We determined the effects of immobilization stress on rat colonic mucus release and mast cell degranulation and examined whether corticotropin releasing factor (CRF) was involved in these responses. After 30-min immobilization, rats were killed, colonic mucosal explants were cultured, and levels of rat mast cell protease II (RMCP II) and prostaglandin E2 (PGE2) were measured. Mucin release from explants was assayed by incorporation of [3H]glucosamine into colonic mucin and by histological evaluation of goblet cell depletion. Stress caused significant increases of colonic RMCP II, PGE2, and mucin release and fecal pellet output and caused an approximately 10-fold increase in colonic mucosal levels of cyclooxygenase-2 (COX-2) mRNA. These stress-associated changes were reproduced by intravenous or intracerebral injection of CRF in conscious, nonstressed rats. Pretreatment of rats with the CRF antagonist alpha-helical-CRF9-41, hexamethonium, atropine, or bretylium, or the mast cell stabilizer lodoxamide inhibited stress-induced release of RMCP II, PGE2, and mucin, whereas indomethacin prevented mucin release but not mast cell degranulation. Hexamethonium and CP-96,345, a substance P antagonist, inhibited fecal pellet output caused by stress. We conclude that CRF released during immobilization stress increases colonic transit via a neuronal pathway and stimulates colonic mucin secretion via activation of neurons and mast cells.

Idiopathic inflammatory bowel disease is a chronic relapsing condition. The role of stress in causing relapses of inflammatory bowel disease remains controversial. We now show that colitis induced in mice by dinitrobenzenesulfonic acid (DNBS) resolves by 6 weeks, but can subsequently be reactivated by stress plus a sub-threshold dose of DNBS, but not by DNBS alone. Stress reduced colonic mucin and increased colon permeability. Susceptibility to reactivation by stress required CD4+ lymphocytes and could be adoptively transferred. We conclude that stress reactivates experimental colitis by facilitating entry of luminal contents that activate previously sensitized CD4 cells in the colon.

Previously we reported that pretreatment of rats with the substance P (SP) antagonist CP-96,345 inhibits the enterotoxic responses following administration of toxin A from Clostridium difficile into ileal loops, indicating that SP participates in the intestinal responses to this toxin. We now report that injection of toxin A into rat ileum causes a rapid increase in SP content in lumbar dorsal root ganglia (DRG) and mucosal scrapings 30-60 min after toxin A administration. Toxin A-mediated f luid secretion, mannitol permeability, and ileal histologic damage is significantly increased only after 2 hr. Toxin A also causes an increase in the abundance of SP mRNA in lumbar DRG and ileal mucosa as measured by reverse transcription-PCR. Lamina propria macrophages (LPMs) obtained from toxin A-injected loops release greater amounts of tumor necrosis factor ␣ (TNF␣) and SP as compared with LPMs isolated from buffer-injected loops (P < 0.01). Pretreatment of rats with the SP antagonist CP-96,345 inhibits toxin A-mediated TNF␣ release from isolated LPMs, whereas an inactive enantiomer (CP-96,344) of the SP antagonist has no effect. LPMs obtained from toxin A-injected ileal loops incubated in vitro with SP (10 ؊8 to 10 ؊9 M) show enhanced TNF␣ secretion, whereas LPMs isolated from buffer-injected loops do not respond to SP. In addition, LPMs obtained from toxin A-injected ileal loops incubated in vitro with CP-96,345 showed a diminished TNF␣ release. Our results indicate that activated LPMs secrete SP during toxin A enteritis that can lead to secretion of cytokines, suggesting an autocrine͞paracrine regulation of cytokine secretion by SP from LPMs during intestinal inf lammation.Substance P (SP), an 11-aa peptide member of the tachykinin family originally isolated by Chang and Leeman (1), is a peptide distributed throughout the central and peripheral nervous system. In the intestine, SP has been found in capsaicin-sensitive sensory neurons (2), enteric neurons (3), as well as intestinal enteroendocrine cells (4). SP is also synthesized in the cell bodies of the dorsal root ganglia (DRG) (5), and evidence indicates that release of SP from DRG to spinal cord mediates nociceptive and inflammatory stimuli (6-8). Recently, Reinshagen et al. (9) reported decreased levels of SP in the DRG of rabbits 48 hr after induction of colitis, suggesting that SP may be released from sensory neurons during the acute phase of inflammation. However, there are no studies specifically evaluating the SP content in the DRG during acute intestinal inflammation mediated by bacterial enterotoxins.In addition to its role as a neurotransmitter, SP also participates in immune and inflammatory responses. Mast cells (10), polymorphonuclear leukocytes (11), T lymphocytes (12, 13), and macrophages (14) can respond to SP, indicating that the effects of SP during inflammation may be mediated by direct activation of these cells. SP levels are also elevated in inflamed tissues (15-17), and increased binding sites for SP have been demonstrated at sites o...