Murray F. Broom

Enterohepatic circulation of bacterial chemotactic peptide in rats with experimental colitis.

The association of hepatobiliary disorders with colonic inflammation is well recognized. Although the pathophysiology is obscure, increased permeation of toxic bacterial products across the inflamed gut to the portal circulation might be one mechanism. Potentially toxic metabolites include N-formylated chemotactic peptides that are produced by several species of intestinal bacteria and can be detected in colonic fluid in vivo. To investigate the metabolic fate of one of these low molecular weight proinflammatory peptides, N-formyl L-methionine L-leucine 125I-L-tyrosine was introduced into colon loops of healthy rats (n = 10) and rats with experimental colitis (n = 15) induced by rectal instillation of 15% (vol/vol) acetic acid. Gut, liver, and blood radioactivity were monitored by external gamma-counting and radioactivity in bile was measured by biliary catheter drainage into a well counter. Bile was processed by high-performance liquid chromatography to determine the amount of intact, bioactive peptide excreted over 3 h. After colonic instillation of 1 nmol of peptide, the mean (+/- SEM) biliary excretion of intact peptide was 6.4 +/- 2.0 pmol in normal rats and 49.0 +/- 20 pmol in rats with colitis (p less than 0.01). An enterohepatic circulation of synthetic N-formyl L-methionine L-leucine L-tyrosine has been demonstrated in the rat. Experimental colitis was associated with an eightfold increase in biliary excretion of this proinflammatory bacterial peptide. Proinflammatory bacterial peptides synthesized by colonic bacteria could be important in the pathophysiology of colon inflammation and its frequently associated hepatobiliary complications.

Bacterial chemotactic oligopeptides and the intestinal mucosal barrier

Intestinal absorption and enterohepatic circulation of N-formyl-methionyl-leucyl-125I-tyrosine, a bioactive synthetic analog of the bacterial chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine has been investigated in the rat. In ileum and proximal and distal colon, dithiothreitol, which increases mucosal permeability, increased peptide absorption and biliary recovery fourfold, 70-fold, and 20-fold over control values, respectively. When dithiothreitol was combined with d-l-benzyl succinate, a potent inhibitor of intestinal carboxypeptidase, absorption and biliary recovery from ileal loops increased markedly to 40-fold over control, whereas there was no further increase in absorption from colon loops. There was a strong correlation between biliary N-formyl-methionyl-leucyl-125I-tyrosine recovery and intestinal absorption of 51Cr-ethylenediaminetetraacetate, a marker of passive mucosal permeability (r = 0.97). We conclude that in the ileum both enzymic degradation and restricted mucosal permeability contribute to the intestinal barrier to luminal bacterial formyl oligopeptides. In the colon, however, enzymic mechanisms are less active and restricted mucosal permeability is the major factor. Abnormalities of the intestinal mucosal barrier to proinflammatory bacterial peptides could play a role in inflammatory disorders of the gut.

Radio‐immunoassay for formyl methionyl leucyl phenylalanine. I. Development and application to assessment of chemotactic peptide production by enteric bacteria

Bacterial chemotactic peptides are low molecular weight peptides which stimulate a wide range of neutrophil functions following binding to specific leucocyte receptors. Formyl methionyl leucyl phenylalanine (FMLP) is the major chemotactic peptide in Escherichia coli culture supematants. This paper reports the development and validation of a radio‐immunoassay (RIA) for FMLP and its application to the analysis of formyl peptide production by enteric bacteria in vitro.

Isolation and purification of N-formylmethionine aminopeptidase from rat intestine

The intestinal mucosal epithelium is exposed to products of intestinal bacteria including potent inflammatory N-formylmethionyl oligopeptides. An N-formylmethionine aminopeptidase has been purified 2300-fold from rat intestine and was shown to degrade natural fMet oligopeptides from Escherichia coli culture supernatants with loss of bioactivity (release of specific granule constituents from human polymorphonuclear leucocytes) and immuno-reactivity (assessed using a polyclonal anti-fMet-Leu-Phe antiserum). The enzyme which was specific for N-terminal acyl-methionine residues had a native Mr of 340,000 and comprised four sub-units of Mr 82,000. The presence of this enzyme in intestinal mucosa could prevent absorption of intact bioactive fMet peptides produced by commensal bacteria in the gut lumen.

Involvement of M1 cholinergic receptors and enteric nerves in the spasmogenic activity of bacterial N‐formyl oligopeptides on guinea‐pig ileum

Bacterial N‐formyl‐methionyl oligopeptides are spasmogenic for guineapig ileum in vitro but the mechanism of this effect is not understood. To investigate this phenomenon further, we have determined pA2 values (the negative logarithm of the concentration of an antagonist reducing a double‐dose agonist response to a single‐dose response) for a number of potential antagonists of N‐formyl‐met‐leu‐phe (F‐met‐leu‐phe) using histamine, acetylcholine, 5HT and substance P as control agonists. Atropine, pirenzepine and tetrodotoxin were potent inhibitors of F‐met‐leu‐phe induced contraction (pA2s 8.4, 8.0 and 7.9, respectively) suggesting involvement of neural and cholinergic pathways in the response. Sulphasalazine, known to block the F‐met‐leu‐phe receptor on neutrophil leucocytes, was also a potent inhibitor. Tachyphylaxis induced by either 5HT, or substance P, did not diminish the response to F‐met‐leu‐phe, suggesting that these potential mediators were not involved. These studies indicate that bacterially synthesized formyl—methionyl oligopeptides bind to cells bearing receptors in guinea‐pig ileum and produce muscle contraction via enteric cholinergic (M1) neural pathways.