Differential regulation of cytochrome P-450 genes along rat intestinal crypt-villus axis

Mammalian small intestine contains cytochrome P-450-dependent monooxygenase enzymes that are capable of metabolizing a wide variety of xenobiotics and activating procarcinogens to mutagenic compounds. The epithelial cells lining the small intestine are separated into a proliferating undifferentiated compartment located in crypts and a nonproliferating differentiated compartment located on villi. The constitutive expression and induction by xenobiotics of genes that encode components of the cytochrome P-450-dependent mono-oxygenase system along the rat intestinal crypt-villus axis were investigated using isolated epithelial cells and in situ hybridization. For each gene examined, hybridization analysis of RNA obtained from isolated epithelial cells correlated with findings on in situ RNA hybridization. Cytochrome P-450IA1 mRNA (CYP1A1), the major aromatic hydrocarbon-inducible P-450, and cytochrome P-450IIB1 mRNA (CYP2B1), the major phenobarbital-inducible P-450, were constitutively expressed in villus cells with no detectable mRNA present in crypts. Treatment with several chemical inducers resulted in a marked increase in CYP1A1 mRNA in both crypt and villus cells. In contrast, although CYP2B1 mRNA was inducible in villus cells, CYP2B1 mRNA was not detected in crypts after treatment with chemical inducers. NADPH cytochrome P-450 reductase, a necessary component for the activity of all P-450 enzymes, was expressed constitutively at low levels only in villus cells. Treatment with dexamethasone induced reductase mRNA in both crypt and villus cells. Taken together, these results demonstrate that there is a complex gene-specific pattern of expression of the microsomal monooxygenase system along the crypt-villus axis of rat small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

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DES2 protein is responsible for phytoceramide biosynthesis in the mouse small intestine

Biochemical Journal ◽

10.1042/bj20031425 ◽

2004 ◽

Vol 379 (3) ◽

pp. 687-695 ◽

Cited By ~ 71

Author(s):

Fumio OMAE ◽

Masao MIYAZAKI ◽

Ayako ENOMOTO ◽

Minoru SUZUKI ◽

Yusuke SUZUKI ◽

...

Keyword(s):

Small Intestine ◽

Epithelial Cells ◽

Intestinal Epithelial ◽

Vitro Assay ◽

Peptide Antibody ◽

Mouse Small Intestine ◽

Mrna Content ◽

Crypt Cells

The C-4 hydroxylation of sphinganine and dihydroceramide is a rate-limiting reaction in the biosynthesis of phytosphingolipids. Mouse DES1 (MDES1) cDNA homologous to the Drosophila melanogaster degenerative spermatocyte gene-1 (des-1) cDNA leads to sphingosine Δ4-desaturase activity, and another mouse homologue, MDES2, has bifunctional activity, producing C-4 hydroxysphinganine and Δ4-sphingenine in yeast [Ternes, Franke, Zahringer, Sperling and Heinz (2002) J. Biol. Chem. 277, 25512–25518]. Here, we report the characterization of mouse DES2 (MDES2) using an in vitro assay with a homogenate of COS-7 cells transfected with MDES2 cDNA and N-octanoyl-sphinganine and sphinganine as substrates. MDES2 protein prefers dihydroceramide as a substrate to sphinganine, and exhibits dihydroceramide Δ4-desaturase and C-4 hydroxylase activities. MDES2 mRNA content was high in the small intestine and abundant in the kidney. In situ hybridization detected signals of MDES2 mRNA in the crypt cells. Immunohistochemistry using an anti-MDES2 peptide antibody stained the crypt cells and the adjacent epithelial cells. These results suggest that MDES2 is the dihydroceramide C-4 hydroxylase responsible for the biosynthesis of enriched phytosphingoglycolipids in the microvillous membranes of intestinal epithelial cells.

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Studies on the rate-limiting enzyme component in the microsomal monooxygenase system. Incorporation of purified NADPH-cytochrome c reductase and cytochrome P-450 into rat liver microsomes.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)62336-7 ◽

1978 ◽

Vol 253 (6) ◽

pp. 1921-1929

Author(s):

G.T. Miwa ◽

S.B. West ◽

A.Y. Lu

Keyword(s):

Cytochrome C ◽

Rat Liver ◽

Liver Microsomes ◽

Monooxygenase System ◽

Rat Liver Microsomes ◽

Cytochrome C Reductase ◽

Microsomal Monooxygenase ◽

Rate Limiting ◽

Cytochrome P 450 ◽

Nadph Cytochrome C Reductase

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Crypt cell isolation in the small intestine of the mouse.

Journal of Histochemistry & Cytochemistry ◽

10.1177/25.7.894003 ◽

1977 ◽

Vol 25 (7) ◽

pp. 554-559 ◽

Cited By ~ 11

Author(s):

V C Kremski ◽

L Varani ◽

C DeSaive ◽

P Miller ◽

C Nicolini

Keyword(s):

Small Intestine ◽

Small Bowel ◽

Epithelial Cells ◽

Single Cell ◽

Cell Isolation ◽

Cell Suspensions ◽

Crypt Cell ◽

Intestinal Crypt ◽

Successful Method ◽

Crypt Cells

A successful method has been developed for isolating viable single cell suspensions of intestinal crypt cells from the small bowel of the mouse. The lumen of the intestine was perfused with a 0.2% trypsin solution that dissociated the lining epithelial cells. Crypt cell isolation, which proves to be extremely critical, occurred under optimal mechanical and chemical configurations about 75 min after the beginning of the procedure.

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Expression and localization of aquaporins in rat gastrointestinal tract

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.3.c621 ◽

1999 ◽

Vol 276 (3) ◽

pp. C621-C627 ◽

Cited By ~ 134

Author(s):

Yu Koyama ◽

Tadashi Yamamoto ◽

Tatsuo Tani ◽

Kouei Nihei ◽

Daisuke Kondo ◽

...

Keyword(s):

Small Intestine ◽

In Situ Hybridization ◽

Gastrointestinal Tract ◽

Epithelial Cells ◽

Cellular Localization ◽

Basolateral Membrane ◽

Rnase Protection Assay ◽

Rnase Protection ◽

Basolateral Membranes

A family of water-selective channels, aquaporins (AQP), has been demonstrated in various organs and tissues. However, the localization and expression of the AQP family members in the gastrointestinal tract have not been entirely elucidated. This study aimed to demonstrate the expression and distribution of several types of the AQP family and to speculate on their role in water transport in the rat gastrointestinal tract. By RNase protection assay, expression of AQP1–5 and AQP8 was examined in various portions through the gastrointestinal tract. AQP1 and AQP3 mRNAs were diffusely expressed from esophagus to colon, and their expression was relatively intense in the small intestine and colon. In contrast, AQP4 mRNA was selectively expressed in the stomach and small intestine and AQP8 mRNA in the jejunum and colon. Immunohistochemistry and in situ hybridization demonstrated cellular localization of these AQP in these portions. AQP1 was localized on endothelial cells of lymphatic vessels in the submucosa and lamina propria throughout the gastrointestinal tract. AQP3 was detected on the circumferential plasma membranes of stratified squamous epithelial cells in the esophagus and basolateral membranes of cardiac gland epithelia in the lower stomach and of surface columnar epithelia in the colon. However, AQP3 was not apparently detected in the small intestine. AQP4 was present on the basolateral membrane of the parietal cells in the lower stomach and selectively in the basolateral membranes of deep intestinal gland cells in the small intestine. AQP8 mRNA expression was demonstrated in the absorptive columnar epithelial cells of the jejunum and colon by in situ hybridization. These findings may indicate that water crosses the epithelial layer through these water channels, suggesting a possible role of the transcellular route for water intake or outlet in the gastrointestinal tract.

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Expression of Viral Proteins in Feline Leukemia Virus-associated Enteritis

Veterinary Pathology ◽

10.1354/vp.37-2-129 ◽

2000 ◽

Vol 37 (2) ◽

pp. 129-136 ◽

Cited By ~ 15

Author(s):

A. Kipar ◽

J. Kremendahl ◽

C. K. Grant ◽

I. von Bothmer ◽

M. Reinacher

Keyword(s):

Epithelial Cells ◽

Viral Infections ◽

Leukemia Virus ◽

Feline Leukemia Virus ◽

Parasitic Infections ◽

Mesenteric Lymph Nodes ◽

Intestinal Crypt ◽

Naturally Occurring ◽

Mesenteric Lymph

Fourteen cases of feline leukemia virus (FeLV)-associated enteritis were immunohistologically examined for the expression of FeLV proteins gp70, p27, and p15E in the jejunum, mesenteric lymph nodes, spleen, and bone marrow. Results were compared with those of FeLV-infected cats without intestinal alterations. Other viral infections and specific bacterial, fungal, and parasitic infections were excluded by standard microbiologic methods, histopathology, immunohistology, and in situ hybridization. In FeLV-associated enteritis, FeLV gp70 and p15E were strongly expressed in intestinal crypt epithelial cells. In contrast, FeLV-positive cats without intestinal alterations showed only faint staining for gp70 and p15E and comparatively strong p27 expression in these cells. Findings suggest a direct relation between FeLV infection and alterations in intestinal crypt epithelial cells that may be attributed to the envelope proteins gp70 and p15E and/or their precursor protein. Distinct similarities to the intestinal changes in the experimentally induced FeLV-feline AIDS syndrome are obvious, suggesting that naturally occurring feline AIDS variants may be responsible for FeLV-associated enteritis.

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Separation and purification of component proteins of the cytochrome P-450-dependent microsomal monooxygenase system by high-performance liquid chromatography

Journal of Chromatography A ◽

10.1016/s0021-9673(01)93877-4 ◽

1989 ◽

Vol 476 ◽

pp. 299-308 ◽

Cited By ~ 2

Author(s):

Hisaaki Taniguchi ◽

Walter Pyerin

Keyword(s):

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

High Performance ◽

Monooxygenase System ◽

Separation And Purification ◽

Microsomal Monooxygenase ◽

Cytochrome P 450

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Sucrase-isomaltase gene expression along crypt-villus axis of human small intestine is regulated at level of mRNA abundance

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1992.262.1.g123 ◽

1992 ◽

Vol 262 (1) ◽

pp. G123-G130 ◽

Cited By ~ 5

Author(s):

P. G. Traber ◽

L. Yu ◽

G. D. Wu ◽

T. A. Judge

Keyword(s):

Gene Expression ◽

Small Intestine ◽

In Situ Hybridization ◽

Epithelial Cells ◽

Stem Cell Population ◽

Specific Gene ◽

Mrna Levels ◽

Human Small Intestine ◽

Crypt Cells

The mucosal lining of the small intestine is a complex epithelium that is continually renewed by division of a stem cell population located in intestinal crypts, migration of daughter cells along the villus, and, finally, extrusion of senescent cells into the lumen. The majority of cells in both crypt and villus cell compartments are enterocytes that acquire differentiated functions as they migrate out of the crypt. Sucrase-isomaltase (SI) is an enterocyte-specific, brush-border enzyme that has little activity in crypt cells and maximal activity in low and mid villus cells. The mechanism by which enterocytes acquire SI enzymatic activity as they move from crypt to villus is controversial. In this study we examined the distribution of SI mRNA along the crypt-villus axis of human small intestine using isolated epithelial cells and in situ hybridization. A complementary DNA to the 5' portion of the human SI mRNA was amplified and cloned using the polymerase chain reaction. Hybridization analysis of RNA extracted from human intestinal epithelial cells showed that the cloned cDNA recognized a single 6.5-kb mRNA. In situ hybridization of duodenal biopsy specimens was performed using a single-stranded RNA probe derived from this cDNA. This analysis showed that there was little SI mRNA in crypt cells and appearance of mRNA in enterocytes located at the crypt-villus junction. The mRNA levels were maximal in lower and mid villus cells with decreased levels noted in villus tip cells. These results are identical to those previously described in rat intestine and suggest that expression of the SI gene as enterocytes emerge from intestinal crypts is regulated primarily at the level of mRNA accumulation. Study of SI gene regulation may provide a useful model to investigate the mechanisms that regulate enterocyte-specific gene expression and intestinal differentiation.

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Electron probe x-ray microanalysis and electron microscopy of amino acid absorption and transport by the small intestine: inhibition by chemical poisons and correlation to the elemental composition of the epithelial cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142311 ◽

1986 ◽

Vol 44 ◽

pp. 134-135

Author(s):

A. J. Tousimis

Keyword(s):

Small Intestine ◽

Amino Acid ◽

Epithelial Cells ◽

Elemental Composition ◽

Isotope Labeling ◽

Structural Components ◽

X Ray ◽

Human Small Intestine ◽

Transport Studies

The elemental composition of amino acids is similar to that of the major structural components of the epithelial cells of the small intestine and other tissues. Therefore, their subcellular localization and concentration measurements are not possible by x-ray microanalysis. Radioactive isotope labeling: I131-tyrosine, Se75-methionine and S35-methionine have been successfully employed in numerous absorption and transport studies. The latter two have been utilized both in vitro and vivo, with similar results in the hamster and human small intestine. Non-radioactive Selenomethionine, since its absorption/transport behavior is assumed to be the same as that of Se75- methionine and S75-methionine could serve as a compound tracer for this amino acid.

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The protease phenotype and crystalline nature of the granules of intraepithelial mast cells in Trichinella spiralis-infected mice

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140464 ◽

1995 ◽

Vol 53 ◽

pp. 818-819

Author(s):

D.S. Friend ◽

N. Ghildyal ◽

M.F. Gurish ◽

K.F. Austen ◽

R.L. Stevens

Keyword(s):

Small Intestine ◽

Mast Cells ◽

Epithelial Cells ◽

Lamina Propria ◽

Trichinella Spiralis ◽

Intestinal Epithelial ◽

Carboxypeptidase A ◽

C3h Mice ◽

Granule Morphology ◽

Crystalline Nature

Trichinella spiralis induces a profound mastocytosis and eosinophilia in the small intestine of the infected mouse. Mouse mast cells (MC) store in their granules various combinations of at least five chymotryptic chymases [designated mouse MC protease (mMCP) 1 to 5], two tryptic proteases designated mMCP-6 and mMCP-7 and an exopeptidase, carboxypeptidase A (mMC-CPA). Using antipeptide, protease -specific antibodies to these MC granule proteases, immunohistochemistry was done to determine the distribution, number and protease phenotype of the MCs in the small intestine and spleen 10 to >60 days after Trichinella infection of BALB/c and C3H mice. TEM was performed to evaluate the granule morphology of the MCs between intestinal epithelial cells and in the lamina propria (mucosal MCs) and in the submucosa, muscle and serosa of the intestine (submucosal MCs).As noted in the table below, the number of submucosal MCs remained constant throughout the study. In contrast, on day 14, the number of MCs in the mucosa increased ~25 fold. Increased numbers of MCs were observed between epithelial cells in the mucosal crypts, in the lamina propria and to a lesser extent, between epithelial cells of the intestinal villi.

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