001) in CCO-fed animals. Leucine-aminopeptidase and sucrase activities were depressed by FO feeding. The activities of PNP-beta-glycosidases were the highest in FO-fed rats. These results indicate that dietary fat quality markedly affects microvillus membrane lipid composition, glycosylation and enzyme functions in rat intestine.

Altered distribution of the nuclear receptor rarβ accompanies proliferation and differentiation changes caused by retinoic acid in Caco-2 cells

All epithelial cells require retinoic acid for growth, maintenance, and differentiation. Although the epithelial cells that line the gastrointestinal tract are exposed to extreme retinoid concentration fluctuations in luminal fluid, whether proliferation and differentiation in these cells are significantly affected is not known. We have investigated this question using Caco-2 cells as a model because, although they are derived from a colon adenocarcinoma, they differentiate spontaneously in a manner similar to enterocytes in the small intestine. We found that retinoic acid caused maximum inhibition of cell growth and ornithine decarboxylase activity during the proliferative period. Retinoic acid increased brush border enzyme activities only in differentiating cells but stimulated transglutaminase activity in cells at all stages. In untreated proliferating cells, we found an early peak of transglutaminase activity that has not been reported before. Retinoic acid in intestinal cells acts through its nuclear receptor, RAR beta. The nuclear distribution of this receptor has not been demonstrated. In this study, we show that RAR beta responds to increasing concentrations of retinoic acid with a shift to the nuclear membrane in undifferentiated cells and progressive aggregation, diffusion, and loss in differentiated cells. We conclude that retinoic acid can inhibit proliferation and stimulate differentiation in Caco-2 cells depending on concentration and cell stage, and that these effects are accompanied by changes in distribution, as well as by the loss of RAR beta.

Maturation of the pancreatic and intestinal digestive functions in sea bass (Dicentrarchus labrax): effect of weaning with different protein sources

The maturation of the digestive functions in sea bass (Dicentrarchus labrax) larvae was evaluated by the enzymatic profile of pancreas and intestine brush border membranes. Sea bass larvae were weaned at day 25 with three simplified diets different by their protein nature: 100% fish meal (FP), 100% casein mixture (CP) and 50% fish meal-50% casein mixture (CFP). The casein mixture contained 35% of hydrolysate. The control group was fed live preys. The specific activity of amylase decreased with age irrespectively of the diets whereas the specific activity of trypsin was enhanced. The casein mixture reduced pancreatic secretion in amylase and trypsin. The CFP group differed from the other groups fed on compound diets, exhibiting as soon as day 32 high activities of brush border enzymes, similar to controls. This sharp increase between day 25 and 32 appeared to be crucial for larval survival. The addition of a protein hydrolysate in a weaning diet seems to facilitate this maturation process.

Effects of concanavalin A and pokeweed lectins on microvillar membrane proteins during the organ culture of rabbit intestinal mucosa

The effects of pokeweed lectin ( PWL) and concanavalin A (Con A) on microvillar membrane ( MVM) proteins during the organ culture of rabbit ileal explants for 24 hours were compared with the known effects of enteropathogenic Escherichia coli ( EPEC). PWL resulted in the accelerated release of brush border enzymes into the culture medium, accompanied by decreased tissue activities and an increase in the total activity present in the tissue and culture medium. Con A had less effect on the release and tissue activities of brush border enzymes and the total activity was not increased. Sucrose density gradient centrifugation of the culture medium showed that MVM enzymes were predominantly particulate, consistent with their release as vesicles. Centrifugation of ileal explants showed that PWL, but not Con A, resulted in a decrease in the modal density of the brush border which was consistent with a lower glycoprotein-to-lipid ratio. These findings suggest that PWL, in common with EPEC, may cause the disruption and vesiculation of microvilli and the compensatory stimulation of MVM protein synthesis.

The Effect of Epidermal Growth Factor on Mucosal Function after Ileal Resection

Since epidermal growth factor (EGF) enhances gut mucosal regeneration, the present study was undertaken to evaluate the effect of EGF on brush-border membrane enzyme activity and glutamine uptake in the intestinal remnant following extensive small bowel resection. Twenty-four adult male New Zealand White rabbits were divided into three groups: Group 1 ( n = 12) served as controls. Groups 2 and 3 ( n = 6 each) underwent a 50-60% mid-jejunoileal resection with anastomosis of the remaining intestine, leaving 90 cm between the pylorus and the ileocecal valve. Group 3 rabbits had a subcutaneous osmotic pump implanted to deliver EGF for 7 days at 0.3 μg/kg/hr. Rabbits from Groups 2 and 3 were sacrificed 3 weeks postoperation. Mucosa from the proximal and distal segments of the remaining intestine was analyzed for wet/dry weight, maltase and aminooligopeptidase activity, and glutamine uptake. There was a twofold increase in mucosal dry weight/cm of intestine in rabbits without EGF at 3 weeks (Group 2) and a fourfold increase in those given EGF (Group 3). The maltase enzyme capacity (UEnzyme/rabbit) increased from 37 ± 10 in controls (Group 1) to 167 ± 30 without EGF and 207 ± 30 with EGF. The aminooligopeptidase enzyme capacity (UEnzyme/ rabbit) increased from 55 ± 10 to 147 ± 20 and 226 ± 30 in Groups 1, 2, and 3, respectively. Glutamine uptake capacity (μ M glutamine/min) also increased significantly, from 63 ± 19 in Group 1 to 88 ± 6 without EGF and 162 ± 18 with EGF ( P < 0.001). We conclude that EGF greatly increases mucosal mass and brush-border membrane enzyme activity in the residual small intestine following extensive bowel resection which significantly increases the absorptive and digestive capacity.

Consecutive use of hormonally defined serum-free media to establish highly differentiated human renal proximal tubule cells in primary culture.

Highly differentiated human proximal tubule (HPT) cells in primary culture were established from heterogeneous suspension of tubules prepared from the human renal cortex by an original two-step procedure. First, gluconeogenic-competent HPT cells were selected by using a hormonally defined serum-free medium without glucose or insulin; then, the selected HPT cells were grown in a medium containing a low concentration of glucose (1 mM) and insulin (0.5 micrograms/mL) but no antibiotics. HPT cells grown on plastic support formed confluent, cobblestone-like monolayers with numerous mitochondria and pinocytosis vacuoles, solitary cilia, junctional complexes, and a well-developed brush border consisting of densely packed microvilli. Compared with cell monolayers on plastic support, HPT cells grown on porous filter membranes showed better morphologic differentiation. HPT cell monolayers expressed the following differentiated functions of the proximal tubule in situ: a low-affinity, high-capacity Na(+)-dependent glucose transport system inhibited by phlorizin, a high-affinity Na(+)-dependent phosphate transport system, a basolateral organic cation uptake inhibited by mepiperphenidol, parathyroid hormone-sensitive cAMP synthesis, brush-border hydrolase activities, gluconeogenesis-associated enzymes, glutathione-S-transferases and N-acetyl-beta-D-glucosaminidase. The medium containing low glucose and insulin concentrations markedly limited the increase in glycolysis but did not prevent the falls in gluconeogenesis and brush-border hydrolase activity at any time of the culture period. Similar decreases of brush border enzyme activities were obtained for HPT cells grown either on plastic or on porous filter membrane. A thorough characterization study demonstrated that this simple and preparative experimental approach makes it possible to establish highly differentiated HPT cells in primary culture suitable for investigating human renal proximal tubular cell function.

Early and delayed effects of hydrocortisone and onapristone on intestinal brush-border enzymes and their sialylation and on thymus growth in suckling rats.

The antigestagen-antiglucocorticoid onapristone (ZK 98.299) was tested on three glucocorticoid-sensitive systems after hydrocortisone (HC) administration to suckling male rats, by determining onapristone (ZK)-induced inhibition of HC-provoked (1) increase of activities of intestinal brush-border enzymes, (2) desialylation of brush-border components and (3) thymolysis. HC acetate (75 mg/kg body weight (b.w.)) was injected s.c. on postnatal days 9 and 10, and ZK (150 mg/kg b.w.) on days 9, 10 and 11. The animals were killed on day 12 for assessing the early effect, or on days 15-17 for determining the delayed effect of HC and ZK. In all three systems the glucocorticoid effects were antagonized by ZK. The most sensitive to HC were systems 1 and 3, which exhibited both the early and the delayed effects. The most sensitive to the counteraction of ZK against administered HC was system 1, where HC was antagonized in both its early and delayed effects, whereas only delayed antagonistic action against administered HC was found in system 2. ZK alone had an early inhibitory effect on the activities of several brush-border enzymes and produced an early increase in thymus weight, accompanied by an increased DNA-protein ratio. No delayed effects of ZK alone on the three systems were observed.

Effects of iodinated x-ray contrast media on renal epithelial cells in culture.

To study cellular mechanisms that cause contrast media nephropathy, an in vitro system for proximal and distal tubular cells has been established to evaluate the influence of x-ray contrast media on tubular function. Confluent cell cultures of the two renal cell lines, proximal tubule (LLC-PK1) and distal tubule (MDCK), were exposed for 20 hours to 0 to 100 mg iodine/mL of the ionic monomer metrizoate, the ionic dimer ioxaglate, and the non-ionic monomer iohexol. Toxicity was assessed by electron microscopy, cell viability, and biochemical assays of brush-border and lysosomal marker enzymes. The results demonstrated a concentration-dependent toxic effect from the contrast media on cellular appearance consisting of an increased vacuolization and on the activity of brush-border and lysosomal marker enzymes in cells and in culture media. The results, in which the nonionic x-ray contrast media iohexol appeared to be less toxic than the ionic x-ray contrast media investigated, demonstrated that defined renal cells in culture are valuable tools in studies regarding renal toxicity of x-ray contrast media.

Serum-free organ culture of suckling rat jejunum: Effect of regulatory hormones

To facilitate the study of regulators of differentiation and proliferation of small intestinal epithelium in the suckling rat we have developed a serum-free organ culture system and used it to examine epithelial responsiveness to various regulatory hormones. These hormones included the insulin-like growth factors (IGFs) whose action can be blocked by binding proteins in serum. Jejunal explants from 5-day-old suckling rats maintained better brush border enzyme activity and better histology when cultured under hyperbaric conditions for 24 h in serum-free Dulbecco's modified Eagle's medium/F12 medium than in RPMI 1640 plus 10% fetal bovine serum. Tissue responsiveness to various regulatory hormones was then tested in the serum-free medium. Insulin had no significant effect on morphology, proliferation rate, or enzyme activity in 5-day explants after 24 h in culture. However, insulin did increase lactase activity and induce the early appearance of sucrase in 10- and 12-day explants after 48 h in culture. Dexamethasone increased specific activities of alkaline phosphatase (30%, P < 0.001) and lactase (15%, P < 0.001), and reduced shedding of alkaline phosphatase into the medium (P < 0.001), in explants of 5-day-old rats cultured over 24 h. Dexamethasone combined with insulin had no obvious effect on the rate of protein or DNA synthesis but did increase villus height (P = 0.04) and crypt depth (P = 0.001) and acted synergistically to further increase lactase activity above levels obtained by either alone. IGF-I and IGF-II, des-(1-3)IGF-I, fibroblast growth factor (FGF), and growth hormone (GH) had no effect on morphology or biochemical activity of explants after 24 or 48 h culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Physiological effects of HIV infection on human intestinal epithelial cells: an in vitro model for HIV enteropathy.

To determine the role of direct infection of intestinal cells with HIV-1 in the pathogenesis of HIV-related enteropathy. We infected HT-29-18-C1 intestinal cells with the IIIB strain of HIV and examined the physiologic effects of enterocyte function. Dipeptidase-IV, aminopeptidase-N, gamma glutamic transferase, and alkaline phosphatase were measured in HIV-infected and control cultures. The cellular second messengers intracellular calcium and cyclic adenosine monophosphate were also measured in infected and control cultures. A persistent infection was established for > 95 days with peak supernatant reverse transcriptase and HIV p24 antigen levels of 5.17 log10 c.p.m./ml and 45 ng/ml, respectively. Brush-border enzyme activity (nmol of product/min/mg protein) tended to be lower in infected cell cultures compared with controls early in infection (P < 0.02). Baseline second messenger concentrations were similar but infected cultures responded to stimulation with a calcium ionophore with an exaggerated increase in intracellular calcium (P = 0.03). These results suggest that absorptive and secretory function of enterocytes may be altered by direct HIV infection and that additional physiologic experiments with this in vitro model may lead to a better understanding of the clinical syndrome of HIV enteropathy.

Development and response to a diet change of some digestive enzymes in sea bass (Dicentrarchus labrax) larvae

Variations in some enzyme activities during larval development of sea bass fed live prey were investigated from hatching to day 40. Fluctuations in the enzyme specific activities (except for trypsin) occurred in three phases: initially a sharp increase until day 12, followed by a plateau and subsequently a decrease around day 23. Then activities remained constant until day 40. Trypsin activity kept rising until day 23, then fell. Enzymatic adaptation to a change in diet was studied by feeding larvae with microparticulate diet from day 25. Adaptation to dietary change was observed for amylase, alkaline phosphatase and leucine aminopeptidase, assayed in whole larvae. In larvae fed microparticulate dry diet, the activities of these three enzymes tended to be higher than in those fed natural prey. Although poor growth was observed in larvae fed microparticles, the brush border enzyme activities purified from whole body homogenate, were not impaired.

Nucleotide Uptake and Metabolism by Intestinal Epithelial Cells

AbstractThe epithelial cells of the gastrointestinal tract are the first to encounter ingested nucleotides. Enterocytes metabolize or transport nucleotides (often partially metabolized) to other cell types. Nucleotides may also affect enterocyte gene expression. These interactions in intestinal cell lines (Caco-2 and IEC-6 cells) are described. Nucleotides and nucleosides are efficiently taken up by neoplastic cells (Caco-2) and substantially metabolized during absorption by epithelial monolayers. In nonmalignant cells (IEC-6), nucleotide pools are smaller than enterocytes of neoplastic origin (Caco-2). Consequently, cell proliferation in IEC-6 cells is more dependent on an external supply of nucleotides. Cell differentiation was examined by measuring brush border enzyme activities (sucrase, lactase and alkaline phosphatase). Nucleotides enhanced the expression of brush border enzymes in carcinoma cells only when stressed by glutamine deprivation. IEC-6 cells, which are poorly differentiated in optimal media, require basement membrane (Matrigel) for expression of brush border enzymes. Under these conditions, adding nucleotides to the culture medium enhanced enzyme activity. In addition to being substrates for intestinal absorption, nucleotides may affect enterocyte differentiation.

The influence of calcium and magnesium on sea bass ( Dicentrarchus labrax) intestinal brush border membrane purification and activity

1. 1. The activity of brush border enzymes (alkaline phosphatase, maltase, sucrase, trehalase, leucine amino peptidase) was higher in purified membranes prepared with calcium. The contamination of these membranes with basolateral membranes was also lower (1.27 for Na-K-ATPase activity ratio). 2. 2. The extraction of brush border lipids was carried out according to Folch adapted method. Two dimensional thin layer chromatography was used to separate the phospholipidic fractions. Fatty acids of phospholipids were analysed using gas chromatography after acid transmethylation (column SP 2330). 3. 3. Phospholipids are composed of phosphatidylcholine (PC: 33%), phosphatidylethanolamine (PE: 30%), sphingomyeline (SM: 21%), phosphatidylserine (PS: 14%) and phosphatidylinositol (PI: 2%). 4. PC, PE and PS are characterized by high levels of unsaturated fatty acids (monounsaturated MUFA: 21.5% and polyunsaturated PUFA: 34.9%). The most abundant PUFA belong to the (n-3) family [18:3 (n-3), 20:5 (n-3) and 22:6 (n-3)]. 4. 5. Fatty acids from sphingomyelin of purified membranes have low proportions of PUFA (13.5%) but higher proportions of MUFA (39.5%). 5. 6. No specific differences were found between calcium and magnesium prepared membranes. 6. 7. The low content in LPC and the absence of LPE confirmed the absence of major structural lipids transformation during the membrane purification with calcium or magnesium. 7. 8. Glycine transport was measured during 10 sec at different temperatures using the rapid filtration technique. Glycine transport was higher with Na + than with K +. In the presence of Na +, this transport increases with temperature. 8. 9. Arrhenius curves were mono phasic without obvious breakpoint and indicated no phase transition in the lipid bilayer. 9. 10. A significant Na + dependent glycine transport has been characterized at low temperatures (0°C) which suggests a possible role of membrane polyunsaturated fatty acids in the control of glycine transport.

Duodenal brush-border mucosal glucose transport and enzyme activities in aging man and effect of bacterial contamination of the small intestine.

Duodenal biopsies were collected from 38 subjects (24 female and 14 male) ranging in age from 55 to 91 years. Evidence of bacterial contamination of the small bowel (BCSB) was sought at the same time by bacterial culture of duodenal aspirates and by hydrogen and [14C]glycocholic acid breath tests; subjects were considered to be positive for BCSB if any one of the three tests was abnormal. Biopsies were analyzed for six brush-border membrane enzyme activities: maltase, sucrase, lactase, alkaline phosphatase, leucine aminopeptidase, and alpha-glucosidase. Analysis of covariance with age as the covariate indicated no significant effect of age on the specific activities of these enzymes. Mucosal Na(+)-dependent glucose transport was quantified in brush-border membrane vesicles prepared from the biopsies. In all groups, glucose transport at 20-30 sec was greater (ranging from mean values of 2.45 to 3.66 times) than at 45 min, consistent with Na(+)-coupled glucose transport, and no significant effect of age was observed. BCSB had no significant effect on specific activities of any of the duodenal mucosal hydrolases but was associated with reduced (P = 0.05) brush-border glucose transport. None of the variables studied was significantly affected by the gender of subjects. In conclusion, these biochemical data do not support the contention that reduced capacity for carbohydrate absorption in the elderly is explained by reductions in duodenal brush-border mucosal disaccharidase activities or glucose transport.

The Extraction of Smooth Muscle Contractile and Noncontractile Proteins from the Rat Small Intestine: Measurement of Protein Synthesis and Effects of Ethanol Toxicity

An investigation was made into the optimum conditions for the extraction and measurement of intestinal smooth muscle contractile proteins of laboratory rats. Isolation of the seromuscular layer was achieved by "mucosal stripping" (using a glass slide); isolation of contractile proteins were achieved by differential solubility (using high and low ionic buffers and ultracentrifugation). Histological evidence revealed that after mucosal striping all cells of the longitudinal and circular muscle layers of the seromuscular region remained intact. Assay of brush-border marker enzyme activities confirmed that mucosal striping did indeed remove the mucosa and there was very little villus contamination of the smooth muscle tissue preparations. Optimum physical conditions for the isolation of seromuscular and mucosal myofibrillary proteins were identified. We ascertained that considerable amounts of myofibrillarly proteins reside in the mucosa, but could be adequately separated by mucosal striping. Improved recoveries of purified contractile proteins necessitated the inclusion of protease inhibitors during all processing steps. Using the optimum method, polyacrylamide gel electrophoresis of contractile proteins showed that the predominant smooth muscle contractile proteins, i.e., myosin heavy chain, actin, tropomyosin, and myosin light chains, were indeed preferentially isolated by our methodology. Using these techniques we demonstrated that the synthesis rates of intestinal contractile proteins were reduced by acute ethanol dosage. These results may be responsible for, or reflect, alcohol-induced defects in intestinal motility.

Effects of Small Peptides as Intraluminal Substrates on Transport Carriers for Amino Acids and Peptides.

The effects of small peptides on brush-border membrane enzyme activities and on transport carriers for amino acids and dipeptides were investigated by measurement of these enzyme activities and the transmural potential difference in the small intestines of guinea pigs fed elemental diets containing either small peptides (SP) or amino acids (AA) as the nitrogen source. Brush-border membrane aminopeptidase activities were significantly higher in the group fed the diet containing SP. In addition, the transmural potential differences induced by L-leucine and glycyl-L-leucine (Gly-Leu) were significantly higher in the SP group. The maximum potential differences for L-leucine and Gly-Leu were significantly higher in the SP group, whereas the half saturation values did not differ between the two groups. The same effect of a SP diet was seen in rats, when the uptake of L-[U-14C]leucylglycine (L-[14C]Leu-Gly) into intestinal brush-border membrane vesicles was measured. Some 70% of L-[14C]Leu-Gly was not hydrolyzed by aminopeptidase, but was found as a dipeptide in the brush-border membrane vesicles. These results indicate that small peptides as intraluminal substrates increase brush-border membrane aminopeptidase activities and the activity of carriers for amino acids and dipeptides.

Small intestinal bacterial overgrowth and enhanced intestinal permeability in healthy Beagles

SUMMARY The small intestine of healthy adult Beagles was examined to determine whether subclinical abnormalities might exist that would be relevant to the use of Beagles in pharmacologic studies. Duodenal juice was obtained for qualitative and quantitative bacteriologic examinations; jejunal mucosa was taken for morphologic and biochemical investigation, and intestinal permeability was assessed by quantification of 24-hour urinary excretion of 51Cr-labeled edta after its oral administration. Comparisons were made with findings in healthy adult dogs of other breeds that served as controls. Small intestinal bacterial overgrowth was found in 14 of the 21 Beagles examined, and represented a mixed flora that included obligate anaerobic bacteria in 8 dogs and exclusively aerobic bacteria in 6 dogs. Intestinal permeability (percentage urinary recovery of 51Cr-labeled edta; mean ± sem) was considerably higher (P &lt; 0.01) in Beagles with anaerobic overgrowth (37.6 ± 3.2%) or aerobic overgrowth (30.5 ± 4.8%), compared with Beagles with no overgrowth (17.3 ± 1.6%) and with controls (11.1 ± 1.0%). In Beagles, significant (r = 0.54, P = 0.03) correlation was observed between 24-hour urinary recovery of 51Cr-labeled edta and bacterial numbers in duodenal juice. Morphologic changes in jejunal mucosa were minimal, and specific activities of brush border enzymes were not significantly decreased, apart from aminopeptidase N, but activities of lysosomal and endoplasmic reticular marker enzymes were higher in the 3 groups of Beagles with anaerobic, aerobic, or no overgrowth, compared with controls. These findings indicate that apparently healthy Beagles can have bacterial overgrowth in the proximal portion of the small intestine, which is associated with enhanced intestinal permeability and may not be suspected by clinical examination or routine histologic examination of mucosa.

Regulation of brush-border enzyme activities and enterocyte migration rates in mouse small intestine.

We adapted the Weiser method, previously used to fractionate enterocytes of rat and rabbit intestine, to the much smaller intestine of mice. By histological, morphometric, enzymatic, histochemical, and immunocytochemical evidence, the method succeeded in removing mouse enterocytes sequentially along the crypt-villus axis while preserving cell viability and minimizing mixing among cell fractions. Activities of three brush-border enzymes [alkaline phosphatase (AP), sucrase, and gamma-glutamyl transpeptidase (GGP)] varied simultaneously with dietary substrate level, intestinal region, and position along the crypt-villus axis. All three enzymes proved to be stimulated by dietary substrate: sucrase by dietary sucrose, AP and GGP by dietary protein. We also studied cell migration rates and life-times by autoradiography and by our modified Weiser method. By both methods, injected [3H]thymidine after short times was virtually confined to crypt cells, whereas after 40-48 h it was distributed from the crypt over the whole villus except for the villus tip. Villus height decreased twofold from duodenum to ileum, parallel to the regional decrease in cell migration rates because the cell lifetime of 68 h was independent of region. When we varied dietary carbohydrate and protein levels reciprocally while maintaining protein above the maintenance level, both cell migration rate and cell lifetime proved independent of diet.