Brush Border Fraction Research Articles

METABOLIC ADAPTATION OF FISH INTESTINAL HELMINTHS: INHIBITORY ABILITY TOWARDS PROTEASES IN CESTODES <i>TRIAENOPHORUS NODULOSUS</i>

The inhibitory ability towards proteases was studied in the extracts of plerocercoids from the liver of perch (Perca fluviatilis Linnaeus), as well as in the excretory-secretory products and extracts of mature and immature cestodes Triaenophorus nodulosus (Pallas, 1781) from the intestine of pike (Esox lucius Linnaeus). It was found that excretory-secretory products and extracts of both mature and immature T. nodulosus display varying degrees of inhibitory ability against proteases. A significant decrease in activity was noted under the action of the studied samples on the activity of commercial trypsin. Tapeworm extracts exhibit a greater inhibitory ability compared to excretory-secretory products. At the same time, the extract of immature worms inhibits the activity of proteases more strongly than that of mature ones. The inhibitory ability is more closely associated with the extract of worms lacking the brush border than with the tegumental brush border fraction.

Localization of the proteinase inhibitor activity in the fish cestode Eubothrium rugosum.

The mechanisms enabling fish tapeworms to avoid proteolytic attacks by digestive enzymes of their fish host have been studied in less detail compared with mammalian cestodes. This study aimed to assess the inhibitory ability towards trypsin and chymotrypsin in Eubothrium rugosum, an intestinal parasite of burbot Lota lota, and establish its localization in the tapeworm. To this end, the worms were treated with Triton X-100 followed by differential centrifugation to isolate the tegumental brush border membrane. The protease inhibitory abilities of the worms were mostly determined by their excretory/secretory products released into the incubation medium. These inhibitory abilities proved to be linked mainly with the brush border fractions. Notably, the per cent inhibition of both studied digestive enzymes (trypsin and chymotrypsin) hardly depended on the duration of the parasite exposure in the incubation medium, probably due to intermittent glycocalyx renewal. Improved knowledge on functions of the excretory/secretory proteins produced by fish tapeworms may contribute to a better understanding of host-parasite relations and development of new approaches to the treatment and prevention of diseases caused by pathogenic helminths.

Nervonic Acid (24:1n-9) is a Dominant Unsaturated Fatty Acid in the Intestinal Brush Border of Atlantic Cod

Atlantic cod is a coldwater teleost of commercial importance. The intestinal epithelium is a large organ in vertebrates serving an important role in nutrient selection and uptake as well as an immunological barrier. Here, we perform lipid and fatty acid analysis of the plasma membrane from the cod intestinal enterocytes after separation of the brush border membrane and the basolateral membrane fractions. Our results show that both membrane fractions contain an unusually high amount of cholesterol and glycolipids but low levels of glycerophospholipids compared with other reported studies on fish. Sphingomyelin was the dominant lipid in the brush border fraction and was also prominent in the basolateral fraction where phosphatidylcholine was the dominant glycerophospholipid. Furthermore, our results show a distinct difference in fatty acids content, where monounsaturated fatty acids (MUFA) were more abundant than polyunsaturated fatty acid (PUFA). Nervonic acid (24:1n-9) was a prominent fatty acid in the BBM at ~50% of the total MUFA. We hypothesize that the high cholesterol content and the presence of this rare fatty acid may serve to maintain membrane fluidity in the cold environment.

Role of a Serotonin Precursor in Development of Gut Microvilli

Monoamines exert diverse functions in various cells in peripheral organs as well as in the central nervous system. 5-Hydroxy-l-tryptophan (5-HTP) has been simply regarded as a precursor of serotonin, and it is believed that the biological significance of 5-HTP is essentially ascribable to the production of serotonin. Systemic treatment with 5-HTP is often applied to patients with low serotonin levels in the brain. Here we show that endogenous and exogenous 5-HTP but not serotonin induced the development of microvilli in the gut villi epithelium. In contrast, serotonin but not 5-HTP regulated phagocytosis by macrophages. 5-HTP specifically induced actin remodeling and decreased phosphorylation of extracellular signal-regulated kinase (ERK) in the gut, whereas serotonin stimulated actin remodeling and increased ERK phosphorylation in macrophages. Functionally, inhibition of ERK activity promoted the development of microvilli in the gut and ameliorated phagocytosis by macrophages. Thus, 5-HTP and serotonin contribute to distinct cell-type-specific functions via common mediators. Our study might create an opportunity to explore the effects of exogenously applied 5-HTP in humans.

γ-Glutamyl Hydrolase, Not Glutamate Carboxypeptidase II, Hydrolyzes Dietary Folate in Rat Small Intestine

Dietary polyglutamyl folates are hydrolyzed to monoglutamyl folate derivatives prior to intestinal transport. In humans and pigs, the reaction occurs at pH 6.5 at the jejunal brush border membrane by folate hydrolase and is encoded by the glutamate carboxypeptidase II (GCPII) gene. Intracellular folate hydrolase with an optimal pH of 4.5 is encoded by the γ-glutamyl hydrolase (γ-GH) gene and predominates in rats. We determined the respective roles of GCPII and γ-GH in dietary folate hydrolysis in rat small intestine. Duodenal, jejunal, and ileal mucosa, pancreas, and duodenal luminal fluid were collected from 10 Sprague-Dawley rats that had not been food deprived. Folate hydrolase was assayed at pH 4.5 and 6.5 with and without parahydroxymercuribenzoate (pHMB), an inhibitor of intracellular folate hydrolase. Folate hydrolase activity occurred at pH 4.5 in all tissues, was significantly inhibited by the addition of pHMB at both pH 4.5 and 6.5, and was virtually absent from brush border fractions at pH 6.5. The highest activity was in the postprandial duodenal luminal fluid at pH 4.5. Rat-specific primers for GCPII and γ-GH were used to detect mRNA expression in pancreas, jejunal mucosa, and liver. GCPII expression was detected only in the liver, whereas γ-GH was expressed in all 3 tissues. These results suggest that the hydrolysis of polyglutamyl folates in rats requires the intracellular folate hydrolase that is expressed by pancreatic γ-GH, in contrast to GCPII that is expressed in the jejunal mucosal brush border in pigs and humans. γ-GH folate hydrolase is abundant in rat postprandial pancreatic secretions and appears to hydrolyze dietary folates in the intestinal lumen prior to intestinal absorption.

Possible involvement of myosin‐X in intercellular adhesion: Importance of serial pleckstrin homology regions for intracellular localization

Subcellular fractionation experiments with mouse hepatocytes, combined with sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)-immunoblot analysis using antibodies against two different tail regions of mouse myosin-X demonstrated a 240 kDa molecular mass to be associated with the plasma membrane-rich P2 fraction. The basolateral plasma membrane fraction, but not the brush border fraction, isolated from renal cortices also contained the 240 kDa form of myosin-X. In an attempt to assess relative contributions of possible functional domains in the tail of myosin-X to localization and function, cDNA corresponding to all three pleckstrin homology (PH) domains and different regions (PH1, 2 and 3, and the two subdomains of PH1: PHS1 and PHS2), as well as the myosin tail homology 4 domain (MyTH4) and the band4.1/ezrin/radixin/moesin-like domain (FERM) were separately inserted into the pEGFP vector and expressed in cultured COS-1 cells. As a result, two distinct regions responsible for localization were identified with regard to PH: one covers all three forms that tends to localize to regions of dynamic actin, such as membrane ruffles, lamellipodia and thick cortical actin bundles at the sites of cell-cell adhesion in a Rac- and Cdc42-dependent manner. The other covers PHS1 and PH2 that localizes to filopodia, filopodial puncta and the sites of intercellular adhesion in a Cdc42-dependent manner. Expression of green fluorescent protein (GFP)-MyTH4 fusion protein resulted in formation of phalloidin-positive granules, while GFP-FERM affected the actin cytoskeletal system in a distinctly different way. Taken altogether, the results lend support to the view that myosin-X is involved in cell-cell adhesion-associated signaling-linked membrane and/or cytoskeleton reorganization.

Intestinal Brush Border Membrane Catalyzes Hydrolysis of Pyridoxine-5′-β-D-Glucoside and Exhibits Parallel Developmental Changes of Hydrolytic Activities Toward Pyridoxine-5′-β-D-Glucoside and Lactose in Rats

Pyridoxine-5'-beta-D-glucoside (PNG) is a major form of vitamin B-6 in plant foods that exhibits partial bioavailability as vitamin B-6 in humans. We previously identified an intestinal mucosal cytosolic PNG hydrolase that catalyzes the partial hydrolysis of PNG absorbed without prior deglycosylation. Recent observations that the brush border membrane also catalyzes PNG hydrolysis led to the hypothesis that PNG hydrolysis may be another function of the beta-glucosidase lactase-phlorizin hydrolase (LPH) and, thus, brush border PNG hydrolysis would undergo a developmental decline similar to that of lactose hydrolysis. In this study, the relationships among hydrolytic activities in small intestinal cytosolic and brush border fractions in rats (n = 9 per group) of various ages (1-2 d and 2, 4, 8, 12 and 24 wk) were examined. In vitro specific activities toward PNG and lactose were greater in brush border than cytosol, and these were greater in newborn rats than in all other age groups (P < 0.01). Brush border activities toward PNG and lactose and were closely correlated (r = 0.84; P < 0.0001). These findings suggest that the hydrolysis of PNG is catalyzed at least partially at the brush border and that the bioavailability of PNG may be influenced by the residual LPH activity in children and adults.

Na(+)/H(+) exchanger 3 is in large complexes in the center of the apical surface of proximal tubule-derived OK cells.

Cell biological approaches were used to examine the location and function of the brush border (BB) Na(+)/H(+) exchanger NHE3 in the opossum kidney (OK) polarized renal proximal tubule cell line. NHE3 epitope tagged with the vesicular stomatitis virus glycoprotein epitope (NHE3V) was stably expressed and called OK-E3V cells. On the basis of cell surface biotinylation studies, these cells had 10-15% of total NHE3 on the BB. Intracellular NHE3V largely colocalized with Rab11 and to a lesser extent with EEA1. The BB location of NHE3V was examined by confocal microscopy relative to the lectins wheat germ aggluttinin (WGA) and phytohemagluttin E (PHA-E), as well as the B subunit of cholera toxin (CTB). The cells were pyramidal, and NHE3 was located in microvilli in the center of the apical surface. In contrast, PHA-E, WGA, and CTB were diffusely distributed on the BB. Detergent extraction showed that total NHE3V was largely soluble in Triton X-100, whereas virtually all surface NHE3V was insoluble. Sucrose density gradient centrifugation demonstrated that total NHE3V migrated at the same size as approximately 400- and approximately 900-kDa standards, whereas surface NHE3V was enriched in the approximately 900-kDa form. Under basal conditions, NHE3 cycled between the cell surface and the recycling pathway through a phosphatidylinositol (PI) 3-kinase-dependent mechanism. Measurements of surface and intracellular pH were obtained by using FITC-WGA. Internalization of FITC-WGA occurred largely into the juxtanuclear compartment that contained Rab11 and NHE3V. pH values on the apical surface and in endosomes in the presence of the NHE3 blocker, S3226, were elevated, showing that NHE3 functioned to acidify both compartments. In conclusion, NHE3V in OK cells exists in distinct domains both in the center of the apical surface and in a juxtanuclear compartment. In the BB fraction, NHE3 is largely in the detergent-insoluble fraction in lipid rafts and/or in large heterogenous complexes ranging from approximately 400 to approximately 900 kDa.

Dietary lipid level affects fatty acid composition and hydrolase activities of intestinal brush border membrane in seabass.

Triplicate groups of juvenile seabass (initial weight of 241 g) were fed during 13 weeks three isonitrogenous experimental diets containing different lipid levels, 12% (LL group), 21% (ML group) and 30% (HL group). At the end of the experiment, fish weight gain was similar among the 3 dietary groups. Intestinal brush border membranes were purified for each dietary group; one part of the brush border fraction was dedicated to enzyme assays, the remaining fraction being used for lipid extraction followed by fatty acid analysis. The fatty acid composition of the brush border membrane differed among the 3 groups, although the 3 experimental diets had the same fatty acid composition. The increase in dietary lipid level resulted in a lowering in (n-3) polyunsaturated fatty acid (PUFA) paralleled with an increase in monounsaturated fatty acid. A significant reduction in the brush border enzyme activities, namely alkaline phosphatase, aminopeptidase N, gamma-glutamyl transpeptidase and maltase, was also observed with the elevation of the dietary lipid level. The change in activity of intestinal digestive enzymes, which are membrane-bound proteins, could be attributed to the modification of fatty acid composition and fluidity of the brush border membranes (BBM). Such lowering in PUFA and increase in monounsaturated fatty acid in BBM, concomitant with a decline in membrane enzymatic activity, has been described as a malnutrition indicator in mammals. It raises the question of possible disorders of gut functions in fish fed increasing lipid levels.

Expression of G protein α subunits in normal rat colon and in azoxymethane-induced colonic neoplasms

Background & Aims: Heterotrimeric G proteins are important in growth-regulating signal transduction. The aim of this study was to characterize the relative expression of G protein α subunits in rat colonocytes, colonocyte antipodal plasma membranes, and colonic neoplasms. Methods: Antipodal plasma membranes were prepared from isolated colonocytes. Azoxymethane was administered to rats to induce colonic neoplasms. K- ras mutations in the neoplasms were determined by oligonucleotide hybridization and confirmed by primer mediated–restriction fragment length polymorphism. Colonocyte and tumor homogenates or membranes were probed for Gα subunits by Western blotting with isoform-specific antibodies. Results: The expressions of Gαi2, αi3, and αq/11 were significantly enriched in the basolateral compared with brush border fraction of colonic antipodal plasma membranes. In neoplasms without K- ras mutations, the expression of Gαi2 increased 4-fold, Gαs(long) increased 2.5-fold, and Gαi3 increased 1.5–2-fold. Expression did not differ among tumor grades. K- ras mutations were associated with lowered expression of G proteins, especially Gαo. Conclusions: In colonocytes, Gα subunits are localized primarily in basolateral plasma membranes. The increased expressions of Gαi2 and, to a lesser degree, Gαi3 and Gαs(long) in tumors was independent of tumor grade but was modulated by the presence of K- ras mutations. GASTROENTEROLOGY 1998;115:1494-1503

Binding of diarrheagenic Escherichia coli to 32- to 33-kilodalton human intestinal brush border proteins.

We have detected human intestinal brush border proteins to which Escherichia coli strains adhere by means of a blotting-nitrocellulose method in which the binding of radiolabeled bacteria to sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated intestinal cell membranes was evaluated. The brush border fraction contained several polypeptides that bound only adherent E. coli strains. The most prominent and consistent of these proteins had apparent molecular masses of 32 to 33 kDa. Additional polypeptides ranging from 50 to 70, from 105 to 130, and from 180 to 200 kDa were also recognized by adherent E. coli strains, although with less intensity (in accordance with the number of bound bacteria to these polypeptides). Independently of the pattern of adherence (localized [LA], diffuse [DA], or aggregative [AggA]) all HEp-2-adhering strains recognized, with different intensities, the 32- to 33-kDa brush border proteins, whereas nonadhesive strains did not. The relative avidity of an LA strain to bind to the 32- to 33-kDa proteins was approximately seven- and sixfold higher than the binding of strains with aggregative and diffuse adherence, respectively. Thus, it is reasonable to think that LA, DA, and AggA strains have a common adhesin that mediates binding to the 32- to 33-kDa bands. Inhibition experiments using HEp-2 cells demonstrated that isolated 32- to 33-kDa proteins or specific antiserum blocked preferentially bacterial adherence of the LA pattern. Delipidization and protein digestion of the human brush borders confirmed that E. coli bound to structures of a proteinaceous nature. Deglycosylation studies and sodium meta-periodate oxidation of the intestinal cell membranes decreased bacterial binding activity significantly, indicating that E. coli bound to carbohydrate moieties in the glycoproteins. These results suggest that binding of E. coli strains, mainly of the LA phenotype, to the 32- to 33-kDa proteins could play a role in colonization through adherence to the intestinal mucosa.

Dipeptide transport and hydrolysis in rat small intestine, in vitro

A range of natural and mixed d-/ l-stereoisomer phenylalanine dipeptides was used to investigate peptide uptake and hydrolysis by isolated rings of rat jejunum. Characterisation of dipeptide hydrolysis by the brush border fraction revealed apparent K m values in the 0.1–1.0 mM range which, except for the charged dipeptides, were significantly higher than those for hydrolysis by the cytosolic fraction. Uptake of l-/ l- dipeptides into jejunal rings, which was followed by HPLC, was unaffected by the presence of peptidase inhibitors in the incubation medium although the absorbed peptides were completely hydrolysed in the cytosol; comparison of the effects of excess leucine on dipeptide uptake and on the uptake of the two constituent amino acids were also consistent with absorption of intact dipeptide followed by cytosolic hydrolysis. The uptake of hydrolysis-resistant mixed d-/ l- dipeptides was also studied and confirmed that peptide uptake preceded hydrolysis; d-alanyl- l-phenylalanine accumulated within the rings to twice the medium concentration.

Enhanced Fe3+-Reducing Capacity Does Not Seem To Play a Major Role in Increasing Iron Absorption in Iron-Deficient Rats

Some eucaryotic organisms, including many plants, yeast and mice, have a higher iron uptake during iron deficiency because the capacity to reduce Fe3+ from the environment to Fe2+ is greatly enhanced. To determine whether this occurs in rats, a common experimental model for iron absorption in humans, we compared the in vivo capacity to reduce intraluminal Fe3+ in iron-deficient and normal rats. We also measured potential Fe(3+)-reducing components within the intestinal lumen and on the mucosal surface. Iron-reducing capacity was higher in iron-deficient rats, by a significant (P = 0.026) but modest 20%, in parallel with higher mucosal weight (R2 = 0.501, P = 0.003). In vitro iron reduction by lumen contents was correlated with mucosal weight, even though mucosal tissue was not present in the assays. This capacity was not related to ascorbic acid, glutathione or other nonprotein sulfhydryls. Mucosal ferric reductase activity was higher in iron-deficient rats in parallel with higher tissue weight, but the specific activity did not differ and the higher total activity was not associated with the brush border fraction. The role of endogenous Fe3+ reduction in regulating iron absorption should be investigated in humans and in other experimental models.

Analysis of lactase processing in rabbit

The proteolytic processing of rabbit intestinal lactase-phlorizin-hydrolase (LPH) was studied by pulse-chase and continuous labeling experiments in organ culture from 15-day-old rabbits in the presence of glycosylation and processing inhibitors. Monensin and brefeldin A inhibited the two proteolytic cleavages of the precursor indicating that they are post-Golgi events as previously reported for the unique cleavage of LPH in man [1]. The inhibition was not related to a concomitant alteration glycosylation; in fact, if trimming was blocked by MDNM the abnormal glycosylated precursor was proteolytically processed normally. Finally the use of the anti-microtubular drug colchicine strongly inhibited both cleavages and caused accumulation of the complex-glycosylated precursor form in the brush border fraction indicating that proteolytic events depend on intact microtubule (transport).

Glutamyl aminopeptidase (aminopeptidase A), the BP-1/6C3 antigen

Summary Glutamyl aminopeptidase (aminopeptidase A, EC 3.4.11.7) was first described in 1961 as a calcium-stimulated aminopeptidase with specificity for N-terminal acidic amino acids. The enzyme was subsequently purified and characterized as a membrane-bound ectoenzyme particularly abundant in the brush border fractions of kidney and intestine. Early interest in this enzyme was stimulated by its conversion of angiotensin II to des-aspartyl angiotensin II (angiotensin III). More recently, glutamyl aminopeptidase has been identified as a pre-B cell differentiation antigen BP-1/6C3. This review focuses on the biochemical properties of this enzyme and its role in the immune and non-immune systems.

Application of two-dimensional gel analysis to identification and characterization of tyrosine phosphorylated substrates for growth factor receptors.

The technique of two-dimensional gel electrophoresis was used for analysis of tyrosine phosphorylated polypeptide substrates after epidermal growth factor (EGF)-induced stimulation of receptor tyrosine kinase activity in a brush border fraction of human placental syncytiotrofoblast cells. After incubation with [gamma 32P]ATP, followed by autoradiography of the gels, 35 phosphorylated components were detected, of which 8 were strongly tyrosine phosphorylated by EGF. Using a more sensitive assay with phosphotyrosine-specific antibody, an additional 12 polypeptide components were found to be strongly tyrosine phosphorylated by EGF. A number of the phosphorylated substrates could be aligned with components in a protein catalog of the human brush border membrane fraction that was characterized by glycoprotein staining, Triton X-114 fractionation, immunoreaction with specific antibodies, and comigration with 35S-labeled AMA (transformed human amnion) cells. Identified components, stimulated by EGF, in addition to well-recognized substrates (calpactin II, ezrin, EGF receptor) included beta-tubulin and serum albumin, while other cytoskeletal proteins and alkaline phosphatase were excluded as substrates. A notable feature of the catalog was that a number of glycoproteins were present in both the membrane and cytoskeletal fraction, suggesting involvement in membrane/cytoskeletal interactions. The data demonstrate the feasibility of using two-dimensional gel electrophoresis in a global way to identify target substrates for tyrosine kinase activity. In addition they suggest that many of these are located in the vicinity of tyrosine kinase at the membrane/cytoskeletal border at a location which is probably involved, at the molecular level, in morphological changes of the plasma membrane associated with cell proliferation.

Localization of capping protein in chicken epithelial cells by immunofluorescence and biochemical fractionation.

We have localized capping protein in epithelial cells of several chicken tissues using affinity-purified polyclonal antibodies and immunofluorescence. Capping protein has a distribution in each tissue coincident with proteins of the cell-cell junctional complex, which includes the zonula adherens, zonula occludens, and desmosome. "En face" views of the epithelial cells showed capping protein distributed in a polygonal pattern coincident with cell boundaries in intestinal epithelium, sensory epithelium of the cochlea, and the pigmented epithelium of the retina and at regions of cell-cell contact between chick embryo kidney cells in culture. "Edge-on" views obtained by confocal microscopy of intact single intestinal epithelial cells and of retinal pigmented epithelium showed that capping protein is located in the apical region of the epithelial cells coincident with the junctional complexes. These images do not resolve the individual types of junctions of the junctional complex. Immunolabeling of microvilli or stereocilia was faint or not detectable. Capping protein was also detected in the cytoplasm of intact intestinal epithelial cells and in nuclei of cells in the pigmented retina and in the kidney cell cultures, but not in nuclei of cells of the intestinal epithelium or sensory epithelium. Biochemical fractionation of isolated intestinal epithelial cells shows capping protein in the brush border fraction, which contains the junctional complexes, and in the soluble fraction. These results are consistent with the results of the immunolabeling experiments. Highly purified microvilli of the brush borders also contained capping protein; this result was unexpected based on the low intensity of immunofluorescence staining of microvilli and stereocilia. The microvilli were not contaminated with junctional complexes, as defined by the absence of several markers for cell junctions. The cause and significance of this discrepancy is not certain at this time. Since capping protein binds the barbed end of actin filaments in vitro, we hypothesize that capping protein is bound to the barbed ends of actin filaments associated with one or more of the junctions of the junctional complex.

Age and serotype dependent binding of K88 fimbriae to porcine intestinal receptors

The porcine small intestine contains several polypeptides that could function as receptors for K88-positive Escherichia coli. The mucus fraction contained three proteins with molecular weights of 25, 35 and 60 kDa respectively, which showed a high affinity for K88-positive E. coli cells, whereas brush borders contained a 16 kDa protein and a set of proteins ranging from 40–70 kDa. Depending on the K88 serotype tested, differences in binding to these proteins were observed. In particular, E. coli cells carrying K88ad fimbriae exhibited only a rather weak binding to mucus proteins. The influence of age of the pig on the presence of K88 receptors was also investigated. One-week-old and 35-days-old post-weaning piglets were shown to contain K88 receptors in their mucus while these receptors were hardly detectable in the mucus of 6-month-old pigs. The presence of receptors in the brush border fraction was shown to be independent of age. The binding of K88 fimbriae to mucus proteins was blocked using a lectin of Euonymus europeaus which specifically recognizes the Galα(1–3)Gal sequence, indicating that this disaccharide forms a significant part of the receptor structure.

Amino acid amides of 2-[(2-aminobenzyl)sulfinyl]benzimidazole as acid-stable prodrugs of potential inhibitors of [formula omitted] ATPase

A series of amino acid amides of 2-[(2-aminobenzyl)sulfinyl]benzimidazole were prepared and found to possess gastric antisecretory activity on oral administration. (Glycylaminobenzyl)sulfinyl compound 23a, stable in artificial gastric juice (pH 1.2), was given orally to dogs. It was absorbed efficiently and converted into aniline derivative 7a which showed a very high plasma concentration. Compound 23a was hydrolyzed by the action of aminopeptidase present in plasma or the brush border fraction of the small intestine to release the terminal glycine. o-Aniline derivatives showed good activity in in vitro, H + K +-ATPase inhibition as well as in the inhibition of histamine stimulated acid secretion in isolated bullfrog gastric mucosa. Although these o-aniline derivatives showed no or weak gastric antisecretory activity in rat by id administration, they were active when administered ip. Therefore, these amino acid amides were considered to be acid stable prodrugs of proton pump inhibiting o-aniline derivatives. The mechanism of H + K +-ATPase inhibition of 7a was also examined.

Temperature adaptation of biological membranes: differential homoeoviscous responses in brush-border and basolateral membranes of carp intestinal mucosa

The effects of temperature acclimation of carp upon the hydrocarbon order of intestinal membranes has been determined. A fractionation technique has been developed for the simultaneous purification of brush-border and basolateral membrane fractions from the intestinal mucosa. The specific activity of alkaline phosphatase in the brush-border fraction was enhanced 6.4-fold over that of the initial homogenate, whilst the (Na +-K +)-stimulated ATPase was enhanced 5.8-fold in the basolateral fraction. The specific activities of NADPH-cytochrome-c reductase, succinate-cytochrome-c reductase and acid phosphatase were not increased in these two fractions. Membrane hydrocarbon order in membranes from 10 and 30°C-acclimated carp has been compared by measuring the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene over a range of temperatures. In the brush-border fraction, polarization was identical in both cold- and warm-acclimated groups, whilst large differences were observed in the basolateral fraction sufficient to offset approx. 75% of the temperature-induced ordering effects of cold. The fatty acid composition of the major phosphoglyceride fractions in the brush-border fraction was also largely unaffected by thermal acclimation, whilst the basolateral fraction showed significant increases in the proportion of unsaturated fatty acids in the cold. It is concluded that whilst the basolateral membrane of intestinal mucosa displays a large homoeoviscous response that correlates with a shift in lipid composition, the brush-border membrane does not. These findings are consistent with evidence of functional adaptations of the basolateral membrane during thermal acclimation (Gibson, J.S., Ellory, J.C. and Cossins, A.R. (1985) J. Exp. Biol. 114, 355–364).