Proline Absorption Research Articles

The role of the imino transporter protein in sepsis-impaired intestinal proline absorption.

Recently, sepsis has been shown to impair intestinal amino acid absorption in addition to gut metabolic and barrier functions. We investigated intestinal proline absorption in a rabbit model of sepsis. Twelve hours after intraperitoneal injection of lipopolysaccharide, proline uptake by everted jejunal sacs prepared from septic animals (480.4 +/- 67.4 nmol per sac per hour) was significantly reduced compared with controls (846.8 +/- 73.5 nmol per sac per hour) (p < .001 by t test). We next investigated whether reduced expression of transporter proteins contributed to the impaired intestinal proline uptake during sepsis. The proline (imino) carrier of rabbit jejunum is covalently bound by fluorescein isothiocyanate (FITC) and/or phenylisothiocyanate with irreversible inhibition of proline uptake. This binding and inhibition is prevented by sodium chloride and L-proline. Single-cell suspensions of rabbit enterocytes were prepared 12 hours after intraperitoneal injection of lipopolysaccharide/saline or saline alone. Enterocytes were incubated for 30 minutes in tris(hydroxymethyl)aminomethane/ethylenediaminetetraacetate (Tris/EDTA) buffer; buffer with 1 mM phenylisothiocyanate; or buffer with 10 mM proline, 100 mM sodium chloride, and 1 mM phenylisothiocyanate. After incubation with 10 microM FITC in Tris/EDTA buffer for 15 minutes, the percent positivity and fluorescent intensity of FITC binding to enterocytes were determined by using flow cytometry. Sepsis significantly reduced the percentage of enterocytes binding FITC and the fluorescent intensity of FITC binding of proline/sodium chloride-pretreated or untreated cells. This suggests that sepsis depresses the expression of imino transporters by rabbit enterocytes, which may explain the reduced intestinal proline absorption.

H +-coupled (Na +-independent) proline transport in human intestinal (Caco-2) epithelial cell monolayers

Previously, absorption of l-proline across the apical membrane of the intestinal enterocyte has been attributed to transport via the Na +-dependent Imino system. However, net (absorptive) transport of proline across intact Caco-2 cell monolayers was enhanced by acidification of the apical environment, under both Na +-containing and Na +-free conditions. This Na +-independent pH-dependent proline flux was associated with H + flow across the apical membrane as determined by continuous measurement of intracellular pH. H +/proline symport was associated with an inward I sc in voltage-clamped Caco-2 epithelial layers demonstrating the electrogenic nature of this transport process. In conclusion Caco-2 cells possess an apically-localised, Na +-independent, electrogenic H +/imino acid transporter which may play an important role in intestinal proline absorption.

Inhibitory effect of neurotensin on proline.

The effect of neurotensin (NT) on proline absorption across rat jejunum was investigated using the single-pass perfusion technique. This study showed that intravenous administration of NT produced a dose-dependent inhibition of proline absorption. Thus, NT at a 0.16 pmol/kg/min concentration gave 10% decrease in proline absorption while 0.32 and 1.6 pmol/kg/min concentration gave 31% and 45% decrease, respectively. In the absence of Na, proline absorption decreased to 77% from control values. No change in proline absorption was noticed when NT at a concentration of 0.32 pmol/kg/min was intravenously injected in the absence of sodium from the perfusion solution. Water absorption did not show significant changes (P > 0.05) in presence or absence of NT. Moreover, NT did not produce a significant change (P > 0.2) in intracellular proline accumulation. NT inhibited proline absorption through an indirect mechanism that is Na-dependent and independent of changes in water absorption.

Effect of chlorpromazine on proline absorption across rat jejunum

The effect of chlorpromazine on proline absorption across jejunum in anaesthetized rats was investigated. Intravenous infusion of chlorpromazine reduced significantly ( P<0.01) proline absorption across the jejunum. Intraluminal perfusion of chlorpromazine into the jejunal segment reversed net absorption of proline to net secretion. Net water absorption was increased significantly ( P<0.01) when chlorpromazine was infused intravenously or perfused intraluminally. Unidirectional influx of proline across the mucosal surface was significantly inhibited ( P<0.01) after preincubation with 1 m m chlorpromazine.

Metabolism of proline by the tiger prawnPenaeus esculentus

The distribution and fate of14C-proline were investigated in immature tiger prawns,Penaeus esculentus Haswell, collected in Moreton Bay, Cleveland, Australia, by trawling during 1986/1987. Initially the prawns were fed14C-proline in food pellets to follow the pathway of proline absorption and distribution in the body.14C-proline was also injected directly into the prawn to provide sufficient tracer to follow the incorporation of14C into other amino acids and into proteins. A comparison was made of the metabolism of injected14C-proline over 48 h in prawns that had been fed and those that had been starved for 10 d. Free amino acids (FAA) in the muscle and protein-bound amino acids were analysed separately. Labelled proline was completely absorbed and distributed within the body 3 h after ingestion, about 80% being in the tissues, mostly in muscle. There was no significant difference between the total CO2 output in fed and starved prawns, but the latter metabolised about twice the amount of labelled proline over 48 h. At this time, in abdominal muscle of fed prawns, about 95% of the total muscle label was in the FAA; of the label in the FAA, 78% was proline and 18% glutamic acid, with the remainder in hydroxyproline, aspartic acid, glutamine, alanine and Kreb's cycle intermediates. In the starved prawns, proline was 58%, glutamic acid 24%, with correspondingly higher amounts in the other compounds. In the muscle protein, the distribution of label was similar in fed and starved prawns, with 72 to 74% as proline. The experiments showed that proline is not very labile in the tiger prawn and its rate of synthesis is slow. It does not appear to be an important source of energy as in some insects and cephalopods, but during starvation is only slowly oxidised for energy.

Ontogenetic development of monosaccharide and amino acid transporters in rabbit intestine

We measured brush-border uptakes of seven sugars and amino acids by rabbit intestine as a function of age from the day of birth to adulthood. Gut dimensions, especially those of the colon and cecum, increase more rapidly with body weight than would be true if rabbits maintained identical proportions as they grew. However, nominal small intestinal area increases in approximately direct proportion to the animal's basal metabolic rate. For all solutes except fructose, uptake per milligram of intestinal tissue is maximal at or near birth and declines to a level 2.5-5 times lower in the adult. Because of small intestinal growth, though, the total uptake capacity of the whole length of the small intestine increases in approximately direct proportion to metabolic rate. Fructose uptake per milligram is unique in increasing steeply at the time of weaning, correlated with the post-weaning first appearance of fructose in the natural diet. Age-related changes in uptake ratios among aldohexoses or amino acids suggest developmental sequences of related transporters. Correlated with the very high protein content of rabbit milk, the proline-to-glucose uptake ratio is higher in suckling rabbits than in other sucking mammals. Remarkably, the ratio for adult rabbits is higher than in other monogastric herbivores and is instead similar to values for carnivores. In explanation, although the transport capacity of the small intestine appears to account for proline absorption in rabbits of all ages and for sugar absorption in suckling rabbits, the hindgut may be a major site of carbohydrate digestion in adult rabbits.

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 ( K 1 = 0.5 mM and V max = 23.3 pmol/mg protein per s). Competition experiments indicated that other amino acids had an affinity for the carrier system with l-leucine > l-alanine > sarcosine > glycine > l-lysine > OH-proline > taurine > β- alanine > d-alanine > d-proline > l-serine > phenylalanine > valine > d-serine > phenylalanine > valine > d-serine > MeAIB > methionine > 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 K t, 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.

Proline transport and oxidation in short-circuited locust rectum: effect of cAMP

Proline absorption from the lumen is the main source of substrate supporting electrogenic Cl− transport and short-circuit current (Isc) in locust rectum. Since cAMP stimulates chloride-dependent Isc by up to 10-fold, we investigated whether this stimulant also increases active transport of the major metabolic substrate proline. A large 40:1 flux ratio of [14C]proline under short-circuited conditions confirmed that net absorption of this amino acid is by active transport. Unexpectedly, cAMP caused a 40% decrease in net transepithelial flux of proline. A 45% increase in the amount of proline oxidized to 14CO2 could account for only 10% of this decrease in proline flux, suggesting that increased Cl− transport after stimulation may competitively reduce the energy supply for proline transport.

Control of ion reabsorption in locust rectum: Implications for fluid transport

AbstractHormonal control of fluid reabsorption in the rectum is a major mechanism of water balance homeostasis in terrestrial insects. We report that steady‐state fluid transport in the absence of, or against, an osmotic gradient can be driven by absorption of Na+, K+, or Cl− from the lumen. Therefore chloride transport stimulating hormone (CTSH), a newly discovered hormone which stimulates KCl reabsorption by elevating tissue cAMP, might enhance fluid absorption indirectly by its action on transport of the major solute. This prediction was not confirmed and explanations are discussed. Rectal tissue volume increases considerably during the first hour in vitro and this epithelia only switches from slow transport of primarily NaCl to rapid absorption to predominantly KCI (the in vivo situation) after stimulation. Metabolic support of KCl transport depends largely on proline absorption from Malpighian tuble fluid entering the rectum. Since these specific requirements and properties of in vitro rectal preparations were not known during earlier investigations, most reports of putative diuretic and antidiuretic factors acting on rectal fluid transport in vitro require reexamination and confirmation.

Absorption of glycine and proline from the small intestine of rats infected with Eimeria nieschulzi.

The absorption of glycine and proline through the jejunum and ileum of rats with an Eimeria nieschulzi infection was impaired when the amino acids were presented to the mucosal surface as either a mixture or the dipeptide, glycyl-proline.

Therapeutic Effect of an Elemental Diet on Proline Absorption across the Irradiated Rat Small Intestine

Active absorption of (/sup 3/H)L-proline across the intestinal wall was used to measure functional change following irradiation of the exteriorized rat small intestine and to see whether an elemental amino acid diet would modify these changes. Segments (15 cm) of the exteriorized upper ileum of male Wistar rats were exposed to 1000 rad. Active transport against a concentration gradient of (/sup 3/H)L-proline from this irradiated segment was measured using the everted sac technique on days 1, 3, 7, 10, 14, 21, and 30 post-irradiation. Irradiated rats maintained on a normal diet showed depression of absorptive function with only partial recovery by day 30. Irradiated rats maintained on an elemental amino acid diet also showed an initial drop in function but then recovered absorptive function completely by day 7.

Changes in Absorption of Glucose and Proline Following Irradiation to the Exteriorized Ileum

upper ileum of rats was measured using the everted sac technique. No significant changes in absorption were found due to aging of normal rats over a 6-month period of time. Single doses of 700 rad to the upper ileum of the exteriorized intestine produced no significant late abnormalities once recovery from acute effects was complete. Doses of 900 rad resulted in full epithelial recovery but caused late depression of active absorption. Higher doses of 1000 and 1100 rad resulted in partial epithelial recovery and also caused reduction of absorption. The level of depressed function was the same for the 900, 1000, and 1100 rad groups in spite of differences in mucosal surface area and degree of epithelial recovery. We feel that the development of the late fibrosis in the bowel wall was the primary reason for depression of absorption as measured by this technique.

Kinetic properties of an imino acid transport system in the chicken intestine

1. 1. The intestinal absorption of γ-aminobutyric acid (GABA), proline and β-alanine has been studied in the chicken. 2. 2. On the basis of measurements made on apparent affinity constants of amino acids for interaction with the brush border epithelium, the following decreasing order of reactivities has been established for the imino acid transport system: proline > β-alanine = GABA> glycine > δ-amino- n-valeric acid > ϵ-amino- n-caproic acid. 3. 3. The magnitude of the individual constants suggests that, of the compounds tested, the site primarily serves to transport proline, β-alanine and GABA.

Type I hyperprolinemia: a study of the intestinal absorption of proline, hydroxyproline, and glycine.

Intestinal absorption of proline, hydroxyproline, and glycine was interpreted by investigation of a type I hyperprolinemia patient and six control subjects. Intestinal perfusion was performed. When proline (Pro), hydroxyproline (OH-Pro), and glycine (Gly) were infused together, an increase in proline concentration did not alter aminoacid uptake in the control subjects; however, in the hyperprolinemia patient, uptake of aminoacids became negliglible (Pro, 17--6 muM/min; OH-Pro, 15--0.3 muM/min; and Gly, 13.5--0 muM/min). When each aminoacid was infused alone at increasing concentrations aminoacid uptake increased in controls; in the hyperprolinemic patient, intestinal absorption was less for glycine and hydroxyproline but aminoacid uptake increased with substrate concentration; however, for proline, the uptake remained constant (1l.5--17 muM/min/20 cm of intestinal test segment) (Table 1). When hydroxyproline was infused with an increased concentration of proline in the hyperprolinemic patient, hydroxyproline uptake first increased (9.8--14.3 muM/min/20 cm) then decreased to its basal value, whereas, in the control subjects, uptake increased without decreasing subsequently.

Effect of prolactin on sugar and amino acid transport by the rat jejunum.

After two daily ovine prolactin injections in rats, significant increases in jejunal absorption of glucose, glycine and proline, as well as of fluid and NaCl, occurred. Although ouabain caused dramatic reductions in fluid, sodium, chloride and glucose transport, prolactin had an effect even in the presence of ouabain. Mucosal hypersomolaity significantly decreased fluid, sodium and chloride absorption but had no significant effect on glucose transport. Addition of prolactin in vitro had no effect on intestinal absorption. Prolactin-induced increases in transport of fluid, NaCl and organic nutrients by the mammalian jejunum may play an important role in nutritional and osmoregulatory adaptations.

Role of epithelial architecture and intracellular metabolism in proline uptake and transtubular reclamation in PRO/re mouse kidney.

The homozygous PRO/Re mouse has less than 1 percent of the very high proline oxidase activity that characterizes normal kidney cortex. In PRO/Re mouse the endogenous proline concentration is eight times normal in plasma and four times normal in kidney cortex cell, but 50 times normal in urine. The integrity of the membrane transport systems for proline uptake at the antiluminal surface of absorbing epithelium is retained in PRO/Re kidney, as determined by the slice method. Clearance studies in vivo under steady-state conditions indicate that the integrity of the luminal uptake system shared by glycine and proline, and serving proline absorption, is also intact. The exaggerated renal clearance of proline in PRO/Re mice (50 times normal) is explained when its raised intracellular concentration, caused by impaired proline oxidation, is considered. Backflux into urine flowing down the nephron will occur under these conditions, thus impairing net reclamation of proline in PRO/Re kidney. The findings reveal that membrane transport and intracellular metabolism of a substrate are, indeed, independent functions, but that metabolism of a substance can influence its transcellular transport.

Distinct components of neutral amino acid transport in chick small intestine

1. 1. The intestinal absorption of alanine, β-alanine, α-aminoisobutyric acid (AIB), glycine, proline and tryptophan has been studied by an in vitro tissue accumulation method in the chick. 2. 2. The use of amino acids as specific blocking agents allowed for the characterization of seven discrete components of neutral amino acid transport which can be grouped into three major categories: (a) those processes inhibited by leucine and glycine; (b) those inhibited by leucine, but not by glycine; (c) those not inhibited by leucine.

Ontogeny of iminoglycine transport in mammalian kidney.

Renal tubular absorption of proline, hydroxyproline, and glycine by the newborn of most mammals is inefficient compared to that of the adult. Cortex slices from seven-day-old rat kidney also transport proline and glycine at reduced initial rates compared to mature kidney. Nonetheless, newborn slices achieve higher intracellular concentrations during prolonged incubation; the latter reflects a reduced rate of efflux, a characteristic peculiar to the membrane of postnatal kidney. The postnatal reduction of initial uptake rates is observed clearly only at substrate concentrations in or below the physiological range; it correlates with the absence of two high-affinity systems which normally serve proline and glycine transport independently at these concentrations in mature kidney, in conjunction with a "common" low-affinity system. The low-affinity system alone performs the observed uptake in the newborn kidney. Specific activity of the high-affinity systems for proline and glycine increases asynchronously after birth, suggesting independent genetic control of the three systems for iminoglycine transport in mammalian kidney.

Studies on membrane transport—VI. Absorption of amino acids by fascioliid trematodes

1. 1. Ligation of Fasciola or Fascioloides did not affect the 2-min absorption of 14C-cycloleucine. 2. 2. Absorption of cycloleucine, arginine, proline or methionine was a linear function of concentration and competitions were not demonstrable. Amino acids entered the tegument by diffusion. 3. 3. Efflux experiments indicated that cycloleucine may be accumulated in an inner “compartment” in Fasciola. 4. 4. Four hours after administration of 14C-cycloleucine to infected hosts, the amino acid was equally distributed in tissue water of host blood and Fasciola.

Impaired intestinal transport of proline in a patient with familial iminoaciduria

Summary Another example of a transport defect involving both renal tubular and intestinal epithelium is provided by the report of a patient with familial iminoaciduria who had impaired absorption of proline from the bowel and increased urinary clearances of iminoacids and glycine.