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.

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.

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.

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.

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.

Nitrergic mechanisms mediating inhibitory control of longitudinal smooth muscle contraction in mouse small intestine.

Studies using genetic manipulation to investigate mechanisms of control of physiologic function often necessitate mouse models. However, baseline functional analysis of murine small intestinal motility has not been well defined. Our aim was to define nitrergic mechanisms regulating mouse small intestinal longitudinal muscle. Endogenous nitric oxide (NO) is an important neuroregulatory substance mediating inhibition of contractile activity in murine small bowel. Full-thickness muscle strips of jejunum and ileum from C57BL/6 mice (n > or =6 mice) cut in the direction of longitudinal muscle were studied. Numerous conditions of electrical field stimulation (EFS) and effects of exogenous NO and NO donors were studied in the absence or presence of inhibitors of nitric oxide synthase (NOS) and 1H-[1,2,4]-oxadiazaolo-[4,3-a]-quinoxalin-1-one (ODQ), a downstream inhibitor of guanylyl cyclase. EFS induced a frequency-dependent inhibition of contractile activity in both jejunum and ileum (P < 0.05). As the voltage of EFS was increased, inhibition turned to excitation in the jejunum; in contrast, the ileum demonstrated a voltage-dependent increasing inhibition (P < 0.05 each). EFS-induced inhibition was blocked by NOS inhibitors and ODQ. NO donors inhibited spontaneous contractile activity abolished by ODQ. NO appears to be an endogenous inhibitory neurotransmitter in murine longitudinal small bowel muscle. Nitrergic mechanisms mediate inhibitory control of murine longitudinal small intestinal muscle. Differences exist in neuroregulatory control between jejunum and ileum that may be related to their known difference in motor patterns.

Postprandial absorptive augmentation of water and electrolytes in the colon requires intraluminal glucose.

Postprandial absorptive augmentation of water and electrolytes occurs in the small intestine and colon. The role of intraluminal nutrients in this response is poorly understood. Our aim was to determine whether postprandial absorptive augmentation of water and electrolytes in the colon requires the presence of intraluminal glucose. Four adult dogs underwent enteric isolation of a 50 cm segment of proximal colon. An ileal-like electrolyte solution (Na(+), 130 mEq/L; K(+), 10 mEq/L; Cl(-), 115 mEq/L; and HCO(3)(-), 25 mEq/L), alone or containing glucose (10 mmol/L), was infused at 4 ml/min into the colonic segment. Experiments were performed during fasting and postprandially after a 400 Kcal mixed-nutrient meal. Effluent was collected in 60-minute intervals after steady state was achieved. Net absorptive flux of water was increased in the presence of intraluminal glucose during the fasted state (11 +/- 0.8 vs 7.4 +/- 0.9 microl/min/cm, P < 0.01). The net absorptive flux of water and electrolytes increased postprandially only in the presence of intraluminal glucose (P < 0.05). Our finding that glucose augments both baseline and postprandial absorption of water and electrolytes in the proximal colon suggests that luminal factors have a role in postprandial absorptive augmentation. Whether this is specific to glucose or occurs with other nutrients remains to be determined.

Role of extrinsic innervation in jejunal absorptive adaptation to subtotal small bowel resection: a model of segmental small bowel transplantation.

Segmental small bowel transplantation offers theoretic advantages over total jejunoileal transplantation, but the regional ability of the transplanted segment to adapt is unknown. Absorption was measured in an 80 cm jejunal segment via a triple-lumen perfusion technique. Separate experiments measuring absorption of four nutrients (glucose, glutamine, oleic acid, and taurocholic acid) were performed before and 2 and 12 weeks after operative intervention. Control dogs (CON, n = 6) underwent distal 50% enterectomy. Experimental dogs (EXT DEN, n = 6), in addition to resection, underwent complete extrinsic denervation of the remaining jejunum. All dogs developed diarrhea, which resolved in all CON dogs but persisted in all EXT DEN dogs. Maximal weight loss was greater in the EXT DEN group. Glucose and oleate absorption was decreased 2 weeks after ileal resection in both the CON and EXT DEN dogs; glutamine absorption was decreased at 2 weeks in EXT DEN dogs only. Taurocholate and water absorption remained unchanged in both groups. Absorption of all solutes returned to baseline at 12 weeks in both groups. Despite greater weight loss and persistent diarrhea in EXT DEN dogs, at 12 weeks there were no differences in net absorptive fluxes between the EXT DEN and the CON group after extrinsic denervation. The extrinsic denervation necessitated by small bowel transplantation does not appear to blunt the net jejunal adaptive response to total ileal resection, but may temporarily alter glutamine 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.

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.

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.

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.

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.

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.

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.

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.