Regulation of amino acid transport across intestines of goldfish acclimatized to different environmental temperatures

1. Serosal transfers of valine and threonine were measured using everted sacs of anterior intestine taken from goldfish acclimatized to different temperatures.

2. Both valine and threonine were actively transported at incubation temperatures equal to or greater than the previous environmental temperature of the fish. There was also a positive serosal transfer of valine, but not threonine, at incubation temperatures below the previous environmental temperature of the fish.

3. The mean stable transmural potentials and amino-acid-evoked potentials depended both on the temperature to which the fish had been acclimatized and on the temperature at which the sacs were incubated.

4. There was a linear relation between the transmural potential and the serosal transfer of amino acid, one additional μmole of valine or threonine being transferred/2 hr incubation period for each 3 mV rise in potential. There was a less obvious correlation between the amino-acid-evoked potential and on serosal transfer of amino acid.

5. Acclimatization of the goldfish intestine from 8 to 25° C, assessed by changes occurring in the transmural potential and serosal transfer of amino acids, tended to stabilize both parameters, but the compensation in each case was only partial.

6. It is possible that the imbalance in transfer of valine-like and threonine-like amino acids, seen at incubation temperatures below the previous acclimatization temperature of the fish, has a special function in initiating the process of acclimatization to the new environmental temperature.

     

The transamination of glutamate and aspartate during absorption in vitro by small intestine of chicken, guinea-pig and rat

1. Procedures are described for direct measurement of the extent and rate of transamination of glutamate and aspartate over periods of up to 90 min, during absorption in vitro by the small intestine of chicken, guinea-pig, and rat.2. During absorption of dicarboxylic amino acids by rat small intestinal segments circulated through the lumen in vitro, alanine contributed up to 85% of the amino acids appearing in the fluid secreted at the serosal surface. In guinea-pig and chicken intestine, the proportion of alanine in the secreted amino acids did not exceed 60%.3. For the different species studied, a relationship was found between the extent to which the dicarboxylic amino acids were transaminated to alanine and the total amount of GPT found in other studies to be present in the intestinal mucosa. In both rat and guinea-pig small intestine, the proportion of alanine in the total amino acids appearing at the serosal surface was similar in the jejunum and ileum. The rate of appearance of alanine in serosal fluid was greater in the ileum than in the jejunum of the rat.4. Reasons are given for supposing that for all the species studied there is a limit to the capacity of the small intestinal mucosa to subject free dicarboxylic amino acids to transamination. It is concluded, however, that it is unlikely that this capacity will be exceeded under in vivo conditions.     

Membrane potentials of epithelial cells in rat small intestine

1. Stripped sacs of rat jejunum in which the outer muscle layers had been removed were found to maintain substantial transport and electrical activities.2. Mucosal and serosal membrane potentials of epithelial cells of normal and stripped everted sacs of rat jejunum were recorded in vitro together with the transmural potential difference.3. The cell interior was negative relative to both serosal and mucosal fluids, the transmural potential being the sum of the two membrane potentials.4. Changes in the transmural potentials in the presence of actively transferred hexoses and amino acids were entirely due to variations in the serosal potential, the mucosal potential being unchanged.5. Serosal and transmural potential increases on the addition of galactose were consistent with Michaelis-Menten kinetics, giving apparent K(m) values of 14.9 and 14.1 mM respectively.6. Phlorrhizin, ouabain, 2,4-dinitrophenol and sodium fluoroacetate inhibited serosal potential changes in the presence of galactose.7. Osmotic potentials resulting from transmural osmotic gradients originated from the serosal layers of the tissue.8. The results are consistent with the concept of a serosally located, electrogenic sodium pump which is stimulated by actively transferred hexoses and amino acids. The sodium-dependent entry mechanism at the mucosal membrane is non-electrogenic.     

The effect of carbohydrates on the intestinal potentials of Cryptochiton stelleri

1. The effect of various monosaccharides on the potential difference across the intestine of the invertebrate, Cryptochiton stelleri, was studied using an everted sac technique.2. D-Glucose, when present in the mucosal solution, increased the transmural potential across the anterior intestine but had no effect on electrical potentials across the posterior intestine.3. D-Galactose, when present in the mucosal solution, increased the transmural potential across the posterior intestine but had no effect on electrical potentials across the anterior intestine.4. D-Fructose and D-mannose, metabolizable but non-transported sugars, increased anterior intestinal potentials but not posterior intestinal potentials.5. 3-O-methylglucose, an actively transported (across the anterior intestine) but non-metabolizable sugar, did not alter potentials across either the anterior or posterior intestine.6. The addition of D-glucose to the serosal solution increased anterior intestinal potentials, but the serosal addition of D-galactose did not affect posterior intestinal potentials.7. NaF, phlorizin and anoxia prevented the increase in potential across the anterior intestine and phlorizin (10(-4) but not 10(-6)M) and NaF but not anoxia prevented the increase in potential across the posterior intestine.8. It was suggested that the effect of glucose on anterior intestinal potentials was a metabolic effect, whereas the effect of galactose on posterior intestinal potentials was related to the active transport of that sugar from mucosa to serosa.     

The use of dietary-restricted rat intestine for active transport studies

1. The effect of dietary restriction (sufficient to produce a loss of about 32% of initial body weight) on intestinal active transport has been studied in the rat by the use of sacs of everted mid-small intestine. Eight D-sugars, four L-sugars and two D-amino acids were employed.2. Dietary restriction enhanced the normally occurring active transport of D-galactose, 3-O-methyl-D-glucose and D-methionine. In addition, sacs of dietary-restricted small intestine were able to concentrate in the serosal fluid D-fucose, D-xylose and D-histidine, which sacs of normal rat intestine could not do. The final (1 hr) serosal/mucosal concentration ratios produced for these actively transported substances were independent of net water movement.3. Sugars which were not concentrated in the serosal fluid of sacs of fully fed or dietary-restricted intestine were D-arabinose, D-fructose, D-glucosamine, D-mannose, L-arabinose, L-fucose, L-sorbose and L-xylose.4. The characteristics of D-fucose and D-xylose active transport suggest that they are transported by the mechanism which actively transports D-glucose. The comparatively low content of D-glucose in dietary-restricted intestine, compared with fully fed intestine, may be part of the explanation for observable active transport of D-fucose and D-xylose by dietary-restricted sacs.5. Thinning of the intestinal wall is believed not to be the cause of the enhanced active transport found during dietary restriction.6. The results show that dietary-restricted rat small intestine may, at times, be more useful than fully fed rat small intestine in the study of intestinal active transport.     

Absorption of amino penicillins from everted rat intestine.

1. Using an in vitro everted gut sac method based on that of Wilson & Wiseman (1954), a number of amino penicillins were tested in order to identify the involvement of any specialized transport mechanisms in their absorption across rat intestine. 2. Only one of the amino penicillins, cyclacillin (1-amino-cyclohexyl penicillin) was shown to be actively transported. The other penicillins appeared to diffuse passively across the intestine. 3. Cyclacillin was found to concentrate against a gradient at 37 degrees C but not at 19 degrees C. 4. Transport of cyclacillin across the mucosal membrane was saturated at mucosal concentrations greater than 1000 microgram/ml. 5. The rate of the forward flux of cyclacillin was many times that of its back flux. 6. No relationship between the active transport of cyclacillin and that of amino acids could be demonstrated.     

Ionic dependence of protein transport across the new-born pig intestine

1. The everted small intestine of the new-born pig transports albumin to its serosal surface at rates which depend on the ionic composition of the bathing medium.2. Albumin transport takes place most rapidly at sodium and potassium concentrations of 120 and 6 mM respectively. Increasing the concentration of sodium or potassium, or decreasing the concentration of sodium, reduces the net transfer of albumin.3. Albumin can increase the serosal transfer of sodium, potassium and water. The calculated ionic composition of fluid transported in the presence of albumin closely resembles that presented to the mucosal surface over a wide range of sodium and potassium concentrations.4. Lowering the concentration of sodium reduces water transfer but only if albumin is present in the mucosal medium. Albumin-stimulated water transfer is only seen when the external concentration of sodium is high.5. Raising the concentration of potassium reduces water transport, whether or not albumin is present, but this reduction is too small to account for the inhibitory effect of potassium on albumin transport.6. The mutual interaction that exists between the transport of albumin and sodium is probably responsible for the increased potassium transport seen at low external concentrations of potassium. Higher concentrations of potassium appear directly to inhibit the interaction between the transport of sodium and albumin.     

Effects of amino acids on chloride transport in Aplysia intestine

This investigation was principally undertaken to examine the mechanism by which organic solutes (amino acids) stimulate chloride transport across the Aplysia californica intestine. Isolated intestine, mounted between identical oxygenated seawater solutions, maintained stable transmural potential differences (serosa negative) and short-circuit currents for several hours at 25 degrees C. The addition of glycine to the mucosal solution stimulated rapid sustained increases in these electrical characteristics. The change in short-circuit increased curvilinearly with increasing concentrations of mucosal glycine. Mucosal glycine stimulated transmural potential difference and short-circuit current after mucosal phlorizin had partially inhibited D-glucose stimulation of the electrical characteristics. Mucosal glycine enhanced the transmural electrical characteristics. Mucosal glycine enhanced the transmural electrical characteristics after serosal ouabain had abolished them. The major portion of the amino acid-induced short-circuit current was carried by a net, active, chloride transfer from mucosa to serosa as determined by flux measurements. These results suggest that the amino acid-induced effect on chloride transport is mediated by a common mucosal membrane carrier for both sodium and the amino acid.     

Transport of neutral amino acids by adult articular cartilage

The transport of three neutral amino acids: alanine, serine and leucine and the non-metabolizable analogue aminoisobutyric acid (AIB) was characterized in bovine articular cartilage slices. Alanine and serine were strongly concentrated intracellularly by a factor of 4.5 and 5 respectively and were transported in a sodium dependent and pH sensitive manner. AIB was concentrated by a factor of 1.8 with respect to the extracellular medium and was also transported in a sodium dependent and pH sensitive manner. Leucine was concentrated weakly across the chondrocyte membrane (X1.1) and its transport was independent of sodium ion concentration and the lowering of the extracellular pH. The apparent Km of transport of alanine, serine, leucine and AIB was determined to be 0.46 mM, 0.69 mM, 0.93 mM and 0.66 mM respectively and the values of Vmax were 35.8, 52.1, 14.8 and 6.8 pmol/min per mg respectively. It was shown that both alanine and serine were transported predominantly by system ASC (63.8% and 67.4% respectively) with a small percentage of transport occurring via system A. AIB was transported mainly by system A (76.0%) and leucine was transported equally via systems L (48.4%) and ASC (45.0%).     

Transport of Neutral Amino Acids by Adult Articular Cartilage

The transport of three neutral amino acids: alanine, serine and leucine and the non-metabolizable analogue aminoisobutyric acid (AIB) was characterized in bovine articular cartilage slices. Alanine and serine were strongly concentrated intracellularly by a factor of 4.5 and 5 respectively and were transported in a sodium dependent and pH sensitive manner. AIB was concentrated by a factor of 1.8 with respect to the extracellular medium and was also transported in a sodium dependent and pH sensitive manner. Leucine was concentrated weakly across the chondrocyte membrane (x 1.1) and its transport was independent of sodium ion concentration and the lowering of the extracellular pH. The apparent K_m of transport of alanine, serine, leucine and AIB was determined to be 0.46 mM, 0.69 mM, 0.93 mM and 0.66 mM respectively and the values of V_max were 35.8, 52.1, 14.8 and 6.8 pmol/min per mg respectively. It was shown that both alanine and serine were transported predominantly by system ASC (63.8% and 67.4% respectively) with a small percentage of transport occurring via system A. AIB was transported mainly by system A (76.0%) and leucine was transported equally via systems L (48.4%) and ASC (45.0%).     

Effect of amino acids on sugar absorption

1. Sacs of everted mid-small intestine of the hamster have been used to study the effect of amino acids on sugar absorption.2. The sugars employed were D-glucose, D-galactose, 3-O-methyl-D-glucose, D-fucose, L-glucose, alpha-glucoheptose, L-fucose, D-mannose and L-sorbose. The amino acids were L- and D-histidine, L- and D-methionine, L- and D-alanine, L- and D-valine, L- and D-glutamic acid, L-leucine, L-proline, L-ornithine and L-aspartic acid.3. Actively absorbed amino acids considerably inhibit the transport of actively absorbed sugars. The results give support for the view that D-histidine and L-glucose are actively transferred. Passively absorbed amino acids and sugars are not involved.4. As L-glutamic and L-aspartic acids in the mucosal fluid have no inhibitory effect on D-glucose absorption, although mucosal fluid L-alanine is quite potent, the step at which the latter exerts its inhibitory action must be before that at which the intracellular transamination of L-glutamic and L-aspartic acids occurs. It would seem likely, therefore, that L-alanine interferes with the process by which epithelial cells capture and concentrate sugars at the luminal border.5. More than one active transfer system may exist for D-glucose.6. The influence of actively absorbed L-amino acids on D-glucose active transport seems to be in some way related to the efficiency with which the amino acids are themselves concentrated.7. Inhibition of D-glucose active absorption by an amino acid may be a simple test of an amino acid's participation in an active transport system.     

Intracellular hydrolysis of short chain glycerides by rat small intestine in vitro and transfer of glycerol

1. When triacetin, tripropionin and tributyrin are incubated with sacs of rat everted intestine, they enter the epithelial cells and are completely hydrolysed to free fatty acids and glycerol.2. The distribution between the mucosal and serosal fluids of the glycerol released is very different from that of the fatty acid. Although both hydrolytic products are accumulated in the serosal fluid, a much higher fraction of the fatty acid appears in this compartment.3. When glycerol is initially present in the mucosal fluid there is no evidence of its movement against a concentration gradient.4. The results confirm the existence of a transport mechanism for fatty acids released intracellularly but do not require the hypothesis of a special mechanism for glycerol transfer.     

Interorgan metabolism of amino acids in streptozotocin-diabetic ketoacidotic rat

Amino acid concentrations in whole blood, liver, kidney, skeletal muscle, and brain were measured and arteriovenous differences calculated for head, hindlimb, kidney, gut, and liver in control and streptozotocin-diabetic rats. In the control rats, glutamine was released by muscle and utilized by intestine, intestine released citrulline and alanine, liver removed alanine, and the kidneys removed glycine and produced serine. In diabetic rats, the major changes from the pattern of fluxes seen in the normal rat were the release of many amino acids from muscle, with glutamine and alanine predominating, and the uptake of these amino acids by the liver. Glutamine removal by the intestine was suppressed in diabetes, but a large renal uptake of glutamine was evident. Branched-chain amino acids were removed by the diabetic brain, and consequently, brain levels of a number of large neutral amino acids were decreased in diabetes.     

Active transport of L-glucose by isolated small intestine of the dietary-restricted rat

1. The effect of semistarvation and complete starvation (sufficient to produce a loss of about 32 and 25% respectively of initial body weight) on the active transport of L-glucose has been studied by the use of sacs of everted mid-small intestine of rats. The animals were allowed free access to water.2. Sacs from animals on a restricted diet transported L-glucose against its concentration gradient, but sacs from fully fed rats did not. Even when sacs from fully fed rats were distended sufficiently to cause them to lose serosal volume, the L-glucose concentration in the final serosal fluid was never greater than that in the final mucosal fluid.3. The L-glucose active transport was independent of net water movement, needed oxygen, was not demonstrable at 27 degrees C, and required Na ions at a concentration of 83 mM or greater. It could be completely inhibited by 10(-6)M phlorrhizin, or 10 mM L-histidine, or 1.39 mM D-glucose. Phlorrhizin at a concentration of 10(-8)M reduced, but did not prevent, L-glucose active transport.4. It seems probable that L-glucose active transport is mediated by the mechanism that actively transports D-glucose.5. Un-incubated mid-small intestine of fully fed rats contained 37.8 mg D-glucose/100 g wet wt. of tissue, whereas semistarved intestine had only 10.8 mg D-glucose/100 g. The lack of demonstrable active transport of L-glucose by normal intestine may possibly have been caused, at least in part, by inhibition of the process by endogenous D-glucose.6. There appeared to be no metabolism of L-glucose by rat intestine, nor conversion to the D-form.7. The hypothesis that sugars require the D-pyranose ring structure for active absorption is no longer tenable.     

Effect of bile salts on amino acid transport by rabbit intestine.

The effect of bile salts on alanine absorption across four regional sites of rabbit intestine was examined using an in vivo single-pass perfusion technique. Na-deoxycholate at a concentration of 3 mM reduced alanine absorption across all levels of the intestine, and a higher concentration (10 mM) of Na-taurodeoxycholate (TDC) caused only a minimal reduction of alanine absorption in the jejunum. TDC, however, was more effective in in vitro experiments, causing an incrase in transmural serosal-to-mucosal flux of alanine and phenylalanine, particularly when present in both the mucosal and serosal media. It also reduced the mucosal-to-serosal alanine flux rate when present only in the mucosal medium. The influx of these amino acids across the mucosal brush border membrane was also decreased by TDC. These amino acid transport changes correlated fairly well with some observed histological changes of the intestinal epithelium. This suggests that bile salt inhibition of amino acid absorption is nonspecific in type and can be mainly explained as being the result of an injurious action of these surface-active agents on the rabbit intestine.     

Handling of glycerides of acetic acid by rat small intestine in vitro

1. When mono-, di- and triacetins are incubated with sacs of rat everted intestine, they enter the epithelial cells and are hydrolysed to free glycerol and acetic acid.2. The rate-limiting step in the process is the entry of glyceride into the epithelial cell.3. The three acetins enter the epithelial cell at the same rate, and the mechanism of this remains unknown.4. The acetate released appears in higher concentrations on the serosal side, and the relation of this to the mechanism for transfer of volatile fatty acids is discussed.5. It is not necessary to postulate a special mechanism for entry of volatile fatty acids into the cell.     

Effects of fasting on intestinal transfer of sugars and amino acids in vitro

1. Transfer of sugars, amino acids and fluid and metabolism of glucose were studied with everted sacs of small intestine prepared from fed and 3-day fasted rats.2. In the absence of glucose there was some evidence for increased intestinal transfer of sugars and amino acids in fasted animals. In the presence of glucose there was in general decrease in transfer of amino acids and fluid.3. In fasted animals glucose transfer was reduced except in the lower ileum, and there was a general reduction in glucose metabolism.4. Because of the large reduction in gut weight in fasted animals, expressing transfer on a weight basis is considered not to be a valid procedure in studying the effects of fasting on intestinal transfer.5. The results have been discussed in relation to effects of fasting on energy availability, efficiency of transfer mechanisms, permeability of the intestine and the value of in vitro methods in the study of physiological absorption.     

Rheogenic property of Na+/acidic amino acid co-transport by guinea pig intestine

The influx of L-glutamate or L-aspartate across the intestinal brush border membrane was absolutely dependent on the presence of Na+ in the mucosal solution and saturable. The addition of these amino acids into the mucosal solution induced a sudden and sustained increase in the transepithelial potential difference (PDt), which was found to be absolutely dependent on the presence of Na+ in the mucosal solution. The increases in PDt induced by L-glutamate or L-aspartate conformed to Michaelis-Menten kinetics against the concentration of each organic substrate. On the other hand, the relationship between the change in short-circuit current induced by acidic amino acid and Na+ concentration did not conform to Michaelis-Menten kinetics but was sigmoid. A kinetic study indicated that 2 Na+ were translocated per molecule of the acidic amino acid. The PDt was not altered by the total replacement of K+ by Na+ in the mucosal solution and showed the optimum pH at around 7. The transfer of L-glutamate from mucosal to serosal solution was examined with everted sac of ileum. A significant increase in serosal appearance was observed for L-glutamate and L-alanine after the incubation with L-glutamate. The serosal appearance of L-glutamate was unaffected by the presence or absence of HCO3-.     

Alterations in neurotransmitter amino acid content in the aging rat striatum are subregion dependent

The topographical distribution of putative neurotransmitter amino acids in both 6- and 20-month-old Fischer 344 rats was studied in eight striatal subregions. Tissue levels of glutamate, aspartate, GABA, and taurine in the 20-month-old rats were elevated in virtually all of the anterior striatal subregions examined. In addition, aspartate levels were higher in all dorsomedial subregions, while glutamate and taurine levels were elevated in all lateral and ventrolateral subregions, respectively. Other amino acids such as glutamate, serine, and alanine did not display any specific subregional changes. These findings demonstrate specific striatal subregion changes in neurotransmitter amino acid content as a function of aging.     

The in vitro transfer of bovine immune lactoglobulin across the intestine of new-born pigs

1. Everted sacs of pig intestine, used soon after birth, maintained transmural potentials and transferred water and glucose to the serosal surface.2. Immune globulin, fed as bovine colostrum to the new-born pig, appeared in the serosal fluid of everted sacs during incubation in bicarbonate saline. The particular segment showing maximum transferring ability varied between limits and appeared to depend on the amount or concentration of colostrum fed to the pig. Sacs from unfed pigs incubated in bovine colostrum also transferred colostral IgG to the serosal fluid. This transfer was dependent on the concentration of colostral IgG in the incubation medium and became more pronounced in the middle third of the small intestine.3. Human serum albumin inhibited the transfer of colostral IgG and about twenty molecules of albumin were transferred for every molecule of colostral IgG, when both were presented together in equal concentration on the basis of weight, to the middle segment of the small intestine.4. Some of the immune globulin collected in vitro after feeding bovine colostrum was found in a degraded form, but the amounts present could not be estimated. There was no apparent degradation of immune globulin in the purely in vitro experiments.5. The in vitro transfer of bovine colostral IgG showed selectivity between molecules of albumin and colostral IgG, the nature of which warrants further study.