Rabbit Small Intestinal Mucosa Research Articles

Effect of Cholera Toxin on ATPase Activities in Rabbit Small Intestinal Mucosa

Cholera toxin induced the appearance of ATPase activity in rabbit small intestinal mucosa. This enzyme significantly differed from other ATPases, including Na(+),K(+)-ATPase and HCO3(-)-ATPase in the small intestinal epithelium of rabbits, by some properties, in particular, by relation to divalent and monovalent cations and anions, pH optimum, substrate specificity, and inhibitory analysis.

Development of Buccoadhesive Systems of Pentarocine for Systemic Drug Delivery

Bucoadhesive patches of Pentazocine (PZ) for unidirectional drug delivery were prepared by casting carboxy methyl cellulose (CMC) with glycerol or propylene glycol and CMC-hydroxy ethyl cellulose (HEC) with glycerol. In vitro mucoadhesivity of the prepared patches were determined using a modified mucoadhesive bond strength apparatus using rabbit small intestine mucosa (SIM). Drug release kinetics was evaluated from composite patches, prepared by covering all but one side of the PZ patches with 3M backing material. Biocompatability / buccoadhesion time and in vivo permeation of placebo and PZ loaded patches were determined using a double blind cross over study in healthy human volunteers. Drug release from CMC-glycerol patches and pure HEC patches showed zero order kinetics with diffusional exponent (n) ranging between 0.79 to 1.046, while that from CMC-HEC and CMC-propylene glycol patches showed an apparent zero order release kinetics. The prepared patches were well tolerated by the human volunteers as they did not produce any side effects at the contact surface. The in vitro mucoadhesivity of CMC-propylene glycol patches were significantly lower than CMC- glycerol based patches. The in vivo permeation of selected PZ patches delivered the drug well above the minimum buccal permeation rate, so as to attain effective blood concentration

A Study of Mucoadhesive Bond Strength of Buccoadhesive Compacts for Systemic Drug Delivery: In-vitro/ In-vivo correlation

Compacts prepared from binary combinations of Carbopol® 934 P (CP), Polycarbophil (Noveon® AA1, PC) and Hydroxy propyl cellulose (Klucel®, HPC) and coated on all but one flat surface with Poly Methyl Methacrylate - PMMA (chloroformic solution) were evaluated for mucoadhesive bond strength on a modified mucoadhesive bond strength apparatus using rabbit stomach mucosa (SM) and small intestine mucosa (SIM). In -vitro mucoadhesion tests indicated that the detachment force increased linearly with concentration of CP/PC in the compacts. Mucoadhesion of the compacts with SIM were higher when compared to SM. The compacts with higher proportions of CPIPC showed longer buccoadhesion time (time the compact remained in contact with the buccal mucosa) than HPC alone in humans. In-vivo buccoadhesevity of the coated compacts was studied in healthy human volunteers. An index was used to study the redness and ulceration of the contact buccal mucosa.
 Compacts with higher proportions of CP/PC showed longer buccoadhesion time than HPC alone. Significant correlation coefficient (r) values (P<0.01) were obtained between in-vitro fracture strength of the compacts and in-vivo buccoadhesion time. Hence, the in-vitro mucoadhesive model developed by us provides useful information on the residence time of the compact for systemic drug delivery in the oral cavity, and compacts containing less than 50% of CP/PC were safer to use in humans.

Purification and characterization of two new cytochrome P-450 related to CYP2C subfamily from rabbit small intestine microsomes

Two forms of cytochrome P-450, designated P-450id and P-450ie, were purified to specific contents of 14.3 and 15.0 nmol of P-450/mg of protein, respectively, from small intestine mucosa microsomes of rabbits. P-450id and P-450ie showed apparent molecular weights of 50 and 49 kDa, respectively, on SDS-PAGE. Both P-450s catalyzed N-demethylation of nitrosodimethylamine. The NH 2-terminal amino acid sequence (first 19 residues) of P-450id exhibited 74–90% identity with those of six members of the rabbit P-450 2C subfamily, except for P-450 2C3. Similarly, the NH 2-terminal sequence (first 22 residues) of P-450ie showed 73–86% identity with those of the same members of the rabbit P-450 2C subfamily. The peptide mapping patterns of the two P-450s were quite different from each other. In addition, P-450id did not cross-react with the guinea-pig antibodies against P-450ie. The results indicate that rabbit small intestine mucosa contain two new distinct forms of P-450s, both of which may be classified into the 2C subfamily.

Molecular cloning of diadenosine tetraphosphatase from pig small intestinal mucosa and identification of sequence blocks common to diadenosine polyphosphate hydrolases and phosphorylases

Diadenosine 5′,5‴- P 1, P 4-tetraphosphate (Ap 4A) pyrophosphohydrolase is the enzyme responsible for reducing intracellular levels of the stress-responsive nucleotide diadenosine 5′,5‴- P 1, P 4-tetraphosphate. In order to gain more information on the relationships between the enzymes hydrolysing diadenosine polyphosphates in different eukaryotes, the Ap 4A hydrolase and a corresponding cDNA have been isolated from pig small intestinal mucosa by standard procedures. The enzyme is a typical mammalian Ap 4A hydrolase ( K m = 0.8 μM), being sensitive to inhibition by fluoride ( K i = 24 μM) and adenosine 5′-tetraphosphate ( K i = 10 nM) and yielding ATP and AMP as products. A low K m Ap 4A hydrolase ( K m = 0.3 μM) was also isolated from rabbit small intestinal mucosa. These enzymes differ from the rat intestinal mucosal hydrolase, which has much higher values of K m for Ap 4A and K i for adenosine 5′-tetraphosphate. A cDNA encoding the pig enzyme was isolated from a pig ileum cDNA library. The derived amino acid sequence of the 16.8 kDa gene product shows 88% identity and 96% similarity to that of the human enzyme. The sequence has the same modification of the MutT motif found in the human enzyme in which a threonine residue replaces a hydrophobic amino acid. Sequence comparisons among eukaryotic diadenosine polyphosphate hydrolases and phosphorylases reveal two blocks of amino acid similarity, including a motif, Z[AD]Gx[ED]AGQ, which may be involved in polyphosphate binding by the hydrolases, and an invariant histidine residue that may be involved in catalysis. These sequence similarities may have arisen by convergent evolution.

Expression of the Na+ dependent uridine transport system of rabbit small intestine: studies with mRNA-injected Xenopus laevis oocytes.

Xenopus laevis oocytes were used as an expression system to prove and characterize the carrier-mediated transport of uridine in the small intestine. Significant Na+ dependency was observed for the uptake of [3H]uridine by Xenopus laevis oocytes injected with poly(A)+RNA prepared from rabbit small intestinal mucosa. By contrast, the uptake of [3H]uridine was negligible in water-injected oocytes. There was no significant difference in the Na+ dependent uptake rates of [3H]uridine among oocytes expressed by using mRNA prepared by three different methods. The uptake of [3H]uridine by mRNA-injected oocytes was enhanced by increasing the culturing time after mRNA injection. Concentration dependency for uridine transport was observed with the Michaelis constant of 8.27 microM, which was comparable to that reported in the study using the brush-border membrane vesicles from rabbit small intestine (6.4 microM). Furthermore, the uptake of [3H]uridine was significantly inhibited by adenosine and thymidine, but not by adenine and uracil. Consequently, the transport system of uridine expressed in mRNA-injected oocytes is clarified to be similar to that functioning in the brush-border membrane of the small intestine.

Confirmation of Carrier-Mediated Intestinal Transport of β-Lactam Antiboitics by Using Gene Expression in <I>Xenopus laevis</I> Oocytes

In the present study, to prove the carrier-mediated transport in the intestinal absorption for orally active β-lactam antibiotics, Xenopus laevis oocytes were employed as exogenous gene expression system. It was demostrated that the uptake of zwitterionic cefadroxil (CDX) was increased by Xenopus laevis oocytes injected with poly(A)+RNA derived from rat, rabbit or human small intestinal mucosa. The uptake of CDX was inhibited by other orally active β-lactam antibiotics and dipeptides significantly, but not by amino acid. Furthermore at acidic extracellular pH, the uptakes of amino-β -lactam antibiotics, CDX, cephalexin and cephradine increased, suggesting that H+ gradient is a driving force for transports of these antibiotics. By size-fractionation of poly(A)+RNA on a sucrose density gradient centrifugation, poly(A)+RNA of which size is approximately 3 kb was identified to encode dipeptide transporter. Photoaffinity labeling of membrane protein with [3H]benzylpenicillin showed an expression of the specific binding protein with a molecular mass of 130 kDa. Stereospecific transport of ceftibuten with cis and trans-isomers was also exhibited in oocytes injectedwith poly(A)+RNA. From these results it was clarified that β-lactam antibiotics are absorbed in the small intestine by a H+ gradient dependent carrier-mediated mechanism which is common to dipeptide transporter.

Expression of the rabbit intestinal N2 Na+/nucleoside transporter in Xenopus laevis oocytes

Polyadenylated [poly(A)+] mRNA isolated from rabbit small-intestinal mucosa was injected into Xenopus laevis oocytes, and expression of the N2 Na+/nucleoside co-transporter was assayed by measuring Na(+)-dependent thymidine uptake. Expression of Na(+)-dependent thymidine uptake steadily increased after mRNA injection and was on average increased 11-fold by day 6 over background. Na(+)-dependent thymidine uptake was saturable (apparent Km approximately 30 microM at 22 degrees C) and inhibited by uridine and cytidine, but not by guanosine and inosine. These properties of the expressed thymidine transport strongly suggest that the epithelial N2 Na+/nucleoside co-transporter can be expressed in X. laevis oocytes.

A novel species of cytochrome P-450 (P-450ib) specific for the small intestine of rabbits. cDNA cloning and its expression in COS cells.

We have isolated a cDNA clone for a P-450, designated P-450ib (Ichihara, K., Kusunose, E., Kaku, M., Yamamoto, S., and Kusunose, M. (1985) Biochim. Biophys. Acta 831, 99-105), from a cDNA library of rabbit small intestine mucosa by using synthetic DNA fragment by the polymerase chain reaction, as a hybridization probe. The cDNA with a 1,829-base pair insert encodes a polypeptide of 501 amino acids. The deduced amino acid sequence contains all of the sequences of the NH2-terminal and 14 tryptic fragments from purified P-450ib. As the NH2-terminal methionine was not found in the sequence from the purified protein, the apoprotein of P-450ib is composed of 500 amino acids with a molecular weight of 57,193. P-450ib shows 35-41% sequence similarity with several members of 8 subfamilies in the P-450 II family, whereas it has a less than 30% sequence similarity with other P-450 families, suggesting that this P-450 is the first member of a novel subfamily within the P-450 II family. RNA blot analysis shows that mRNA hybridized to the cDNA is expressed in the small intestine, but not significantly in other tissues including liver, colon, kidney, lung, spleen, brain, stomach, and cecum, indicating that P-450ib is a P-450 specific to the small intestine. The protein expressed in COS-7 cells using the cDNA in an expression vector, pKCRH2, shows benzphetamine N-demethylase activity and gives a band identical with that of P-450ib in its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Somatostatin content and binding in small intestinal mucosa from fed, fasted, and refed rabbits

The present study is an investigation of the effects of 12- to 96-hours' starvation and 96-hours' starvation plus 48-hours' refeeding on both somatostatin-like immunoreactivity (SLI) and cytosolic somatostatin binding sites in rabbit small intestinal mucosa. The SLI concentration increased after 24 h in duodenal and jejunal mucosa, but not in ileal mucosa, and reached its highest value after 96 h of fasting. The number of specific high and low-affinity somatostatin binding sites, but not their affinity, decreased with the duration of fasting in the same gut segments, refeeding of fasted animals resulted in a return to normal control values for small intestine mucosal SLI and somatostatin binding.

Adherence targets of Vibrio parahaemolyticus in human small intestines

Formalin-fixed human small intestinal mucosa with mucus coating, villi, and lymphoid follicle epithelium at the mucosal surface was used to test the adherence sites of clinically isolated (Kanagawa phenomenon-positive) strains of Vibrio parahaemolyticus. V. parahaemolyticus strains grown on CFA agar (supplemented with 3% NaCl) for ca. 3 h at 37 degrees C possessed various levels of cell-associated hemagglutinins (HAs) which were detected with human or guinea pig erythrocytes. The observed adherence abilities of V. parahaemolyticus strains to human small intestinal mucosa correlated roughly with the HA levels of the strains. Under the test conditions, ileal lymphoid follicle epithelium (especially M cells) provided the best adherence target for V. parahaemolyticus. Adherence to villus absorptive cells or to mucus coating was observed at lower levels. In addition, all 3-h-grown V. parahaemolyticus strains tested produced high levels of HAs as detected with rabbit erythrocytes. The strains were all strikingly motile. In contrast, V. parahaemolyticus strains grown on CFA agar (supplemented with 3% NaCl) for ca. 20 h at 37 degrees C had much lower levels of HAs, adherence abilities, and motility. In contrast to the above observations, piliation of V. parahaemolyticus was more extensive at ca. 20 h of incubation at 37 degrees C than at ca. 3 h of incubation at 37 degrees C. The remarkable ability of V. parahaemolyticus to adhere to lymphoid follicle epithelium was also confirmed by using rabbit small intestinal mucosa.

Flow cytometry, a very useful technique for the characterization of intestinal membrane vesicles.

The plasma membrane of enterocytes comprises two structurally and functionally distinct domains. These are the apical brush border, containing digestive hydrolases and glycocalyx, and the basolateral domain, characterized by other specific markers. Using a fast and easy subcellular fractionation, we purified four membrane vesicle fractions from rabbit small intestinal mucosa: brush border, basolateral, rough endoplasmic reticulum and Golgi + smooth endoplasmic reticulum. Using flow cytometry, the fluorescence polarization of diphenylhexatriene was determined in brush border and in basolateral + Golgi + smooth endoplasmic reticulum membrane fractions in order to investigate changes in the membrane fluidity of both fractions and to compare the results obtained with those of spectroscopic techniques. Moreover, it was possible with flow cytometry to detect and quantify basolateral and brush border markers by using polyclonal and monoclonal antibodies. The advantages of flow cytometry in the detection of brush border membrane markers found in small amounts in the basolateral domain are discussed. Finally, flow cytometry holds great promise for the analysis and sorting of subcellular fractions.

Purification and characterization of cytochrome P-450 specific for prostaglandin and fatty acid hydroxylase activities from the microsomes of rabbit small intestinal mucosa.

Earlier studies (Kusunose, E., Kaku, M., Ichihara, K., Yamamoto, S., Yano, I., & Kusunose, M. (1984) J. Biochem. 95, 1733-1739) showed that a form of cytochrome P-450 isolated from microsomes of rabbit small intestinal mucosa had the highest prostaglandin A1 (PGA1) hydroxylase activity so far reported among cytochrome P-450s. The present paper describes the procedure for the purification and further characterization of this cytochrome (designated as cytochrome P-450ia). Cytochrome P-450ia had a monomeric molecular weight of 53,000. The CO-difference spectra of its reduced form showed a maximal absorption at 451 nm, and the absolute spectra of its oxidized form indicated that cytochrome P-450ia was present largely in the low-spin state, and partially in the high-spin state. The cytochrome efficiently catalyzed the hydroxylation of fatty acids as well as prostaglandins in a reconstituted system containing cytochrome P-450, NADPH-cytochrome P-450 reductase, phospholipid, and cytochrome b5. PGA1 was the most efficient substrate, followed by myristate, laurate, palmitate, caprate, and PGE1 or PGE2. Among phospholipids, didecanoyl- and dilauroylphosphatidylcholines had the most stimulatory effect for both activities. 20-Hydroxy PGA1 was identified as the hydroxylation product of PGA1 by gas chromatography-mass spectrometry and mass fragmentography; the possibility of 19-hydroxy PGA1 being the product was excluded. In contrast, both omega- and (omega-1)-hydroxy fatty acids were identified as hydroxylation products of fatty acids. Cytochrome P-450ia had no detectable activity toward aminopyrine, benzphetamine, p-nitroanisole, 7-ethoxycoumarin, benzo(a)pyrene, or hexadecane.

Hydroxylation of prostaglandin A1 by the microsomes of rabbit intestinal mucosa.

Microsomes from rabbit small intestine mucosa were found to catalyze the hydroxylation of PGA1 in the presence of NADPH. The major product was identified as 20-hydroxy PGA1 by using high performance liquid chromatography and gas chromatography-mass spectrometry, and the minor product was assumed to be 19-hydroxy PGA1. The ratio of the former product to the latter was about 24.1. The specific PGA1 omega-hydroxylase activity of small intestine microsomes was comparable to that of liver microsomes, and was significantly higher than those of microsomes from other tissues such as kidney cortex and lung. Microsomes from rabbit colon mucosa also catalyzed the hydroxylation of PGA1 in the presence of NADPH, with the ratio of omega- to (omega-1)-hydroxy PGA1 formed being 33.0. The PGA1 hydroxylase activities of the microsomes from both small intestine and colon were inhibited markedly by carbon monoxide, indicating the participation of cytochrome P-450. A cytochrome P-450 was solubilized from small intestine microsomes, and purified to a specific content of 10.5 nmol of cytochrome P-450/mg of protein. This cytochrome hydroxylated PGA1 at the omega-position with a turnover rate of 38.2 nmol/min/nmol of cytochrome P-450 in the reconstituted system containing cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and phosphatidylcholine. It is suggested that this cytochrome P-450 is specialized for the omega-hydroxylation of PGA1 in small intestine microsomes.

Effect of bivalent cations on properties of NaCl-stimulated ATPase activity in rabbit small intestinal mucosa

Effect of bivalent cations on properties of NaCl-stimulated ATPase activity in rabbit small intestinal mucosa

Isolation and properties of the catalytic subunit of cyclic AMP-dependent protein kinase from rabbit small intestinal mucosa

The protein kinases constitute a large class of enzymes catalyzing the phosphorylation of protein substrates and performing important regulatory functions in the cell. There are several different types of protein kinases, differing in their method of regulation and in their substrate specificity. There are cyclic AMPand cyclic GMP-dependent protein kinases, and also protein kinases independent of the action of cyclic nucleotides.

Properties of rabbit intestinal fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphatase has been isolated in high yield from rabbit intestinal mucosa by Blue Sepharose chromatography. The isolated enzyme preparation was homogeneous in gel electrophoresis and sedimentation analysis. The molecular weight and subunit molecular weight of the enzyme were 145,000 and 37,000, respectively. Intestinal fructose 1,6-bisphosphatase could not be distinguished from the liver enzyme. The amino acid composition of the intestinal enzyme was generally similar to that of the liver enzyme. No tryptophan residue was detected. Rabbit liver enzyme cross-reacted with the antiserum against rabbit intestinal enzyme, whereas mouse intestinal enzyme did not react with the antiserum against mouse liver enzyme. The purified intestinal fructose 1,6-bisphosphatase hydrolyzed ribulose 1,5-bisphosphate ( K m = 21 μM), as well as fructose 1,6-bisphosphate ( K m = 2.6μM) and sedoheptulose l,7-bisphosphate ( K m = 8.0 μM). The K m values of mouse intestinal enzyme for the three ketose bisphosphates were different from those of mouse liver enzyme. The K i value of rabbit intestinal fructose 1,6-bisphosphatase for AMP was 17 μ m in the presence of 2 m m Mg 2+ at pH 7.5, and the Hill coefficient for AMP was 1.72. The total activity of rabbit intestinal fructose 1,6-bisphosphatase was approximately 30% of that of the enzyme in rabbit liver, and approximately threefold higher than that of the enzyme in mouse intestinal mucosa. The amount of the enzyme in rabbit small intestinal mucosa was around 30% of that in rabbit liver, while the total amount of mouse intestinal enzyme was approximately 10% of mouse liver enzyme.

Model for mucosal circulation of rabbit small intestine.

A theoretical model of the vascular network of normal rabbit small intestine mucosa was constructed from tissues injected with silicone latex. The segmental resistances of the network were calculated from Poiseuille's law by use of lengths and radii measured microscopically and a value of 0.03 P for the viscosity of blood. Where required for the theoretical calculations, published values were used for pressures in arteries down to 20 micrometer in diameter. Values for total-organ blood flow, its distribution between mucosa and nonmucosa, and the partition of mucosal flow between crypts and villi were similar to values measured in the same animals with microspheres. Blood pressures in arteries smaller than 20 micrometer in diameter were also similar to published values. Mean mucosal capillary pressures were close to published experimental estimations. The model seems to provide a useful starting point for describing the magnitudes and patterns of pressures and flow in the small intestine and for consideration of specific location of the sites of their control.

Specific Inhibition of Cell Proliferation In the Mouse Intestine By an Aqueous Extract of Rabbit Colon

ABSTRACTAqueous extracts from rabbit colon, kidney, testis and small intestinal mucosa were prepared by homogenization and centrifugation at 105,000 g. After precipitation with ammonium sulphate. the 0–50 fraction (F1) and the supernatant (F2) were collected, dialysed against a phosphate buffer and tested on mice in vivo.1 hr after a single injection of F1 (15 mg content) from colon, the uptake of tritiated thymidine was decreased in jejunal and colonic DNA in mice. This effect, maximal after 3 hr and totally reversible after 7 hr, was found in neither the kidney nor the testis. the F1 fractions of non‐digestive organs (kidney, testis) were also found to exert a significant inhibition on thymidine incorporation into intestinal DNA in vivo. F1 fractions of intestinal contents, prepared under the same conditions, exerted no significant effects on DNA synthesis in mouse intestine. Conversely, the colon F2 fraction did not inhibit the synthesis of jejunal and colonic DNA in vivo. A slowing of cellular migration was also noticed in the jejunum and colon of mice injected with colon or small intestine F1, as ascertained radioautographically by determining the position of the leading edge of the labelled cells in jejunal or colonic F1‐injected mice.Our results suggest that the F1 fraction of the aqueous extract of rabbit colon contains one or more substances, which may act either on intestinal DNA synthesis or on the G1‐S transition of the cellular cycle in the mouse intestine. This reversible and tissue‐specific intestinal action appears to inhibit cell proliferation and presents several of the characteristics defining a chalone, as does the action of small intestinal F1 previously reported (Sassier & Bergeron, 1977). However, because of a relative lack of origin specificity of this effect, the physiological significance of our data remains to be ascertained.

50: Peptidases of brush border of human and rabbit small intestinal mucosa

Three peptidase activities were found to be present in human and rabbit small intestinal mucosa: the aminopeptidase A, hydrolyzing the α-L-glutamyl-B-naphthylamide (BNA); the dipeptidyl aminopeptidase IV, hydrolyzing glycyl-L-proline from glycyl-L-prolyl-BNA; a carboxypeptidase, hydrolyzing N-CBZ-L-prolyl-L-alanine. These enzymatic activities are almost totally localized in the brush border and have maximal activity in the distal ileum: they were due to three different enzymes, which have been separated each from the other, as well as from the oligoaminopeptidase (hydrolyzing L-leucyl-BNA), and partially purified. The identification of these new peptidases of the brush border demonstrates a previously unrecognized importance of this subcellular organelle in the digestion of proteins and peptides, in a complementary way to intraluminal and intracellular digestion.