The significance and future prospects of study of the small intestine.
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Adaptation, Physiological↗
Biomedical subjects
D H Alpers
Publications and source records attributed to D H Alpers.
The secretion of intestinal alkaline phosphatase (IAP) from the enterocyte.
Rat intestinal alkaline phosphatase exists in two separate forms, differing markedly in the carboxyterminal coding region, as well as in the 3' untranslated regions. It is secreted bound to a phospholipid-rich particle (surfactant-like particle) which appears to have unique properties, but whose role is still uncertain. Evidence is presented to suggest that intestinal alkaline phosphatase secretion is mediated by this particle. The challenge for the future is to study the biology of the surfactant-like particle, to ascertain its role in gut physiology, and to determine how the dual alkaline phosphatases are involved in this specialized secretory apparatus.
Alkaline Phosphatase↗
Isolation and sequence analysis of variant forms of human transcobalamin II.
Two cDNA clones (1.9 kb and 1.5 kb, respectively) encoding full length human TC II have been isolated from a human endothelial cell cDNA library and sequenced. The differences between the two clones are the length of the 5' end and the 3' end non-coding regions and the codon at position 198 and 219. Both the clones differ from the recently isolated (human endothelial cell) cDNA for TC II (Platica, O., Janecko, R., Quadros, E.V., Regee, A., Romain, R. and Rothenberg, S.P. (1991) J. Biol. Chem. 266, 7860-7863) in codon 259 and 376 and in their calculated pI values. In vitro transcription followed by translation in a reticulocyte lysate system and SDS-PAGE revealed that the isolated cDNA clones encode a protein of 43 kDa. Upon treatment with canine pancreatic microsomes, the molecular mass of the in vitro translated product was reduced to 41.5 kDa, indicating the presence of an approximately 1.5 kDa signal peptide. This translation product was immunoprecipitated with rabbit anti-serum to human TC II and was able to bind to Cbl-Sepharose beads. The amino acid sequence alignment of TC II with that of other Cbl binding proteins (rat intrinsic factor, human transcobalamin I and porcine haptocorrin) revealed only 33% overall homology. However, there were four regions of greater than 80% homology and two regions of about 60% homology. These regions encompass the majority of the hydrophobic areas of the Cbl-binders. Based on these studies, we suggest that structural basis for the expression of different polymorphic forms of TC II may be due to single point mutations and that TC II, like other mammalian Cbl-binders, have evolved from a common ancestral gene. Furthermore, the Cbl-binding functional domain most probably resides in a hydrophobic pocket which is formed by all or some of the six regions of high homology.
Amino Acid Sequence↗
Characterization of proteins in rat and human intestinal surfactant-like particles.
Phospholipid-rich particles isolated from the apical surface of rat enterocytes have surfactant-like properties and are enriched for intestinal alkaline phosphatase. Purified intact rat particles were used to produce antibodies in rabbits. Antiserum against the rat particle identified major proteins of 48, 68, 98, and 118 kDa on Western blots of isolated rat surfactant-like particle and did not detect any protein in rat intestinal basolateral membranes, rat brush border membranes, or human particles, but did detect a single 180-kDa protein in a preparation of rat milk fat globules, and two proteins (66 and 103 kDa) in rat pulmonary surfactant. The proteins detected on Western blot corresponded with the major proteins identified by Coomassie blue staining. Similar particles were isolated from the apical surface of human intestine and had an enzyme composition, buoyant density, and lipid content similar to those of the rat particles. Antiserum raised against the human jejunal particle detected proteins in the human particle by Western blot that were similar in size with the rat particle proteins (33, 52, 75, 82, and 118 kDa), but did not cross-react with human brush border or rat particle proteins. These studies demonstrate that the surfactant-like particles are present in human as well as rat intestinal tissue, demonstrate similar enzyme and protein content, and confirm their unique identity, distinct from apical brush border or basolateral membranes.
Animals↗
Intractable neurological Wilson's disease treated with orthotopic liver transplantation.
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Adult↗
Asialoglycoprotein receptor mRNAs are expressed in most extrahepatic rat tissues during development.
The expression of an asialoglycoprotein receptor, comprised of a major (R-1) and minor (R-2/3) component, was characterized in various rat tissues during postnatal development. Two R-1 mRNA subspecies were identified in the liver by Northern blots. The 1.4-kb form, which was the predominant subspecies in the liver, was detected in small amounts only in the stomach and kidney. The 1.7-kb mRNA, however, was present in all extrahepatic tissues examined. R-1 mRNA content in the liver increased by only 38% during the suckling period, but the extrahepatic R-1 mRNA had a dramatic biphasic developmental pattern with the highest levels at birth and after weaning. The R-2/3 mRNA was present as a single species on Northern blot in the liver and in a restricted number of extrahepatic tissues (salivary glands, ileum, and kidney) but was detected in most tissues by polymerase chain reaction. The R-2/3 mRNA content in the liver increased fourfold after birth but was consistently low in the other tissues. [57Co]cobalamin-haptocorrin binding activity was present in all extrahepatic tissues examined, although only at a level 2-5% of hepatic binding. Binding affinities of the adult kidney brush-border membranes (1.9 x 10(9) M-1) and the adult liver plasma membranes (2.4 x 10(9) M-1) were comparable. On Western blot, < 4% of adult liver R-1 content and a relatively larger amount of R-2/3 were detected in all four extrahepatic tissues examined. Thus the low binding activity in extrahepatic tissues correlates with the presence of both receptor subunits, which are closely related to or identical with the hepatic receptor subunits.
Aging↗
Cellular localization and hormonal regulation of pancreatic intrinsic factor secretion in dogs.
Gastric mucosal cells are considered to be the principal site of intrinsic factor (IF) production in most mammals. Recent observations in dogs suggest that the pancreas is the major site of IF production in this species. The present study was undertaken to determine the cellular origins of canine pancreatic IF by combining in situ hybridization with immunocytochemistry and to examine the potential role of physiological pancreatic secretagogues, cholecystokinin octapeptide (CCK-8) and secretin, as mediators of canine pancreatic IF secretion. A human IF cDNA probe (J. Hewitt et al., Genomics 10: 432-440, 1991), validated for use in the dog, identified IF mRNA in parietal cells in the gastric fundus, gastric gland cells in the pyloric antrum, and in secretory duct cells of the pancreas. Immunocytochemistry using antibody against rat IF confirmed that these cells, as well as secretory ducts of salivary glands, synthesized an immunoreactive protein. In stimulated secretions of anesthetized dogs, mean 45-min outputs of IF, haptocorrin, and trypsinogen were 13-, 8-, and 16-fold greater during stimulation with CCK-8 than with secretin. No synergistic effects of combined stimulation were observed for IF or haptocorrin, although a synergistic effect was observed for trypsinogen. These findings demonstrate that IF is synthesized in the canine stomach, pancreas, and probably salivary glands and that CCK-8 mediates IF secretion from pancreatic duct cells.
Animals↗
The intrinsic factor (IF)-cobalamin receptor binding site is located in the amino-terminal portion of IF.
Intrinsic factor has two binding sites, one each for cobalamin and for the ileal receptor recognizing the intrinsic factor-cobalamin complex. To obtain initial functional mapping of these domains, cDNAs encoding intact rat and human intrinsic factor or fragments thereof were expressed transiently in COS-1 cells or in an in vitro transcription/translation system. Deletion of as little as 12% of the amino acids from the carboxyl terminus resulted in loss of cobalamin binding activity. On the other hand, the receptor binding region of intrinsic factor appears localized to a restricted region in the amino-terminal portion of the protein. Only those transcription/translation fragments of rat or human intrinsic factor tested that contained amino acid residues 25 to 62 (out of 399) showed calcium-dependent binding to isolated kidney brush borders, the shortest sequence corresponding with 20 consecutive amino acids. In contrast, a 232-amino acid carboxyl-terminal fragment of rat intrinsic factor and 243- and 338-amino acid carboxyl-terminal fragments of human intrinsic factor showed no receptor binding activity.
Animals↗
In vitro expression and secretion of functional mammalian intrinsic factor using recombinant baculovirus.
Intrinsic factor was produced at levels of 1-2 mg per 1 (0.25 micrograms per 10(6) cells) by growth of recombinant baculovirus-infected Sf9 cells in spinner culture. The recombinant IF showed a binding affinity for cobalamin (2.6.10(-10) M) and for the intrinsic factor-cobalamin receptor (3.5.10(-10) M) nearly identical with native IF. Purification of the recombinant intrinsic factor could be accomplished by affinity chromatography, but final purification by gel chromatography (FPLC) was necessary to separate intrinsic factor from a 62 kDa protein secreted from uninfected Sf9 cells. This protein binds selectively to the cobalamin-Sepharose column, but demonstrates no cobalamin binding activity after elution. Microgram quantities of radiolabelled protein could be produced for metabolic and autoradiographic studies. The stability of intrinsic factor to pancreatic proteinases was nearly identical with human gastric intrinsic factor, both native and recombinant as produced in mammalian cells. Glycosylation of the intrinsic factor was demonstrated by lectin binding to the recombinant protein separated on SDS-PAGE, and by a shift in apparent molecular mass from 47 kDa to 43 kDa following treatment of Sf9 cells with tunicamycin. Most of the recombinant IF was produced by Sf9 cells in the first 48 h post infection.
Animals↗
Pericrypt eosinophilic enterocolitis and chronic diarrhea.
An unusual pattern of eosinophilic infiltration around intestinal crypts was detected in mucosal biopsy specimens of 10 patients with chronic diarrhea, half of whom had evidence of systemic connective tissue disease. The median duration of symptoms was 11 months, and no other explanation for diarrhea could be determined in any case. The cellular infiltrate on biopsy specimens was present deep in the mucosa of small and large intestinal specimens, separating crypt bases from the muscularis mucosae and penetrating the latter. Consistent with the microscopic findings, surface mucosal appearance by endoscopy was uniformly normal. These histological features of colonic biopsy specimens were statistically differentiated from those of asymptomatic subjects (n = 8), subjects with diarrhea-predominant irritable bowel syndrome (n = 6), and subjects with collagenous colitis (n = 7) or lymphocytic colitis (n = 5). Diarrhea improved in five of seven subjects treated with oral prednisone or prednisone in conjunction with azathioprine (median follow-up period, 2.2 years). Histological changes on subsequent biopsy specimens correlated closely with symptomatic status. These findings strongly suggest that chronic diarrhea is related to this pericrypt eosinophilic enterocolitis, a pathological lesion often associated with features of systemic connective tissue disease. The disorder appears responsive to corticosteroid therapy in some cases.
Adrenal Cortex Hormones↗
Apolipoprotein expression and cellular differentiation in Caco-2 intestinal cells.
Caco-2 cells, cultured for 18 days on porous filter supports and conventional plastic culture dishes, were used to study the effects of cellular differentiation on the expression of apolipoprotein (apo) genes. Media of filter-grown cells accumulated more apo B as apo B-48 and contained three times the amount of edited apo B mRNA compared with plastic-grown cells. The accumulation of apo A-I by media of plastic-grown cells was higher than accumulation by filter-grown cells, despite similar concentrations of apo A-I mRNA. The apo A-IV was detectable in the culture media earlier with filter-grown cells compared with plastic-grown cells, despite similar apo A-IV mRNA concentrations. Plastic-grown cells contained more apo E mRNA, and their media accumulated more apo E than filter-grown cells. With the exception of apo A-I, apo gene expression changed with Caco-2 cell differentiation to resemble more closely the patterns seen in adult enterocytes. There were no effects or minimal effects of added retinoic acid, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or thyroid hormone on apo accumulation in media of filter-grown cultures of Caco-2 cells. However, 1,25(OH)2D3 and thyroid hormone increased apo B, apo A-IV, and apo A-I mRNA concentrations, retinoic acid increased apo B mRNA concentrations alone, and all three reduced apo E mRNA concentrations. Ratios of edited to unedited apo B mRNA were unaffected. In conclusion, culture substratum importantly influences Caco-2 cell differentiation. Soluble factors that influence cellular differentiation may affect apo gene expression over and above effects mediated by the culture substratum.(ABSTRACT TRUNCATED AT 250 WORDS)
Apolipoprotein A-I↗
Caco-2 cell transfection by rat intestinal alkaline phosphatase cDNA increases surfactant-like particles.
The rat enterocyte produces a particle with surfactant-like properties (including a whorled appearance, enrichment for dipalmitoyl phosphatidylcholine, and ability to lower surface tension) that also is enriched for intestinal alkaline phosphatase. Human Caco-2 cells grown on polycarbonate filters were utilized to study the secretion of these particles and exhibited whorls and strands of unilamellar membranes, particularly concentrated at the apical pole or near junctional complexes. Concentrated culture medium from these cells separated on continuous NaBr gradients revealed a fraction at density = 1.07 g/l enriched for phosphatidylcholine and intestinal alkaline phosphatase. This fraction contained membranous sheets containing alkaline phosphatase, detected by immunolocalization. Phosphatidylcholine comprised 54% of phospholipid in this fraction, compared with 20% in brush borders. When Caco-2 cells were transfected with cDNA encoding rat intestinal alkaline phosphatase, cellular phosphatase activity increased twofold, but activity in the medium increased 14-fold to > 200 (average 32)-fold. Ultrastructurally, compared with mock-transfected cells or cells transfected with human placental alkaline phosphatase, transfection with rat intestinal alkaline phosphatase cDNA led to intracellular and extracellular accumulation of surfactant-like particles. We conclude that surfactant-like particles are produced by Caco-2 cells, and their production can be enhanced by transfection with a cDNA encoding a protein known to be associated with such particles.
Alkaline Phosphatase↗
The two mRNAs encoding rat intestinal alkaline phosphatase represent two distinct nucleotide sequences.
Rat duodenal mucosa contains two mRNAs (2.7 and 3.0 kb) encoding intestinal alkaline phosphatase (IAP), but the mechanism for their production has not been clear. By means of the polymerase chain reaction (PCR), we isolated a fragment that identifies a second rat IAP (rIAP-II), differing from the rIAP-I sequence in the coding and 3' untranslated regions and encoding a COOH-terminal sequence predicted to be hydrophilic. By means of probes unique to each sequence, rIAP-I identified the 2.7-kb mRNA, and rIAP-II the 3.0-kb mRNA. The different structures and differential regulation of the mRNAs after fat feeding demonstrate the presence of two rat IAP transcripts.
Alkaline Phosphatase↗
Mast cell granules within endothelial cells: a possible signal in the inflammatory process?
Mast cell-endothelial cell interactions have long been suspected to be important in inflammation. The detail of the pathways of communication have yet to be elucidated. In this report we describe, for the first time, mast cell granules within endothelial cells of colonic capillaries in a patient with florid colitis, as well as rats with experimentally induced mast cell degranulation. We postulate that this phenomenon may occur more generally during immunologically mediated inflammatory cell degranulation, and may be one mechanism of communication to endothelium.
Adult↗
Do life events or depression exacerbate inflammatory bowel disease? A prospective study.
OBJECTIVE: To determine whether depressed mood or life events are associated with an exacerbation of inflammatory bowel disease. DESIGN: A prospective study of a consecutive sample of patients with relapsing inflammatory bowel disease, followed by monthly questionnaires and periodic office visits. SETTING: A referral-based gastroenterology clinic at a medical school. PATIENTS: A consecutive sample of 32 patients with inflammatory bowel disease who had had at least one relapse in a 2-year period after entry into the study. MEASUREMENTS AND MAIN RESULTS: The Social Readjustment Rating Scale (measuring life events), the Beck Depression Inventory (a visual analog scale for depressed mood) and an inventory of intestinal symptoms were completed monthly by each subject with a 78% rate of compliance. A mean of 2.2 exacerbations was seen per subject during the study period. Life events were not temporally associated with changes in intestinal symptoms. Significant associations were found between intestinal symptoms and the two mood scales (P less than 0.05 for each), but no directionality in symptom occurrence could be detected in a time-lagged analysis. The results were similar when the months preceding exacerbations of inflammatory bowel disease were analyzed separately. CONCLUSIONS: Although these findings suggest that mood changed concurrently with exacerbation of inflammatory bowel disease, no evidence indicated that stressful life events or depressed mood precipitated exacerbations in this study group.
Colitis, Ulcerative↗
Rat intestinal alkaline phosphatase secretion into lumen and serum is coordinately regulated.
We have reported the presence of intestinal alkaline phosphatase on particles with surfactant-like properties within enterocytes, on the luminal surface (light mucosal scrapings) and in the lumen of adult fat-fed rat intestines ((1989) J. Clin. Invest. 84, 1355). To test the physiological role of these particles, we compared the effect on particle secretion of a known inducer of luminal and serum alkaline phosphatase secretion (fat), with the effect of pharmacological stimulators (cholecystokinin and bethanecol). Fat induced a 2-3-fold increase in membrane-free phosphatase activity in serum, and in particle-bound alkaline phosphatase activity in proximal luminal washings and light mucosal scrapings, reaching a peak in both compartments 7 h after a corn oil feed. Bethanecol given subcutaneously induced a quantitatively similar increase in serum alkaline phosphatase activity and in particle-bound phosphatase activity in proximal light mucosal scrapings, reaching a peak 7.5 min after injection. Cholecystokinin also had a 2-3-fold stimulatory effect, 30 min after injection, on particle-bound phosphatase activity in proximal intestinal light mucosal scrapings and distal intestinal luminal washings. The increase in alkaline phosphatase activity in serum samples reached a peak 60 min after cholecystokinin injection. Thus, three independent stimuli increase both luminal and serum appearance of intestinal alkaline phosphatase. These data support the earlier findings that intestinal alkaline phosphatase secretion into the lumen is mediated by a secreted particle, further show that secretion into serum and lumen is coordinately regulated, and are consistent with the hypothesis that the rise in serum alkaline phosphatase activity could be related to extracellular release of the enzyme from the particles.
Alkaline Phosphatase↗
A five-year plan of the American Gastroenterological Association.
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Forecasting↗
The minor components of the rat asialoglycoprotein receptor are apically located in neonatal enterocytes.
Because specific uptake of the asialoglycoprotein haptocorrin has been reported in suckling distal intestine, evidence of the asialoglycoprotein receptor in rat ileum was sought. On Western blot, two different polyclonal antisera against purified rat holoreceptor recognized only proteins of the size of the minor receptor components (51 and 55 kilodaltons) in both suckling and adult rat intestine. Messenger RNA encoding the minor component (RHL-2/3) was detected in total RNA extracted from rat ileum. Calcium-specific binding of porcine or rat haptocorrin-[57Co]cobalamin complexes was detected in the brush border membranes of distal suckling rat and porcine small intestine. This binding was almost completely blocked by another asialoglycoprotein, asialofetuin. The pH optimum for binding was 6-9, with a Ka of 0.25 nmol/L and a capacity of 4.6 fmol/mg protein. These properties, with the exception of the low capacity, are all consistent with those of the intact receptor on hepatocytes. The intestinal receptor was localized not to the basolateral membrane, as in the liver, but to the apical brush border, as suggested by the binding data. Furthermore, immunoperoxidase stains of paraffin-embedded tissue detected strong binding to the brush border and apical phagolysosome of mid and distal suckling rat intestine. These data show that, contrary to expectations, the minor components of the asialoglycoprotein receptor are functional and expressed in the apical membrane of the suckling intestine. The abundance of the protein by Western blot suggests possible roles for it in the neonatal gut other than haptocorrin binding.
Animals↗