Lack of functionally active sweet taste receptors in the jejunum in vivo in the rat.

BACKGROUND: When studied in enterocyte-like cell lines (Caco-2 and RIE cells), agonists and antagonists of the sweet taste receptor (STR) augment and decrease glucose uptake, respectively. We hypothesize that exposure to STR agonists and antagonists in vivo will augment glucose absorption in the rat. MATERIALS AND METHODS: About 30-cm segments of jejunum in anesthetized rats were perfused with iso-osmolar solutions containing 10, 35, and 100 mM glucose solutions (n = 6 rats, each group) with and without the STR agonist 2 mM acesulfame potassium and the STR inhibitor 10 µM U-73122 (inhibitor of the phospholipase C pathway). Carrier-mediated absorption of glucose was calculated by using stereospecific and nonstereospecific (14)C-d-glucose and (3)H-l-glucose, respectively. RESULTS: Addition of the STR agonist acesulfame potassium to the 10, 35, and 100 mM glucose solutions had no substantive effects on glucose absorption from 2.1 ± 0.2 to 2.0 ± 0.3, 5.8 ± 0.2 to 4.8 ± 0.2, and 15.5 ± 2.3 to 15.7 ± 2.7 µmoL/min/30-cm intestinal segment (P > 0.05), respectively. Addition of the STR inhibitor (U-73122) also had no effect on absorption in the 10, 35, and 100 mM solutions from 2.3 ± 0.1 to 2.1 ± 0.2, 7.7 ± 0.5 to 7.2 ± 0.5, and 15.7 ± 0.9 to 15.2 ± 1.1 µmoL/min/30-cm intestinal segment, respectively. CONCLUSIONS: Provision of glucose directly into rat jejunum does not augment glucose absorption via STR-mediated mechanisms within the jejunum in the rat. Our experiments show either no major role of STRs in mediating postprandial augmentation of glucose absorption or that proximal gastrointestinal tract stimulation of STR or other luminal factors may be required for absorption of glucose to be augmented by STR.

Mechanisms of glucose uptake in intestinal cell lines: role of GLUT2.

BACKGROUND: GLUT2 is translocated to the apical membrane of enterocytes exposed to glucose concentrations >∼50 mM. Mechanisms of GLUT2-mediated glucose uptake in cell culture models of enterocytes have not been studied. AIM: To explore mechanism(s) of glucose uptake in 3 enterocyte-like cell lines. METHODS: Glucose uptake was measured in Caco-2, RIE-1, and IEC-6 cell lines using varying concentrations of glucose (0.5-50 mM). Effects of phlorizin (SGLT1 inhibitor), phloretin (GLUT2 inhibitor), nocodazole and cytochalasin B (disrupters of cytoskeleton), calphostin C and chelerythrine (PKC inhibitors), and phorbol 12-myristate 13-acetate (PKC activator) were evaluated. RESULTS: Phlorizin inhibited glucose uptake in all 3 cell lines. Phloretin inhibited glucose uptake in Caco-2 and RIE-1 cells. Starving cells decreased glucose uptake in Caco-2 and RIE-1 cells. Glucose uptake was saturated at >10 mM glucose in all 3 cell lines when exposed briefly (<1 min) to glucose. After exposure for >5 min in Caco-2 and RIE-1 cells, glucose uptake did not saturate and K(m) and V(max) increased. This increase in glucose uptake was inhibited by phloretin, nocodazole, cytochalasin B, calphostin C, and chelerythrine. PMA enhanced glucose uptake by 20%. Inhibitors and PMA had little or no effect in the IEC-6 cells. CONCLUSION: Constitutive expression of GLUT2 in the apical membrane along with additional translocation of cytoplasmic GLUT2 to the apical membrane via an intact cytoskeleton and activated PKC appears responsible for enhanced carrier-mediated glucose uptake at greater glucose concentrations (>20 mM) in Caco-2 and RIE-1 cells. IEC-6 cells do not appear to express functional GLUT2.

Acute enterocyte adaptation to luminal glucose: a posttranslational mechanism for rapid apical recruitment of the transporter GLUT2.

BACKGROUND: Glucose absorption postprandially increases markedly to levels far greater than possible by the classic glucose transporter sodium-glucose cotransporter 1 (SGLT1). HYPOTHESIS: Luminal concentrations of glucose >50 mM lead to rapid, phenotypic, non-genomic adaptations by the enterocyte to recruit another transporter, glucose transporter 2 (GLUT2), to the apical membrane to increase glucose absorption. METHODS: Isolated segments of jejunum were perfused in vivo with glucose-containing solutions in anesthetized rats. Carrier-mediated glucose uptake was measured in 10 and 100 mM glucose solutions (n = 6 rats each) with and without selective inhibitors of SGLT1 and GLUT2. RESULTS: The mean rate of carrier-mediated glucose uptake increased in rats perfused with 100 mM versus 10 mM glucose to 13.9 ± 2.9 µmol from 2.1 ± 0.1 µmol, respectively (p < 0.0001). Using selective inhibitors, the relative contribution of GLUT2 to glucose absorption was 56% in the 100 mM concentration of glucose compared to the 10 mM concentration (27%; p < 0.01). Passive absorption accounted for 6% of total glucose absorption at 100 mM glucose. CONCLUSION: A small amount of GLUT2 is active at the lesser luminal concentrations of glucose, but when exposed to concentrations of 100 mM, the enterocyte presumably changes its phenotype by recruiting GLUT2 apically to markedly augment glucose absorption.

Role of hydrogen sulfide as a gasotransmitter in modulating contractile activity of circular muscle of rat jejunum.

AIM: Our aim was to determine mechanisms of action of the gasotransmitter hydrogen sulfide (H(2)S) on contractile activity in circular muscle of rat jejunum. METHODS: Jejunal circular muscle strips were prepared to measure isometric contractions. Effects of sodium hydrosulfide (NaHS), a H(2)S donor, were evaluated on spontaneous contractile activity and after pre-contraction with bethanechol. L-cysteine was evaluated as an endogenous H(2)S donor. We evaluated extrinsic nerves, enteric nervous system, visceral afferent nerves, nitric oxide, K(ATP)+ and K(Ca)+ channels, and myosin light chain phosphatase on action of H(2)S using non-adrenergic/non-cholinergic conditions, tetrodotoxin, capsaicin, L-N(G)-nitro arginine (L-NNA), glibenclamide, apamin, and calyculin A, respectively, and electrical field stimulation (EFS). RESULTS: NaHS dose-dependently and reversibly inhibited spontaneous and bethanechol-stimulated contractile activity (p < 0.05). L-cysteine had a dose-dependent inhibitory effect. Non-adrenergic/non-cholinergic conditions, tetrodotoxin, capsaicin, L-NNA, or apamin had no effect on contractile inhibition by NaHS; in contrast, low-dose glibenclamide and calyculin A prevented NaHS-induced inhibition. We could not demonstrate H(2)S release by EFS. CONCLUSIONS: H(2)S inhibits contractile activity of jejunal circular muscle dose-dependently, in part by K(ATP)+ channels and via myosin light chain phosphatase, but not via pathways mediated by the extrinsic or enteric nervous system, visceral afferent nerves, nitric oxide, or K(Ca)+ channels.

Peptide absorption after massive proximal small bowel resection: mechanisms of ileal adaptation.

BACKGROUND: Protein absorption occurs as di- and tri-peptides via H(+)/peptide co-transporter-1 (PepT1). AIM: The aim of this study is to identify mechanisms of ileal adaptation after massive proximal enterectomy. HYPOTHESIS: Ileal adaptation in uptake of peptides is mediated through upregulation of PepT1 gene expression. STUDY DESIGN: Rats underwent 70% jejunoileal resection. Total mucosal cellular levels of messenger RNA (mRNA) and protein and transporter-mediated uptake per centimeter of the di-peptide glycyl-sarcosine (Gly-Sar) were compared in remnant ileum 1 and 4 weeks postoperatively to control and to 1-week sham laparotomy rats. Histomorphology, food consumption, and weights of rats were monitored. RESULTS: After 70% resection, although mRNA per cell for PepT1 decreased at 1 week (p = 0.002), expression of mRNA at 4 weeks and protein at 1 and 4 weeks in remnant ileum were unchanged (p > 0.1). Ileal Gly-Sar uptake (V (max)-nanomoles per centimeter per minute, i.e., number of transporters per centimeter) increased at 1 and 4 weeks compared to control and 1-week sham (p < 0.05 each); K (m) (i.e., transporter function) was unchanged. Villous heights (millimeters) in remnant ileum increased at 1- and 4-week time points over controls (0.45 and 0.57 vs 0.21, resp; p < 0.001). CONCLUSIONS: Ileal adaptation to proximal resection for peptide absorption occurs through cellular proliferation (hyperplasia) and not through cellular upregulation of PepT1 mRNA or protein per enterocyte.

Differentiating passive from transporter-mediated uptake by PepT1: a comparison and evaluation of four methods.

BACKGROUND: To quantify transmembrane transport of dipeptides by PepT1, passive uptake (non-PepT1 mediated) must be subtracted from total (measured) uptake. Three methods have been described to estimate passive uptake: perform experiments at cold temperatures, inhibit target dipeptide uptake with a greater concentration of a second dipeptide, or use modified Michaelis-Menten kinetics. We hypothesized that performing uptake experiments at pH 8.0 would estimate passive uptake accurately, because PepT1 requires a proton gradient. Our aim was to determine the most accurate method to estimate passive uptake. METHODS: Caco-2 cells were incubated with various concentrations of glycyl-sarcosine (gly-sar) at pH 6.0 and at 37°C to measure total uptake. Passive uptake was estimated: (1) by incubating Caco-2 cells with varying concentrations of gly-sar at 4°C, (2) in the presence of 50 mM glycyl-leucine, (3) in solution at pH 8.0, or (4) using modified Michaelis-Menten kinetics. PepT1-mediated uptake was calculated by subtracting passive uptake from total uptake. K(m), V(max), and % gly-sar transported by PepT1 were calculated and compared. RESULTS: K(m), V(max), and % gly-sar transported by PepT1 varied from 0.7 to 2.4 mM, 8.4 to 21.0 nmol/mg protein/10 min, and 69% to 87%, respectively. Uptakes calculated with cold, 50 mM gly-leu and using modified Michaelis-Menten kinetics were similar but differed significantly from uptake at pH 8.0 (P < 0.001). CONCLUSIONS: Estimating passive uptake at pH 8.0 does not appear to be accurate. Measuring uptake at cold temperatures or in the presence of a greater concentration of a second dipeptide, and confirming results with modified Michaelis-Menten kinetics is recommended.

Absence of evidence of translocation of GLUT2 to the apical membrane of enterocytes in everted intestinal sleeves.

INTRODUCTION: Traditional models of intestinal glucose absorption confine GLUT2 to the basolateral membrane. Evidence suggests that GLUT2 is translocated to the apical membrane when the enterocyte is exposed to high luminal glucose concentrations. HYPOTHESIS: GLUT2 translocates to the apical membrane by a PKC signaling mechanism dependent on activity of SGLT1 and the cellular cytostructure. METHODS: Transporter-mediated glucose uptake was studied in rat jejunum using everted sleeves under seven conditions: Control, SGLT1 inhibition (phlorizin), GLUT2 inhibition (phloretin), both SGLT1 and GLUT2 inhibition, PKC inhibition (calphostin C or chelerythrine), and disruption of cellular cytostructure (nocodazole). Each condition was tested in iso-osmotic solutions of 1, 20, or 50 mM glucose for 1 or 5 min incubations (n = 6 rats each). RESULTS: Control rats exhibited a saturable pattern of uptake at both durations of incubation. Phlorizin (P ≤ 0.006 each) inhibited markedly and phloretin (P ≤ 0.01 each) inhibited partially glucose uptake in all concentrations and time. Phloretin and phlorizin together completely inhibited uptake (P = 0.004 each). Calphostin C, chelerythrine, and nocodazole had little effect on glucose uptake at either 1 or 5 min. Inhibition of SGLT1 led to near complete cessation of transporter-mediated glucose uptake, while GLUT2 inhibition led to partial inhibition, suggesting some constitutive expression of GLUT2 in the apical membrane. Disruption of PKC signaling or cytoskeletal integrity partially inhibited transporter-mediated glucose uptake only in 1 mM glucose, suggesting a non-specific effect. CONCLUSIONS: Under these conditions, it does not appear that GLUT2 is translocated to the apical membrane on the cellular cytostructure in response to PKC signaling.

Intestinal adaptation for oligopeptide absorption via PepT1 after massive (70%) mid-small bowel resection.

INTRODUCTION: Proteins are absorbed primarily as short peptides via peptide transporter 1 (PepT1). HYPOTHESIS: Intestinal adaptation for peptide absorption after massive mid-small intestinal resection occurs by increased expression of PepT1 in the remnant small intestine and colon. METHODS: Peptide uptake was measured in duodenum, jejunum, ileum, and colon using glycyl-sarcosine 1 week (n = 9) and 4 weeks (n = 11) after 70% mid-small bowel resection and in corresponding segments from unoperated rats (n = 12) and after transection and reanastomosis of jejunum and ileum (n = 8). Expression of PepT1 (mRNA, protein) and villus height were measured. RESULTS: Intestinal transection/reanastomosis did not alter gene expression. Compared to non-operated controls, 70% mid-small bowel resection increased jejunal peptide uptake (p < 0.05) associated with increased villus height (1.13 vs 1.77 and 1.50 mm, respectively, p < 0.01). In ileum although villus height increased at 1 and 4 weeks (1.03 vs 1.21 and 1.35 mm, respectively; p < 0.01), peptide uptake was not altered. PepT1 mRNA and protein were decreased at 1 week, and PepT1 protein continued low at 4 weeks. Gene expression, peptide uptake, and histomorphology were unchanged in the colon. CONCLUSIONS: Jejunal adaptation for peptide absorption occurs by hyperplasia. Distal ileum and colon do not have a substantive role in adaptation for peptide absorption.

Mechanisms of action of the gasotransmitter hydrogen sulfide in modulating contractile activity of longitudinal muscle of rat ileum.

AIM: This study aims to determine mechanisms of action of the gasotransmitter hydrogen sulfide (H(2)S) on contractile activity in longitudinal muscle of rat ileum. METHODS: Ileal longitudinal muscle strips were prepared to measure isometric contractions. Effects of sodium hydrosulfide (NaHS), a donor of H(2)S, were evaluated on spontaneous contractile activity and after enhanced contractile activity with bethanechol. L-cysteine was evaluated as a potential endogenous donor of H(2)S. We evaluated involvement of extrinsic nerves, enteric nervous system, visceral afferent nerves, nitric oxide, and K(ATP)(+) channel and K(Ca)(+) channel activity on the action of H(2)S using non-adrenergic/non-cholinergic conditions, tetrodotoxin, capsaicin, L-N(G)-nitro arginine (L-NNA), glibenclamide, and apamin, respectively, as well as electrical field stimulation. RESULT: NaHS dose-dependently and reversibly inhibited spontaneous and bethanechol-stimulated contractile activity (p < 0.05). L-cysteine had no inhibitory effect. Non-adrenergic/non-cholinergic conditions, tetrodotoxin, capsaicin, L-NNA, glibenclamide, or apamin had no major effect on total contractile activity by NaHS, although both tetrodotoxin and apamin decreased the frequency of bethanechol-enhanced contractile activity (p < 0.05). We could not demonstrate H(2)S release by electrical field stimulation but did show that inhibition of cystathionine ß synthase, an endogenous source of H(2)S, augmented the inhibitory effect of low-frequency electrical field stimulation. CONCLUSION: H(2)S inhibits contractile activity of ileal longitudinal muscle dose-dependently but not through pathways mediated by the extrinsic or enteric nervous system, visceral afferent nerves, nitric oxide, K(ATP)(+) channels, or K(Ca)(+) channels.

Age-related changes in functional NANC innervation with VIP and substance P in the jejunum of Lewis rats.

Age-related changes in non-adrenergic, non-cholinergic (NANC) neurotransmission might contribute to differences in gastrointestinal motility. Our aim was to determine age-related changes in functional innervation with vasoactive intestinal polypeptide (VIP) and substance P (Sub P) in rat jejunum. We hypothesized that maturation causes changes in neurotransmission with these two neuropeptides. Longitudinal and circular jejunal muscle strips from young (3 months) and middle-aged (15 months) rats (total: 24 rats) were studied; the response to exogenous VIP and Sub P and the effect of their endogenous release from the enteric nervous system during electrical field stimulation (EFS) were evaluated. In longitudinal muscle, response to exogenous VIP and endogenously released VIP during EFS were increased in middle-aged rats, while the effect of endogenously released Sub P was decreased. In the circular muscle, the response to endogenously released VIP was increased in middle-aged rats, while the effects of exogenous VIP and endogenously released Sub P were unchanged. Response to exogenous Sub P was unaffected by maturation in both muscle layers. Spontaneous contractile activity was increased in the longitudinal and circular muscle of the older rats. In the jejunum of middle-aged rats, participation of VIP in functional NANC innervation was increased, while functional innervation with Sub P was decreased. These changes in the balance of inhibitory and excitatory neurotransmission occur during the year of maturation in rats and demonstrate an age-dependant plasticity of neuromuscular bowel function.

Role of vagal innervation in diurnal rhythm of intestinal peptide transporter 1 (PEPT1).

BACKGROUND: Protein is absorbed predominantly as di/tripeptides via H(+)/peptide cotransporter-1 (PEPT1). We demonstrated previously diurnal variations in expression and function of duodenal and jejunal but not ileal PEPT1; neural regulation of this pattern is unexplored. HYPOTHESIS: Complete abdominal vagotomy abolishes diurnal variations in gene expression and transport function of PEPT1. METHODS: Twenty-four rats maintained in a 12-h light/dark room [6AM-6PM] underwent abdominal vagotomy; 24 other rats were controls. Four weeks later, mucosal levels of mRNA and protein were measured at 9AM, 3PM, 9PM, and 3AM (n = 6 each) by quantitative real-time PCR and Western blots, respectively; transporter-mediated uptake of dipeptide (Gly-Sar) was measured by the everted-sleeve technique. RESULTS: Diurnal variation in mRNA, as in controls, was retained post-vagotomy in duodenum and jejunum (peak at 3PM, p < 0.05) but not in ileum. Diurnal variations in expression of protein and Gly-Sar uptake, however, were absent post-vagotomy (p > 0.3). Similar to controls, maximal uptake was in jejunum after vagotomy (V (max), nmol/cm/min: jejunum vs. duodenum and ileum; 163 vs. 88 and 71 at 3AM; p < 0.04); K (m) remained unchanged. CONCLUSIONS: Vagal innervation appears to mediate in part diurnal variations in protein expression and transport function of PEPT1, but not diurnal variation in mRNA expression of PEPT1.

Diurnal expression and function of peptide transporter 1 (PEPT1).

BACKGROUND: Protein is absorbed primarily as di/tripeptides, which are transported into the enterocyte exclusively by H(+)/peptide cotransporter 1 (PEPT1). Diurnal changes in expression and function of several other mucosal transporters occur in rat. Diurnal variations in mRNA, protein, and transport function of PEPT1 occur in rat duodenum and jejunum, but not in ileum. METHODS: Mucosal levels of mRNA and protein were determined at 9 AM, 3 PM, 9 PM, and 3 AM (n=6 each) by real time RT-PCR and Western blotting, respectively, in rats maintained in a 12-h light/dark room [light 6 AM to 6 PM]; transporter-mediated uptake of dipeptide (Gly-Sar) was also measured by everted sleeve technique. RESULTS: mRNA transcripts of PEPT1 and Gly-Sar uptake varied diurnally in duodenum and jejunum (peak at 3 PM, P<0.05), but not in ileum; maximal uptake was in jejunum. V(max) (nmol/cm/min) was greater at 3 PM and 9 PM compared with 9 AM (3 PM versus 9 AM: 104 versus 62 in duodenum, and 185 versus 101 in jejunum; P<0.03); K(m) was unchanged across time points or locations. Protein levels varied minimally in jejunum and ileum with peaks at 9 PM and 3 AM. CONCLUSION: Gene expression and transport function of PEPT1 vary diurnally in duodenum and jejunum in temporal association with nocturnal feeding of rats.

Hexose transporter expression and function in mouse small intestine: role of diurnal rhythm.

BACKGROUND: Expression and function of hexose transporters vary diurnally in rat small intestine; however, this subject remains unexplored in mice. AIM: The aim of the study was to investigate the diurnal expression and function of hexose transporters SGLT1, GLUT2, and GLUT5 in mouse small bowel. METHODS: Twenty-four c57bl6 mice maintained in a 12-h light/dark room (6 AM: -6 PM: ) were sacrificed at 9 AM: , 3 PM: , 9 PM: , and 3 AM: (n = 6 each). In duodenal, jejunal, and ileal mucosa, total cellular mRNA and protein levels were quantitated by real-time PCR and semiquantitative Western blotting, respectively. The everted sleeve technique measured transporter-mediated glucose uptake at 9 AM: and 9 PM: . RESULTS: mRNA expression of SGLT1, GLUT2, and GLUT5 varied diurnally in all three intestinal segments (p 70% of food intake occurred; glucose transport followed a similar pattern with increased uptake at 9 PM: . CONCLUSION: Hexose transporter expression and function vary diurnally with nocturnal feeding patterns of mice.

Coordinated, diurnal hexose transporter expression in rat small bowel: implications for small bowel resection.

BACKGROUND: Hexose transporter mRNA and protein levels follow a diurnal rhythm in rat jejunum. Their coordinated expression and resultant function throughout the small bowel is not well understood. We hypothesized that hexose transporter levels and glucose absorption follow a coordinated, site-specific diurnal rhythm in rat duodenum and jejunum, but not in ileum. METHODS: Sprague-Dawley rats were housed in a strictly maintained, 12-h, light/dark room [light 6 am to 6 pm] with free access to water and chow. Mucosa was harvested from duodenum, jejunum, and ileum at 3 am, 9 am, 3 pm, and 9 pm, and full thickness 1-cm segments were harvested at 9 am, and 9 pm (n = 6 for each segment at each time point). mRNA levels were determined by reverse-transcription, real-time polymerase chain reaction (n > or = 5), protein levels by semiquantitative Western blotting (n > or = 5), and transporter-mediated glucose uptake by everted sleeve technique (n = 6). RESULTS: mRNA levels of SGLT1 and GLUT5 followed a temporally coordinated, diurnal rhythm in all 3 segments (P < .01), while mRNA for GLUT2 and protein levels for SGLT1 and GLUT2 varied diurnally only in duodenum and jejunum (P > .05) but not in ileum (P > .10). SGLT1 and GLUT5 mRNA induction decreased aborally. Baseline SGLT1 and GLUT5 mRNA levels and SLGT1 and GLUT2 protein levels did not vary aborally (P > .05 for all). GLUT2 mRNA baseline levels were decreased in ileum (P < .01). Glucose uptake varied diurnally in duodenum and jejunum with no difference in ileum. Transporter-mediated glucose uptake was greater in duodenum and jejunum compared with ileum. CONCLUSION: Regulation of hexose absorption in rat small bowel seems to be site-specific and mediated by multiple mechanisms.

Long-term effects of extrinsic denervation on VIP and substance P innervation in circular muscle of rat jejunum.

Intestinal denervation contributes to enteric motor dysfunction after small bowel transplantation (SBT). Our aim was to determine long-term effects of extrinsic denervation on function of nonadrenergic, noncholinergic innervation with substance P and vasoactive intestinal polypeptide (VIP). Contractile activity of jejunal circular muscle strips from six age-matched, naive control rats (NC) and eight rats 1 year after syngeneic SBT was studied in tissue chambers. Spontaneous contractile activity did not differ between groups. Exogenous VIP inhibited contractile activity dose-dependently to a comparable degree in both groups. The VIP antagonist ([D-p-Cl-Phe(6),Leu(17)]-VIP) and the nitric oxide synthase inhibitor L-NG-nitro-arginine did not affect VIP-induced inhibition but increased contractile activity during electrical field stimulation (EFS) in both groups. Exogenous substance P increased contractile activity dose-dependently, greater in NC than SBT. The substance P antagonist ([D-Pro(2),D-Trp(7,9)]-substance P) inhibited effects of exogenous substance P and decreased the excitatory EFS response. Immunohistofluorescence showed tyrosine hydroxylase staining after SBT indicating sympathetic reinnervation. In jejunal circular muscle after chronic denervation, response to exogenous substance P, but not VIP, is decreased, whereas endogenous release of both neurotransmitters is preserved. Alterations in balance of excitatory and inhibitory pathways occur despite extrinsic reinnervation and might contribute to enteric motor dysfunction after SBT.

Role of VIP and substance P in NANC innervation in the longitudinal smooth muscle of the rat jejunum - influence of extrinsic denervation.

BACKGROUND: This study was designed to determine changes in nonadrenergic, noncholinergic (NANC) neurotransmission mediated by Vasoactive Intestinal Polypeptide (VIP) and Substance P after small bowel transplantation (SBT). MATERIALS AND METHODS: Six groups of rats (n > or = 6 per group) were studied: naïve controls (NC); 1 wk after anesthesia/sham celiotomy (SC-1); 1 or 8 wk after jejunal and ileal transection/reanastomosis (TA-1, TA-8), or syngeneic, orthotopic SBT (SBT-1, SBT-8). Jejunal longitudinal muscle strips were studied under NANC-conditions for spontaneous contractile activity, response to exogenous VIP and Substance P, and electrical field stimulation (EFS). RESULTS: Spontaneous activity did not differ between the six groups. VIP inhibited contractile activity in all groups 1 wk postoperatively (P < 0.05), which was prevented by the NO synthase inhibitor L-N(G)-nitro arginine (L-NNA). In contrast, VIP had no effect in the other groups. Precontraction with Substance P exposed an inhibitory effect of VIP in all groups (P < 0.05 each). Substance P increased contractile activity in all groups, but to a lesser extent in SBT-8 compared with NC, TA-8, and SBT-1 (P < 0.05). The inhibitory effect of EFS at 6 Hz was prevented by L-NNA in NC and TA-8; addition of the VIP antagonist ([D-p-Cl-Phe(6), Leu(17)]-VIP) increased contractile activity in NC, but not in TA-8 and SBT-8. The Substance P antagonist ([D-Pro(2), D-Trp(7,9)]-Substance P) decreased contractile activity during EFS at 50 Hz in NC and SBT-8. CONCLUSIONS: SBT decreased response to exogenous Substance P, although release of endogenous Substance P (EFS) is preserved. Changes in VIP signaling are acute and reversible and not caused by effects of SBT.

The diurnal periodicity of hexose transporter mRNA and protein levels in the rat jejunum: role of vagal innervation.

BACKGROUND: Protein and messenger RNA (mRNA) levels of the hexose transporters sodium-dependent glucose transporter-1, glucose transporter 2, and glucose transporter 5 follow a (daily) diurnal rhythm in rat jejunum. Because vagal innervation mediates the diurnal activity of other proteins in the rat small bowel, we hypothesized that the diurnal variation of mRNA and protein levels of these hexose transport proteins are mediated by vagal innervation. METHODS: Forty-eight rats kept in a strictly maintained, alternating 12-hour light-dark room underwent either sham laparotomy (n = 24) or bilateral total abdominal vagotomy (n = 24). Four weeks postoperatively, jejunal mucosa was harvested from 6 rats in each group at 3 am, 9 am, 3 pm and 9 pm; mRNA levels were determined by reverse transcription real-time polymerase chain reaction and protein levels by semiquantitative Western blot analysis. Transporter mRNA and protein levels were expressed as a ratio to the corresponding mRNA and protein levels of the stably expressed housekeeping gene glyceraldehyde-6-phosphate dehydrogenase. RESULTS: mRNA and protein levels for all 3 hexose transporters showed diurnal variation in sham controls (P < or = .01 for all). After vagotomy, although mRNA levels of all 3 transporters showed diurnal variation (each P < .01), diurnal variation in all 3 hexose transporter protein levels was abolished (P > .10 for all). CONCLUSIONS: Vagal innervation appears to differentially mediate the diurnal changes in hexose transporter mRNA and protein expression in the rat jejunum by posttranscriptional, and/or posttranslational processes.

Postprandial augmentation of absorption of water and electrolytes in jejunum is neurally modulated: implications for segmental small bowel transplantation.

Postprandial augmentation of absorption of water and electrolytes is believed to occur in the jejunum. Neural mechanisms of control, however, have not been studied in the in situ jejunum or in the transplanted bowel. The aim of this study was to determine if postprandial augmentation of absorption occurs in the in situ jejunum and to evaluate neural mechanisms controlling postprandial jejunal absorption. Based on our previous work, we hypothesized that postprandial augmentation of absorption does not occur in the jejunum in situ and that extrinsic denervation of the jejunum is associated with decreased postprandial absorption. Absorption was studied in an 80 cm, in situ jejunal segment in six dogs by using an isosmolar electrolyte solution alone, or with 80 mmol/L glucose before and after jejunal transection to disrupt intrinsic neural continuity of the study segment with the remaining gut. Net absorptive fluxes of water and electrolytes were measured in the fasted state and after a 400-kcal meal. Another six dogs were studied 3 weeks after our validated model of extrinsic denervation of jejunoileum; identical fasting and postprandial absorptive states were evaluated. Postprandial augmentation of absorption of water and electrolytes did occur in the jejunum (P < 0.03) both in the absence and in the presence of intraluminal glucose. After intrinsic neural transection or extrinsic denervation, no postprandial augmentation of absorption occurred, with or without glucose. Postprandial augmentation of absorption of water and electrolytes occurs in the in situ jejunum. Disrupting intrinsic neural continuity or extrinsic denervation (as after intestinal transplantation) abolishes postprandial augmentation.

Inhibition by nitric oxide and nonadrenergic, noncholinergic (NANC) nerves is preserved in a canine model of extrinsic denervation: implications for small bowel transplantation.

BACKGROUND: Small bowel transplantation (SBT) is complicated by changes in graft motility, especially in the early postoperative period. This dysmotility may be related in part to the extrinsic denervation necessitated by the procedure, but specific neurotransmitter response to SBT is incompletely understood. The aim of this study was to evaluate the role of nitric oxide and nonadrenergic, noncholinergic (NANC) enteric neural input in the nonimmunologic etiology of the dysmotility seen after SBT. METHODS: A technique of jejunoileal extrinsic denervation (without disruption of mesenteric vascular supply) was used as a model of canine jejunoileal autotransplantation to avoid potential confounding factors such as ischemia-reperfusion and postallotransplant immunologic effects. Longitudinal smooth muscle strips from ileum and jejunum were studied with in vitro tissue chamber methodology at 0, 2, and 8 weeks after this experimental model to explore early and late effects of denervation. Effects of exogenous nitric oxide (NO) and electric field stimulation (EFS), which releases native, endogenous enteric neurotransmitters) were evaluated in neurally intact control dogs and those undergoing extrinsic denervation. RESULTS: Exogenous NO caused a dose-dependent inhibition of spontaneous contractile activity and in some muscle strips a decrease in basal tone in both groups of dogs. These effects were unchanged by neural blockade with tetrodotoxin and preserved after extrinsic denervation. EFS produced inhibition of spontaneous contractile activity in ileum and a complex, inconsistent response in jejunum. The response to EFS in both ileum and jejunum was unchanged after extrinsic denervation. CONCLUSIONS: Nitric oxide inhibits contractile activity in canine longitudinal muscle of small bowel. Motility changes seen after this large animal model of extrinsic denervation are not caused by changes in NO or NANC neural function. The variability observed between different segments of intestine is important to consider in the context of SBT.

Extrinsic denervation alters postprandial absorption of glucose and glutamine in the ileum: implications for small bowel transplantation.

In the postprandial period, augmentation of absorption of water, electrolytes, and taurocholate is believed to occur in the ileum. The role of extrinsic innervation in this postprandial augmentation has not been well studied and may be an important concept in small bowel transplantation. Our aim was to investigate extrinsic neural mechanisms mediating postprandial absorptive patterns. The study hypothesis was that postprandial augmentation of absorption in the ileum is blunted in transplanted (extrinsically denervated) bowel. Ileal absorption was studied in six dogs with an 80-cm in situ ileal segment via a triple-lumen perfusion technique using an iso-osmolar, ileal-like electrolyte solution alone and containing either glucose 2.5 mM, glutamine 2.5 mM, oleic acid 5 mM, or taurocholate 5 mM. Net absorptive fluxes of each substrate, as well as water and electrolytes, were measured in both the fasted state and after a 400-Kcal mixed meal before and at 2 and 12 weeks after our validated model of complete extrinsic denervation of the jejunoileum. At baseline, there were no differences in absorption of water, electrolytes, or any nutrient postprandially compared with the fasted state. Two weeks after extrinsic denervation, absorption of glucose at both 1 and 2 hours postprandially was decreased compared with absorption during fasting. Glutamine absorption was also decreased at 2 hours postprandially. At 12 weeks after extrinsic denervation, net postprandial absorption of glucose and glutamine returned toward normal and was not different from fasting absorption. No differences were noted in postprandial absorption of oleic acid or taurocholate at any time point. Decreases in absorption of nutrients postprandially after extrinsic denervation (which is necessitated by small bowel transplantation) may play an important role in post-transplant enteric absorptive dysfunction. The previously described postprandial augmentation in net absorption may be a function of enterically isolated gut and does not appear to occur in the in situ ileum.