Rat Small Intestinal Mucosa Research Articles

17β-hydroxysteroid dehydrogenase activity in the mucosa of rat and human small intestine

Although circulating plasma concentrations of androgens are determined in pan by peripheral interconversion in a variety of non-endocrine tissues, the role of the gastrointestinal tract in the peripheral metabolism of testosterone has not been established. Oxidation of testosterone to androstenedione was the predominant conversion by homogenates of rat and human small intestinal mucosa. Reduction of testosterone was not observed. The conversion of testosterone to androstenedione was independent of intestinal bacteria since it was effected by homogenates of germ-free rat jejunal mucosa. The conversion conformed to the kinetics of an enzyme-mediated reaction, with an apparent Michaelis constant for testosterone of 2.8 × 10 −6 M. Assay conditions for the enzyme responsible for this conversion, 17β-hydroxysteroid dehydrogenase (17β-HSD; E.C. 1.1.1.51) were determined. Specific activity of 17β-HSD in the intestinal mucosa was similar to testis and lung but greater than skeletal muscle or ventral prostate. The estimated total 17β-HSD capacity of the rat small intestinal mucosa was considerably greater than both testis and lung, suggesting that the small intestine may make significant contribution to the peripheral oxidative metabolism of testosterone. In addition, the high specific activity of 17β-HSD in the small intestinal mucosa may be an important factor leading to testosterone's lack of oral potency.

Amygdalin (Laetrile) and prunasin beta-glucosidases: distribution in germ-free rat and in human tumor tissue.

Amygdalin, the gentiobioside derivative of mandelonitrile commonly referred to as Laetrile, is presently under intensive investigation as a potential cancer chemotherapeutic agent. Because of this interest, we investigated the activity of beta-glucosidases that cleave glucose from amygdalin and from prunasin (mandelonitrile monoglucoside) in tissues from germ-free rats and in normal and neoplastic human tissues. Rat and human small intestinal mucosa contain high levels of activity of glucosidases that act on both of these cyanogenic glucosides. Release of glucose from these compounds was not detected in any of the human neoplastic tissues examined in the present study. These observations are consistent with reports of cyanide toxicity through the oral use of amygdalin or prunasin and pose serious questions concerning the alleged tumoricidal effect of amygdalin.

Effects of proteolytic enzymes on vitamin B12 uptake by rat intestinal mucosa homogenates and of proteolytic enzymes and anti-rat intrinsic factor antibodies on vitamin B12 absorption in total gastrectomized rats.

Trypsin inhibited 57CoB12-rat IF uptake by rat small intestinal mucosa homogenates in vitro. This tryptic inhibition was inactivated by rat serum and normal human serum, as well as soybean trypsin inhibitor (STI). The effects of trypsin dissolved in distilled water or 0.0025 N HCl, 0.15 M saline extract of rat pancreas and STI on vitamin B12 absorption were studied in vivo, using gastrectomized rats. IF-mediated B12 absorption was significantly decreased by 5 mg of trypsin dissolved either in distilled water or 0.0025 N HCl. However, the inhibition of trypsin was much more remarkable when it was dissolved in 0.0025 N HCl, whereas B12 absorption was insignificantly decreased by administering 10 mg of trypsin dissolved in 0.0025 N HCl. The possibilities of this phenomenon was discussed. The inhibitory effect of 0.5 ml of 0.15 M saline extract of rat pancreas was investigated as well. Soybean trypsin inhibition showed no effect on B12 absorption when it was given with 57CoB12-rat IF at the same time. However, the B12 absorption was significantly increased when STI was given one hour prior to the administration of 57CoB12-rat IF. This suggested that endogenous trypsin bound to rat small intestine had been inactivated and pancreatic secretion had been stimulated by STI. IF-mediated B12 absorption was diminished to almost the same level of 57CoB12 administration alone by simultaneous intubation of both 57CoB12-rat IF and rabbit anti-rat IF serum in total gastrectomized rats.

Methylprednisolone stimulation of guanylate cyclase activity in rat small intestinal mucosa: Possible role in electrolyte transport

A single, intramuscular dose of the glucocorticoid methylprednisolone (3 mg/100 g) significantly increased rat jejunal and ileal mucosal guanylate cyclase (GTP pyrophosphatelyase [cyclizing]; EC 4.6.1.2) activity and cyclic GMP concentration 6 h after treatment. This increase in guanylate cyclase activity was maximal 6 h after injection, was not associated with changes in mucosal cyclic AMP concentration, and occurred before any increase in NaK-activated adenosine triphosphatase activity could be detected. In contrast to the findings in the small intestine, no alterations in guanylate cyclase activity were present in the rat colon 6 or 72 h after methylprednisolone treatment. The increase in ileal guanylate cyclase activity and cGMP concentration coincided with increases in ileal short-circuit current, potential difference, net Cl secretion, and serosal-to-mucosal unidirectional Cl flux. In contrast, in the colon, where no changes in guanylate cyclase activity were observed, methylprednisolone did not alter electrolyte transport in vitro. A similar correlation was observed between various methylprednisolone doses, the increase in guanylate cyclase activity and the alteration in ileal short-circuit current and net Cl transport. The methylprednisoloneinduced increase in ileal guanylate cyclase activity was not prevented by pretreatment with the mineralocorticoid competitive inhibitor spironolactone, nor did the mineralocorticoid deoxycorticosterone acetate (0.5 mg/100 g) affect ileal or colonic mucosal guanylate cyclase activity. In addition, isolated enterocytes from methylprednisolone-treated animals exhibited significant increases in guanylate cyclase activity with the greatest percentage increase occurring in the crypt cell population. These results suggest that the changes in chloride transport which occurred in the small intestine 6 h after methylprednisolone administration may be due to alterations in guanylate cyclase activity, and further support the role of cGMP as an intracellular mediator of ileal chloride secretion.

Subcellular localization and properties of N-acetylglutamate synthase in rat small intestinal mucosa.

N-Acetylglutamate synthase [EC 2.3.1], which catalyzes the synthesis of N-acetylglutamate, a key effector of carbamoyl-phosphate synthase (ammonia) [EC 6.3.4.16] in the liver of ureotelic animals, was demonstrated to be present in rat small intestinal mucosa. The activity of the enzyme was estimated to be 0.17 nmol N-acetylglutamate formed X (g mucosa)-1 X min-1 at 25 degrees C. Little activity was found in the muscle layer and serosa of the small intestine. The intestinal villous cells were separated from everted intestine, disrupted by nitrogen cavitation, and fractionated into nuclear, mitochondrial, microsomal, and soluble fractions. The mitochondria isolated by this method retained integrity of respiratory function. The mitochondrial fraction was further subjected to isopycnic centrifugation using Percoll (colloidal silica coated with polyvinylpyrrolidone). The activities of N-acetylglutamate synthase and the first two urea cycle enzymes, carbamoylphosphate synthase (ammonia) and ornithine carbamoyltransferase [EC 2.1.3.3], were cofractionated with mitochondrial marker enzymes during the cell fractionation and the isopycnic centrifugation. N-Acetylglutamate synthase, purified 8-fold from the acetone powder extract of small intestinal mucosa, had a high substrate specificity for L-glutamate and acetyl-CoA. The synthase reaction fitted normal Michaelis-Menten kinetics with respect to both L-glutamate (apparent Km, 2.5 mM) and acetyl-CoA (apparent Km, 0.8 mM). L-Arginine stimulated the enzyme activity by increasing the maximal velocity with no effect on apparent Km values for the substrates. These properties were similar to those of the rat liver enzyme (Shigesada & Tatibana (1978) Eur. J. Biochem. 84, 285-291). These results suggest that a function of the intestinal N-acetylglutamate is to activate carbamoyl-phosphate synthase (ammonia) and to allow citrulline synthesis in the tissue.

Effect of short - chain fatty acids on electrical activity of the small intestinal mucosa of rat.

Abstract Intravenous administration of short-chain fatty acid (SCFA), such as propionate, butyrate, valerate and caproate, caused a transient increase in transmural potential difference (p.d.) across the small intestine of rat in vivo . There was a sigmoid relationship between the change in the p.d. and the logarithm of the dose of SCFA. The median effective dose of propionate, n-butyrate, n-valerate and n-caproate, which was calculated from the each dose-response curve obtained from the terminal ileum, 1.31, 1.43, 0.83 and 0.81 μmole, respectively. Repeated administrations of the same dose of propionate evoked progressively smaller response. The dose-response curve of propionate was shifted to the left by neostigmine and to the right by atropine, suggesting that the action of SCFA may be mediated by acetylcholine, which was released from a nerve ending.

Paramètres cinétiques d'une fucosyl-transférase intestinale soluble purifiée

The soluble fucosyl-transferase of the rat small intestinal mucosa was purified by passing through a Sephadex G-100 column, then resolved in two isoenzymes F1 (pHi = 4,47) and F2 (pHi = 4,96) by isoelectric focusing. The use in the first step of DEAE-cellulose instead of Sephadex G-100 leads to another component F3 (pHi = 8,70). Kinetic parameters (KM et Vm) for the three isoenzymes are given. The enzymatic mechanism seems to be a bi-bi random type for the three isoenzymes, and F3 appears as the most active species.

Temporal relationship between cyclooxygenase inhibition, as measured by prostacyclin biosynthesis, and the gastrointestinal damage induced by indomethacin in the rat

Inhibition of cyclooxygenase in the rat small intestine and gastric mucosa after subcutaneous administration of indomethacin has been investigated using prostacyclin (PGI2) production ex vivo as an index of prostaglandin biosynthesis. This has been compared with the formation of intestinal lesions and gastric erosions. Indomethacin caused marked inhibition of prostacyclin formation in the gastric mucosa, and this was accompanied by the development of gastric erosions, although after 48 h both inhibition of cyclooxygenase and gastric erosions were no longer apparent. There was no such temporal relationship between prostaglandin inhibition and the formation of lesions in the small intestine since the lesions became macroscopically apparent and developed at a time when cyclooxygenase inhibition was already declining. Aspirin caused a prolonged inhibition of small-intestinal cyclooxygenase activity, yet failed to cause intestinal damage. Thus, inhibition of prostaglandin synthesis alone may not be sufficient to initiate the processes which ultimately result in intestinal lesions. The prostaglandin-independent processes affected by indomethacin which lead to intestinal damage are as yet unknown.

Electron microscopic studies on the small intestinal mucosa of rats after mechanical intestinal obstruction and ischemia

Ultrastructural changes in the small intestine during the early phase following mechanical obstruction were compared with those after vascular ligation. In both experiments (the early phase of mechanical ileus and ischemia) intestinal epithelial cells at the tips of villi showed common features. One of the most significant changes was an alteration in microvilli, with fragmentation into vesicles, narrowing of apical microvilli and decrease in number. The other change was fatty degeneration of the epithelial cells, which was accompanied by vesiculation of smooth endoplasmic reticulum beneath the terminal webs, fat deposition and dilated Golgi complex containing fat. These observations suggest that in the early phase of mechanical ileus, ischemic damage plays an important role.

The effect of long term treatment with disulfiram on content of cytochrome P-450 and on benzo(a)pyrene monooxygenase activity in microsomes isolated from the rat small intestinal mucosa

Disulfiram (DS) was administered perorally once daily to rats for 30 days to investigate the effects on cytochrome P-450 content and benzo(a)pyrene (BP) monooxygenase activity in microsomes isolated from the small intestinal mucosa. 50 mg or 100 mg DS/kg body weight caused a dose-related increase in BP monooxygenase activity, whereas the content of cytochrome P-450 was increased at the higher dose only. Similar absorption characteristics of cytochrome P-450 and turnover rates for BP on the basis of cytochrome P-450 was observed among the different microsomal preparations. The addition of DS or diethyldithiocarbamate (DDTC) to incubates of intestinal microsomes inhibited BP monooxygenase activity. Microsomes isolated from DS-treated rats were however less sensitive to in vitro inhibition by DS.

Phosphatidylcholine exchange between brush border membrane vesicles and sonicated liposomes

The brush border membrane is that part of the plasma membrane of the enterocytes which is most highly specialized in both digestive and transport functions. For a better understanding of its physiolog- ical functions a knowledge of the membrane architec- ture is essential. Phospholipid exchange activity is present in rat small intestine [l] and the isolation of phospholipid exchange proteins from rat small intestinal mucosa has been reported [2]. Phospholipid exchange has been assumed to play a role in the distribution of newly synthesized phospholipids between subcellular membranes. It may also be important for the process of fat absorption in the intestine [I]. Here we present evidence for the first time that in the presence of phosphatidylcholine exchange protein significant amounts of phosphatidylcholine can be incorporated into brush border membranes from rabbit small intestine when incubated with sonicated phosphatidylcholine liposomes. The extent of lipid transfer observed can be accounted for if it is assumed that practically all of the phosphati- dylcholine in brush border membrane is available for exchange as a single pool. Measurements of D-glucose uptake show that the brush border membrane vesicles remained intact and sealed under the conditions of intermembrane phospholipid exchange. Possible inter- pretations of our results are either that all phosphati- dylcholine is located on the outer surface of the mem- brane and readily accessible to the exchange protein or, that, if some phospholipid is present on the inner

Effect of fasting on Na-K-ATPase activity in rat small intestinal mucosa.

The effect of fasting on mucosal Na-K-ATPase activity in various regions of rat small intestine was investigated. Fasting (17--48 h) was associated with a consistent decrease in specific and total activity of Na-K-ATPase in the jejunum, the levels tending to rise more distally. No effect on the specific activities of Mg-ATPase or alkaline phosphatase was found. Fasting was also associated with incresed adrenocortical activity and with decreases in mucosal mass, protein content, and histological dimensions of the jejunum, no similar changes being found in the distal small intestine. Glucose ingestion prevented the decrease in jejunal enzyme activity associated with fasting and elevated levels in the mid and terminal small intestine of fed animals. These effects suggest that Na-K-ATPase activity in small intestinal mucosa may be, in part, inducible.

Differential effects of disulfiram and diethyldithiocarbamate on small intestinal and liver microsomal benzo[ a]pyrene metabolism

Microsomes isolated from rat small intestinal mucosa and liver were used to study the effects of disulfiram and diethyldithiocarbamate on benzo[ a]pyrene monooxygenase activity. This activity was decreased in the intestinal microsomes to 25 per cent of control 24 hr after a single oral dose of disulfiram. In contrast, daily administration of disulfiram for 5 days produced a dose related increase of benzo[ a]pyrene monooxygenase activity, above control level. The elevated activities were accompanied by a concomitant increase in the concentration of cytochrome P-450. This benzo[ a]pyrene monooxygenase activity was further stimulated by addition of α-naphthoflavone to the incubation medium. Furthermore, the absorption maximum of this cytochrome was at 450 nm in the CO bound reduced difference spectrum. These observations indicate that the disulfiram induced cytochrome P-450 was of the control type. Daily pretreatment with diethyldithiocarbamate impaired both intestinal and liver microsomes at benzo[ a]pyrene monooxygenase activities. Pretreatment with a single dose of 3-methylcholanthrene resulted in a more than 10-fold increase of intestinal benzo[ a]pyrene monooxygenase activity after 24 hr. Administration of disulfiram 24 hr before treatment appeared to potentiate the 3-methylcholanthrene induced increase of intestinal benzo[ a]pyrene monooxygenase activity. In vitro addition of disulfiram and diethyldithiocarbamate to incubates of intestinal or liver microsomes inhibited benzo[ a]pyrene metabolism to various extents; the liver being more sensitive. Disulfiram was approximately 50-fold more potent as an inhibitor than diethyldithiocarbamate. The in vitro inhibition of intestinal benzo[ a]pyrene monooxygenase activity obtained with disulfiram appeared to be caused both by direct interaction with the monooxygenase system and through NADPH dependent metabolic activation of disulfiram, while the inhibition of diethyldithiocarbamate may be a result of the latter process only.

Quantitation of hemoproteins in rat small intestinal mucosa with identification of mitochondrial cytochrome P-450.

Quantitation of hemoproteins in rat small intestinal mucosa with identification of mitochondrial cytochrome P-450.

Purification of an Enzyme with Lysophospholipase Activity from Rat Intestinal Mucosa by Hydrophobic Chromatography

A method based on hydrophobic chromatography is described for the purification of a lysophospholipase from the mucosa of rat small intestine. Under the conditions used, one single chromatographic run on phenyl-Sepharose CL-4B yielded a 582-fold purification with a 79% overall recovery of enzyme activity. A subsodium dodecyl sulphate gel electrophoresis; its isoelectric point is 5.6.

Properties of mitochondrial Mg 2+-HCO −3-stimulated and SCN −-inhibited ATPase in rat liver, kidney and small intestinal mucosa and some relationships between ATPase and supernatant carbonic anhydrase

1. 1. The optimal concentration of Mg 2+ for mitochondrial Mg 2+-ATPase was 1.5 mM in all organs tested. 2. 2. The optimal concentration of HCO − 3 for MG 2+-HCO − 3-ATPase was 30 mM of liver, kidney, ileum and 60 mM of jejunum. 3. 3. The optimal pH value for maximum Mg 2+-and Mg 2+-HCO − 3-ATPase activities was about 9.2 at 37°C. 4. 4. SCN − inhibited both Mg 2+- and Mg 2+-HCO − 3-ATPase at concentrations above 10 −3 M. 5. 5. Acetazolamide inhibited intestinal Mg 2+-HCO − 3-ATPase activity at concentrations above 10 −3 M with no effect on hepatic and renal ATPase activities. 6. 6. Mg 2+- and Mg 2+ -HCO − 3-ATPase activities in duodenal mucosa were highest in all organs tested and may have some correlation with carbonic anhydrase.

Purification and Properties of Alkaline Phosphatase from the Mucosa of Rat Small Intestine1

Alkaline phosphatase [orthophosphoric monoester phosphohydrolase, EC 3.1.3.1] was purified from the mucosa of rat small intestine by butanol extraction, ethanol fractionation, gel filtration, with controlled-pore glass-10 and DEAE-cellulose column chromatography. On the gel filtration, the enzyme activity was separated into three peaks; A in the void volume, B and C at lower molecular weight positions. Enzyme A was purified to homogeneity. The activity of enzymes A, B, and C was detected even on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at the position of the protein of enzyme A, which had a molecular weight of 110,000 daltons. Enzymatic properties such as pH optimum, Km value for the substrate, heat inactivation and inhibition by amino acids were the same in all three enzymes. Based on these findings, together with the elution positions on gel filtration, enzyme A was regarded as an aggregate, and enzymes B and C as dimer and monomer molecules, respectively.

ATPase and alkaline phosphatase activities of chick and rat small intestinal mucosa

The alkaline phosphate and (Ca 2+ + Mg 2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of chick and rat small intestine have been investigated. The same pH optimum was found for membrane-bound and solubilized alkaline phosphatase, whereas those of the corresponding ATPase differed. The solubilised ATPases had inhibition and activation characteristics similar to those of alkaline phosphate but markedly different from those of the membrane-bound ATPase. These results suggest that membrane-bound alkaline phosphatase and ATPase are not the same enzyme.

An automated continuous flow procedure for the determination of enterokinase

A continuous flow method has been developed for the automatic determination of enterokinase in rat small intestine mucosa and/or luminal content. Trypsinogen was first hydrolysed by enterokinase under conditions which minimize autocatalytic activation. l-benzoyl-arginine paranitroanilide was then added and split to paranitroaniline by the trypsin so formed. Liberated paranitroalinine was diazotized and converted by the Bratton-Marshall reagent ( N-naphthyl ethylene diamine) to an azodye, with maximum absorption at 550 nm. This method of determination was found to be six times more sensitive than the direct p-nitroaniline determination method. 36 determinations can be made hourly.

Improved isolation of villus and crypt cells from rat small intestinal mucosa.

An enzymic method is described which allows the isolation under comparable conditions of crypt and villus cells from rat jejunum with normal morphologic appearance and high metabolic activity when compared with previous preparations. The method is based on a differential scraping of short lengths of everted small intestine to yield two villus cell fractions and a gut wall residue. The scrapings and the gut tube are incubated for the same length of time in a HEPES-buffered modified Hanks' balanced salt solution containing hyaluronidase, DNase, and soybean trypsin inhibitor. The cells of the crypt region are recovered by a further scraping of the digested gut wall. Cells from all fractions are dispersed by gentle agitation, washed, and harvested by centrifugation. The final crypt and villus cells are 95--99% viable by dye exclusion and exhibit 5--20% cross-contamination on the basis of differential marker enzymes. The isolated crypt and villus cells prepared by the new procedure are suitable for comparative studies of metabolic activity in the absence of chelation-induced structural and metabolic abnormalities.