Alanine Absorption Research Articles

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.

Effects of deoxynivalenol and lipopolysaccharide on electrophysiological parameters in growing pigs

Deoxynivalenol (DON) is a major B-trichothecene that draws importance from its natural occurrence in cereals worldwide. It has many effects on rapidly dividing cells. Lipopolysaccharide (LPS) is an endotoxin released from most Gram-negative bacteria, which plays a major role in induction of inflammation and sepsis under certain conditions. In our experiments we aimed to study the effects of different concentrations of DON (up to 8,000ng/ml) on the electrogenic transport of nutrients and on tissue conductances in growing pigs using the Ussing chamber technique. The effect of DON-contaminated feed (2.9mg/kg feed) on the respective parameters, as well as the interactions between DON and intraperitoneal (i.p.) LPS were assessed using porcine jejunal tissues. In vitro DON inhibited the absorption of alanine and glucose across the pig jejunum at concentrations of 4,000 and 8,000ng/ml, suggesting that DON had an inhibitory effect on the electrogenic transport of nutrients across porcine small intestines. Electrogenic transport of alanine and glucose across porcine small intestines varied regionally among intestinal segments with higher response in ileal tissues. A synergistic effect was observed between DON in feed and injected LPS on tissue conductance. In response, glucose with higher short circuit currents was observed across porcine jejunal mucosa in nutrient stimulated conditions.

Experimental determination of the energy response of alanine pellets in the high dose rate 192Ir spectrum

An experimental determination of the energy correction factor for alanine/paraffin pellets in the 192Ir spectrum at varying distances from the source is presented. Alanine dosimeters were irradiated in water under full scatter conditions with a high dose rate (HDR) 192Ir source (Flexisource), using a dedicated holder. Up to six line sources (catheters) fit in a regular pattern at fixed radial distances from the holder axis, the alanine detector being placed at the centre of the holder. The HDR source was stepping every 0.5 cm within a trocar needle within ± 3.0 cm around the medial plane through the detector in order to achieve dose homogeneity within the detector volume. The energy correction factor of alanine/paraffin pellets in 192Ir relative to 60Co was experimentally determined as the inverse ratio of the dose to water measured in water around the 192Ir source to the dose to water calculated in water using the TG-43 formalism. The pellets were read out with a Bruker EMXmicro spectrometer (X-band). The amplitude of the central line in the alanine absorption spectrum from pellets irradiated within the 192Ir spectrum was directly compared with the amplitude from 60Co-irradiated pellets. The energy correction factors of Harwell pellets irradiated in the 192Ir spectrum are 1.029 ± 0.02, 1.027 ± 0.02 and 1.045 ± 0.02 at a mean weighted source–detector distance of 2.0, 2.9 and 5.3 cm, respectively. The experimentally obtained values for the energy response are 1.3% lower compared to the theoretical values for radial distances smaller than 3 cm.

Capsaicin‐Sensitive Primary Afferent (CSPA) Fibers are Involved in the Regulation of Glucose Transport and the Expression of its Transporters in the Small Intestine in Rats

Previous studies have shown that CSPA fibers are involved in the regulation of alanine absorption in the small intestine. However, their effect on glucose absorption has not been yet fully characterized. This study examines the effect of CSPA fibers on glucose absorption and determines their effect on mRNA expression of glucose transporters (GLUT). Glucose absorption was measured in a jejunal segment perfused with 20mM glucose using the single-pass perfusion technique. CSPA ablation was achieved by subcutaneous injections of 1% capsaicin under light anesthesia at different time intervals. Sham rats received vehicle injections. Two weeks post-treatment, verification of desensitization was done by standard method. Glucose absorption in the jejunum showed a 68% increase in capsaicin-treated rats compared to sham. mRNA expressions of sodium-glucose cotransporter (SGLT1), GLUT2 and Na+-K+-ATPase were determined in jejunal samples by real-time PCR with actin as the housekeeping gene. A 14-fold increase in SGLT1 and a 7-fold increase in Na+-K+-ATPase mRNA expression were detected in capsaicin-treated rats as compared to sham. No significant increase in GLUT2 mRNA expression was noted in capsaicin-treated rats. Our results show that ablation of CSPA fibers increased glucose absorption by increasing the expression of its transporter. This indicates that CSPA fibers exercise an inhibitory tone in the small intestine that regulates epithelial absorption of glucose. This work is partially supported by the Lebanese National Council for Scientific Research.

Troubleshooting in animal models of colitis: The use of a novel electrocautery model

Introduction: Experimental colitis induced by chemical agents leads to upregulation of inflammatory cytokines in distant unaffected small intestine and to a decrease in nutrient absorption. To preclude any possible proximal diffusion of these chemicals, we designed a novel method for ulcer induction in the colon by electrocautery. Methods: Under light anesthesia, a colonic ulcer was induced in rats by a special electrocautery probe introduced in the descending colon through the rectum allowing the injection of a controlled electrolytic current. A direct current (3–7 mA) was delivered through the electrodes for 30 s and then for another 30 s after reversing the polarity of the electrodes. Then, the probe was moved for a distance of ± 0.5 cm and the current injection was repeated. Rats were sacrificed at various time intervals after ulcer induction (3–96 h). Samples from colon and jejunum were taken for histological assessment and determination, by ELISA, of the levels of interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α). In other groups of animals, jejunal amino acid absorption was determined in vivo at 24 and 48 h post electrocautery. Results: A colonic ulcer persisted for 72 h after cauterization. A significant upregulation of the levels of different cytokines was observed in the colon and jejunum post cauterization and persisted for at least 48 h. In the jejunum, IL-1β increased from 81 ± 9 to 652 ± 110 ( p < 0.01) and 243 ± 47 ( p < 0.05) pg/mg protein at 24 and 48 h, respectively. Similarly, jejunal TNF-α levels increased by approximately 2 folds at 24 and 48 h post ulcer induction ( p < 0.05). A similar but higher increase in cytokines was observed in the colon. Jejunal alanine absorption (0.2 ± 0.02 µmol/20 min/cm) decreased significantly at 24 and 48 h after colitis induction (0.12 ± 0.01 and 0.14 ± 0.02, respectively; p < 0.01). Discussion: This model may be used as an alternative or a complement to chemical models of colitis.

Characterisation of electrogenic nutrient absorption in the Cftr TgH(neoim)Hgu mouse model

Most cystic fibrosis (CF) patients show an exocrine pancreatic insufficiency that results in lower enzyme and bicarbonate secretion. To test whether an altered function of nutrient transporters might additionally attribute to the lower bodyweight of CF patients we investigated electrogenic absorption of alanine, glycyl-glutamine, glucose and the effect of pH on nutrient absorption by Ussing chambers in a CF mouse model carrying the Cftr TgH(neoim)Hgu mutation. The electrogenic transport of all three nutrients was similar between the D2.129P2(CF/3)-Cftr TgH(neoim)Hgu congenic strain and DBA/2J mice as well as between the B6.129P2(CF/3)-Cftr TgH(neoim)Hgu congenic strain and C57BL/6J mice. This indicates that the Cftr TgH(neoim)Hgu mutation does not affect the electrogenic absorption of alanine, glycyl-glutamine and glucose. In contrast, electrogenic nutrient absorption was reduced in the CF/1-Cftr TgH(neoim)Hgu and CF/3-Cftr TgH(neoim)Hgu inbred strains compared to the HsdOla:MF1, D2.129P2(CF/3)-Cftr TgH(neoim)Hgu and B6.129P2(CF/3)-Cftr TgH(neoim)Hgu strains, whereas no difference was found among the wild-type strains. This indicates that not the Cftr TgH(neoim)Hgu mutation but differences in the genetic background of the CF/1-Cftr TgH(neoim)Hgu and CF/3-Cftr TgH(neoim)Hgu strains compared to HsdOla:MF1, D2.129P2(CF/3)-Cftr TgH(neoim)Hgu and B6.129P2(CF/3)-Cftr TgH(neoim)Hgu strains are associated with the differences in electrogenic nutrient absorption. The electrogenic absorption of alanine, glycyl-glutamine and glucose was not influenced by an acidic pH (5.4) compared to absorption at pH 7.4.

Enterogenesis in a clinically feasible model of mechanical small-bowel lengthening

Recent work indicates that mechanical force induces small-bowel growth, although methods reported do not have direct clinical application. We report a clinically feasible technique of enterogenesis and describe intestinal function in this model. Using a pig model (n = 11), we stretched isolated small intestinal segments mechanically for 7 days in vivo with an intraluminal device. Control segments were not stretched. Morphology, histology, and epithelial proliferation were assessed. Absorption and epithelial barrier function were examined in an Ussing chamber. Stretch segments were significantly longer than Control segments and had nearly 2-fold greater surface area (P < .001). Mucosal thickness was much greater in Stretch than Control segments (772 +/- 134 vs. 647 +/- 75 microm, P = .02). Although villus height was reduced in Stretch and Control segments (353 +/- 76 vs. 324 +/- 76 microm, P = .6) versus native jejunum (522 +/- 87, P < .0005), crypt depth was increased dramatically in Stretch (450 +/- 95 microm) versus Control segments (341 +/- 64, P = .005). This observation was accompanied by a 2-fold increase in cellular proliferation (26.3 +/- 3.8 vs 12.1 +/- 6.6 % bromodeoxyuridine+, P < .05). Barrier function was intact ([3H]-mannitol permeation, 0.16 +/- 0.08%, vs native jejunum, 0.17 +/- 0.08%, P = .81). Glucose-mediated sodium transport was similar in Stretch versus native jejunum segments (60.0 +/- 23.5 vs 82.3 +/- 47.3 microA/cm2, P = .31), as was carbachol-induced chloride transport (82.4 +/- 72.2 vs 57.2 +/- 33.4 microA/cm2, P = .54) and alanine absorption (16.46 +/- 12.94 vs 23.53 +/- 21.31 microA/cm2, P = .53). Mechanical stretching induces small intestinal growth, while maintaining function. Epithelial architecture does change, such that a decrease in villus height is offset by a marked increase in crypt depth and a 2-fold increase in epithelial proliferation. Epithelial barrier and absorptive functions remain intact. The device described may have direct clinical applicability.

Na+ and pH dependence of proline and β‐alanine absorption in rat small intestine

Early characterization of intestinal absorption of imino acids in mammals has demonstrated the existence of a Na+-dependent, Cl- -independent transport system in rat small intestine, which is the only carrier for beta-alanine. Based on the substrate selectivity, it was proposed that the Proton Amino Acid Transporter 1 (PAT1) could be the same as this imino acid carrier. The present study characterizes the pH and Na+ dependence of proline and beta-alanine uptake in rat small intestine. Intestinal uptake of radiolabelled l-proline or beta-alanine was measured in brush border membrane vesicles and everted intestinal rings, in the presence and absence of Na+ and at different pH values. The existence of an inwardly directed H+ gradient in the absence of Na+ enhanced the initial entry of proline and beta-alanine in brush border membrane vesicles, that reached a transient overshoot with maximal value around 30 s. In the absence of pH gradient, no overshoot was shown. In entire tissue, there was an increase of proline and beta-alanine uptake at acidic pH that was higher in the presence of Na+ than in its absence. This ion dependence and pH effect of the amino acids uptake also increased with the incubation period. Substrate inhibition studies confirmed that intestinal proline absorption in rat occurs mainly by system B and PAT1-like transporter. There is a Na+ -independent, H+ -dependent transporter of amino acids at the apical membrane of the rat enterocytes.

Effect of glutamine on water and sodium absorption in human jejunum at baseline and during PGE1-induced secretion

Glutamine, a major fuel for enterocytes, stimulates water and sodium absorption in animal models of secretory diarrhea, but data in humans are still limited. The aim of this study was to investigate the effect of glutamine on jejunal absorption during hypersecretion in humans. In six healthy adults, the effects of glutamine on jejunal absorption were assessed with a triple-lumen tube on two occasions, at baseline and during PGE(1)-induced hypersecretion (0.1 microg.kg(-1).min(-1)) in a random order. Isoosmolar solutions containing polyethylene glycol 4000 as nonabsorbable marker were infused in the jejunum at 10 ml/min over 1-h periods: saline (sodium chloride 308 mmol/l), glucose-mannitol 45:45 mM, glucose 90 mM, alanine-glucose 45:45 mM, glutamine-glucose 45:45 mM, and glutamine 90 mM. Net absorptive and secretory fluxes were measured at steady state. At baseline, glutamine- and alanine-containing solutions induced a threefold increase of water and sodium absorption (P < 0.05); 90 mM glutamine stimulated water absorption more than 90 mM glucose (3.6 +/- 0.6 vs. 1.9 +/- 0.3 ml.min(-1).30 cm(-1), P < 0.05). PGE(1)-induced hypersecretion was reduced (P < 0.05) by solutions of alanine-glucose, glutamine-glucose, and glutamine 90 mM (P < 0.05) and reversed to absorption by alanine-glucose and glutamine-glucose. Glutamine and alanine absorption was nearly complete and was not influenced by PGE(1). In conclusion, glutamine stimulates water and electrolyte absorption in human jejunum, even during experimental hypersecretion. In addition to the metabolic effects of glutamine, these results support the evaluation of glutamine-containing solutions for the rehydration and the nutritional support of patients with secretory diarrhea.

Effects of vasoactive intestinal polypeptide microinjected into the nucleus tractus solitarius on jejunal electrolytes absorption in rats

It has been shown that vasoactive intestinal polypeptide (VIP) injected into the nucleus tractus solitarius inhibits alanine absorption across the jejunum. The aim of the present study was to investigate the effects of VIP injection into the nucleus tractus solitarius on jejunal absorption of electrolytes in the rat. Fifty-three Wistar rats were submitted to midline laparotomy to expose and isolate 20 cm of jejunal loop and to perform a subdiaphragmatic troncular vagotomy. Saline or VIP (10 pg 100 nl(-1)) was injected into the rostral nucleus tractus solitarius using a stereotaxic instrument. Tyrode solution, pH 8, containing twice glucose, sodium and potassium concentration was infused (0.5 ml min(-1)) into the jejunal loop. Samples were taken at 10-min intervals during the 40-min-experiment. Injection of VIP into the nucleus tractus solitarius increased jejunal potassium absorption. Moreover, VIP associated with vagotomy resulted in inhibition of jejunal potassium absorption by VIP alone at 40 min after perfusion (5.99 +/- 0.74 vs. 9.83 +/- 0.57 microM). There was no change in jejunal sodium absorption in any of the experimental groups. VIP had a modulatory action on jejunal potassium absorption when injected into the nucleus tractus solitarius.

Effects of vasoactive intestinal polypeptide microinjected into the nucleus tractus solitarius on jejunal glucose absorption in rats

It has been shown that vasoactive intestinal polypeptide (VIP) injected into the nucleus tractus solitarius and into the dorsal motor nucleus of the vagus inhibits alanine absorption across the jejunum. The aim of the present study was to investigate the effects of VIP injection into the nucleus tractus solitarius on jejunal absorption of glucose in the rat. Forty Wistar rats were submitted to midline laparotomy to expose and isolate 20 cm of jejunal loop and to perform a subdiaphragmatic troncular vagotomy. Saline or VIP (10 pg 100 nl −1) was injected into the rostral nucleus tractus solitarius using a stereotaxic instrument. Tyrode solution, pH 8, containing twice glucose, sodium, and potassium concentrations was infused (0.5 ml min −1) into the jejunal loop. Samples were taken at 10-min intervals during the 40-min experiment. Injection of VIP into the nucleus tractus solitarius associated with vagotomy resulted in inhibition of jejunal glucose absorption by VIP alone at 10 and 40 min after perfusion (2.75±0.19 vs. 3.53±0.29 mg). The vagal outflow tract maintained jejunal glucose absorption even when VIP was microinjected into the nucleus tractus solitarius.

2003 Harry M. Vars Research Award. Keratinocyte growth factor improves epithelial function after massive small bowel resection.

Massive small bowel resection with subsequent short bowel syndrome (SBS) leads to the acute loss of epithelial cell (EC) absorptive function. Keratinocyte growth factor (KGF) has been shown to improve EC growth, although little is known about KGF activity on EC function after SBS. We hypothesized that KGF would improve epithelial function in a mouse SBS model. Adult C57BL/6J mice were randomized to a 55% mid-small bowel resection (SBS), SBS with KGF administration (SBSKGF), or sham-operated (Control) group, and were killed at 7 days. Ussing chambers were used to study epithelial function. Short circuit current (Isc) was monitored. EC absorption was studied by measuring (1) glucose [3-O-methyl-D-[1-3H]glucose (3-OMG)] absorption; (2) sodium coupled amino acid (alanine) absorption; and (3) changes in Isc by using the absorptive agent D-glucose (stimulated Na+ absorption). Epithelial barrier function was measured with transepithelial resistance (TER) and transmural passage of 3H-mannitol (Papp). ECs were separated along the crypt-villus axis with laser capture microdissection. Epithelial KGF receptor (KGFR) mRNA expression was studied using real time reverse transcriptase-polymerase chain reaction (RT-PCR). KGF administration increased the basic ion transport activity and net transepithelial absorption of 3-OMG and sodium-coupled alanine absorption. SBS significantly decreased epithelial ion transport, including the Na+ absorption stimulated by D-glucose and L-alanine. KGF administration partially improves Na+ absorption. KGF had no apparent effect on the TER and 3H-mannitol permeability in this study. KGF upregulated EC KGFR mRNA expression, predominately in the crypt and lower portion of the villus. KGF administration improves epithelial absorptive function and stimulates intestinal proliferation after SBS. This suggests that KGF improves intestinal adaptation after SBS and may have clinical applicability.

Experimental colitis decreases rat jejunal amino acid absorption: Role of capsaicin sensitive primary afferents

Ulcerative colitis and experimental colitis are known to be associated with functional and structural abnormalities of the small intestine. The aim of this study was to determine whether experimental colitis in the rat has any effect on jejunal amino acid absorption and to investigate the neural mechanisms involved. In Sprague Dawley rats, colitis was induced by intracolonic administration of 0.1 ml of 6% iodoacetamide. Alanine absorption in the jejunum was measured using the single pass intraluminal perfusion technique in vivo and the three-compartment model in vitro. Experiments were done in normal and sham treated rats, as well as in rats that underwent neonatal capsaicin treatment, adult capsaicin treatment, or subdiaphragmatic vagotomy. Colitis was more severe in rats subjected to neonatal or adult capsaicin treatment, but was not affected by subdiaphragmatic vagotomy. In rats with colitis, jejunal alanine absorption was reduced by 2% (P>0.05), 28%, 40%, and 18% (P<0.001) at 1, 1.5, 2, and 3 days post rectal iodoacetamide administration. A rebound increase of 12% above baseline was noted at 4 days (P<0.05). Similar results were noted in vitro. In rats that received two consecutive injections of iodoacetamide, the decrease in jejunal alanine absorption occurred earlier, was more severe, and persisted for more than 30 days. Neonatal as well as adult capsaicin treatment aggravated both the colitis and the decrease in jejunal alanine absorption. On the other hand, subdiaphragmatic vagotomy attenuated the decrease in jejunal alanine absorption, but had no significant effect on colitis severity. It is concluded that iodoacetamide induced colitis impairs jejunal amino acid absorption and that this effect involves vagal efferents as well as capsaicin sensitive primary afferents.

Absorption of leucine, alanine and lysine from the rumen.

The absorption of three amino acids (leucine, alanine and lysine) from the washed, closed rumen was studied in a short-term (75 min) experiment in situ. The concentration of leucine and alanine did not change in the rumen during the experiment, while that of lysine continuously decreased, and 40% of the total lysine placed in the rumen was absorbed during the experimental period. The rate of absorption decreased in proportion to the fall of amino acid concentration.

Absorption of leucine, alanine and lysine from the rumen

Absorption of leucine, alanine and lysine from the rumen

Effect of cinnamon, clove and some of their constituents on the Na +-K +-ATPase activity and alanine absorption in the rat jejunum

The effect of a water extract of some spices on the in vitro activity of the rat jejunal Na +-K +-ATPase was investigated. Extracts of nutmeg, cinnamon, clove, cumin, coriander, turmeric and caraway all inhibited the ATPase, while aniseed and white pepper exerted no significant effects. The extracts of clove and cinnamon had the most potent inhibitory effect on the intestinal ATPase as compared to extracts of other spices. They also inhibited the in vitro Na +-K +-ATPase activity in a crude kidney homogenate and the activity of an isolated dog kidney Na +-K +-ATPase. The alcoholic extract of cinnamon, compared to the aqueous extract, had a stronger inhibitory action on the jejunal enzyme and a lower IC 50 value, which was not significantly different from the one observed with cinnamaldehyde, the major volatile oil present cinnamon, suggesting that in alcoholic extracts cinnamaldehyde is the major inhibitory component. The IC 50 values of eugenol, aqueous clove extract and ethanolic clove extract all fell within the same range and were not significantly different from each other, suggesting that eugenol is the major inhibitory component in both alcoholic and aqueous extracts. Based on the IC 50 values, the order of sensitivity of the enzyme to the spices extracts is as follows: isolated dog kidney ATPase>rat kidney ATPase>rat intestine ATPase. The aqueous extracts of clove and cinnamon also significantly lowered the absorption of alanine from the rat intestine. It was concluded that the active principle(s) in clove and cinnamon can permeate the membrane of the enterocytes and inhibit the Na +-K +-ATPase that provides the driving force for many transport processes.

Calcitonin gene-related peptide regulates amino acid absorption across rat jejunum

The calcitonin gene related peptide (CGRP) is widely distributed in the enteric nervous system and gut afferents. Its role in normal digestion and absorption is not characterised. This study is conducted to elucidate whether CGRP regulates amino acid absorption in the small intestine. In in vivo experiments using the single-pass perfusion technique, intravenous infusion of CGRP (250–750 pmol/kg-min) reduced alanine absorption by 35–40%. The effects were completely blocked by the antagonist hCGRP (8–37). Moreover, intravenous infusion of CGRP antagonist blocked the inhibitory effect of intraluminal capsaicin perfusion on alanine absorption. Similarly, intracerebral injection of CGRP decreased alanine absorption, an effect which was reduced by vagotomy. In vitro experiments using isolated jejunal strips showed that CGRP reduced alanine absorption in a dose-dependent manner. At 6 pM, CGRP decreased alanine absorption by 33%. Similarly, CGRP reduced the absorption of proline and taurine by 20 and 11.5%, respectively. Kinetic studies revealed that CGRP reduces alanine influx into intestinal epithelial cells by inhibiting the affinity of the carriers. It is demonstrated that CGRP is involved in the regulation of jejunal amino acid absorption through intrinsic (enteric) and extrinsic (central) neural mechanisms.

Neural mediation of vasoactive intestinal polypeptide inhibitory effect on jejunal alanine absorption.

It was recently shown that vasoactive intestinal polypeptide (VIP) inhibits rat jejunal alanine absorption, an effect that was significantly reduced by vagotomy. This study assesses the role of capsaicin-sensitive primary afferents (CSPA) and the myenteric plexus in the inhibition of rat jejunal alanine absorption by VIP. Continuous intravenous infusion of VIP (11.2 ng . kg-1 . min-1) reduced alanine absorption by 60% in sham control rats and by 20% in rats neonatally treated with capsaicin (P < 0.01). In in vitro experiments, VIP decreased alanine uptake by jejunal strips isolated from sham control rats in a dose-dependent manner. In the presence of 40 nM VIP, alanine uptake by full-thickness jejunal strips was reduced by 54% in sham control rats and by 25% in rats neonatally treated with capsaicin (P < 0.001). On the other hand, VIP reduced alanine uptake by mucosal scrapings by 25% in sham rats compared with 9% reduction in neonatally treated rats. Chemical ablation of the extrinsic innervation and jejunal myenteric plexuses by pretreatment with benzalkonium chloride significantly (P < 0.001) reduced basal alanine absorption and the inhibitory effect of VIP. Moreover, incubation of intestinal strips with tetrodotoxin and atropine reduced significantly (P < 0.05) the inhibitory effect of VIP on alanine absorption. These data suggest that VIP exerts its inhibitory effect on alanine absorption through the CSPA fibers and the myenteric plexus. The neuronal circuitry of this inhibitory process may involve cholinergic muscarinic mechanisms.

Acute and neonatal capsaicin treatment inhibit jejunal amino acid absorption through a Na+-dependent mechanism.

It has recently been shown that capsaicin inhibits alanine absorption in rat jejunum via mechanisms that involve intestinal capsaicin-sensitive primary afferent (CSPA) fibers. This study provides further evidence that the effect of capsaicin is neurally mediated and demonstrates that CSPA fibers regulate Na+-dependent amino acid absorption. In vivo, basal alanine absorption in rats neonatally treated with capsaicin was reduced by 35% below control. Furthermore, intraluminal perfusion of 400 microM capsaicin reduced jejunal alanine absorption by 31% in sham rats but had no significant effect in rats neonatally treated with capsaicin. In vitro, capsaicin significantly reduced uptake of alanine and proline by jejunal strips but had no effect on uptake of lysine. Tetrodotoxin (0.2 microM) partially blocked the effects of capsaicin but did not itself affect alanine absorption. Capsaicin reduced unidirectional mucosal-to-serosal alanine (1 mM) influx by 33%, an effect that becomes significant after 5 min of preincubation with capsaicin. Neonatal capsaicin treatment reduced basal alanine influx in jejunal strips by 37%; however, preincubation of these strips with capsaicin had no significant effect. Kinetic analysis of alanine steady-state uptake and influx by jejunal strips incubated with capsaicin revealed that capsaicin reduced the Na+-dependent component of alanine influx into intestinal epithelial cells. Long-term sensory denervation by capsaicin also decreased the Na+-dependent component of alanine absorption. These data suggest that intestinal capsaicin-sensitive primary afferent fibers regulate Na+-dependent amino acid absorption.

Effect of cationic drugs on Na&lt;SUP&gt;+&lt;/SUP&gt;-dependent carrier mediated transport and its evaluation

We investigated the effect of cationic drugs on carrier mediated transport using in vitro and in situ techniques. The initial uptake of glucose, alanine and glutamic acid by rat intestinal brush border membrane vesicles(BBMV) was distinctly inhibited by imipramine in the presence of Na+ gradient, but not inhibited in the absence of Na+ gradient. This result indicates that imipramine inhibited carrier mediated transport. Imipramine exhibited competitive inhibition with respect to Na+ concentration, while exhibited non-competitive inhibition with respect to glutamic acid concentration. This finding suggests that imipramine affected carrier mediated transport by competitive inhibition to Na+ binding site of the carrier. On the other hand, in situ single pass perfusion method showed that absorption of alanine was also inhibited by imipramine and other cationic drugs in the presence of Na+, but not inhibited in the absence of Na+. This result is agreement with that of BBMV study. The inhibition of carrier mediated alanine transport by cationic drugs in BBMV corresponded to that in situ. There was a relatively good correlationship between the BBMV study and in situ perfusion study in the inhibitory effects.