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

K. A. Barada, N. E. Saadé, S. F. Atweh, and C. F. Nassar

Departments of Physiology, Human Morphology, and Internal Medicine, American University of Beirut, Beirut, Lebanon

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

capsaicin-sensitive primary afferents; myenteric plexus; intestinal absorption

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THERE IS ACCUMULATING EVIDENCE that the action of several peptides and molecules in the gut is partly or totally mediated by the enteric nervous system (ENS) and by the afferent and/or efferent components of the vagus nerve. Such evidence includes cholera toxin-induced fluid and mucin secretion (18, 26), substance P-induced sodium and bicarbonate secretion in the jejunum (5), secretin and cholecystokinin (CCK) inhibition of gastric emptying (34, 35), neurokinin- and norepinephrine-induced jejunal ion transport (13, 31), and vasoactive intestinal polypeptide (VIP)-induced intestinal fluid and electrolyte secretion (29, 36, 45).

VIP is a neurotransmitter present in the neurons of the ENS that innervate the epithelium of the gut (20). It is also present in the nerve endings of capsaicin-sensitive primary afferent (CSPA) fibers (19, 24, 36), and it is released in response to parasympathetic nerve stimulation (16, 30). It has been reported that VIP receptors are present in the myenteric plexus (39, 47) and on submucosal synaptosomes (46).

Recently, we (28) demonstrated that acute stimulation of the vagal CSPA fibers produces a significant decrease in jejunal alanine absorption, whereas chronic blockade of these fibers results in an increase of the basal level of alanine absorption. These effects were attributed to the local effector mechanisms of CSPA fibers that are known to release various peptides including VIP (14, 24). Moreover, it was shown that the intravenous administration of VIP inhibits amino acid absorption across the rat small intestine (6, 27) and that this inhibitory effect is markedly reduced by subdiaphragmatic vagotomy (27). Similar findings were noted with injection of VIP into the dorsal motor nucleus-nucleus tractus solitarii complex (37). However, the neural mechanisms and pathways mediating the effects of VIP are not well defined.

The aim of this study is to further investigate the neural mechanisms involved in amino acid transport and to assess the role of CSPA fibers and the ENS in mediating the effects of VIP on amino acid absorption.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Sprague-Dawley rats of either sex weighing 150-200 g were used in all experiments. They were fed regular lab chow ad libitum and were fasted for 24 h before the day of the experiment, with free access to water.

Capsaicin treatment. Newborn rats were subcutaneously injected with capsaicin (50 mg/kg dissolved in 10% Tween 80, 10% ethanol, and 80% PBS) on the second day after birth. Sham control rats received a vehicle injection. Verification of desensitization to capsaicin was done by using the eye wiping test in response to capsaicin (12).

Benzalkonium chloride treatment. Rats were anesthetized with pentobarbital sodium (50 mg/kg), and the abdominal cavity was opened by a midline incision. A segment of the proximal jejunum was identified, exteriorized, and soaked in a sterile petri dish containing 2 mM benzalkonium chloride (BAC) for 30 min. The jejunum was then thoroughly washed with saline and returned to the abdominal cavity. The abdominal wall was closed, and the rats were left to recover for a period of 2-3 wk. This procedure results in chemical ablation of the myenteric plexus and extrinsic innervation of the treated segment (11, 33, 38). Sham rats underwent the same surgical procedure, and the jejunal segment was soaked with saline for an equal time interval.

In vivo studies. As described previously (27), the single-pass intraluminal perfusion technique was used to measure alanine absorption. Briefly, the rats were anesthetized with an intraperitoneal injection of 50 mg pentobarbital sodium per kilogram of body weight and tracheostomized to allow for adequate ventilation. The femoral vein was cannulated and used for injections. The abdominal cavity was opened by a midline incision, and the jejunum was exposed with its blood supply kept intact. A jejunal loop was isolated and cannulated by an inlet inserted 5 cm caudal to the ligament of Treitz, and an outlet was inserted 15-20 cm distal to the inlet. This loop was then replaced in the abdominal cavity and covered with warm paraffin oil. The cannulated jejunal segment was perfused by a peristaltic pump at a rate of 0.70 ml/min for a period of 160 min. The perfusate consisted of PBS (0.14 M NaCl and 0.01 M K H2PO4 with pH adjusted to 7.4 with K2HPO4) containing 1 mM "cold" alanine, 10 µCi [14C]alanine, as well as 15 mg/l phenol red. The nonabsorbable indicator (phenol red) was used to correct for any changes in alanine absorption resulting from intestinal water transport. During all the experiments the perfusate was kept at a constant temperature of 37°C. The effluent solution was collected at 20-min intervals for a maximum period of 160 min. One-milliliter aliquots of the initial and effluent solutions were assayed for phenol red concentration and radioactivity content. Absorption was calculated from the rate of disappearance of labeled amino acid from the perfusate solution, taking into account water transport as measured by the change of phenol red concentrations.

To study the effects of VIP on jejunal L-alanine absorption in sham control, capsaicin-treated, and BAC-treated rats, a solution of the peptide or its antagonist was continuously infused through the femoral vein. The infusion was regulated by a pump that maintained a constant rate of 11.2 ng · kg-1 · min-1 throughout the experiment. This concentration of VIP was shown to produce maximal inhibition of alanine absorption (27). In another set of experiments, VIP antagonist was continuously infused through the femoral vein and capsaicin was perfused intraluminally at a concentration of 400 µM 30 min after the VIP antagonist infusion was started.

In vitro studies. The methods used in this set of experiments were described previously (28). In anesthetized rats the abdominal cavity was opened, and the jejunum was removed and placed immediately in oxygenated ice-cold PBS solution. The jejunum was freed from the mesenteric and fat attachments, opened along the mesenteric line, and cut longitudinally into strips each 1 cm in length.

Isolated jejunal strips (weighing 50-80 mg) from sham control rats, BAC-treated rats, and rats neonatally treated with capsaicin were incubated in PBS containing 10 µCi of 14C-labeled alanine and 1 mM cold alanine, and shaken at 60-65 cycles/min in a water bath at a temperature of 37°C for 30 min. After incubation, the strips were removed and immediately dipped into ice-cold isotonic mannitol solution. Each strip was blotted with Whatman no. 1 filter paper, its wet weight was determined, and then the tissue was extracted in 2 ml of 0.1 M HNO3 for at least 4 h. Aliquots of the tissue extracts and incubation media were counted for their [14C]alanine content in a liquid scintillation counter. From the data, the intracellular concentration of alanine was calculated after accounting for the extracellular space that was measured by using labeled inulin as an extracellular probe. The effect of adding VIP and other drugs to the incubation media on alanine absorption was determined.

The effect of VIP on alanine uptake by mucosal scrapings was also studied. Jejunal segments were cut open along the mesenteric border, flattened, and scraped using glass slides. Aliquots of scrapings weighing 80-120 mg were incubated in PBS in the presence and absence of VIP. The details of the experiment are similar to those described for intestinal strips. To determine whether these mucosal preparations retained their ability to actively transport alanine, the same experimental procedure was repeated with 5 mM ouabain added to the incubation medium.

The effect of VIP on alanine influx was studied according to the method described previously (2). Briefly, jejunal strips isolated from normal sham rats and rats neonatally treated with capsaicin were prepared and mounted on Lucite chambers (modified Schultz chambers) in such a way that the mucosal side only would be exposed to the incubation medium. The strips were preincubated in PBS (without alanine) with and without 40 nM VIP. Different concentrations of alanine (0.5-5 mM) were then added to the mucosal surface for 1 min. The intracellular alanine concentration (a measure of unidirectional influx) was then determined.

To define the neural mechanism involved in mediating the effect of VIP on alanine absorption, independent sets of experiments were performed to study the effects of tetrodotoxin (TTX; 0.2 µM) and atropine (1 µM) on alanine absorption with and without 40 nM VIP.

Data analysis. Statistical significance of the difference between the control and the experimental groups was determined using ANOVA followed by a Bonferroni post hoc test (Graph Pad Instat) taking 0.05 as the limit of significance.

Chemicals. The following chemicals were obtained from Sigma Chemical (St. Louis, MO): VIP (human, porcine, and rat), [D-p-Cl-Phe6, Leu17]VIP (antagonist, human, porcine, and rat), 8-methyl-N-vanillylnonanamide (dihydrocapsaicin), BAC, and TTX. Atropine was purchased from Laboratoire Aguettant (Lyon, France). Radioactive alanine was obtained from Amersham International. All other chemicals were of reagent grade.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of VIP on jejunal alanine absorption in sham, capsaicin-treated, and BAC-treated rats. Basal alanine absorption was 0.20 ± 0.01 µmol · cm-1 · 20 min-1 in sham control rats, whereas it was 0.13 ± 0.01 µmol · cm-1 · 20 min-1 in rats neonatally treated with capsaicin (P < 0.001). The effect of intravenous VIP infusion (11.2 ng · kg-1 · min-1) on alanine absorption is shown in Fig. 1. VIP decreased alanine absorption by 60% in sham rats and by 20% in rats neonatally treated with capsaicin (P < 0.05). This effect was maintained throughout the experiment in sham rats and appeared after 40 min of infusion in neonatally treated rats.


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Fig. 1.   Time course of alanine absorption by jejunal segments in sham and capsaicin-treated rats (n = 7 each) and response to intravenous vasoactive intestinal polypeptide (VIP) infusion. Intravenous VIP (11.2 ng · kg-1 · min-1) was started at 80 min (arrows) and continued throughout the experiment. Alanine absorption was determined every 20 min and averaged for each time interval in each group of rats. Statistical significance was determined for each group compared with control sham group.

In BAC-treated rats basal alanine absorption was significantly reduced from 33.6 ± 0.25 µmol · h-1 · g dry wt-1 in control sham rats to 25.7 ± 0.38 µmol · h-1 · g dry wt-1 (P < 0.001). As shown in Fig. 2 VIP reduced alanine absorption from 33.6 ± 0.25 to 13.45 ± 0.35 µmol · h-1 · g dry wt-1 in sham rats (P < 0.001) and from 25.7 ± 0.38 to 22.14 ± 0.36 µmol · h-1 · g dry wt-1 in BAC-treated rats (P < 0.001). Thus the inhibition of alanine by VIP was decreased from 60% in control rats to 14% in BAC-treated rats.


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Fig. 2.   Alanine absorption in sham and benzalkonium chloride (BAC)-treated rats with and without intravenous infusion of VIP. Alanine absorption was determined every 20 min in each group (n = 7 rats) and plotted as absorption per hour per gram dry weight of intestinal tissue. Note significant decrease of basal alanine absorption in BAC-treated rats.

To characterize further the mechanism of release of VIP, a group of rats was infused intravenously with VIP antagonist at a rate of 11.2 ng · kg-1 · min-1. As shown in Fig. 3 basal alanine absorption was not significantly altered by the VIP antagonist. This result obviates the possibility of tonic VIP release under our experimental conditions. Furthermore, because capsaicin is known to stimulate peptidergic neurons and to cause the release of neurotransmitters including VIP, another group of rats was perfused intraluminally with 400 µM capsaicin in addition to the VIP antagonist as described above. Figure 3 shows that the VIP antagonist reduced the inhibitory effect of capsaicin on alanine absorption by 70% (P < 0.01).


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Fig. 3.   Time course of alanine absorption by jejunal segments in sham rats, rats infused with VIP antagonist, rats perfused intraluminally with capsaicin only, and rats perfused with capsaicin preceded by infusion of VIP antagonist. Antagonist infusion was started at beginning of experiment and continued throughout. Arrow indicates time of initiation of capsaicin perfusion. Alanine absorption was determined every 20 min and averaged for each time interval in each group of rats (n = 5). Statistical significance was determined for each experimental group compared with control sham group.

Effect of VIP on alanine absorption by jejunal strips in rats neonatally treated with capsaicin. In full-thickness jejunal strips, basal alanine uptake was 11.33 ± 0.65 µmol · g dry wt-1 · 30 min-1. As shown in Fig. 4, VIP reduced alanine uptake in a dose-dependent manner. At 20 nM VIP, the maximal effective concentration, alanine uptake was 5.2 ± 0.34 µmol · g dry wt-1 · 30 min-1, which is 54% below control. In capsaicin-treated rats, basal alanine uptake was 6.85 ± 0.4 µmol · g dry wt-1 · 30 min-1 and was reduced to 5.10 ± 0.30 µmol · g dry wt-1 · 30 min-1 by 30 nM VIP (P < 0.001), which represents a drop of 25% below control.


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Fig. 4.   Dose-dependent inhibition of jejunal alanine uptake by VIP. Jejunal strips from sham and capsaicin-treated rats were incubated with 1 mM alanine and different concentrations of VIP. Each point is the mean ± SE of 21 determinations from 7 animals.

The unidirectional influx of alanine across the mucosal membrane was measured in control sham and neonatally treated rats with capsaicin. Figure 5 demonstrates an inhibitory effect of VIP on alanine influx that amounts to ~40% below control. However, in rats neonatally treated with capsaicin VIP did not elicit significant alteration in alanine influx (Fig. 5).


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Fig. 5.   Concentration dependence of total alanine unidirectional influx in sham rats (A) and in rats neonatally treated with capsaicin (B). Jejunal strips were incubated with different concentrations of alanine in presence and absence of 40 nM VIP. Each point represents mean ± SE of 12 determinations from 6 rats.

Effect of VIP on alanine uptake in rats treated with BAC. Alanine uptake by jejunal strips of rats pretreated with BAC was 5.60 ± 0.31 µmol · g dry wt-1 · 30 min-1, which is 50% below control (P < 0.001, Table 1). VIP had no significant effect on alanine uptake in these strips.

                              
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Table 1.   Effect of VIP on alanine uptake in intestinal strips of sham rats and rats pretreated with BAC

To ensure the viability of cells in isolated intestinal tissues, jejunal strips from rats of all experimental groups were incubated with 5 mM ouabain for 30 min. Alanine uptake was reduced by 67% in sham rats, 48% in rats neonatally treated with capsaicin, and 40% in BAC-treated rats.

Effects of VIP on alanine uptake by jejunal scrapings. Alanine uptake in scrapings of sham control rats was 3.36 ± 0.03 µmol · g dry wt-1 · 30 min-1 (Table 2). VIP (40 nM) decreased alanine uptake to 2.51 ± 0.06 µmol · g dry wt-1 · 30 min-1, a 25% reduction (P < 0.01). On the other hand, uptake in scrapings prepared from rats neonatally treated with capsaicin was 2.79 ± 0.07 µmol · g dry wt-1 · 30 min-1 (P < 0.05) and was decreased by VIP to 2.54 ± 0.08 µmol · g dry wt-1 · 30 min-1, a 9% reduction (P < 0.05). The presence of active transport was established by incubating scrapings in PBS containing 5 mM ouabain, which decreased alanine uptake in preparations from sham rats and neonatally treated rats by 36%.

                              
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Table 2.   Effect of VIP on steady-state alanine uptake by mucosal scrapings isolated from sham and capsaicin-treated rats

Effect of TTX and atropine on alanine uptake with and without VIP. Independent sets of experiments were performed to study the effects of TTX and atropine on alanine uptake by jejunal strips in the presence and absence of 40 nM VIP. As shown in Table 3, 0.2 µM TTX had no significant effect on alanine uptake. Moreover, the inhibitory effect of VIP on alanine uptake was significantly reduced from 55% in control rats to 15% in rats treated with TTX at a dose that is reported to block neuronal conduction (48). On the other hand, atropine reduced basal alanine uptake by 27% (P < 0.05). However, when VIP and atropine were added together no further significant decrease in alanine absorption was noted.

                              
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Table 3.   Effect of 0.2 µM TTX and 1 µM atropine on alanine uptake in jejunal strips of sham rats in presence and absence of 40 nM VIP

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The results of the present study show that VIP inhibits alanine absorption across the rat jejunum in vivo and in vitro and demonstrate that this inhibitory effect is dose dependent. We (27, 37) previously showed that subdiaphragmatic vagotomy markedly reduced the inhibitory effect of intravenous and intracerebral VIP injections on alanine absorption. These results provide an indication of a possible neural mediation of the effect of VIP. Furthermore, we (28) demonstrated the involvement of vagal CSPA fibers in the regulation of amino acid absorption. These fibers as well as the enteric neurons are known to release VIP in response to certain stimuli (16, 30, 36). As a matter of fact, VIP and other neuropeptides were shown to be released through local effector mechanisms by CSPA fibers (for review see Refs. 14 and 23). In addition, several authors reported that VIP is released by vagal stimulation that might involve local effector mechanism, reflex mechanism, or both (3, 22, 23, 36). In this study we examined the hypothesis that the inhibitory effect of VIP is mediated by the CSPA fibers and the myenteric plexus of the small intestine.

Different experimental paradigms were designed to demonstrate the possible mediation of VIP effects by CSPA fibers, i.e., through local effector mechanism, or by reflex activation of the enteric neurons through the vagal system or through local intestinal mechanisms.

The results reported here show that neonatal capsaicin treatment reduced the inhibitory effect of VIP on jejunal alanine absorption from 62 to 20% in vivo and from 54 to 25% in vitro. Furthermore, the results of the experiments on the unidirectional influx of alanine across the mucosal membrane showed that the effect of VIP is exerted at the mucosal level and is abolished by neonatal capsaicin treatment. Accordingly, it can be speculated that intact CSPA fibers are necessary for a complete effect of VIP.

Previous results from our laboratory (2) showed that activation of CSPA fibers inhibited alanine absorption through the sodium-dependent component at the brush- border membrane. In the present work we have demonstrated that pretreatment with VIP antagonist blocked the inhibitory effect of CSPA fibers activated by capsaicin (Fig. 3). Thus ablation of CSPA fibers blocks the effect of VIP, and VIP antagonist alters the effects of activation of CSPA fibers on alanine absorption.

This assumption receives further support from our observation that VIP inhibits alanine uptake in mucosal scrapings by only 25%, compared with 54% inhibition in full-thickness jejunal strips. Although mucosal scrapings are essentially devoid of most of the neural elements (4), the remaining effect of VIP may be caused by the presence of a few nerve endings left in these tissues (15) and/or by the presence of VIP receptors on the mucosal cells (17, 25, 39, 47). Moreover, the further reduction of the effect of VIP on alanine uptake by mucosal scrapings isolated from rats neonatally treated with capsaicin appears to warrant further explanation. First, this reduction was not caused by a loss of active transport mechanisms. Evidence about the persistence of active transport of alanine in mucosal scrapings was demonstrated by the inhibition of alanine uptake on incubation with ouabain in the presence and absence of VIP. Second, chronic capsaicin treatment destroys CSPA terminals that may remain in mucosal preparations. Third, as discussed previously (2), chronic ablation of CSPA fibers may lead to long-term plastic and trophic changes (1, 12, 32); a possible downregulation of VIP mucosal receptors could be a part of these changes. However, unpublished data from our laboratory showed no evident morphological changes in the intestine of rats neonatally treated with capsaicin.

Further support for our hypothesis is provided by the results of BAC treatment, which was used as another model for selective denervation. Several studies reported that BAC treatment of intestinal tissue produced selective ablation of the myenteric plexus in addition to its extrinsic innervation (11, 33, 38). This treatment is accompanied by trophic changes in the smooth muscle layer and an increase in epithelial cell proliferation. Submucosal neurons were not affected by serosal or by intraluminal BAC treatment (43). The effects of such changes on amino acid absorption were not previously studied. BAC treatment reduced basal alanine absorption, decreased VIP inhibitory effect in vivo (Fig. 2), and abolished it in vitro (Table 1). These results suggest that the effect of VIP requires the presence of an intact intrinsic innervation in addition to the extrinsic intestinal innervation. This assumption is supported by a recent report showing that isolated villous cells, which are devoid of neural elements, did not have any response to VIP (21). The residual effect of VIP noted in vivo could be attributed to central (neuroendocrine) mechanisms affecting the remaining enteric neurons. Furthermore, the reduction of basal alanine uptake in rats treated with BAC or capsaicin suggests the presence of a proabsorptive tone on alanine absorption that is exerted by the extrinsic innervation and the myenteric plexus in the gut.

To define further the neural mediation of the effect of VIP, experiments were performed using TTX. The inhibitory effect of VIP on alanine absorption in normal rats was reduced by 71% by the neuronal conduction blocker TTX. The data suggest that the effect of VIP requires action potential-dependent neurotransmitter release in the enteric nervous system. Moreover, treatment with TTX (0.2 µM) did not change alanine uptake by the jejunal strips. The concentration of TTX used in these experiments is known to interfere with synaptic transmission without interfering with the function of the enterocytes (48). These observations demonstrate the key role of neuronal mechanisms in the mediation of the effects of VIP.

The results of the experiments with atropine (Table 3) reveal a possible interaction between VIP and cholinergic mechanisms that control nutrient absorption. The concentration used (1 µM) was shown to interfere with muscarinic mechanisms (48). Consequently, these results indicate that VIP effects may be expressed through muscarinic receptors. This assumption is in line with several reports that correlate between VIPergic and cholinergic mechanisms (10, 16, 42).

There is experimental evidence that VIPergic mechanisms are activated by stimulation of CSPA fibers (16, 40, 42, 44). VIPergic neurons and/or receptors are located in the extrinsic innervation of the gastrointestinal tract and in the enteric nervous system (8, 9). Moreover, it has been demonstrated that VIP receptors are present on mucosal cells (17, 25, 39, 47) and on nerve terminals in the villi (15). Accordingly, VIP effects can be expressed either indirectly at the level of the extrinsic and/or the intrinsic innervation or directly at the level of the mucosa. Our data suggest that a major component of the effect of VIP is expressed through neural mediation.

VIP is released into the mesenteric veins after a meal and in response to either distension or mechanical stimulation of the mucosal surface (7, 41, 42). Accordingly, an argument can be made for a VIP regulatory mechanism of amino acid absorption in the small intestine that is neurally mediated. It remains to be determined whether this mediation is preferential through VIP or involves other neurotransmitters and/or peptides.

    ACKNOWLEDGEMENTS

The authors thank Carmen Khoury for technical assistance in this study. This research was funded by the University Research Board.

    FOOTNOTES

Address for reprint requests: K. Barada, Dept. of Internal Medicine, American Univ. of Beirut, Beirut, Lebanon.

Received 3 December 1997; accepted in final form 2 July 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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