Effect of various methods of oxygenation in the isolated small intestine on the tryptophan and glycine accumulation

The impact of various oxygenation types of intestinal preparations (mucosal, serosal and bilateral) on the transepithelial transport of amino acids and peptide according to the character of amino acids, their stereoisometry, incubation time, intestinal gradient and other factors, was investigated in fowl. Against-the-gradient-transport of L-tryptophan and glycine was observed at various oxygenation types, and its intensity decreased in the following sequence: bilateral, mucosal, serosal oxygenation. At various oxygenation types, the accumulation of L-tryptophan occurs more efficiently than that of DL-tryptophan and D-tryptophan. Using an in vitro bilateral intestinal oxygenation model system, we have demonstrated the plasma membrane of enterocyte microvilli to be rapidly impaired after oxidative stress. Glycine, but not L-tryptophan, attenuates oxidative injury in brush border membrane and alterations in amino acid transport activity. Overall, our data indicates that in vitro serosal oxygenation of the duodenum markedly improves glycine absorption, possibly involving the basolateral transporters.     

Coupling of protease activity and sodium loading with intestinal absorption of amino acids

Membrane-bound serine proteases to play a certain role in activation of sodium transport in epithelial cells. To were found explain the protease activity and sodium-dependent L-tryptophan transport across chicken small intestine interaction, four experiments were conducted. One hundred chicks were fed diets that contained 0; 0.3; 3 or 6% of supplemental NaCl and were given distillated water ad libitum. Signs of salt toxicity observed were as follows: a decreased body weight, increased heart and kidney weights, formation of secondary lysosomes in enterocytes and lymphocytes. Such chickens were in the state of negative nitrogen balance. Intestinal absorption of L-tryptophan correlated with mucosal protease activity during increased dietary sodium chloride intake. Recent in vitro and in vivo experiments indicate that enterocyte proteases may be of critical importance in activation of sodium-dependent intestinal transporters for L-tryptophan.     

Inhibition of intestinal uptake of amino acids by unconjugated bile salt.

The unconjugated bile salt, sodium deoxycholate, at a concentration of 0.5 mM was shown to inhibit the intestinal uptake of the amino acids L-glycine, L-leucine, L-proline, L-lysine and L-tyrosine in rats in vitro. This effect was acutely reversible except for the basis amino acid L-lysine and is therefore not simply due to tissue damage. These results, and the recent finding that sodium deoxycholate inhibits intestinal absorption of amino acids in vivo, suggest that impaired intestinal amino acid transport may contribute to hypoproteinaemia in patients with bacterial overgrowth in the upper small intestine in whom deoxycholate is present in the small intestinal lumen in excessive concentrations.     

Stimulation of L-tryptophan transport by di- and polysaccharides in the small intestine of chicks

In in vivo and in vitro experiments the effect of various carbohydrates on the absorption of L-tryptophan in the chick small intestine was investigated. On feeding the chicks with semisynthetic diet containing 58.5% of carbohydrates a stimulatory effect of glucose, particularly of starch and saccharose, on the entry of L-tryptophan into the portal vein from the chick gastrointestinal tract has been found. Using an in vitro technique the activating effect of starch and disaccharides (maltose, saccharose) on the intestinal transport of L-tryptophan was detected while monosaccharides (glucose, fructose) at different concentrations had no effect on this process or inhibited it. The possibility that energy of disaccharide hydrolysis is used to stimulate transport process is discussed.     

A new role for enteric glucagon-37: acute stimulation of glucose absorption in rat small intestine

Glucagon-37 is secreted by intestinal L-cells following carbohydrate uptake. It is known to inhibit gastric acid secretion (hence also named oxyntomodulin) and appears to increase intracellular cyclic AMP concentrations. Since cyclic AMP could enhance intestinal glucose absorption, a possible stimulatory effect of glucagon-37 on glucose transport was examined. Glucagon-37 acutely increased glucose absorption in the isolated, vascularly perfused small intestine and in isolated enterocytes of the rat. In these cells the stimulation by glucagon-37 could be completely blocked by the cAMP antagonist Rp-cAMPS and was therefore mediated by cAMP. The stimulation of intestinal glucose absorption by glucagon-37 appears to be a major new physiological function.     

Postnatal amino acid uptake by the rat small intestine. Energetics of membrane transport systems for amino acids in the developing jejunum

The energetics of amino acid uptake by the developing small intestine was investigated in vitro. L-valine, L-leucine, L-phenylalanine, L-methionine, L-lysine and L-arginine were all actively transported by the newborn rat jejunum. Metabolic inhibitors (e.g. 2,4-dinitrophenol) significantly reduced uptake of all amino acids but uptake against a concentration gradient was not totally abolished. Uptake of all amino acids was reduced at low[Na+]. Inhibition of transport of neutral amino acids by reduced luminal [Na+] was greater than that of basic amino acids, and the tissue was barely able to concentrate the neutral amino acids. [Na+] affected the Michaelis constant (Km) of neutral transport systems for their substrates; for the basic amino acids Km values were unaffected by the presence or absence of Na+. Ouabain significantly inhibited neutral amino acid uptake but had no effect on L-lysine or L-arginine uptake. These results are discussed in terms of the Na+ gradient hypothesis for amino acid transport, and the site of energy input to active transport. The role of glycolysis in providing energy for intestinal transport in the neonatal rat and the efficiency of Na+ dependent and independent transport mechanisms are considered. It is concluded that the energetics of amino acid transport systems in neonatal and adult rats are essentially similar.     

Intestinal transport of fructose and glycine in Ophiocephalus punctatus.

The regional variations in the absorption of fructose and glycine have been studied in the intestine and pyloric caeca of the fish, Ophiocephalus punctatus in vivo, by preparing intestinal sacs. Diffusion of amino acids out of the intracellular compartment of the intestine was observed in all the portions of the intestine. The pyloric caeca showed maximum transport of fructose. Absorption of fructose was slightly more in the posterior intestine than in the anterior intestinal sac. Maximum transport of glycine took place in the anterior intestinal sac. Pyloric caeca adsorbed more glycine than did the posterior sac. For both nutrients, the rate of uptake did not differ significantly in the upper and the lower portion of the intestine. In all the portions, the transport of fructose was more rapid than that of glycine. The experiments revealed that though the nutrients are absorbed in the entire length of the intestine, the quantity absorbed varies from one portion to another and is dependent on the nature of the compound absorbed.     

Postnatal amino acid uptake by the rat small intestine. Changes in membrane transport systems for amino acids associated with maturation of jejunal morphology.

The uptake of a number of amino acids by the developing small intestine of the rat was investigated in vitro. L-valine, L-leucine, L-methionine, L-phenylalanine, L-arginine and L-lysine were all taken up by active transport and concentrated within the jejunal mucosa. GABA was not actively transported by the jejunum. The kinetics of carrier transport of amino acids was determined from birth to maturity. The Michaelis constant (Km) of the L-leucine, L-methionine, L-arginine and l-lysine transport systems was found to be low postnatally and increased with age, particularly after the time of weaning. The rate of l-leucine, L-methionine, L-phenylalanine and L-lysine transport (Vmax) was high postnatally but decreased after weaning. Neutral amino acids were transported at higher rates than basic amino acids. l-arginine was poorly transported by the jejunum. The specificity of transport systems for amino acids was investigated in inhibition studies. Amino acid transport systems appeared to be polyfunctional in the postnatal period but were more specific in post-weaned animals. The changes in kinetics and specificity of amino acid transport in the small intestine are discussed with reference to their possible functional significance and to the maturational changes in the jejunum, particularly with the appearance of a functionally distinct absorptive cell lining the intestinal villi during the third postnatal week (the time of weaning).     

The role of calcium binding protein in the mechanism of action of cholecalciferol (vitamin D3).

A role has been sought for the calcium binding protein (CaBP) which is synthesised de novo after giving cholecalciferol (CC, vitamin D3) to rachitic chicks. After homogenation of mucosal cells in sucrose media, the CaBP was found in the 78,000 X g supernatant. Therefore, the CaBP is either present in the cytoplasm or in some labile membrane structure, e.g. the microvilli, that is disrupted by homogenation. This intracellular CaBP may facilitate diffusion of Ca into intestinal cells. No secretion of CaBP into the lumen could be detected nor did excess CaBP placed in the lumen increase Ca absorption of rachitic chicks. The mitochondria of duodenal mucosal cells contained most of the Ca being translocated by the small intestine. CaBP caused release of Ca already present in mitochondria and diminished Ca uptake by mitochondria and it appreared to do this by increasing the rate of Ca flux across the mitochondrial membrane. This would explain the greater "turnover" of Ca in mucosal cells of cholecalciferol-treated chicks. These and previous findings have been used to propose a scheme for the effect of cholecalciferol on Ca transport from the small intestine.     

Experimental cystinuria: the cycloleucine model. I. Amino acid interactions in renal and intestinal epithelia.

The injection of cycloleucine (1-aminocyclopentanecarboxylic acid (ACPC) into rats produces a hyperexcretion of dibasic amino acids and cystine, an aberration resembling cystinuria. This may constitute a model of experimental cystinuria, and the transport of amino acids involved in this disease was studied with the techniques of everted intestinal sacs (in vitro) and microinjections into renal tubules (in vivo). In verted sacs from normal rats, there was a decrease in transfer and in accumulation of L-cystine (0.03 mM) and L-valine (0.065 mM) when ACPC was on the mucosal (luminal) side. Dibasic amino acids such as L-arginine and L-lysine caused a similar inhibition of the transport of L-cystine. However, when ACPC was on the serosal (antiluminal) side, a lesser effect was noted while arginine and lysine had no effect. Intestinal sacs from treated rats (ACPC, 300 mg/kg X 3 days) transferred and accumulated as much L-cystine as those from control rats. The interaction between cycloleucine and L-cystine was competitive at the luminal and non-competitive at the antiluminal side of the intestine. Cycloleucine inhibited L-lysine transport in a non-competitive fashion at either side of the intestine. L-Lysine also interacted in a non-competitive fashion with L-cystine transport at the luminal membrane. In proximal convoluted tubules, the presence of L-arginine or ACPC caused a decrease in the transport of L-cystine and L-lysine. L-Valine exerted no effect. Furthermore, L-lysine and ACPC did not impair the reabsorption of L-valine significantly. These results suggest a functional heterogeneity between luminal and antiluminal membranes of renal and intestinal epitehlia and the existence, at both membranes, of different transport sites for cystine and dibasic amino acids.     

Exogenous ATP-stimulated calcium uptake in isolated rat intestinal epithelial cells

ATP in the extracellular medium is known to stimulate Ca uptake into avian intestinal epithelial cells. We have now demonstrated a similar effect of ATP in mammalian intestinal epithelial cells and have further characterized this effect. Exogenous ATP increased 45Ca uptake 2-6 fold in isolated rat small intestinal epithelial cells, with a maximal effect at 1 mM and an ED50 of 290 microM. A strict structural requirement for nucleotide-stimulated 45Ca uptake was observed. ADP was much less effective than ATP and gamma-thio-ATP, and 5'-AMP, cyclic AMP, adenosine, non-adenine nucleotides, non-hydrolyzable ATP analogs and ATP analogs with ring substitutions at the 8 position were inactive. Prenylamine (100 microM) completely inhibited ATP-stimulated 45Ca uptake, while verapamil (100 microM) had only a small effect. In the intact intestine, ATP increased short-circuit current (Isc) when added to the mucosal side of the tissue. This effect was reduced by 10 microM and abolished by 100 microM prenylamine. The effect of ATP on Isc was markedly reduced in Cl-free solutions and in reduced-Ca solutions. Serosal and mucosal addition of the nonhydrolyzable ATP analog, beta, gamma-methylene-ATP, and serosal addition of ATP had little or no effect on Isc. The similarities between the effects of ATP in isolated cells and in the intact intestine suggest that the effect of ATP on Isc may be at least partially mediated through stimulation of Ca uptake into the epithelial cells.     

Effect of phloretin and phloridzin on properties of digestion and absorption in the rat small intestine

Experiments in vitro on everted sacs of rat small intestine have shown that phloretin (an inhibitor of basolatheral glucose GLUT2 transporter) added from mucosal side of the sacs decreases release of glucose from enterocytes into serosal fluid without changing glucose accumulation in tissue of the preparations. Addition of phloridzin (an inhibitor of Na⁺ and glucose co-transporter SGLT1) from mucosal side inhibited both glucose accumulation in the tissue and its release into serosal fluid. Unspecific effects of phloretin and phloridzin on activities of several digestive enzymes (in particular, alkaline phosphatase, amino peptidase, and glycyl-L-leucine dipeptidase) has been revealed in homogenates of the rat small intestine mucosa. In chronic experiments on rats, absorption of glycine from the isolated small intestinal loop was inhibited in the presence of phloretin in perfusate. The obtained results indicate that the experimental approach of inhibition of glucose absorption by phloretin used from mucosal side in vitro appears to give a significant overestimation of contribution of facilitated diffusion (with participation of the GLUT2 transporter inserted in the apical enterocyte membrane) to glucose transport across this membrane. Thus, the role of the GLUT2 transporter in the mechanism of glucose absorption in the small intestine under its physiological conditions does not seem to be as great as it is thought by the authors of the recently proposed hypothesis.     

A novel imino-acid carrier in the enterocyte basolateral membrane

Basolateral membrane vesicles prepared from rat small intestinal epithelial cells were used to study the sodium-independent transport of L-proline. The uptake of L-proline was unaffected by the presence of sodium and showed saturation kinetics (Kt = 0.5 mM and Vmax = 23.3 pmol/mg protein per s). Competition experiments indicated that other amino acids had an affinity for the carrier system with L-leucine greater than L-alanine greater than sarcosine greater than glycine greater than L-lysine greater than OH-proline greater than taurine greater than beta-alanine greater than D-alanine greater than D-proline greater than L-serine greater than phenylalanine greater than valine greater than D-serine greater than phenylalanine greater than valine greater than D-serine greater than MeAIB greater than methionine greater than threonine. This pathway does not resemble those previously described either in the brush-border membrane of intestinal epithelial cells or the plasma membrane of other cell types. The lack of effect of methionine and threonine indicate that proline is not using the L-type system, while the very low affinity for MeAIB and the Na+ independence suggest that this is a novel system for imino acids. The relatively high capacity of this system and its low Kt, which is almost identical to the proline system in the brush-border membrane, strongly suggest that this is an important pathway in the final step for proline absorption by the small intestine.     

Cell membrane amino acid transport processes in the domestic fowl (Gallus domesticus)

Intestinal absorption of amino acids in the chicken occurs by way of processes which are concentrative, Na+-dependent and dependent upon metabolic energy in the form of ATP. Intestinal transport is carrier-mediated, subject to exchange transport (trans-membrane effects) and is inhibitable by sugars, reagents which inactivate sulfhydryl groups, potassium ion, and by deoxpyridoxine, an anti-vitamin B6 agent. It is stimulated by phlorizin, a potent inhibitor of sugar transport, and in Na+-leached tissue by modifiers of tissue cyclic AMP levels, e.g. theophylline, histamine, carbachol and secretin. Separate transport sites with broad, overlapping specificities function in the intestinal absorption of the various classes of common amino acids. A simple model for these sites includes one for leucine and other neutral amino acids, one for proline, beta-alanine and related imino and amino acids, one for basic amino acids, and one for acidic amino acids. Absorption of amino acids appears to be widespread in occurrence in the digestive tract of the domestic fowl; transport has been reported to be present in the crop, gizzard, proventriculus, small intestine and in the colon. By the end of the first week of life post-hatch, the caecum loses its ability to transport. Similarly, the yolk sac loses its ability by the second day post-hatch. Intestinal transport was noted before hatch and was found to be maximal immediately post-hatch. A requirement for Ca2+ appears to be lost after the first week of life post-hatch. The cationic amino acids appear to be reabsorbed by a common mechanism in the kidney. Transport rates of leucine measured in the intestine or in the erythrocyte were found to cluster about discrete values when many individual chickens were surveyed; such patterns may be an expression of gene differences between individuals. Two lines of chickens have been developed, one high and the other low uptake, through selective breeding based on the ability of individual birds to absorb leucine in erythrocytes. High leucine absorbing chickens were found to be more effective in absorbing lysine and glycine, were more effectively stimulated by Na+, had greater erythrocyte Na+, K+-ATPase activity, and their erythrocytes contained about 20% less Na+ than low line erythrocytes. The underlying genetic difference between these lines may reside at the level of the Na+, K+-ATPase and (or) with a regulatory gene determining carrier copies. Amino acid transport in erythrocytes was noted to be highest in pre-hatch chicks and to diminish during post-hatch development.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of bile acid active transport related to increased mucosal cyclic AMP content in rat ileum in vitro

The regulation of bile acid transport in rat ileum was studied in vitro using the adenylate cyclase stimulator forskolin, or 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Forskolin 20 microM as well as 100 microM IBMX enhanced mucosal cyclic AMP to 3-fold the control levels. As a physiological response, net fluid absorption in everted ileal sacs was reduced. Taurocholate (10-500 microM) transfer in everted perfused segments of rat ileum was measured using a three compartment dual label method suitable for measuring active transport. Transport asymmetry with absorption exceeding its counterflux by 26-fold, was measured at 500 microM taurocholate. Forskolin increased absorption of taurocholate still further, by 68%, and reduced the serosal to mucosal flux. Enhanced intracellular accumulation of taurocholate indicated a stimulatory action of forskolin on active transport at the mucosal brush-border membrane. In uptake studies, accumulation of taurocholate was enhanced by 100 microM IBMX also. Forskolin-induced uptake stimulation could also be shown for chenodeoxycholate and cholate. In the presence of the neuronal blocker tetrodotoxin, uptake stimulation was still effective. Results indicate that the ileal bile acid transporter is included within the group of sodium-dependent cotransporters of the rat small intestine which are subject to a cyclic AMP-related stimulation at the mucosal cellular level.     

Mechanism of L-Lysine Transport by Pea Protoplasts

Dureja, I., Guha-Mukherjee, S. and Prasad, R. 1986. Mechanismof L-lysine transport by pea protoplasts.—J. exp. Bot.37: 549–555. L-Lysine uptake was studied in pea protoplasts to characterizethe transport process. The uptake was pH dependent with optimumat pH 5?8. A kinetic analysis of uptake showed that L-lyslneuptake was biphasic. The respiratory inhibitors, sodium arsenate,azide, iodoacetate and 2, 4, dinitrophenol, inhibited the uptakeof L-lysine at a final concentration of 0?1 mol m^–3 suggestingit to be mediated in part by an active process. Competitiveinhibition of L-lysine uptake by only L-arglnine and of L-leucineand glycine uptake by several amino acids indicated that L-lysineuptake occurs via a specific system whereas the uptake of L-leucineand glycine was mediated through a relatively non-specific permease. Key words: Pea protoplasts, L-lysine transport, active transport, specific system     

Intestinal iodide secretion and its dependence upon mucosal I- permeability

Iodide secretion across different regions of rat small intestine has been investigated in vitro using the standard Wilson-Wiseman technique. Net I- secretion was observed along the entire small intestine, being significantly higher in the central region. Anaerobic conditions, ouabain (2 mM) and Na+ free Ringer solution prevented net I- secretion, whilst both theophylline (1 mM) and carbachol (0,1 mM) enhanced the observed basal intestinal I- secretion. Furthermore, Ca2+-deprived bathing solutions significantly reduced intestinal I- secretion. Epithelial I- uptake from both mucosal and serosal sides was measured by using a Ussing-type chamber technique. The initial rate of I- uptake across the mucosal membrane was significantly higher in the central region than in the proximal part of rat small intestine. No significant differences were observed in the rate of I- uptake from the serosal side. These studies suggest that mucosal I- permeability might determine the direction of net I- intestinal transport and that cytosolic Ca2+ may be a physiological regulator of intestinal I- transport.     

Amino acid transport in the intestine of the caiman

Seventeen amino acids were fed singly to small caimans and the rates of their disappearance from the gut lumen, and of their appearance in intestinal mucosa, whole intestine, whole stomach, and plasma were determined. The results were compared with those in which massive amounts of protein were fed. When single amino acids were fed, only traces of arginine, ornithine, lysine, aspartate and asparagine were absorbed intact. Glycine, alanine and serine were absorbed rapidly reaching mucosal concentrations as high as 40 mM. The others were not concentrated as highly and most were absorbed by the mucosa more slowly than the glycine group. Protein feeding did not result in high amino acid concentrations in the mucosa. Whether amino acids were ingested as protein or in the free state, glycine, alanine and glutamine increased in the mucosa, suggesting these three incorporate nitrogen released from the others. It appeared that several transport systems operate if amino acids are given singly, and that a different more efficient transport system operates during protein digestion.     

The effect of prostacyclin on intestinal ion transport in the rat

The actions of PGI₂ and PGE₂ on electrically monitored ion transport in rat jejunum and colon have been determined both $\text{in vivo}$ and $\text{in vitro}$. Whilst PGE₂ was shown to induce a marked change in ion transport PGI₂ was relatively ineffective. The ability of the prostanoids to influence ion transport is related to their capacity to change mucosal cyclic AMP levels since in isolated small intestinal enterocytes PGE₂ caused a marked stimulation in cyclic AMP levels whilst PGI₂ had little effect. In colonic mucosal scrapes PGE₂ was more effective than PGI₂ in stimulating changes in cyclic AMP levels. It appears doubtful that PGI₂ plays a direct role in the regulation of intestinal ion transport.     

Adult worms of the rodent intestinal nematode,Strongyloides venezuelensis,successfully invade chick intestinal mucosa

In order to study the mucosal invasion of a rodent intestinal nematode in bird intestine, chicks were infected with the intestinal nematode of rodents, Strongyloides venezuelensis, by subcutaneous larva inoculation and adult worm implantation. No evidence was obtained for larvae reaching the lungs or the intestine after infective larva inoculation. Adult worms implanted in the small intestine invaded the mucosa and remained there at least for 24 h, whereas those implanted in the caecum were trapped by mucus, and did not invade the mucosa. Mucosal invasion of adult worms in the small intestine was confirmed by histological examination. The number of adult worms in the intestinal mucosal tissue dropped rapidly within the first 24 h, which was associated with infiltrating granulocytes around the worms. The present study suggests that S. venezuelensis adult worms are able to invade the intestinal tissue of chicks, which do not belong to the vertebrate class of its normal definitive host, but that they are eliminated rapidly by mucosal defense system of the bird.