Vagal innervation and early postoperative ileus in mice.

INTRODUCTION: Postoperative ileus is characterized by infiltrates of leukocytes in the gut wall 24 h after surgery, which is subject to vagal modulation. We hypothesized that vagal modulation is irrelevant during earlier hours of postoperative ileus and aimed to determine whether afferent neuronal feedback to the central nervous system is altered by vagal innervation during this early period. METHODS: C57BL6 mice were laparotomized and received standardized small bowel manipulation to induce postoperative ileus. Subgroups were vagotomized 3-4 days prior to experiments while control animals were sham-operated. Three or 9 h later a 2-cm jejunal segment was harvested for multi-unit mesenteric afferent nerve recordings in vitro. Intestinal motility was monitored continuously and intestinal muscularis was stained for myeloperoxidase to determine infiltration of leukocytes. RESULTS: Peak amplitudes of intestinal motility and afferent nerve discharge at baseline were not different in all subgroups. Afferent discharge to 5-HT (500 µM) was virtually absent following vagotomy at 3 and 9 h of postoperative ileus (POI) compared to controls (p < 0.05). Maximum afferent nerve discharge to bradykinin and peak firing during maximum distension at 60 mmHg was not different in all subgroups while luminal distension from 10 to 30 mmHg was lower at 3 h of POI following vagotomy compared to controls (p < 0.05). The number of myeloperoxidase positive cells was similar at 3 h of POI in both subgroups; however, at 9 h of POI, ileus counts were increased to 713 ± 99 cells following vagotomy compared to 47 ± 6 cells per square millimeter in control animals. CONCLUSIONS: Vagal afferents mediate sensitivity to low-threshold distension and 5-HT during postoperative ileus but not to high-threshold distension and bradykinin. Vagal inhibition of the intestinal immune response is present at 9 h but not detectable earlier, i.e., at 3 h of postoperative ileus when spinal reflex inhibition may prevail.

Role of the vagus nerve on the development of postoperative ileus.

INTRODUCTION: Postoperative ileus involves reflex inhibition of intestinal motility within hours after surgery and a subsequent intestinal inflammatory response that is characterized by efferent vagal modulation via acetylcholine receptors on intestinal macrophages. We aimed to characterize the role of vagal modulation of intestinal motility during the early hours after surgery. METHODS: C57BL6 mice underwent laparotomy and standardized small bowel manipulation to induce postoperative ileus. Subgroups were vagotomized 3-4 days prior to experiments or received pharmacological inhibition of the acetylcholine alpha7 subunit with the inhibitor alpha-bungarotoxin, while control animals were sham operated and remained otherwise untreated. Three hours later, a 2-cm jejunal segment was harvested with the mesentery attached. Mesenteric afferent nerve recordings were established in an organ bath generating a multiunit signal with subsequent computerized analysis. Intraluminal pressure was continuously recorded to assess intestinal motility. Afferent nerve responses were quantified at baseline and to chemical stimulation with bradykinin (0.5 microM) or serotonin (5-HT; 500 microM) and following mechanical stimulation by continuous ramp distension to 60 mmHg. RESULTS: Peak amplitudes of intestinal motility and afferent nerve discharge at baseline were not different following chronic vagotomy, alpha-bungarotoxin or sham operation. Maximum afferent discharge to 5-HT following alpha-bungarotoxin was comparable to sham controls, while the response was reduced in chronically vagotomized animals (p < 0.05). Maximum afferent nerve discharge to bradykinin and peak firing during maximum distension at 60 mmHg was similar in the different subgroups. At luminal distension from 10 to 30 mmHg, afferent discharge was lower in vagotomized animals compared to sham controls (p < 0.05) but unchanged after alpha-bungarotoxin. CONCLUSIONS: Sensitivity to low-threshold distension and 5-HT is mediated via vagal afferents during postoperative ileus, while sensitivity to high-threshold distension and bradykinin is independent of vagal afferent innervation. Early inhibition of intestinal motility at 3 h after onset of postoperative ileus does not appear to depend on vagal innervation.

Two novel monothiol glutaredoxins from Saccharomyces cerevisiae provide further insight into iron-sulfur cluster binding, oligomerization, and enzymatic activity of glutaredoxins.

Two novel monothiol glutaredoxins from yeast (ScGrx6 and ScGrx7) were identified and analyzed in vitro. Both proteins are highly suited to study structure-function relationships of glutaredoxin subclasses because they differ from all monothiol glutaredoxins investigated so far and share features with dithiol glutaredoxins. ScGrx6 and ScGrx7 are, for example, the first monothiol glutaredoxins showing an activity in the standard glutaredoxin transhydrogenase assay with glutathione and bis-(2-hydroxyethyl)-disulfide. Steady-state kinetics of ScGrx7 with glutathione and cysteine-glutathione disulfide are similar to dithiol glutaredoxins and are consistent with a ping-pong mechanism. In contrast to most other glutaredoxins, ScGrx7 and ScGrx6 are able to dimerize noncovalently. Furthermore, ScGrx6 is the first monothiol glutaredoxin shown to directly bind an iron-sulfur cluster. The cluster can be stabilized by reduced glutathione, and its loss results in the conversion of tetramers to dimers. ScGrx7 does not bind metal ions but can be covalently modified in Escherichia coli leading to a mass shift of 1090 +/- 14 Da. What might be the structural requirements that cause the different properties? We hypothesize that a G(S/T)x3 insertion between a highly conserved lysine residue and the active site cysteine residue could be responsible for the abrogated transhydrogenase activity of many monothiol glutaredoxins. In addition, we suggest an active site motif without proline residues that could lead to the identification of further metal binding glutaredoxins. Such different properties presumably reflect diverse functions in vivo and might therefore explain why there are at least seven glutaredoxins in yeast.

Allosteric coupling of two different functional active sites in monomeric Plasmodium falciparum glyoxalase I.

Glyoxalase I (GloI) catalyzes the glutathione-dependent conversion of 2-oxoaldehydes to S-2-hydroxyacylglutathione derivatives. Studies on GloI from diverse organisms such as man, bacteria, yeast, and different parasites show striking differences among these potentially isofunctional enzymes as far as metal content and the number of active sites per subunit are concerned. So far, it is not known whether this structural variability is linked to catalytic or regulatory features in vivo. Here we show that recombinant GloI from the malaria parasite Plasmodium falciparum has a high- and a low-affinity binding site for the diastereomeric hemithioacetals formed by addition of glutathione to methylglyoxal. Both active sites of the monomeric enzyme are functional and have similar k(cat)(app) values. Proteolytic susceptibility studies and detailed analyses of the steady-state kinetics of active-site mutants suggest that both reaction centers can adopt two discrete conformations and are allosterically coupled. As a result of the positive homotropic allosteric coupling, P. falciparum GloI has an increased affinity at low substrate concentrations and an increased activity at higher substrate concentrations. This could also be the case for GloI from yeast and other organisms. Potential physiologically relevant differences between monomeric GloI and homodimeric GloI are discussed. Our results provide a strong basis for drug development strategies and significantly enhance our understanding of GloI kinetics and structure-function relationships. Furthermore, they extend the current knowledge on allosteric regulation of monomeric proteins in general.