Camille F. Nassar

Interplay between Nitric Oxide and Vasoactive Intestinal Polypeptide in Inducing Fluid Secretion in Rat Jejunum

Nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) interact in the regulation of neuromuscular function in the gut. They are also potent intestinal secretogogues that coexist in the enteric nervous system. The aims of this study were: (1) to investigate the interaction between NO and VIP in inducing fluid secretion in the rat jejunum, and (2) to determine whether the NO effect on intestinal fluid movement is neurally mediated. The single pass perfusion technique was used to study fluid movement in a 25 cm segment of rat jejunum in vivo. A solution containing 20 mml‐arginine, a NO precursor, was perfused into the segment. The effect of the NO synthase inhibitors (l‐NAME and l‐nitroindazole (l‐NI)) and the VIP antagonist ([4Cl‐D‐Phe6,Leu17]VIP (VIPa)) on l‐arginine‐induced changes in fluid movement, expressed as μl min−1 (g dry intestinal weight)−1, was determined. In addition, the effect of neuronal blockade by tetrodotoxin (TTX) and ablation of the myenteric plexus by benzalkonium chloride (BAC) was studied. In parallel groups of rats, the effect of l‐NAME and l‐NI on VIP‐induced intestinal fluid secretion was also examined. Basal fluid absorption in control rats was (median (interquartile range)) 65 (45–78). l‐Arginine induced a significant fluid secretion (−14 (−20 to −5); P < 0.01). This effect was reversed completely by l‐NAME (60 (36–65); P < 0.01) and l‐NI (46 (39–75); P < 0.01) and partially by VIPa (37 (14–47); P < 0.01). TTX and BAC partially inhibited the effect of l‐arginine (22 (15–32) and 15 (10–26), respectively; P < 0.05). The effect of VIP on fluid movement (−23 (−26 to −14)) was partially reversed by l‐NAME (24 (8.4–35.5); P < 0.01) and l‐NI (29 (4–44); P < 0.01). The inhibition of VIP or NO synthase prevented l‐arginine‐ and VIP‐induced intestinal fluid secretion through a neural mechanism. The data suggest that NO enhances the release of VIP from nerve terminals and vice versa. Subsequently, each potentiates the others effect in inducing intestinal fluid secretion.

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.

Effects of intravenous vasoactive intestinal peptide injection on jejunal alanine absorption and gastric acid secretion in rats.

The effect of intravenous vasoactive intestinal polypeptide (VIP) injection on jejunal L-alanine absorption and gastric acid secretion in the rat was investigated. Continuous intravenous VIP infusion (11.2 ng/kg per min) throughout the experimental period (160 min) produced 60% decrease in alanine absorption and 40% decrease in gastric acid secretion during the second hour of the experiment. Subdiaphragmatic vagotomy reduced alanine absorption to 91% (P > 0.05) and 71.3% (P < 0.05) of control value during the first and second hours of perfusion, respectively. VIP infusion following vagotomy elicited a reduced effect when compared to that produced by similar injections in normal rats. Gastric secretion in vagotomized rats was reduced by 40% (P < 0.05) below control. VIP infusion in vagotomized rats exerted a significant decrease (P < 0.05) of gastric acid secretion. Moreover, water absorption was decreased by almost 10% (P < 0.05) after i.v. injection of VIP and was increased by 20-24% above control value following vagotomy. However, i.v. administration of VIP following vagotomy did not elicit any further change in water absorption. It can be concluded that VIP inhibits alanine absorption and gastric acid secretion in the rat and that these inhibitory effects might be partially mediated by the vagus nerve.

Amino acids in the rat intestinal lumen regulate their own absorption from a distant intestinal site

Intestinal nutrient transport is altered in response to changes in dietary conditions and luminal substrate level. It is not clear, however, whether an amino acid in the intestinal lumen can acutely affect its own absorption from a distant site. Our aim is to study the effect of an amino acid present in rat small intestinal segment on its own absorption from a proximal or distal site and elucidate the underlying mechanisms. The effect of instillation of alanine (Ala) in either jejunum or ileum on its own absorption at ileal or jejunal level was examined in vivo. The modulation of this intestinal regulatory loop by the following interventions was studied: tetrodotoxin (TTX) added to Ala, subdiaphragmatic vagotomy, chemical ablation of capsaicin-sensitive primary afferent (CSPA) fibers, and IV administration of calcitonin gene-related peptide (CGRP) antagonist. In addition, the kinetics of jejunal Ala absorption and the importance of Na+-dependent transport were studied in vitro after instilling Ala in the ileum. Basal jejunal Ala absorption [0.198 +/- 0.018 micromol x cm(-1) x 20 min(-1) (means +/- SD)] was significantly decreased with the instillation of 20 mM Ala in the ileum or in an adjacent distal jejunal segment (0.12 +/- 0.015; P < 0.0001 and 0.138 +/- 0.014; P < 0.002, respectively). Comparable inhibition was observed in the presence of proline in the ileum. Moreover, basal Ala absorption from the ileum (0.169 +/- 0.025) was significantly decreased by the presence of 20 mM Ala in the jejunum (0.103 +/- 0.027; P < 0.01). The inhibitory effect on jejunal Ala absorption was abolished by TTX, subdiaphragmatic vagotomy, neonatal capsaicin treatment, and CGRP antagonism. In vitro studies showed that Ala in the ileum affects Na+-mediated transport and increases K(m) without affecting Vmax. Intraluminal amino acids control their own absorption from a distant part of the intestine, by affecting the affinity of the Na+-mediated Ala transporter, through a neuronal mechanism that involves CSPA and CGRP.

Effects of intracerebral injections of VIP on jejunal alanine absorption and gastric acid secretion in rats

The effects of intracerebral injections of VIP on jejunal alanine absorption and gastric acid secretion, and its association with vagal outflow were examined in Sprague-Dawley rats. Intracerebroventricular injection of VIP (2 ng) decreased significantly (P < 0.05) alanine absorption across the jejunum, whereas similar injections in vagotomized rats did not show further decrease in absorption beyond that noticed by vagotomy only. Moreover, VIP injected in the Nucleus Tractus Solitarius-Dorsal Motor Nucleus (NTS-DMN) complex (1 ng) produced also a significant inhibition of Ala absorption which was reduced but remained significant (P < 0.05) after vagotomy. Water movement was not affected by VIP injection in the lateral ventricle, while VIP injections in the NTS-DMN inhibited significantly (P < 0.05) jejunal water absorption by 10-12%. Vagotomy increased water absorption by 15-20% above control (P < 0.05) which was not altered by injecting VIP in the NTS-DMN complex. On the other hand, VIP injection in the NTS-DMN produced a 25.7% increase in gastric acid output in the first hour of the experiment followed by a non-significant decrease (P > 0.05) in the second hour. Same injections done in vagotomized animals produced similar effects to those elicited by vagotomy only. It can be suggested that NTS-DMN complex could be a site of action of VIP since injection of VIP in it produced a more pronounced inhibitory effect on water and Ala absorption than that produced by VIP injection in the LV. These effects were reduced or abolished by vagotomy.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Protective effect of the nitric oxide donor molsidomine on indomethacin and aspirin-induced gastric injury in rats

Objective To study the effect of the nitric oxide donor, molsidomine, on gastric and duodenal injury induced by indomethacin and aspirin. Methods Sprague‐Dawley rats weighing 180‐200 g were used after 24 h fasting. Indomethacin (5 mg/kg) was given subcutaneously as a single dose and followed by multiple injections of histamine. Molsidomine (0.05 mg/kg) or distilled water was given by gavage 30 min before indomethacin and repeated at 3 h intervals for two doses. Rats were killed 2 h after the last dose of molsidomine. Aspirin (500 mg/kg) was given by gavage and repeated 2.5 h later. Molsidomine or distilled water was given 30 min before the initial aspirin dose and repeated after 2 h. Animals were killed 2.5 h after the second dose of aspirin. The severity of the gastric mucosal damage was graded from 0 to 3, and the duodenal bulb ulcer surface area calculated by two independent observers using a dissecting microscope. Results Indomethacin and aspirin resulted in significant gastric mucosal damage with median scores of 2 (interquartile ranges 1.4‐3, n = 16 and 2‐3, n = 10, respectively). Molsidomine significantly ameliorated indomethacin‐ and aspirin‐induced damage with median scores of 1 (interquartile ranges 0.5‐1.5, n = 19 and 0.6‐1.9, n = 10, respectively; P < 0.008 and P < 0.02, respectively (Mann‐Whitney U test)). Molsidomine had no effect on duodenal bulb ulcerations caused by indomethacin. Conclusion Oral molsidomine has a protective effect on gastric mucosa against damage induced by ulcerogenic agents. This could have an important clinical benefit, especially in cardiac patients taking aspirin in addition to a nitric oxide donor such as molsidomine. Eur J Gastroenterol Hepatol 12:81 ‐ 84

Cytoskeletal control of alanine transport across the rat and turtle small intestine

Procaine inhibited significantly (P less than 0.01) alanine accumulation in the rat intestinal strips in a concentration-dependent pattern, whereas it showed no effect on alanine uptake by the turtle intestinal cells. Colchicine and Vinca alkaloids at 5 X 10(-4) and 1.5 X 10(-6) M respectively caused a significant inhibition (P less than 0.01) of intracellular alanine concentration in the rat with no effect noticed in the turtle. Unidirectional influx of alanine across the brush border membrane of the rat was significantly (P less than 0.01) reduced in the presence of procaine, colchicine and vincristine in the preincubation medium. The same drugs did not show any effect on alanine influx into the turtle small intestine. Electron microscopy showed major structural alterations in the cytoskeletal organization of the turtle intestine in response to procaine, colchicine or vincristine treatment. It is proposed that microtubular system may participate in the overall transport mechanism of alanine across the small intestine.

Taurine transport across the small intestine of adult and suckling rats

1. Taurine accumulation in intestinal cells of adult and suckling rats reached steady-state after 60 min with an In/Out ratio of 1.46 and 4.66 in the adult and suckling rats respectively. 2. The accumulative capacity of the intestinal strips isolated from suckling rats is almost four times higher than that of adult rats. 3. The steady-state uptake of taurine by the adult and suckling rats intestinal cells is saturable, sodium-dependent and inhibited by ouabain. 4. The calculated Vmax of the mediated component of the steady-state uptake in the suckling rats is three times greater than that of the adult rats, and the affinity is seven fold greater in the suckling as compared to the adult. 5. Taurine influx across the mucosal membrane in the suckling rat is significantly greater than that of the control adult.

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.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.