A novel marker glycoprotein for the microvillus membrane of surface colonocytes of rat large intestine and its presence in small-intestinal crypt cells.

Murine mAbs were produced against purified microvillus membranes of rat colonocytes in order to establish a marker protein for this membrane. The majority of antibodies binding to the colonic microvillus membrane recognized a single protein with a mean apparent Mr of 120 kD in both proximal and distal colon samples. The antigen is membrane bound as probed by phase-partitioning studies using Triton X-114 and by the sodium carbonate extraction procedure and is extensively glycosylated as assessed by endoglycosidase F digestion. Localization studies in adult rats by light and electron microscopy revealed the microvillus membrane of surface colonocytes as the principal site of the immunoreaction. The antigen was not detectable in kidney or liver by immunoprecipitation but was present in the small intestine, where it was predominantly confined to the apical membrane of crypt cells and much less to the microvillus membrane of differentiated enterocytes. During fetal development, the antigen appears first in the colon at day 15 and 1-2 d later in the small intestine. In both segments, it initially covers the whole luminal surface but an adult-like localization pattern develops soon after birth. The antibodies were also used to develop a radiometric assay for the quantification of the antigen in subcellular fractions of colonocytes in order to assess the validity of a previously developed method for the purification of colonic brush-border membranes (Stieger, B., A. Marxer, and H.P. Hauri. 1986. J. Membr. Biol. 91:19-31.). The results suggest that we have identified a valuable marker glycoprotein for the colonic microvillus membrane, which in adult rats may also serve as a marker for early differentiation of enterocyte progenitor cells in small-intestinal crypt cells.

Localization of actin and microfilament-associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy.

Indirect immunofluorescence microscopy was used to localize microfilament-associated proteins in the brush border of mouse intestinal epithelial cells. As expected, antibodies to actin decorated the microfilaments of the microvilli, giving rise to a very intense fluorescence. By contrast, antibodies to myosin, tropomyosin, filamin, and alpha-actinin did not decorate the microvilli. All these antibodies, however, decorated the terminal web region of the brush border. Myosin, tropomyosin, and alpha-actinin, although present throughout the terminal web, were found to be preferentially located around the periphery of the organelle. Therefore, two classes of microfilamentous structures can be documented in the brush border. First, the highly ordered microfilaments which make up the cores of the microvilli apparently lack the associated proteins. Second, seemingly less-ordered microfilaments are found in the terminal web, in which region the myosin, tropomyosin, filamin and alpha-actinin are located.

Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon.

The primary structure of human apolipoprotein (apo) B-48 has been deduced and shown by a combination of DNA excess hybridization, sequencing of tryptic peptides, cloned complementary DNAs, and intestinal messenger RNAs (mRNAs) to be the product of an intestinal mRNA with an in-frame UAA stop codon resulting from a C to U change in the codon CAA encoding Gln2153 in apoB-100 mRNA. The carboxyl-terminal Ile2152 of apoB-48 purified from chylous ascites fluid has apparently been cleaved from the initial translation product, leaving Met2151 as the new carboxyl-terminus. These data indicate that approximately 85% of the intestinal mRNAs terminate within approximately 0.1 to 1.0 kilobase downstream from the stop codon. The other approximately 15% have lengths similar to hepatic apoB-100 mRNA even though they have the same in-frame stop codon. The organ-specific introduction of a stop codon to a mRNA appears unprecedented and might have implications for cryptic polyadenylation signal recognition and RNA processing.

Dopamine protects mice against whole-body irradiation.

Injection of dopamine before whole-body x-irradiation of mice resulted in 80 percent survivors whereas no irradiated controls survived; injection after exposure had no effect. D,L-Dihydroxyphenylalanine, the precursor of dopamine, had no effect on survival when injected either before or after irradiation.

Molecular characterization of a functional cDNA encoding the rat substance P receptor.

Substance P is a member of the tachykinin peptide family and participates in the regulation of diverse biological processes. The polymerase chain reaction and conventional library screening were used to isolate a complementary DNA (cDNA) encoding the rat substance P receptor from brain and submandibular gland. By homology analysis, this receptor belongs to the G protein-coupled receptor superfamily. The receptor cDNA was expressed in a mammalian cell line and the ligand binding properties of the encoded receptor were pharmacologically defined by Scatchard analysis and tachykinin peptide displacement as those of a substance P receptor. The distribution of the messenger RNA for this receptor is highest in urinary bladder, submandibular gland, striatum, and spinal cord, which is consistent with the known distribution of substance P receptor binding sites. Thus, this receptor appears to mediate the primary actions of substance P in various brain regions and peripheral tissues.

Sources of cyclic nucleotides in plasma.

In order to determine the sites of net production and removal of the cyclic nucleotides in plasma, various blood vessels were catheterized in 17 anesthetized dogs and arterial and venous concentrations of adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5'-monophosphate (cGMP) were measured by radioimmunoassay. Aortic cAMP was 30+/-2 nM (mean+/-SE) and cGMP was 13+/-1 nM. There were no significant differences for either cyclic nucleotide between the concentration in the aorta and that in the inferior vena cava, coronary sinus, hepatic vein, and femoral vein. The concentration of cAMP in renal venous plasma was 25% lower than in aortic plasma, and renal venous cGMP was 51% lower than in the aorta. The pulmonary arterial concentrations of cAMP and cGMP were slightly lower than in the aorta. The concentration of cGMP in the superior mesenteric vein plasma was 83% greater than in aortic plasma; the concentration of cAMP in this vessel was only 16% greater than that in the aorta. Superior vena cava concentrations of both cyclic nucleotides were slightly greater than arterial concentrations. THE RESULTS SUGGEST THAT: (a) the kidneys are a major site of removal of both cyclic nucleotides from plasma. (b) The lungs may be a site of net addition of both cyclic nucleotides to plasma. (c) The small intestine is a site of net production of both cyclic nucleotides, particularly cGMP. (d) The liver probably removes cyclic nucleotides from plasma. (e) Since no other organs or regions studied added detectable net amounts of cyclic nucleotides to plasma, and since the turnover of these compounds in plasma is known to be rapid, the production of plasma cyclic nucleotides under basal conditions may well be the result of small net contributions may well be the result of small net contributions from many tissues or bidirectional fluxes between tissues and plasma, or both.

Glycolipids of rat large intestine. Characterization of a novel blood group B-active tetraglycosylceramide absent from small intestine.

A novel blood group B-active tetraglycosylceramide has been isolated from rat large intestine. It is probably present only in the epithelial cells. Although the compound was not obtained pure, the structure was conclusively established by mass spectrometry, proton NMR spectroscopy and degradative studies to be Galp alpha 1 leads to 3Galp(2 comes from 1 alpha Fucp)beta 1 leads to 4Glcp beta 1 leads to 1Cer. The ceramide was composed of mainly trihydroxy long-chain base (18:0) and non-hydroxy fatty acids (16:0-24:0). The glycolipid was absent from small intestine.

Identification of a novel hepraglycosylceramide with two fucose residues and a terminal hexosamine.

A polar fucose-containing glycosphingolipid fraction isolated from dog small intestine has been characterized by mass spectrometry of intact methylated, and methylated and reduced (LiAlH4) glycolipid. The native fraction, which was homogenous on thin-layer chromatography, was shown after methylation to be a mixture of two compounds. One was identified as a hexaglycoslyceramide with the following composition and sequence: fucose-hexose(fucose)-hexosamine-hexose-hexose-ceramide, with a terminal saccharide structure similar to blood group Leb determinants. The second compound was a novel heptaglycosyceramide with the sequence: hexosamine(fucose)-hexose-tfucose)-hexosamine-hexose-hexose-ceramide. This glycolipid was also detected in human small intestine and pancreas. The dog intestinal fraction had phytosphingosine as its major base and contained almost exclusively 2-hydroxy fatty acids (16 : 0--24 : 0). The fraction of human pancreas differed in having spingosine as its major base and normal fatty acids (16 : 0--24 :0) as major acids.

Rat proximal small intestinal Golgi membranes: lipid composition and fluidity.

The present studies were conducted to examine and characterize the lipid composition and physical state of the membrane lipids of rat proximal small intestinal Golgi membranes. Golgi membranes were purified from isolated enterocytes; lipids were extracted from these membranes and analyzed by thin-layer and gas-liquid chromatography. The 'static' and 'dynamic' components of fluidity of Golgi membranes and their liposomes were assessed by steady-state fluorescence polarization techniques utilizing r infinity and S values of 1,6-diphenyl-1,3,5-hexatriene and r values of DL-2-(9-anthroyl)- and DL-12-(9-anthroyl)stearic acid, respectively. Additional studies were also performed on these membranes, using benzyl and methyl alcohol, to examine the relationship between alterations in lipid fluidity and glycosphingolipid glycosyltransferase activities. The results of these studies demonstrated that: (1) the principal phospholipids and neutral lipids of intestinal Golgi membranes, respectively, were phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, and unesterified cholesterol and fatty acids; (2) the major fatty acids of Golgi membranes were palmitic (16:0), stearic (18:0), linoleic (18:2), arachidonic (20:4) and oleic (18:1) acids; (3) fluorescence polarization studies using diphenylhexatriene detected a thermotropic transition at 24-26 degrees C in Golgi membranes and liposomes prepared from lipid extracts of these membranes; (4) benzyl alcohol (25 and 50 mM) but not methyl alcohol (50 mM) significantly increased the fluidity of these membranes; and (5) at these same concentrations, benzyl alcohol was also found to increase significantly the specific activity of UDP-galactosyllactosylceramide galactosyltransferase but not CMP-acetylneuraminic acid: lactosylceramide sialyltransferase. Methyl alcohol was not found to influence either enzyme's activity in these membranes.

Purification and amino acid sequence of vasoactive intestinal peptide, peptide histidine isoleucinamide (1-27) and secretin from the small intestine of guinea pig.

The neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucinamide (1-27) (PHI) and the hormone secretin were purified from the small intestine of guinea pig, being detected by radioimmunoassay and radioreceptor assay throughout six to seven chromatographic steps. After elution on a reverse-phase C18 column, the three peptides were separated on a Fractogel column. After cation-exchange chromatography of each peptide on Mono S, the final steps were performed using a reverse-phase RP8-e column. Guinea pig PHI differed from porcine PHI in having Tyr and Arg residues instead of Phe and Lys in, respectively, position 10 and 20. We confirmed the original sequence of guinea pig VIP previously documented (with Leu5, Thr9, Met19 and Val26). We also established the similarity of the primary structure of guinea pig secretin with that of porcine and bovine.

A new solid-state microelectrode for measuring intracellular chloride activities.

Solid-state microelectrodes from measuring intracellular Cl activity (alphaiCl) were made by sealing the tips of tapered glass capillaries (tip diameter 0.3 mum), coating them under vacuum with a 0.2-0.3 mum thick layer of spectrscopic grade silver, and sealing them (except for the terminal 2-5 mum of the tip) inside tapered glass shields. 106 microelectrodes had an average slope of 55.0+/- 0.6 m V (S,E,) per decade c hange in alphaCl. Tip resistance was (77.1+/- 3.1) x 10(9) omega(n=30). Electrode response was rapid (10-20 s), was unaffected by HCO3, H2PO4, HPO42 or protein, and remained essentially unchanged over a 24-h period. AlphaiCl in frog sartorius muscle fibers and epithelial cells of bullfrog small intestine was measured in vitro. In both tissues, alphaiCl significantly exceeded the value corresponding to equlibrium ditribution of Cl across the cell membrane.

Structural and compositional difference in the neutral glycolipids between epithelial and non-epithelial tissue of the mouse small intestine.

Five major neutral glycolipids, GL-1-GL-5, were isolated from the mouse small intestine. Their structures and distribution were determined by permethylation analysis, sequential degradation with exoglycosidases and/or immunohistochemistry. The molar ratio of GL-1, GL-2, GL-3, GL-4 and Gl-5 in the whole small intestine was 1:0.04:0.03:0.42:0.02. The structures of GL-1 and GL-4 present in epithelial cells were reported previously to be glucosyl ceramide and asialo GM1, respectively (Umesaki, Y., Suzuki, A., Kasama, T., Tohyama, K., Mutai, M. and Yamakawa, T. (1981) J. Biochem. 90, 1731-1738). GL-5, also present in the epithelial cells, was fucosyl asialo GM1, and fucose was shown to be linked to terminal galactose of asialo GM1 in the manner of alpha(1-2) bond. GL-2 and GL-3, present in the residual tissue after scraping the mucosa, were determined to be globoside and Forssman glycolipid, respectively. Both globoside and Forssman glycolipid of the non-epithelial tissue had non-hydroxy fatty acid (C16-C24) in combination with sphingosine (C18) as the ceramide components, in contrast with the ceramide structures of the epithelial glycolipids, which contained alpha-hydroxy fatty acids in combination with phytosphingosine. Immunohistochemical staining using anti-glycolipid antibodies confirmed the distribution of asialo GM1 and fucosyl asialo GM1, and Forssman glycolipid in the epithelial and non-epithelial tissue, respectively.

Bile acids in deermice lacking liver alcohol dehydrogenase.

Liver alcohol dehydrogenase (EC 1.1.1.1) is believed to catalyze the oxidation of 26-hydroxylated intermediates in the biosynthesis of bile acids from cholesterol. We have therefore analyzed the composition and size of the bile acid pool in deer-mice genetically lacking alcohol dehydrogenase. Cholic acid was found to be the major primary bile acid accompanied by small amounts of chenodeoxycholic acid. Variable amounts of secondary bile acids were also present, mainly deoxycholic acid and 3 alpha, 12 alpha-dihydroxy-7-oxo-5 beta-cholanoic acid. The same bile acids were found in animals with normal levels of alcohol dehydrogenase. The pool of bile acids in the gallbladder, small intestine and large intestine varied between 4.2 and 8.4 mumol in four animals lacking alcohol dehydrogenase and between 6.0 and 8.4 mumol in four control animals. Ethanol did not influence pool size or composition of bile acids in the animal studied. It is concluded that alcohol dehydrogenase is not obligatory for normal bile acid biosynthesis.

Membrane fluidity and lipid composition of rat small intestinal brush-border membranes during postnatal maturation.

Fluidity and lipid composition of rat small intestinal brush-border membranes (BBM) were studied during maturation in five age groups: newborns, sucklings (1-3 weeks), weaned (4-6 weeks), juveniles (8-10 weeks), and adults (12 weeks). Brush-border membrane fluidity was measured by steady-state fluorescence polarization. Fluorescent probes used were: 1,6-diphenyl-1,3,5-hexatriene, 1-(4-trimethylammonium)phenyl)-6-phenyl-1,3,5-hexatriene, and a set of n-(9-anthroyloxy) fatty acids. Fluorescence anisotropy measured with all fluorophores was increased in adult versus newborn rats (P less than 0.004). The weight ratio of saturated to cis-unsaturated fatty acids increased from birth to the suckling age (P less than 0.0004). The cholesterol to phospholipid molar ratio increased from birth to the weaned age (P less than 0.0001). Cholesterol to protein ratio and phospholipid to protein ratio decreased after the weaned age (P less than 0.004). The results not only describe maturational changes of brush-border membranes but also give a better understanding of the correlations between biophysical and biochemical data in biological membranes.

Glycosphingolipid patterns of fetal and adult small intestinal mucosa in the sheep.

The ganglioside and neutral glycosphingolipid composition of fetal and adult sheep small intestinal mucosa were characterized and compared. Mono- and tetrahexosylceramide were the major neutral glycolipids of both fetal and adult tissue. Fetal mucosa, however, possessed a higher content of monohexosylceramide than its adult counterpart. Similarly, GD1a, GM3 and GM2 were found to be the principal gangliosides in fetal and adult tissue. Adult intestinal mucosa possessed significant amounts of GT1a not present in fetal tissue. Analysis of the hydroxy and nonhydroxy fatty acids as well as the long-chain bases of the major glycosphingolipids revealed differences between these lipophilic components of glycolipids in fetal and adult intestinal mucosa. The results, therefore, indicate that both quantitative and qualitative differences in glycosphingolipid composition exist between fetal and adult sheep small intestinal mucosa.

Measurement of sodium ion concentration in the unstirred layer of rat small intestine by polymer Na+-sensitive electrodes.

The concentration of sodium ion at the surface of rat small intestine both in vitro and in vivo was measured with plastic polymer sodium ion sensitive electrodes. In vitro the surface sodium ion concentration [Nas+] was found to be significantly higher than bulk bathing solutions of 25 mM concentration. This value could be increased by the addition of glucose to the medium and was significantly higher in the jejunum than in the ileum. Ouabain, deoxycholic acid and dithiothreitol all reduced the [Nas+] under in vitro conditions. In vivo, very high values for [Nas+] were found in the jejunum (approx. 80 mM) when the bulk concentration was 25 mM, indicating a substantial local accumulation of sodium ion at or near the brush border. This could be reduced by omission of glucose from the buffer and further reduced when magnesium was the substituent cation rather than choline. Consequently, in vivo, an appropriately orientated sodium ion gradient persists in the face of adverse tissue to bulk solution concentration gradients and potentially explains why solute absorption occurs under these circumstances. The further reduction of the correctly aligned sodium ion gradient, as required by the gradient hypothesis, by magnesium-substituted buffers and indicates that there is no real need to postulate additionally a sodium-independent magnesium-sensitive glucose transport system in vivo.

Chemical modification of the small intestinal Na+/D-glucose cotransporter by amino group reagents. Evidence for a role of amino group(s) in the binding of the sugar.

Some amino group reagents inactivate the small-intestinal Na+/D-glucose cotransporter, as measured either as a catalyst of Na+-dependent D-glucose transport or as a Na+-dependent phlorizin ligand. The amino group(s) studied in this paper are not identical with those investigated previously (Biber, J., Weber, J. and Semenza, G. (1983) Biochim. Biochim. Acta 728, 429-437): these are protected from inactivation by the simultaneous presence of Na+ plus sugar substrates. They are thus likely to be located within the substrate-binding site.

The subcellular localization of the glycosphingolipids in the epithelial cells of rat small intestine.

Analysis of brush-border and basolateral membrane prepared from the epithelial cells of rat small intestine showed that the brush border contained less of some phospholipids and more glycosphingolipids per protein. The N-glycoloylneuraminosyllactosylceramide was enriched in the brush border, while cholesteryl sulphate and fucosyllactosylceramide were more prominent in the basolateral membrane. By periodate-NaB3H4 labelling of the intact small intestine, it was shown that N-glycoloylneuraminosyllactosylceramide is exposed at the luminal cell surface of the epithelial cell.

Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420.

The presence of cytochromes b5, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c redutases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b5. In addition, cytochrome b5 fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with Mr values in the range 10 000-13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.

Glycosphingolipid patterns of the epithelial and non-epithelial compartments of rat large intestine.

The epithelial cells and the non-epithelial residue from large intestine of two inbred rat strains were separated and the glycosphingolipids characterized in comparison with earlier detailed data from small intestine of the same strains. Total acid and non-acid glycolipids were prepared and the non-acid glycolipids were further fractionated into subgroups as acetylated derivatives on silicic acid. The fractions obtained were characterized mainly by thin-layer chromatography, including binding of monoclonal anti-A and anti-B antibody to the chromatogram, and by direct-inlet mass spectrometry after derivatization. This combined technology allowed an overall conclusion from a small number of animals concerning relative amounts of glycolipids, microheterogeneity of blood group glycolipids and carbohydrate sequence and lipophilic components of major species of each subfraction. As for the small intestine, the two separated compartments differed distinctly in composition, with blood group fucolipids being confined to the epithelial cells, and a series of glycolipids with probably internal Gal alpha being restricted to the non-epithelial part. The main difference between large and small intestine concerned fucolipids of the epithelium. Three blood group B active glycolipids with four, six and seven sugars were detected which were absent from the small intestine. The four-sugar glycolipid was a major glycolipid with the structure Gal alpha 1----3Gal(2----1 alpha Fuc) beta 1----4Glc beta 1----1Cer. as reported before. The six-sugar glycolipid was shown by mass spectrometry and NMR spectroscopy to have the probable structure Gal alpha 1----3Gal(2----1 alpha Fuc)beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1Cer. The seven-sugar glycolipid had an additional fucose linked to N-acetylhexosamine, as shown by mass spectrometry. Three blood group A active glycolipids with four, six and seven sugars were found in both rat strains, with sequences analogous to the B glycolipids but with a terminal GalNAc instead of Gal. The four- and six-sugar blood group A compounds, but not the seven-sugar glycolipid, have been found before in the small intestine of one of the rat strains. In the small intestine, on the other hand, a branched-chain twelve-sugar blood group A active glycolipid has been found which was absent from the large intestine. Therefore large intestine of both rat strains expressed glycolipid-based blood group A and B activity, while small intestine lacked B activity and showed A activity only in one of the strains.(ABSTRACT TRUNCATED AT 400 WORDS)