Judith A. Duenes

Jejunal and ileal absorptive function after a model of canine jejunoileal autotransplantation.

The effects of intestinal transplantation on the physiologic functions of the gut are not well understood. Our aim was to determine the effect of a large animal model of small intestinal transplantation (disruption of all neural and lymphatic continuity) on selected absorptive functions of the jejunoileum. Seven dogs were studied before and at 1, 4, and 12 weeks after a model of jejunoileal autotransplantation, which avoids confounding factors of immune rejection, immunosuppression, and harvest ischemia. Jejunal function was assessed by quantitative [3H]-folate and D-xylose absorption and ileal function by quantitative 57Co-vitamin B12 absorption. The role of lymphatic continuity was assessed by fecal fat recovery following 5 days of a controlled, high fat diet (75 g/day). All dogs developed a profuse, watery diarrhea that persisted for 6 to 12 weeks and lost about 15% body weight; however, absorption of D-xylose, folate, and vitamin B12 was unaffected at any time point. Fat absorption postoperatively was only mildly abnormal (less than or equal to 8 g/day) at all time points in five of seven dogs despite complete lymphatic disruption. We concluded that jejunoileal autotransplantation does not markedly affect these specific jejunoileal absorptive functions. Fat absorption in most dogs surprisingly remains almost normal. Anatomic and physiologic consequences of intestinal transplantation do not appear to induce global abnormalities in all absorptive functions in the nonrejecting jejunoileum.

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

AimThis study aims to determine mechanisms of action of the gasotransmitter hydrogen sulfide (H2S) on contractile activity in longitudinal muscle of rat ileum.MethodsIleal longitudinal muscle strips were prepared to measure isometric contractions. Effects of sodium hydrosulfide (NaHS), a donor of H2S, were evaluated on spontaneous contractile activity and after enhanced contractile activity with bethanechol. l-cysteine was evaluated as a potential endogenous donor of H2S. We evaluated involvement of extrinsic nerves, enteric nervous system, visceral afferent nerves, nitric oxide, and KATP+ channel and KCa+ channel activity on the action of H2S using non-adrenergic/non-cholinergic conditions, tetrodotoxin, capsaicin, l-NG-nitro arginine (l-NNA), glibenclamide, and apamin, respectively, as well as electrical field stimulation.ResultNaHS 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 H2S release by electrical field stimulation but did show that inhibition of cystathionine β synthase, an endogenous source of H2S, augmented the inhibitory effect of low-frequency electrical field stimulation.ConclusionH2S 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, KATP+ channels, or KCa+ channels.

Small bowel transplantation induces adrenergic hypersensitivity in ileal longitudinal smooth muscle in rats

Our aim was to determine the effects of small bowel transplantation on contractility of longitudinal muscle in the rat ileum. Full-thickness longitudinal muscle strips from four groups of rats (naive controls, sham-operated controls, and 1 week and 8 weeks after syngeneic orthotopic small bowel transplantation) were studied in vitro. Neither baseline contractility nor response to neural blockade (tetrodotoxin) or adrenergic/cholinergic blockade differed among the groups. Although the dose response to the cholinergic agonist bethanechol and to nitric oxide did not differ among groups, the ED50 (negative log of concentration giving half-maximal effect) for the adrenergic agonist norepinephrine was increased 1 week and 8 weeks after transplantation, indicating a hypersensitivity response not blocked by tetrodotoxin. Nonadrenergic, noncholinergic inhibitory responses to electrical field stimulation were of greater amplitude and occurred at lesser frequencies (≤5 Hz) 1 week after small bowel transplantation, but returned to control values 8 weeks postoperatively. These inhibitory responses were blocked by the nitric oxide synthase inhibitor L-NMMA but not by methylene blue, a nonspecific inhibitor of guanylate cyclase. Small bowel transplantation induces a persistent adrenergic denervation hypersensitivity at the muscle and appears to upregulate, at least transiently, other inhibitory mechanisms mediated by neural release of nitric oxide. Small bowel transplantation does not alter muscle response to cholinergic pathways. These alterations in smooth muscle contractility may affect gut function early after clinical small bowel transplantation.

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 ≥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 frequencydependent 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.

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.

Functional changes in nonadrenergic, noncholinergic inhibitory neurons in ileal circular smooth muscle after small bowel transplantation in rats

This experiment was designed to determinemechanisms of change in nonadrenergic, noncholinergic(NANC) inhibitory neurons in the ileum after small boweltransplantation (SBT) in the rat and whether nitric oxide (NO) serves as an important NANCinhibitory neurotransmitter in the rat ileum. Eightgroups of rats (N ≥ 8 rats/group) were studied:neurally intact unoperated controls; rats one week afteranesthesia and sham celiotomy; and separate groups one andeight weeks after either 40 min of cold ischemia of thejejunoileum, combined jejunal and ileal intestinaltransection/reanastomosis, or orthotopic SBT of the entire jejunoileum. Contractile activitywas evaluated in full-thickness ileal circular musclestrips under isometric conditions. Spontaneous activitydid not differ among groups. In all groups, exogenous NO, NG-monomethyl-L-arginine(L-NMMA, an NO synthase inhibitor), and methylene blue(soluble guanylate cyclase inhibitor) had no effect onspontaneous activity, while 8-bromocyclic guanosinemonophosphate (8Br-cGMP) inhibited contractile activity inall groups. Low frequency (2-10 Hz) electrical fieldstimulation (EFS) inhibited contractile activity only incontrol and SBT groups; L-NMMA and methylene blue did not alter the response to EFS in any group.These results suggest that each aspect of the SBTprocedure, ischemia/reperfusion injury, disruption ofenteric neural continuity by intestinal transection, and extrinsic denervation, alter function ofenteric ileal inhibitory neurons separately early (oneweek) after operation. NO, a known inhibitoryneurotransmitter in other gut regions, does not affectileal circular muscle in neurally intact tissue normediate functional changes in inhibitory nerve functionnor smooth muscle contractility after SBT.

Role of Hydrogen Sulfide as a Gasotransmitter in Modulating Contractile Activity of Circular Muscle of Rat Jejunum

AimOur aim was to determine mechanisms of action of the gasotransmitter hydrogen sulfide (H2S) on contractile activity in circular muscle of rat jejunum.MethodsJejunal circular muscle strips were prepared to measure isometric contractions. Effects of sodium hydrosulfide (NaHS), a H2S donor, were evaluated on spontaneous contractile activity and after pre-contraction with bethanechol. l-Cysteine was evaluated as an endogenous H2S donor. We evaluated extrinsic nerves, enteric nervous system, visceral afferent nerves, nitric oxide,

Role of extrinsic innervation in carbohydrate-induced ileal modulation of pancreatic secretion and upper gut function

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