Jejunal Absorption Research Articles

Angiotensin II exerts dual actions on sodium-glucose transporter 1-mediated transport in the human jejunal mucosa

Objectives. Intestinal glucose absorption is mainly mediated via the sodium-glucose transporter 1 (SGLT1) at the apex of the enterocytes, whereas the glucose transporter 2 (GLUT2) provides a basolateral exit. It has been shown in rats that Angiotensin II (AngII), the principal mediator of renin-angiotensin system (RAS), inhibits jejunal SGLT1-mediated glucose absorption. The aim of the present study was to investigate if a similar mechanism exists also in the human jejunal mucosa. Material and methods. Enteroscopy with mucosal biopsy sampling was performed in 28 healthy volunteers. Functional assessments were performed in Ussing chambers using a pharmacological approach. Western blotting and immunohistochemistry were used to assess the presence of the AngII type 1 (AT1R) and type 2 receptor (AT2R), as well as the glucose transporters SGLT1 and GLUT2. Results. Exposure of the mucosa to 10 mM glucose elicited a ≈50% increase in the epithelium-generated current (Iep). This glucose-induced electrogenic response was sensitive to the competitive SGLT1 inhibitor phlorizin, but not to AngII when given alone. AngII combined with the AT2R blocker PD123319 markedly inhibited the response. AngII in combination with the AT1R antagonist losartan tended to increase the electrogenic response, whereas direct activation of AT2R using the agonist C21 significantly enhanced the mucosal response to glucose. The AT1R and AT2R as well as SGLT1 and GLUT2 were detected inside the human enterocytes. Conclusions. The pharmacological analysis indicated that activation of AT1R inhibits, whereas activation of AT2R enhances SGLT1-mediated glucose transport in the human jejunal mucosa.

Bioavailability enhancement of paclitaxel via a novel oral drug delivery system: paclitaxel-loaded glycyrrhizic acid micelles.

Paclitaxel (PTX, taxol), a classical antitumor drug against a wide range of tumors, shows poor oral bioavailability. In order to improve the oral bioavailability of PTX, glycyrrhizic acid (GA) was used as the carrier in this study. This was the first report on the preparation, characterization and the pharmacokinetic study in rats of PTX-loaded GA micelles The PTX-loaded micelles, prepared with ultrasonic dispersion method, displayed small particle sizes and spherical shapes. Differential scanning calorimeter (DSC) thermograms indicated that PTX was entrapped in the GA micelles and existed as an amorphous state. The encapsulation efficiency was about 90%, and the drug loading rate could reach up to 7.90%. PTX-loaded GA micelles displayed a delayed drug release compared to Taxol in the in vitro release experiment. In pharmacokinetic study via oral administration, the area under the plasma concentration-time curve (AUC0→24 h) of PTX-loaded GA micelles was about six times higher than that of Taxol (p < 0.05). The significant oral absorption enhancement of PTX from PTX-loaded GA micelles could be largely due to the increased absorption in jejunum and colon intestine. All these results suggested that GA would be a promising carrier for the oral delivery of PTX.

Structure–retention behaviour of biologically active fused 1,2,4-triazinones – Correlation with in silico molecular properties

The chromatographic behaviour and significant lipophilicity/hydrophobicity indices (log kw, S, φ0) are presented for 21 biologically active fused 1,2,4-triazinones based on the linear relationship: log k=log kw−Sφ established for the retention on LC-18 HPLC column, using as mobile phases mixtures of three organic modifiers with water. The effect of these mobile phase modifiers on the chromatographic behaviour of solutes was established and the organic modifier of choice is suggested. The complex correlation of slopes versus intercepts obtained for acetonitrile, contrary to linear ones obtained for methanol and dioxane are disclosed. The observed difference in retention mechanism for acetonitrile compared to methanol and dioxane is explained by intermolecular interactions encoded in lipophilicity. Linear correlations with statistically significant levels between log kw values determined from three different chromatographic systems were obtained. The relationships between log kw constants (derived from the linear model for methanol–water mobile phases) and predicted log P and log S values by the use of various computational methods were investigated and these were established with high correlation coefficients. The predicted log P values plotted against φ0 (MeOH) indices showed the best fit. Principal component analysis was used to compare various lipophilicity parameters of the solutes and their in silico biological descriptors relevant to optimal pharmacokinetics profile. The similarities and dissimilarities between all the variables and molecular structures of solutes are presented. Statistically significant correlations were found between the chromatographic lipophilicity indices and the calculated pharmacokinetic descriptors: fraction unbound in brain (fu, brain), oral bioavailability (%F), permeability and intestinal absorption in jejunum (Caco-2), skin permeation (log Kp) and blood/brain concentration (log BB).

Enhancing effect of bile salts on gastrointestinal absorption of insulin

Purpose: To investigate the effect of co-administration of two absorption enhancing bile alts, sodium glycocholate (NaGc) and sodium salicylate (NaSal), on insulin absorption via intestinal targeted delivery system.Methods: Insulin (10 IU/kg), associated with and without absorption enhancers (5 % enhancer solution of NaGc or NaSal), was administered to the duodenum, jejunum, and ileum part of the diabetic rat's gastrointestinal (GI) tract by surgical technique. The insulin absorbed from the GI tract was evaluated by its hypoglycemic effect at 45 and 60 min post-administration.Results: The results showed that insulin formulations containing NaGc or NaSal administered into the duodenum and with little quantity in the jejunum decreased blood glucose levels, compared to the reference formulations (p &lt; 0.05). It was also observed that formulations containing cellulose acetate phthalate (CAP) protectors and enhancers may protect insulin during transit through the stomach for 180 min.Conclusion: Thus, the results of this study demonstrate that duodenum-specific delivery of insulin with NaGc and NaSal was achievable by oral administration compared to the other parts of small intestine. Furthermore, NaGc has a greater enhancing effect on duodenal, and to some extent, jejunal absorption of insulin.Keywords: Bile salts, Sodium glycocholate, Sodium salicylate, Insulin, Gastrointestinal administration

Electrocautery-induced localized colonic injury elicits increased levels of pro-inflammatory cytokines in small bowel and decreases jejunal alanine absorption

BackgroundColitis is associated with functional abnormalities in proximal non-inflamed gut areas, but animal models to study small bowel dysfunction in colitis have limitations. This study aims to determine small intestinal alanine absorption and cytokine expression in a novel model of colonic ulceration induced by electro-cautery. MethodsA descending colon ulcer was induced in rats by a bipolar electro-cautery probe. Ulcer score was determined using Satoh’s criteria. Jejunal alanine absorption was measured immediately and at different time intervals post ulcer induction. Levels of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) protein and m-RNA were determined in mucosal scrapings obtained from the colon, duodenum, jejunum and ileum at various time intervals after colonic ulcer induction. ResultsThe mean ulcer score was 3 up to 48h, followed by healing by 96h post ulcer induction. Small bowel histology was normal throughout. Jejunal alanine absorption was reduced by 12–34% immediately and up to 72h after cautery and returned to normal at 96h. IL-1 and TNF-α mRNA increased significantly in the colon, duodenum, jejunum and ileum 3h post electro-cautery and returned to normal at 48h, while that of IL-6 increased significantly at 48h post ulcer induction. Similarly, IL-1, IL-6 and TNF-α protein levels increased in the duodenum, jejunum, ileum and colon up to 48h post ulcer induction. ConclusionsElectrically induced localized colonic injury increased production of pro-inflammatory cytokines in non-inflamed segments of the small intestine and was associated with derangements of jejunal absorptive function.

In vitro and in silico determination of oral, jejunum and Caco-2 human absorption of fatty acids and polyphenols. Micellar liquid chromatography

In this investigation chosen saturated, mono- and polyunsaturated fatty acids as well as polyphenols have been analyzed.The main aim of this study was to determine oral, jejunum and Caco-2 human absorption of chosen fatty acids and polyphenols using in vitro and in silico methods. For in vitro determination of human drug absorption, the usefulness of Micellar Liquid Chromatography (MLC) with mobile phases containing different surfactants (including Brij35–Biopartitioning Micellar Chromatography (BMC)) has been confirmed.On the basis of Foley׳s equation, 1/k vs. CM correlations for the tested compounds have been done. Satisfactory linearity of the relationships was found over the whole eluents composition range studied with R2 approximately 0.99 in each case. Moreover, the analyte–micelle association constants (Kma) from Foley׳s equation have been compared for different micellar environments, containing Brij35, SDS and CTAB as a main component of micellar mobile phases. Completely new models describing human oral as well as Caco-2 and jejunum absorption have been constructed and compared with the cited models. These models are based on the Abraham descriptors and lipophilicity parameters as well as steric descriptors. Furthermore, many different correlations between physicochemical parameters and human intestinal absorption have been done, e.g. the correlation between human jejunum permeability estimated in silico and received using LSER parameters was excellent (R2 nearly 0.99).Chromatographic parameters have been collated with steric, electronic and physicochemical ones using QRAR (Quantitative Retention – Activity Relationships) and QSAR (Quantitative Structure – Activity Relationships) models. Moreover, retention BMC data have been compared with lipophilicity parameter logPo/w (n-octanol–water partition coefficient). The influence of lipophilicity on oral absorption (%) has been checked. The correlation between predicted oral absorption (%) and logPo/w has been done. Obtained R2 was 0.82.On the basis of chromatographic, lipophilicity, steric and different physicochemical parameters, the principal components analysis (PCA) has been done.

GIP regulates glucose transporer‐2 to augment jejunal glucose absorption in obese mouse prior to the onset of diabetes (908.5)

Background: Jejunal glucose absorption is mediated by apical SGLT1 in normal‐, and by GLUT2 in obese mice. GIP, an incretin hormone, activates SGLT1 in normal mice. Aim: To determine if GIP regulates glucose absorption via GLUT2 in the obese jejunum. Methods: Short circuit current (Isc, a measure of SGLT1 activity) and glucose flux (measure of SGLT1 &amp;amp; GLUT2) were obtained under voltage clamp conditions at 0, 1, 2 and 5 mo in jejunums from mice fed a high‐fat diet. Results: In obese mice, GIP significantly activated SGLT1‐mediated glucose absorption for up to 2 mos, but was drastically decreased by 5 mos (2 vs. 5 mos = 725 vs. 49 µA/cm2). In contrast, the rate of GIP‐activated glucose absorption was steadily increased for up to 5 mos on a high‐fat diet (2 vs. 5 mos = 1.4 vs. 2.3 µEq/h.cm2). Western‐blot analyses of biotinylated proteins revealed that GIP exposure enhanced GLUT2 expression in apical membranes of jejunum at 5 mos. Conclusions: GIP both activates and recruits sub‐apical GLUT2 to apical membranes to augment glucose absorption in the jejunum of obese mice.Grant Funding Source: Supported by NIH CTSI grant number UL1TR000157

The distinct roles of anion transporters Slc26a3 (DRA) and Slc26a6 (PAT-1) in fluid and electrolyte absorption in the murine small intestine

The mixing of gastric and pancreatic juice subjects the jejunum to unique ionic conditions with high luminal CO2 tension and HCO3 − concentration. We investigated the role of the small intestinal apical anion exchangers PAT-1 (Slc26a6) and DRA (Slc26a3) in basal and CO2/HCO3 −-stimulated jejunal fluid absorption. Single pass perfusion of jejunal segments was performed in anaesthetised wild type (WT) as well as in mice deficient in DRA, PAT-1, Na+/H+ exchanger 3 (NHE3) or NHE2, and in carbonic anhydrase II (CAII). Unbuffered saline (pH 7.4) perfusion of WT jejunum resulted in fluid absorption and acidification of the effluent. DRA-deficient jejunum absorbed less fluid than WT, and acidified the effluent more strongly, consistent with its action as a Cl−/HCO3 − exchanger. PAT-1-deficient jejunum also absorbed less fluid but resulted in less effluent acidification. Switching the luminal solution to a 5 % CO2/HCO3 − buffered solution (pH 7.4), resulted in a decrease in jejunal enterocyte pHi in all genotypes, an increase in luminal surface pH and a strong increase in fluid absorption in a PAT-1- and NHE3- but not DRA-, CAII, or NHE2-dependent fashion. Even in the absence of luminal Cl−, luminal CO2/HCO3 − augmented fluid absorption in WT, CAII, NHE2- or DRA-deficient, but not in PAT-1- or NHE3-deficient mice, indicating the likelihood that PAT-1 serves to import HCO3 − and NHE3 serves to import Na+ under these circumstances. The results suggest that PAT-1 plays an important role in jejunal Na+HCO3 – reabsorption, while DRA absorbs Cl− and exports HCO3 − in a partly CAII-dependent fashion. Both PAT-1 and DRA significantly contribute to intestinal fluid absorption and enterocyte acid/base balance but are activated by different ion gradients.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-013-1381-2) contains supplementary material, which is available to authorized users.

Intestinal absorption, metabolism, and excretion of (–)-epicatechin in healthy humans assessed by using an intestinal perfusion technique

(-)-Epicatechin is a dietary flavonoid present in many foods that affects vascular function, but its action is limited by incomplete absorption, conjugation, and metabolism. Factors that influence this activity may be attributed to instability in the gastrointestinal lumen, low permeability across the intestinal wall, or active efflux from enterocytes and extensive conjugation. With the use of a multilumen perfusion catheter, we investigated the jejunal absorption, systemic availability, metabolism, and intestinal, biliary, and urinary excretion of (-)-epicatechin in humans. In a single-center, randomized, open, controlled study in 8 healthy volunteers, 50 mg purified (-)-epicatechin was perfused into an isolated jejunal segment together with antipyrine as a marker for absorption. (-)-Epicatechin and conjugates were measured in intestinal perfusates, bile, plasma, and urine. Forty-six percent of the dose was recovered in the perfusate either as unchanged (-)-epicatechin (22 mg) or conjugates (0.8 mg); with stability taken into account, this result indicates that ∼46% of the dose had apparently been absorbed. The conjugates were predominantly sulfates, which indicated conjugation by sulfotransferases followed by efflux from the enterocytes. In contrast, epicatechin glucuronides were dominant in plasma, bile, and urine. Almost one-half of the (-)-epicatechin is apparently absorbed in the jejunum but with substantial interindividual differences in the extent of absorption. The data suggest that the nature and substitution position of (-)-epicatechin conjugation are major determinants of the metabolic fate in the body, influencing whether the compound is effluxed into the lumen or absorbed into the blood and subsequently excreted.

Increases in Bioavailability of Poorly Absorbed Drug by Acylcarnitine

We examined the effect of acylcarnitines on the in situ bioavailability of lucifer yellow (LY) from the loops of small and large intestines of rats. The area under the blood concentration of LY versus time curve (AUC) from the jejunum was significantly increased by the treatments of the loop with 100 µM lauroylcarnitine (LC) or 100 µM palmitoylcarnitine (PC) (fourfold and 17-fold, respectively). No marked change in the expression of claudin-4 protein was observed by the treatments. On the contrary, the expression of P-glycoprotein (P-gp) was decreased by the treatment, more greatly by PC than by LC, suggesting that increases in the bioavailability of LY by LC and PC are associated with the decreased expression of P-gp in jejunum. The increase in the bioavailability was also observed for colon by the treatment of LC, but not that of PC. LC decreased the expression of claudin-4 protein, whereas PC decreased the expression of P-gp in colon. Therefore, LC and PC appear to have different impact on the intestinal transporters depending on the site (i.e., jejunum and colon).

Comparison of mucosal absorption-enhancing activity between a claudin-3/-4 binder and a broadly specific claudin binder

Intercellular spaces between adjacent mucosal epithelial cells are sealed by tight junctions (TJs) that prevent the free movement of solutes across the epithelium. Claudins (CLs), a family of 27 integral membrane proteins, are essential components for TJ seals. We previously used a CL-3/-4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), to show that CL modulation is a promising method to enhance mucosal absorption. Recently, by using a C-CPE mutant library, we developed a CL binder (m19) with broad specificity to CL-1, -2, -4, and -5. Here, we compared the mucosal absorption-enhancing activity of C-CPE and m19. Both CL binders enhanced jejunal absorption of dextran with a molecular mass of 4000 and 150,000Da and nasal absorption of dextran with a mass of 4000Da but not 150,000Da in rats. Although both binders showed similar nasal absorption-enhancing activity of dextran (4000Da), m19 exhibited a more potent jejunal absorption-enhancing effect than that of C-CPE. These findings suggest that mucosal absorption-enhancing activity may be modified by modulating CL specificity.

Creation and biochemical analysis of a broad-specific claudin binder

Claudins (CL) are a family of tetra-transmembrane proteins that are the structural and functional components of tight junctions (TJ). CLs are promising targets for drug development because of their role in mucosal drug absorption and cancer. However, CL-targeted drug development has been delayed because CLs have low antigenicity and preparing CL proteins is difficult. We developed a CL binder by using the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) and a baculoviral display system. After screening CL binders from a C-CPE mutant-displaying library by using CL-displaying budded baculovirus (BV) we isolated a C-CPE mutant called m19, which bound to CL1, CL2, CL4 and CL5. A 3-dimensional analysis showed that m19 has a structural backbone similar to C-CPE. The charge density of the CL-binding domains of m19 and C-CPE differed, suggesting that electrostatic interactions may occur between m19 and CLs. Treatment of epithelial cells with m19 decreased the paracellular but not transcellular integrity, and m19 enhanced jejunal absorption. Thus, we successfully created a CL binder with broad specificity. These findings will contribute to future preparation of CL binders for CL-targeted drug development.

Does pH of tyrode solution modify glucose and electrolyte jejunal absorption in rats?

Studies have shown that glucose is able to decrease the pH of the surface epithelium jejunal preparations when added in vitro and the existence of a high concentration of protons in the immediate area of the mucosa could be of considerable significance for absorption of electrolytes. The aim of this study is to assess whether the change in pH of Tyrode (solution used for perfusion of the jejunum) interferes with the absorption of glucose and electrolytes. Male Wistar rats weighing 200 to 220 g (n = 6) were utilized. Jejunal absorption of glucose and electrolytes was investigated in rats. A Tyrode solution containing twice glucose, sodium and potassium concentration (pH 7.0, 7.4, 8.0 and 8.5) was infused through the jejunal loops during 40 minutes. The glucose absorption was not significantly affected by Tyrode. However, there was significantly decrease in sodium absorption at pH 7.0 and 8.5 (41.13 ± 2.79 and 41.37 ± 1.71, respectively, P < 0.05) when compared with the uptake at pH 7.4 and 8.0 (61.06 ± 6.50 and 56.28 ± 7.03, respectively, P < 0.05). Moreover, potassium absorption increased at pH 8.0 (1.04 ± 0.07) when compared with the uptake at pH 7.0 (0.59 ± 0.04), 7.4 (0.78 ± 0.08) and 8.5 (0.54 ± 0.05) (P < 0.05). These data indicate that the pH of Tyrode has no influence on glucose absorption. However, the major potassium uptake occurs at pH 8.0, while the absorption of sodium is impaired at pH 7.0 and 8.5.

Increased Jejunal Absorption of Glucose in Rats Submitted to Blockade of GABAA Receptors in the Hypothalamic Paraventricular Nucleus

In this study we investigated the status of jejunal absorption and peripheral metabolism of glucose following disinhibition of paraventricular nucleus (PVN) induced by the GABAA antagonist bicuculline methiodide (BMI). Adult male Wistar rats (270-300g) were anesthetized to implant unilateral guide cannula targeted to PVN for later microinjec- tion of BMI (10 pmol/100 nl, n=6) or vehicle (NaCl 0.9 % /100 nl, n=5). The jejunal loop was isolated and perfused (0.5 mL/min) with Tyrode solution containing twice the normal concentrations of glucose, sodium, and potassium. After mi- croinjections into PVN, perfusate and blood samples were taken every 10 min over a 40 min period. In comparison with vehicle, BMI into PVN increased glucose absorption at 30 min (1.2 ± 0.1 vs. 1.8 ± 0.1 � mol/min; *P<0.05) and at 40 min (1.3 ± 0.2 vs. 2.4 ± 0.3 � mol/min; *P<0.05), whereas plasma insulin was significantly reduced (0.5 ± 0.04 vs. 0.3 ± 0.04 ng/ml, *P<0.01). At the end of the experiment, samples from the liver and gastrocnemius muscle were taken to measure levels of glycogen, intermediate metabolites of the glycolytic pathway and ATP. Compared to control, muscle glucose-6- phosphate (0.380 ± 0.063 vs. 0.253 ± 0.020 � mol/g; P<0.05) and ATP (0.166 ± 0.065 vs. 0.038 ± 0.013 � mol/g; *P<0.05) were reduced in the group microinjected with BMI into PVN. We conclude that PVN disinhibition changes the absorption and peripheral glucose metabolism.

The response of the intestinal mucosa to prostaglandin E2 during withdrawal from morphine.

Experiments were designed to determine whether the diarrhoea characteristic of morphine withdrawal results from an enhanced sensitivity of the intestinal mucosa to PGE2. Rats (250-300 g) were made morphine-dependent by subcutaneous injection of an emulsion releasing 300 mg morphine HCl over 48 h. In-vivo, the transintestinal potential difference (PD) responses to PGE2 (4.6-46 micrograms kg-1 i.v.), which reflect increased Cl secretion, were significantly larger in withdrawn (morphine emulsion, 10 mg kg-1 naloxone s.c.) compared with non-dependent animals (emulsion only, naloxone s.c., P less than 0.05). Muscle-stripped intestinal sheets from dependent animals incubated with naloxone (10(-5) mol L-1) in-vitro did not demonstrate a greater electrical response (PD, short circuit current) to PGE2 (1.4 x 10(-6) mol L-1) than sheets taken from non-dependent animals. In-vitro preparations from animals withdrawn in-vivo did not respond differently from tissue taken from non-dependent animals (naloxone 10 mg kg-1 s.c., 10(-4) mol L-1 in medium, in both groups). This occurred in whole sheets of intestine as well as sheets without attached muscle. Jejunal fluid absorption in-vivo was lower in withdrawn animals than in non-dependent animals. However, the responses to intra-arterial infusion of PGE2 were similar in both groups with 2 micrograms min-1 inhibiting absorption and 4 micrograms min-1 inducing secretion. In-vivo, PGE2-induced Cl secretion appears to be enhanced during withdrawal although net fluid transport is not altered, suggesting different effects of the withdrawal process on the electrogenic Cl secretory and neutral NaCl absorptive mechanisms.

Enhancement of jejunal and colonic absorption of fosfomycin by promoters in the rat.

A means of enhancing absorption of the antibiotic, fosfomycin, has been investigated using promoters in rat jejunum and colon. Polyoxyethylene lauryl ether (BL-9EX), saponin, the sodium salts of fatty acids and mixed micelles were effective at 1% in increasing fosfomycin absorption. Of the sodium salts of saturated medium-chain fatty acids examined, the strength of this effect was in the order caprate greater than laurate greater than caprylate. Mixed micelles, consisting of fusogenic lipids and sodium taurocholate, enhanced fosfomycin absorption independently of the degree of unsaturation of the lipids; their effectiveness far exceeded that of sodium taurocholate alone. The action of these promoters was more evident in the colon than in the jejunum, except for the sodium salts of bile acids and disodium ethylenediaminetetraacetate (EDTA-2Na). The effects of glycocholate and taurocholate were essentially the same at both absorption sites, but that of EDTA-2Na was much greater in the jejunum than the colon. Improved fosfomycin absorption was observed at more than 0.5% sodium caprate concentrations in both the jejunum and colon. BL-9EX was effective at 0.1% in the jejunum or at 0.05% in the colon. The effectiveness at these low concentrations demonstrates the practicality of promoters for improving fosfomycin absorption with only minor membrane damage, especially in the colon.

Claudin as a Target for Drug Development

Tight junctions (TJs) play pivotal roles in the fence and barrier functions of epithelial and endothelial cell sheets. Since the 1980s, the modulation of the TJ barrier has been utilized as a method for drug absorption. Over the last decade, the structural and functional biochemical components of TJs, such as occludin and claudin, have been determined, providing new insights into TJ-based pharmaceutical therapy. For example, the modulation of the claudin barrier enhances the jejunal absorption of drugs, and claudin expression is deregulated in cancer cells. Claudin is a co-receptor for the hepatitis C virus. Moreover, claudin is modulated during inflammatory conditions. These findings indicate that claudins are promising drug targets. In this review, we discuss the seeds of claudin-based drug development, which may provide potential pharmaceutical breakthroughs in the future.

Gastrointestinal calcium absorption in sheep is mostly insensitive to an alimentary induced challenge of calcium homeostasis

For ruminants, marked differences to monogastric species have been described concerning the localisation and vitamin D sensitivity of gastrointestinal calcium absorption, particularly with respect to the forestomach compartment. Therefore, we investigated gastrointestinal calcium transport of sheep as influenced by a dietary calcium restriction and/or a supraphysiological dosage of exogenous calcitriol. Using the Ussing chamber technique, we determined calcium and mannitol flux rates to differentiate between para- and transcellular calcium transport in rumen, duodenum, jejunum and colon. Expression of epithelial calcium channels, calbindin-D(9K), and basolateral extrusion mechanisms was determined by quantitative RT-PCR and Western blot analysis. Active calcium transport could be demonstrated in jejunum and rumen. A significant stimulation of jejunal calcium absorption was only observed in animals treated with calcitriol. The alimentary calcium restriction alone did not result in significant effects indicating a less effective intestinal adaptation to alimentary calcium restriction than observed in monogastric animals. The observed ruminal calcium transport was not affected at all, neither by the diet nor the calcitriol treatment. Furthermore, no significant expression of epithelial calcium channels or calbindin-D(9K) could be detected in the rumen; therefore it is concluded that calcium transport in the forestomachs is probably mediated by a different, so far unknown mechanism.

Gene ablation for PEPT1 in mice abolishes the effects of dipeptides on small intestinal fluid absorption, short-circuit current, and intracellular pH

PEPT1 function in mouse intestine has not been assessed by means of electrophysiology and methods to assess its role in intracellular pH and fluid homeostasis. Therefore, the effects of the dipeptide glycilsarcosin (Gly-Sar) on jejunal fluid absorption and villous enterocyte intracellular pH (pH(i)) in vivo, as well as on enterocyte[(14)C]Gly-Sar uptake, short-circuit current (I(sc)) response, and enterocyte pH(i) in vitro were determined in wild-type and PEPT1-deficient mice and in mice lacking PEPT1. Immunohistochemistry for PEPT1 failed to detect any protein in enterocyte apical membranes in Slc15a1(-/-) animals. Saturable Gly-Sar uptake in Slc15a1(-/-) everted sac preparations was no longer detectable. Similarly, Gly-Sar-induced jejunal I(sc) response in vitro was abolished. The dipeptide-induced increase in fluid absorption in vivo was also abolished in animals lacking PEPT1. Since PEPT1 acts as an acid loader in enterocytes, enterocyte pH(i) was measured in vivo by two-photon microscopy in SNARF-4-loaded villous enterocytes of exteriorized jejuni in anesthetized mice, as well as in BCECF-loaded enterocytes of microdissected jejunal villi. Gly-Sar-induced pH(i) decrease was no longer observed in the absence of PEPT1. A reversal of the proton gradient across the luminal membrane did not significantly diminish Gly-Sar-induced I(sc) response, whereas a depolarization of the apical membrane potential by high K(+) or via Na(+)-K(+)-ATPase inhibition strongly diminished Gly-Sar-induced I(sc) responses. This study demonstrates for the first time that proton-coupled electrogenic dipeptide uptake in the native small intestine, mediated by PEPT1, relies on the negative apical membrane potential as the major driving force and contributes significantly to intestinal fluid transport.

A claudin‐4 modulator enhances the mucosal absorption of peptide

ObjectiveParacellular pathway is a principal route for absorption of biologics (peptides, proteins and nucleic acids); however, tight junctions (TJs) restrict passing of biologics across epithelium. Claudin is known as a key component in TJs‐barrier. We previously found that a claudin‐4 modulator, C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE), enhanced jejunal absorption of dextran. In this study, we investigated whether a claudin‐4 modulator is a potent mucosal absorption‐enhancer of a peptide drug.MethodsMucosal absorption was evaluated by rat jejunal, pulmonary and nasal administration of human parathyroid hormone derivative, hPTH(1–34) and C‐CPEs. Plasma hPTH(1–34) levels were measured by RIA. Binding of C‐CPEs to claudin‐4 was assayed by ELISA and BIAcore analysis.ResultsPre‐treatment with C‐CPE enhanced mucosal absorption of hPTH(1–34). However, the jejunal and pulmonary absorption‐enhancing effects were not observed by co‐treatment with C‐CPE. The deletion of 10 amino acids at the N‐terminal of C‐CPE increased its solubility by 30‐fold. Moreover, the truncated C‐CPE bound to claudin‐4, modulated the TJ‐barrier and enhanced jejunal absorption of dextran. Co‐treatment of the truncated C‐CPE also enhanced jejunal and pulmonary absorption of hPTH(1–34).ConclusionClaudin‐4 modulator may be a promising absorption‐enhancer of a peptide drug.