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M R Neutra

Publications and source records attributed to M R Neutra.

At least 91 records · Page 5Linked to original sources
PubMed1987

Transepithelial transport of epidermal growth factor by absorptive cells of suckling rat ileum.

Epidermal growth factor (EGF), an acid-stable peptide present in rodent and human milk, is absorbed and promotes intestinal growth when fed to suckling rats. To determine whether absorptive cells of suckling rat ileum conduct selective transepithelial transport of EGF, we followed uptake of 125I-EGF from ileal loops by autoradiography and biochemical methods. Specific binding sites for 125I-EGF were localized by electron microscope autoradiography on apical membranes of ileal epithelial sheets in vitro. During uptake in vivo, radiolabeled molecules were concentrated in apical endosomal compartments and were also associated with lysosomal vacuoles, basolateral cell surfaces, and lamina propria. Excess cold EGF reduced basolateral label by 44% and TCA precipitable serum label by 38%. After 30 and 60 min of continuous uptake, radiolabeled molecules in epithelium, denuded mucosa, blood, and liver were analyzed under reducing conditions by reversed-phase high-pressure liquid chromatography (HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although considerable degradation of 125I-EGF occurred after uptake from the lumen, a portion of radiolabel in epithelium and mucosa represented 125I-EGF which eluted somewhat more rapidly from C18 HPLC columns and showed a slight decrease in apparent molecular weight by SDS-PAGE. All radiolabel in blood and liver represented breakdown products. Thus, EGF is selectively transported across the ileal epithelium in suckling rats but is modified during transport. Milk EGF may accumulate in the lamina propria where it could influence growth and maturation of the suckling intestine.

Animals↗
PubMed1986

Functional expression of the polymeric immunoglobulin receptor from cloned cDNA in fibroblasts.

The polymeric immunoglobulin receptor, a transmembrane protein, is made by a variety of polarized epithelial cells. After synthesis, the receptor is sent to the basolateral surface where it binds polymeric IgA and IgM. The receptor-ligand complex is endocytosed, transported across the cell in vesicles, and re-exocytosed at the apical surface. At some point the receptor is proteolytically cleaved so that its extracellular ligand binding portion (known as secretory component) is severed from the membrane and released together with the polymeric immunoglobulin at the apical surface. We have used a cDNA clone coding for the rabbit receptor and a retroviral expression system to express the receptor in a nonpolarized mouse fibroblast cell line, psi 2, that normally does not synthesize the receptor. The receptor is glycosylated and sent to the cell surface. The cell cleaves the receptor to a group of polypeptides that are released into the medium and co-migrate with authentic rabbit secretory component. Cleavage and release of secretory component do not depend on the presence of ligand. The cells express on their surface 9,600 binding sites for the ligand, dimeric IgA. The ligand can be rapidly endocytosed and then re-exocytosed, all within approximately 10 min. Very little ligand is degraded. At least some of the ligand that is released from the cells is bound to secretory component. The results presented indicate that we have established a powerful new system for analyzing the complex steps in the transport of poly-Ig and the general problem of membrane protein sorting.

Animals↗
PubMed1985

Glycoconjugate distribution and mobility on apical membranes of absorptive cells of suckling rat ileum in vivo.

The luminal membrane of ileal absorptive cells in suckling rats includes two domains: microvillar membranes and deep invaginations between microvilli. We examined the fates of foreign macromolecules that bind to anionic or saccharide sites on these domains after infusion into ligated loops in vivo. Cationized ferritin (CF) and ferritin-RCAI (beta-galactosyl) binding sites were distributed over the entire apical membrane. Ligands bound to apical invaginations were rapidly endocytosed, but ligands on microvilli were not. After CF binding, anionic sites on microvilli were mobile in the plane of the membrane and formed CF clusters at the tip and base of each microvillus. RCAI binding sites did not cluster. Wheat germ agglutinin (WGA, sialic acid) labeling was restricted to microvillus tips of mature cells but was dispersed over the microvillar surfaces of lower villus cells. Ferritin conjugates of Concanavalia ensiformis (Con A), Ulex europaeus agglutinin (UEA), and Dolichos biflorus agglutinin (DBA) did not bind to cell surfaces in vivo. Aldehyde fixation dramatically altered lectin binding patterns, resulting in unmasking and labeling of Con A, WGA, and DBA binding sites that were unavailable in vivo.

Animal Population Groups↗
PubMed1985

Macromolecular transport in the fetal rat intestine.

Macromolecular barrier function of the fetal rat small intestine and colon was analyzed from 16 to 22 days gestation (birth). During this period the epithelium is converted from stratified to simple columnar. To assess permeability, horseradish peroxidase (HRP) was introduced by microinjection into the lumen or into the umbilical circulation. Proximal small intestine, distal small intestine, and colon were examined after 10-20 min. Paracellular passage of HRP through occluding junctions was not observed after either intraluminal or intravascular injection. After intraluminal injection, transepithelial transport of HRP from lumen to blood occurred in all regions at all ages studied. Horseradish peroxidase was present in cytoplasmic vesicles of most cells in the primitive stratified epithelia, during epithelial conversion, and in simple columnar epithelia. After intravascular injection, HRP was present in the lamina propria and in intercellular spaces of the epithelium, but HRP did not enter tight junctions. Tracer was taken up into cytoplasmic vesicles of both stratified and simple columnar epithelial cells, but was only rarely seen in the lumen. We conclude that there is rapid transcellular, vesicle-mediated transport from lumen to blood across both stratified and simple columnar epithelia of fetal rat small intestine and colon; after intravascular injection, macromolecules may be taken up into vesicles at basolateral epithelial cell surfaces but are not rapidly transported into the lumen; paracellular passage does not occur in the fetal ages studied.

Animals↗
PubMed1985

Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis.

Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid-phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR-gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle.

Asialoglycoproteins↗
PubMed1984

Regulation of intestinal goblet cells in situ, in mucosal explants and in the isolated epithelium.

Cholinergic secretagogues were previously shown to accelerate mucin secretion from intestinal goblet cells of adult rats and rabbits, both in vitro and in mucosal explants. This rapid secretory response occurs only in crypt cells; surface goblet cells are not affected. Rapid secretion involves the sequential fusion of secretory granule membranes with the plasma membrane and with each other, but does not require granule movement. In unstimulated cells, slow transport of secretory granules towards the luminal cell surface depends on functional microtubules. Goblet cells appear in the rat fetal intestine three to four days before birth but they are insensitive to cholinergic agents in the fetus and neonate. The secretory response of crypt goblet cells to carbachol, both in vivo and in mucosal slices in vitro, is established throughout the intestines only after weaning (20-25 days after birth). To determine whether acetylcholine from nerve endings in the intact mucosa may mediate a mucus secretory response in the absence of exogenous secretagogues, mucosal sheets were mounted in modified Ussing chambers and goblet cell secretion was assessed after electrical field stimulation. Electrical field stimulation elicited mucus secretion from crypt (but not surface) goblet cells. Secretion was inhibited by prior treatment of the mucosa with 500 nM-tetrodotoxin or 100 microM-atropine, but not by 10 microM-atropine. Thus, endogenous nerves may regulate mucus secretion from crypt goblet cells in the intact mucosa. When intact sheets of epithelium were isolated from adult rat ileum and colon, then maintained in vitro and exposed to 20 microM-carbachol, crypt goblet cells released mucin in response to the secretagogue but goblet cells in in portions of the epithelium derived from villi or mucosal surfaces were unresponsive. This suggests that crypt epithelial cells respond directly to cholinergic agents and that they lose this sensitivity as they migrate out of the crypts.

Animals↗
PubMed1984

Membrane-bound and fluid-phase macromolecules enter separate prelysosomal compartments in absorptive cells of suckling rat ileum.

The absorptive cell of the suckling rat ileum is specialized for the uptake and digestion of milk macromolecules from the intestinal lumen. The apical cytoplasm contains an extensive tubulocisternal system, a variety of vesicles and multivesicular bodies (MVB), and a giant phagolysosomal vacuole where digestion is completed. To determine if sorting of membrane-bound and fluid-phase macromolecules occurs in this elaborate endocytic system, we infused adsorptive and soluble tracers into ligated intestinal loops in vivo and examined their fates. Lysosomal compartments were identified by acid phosphatase histochemistry. Native ferritin and two ferritin-lectin conjugates that do not bind to ileal membranes (Con A, UEAI) served as soluble tracers. Horseradish peroxidase binds to ileal membranes and thus was not useful as a fluid-phase tracer in this system. Cationized ferritin and a lectin that binds to terminal B-D-galactosyl sites on ileal membranes (Ricinus communis agglutinin [RCAI]-ferritin) were used as tracer ligands. All tracers entered the wide apical invaginations of the luminal cell surface and were transported intracellularly. Membrane-bound tracers were found in coated pits and vesicles, and throughout the tubulocisternal system (where cationized ferritin is released from the membrane) and later, in large clear vesicles and MVB. In contrast, fluid-phase tracers appeared within 5 min in vesicles of various sizes and were not transported through the tubulocisternae, rather, they were concentrated in a separate population of vesicles of increasing size that contained amorphous dense material. Large clear vesicles, large dense vesicles, and MVB eventually fused with the giant supranuclear vacuole. Acid phosphatase activity was present in MVB and in the giant vacuole but was not present in most large vesicles or in the tubulocisternae. These results demonstrate that membrane-bound and soluble protein are transported to a common lysosomal destination via separate intracellular routes involving several distinct prelysosomal compartments.

Animals↗
PubMed1984

Regulation of intestinal goblet cell secretion. III. Isolated intestinal epithelium.

Cholinergic secretagogues evoke mucus secretion from goblet cells in the crypts of small and large intestinal mucosa in vivo and in organ culture. It was not known whether this response reflected a direct action on epithelial cell receptors or an indirect effect involving intermediate neurons of the enteric nervous system. To resolve this, carbachol was applied to isolated intestinal epithelium maintained in vitro. Intact sheets of epithelium, measuring 10-200 mm2, were isolated from the ileum and colon of adult rats following short intravascular perfusion with 30 mM EDTA. The isolated epithelia lacked a basal lamina and cytoplasmic blebs formed on the basal cell surfaces, but cell ultrastructure was normal and intercellular junctions were intact. Autoradiography revealed that both goblet and columnar cells continued to incorporate [3H]glucosamine into nascent secretory macromolecules for at least 45 min after isolation. When exposed to 20 microM carbachol for 5 min, crypt goblet cells discharged their stored mucin granules by compound exocytosis, whereas goblet cells in portions of the epithelium derived from villi or mucosal surfaces were unresponsive. We conclude that cholinergic secretagogues act directly on crypt epithelial cells to elicit mucus secretion.

Animals↗
PubMed1984

Regulation of intestinal goblet cell secretion. IV. Electrical field stimulation in vitro.

To determine whether transmitters released from enteric neurons can elicit secretion from goblet cells, full-thickness sheets of adult rat distal ileum or descending colon were mounted in modified Ussing chambers, and mucus secretion was assessed morphologically after electrical field stimulation (EFS). Square-wave pulses (56 V, 2 ms duration) were delivered at 10 Hz for 5 min. Goblet cells in colonic crypts, but not those on the mucosal surface, secreted mucus in response to EFS. This secretion was at least in part atropine insensitive, indicating a noncholinergic mechanism. In the ileum goblet cells located in the crypts, but not on villi, secreted mucus when tissue was mounted in the chamber, even in the absence of EFS. This "unelicited" secretion did not occur in unmounted control tissue in vitro, and it could be prevented by preincubating ileal tissue in 1 microM tetrodotoxin (TTX) or 10 microM atropine for 15 min before mounting. Furthermore, following preincubation with either TTX or atropine, EFS' failed to elicit secretion. Incubation of unmounted tissue with TTX, however, did not block the secretory response of crypt goblet cells to 20 microM carbachol. Thus, intrinsic cholinergic neurons may be stimulated during the mounting of the ileum in the chamber. Taken together, these data demonstrate that mucus secretion from crypt goblet cells may be regulated by cholinergic (in ileum and perhaps colon) and noncholinergic (in colon) elements of the enteric nervous system.

Animals↗
PubMed1984

Cytoskeleton of intestinal goblet cells in rabbit and monkey. The theca.

The ultrastructure and function of the cytoskeleton in intestinal goblet cells was investigated in colonic mucosa from rabbits and monkeys. This exocrine cell is unusual in that its secretory granules are stored as a compact apical mass limited by a dense, cup-shaped layer of cytoplasm called the "theca." Ultrastructural analysis of this cytoplasmic layer in rabbit goblet cells permeabilized with Triton X-100, treated with S4 fragments of heavy meromyosin, and fixed in the presence of tannic acid revealed that it contains an orderly arrangement of microtubules and intermediate filaments, but no detectable actin filaments. Microtubules are arranged vertically, like barrel staves, along the inner aspect of the theca. Intermediate filaments are arranged in two contiguous layers: an inner, basket-like network and an outer series of circumferential bundles resembling the hoops of a barrel. Autoradiography of [3H]glucosamine-labeled human and rabbit cells maintained in organ culture without secretagogues had provided preliminary data suggesting that labeled secretory granules migrate preferentially along the periphery of the apical granule mass, adjacent to the theca, toward the luminal cell surface. In this study, we confirm this observation and show that colchicine inhibits this movement. Cholinergic secretagogues induce rapid release of mucin by compound exocytosis of the granules stored in the theca. This secretory activity is not inhibited by colchicine, presumably because it does not require granule movement. The cuplike shape of the theca is unaltered during rapid release of stored granules. Because the shape is unaltered after 6 h of colchicine treatment, it appears to be maintained by the intermediate filament layers.

Animals↗
PubMed1983

Epithelial differentiation in the fetal rat colon. I. Plasma membrane phosphatase activities.

During the last week of gestation of the fetal rat, the epithelium of the colon is rapidly remodeled. At 16 days a primitive stratified epithelium surrounds a small central lumen. Over the next 3 days, the main lumen extends narrow clefts down to the basal cell layer and small secondary lumina appear within the stratified epithelium between these clefts. At 19 and 20 days, secondary lumina enlarge but remain discrete; an infusion of cationic ferritin into the main lumen does not enter secondary lumina. During the 2 days prior to birth (21-22), the secondary lumina join the main lumen as superficial cells are sloughed, and the epithelium becomes simple columnar. Freeze-fracture replicas indicate that luminal and nonluminal membrane domains of epithelial cell plasma membranes are separated by continuous tight junctions throughout the conversion process. Cytochemical analysis of tissue slices from 16- to 22-day fetal colon demonstrated the appearance and segregation of two phosphatases on apical and basolateral membrane domains during epithelial conversion. Cysteine-sensitive pH 9.0 (alkaline) phosphatase activity was first detected along the luminal membranes of cells bordering both primary and secondary lumina at 18 days gestation and increased to a maximum at 20-21 days; weaker activity was present on basolateral membranes. Phosphatase activity at pH 8.0 also appeared at 18 days and increased thereafter, but was localized primarily on nonluminal membranes. At pH 8.0, reaction product appeared on both inner and outer sides of the membrane, and was only partially abolished by omission of K+ or addition of ouabain; thus the reaction may be only partially due to K+-dependent ATPase activity. Biochemical analysis of the cytochemical media confirmed the appearance of phosphatase activities at 18 days. Thus, plasma membrane phosphatase activities appear while the epithelium is still stratified, but are segregated to luminal and nonluminal membrane domains at the onset of activity. Segregation is maintained throughout the process of conversion of a simple columnar epithelium.

Animals↗
PubMed1983

The role of pili and capsule in the pathogenesis of neonatal infection with Escherichia coli K1.

The role of pili and capsule was studied in neonatal infection with Escherichia coli K1. E coli strains were selectively cultured into three phases: mannose-sensitive (MS) piliated, non-mannose-sensitive (NMS) piliated, and nonpiliated. A high percentage of neonatal rats fed each phase of K1 strains developed bacteremia; there was no bacteremia with non-K1 strains or an acapsular mutant of K1 strain C94 (C94K-). Oral cavity colonization was noted in nearly 100% of rats fed K1 strains, non-K1 strains, or C94K-, regardless of the phase of piliation at feeding. Only MS piliated bacteria were found on oral cavity culture, indicating a rapid shift of NMS piliated and nonpiliated bacteria to the MS piliated phase. Conversely, only nonpiliated bacteria were found on blood culture when neonatal rats were fed piliated bacteria. Colonization of ileal epithelium was not observed. Thus, in vivo phase variation may be important in colonization and bacteremia with E coli K1.

Adhesiveness↗
PubMed1982

Regulation of intestinal goblet cell secretion. I. Role of parasympathetic stimulation.

The in vivo effects of the parasympathomimetic drug pilocarpine on rat intestinal goblet cells were analyzed by autoradiography, light microscopy (LM), and electron microscopy (EM). Pilocarpine accelerated the release of mucus by compound exocytosis from crypt (but not surface) goblet cells throughout the small and large intestine. Pilocarpine-induced mucus secretion was blocked by atropine alone in ileum and colon, but total inhibition in proximal small intestine required a combination of atropine and tubocurarine. The sensitivity of morphological-autoradiographic methods for detection of goblet cell secretion was compared with that of a biochemical detection method, separation of labeled high-molecular-weight glycoproteins by Sepharose 4B gel filtration of luminal washings. Even when secretion of labeled mucus by compound exocytosis was clearly demonstrated by LM, EM, and autoradiography, gel filtration assay of luminal washings from pilocarpine-injected rats failed to reveal an increase in labeled high-molecular-weight glycoproteins. Autoradiographs of mucosal tissue after luminal washing showed that newly secreted, labeled mucus was retained in the crypts and was thus unavailable to the biochemical assay. Thus, direct observation of exocytosis in individual goblet cells provides a qualitative, but sensitive, assay for short-term acceleration of intestinal mucus secretion.

Animals↗
PubMed1982

Regulation of intestinal goblet cell secretion. II. A survey of potential secretagogues.

The factors that regulate the rate of mucus secretion in intestinal goblet cells are only partially defined. Autoradiographic and ultrastructural studies demonstrated that muscarinic cholinergic agents accelerate the exocytosis of mucus from goblet cells in the crypts throughout the small and large intestine, both in vivo and in mucosal organ culture. The present study seeks to identify other factors that may alter mucous secretory rates. Mucosal explants were exposed to potential secretagogues and inhibitors in the organ-culture system and analyzed by light and electron microscopy, alpha- and beta-Adrenergic agents, gastrointestinal regulatory peptides, serotonin, histamine, and dibutyryl cyclic nucleotides were tested over wide concentration ranges. With the exception of histamine, none of these agents accelerated or inhibited the exocytosis of mucous granules. Histamine was effective at the concentration of 10(-4) M and induced rapid, compound exocytosis by crypt goblet cells in mucosal explants from the colon but not from small intestine. The response to histamine was unaffected by atropine. Goblet cells on the mucosal surface released mucus by compound exocytosis when exposed to mustard oil, a nonspecific chemical irritant, but not when exposed to histamine or cholinergic agents.

Acetylcholine↗
PubMed1981

The surface topography of the colonic crypt in rabbit and monkey.

Scanning electron microscopy (SEM) was used to investigate the epithelial topography of the surface and crypt in rabbit and monkey colon. Crypt openings in monkey colon are arranged in a hexagonal pattern, in sharp contrast to rabbit colon where they are randomly arrayed and frequently hidden by epithelial folds. Crypt lumens were exposed by freezing ethanol-dehydrated tissue in liquid nitrogen and fracturing the tissue with a razor blade. The resulting overview of crypt-cell luminal surfaces showed that as columnar cells mature and migrate up the crypt and onto the colonic surface, their microvilli become progressively more abundant. Goblet cells were readily identified in the cross-fractured crypt epithelium; their luminal surfaces are characterized by short, sparse microvilli. The changing appearance of the luminal surface of goblet cells was visualized by SEM during the exocytosis of single mucous granules from unstimulated crypt goblet cells, and during the compound exocytosis of multiple granules in response to acetylcholine.

Animals↗
PubMed1980

Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine.

The parasympathetic control of goblet cell secretion and the membrane events accompanying accelerated mucus release were studied in large intestinal mucosal biopsies maintained in an organ culture system. The secretory response of individual goblet cells to 10(-6) M acetylcholine chloride with 3 x 10(-3) M eserine sulfate (a cholinesterase inhibitor) was assessed by light microscopy and autoradiography, by scanning and transmission electron microscopy, and by freeze-fracture. Goblet cells on the mucosal surface are unaffected by acetylcholine. In crypt goblet cells acetylcholine-eserine induces rapid fusion of apical mucous granule membranes with the luminal plasma membrane (detectable by 2 min), followed by sequential, tandem fission of the pentalaminar, fused areas of adjacent mucous granule membranes. These events first involve the most central apical mucous granules, are then propagated to include peripheral granules, and finally spread toward the most basal granules. By 60 min, most crypt cells are nearly depleted. The apical membrane, although greatly amplified by these events, remains intact, and intracellular mucous granules do not coalesce with each other. During rapid secretion membrane-limited tags of cytoplasm are observed attached to the cavitated apical cell surface. These long, thin extensions of redundant apical membrane are rapidly lost, apparently by being shed into the crypt lumen.

Acetylcholine↗