Gluten-sensitive enteropathy in Irish setter dogs: characterisation of jejunal microvillar membrane proteins by two-dimensional electrophoresis.

This study investigated whether gluten-sensitive enteropathy (GSE) in Irish setter dogs was associated with underlying structural abnormalities of microvillar membrane proteins. Jejunal biopsies taken from eight-month-old GSE-affected dogs reared on a normal, gluten-containing diet exhibited partial villous atrophy and contained more intra-epithelial lymphocytes than controls. The morphological abnormalities were reversed by feeding a gluten-free diet for five months and the changes were accompanied by an increase in the mucosal activity of the microvillar hydrolases, particularly aminopeptidase N and dipeptidyl aminopeptidase IV, which reverted to pre-treatment levels after a gluten challenge. Two-dimensional electrophoresis of microvillar membrane proteins isolated from GSE-affected dogs revealed an essentially normal protein map that was comparable to controls. The exception was an intense 85 kDa protein spot that diminished when the affected dogs were fed a gluten-free diet and re-intensified after a gluten challenge.

An aminopeptidase N deficiency in dog small intestine.

This study has identified a naturally occurring, specific deficiency of a brush border aminopeptidase N (ApN) in the small intestines of five clinically healthy dogs. ApN activity in mucosal homogenates of dog small intestine was reduced significantly in deficient animals (13.4 (1.1) nmol min-1 mg-1 protein, n = 5, P < 0.002) compared to healthy control dogs (95.1 (6.7), n = 22). Alkaline phosphatase, gamma-glutamyl transferase, zinc-resistant alpha-glucosidase, maltase, sucrase and lactase in the ApN deficient dogs exhibited comparable activities to those in the control dogs. Microvillar membranes were analysed by one- and two-dimensional electrophoresis. ApN was represented by a single 145kDa band in all control dogs, identified by immunoblotting and immunoprecipitation. Protein maps from deficient dogs were normal apart from the virtual absence of an ApN spot and there were no apparent abnormalities in the glycosylation of microvillar proteins. The findings suggest that intestinal ApN deficiency in these dogs is a primary lesion involving diminished expression of an otherwise normal enzyme protein.

Longitudinal variation in the activities of mucosal enzymes in the small intestine of suckling rats.

The extent of positional variation in mucosal enzyme activity along the small intestine was investigated in 14-day-old suckling rats. Samples were taken from ten equally spaced sites along the intestine in 11 rat pups and the activities of the enzymes alkaline phosphatase, neutral aminopeptidase, gamma-glutamyl transferase, lactase and sucrase were measured. All the enzymes except sucrase were subject to considerable positional variation. Alkaline phosphatase and aminopeptidase activities were distributed throughout the intestine, with a broad maximum in the distal intestine. Lactase was also broadly distributed but with greatest activity in the mid intestine. gamma-glutamyl transferase exhibited a novel profile, with a very high proportion of the total activity (78%) present in the distal intestine. Sucrase was essentially absent throughout the intestine.

Characterization of microvillar membrane proteins of dog small intestine by two-dimensional electrophoresis.

A method for analysing microgram amounts of microvillar membranes by two-dimensional electrophoresis (protein mapping) is described, and has been used to characterize the microvillar proteins of the small intestine of German shepherd, corgi, and beagle dogs. Detergent-solubilized microvillar membranes were radiolabelled with 14C and separated by isoelectric focussing followed by SDS-PAGE. Proteins were detected fluorographically and glycoproteins by lectin-affinity staining. The microvillar hydrolases alkaline phosphatase and dipeptidyl aminopeptidase IV were identified by active-site labelling and aminopeptidase N by immunoprecipitation. Changes following pancreatic duct diversion were consistent with accumulation of pro-sucrase-isomaltase and diminished expression of the sucrase and isomaltase subunits. Cytoskeletal proteins were concentrated in the core fraction remaining after extraction of microvillar membranes with Triton X-100. There were no consistent differences between dogs of different breed, and the canine protein maps were similar to the human.

Basic fibroblast growth factor increases the multiplication and migration of a serum-free derivative of CACO-2 but does not affect differentiation.

Basic fibroblast growth factor (bFGF) is found in the extracellular matrix and around the endothelial and epithelial cells of some human colon carcinomas. It is believed to play a role in angiogenesis, but in addition, recent data suggest that it can directly stimulate mitogenesis in some colon carcinoma cell lines. To clarify the role of bFGF in human colon carcinoma, we developed a model of Caco-2 which grew in serum-free conditions so that the effect of bFGF on multiplication, migration, and differentiation could be studied in defined conditions. Through morphological and biochemical studies in serum-free conditions, we demonstrated that this subline of Caco-2 differentiated spontaneously on reaching confluence. Using this model, we found that bFGF did not affect differentiation but that multiplication and migration were increased. The implication of these findings is that bFGF, released from the extracellular matrix by invading cells or produced by neovascular endothelial cells, can increase the mitogenic rate and migratory potential of colon carcinoma cells. In addition, the dual role of bFGF in stimulating colon carcinoma cells directly and promoting angiogenesis suggests that anti-bFGF strategies could form the basis of a novel approach to the treatment of colon carcinoma.

Acid-base transport systems in a polarized human intestinal cell monolayer: Caco-2.

Acid-base transport systems have been incompletely characterized in intact intestinal epithelial cells. We therefore studied the human cell line Caco-2, cultured on Teflon membranes to form confluent monolayers with apical microvilli on transmission electron microscopy and progressive enrichment in microvillar hydrolases. Monolayers (16- to 25-day-old), loaded with the pH-sensitive dye BCECF-AM (2',7'-bis (carboxyethyl)-5-carboxyfluorescein), were mounted in a spectrofluorometer cuvette to allow selective superfusion of apical and basolateral surfaces with Hepes- or HCO(3-)-buffered media. Intracellular pH (pHi) was measured by dual-excitation spectrofluorimetry; calibration was with standards containing nigericin and 110 mM K+ corresponding to measured intracellular [K+] in Caco-2 cell monolayers. In HCO(3-)-free (Hepes-buffered) media, bilateral superfusion with 1 mM amiloride or with Na(+)-free media reversibly inhibited pHi recovery from an intracellular acid load (NH4Cl pulse) by 86 and 98% respectively. Selective readdition of Na+ to the apical or basolateral superfusate also induced a pHi recovery, which was inhibited by ipsilateral but not by contralateral amiloride (1 mM). The pHi recovery induced by apical Na+ readdition had a Michaelis constant (Km) for Na+ of 30 mM and a relatively high inhibitor constant (Ki) for amiloride of 45.5 microM. Initial pHi in HCO(3-)-buffered media was lower than in the absence of HCO3- (7.35 vs. 7.80). pHi recovery from an acid load in HCO3- was Na- dependent but was inhibited only 18% by 1 mM amiloride. The amiloride-independent pHi recovery was inhibited 49% by pre-incubation of cells in 5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). These data suggest that Caco-2 cells possess: (a) both apical and basolateral membrane Na(+)-H+ exchange mechanisms, the apical exchanger being relatively resistant to amiloride, similar to apical Na(+)-H+ exchangers in several normal epithelia; and (b) a Na(-)-dependent HCO3- transport system, either Na(+)-HCO3- cotransport or Na(-)-dependent Cl(-)-HCO3- exchange.

Cytokeratin expression in epithelial cells isolated from the crypt and villus regions of the rodent small intestine.

Many physiological and structural features of epithelium in the small intestine are regulated during their transit from the crypt base to the villus tip. This crypt-villus axis is an important model for the study of the regulation of cell proliferation and differentiation. We have investigated the expression of cytokeratins in purified epithelial cells from the proliferative (crypt) and differentiated (villus) regions of this tissue. Three polypeptides were identified (cytokeratins 8, 18 and 19) as well as a fourth, 46 kDa polypeptide with similar electrophoretic characteristics to the recently identified cytokeratin 20. The distribution of these molecules was found to vary along the crypt-villus axis, with cytokeratin 18 being restricted to the proliferative crypt and cytokeratins 8 and 19 demonstrating more uniform distributions. The 46 kDa component was found to be expressed predominantly within the villus epithelium. Although there is no substantial evidence of a direct role for cytokeratins in the process of epithelial differentiation, these data suggest that differential expression of cytokeratins is associated with changes in intestinal epithelial differentiation.

Effects of urogastrone-epidermal growth factor and age at administration on five enzymes in the small intestine of suckling rats.

Suckling rats were given urogastrone-epidermal growth factor (EGF: 1,000 micrograms/kg body weight) or vehicle by gavage at one of three stages of development: 8 to 10, 11 to 13 or 14 to 16 days of age. Intubation was carried out at 8-hourly intervals over these periods. Fourteen to 16 h after the last intubation the rats were killed; that is, at 11, 14 and 17 days respectively. Samples of proximal and distal small intestine (SI) were taken for enzyme analysis. Five enzymes were assayed; sucrase, lactase, gamma-glutamyl transferase, alkaline phosphatase and neutral amino-peptidase, and their activities expressed per g protein. Treatment with EGF had no effect on body weight or on the length of the small intestine at any age. The nature of the effects on enzyme activities depended on the specific enzyme concerned, the site within the small intestine and the timing of the treatment. Lactase was increased by EGF at both sites only on day 14, whereas gamma-glutamyl transferase was increased in proximal samples at 11 and 14 days, and in distal samples at 17 days. Nor was the outcome always to increase activity. On day 11 alkaline phosphatase was increased in proximal SI, but decreased in distal SI; and so too was aminopeptidase N decreased in distal SI at 11 days. Sucrase showed no response at all. The pattern is complex. Certainly it does not indicate accelerated functional maturation.

Spectrofluorimetric determination of urinary p-aminobenzoic and p-aminosalicylic acids in the BT-PABA/PAS test of pancreatic function.

Spectrofluorimetry was investigated as an alternative to HPLC for determining p-aminobenzoic acid and p-aminosalicylic acid in the N-benzoyl-L-tyrosyl-p-aminobenzoic acid/p-aminosalicylic acid test of pancreatic exocrine function. Urine specimens were hydrolysed for 30 min in 4 M NaOH at 100 degrees C. The fluorescence of p-aminobenzoic acid was measured in dimethyl sulphoxide solution (lambda ex = 300 nm, lambda em = 340 nm) and that of p-aminosalicylic acid in sodium acetate buffer, pH 4.0 (lambda ex = 297 nm, lambda em = 394 nm). The linear range was 0.038-8 mM for p-aminobenzoic acid and 0.051-12 mM for p-aminosalicylic acid, within-batch precision was 2.2% and 5.5%, respectively, and the entire analysis could be completed within 40 min. Although not eliminated, drug interference was greatly reduced in comparison with colorimetry. In 23 consecutive pancreatic function tests there was an excellent correlation between the p-aminobenzoic acid/p-aminosalicylic acid excretion index obtained by fluorimetry and the results from HPLC analysis (y = 0.914x + 0.070, r = 0.987, p less than 0.001). The method is simple, cost-effective and may be particularly valuable in developing countries having a high incidence of chronic pancreatitis.

Simultaneous assessment of intestinal permeability and lactose tolerance with orally administered raffinose, lactose and L-arabinose.

1. In order to develop an improved differential sugar absorption test for simultaneously assessing intestinal permeability and lactose intolerance, methods were established for determining raffinose, lactose and L-arabinose in human urine. Using NAD(P)H-coupled enzymatic assays and fluorimetry, each sugar was measurable over a concentration range of approximately 3-300 mumol/l in diluted urine specimens. 2. After an overnight fast, 40 normal volunteers drank an iso-osmotic solution containing raffinose, lactose and L-arabinose. The median 5 h urinary sugar excretion was 0.26% of the ingested raffinose, 0.05% of lactose and 17.5% of L-arabinose. 3. In 143 patients with gastrointestinal disease, excretion of both ingested raffinose and lactose was significantly increased in coeliac disease in relapse or in partial remission and in Crohn's disease, but not in the irritable bowel syndrome, coeliac disease in remission or ulcerative colitis. Excretion of lactose, but not raffinose, was increased in patients with mucosal lactase deficiency, whereas excretion of L-arabinose was reduced in all disease groups except ulcerative colitis. 4. Discrimination between diseases was poor when based on individual sugar recoveries, but improved dramatically when excretion was expressed relative to that of L-arabinose. The raffinose/L-arabinose excretion ratio, an index of intestinal permeability, was greater than 0.08 in 15/15 untreated coeliac patients but less than 0.06 in all normal subjects and in 9/9 lactase-deficient patients, 15/16 recovered coeliac patients, 5/6 patients with ulcerative colitis, 13/16 patients with Crohn's disease and 61/62 patients with irritable bowel syndrome.

Investigation of the physical properties of dog intestinal microvillar membrane proteins by polyacrylamide gel electrophoresis: a comparison between normal dogs and dogs with exocrine pancreatic insufficiency.

Procedures have been validated for the investigation of the physical properties of canine microvillar membrane proteins by SDS-polyacrylamide gel electrophoresis. These have been used to examine mucosal samples from eight control dogs and from five dogs with naturally occurring exocrine pancreatic insufficiency (EPI) in order to evaluate the potential role of the pancreas in the normal turnover of microvillar membrane proteins in the dog. Gel scanning showed that the proportion of total membrane protein in bands corresponding to a molecular mass greater than 200 kDa was up to 20-times higher in dogs with EPI than in control dogs. In particular, a band of apparent molecular mass 218 kDa represented between 8 and 28% of membrane protein in all affected dogs, compared with only 0.5 to 1.8% in controls, and is most likely to contain single chains of both pro-maltase-glucoamylase and pro-sucrase-isomaltase. Incubation of microvillar membranes in vitro with either trypsin or canine pancreatic juice resulted in degradation of this high molecular mass band and a corresponding increase in the amount of protein in three bands representing molecular masses of 150, 133 and 106 kDa. In samples from control dogs aminopeptidase N was identified in the 133 kDa band by Western blotting and incubation with monospecific antiserum. These findings suggest that pancreatic enzymes play a major role in the normal post-translational processing of intestinal microvillar membrane proteins in the dog.

Changes in jejunal permeability and passive permeation of sugars in intestinal biopsies in coeliac disease and Crohn's disease.

1. The relative effects of changes in mucosal surface area and mucosal permeability on the passive uptakes of mannitol and raffinose have been studied in vitro using jejunal biopsies from 48 controls, 32 patients with coeliac disease and 11 patients with Crohn's disease. Total mucosal permeation was corrected for surface area measured morphometrically to provide an index of mucosal permeability. 2. In untreated coeliac disease, permeation of mannitol was reduced by 35% (P = 0.006) and that of raffinose was increased by 66% (P = 0.0095) compared with controls, whereas mucosal permeability to mannitol was increased twofold (P = 0.009) and to raffinose fivefold (P = 0.0001). Mucosal permeability was similar for each sugar. 3. In treated coeliac disease, permeation and permeability for mannitol were normal, but remained elevated for raffinose by 23% (P = 0.036) and 41% (P = 0.024), respectively. 4. In Crohn's disease, permeation of mannitol was reduced by 21%, but that of raffinose and mucosal permeability to both sugars were normal. 5. These findings suggest that surface area is quantitatively more important than mucosal permeability in determining the total permeation of mannitol, while the converse is true for raffinose. The findings are compatible with paracellular uptake of raffinose, but with both paracellular and transcellular uptake of mannitol. Both pathways are affected in coeliac disease, whereas only transcellular uptake is affected in Crohn's disease.

The kinetics of monosaccharide absorption by human jejunal biopsies: evidence for active and passive processes.

The kinetics of initial rates of uptake of glucose, galactose, arabinose and mannitol have been measured in jejunal biopsies from normal subjects in order to investigate the existence of multiple uptake systems. Glucose kinetics fitted best a model of a saturable uptake system (app Kt = 2.06 +/- 0.33 mM, app Jmax = 93.85 +/- 1.19 nmol/10 min/mg dry weight), together with a linear uptake indistinguishable from the passive uptake of arabinose and mannitol (app Kd = 0.80 +/- 0.05 nmol/10 min/mg dry weight/mM for glucose, 0.75 +/- 0.03 for arabinose, and 0.83 +/- 0.03 for mannitol). The saturable uptake, but not the linear uptake, was inhibited by phlorizin and by the absence of sodium. Cytochalasin B and phloretin had no effect on overall uptake. Galactose kinetics in the absence of inhibitors fitted best a model of a single saturable uptake system (app Kt = 11.05 +/- 0.12, app Jmax = 201.9 +/- 1.13) with no evidence of linear uptake. In the presence of phlorizin, or in the absence of sodium, uptake was predominantly linear with app Kds of 0.84 +/- 0.03 and 0.79 +/- 0.01, not significantly different from the linear component of glucose uptake. We conclude that hexose uptake in human jejunum in vitro occurs by both active and passive routes, and that the active uptake of galactose appears to be inhibited at high galactose concentration.

Lactose digestion by human jejunal biopsies: the relationship between hydrolysis and absorption.

The relationship between lactose hydrolysis and absorption of released glucose was investigated by determining the kinetics of lactose digestion by jejunal biopsies incubated in vitro. Lactase activity in intact biopsies correlated with conventional assay of tissue homogenates (r = 0.85, p less than 0.001), and glucose uptake from 28 mM lactose was directly proportional to lactase activity (r = 0.95, p less than 0.001) in 21 subjects with normal lactase levels, six with hypolactasia (primary or secondary to coeliac disease) and two with lactose intolerance but normal lactase activity. Kinetic analysis at 0.56-56 mM lactose in five normal subjects showed saturable kinetics for hydrolysis (app Km = 33.9 +/- 2.2 mM; app Vmax = 26.5 +/- 1.1 nmol/min/mg dry weight) but glucose uptake could be fitted to a model either of saturable uptake (app Kt = 47.2 +/- 0.3 mM; app Jmax = 14.1 +/- 0.2 nmol/min/mg) or saturable uptake plus a linear component (app Kt = 21.3 +/- 1.15; app Jmax = 4.59 +/- 0.12; app Kd = 0.093 +/- 0.010 nmol/min/mg/mM). The proportion of glucose taken into the tissue did not significantly exceed 50% of the total released at any lactose concentration suggesting the lack of an efficient capture mechanism for the released glucose. The results suggest that lactose hydrolysis is the rate limiting step in the overall absorption of glucose from lactose in vitro, and that the relationship between hydrolysis and absorption is the same in normal subjects and in hypolactasic subjects.

Monosaccharide absorption and water secretion during disaccharide perfusion of the human jejunum.

During jejunal perfusion of normal subjects with isotonic mannitol-saline containing glucose, maltose or sucrose (112 mmol/l), monosaccharide absorption from disaccharides was greater than expected from the intraluminal concentration of free monosaccharides, and water secretion was greater than with glucose. With equimolar (112 mmol/l) glucose + disaccharide mixtures, intraluminal glucose concentrations were similar to but total glucose absorption less than expected from experiments with the individual sugars. The results favour sequential hydrolysis-absorption whereby high concentrations of monosaccharide accumulate at the brush border and promote sugar uptake by conventional rather than 'disaccharidase-related' transport, and stimulate water secretion by local osmotic effects.