Serosal Transfer Research Articles

Effect of Thyroidectomy and Thyroxine on Glucose Transport in the Everted Small Intestine of Rat

Thyroid hormone has been shown to alter glucose metabolism, however, there seems to be controversial reports on the effect of thyroxine on intestinal glucose transport. This study was conducted to investigate the effect of thyroidectomy and thyroxine on glucose transport in the small intestine. Forty rats were randomly selected into four groups of ten rats each. Groups one and two rats were thyroidectomised to make them hypothyroid after which group two rats were given thyroxine replacement of 10ug/100g b/w for thirty five days to make them euthyroid. Rats in groups three and four were sham operated thereafter group three rats were given 10ug/100g b/w thyroxine for thirty five days to make them hyperthyroid. Group four served as the control. 10mg/kg b/w Ketamine was administered intraperitoneally as anesthesia for the surgeries. On the thirty-fifth day post-surgery all the animals were killed via cervical dislocation and their small intestines were harvested. 10cm length of jejunum and ileum respectively were used to make everted sacs for the invitro study. Mucosa glucose transfer (MGT), Final glucose concentration gradient (FCG) and Gut glucose uptake (GGU) were significantly higher (P< 0.05) in the hyperthyroid group and lower (P< 0.05) in the hypothyroid group compared with the control with transport in the jejunum greater (P< 0.05) than the ileum in all groups. Serosal glucose transfer (SGT) was Negative in the hyperthyroid group. SGT was significantly greater (P< 0.05) in the jejunum than the ileum. These findings suggest that thyroidectomy causes reduced glucose transport while thyroxine increases glucose transport in different segments of the small intestine with the transport in the jejunum greater than that of the ileum.

Effect of Thrombin on Permeability of Human Epithelial Cell Monolayers

Epithelial cells play an important role in maintaining the airway barrier, which is impaired in inflammatory conditions. Recently, thrombin was reported to be increased in the airway of patients with asthma, and thrombin has been shown to increase the permeability of endothelial cell monolayers. Therefore, we suspected that thrombin affects airway permeability. Calu-3 cell monolayers were established on microporous membranes of tissue culture cell inserts. We examined the effects of topically applied thrombin or thrombin receptor-activating peptide (TRAP) on: (1) transepithelial permeability (luminal to serosal transfer) of radiolabeled mannitol and albumin, (2) changes in electrical resistance, and (3) actin fiber content as assessed by fluorescence microscopy. Compared with untreated control cultures, treatment of the monolayers for 24 h with thrombin or TRAP significantly decreased the electrical resistance and increased the permeability to mannitol and albumin. In addition, these treatments enhanced the appearance of actin stress fibers, and small gaps became visible at areas of cell-cell contact. Thrombin appears to increase epithelial permeability by receptor-mediated reorganization of the actin network in airway epithelial cells. This is likely to contribute to the impairment of the airway barrier function.

Inhibitory effects of dietary calcium on the initial uptake and subsequent retention of heme and nonheme iron in humans: comparisons using an intestinal lavage method

Calcium is the only reported dietary inhibitor of both heme- and nonheme-iron absorption. It has been proposed that the 2 forms of iron enter a common pool in the enterocyte and that calcium inhibits the serosal transfer of iron into blood. We aimed to ascertain whether the inhibitory effect of calcium occurs during initial mucosal uptake or during serosal transfer and to compare the serosal transfer of heme and nonheme iron, which should not differ if the 2 forms have entered a common mucosal iron pool. Whole-gut lavage and whole-body counting were used to measure the initial uptake (8 h) and retention (2 wk) of heme and nonheme iron with and without a calcium supplement (450 mg). Two experiments tested basal meals with low iron bioavailability and 360 mg Ca (n = 15) or with high iron bioavailability and 60 mg Ca (n = 12). Added calcium reduced the initial uptake of heme iron by 20%, from 49% to approximately 40% from both meals (P = 0.02), and reduced the total iron absorbed from the low- and high-bioavailability meals by approximately 25% [from 0.033 to 0.025 mg (P = 0.06) and from 0.55 to 0.40 mg (P < 0.01), respectively]. Calcium did not affect the serosal transfer of either form of iron. Calcium supplementation reduced heme and total iron without significantly affecting nonheme-iron absorption, regardless of meal bioavailability. Calcium inhibited the initial mucosal uptake rather than the serosal transfer of heme iron. Differences in serosal transfer indicate that heme and nonheme iron did not enter a common absorptive pool within 8 h after a meal.

Initial uptake and absorption of nonheme iron and absorption of heme iron in humans are unaffected by the addition of calcium as cheese to a meal with high iron bioavailability

Quantitative data on the mucosal uptake and serosal transfer of nonheme-iron absorption in humans and the effects of calcium on these components are limited. Our objective was to measure the initial mucosal uptake and the subsequent serosal transfer of nonheme iron and to determine the effects of adding calcium to a meal on both heme- and nonheme-iron retention. Whole-gut lavage and whole-body scintillation counting methods were applied to determine the 8-h uptake of nonheme iron and the 2-wk retention (absorption) of heme and nonheme iron in healthy adults (n = 17) after the consumption of meals of radiolabeled food. The initial uptake and absorption of nonheme iron were 11% and 7%, respectively, and the absorption of heme iron was 15%. Two-thirds of the nonheme iron taken up by the mucosa within 8 h was retained by the body after 2 wk (serosal transfer index: 0.63). Serum ferritin correlated inversely with the initial uptake and absorption of nonheme iron, but not with the nonheme serosal transfer index or the absorption of heme iron. Adding calcium (127 mg in cheese) to the meal did not affect absorption. On the basis of its association with serum ferritin, the initial mucosal uptake was the primary control point for nonheme-iron absorption. An apparent reduction in heme-iron absorption associated with the lavage procedure suggested that uptake of heme iron may take longer and proceed further through the intestine than that of nonheme iron. The absorption of both forms of iron was unaffected by the addition of cheese to this meal with high iron bioavailability.

Decreased mucosal iron induces an increase in intestinal iron absorption in the early stage of iron-deficiency

To investigate the contribution of non-heme iron and ferritin in the intestinal mucosa in regulating intestinal iron absorption during the early stage of iron-deficiency, we performed an in vivo study using rats fed an iron-deficient diet. Intestinal iron absorption was measured using the ligated intestine method. Rats fed the iron-deficient diet for 2 days showed no change in hematological parameter values (blood hemoglobin, plasma iron, the total iron binding capacity) and no change in the content of non-heme iron and ferritin in the liver. However, uptake of iron into the mucosa (mucosal uptake) and transfer of iron from mucosal cells to blood (serosal transfer) was significantly increased compared with the initial levels. At that time, the content of non-heme iron in the mucosa significantly decreased, however, no significant change in mucosal ferritin was observed. Extending the period of feeding the iron-deficient diet resulted in a small additional decrease in the level of mucosal non-heme iron, but led to no significant increase in mucosal uptake and serosal transfer. These findings suggest that a decrease in non-heme iron content in the intestinal mucosa may induce an increase in intestinal iron absorption during the early stage of iron-deficiency. In addition, it was evident that there is an upper limit in intestinal iron absorption. This limit in iron absorption may play a role in preventing iron-overload in the body.

Anionic PAMAM dendrimers rapidly cross adult rat intestine in vitro: a potential oral delivery system?

To investigate systematically the effect of polyamidoamine (PAMAM) dendrimer size, charge, and concentration on uptake and transport across the adult rat intestine in vitro using the everted rat intestinal sac system. Cationic PAMAM dendrimers (generations 3 and 4) and anionic PAMAM dendrimers (generations 2.5, 3.5, and 5.5) that were modified to include on average a single pendant amino group were radioiodinated using the Bolton and Hunter Reagent. 125I-Labelled dendrimers were incubated with everted sacs in vitro and the transfer of radioactivity into the tissue and serosal fluid was followed with time. The serosal transfer rates seen for all anionic generations were extremely high with Endocytic Indices (EI) in the range 3.4-4.4 microL/mg protein/h. The concentration-dependence of serosal transfer was linear over the dendrimer concentration range 10-100 microg/mL. For 125I-labelled generation 5.5 the rate of tissue uptake was higher (EI = 2.48+/-0.51 microL/mg protein/h) than seen for 125I-labelled generations 2.5 and 3.5 (0.6-0.7 microL/mg protein/h) (p < 0.05). The 125I-labelled cationic PAMAM dendrimers (generations 3 and 4) displayed a tissue uptake (EI = 3.3-4.8 microL/mg protein/h) which was higher (p < 0.05) than the rate of serosal transfer (EI = 2.3-2.7 microL/mg protein/h), probably due to nonspecific adsorption of cationic dendrimer to the mucosal surface. As the anionic PAMAM dendrimers displayed serosal transfer rates that were faster than observed for other synthetic and natural macromolecules (including tomato lectin) studied in the everted sac system, these interesting nanoscale structures may have potential for further development as oral drug delivery systems.

Evaluation of N-(2-hydroxypropyl)methacrylamide copolymer-peptide conjugates as potential oral vaccines. Studies on their degradation by isolated rat small intestinal peptidases and their uptake by adult rat small intestinal tissue in vitro

Oral administration of therapeutic peptides and peptide antigens has achieved limited success owing to their degradation and poor transport across the gastrointestinal tract. In this study covalent coupling of peptides to the water soluble polymer N-(2-hydroxypropyl)methacrylamide (HPMA) is explored as a means to overcome these problems. A model peptide, b-chain of insulin (b-chain), and the human rhinovirus antigenic determinant, peptide VP2, were covalently bound to HPMA copolymers of molecular weight 23 200 to give a peptide content of approximately 25% (w/w). Conjugation resulted in a marked reduction in the rate of degradation of both peptides during in vitro incubation with small intestinal brush border (BBM) and luminal enzymes. In the case of b-chain, reductions of up to 80% and 60% were observed with BBM and luminal peptidases, respectively. For peptide VP2, reductions up to a maximum of 80% and 55% were observed with BBM and luminal peptidases, respectively. Incubation of 125I-labelled b-chain with everted rat jejunal sacs in vitro showed no serosal transfer of intact free 125I-labelled b-chain as a result of peptide degradation. In contrast, the 125I-labelled HPMA copolymer-peptide conjugate displayed transfer of intact b-chain into the serosal fluid, and sacs with or without Peyer's Patches (PP) displayed transfer of 66 and 58 ng of conjugated b-chain per mg tissue protein. As polymer conjugation both protects against peptide degradation and promotes peptide uptake, HPMA copolymer conjugation has the potential to improve oral vaccination using peptide antigens.

Uptake and transport of poly( N-vinylpyrrolidone-co-maleic acid) by the adult rat small intestine cultured in vitro: Effect of chemical structure

To study the structure-activity relationships of polymer uptake and transfer across the gastrointestinal mucosa, poly( N-vinylpyrrolidone-co-maleic acid) (NVP MA) polymers of standardised molecular mass (20000 Da) were synthesised to contain side-chains of different charge or hydrophobicity. All polymers additionally contained tyrosinamide residues to permit radioiodination. Using an improved everted gut sac prepared from adult rat small intestine, the tissue accumulation and serosal transport of the polymers was measured in vitro over a 2 h incubation period. All polymers were captured by the tissue linearly with time (endocytic indices between 1.6 and 16 μl/mg protein per h), and then transferred slowly to the serosal fluid (endocytic indices between 0.18 and 2 μl/mg protein per h). The neutral NVP MA polymer showed the lowest rate of tissue association, but this was increased 5-fold by the presence of either negatively or positively charged groups. The maximum rate of transport across the mucosa was seen for the most negatively charged polymer derivative, this being equivalent to approx. 26% its rate of tissue accumulation. Increasing hydrophobicity of the polymer derivatives had a more pronounced effect on the rate of tissue capture, increasing it by up to 10-fold for the most hydrophobic derivative. However, in this case, the serosal transfer was only 10–15% the rate of tissue uptake. The data presented indicate that NVP MA polymers can be tailor-made for use in oral delivery systems. Substituent groups can be incorporated to promote tissue uptake or translocation across the gastrointestinal mucosa, or a combination of the two.

Absorption of [formula omitted] by rat everted intestinal sacs

Absorption of the N 4- d-glucose conjugate of sulphamethazine (glucose-SMZ, 0.5 mM) by isolated everted sacs of the rat small intestine was studied at 37° and pH 6.6. Phlorizin (0.5–2.0 mM) significantly reduced (P < 0.05) both mucosal and serosal transfer of glucose-SMZ and inhibition of mucosal transfer appeared to be concentration-dependent. Phloretin (0.5 mM) and removal of Na + from the incubation medium also diminished absorption of glucose-SMZ. Furthermore, d-glucose (0.5 and 5.0 mM) inhibited mucosal and serosal transfer of the glycoside. The results suggest the d-glucose/Na + cotransporter mediates absorption of glucose-SMZ from the small intestine of the rat. Thus, glucose-SMZ might be bioavailable from ingested tissues in which it is present.

Intestinal zinc transfer by everted gut sacs from rats given diets containing different amounts and types of dietary fibre

Two experiments were carried out in which rats were offered diets containing different amounts and types of dietary fibre, i.e. commercial stock diet and three semi-purified diets containing no fibre, 200 g wheat bran or 200 g pectin/kg. Dietary inclusion of fibre, and especially pectin, stimulated large bowel fermentation, as indicated by caecal hypertrophy and reduced caecal pH. After 3 weeks, mucosal:serosal zinc transfer and Zn accumulation by tissue were measured using the everted-gut-sac technique. In Expt 2, incubations were carried out in the presence and absence of 0.25 mM-ouabain to assess the importance of transfer by Na+,K+-ATPase-dependent mechanisms, and some observations on glucose transport were also made. Ouabain reduced rates of transfer of both Zn and glucose and also tissue Zn accumulation. There were no significant differences in rates of Zn transfer by everted sacs from duodenal, ileal and colonic sites, but accumulation of Zn by tissue was a more important fate than transfer across the serosal surface, and accumulation by duodenal tissue was approximately twice as great as by other tissues. Mucosal:serosal transfer of glucose by ileal tissue was much more sensitive to ouabain than was Zn transfer. Previous diet appeared to alter the capacity of the intestinal tissue to transfer Zn, with the highest rates of transfer being by colonic tissue from pectin-fed rats.

The effect of immunization with protein-hapten conjugate on the intestinal uptake of p-aminobenzoic acid as a hapten

Intestinal uptake of p-aminobenzoic acid was examined by means of an in vitro everted sac technique in rats immunized with ovalbumin-p-amino-benzoic acid conjugate. A dose-dependent and antigen-specific decrease in the serosal transfer of p-aminobenzoic acid was observed in rats immunized 6 times with protein-hapten conjugate compared with the control. There was a significant increase in the recovery of p-acetamidobenzoic acid, a metabolite of p-aminobenzoic acid, in mucosal fluid, tissue, and serosal fluid in the jejunum. In the case of ileum, increase of p-acetamidobenzoic acid was observed in mucosal fluid. However, there was no significant effect in the ileal p-acetamidobenzoic acid in tissue and serosal fluid between immunized and non-immunized rats. To examine the increased metabolism of immunized rats, N-acetyltransferase activity of the small intestinal mucosa was examined. There was a significant increase in mucosal N-acetyltransferase activity in immunized rats compared with the control animals. These observations suggested that the mucosal immune system may play an important role in regulating the intestinal uptake of the low molecular weight compounds.

Kinetic analysis of zinc uptake and serosal transfer by vascularly perfused rat intestine

Transport kinetics were examined for uptake of 65Zn from the lumen and for transport of mucosal 65Zn subsequent to uptake in the isolated, vascularly perfused intestines of rats fed either a zinc-deficient or zinc-adequate diet. Zinc depletion influenced the intestinal transport of zinc by stimulating a saturable uptake mechanism, reducing secretion of mucosal 65Zn into the lumen, and increasing the rate of 65Zn turnover in a rapidly absorbed mucosal zinc compartment. Uptake of 65Zn involved both saturable and nonsaturable processes. The saturable process was stimulated by zinc depletion with the apparent maximal transport rate for the saturable mechanism increasing from 60 to 180 nmol Zn X g-1 X 30 min-1. Most of the 65Zn taken up was not involved in the short-term secretion or absorption, and mucosal 65Zn retention was independent of dietary zinc status. Absorption of mucosal 65Zn was nonsaturable, involved a rapid exchanging zinc compartment, and was stimulated by zinc depletion. The half-life for 65Zn in this mucosal zinc compartment was approximately 24 min in the zinc-adequate group and 13 min in the zinc-depleted group.

Effect of allopurinol on the intestinal absorption of 6-mercaptopurine in rats.

The effect of allopurinol, a xanthine oxidase inhibitor, on the intestinal absorption characteristics of 6-mercaptopurine was investigated by in situ and in vitro absorption techniques in rats. In the in situ experiment, the percent disappearance of 6-mercaptopurine from the lumen showed a dependency on initial drug concentration. Most of the 6-mercaptopurine that was lost from the lumen appeared as its metabolite, 6-thiouric acid, in the lumen. Dose-dependency of 6-mercaptopurine disappearance from the lumen and the biotransformation of drug to 6-thiouric acid were not observed in the presence of allopurinol. The in vitro data supported the results of the in situ experiment. In addition, allopurinol increased the serosal transfer in vitro and the mesenteric appearance of 6-mercaptopurine in situ by inhibiting the biotransformation to 6-thiouric acid. These results suggested that the type of absorption characteristics observed with 6-mercaptopurine was due to its metabolism by xanthine oxidase in the intestine.

Modes of Cl- transport across the mucosal and serosal membranes of urodele intestinal cells.

The characteristics of Cl- movement across luminal and basolateral membranes of Amphiuma intestinal absorptive cells were studied using Cl(-)-sensitive microelectrodes and tracer 36Cl techniques. Intracellular Cl- activity (aiCl) was unchanged when serosal Cl- was replaced; when luminal Cl- was replaced cell Cl- was rapidly lost. Accordingly, the steady state aiCl could be varied by changing the luminal [Cl]. As luminal [Cl] was raised from 1 to 86 mM, aiCl rose in a linear manner, the mucosal membrane hyperpolarized, and the transepithelial voltage became serosa negative. In contrast, the rate of Cl- transport from the cell into the serosal medium, measured as the SITS-inhibitable portion of the Cl- absorptive flux, attained a maximum when aiCl reached an apparent value of 17 mM, indicating the presence of a saturable, serosal transport step. The stilbene-insensitive absorptive flux was linear with luminal [Cl], suggestive of a paracellular route of movement. Intracellular aCl was near electrochemical equilibrium at all but the lowest values of luminal [Cl] after interference produced by other anions was taken into account. aiCl was unaffected by Na replacement, removal of medium K, or elevation of medium HCO-3. Mucosae labeled with 36Cl lost isotope into both luminal and serosal media at the same rate and from compartments of equal capacity. Lowering luminal [Cl] or addition of theophylline enhanced luminal Cl- efflux. It is concluded that a conductive Cl leak pathway is present in the luminal membrane. Serosal transfer is by a saturable, stilbene-inhibitable pathway. Luminal Cl- entry appears to be passive, but an electrogenic uptake cannot be discounted.

In vivo studies of mucosal-serosal transfer in rat jejunum.

In anesthetized rats, the appearance rates of a series of labeled substances in jejunal venous blood (phi B) and serosal bath (phi S) were measured in vivo (intestinal blood flow rate 1.5 ml min-1 g-1) after intraluminal administration of 0.5 ml buffer solution (initial concentration 1 mmol/1 or 1 GBq/1 tritiated water) into a closed jejunal segment (length 4-5 cm). Between 32% (erythritol) and 93% (salicylic acid) of the administered activity (unchanged substance and possible metabolites) appeared in the intestinal venous blood within 60 min. The fraction recovered from the serosal bath after 15 (60) min was 11 (6)% for tritiated water, 7 (4)% for aniline, 3 (7)% for aminopyrine, 5 (4)% for butanol, 3 (3)% for benzyl alcohol, 2 (4)% for benzylamine, 1-2% for benzoic acid, theophylline, methyl-alpha-D -glucopyranoside, L-lysine, antipyrine, and urea, and less than 1% for L-phenylalanine, D-galactose, erythritol, and salicylic acid. During single pass perfusion of a jejunal segment (length 3-4 cm) the fraction of serosal transfer phi S/(phi B + phi S) was 19% for tritiated water, 4.9% for antipyrine, 0.5% for benzoic acid, and 0.08% for salicylic acid. Distension of the intestinal wall by administration of 1 ml buffer solution instead of 0.5 ml increased the appearance rate of benzoic acid and antipyrine in intestinal venous blood by a factor of 2 and serosal transfer by a factor of approximately 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Active transport of lead-210 by everted segments of rat duodenum.

Everted gut sacs prepared from rat duodenum can transfer 210Pb from mucosal to serosal surfaces against concentration gradients in vitro. The mechanism requires oxygen and is significantly inhibited by sodium azide, sodium iodoacetate, 2,4-dinitrophenol, sodium cyanide, and sodium fluoride. Lack of oxygen or the presence of metabolic inhibitors produced greater relative inhibition of net serosal transfer but greater absolute decrements of net mucosal uptake. Treatment of rats prior to sac preparation with either intravenous FeCl2 or endotoxin, or the addition of FeCl2 to the mucosal medium, selectively inhibited net serosal transfer. Net mucosal uptake and net serosal transfer were significantly negatively correlated with animal weight. Jejunal or ileal sacs did not actively transport 210Pb.

In vitro intestinal absorption of glycine and l-alanine by rainbow trout, Salmo gairdnerii, Rich.

1. 1. Tissue accumulation and serosal transfer of glycine and l-alanine have been studied in vitro in trout with duodenal, ileal and posterior portions of the intestine. 2. 2. Neither tissue accumulation nor serosal transfer was stimulated by mucosal glucose. 3. 3. The absorptive capacity of the three portions was different. 4. 4. The various modes of expression of the results (i.e. with respect to fresh weight, length or surface) lead to different conclusions regarding the so-called specialization of the portions.

Effects of Zinc on Mucosal Copper Binding and on the Kinetics of Copper Absorption

The kinetics of serosal copper transfer and the distribution of copper between metallothionein (MT) and a high-molecular-weight protein fraction (HMWPF) within the mucosal cells were examined, using isolated duodenal segments from rats fed different amounts of zinc. No difference in the Vmax or Kon for serosal transfer could be detected between the different zinc groups, suggesting that zinc did not affect this aspect of copper absorption. When intestinal segments from rats fed the low zinc diet were incubated in high copper media, the majority of the copper was associated with the HMWPF, while those from rats fed the high zinc diet had the largest proportion bound to MT. All the intestines incubated in the low copper media, regardless of rat zinc status, had the majority of copper associated with MT. These results suggested that with the low zinc diet, the limited amount of MT present was saturated, and the excess copper was bound to the HMWPF. With larger intakes of zinc, MT synthesis was induced, and the larger amount of this protein was not as readily saturated, resulting in less copper binding to the HMWPF and more to MT. It is suggested that zinc interferes with copper absorption by inducing MT, which sequesters copper in the mucosal cells, making it unavailable for serosal transfer. The copper bound to the HMWPF is available for transfer.

The effect of dietary zinc on intestinal copper absorption

Everted duodenal segments, tied into sacs, taken from animals fed different amounts of zinc were used to investigate the antagonistic effect of dietary zinc on copper absorption. The intestinal segments taken from animals fed low amounts of zinc transferred more copper from a nutrient medium across the mucosal cells than did intestines from rats fed high levels of zinc. The mucosal cells from animals fed low amounts of zinc retained less copper than the cells from animals fed high amounts of the element. This retained copper was bound to a protein fraction having a molecular weight similar to that of metallothionein. The data suggest that zinc exerts its antagonistic effect by inducing the synthesis of a copper-binding ligand, probably a thionein, in the mucosal cells which sequesters copper from the nutrient medium, making it unavailable for serosal transfer. This may be a possible mechanism by which dietary zinc decreases copper absorption and leads to a decreased copper status.

Polybrominated biphenyl: Acute and chronic effect on iron absorption and benzo( a)pyrene hydroxylase

A single oral dose of 200 mg/kg polybrominated biphenyl (PBB) maximally increased the serosal transfer of iron about 50% in duodenal gut sacs prepared from treated rats. Smaller doses caused inconstant changes in iron transport. The effect, which was maximal at 2 days after treatment, returned to normal at 4 days, and then was increased 16 and 24% at 7 and 18 days, respectively. Iron absorption in vivo increased 29% in rats given 0.1 mg orally five times weekly for 5 weeks. The stimulation of iron absorption was accompanied by an 18% increase in serum iron. The activities of the intestinal and hepatic aryl hydrocarbon hydroxylase was increased in a manner that did not directly correlate with the effects on iron transport, suggesting that the control mechanism for the different biochemical effects are not directly related.