Fluid secretion caused by aerolysin-like hemolysin of Aeromonas sobria in the intestines is due to stimulation of production of prostaglandin E2 via cyclooxygenase 2 by intestinal cells

Abstract

To clarify the mechanisms of diarrheal disease induced by Aeromonas sobria, we examined whether prostaglandin E2 (PGE2) was involved in the intestinal secretory action of A. sobria hemolysin by use of a mouse intestinal loop model. The amount of PGE2 in jejunal fluid and the fluid accumulation ratio were directly related to the dose of hemolysin. The increase over time in the level of PGE2 was similar to that of the accumulated fluid. In addition, hemolysin-induced fluid secretion and PGE2 synthesis were inhibited by the selective cyclooxygenase 2 (COX-2) inhibitor NS-398 but not the COX-1 inhibitor SC-560. Western blot analysis revealed that hemolysin increased the COX-2 protein levels but reduced the COX-1 protein levels in mouse intestinal mucosa in vivo. These results suggest that PGE2 functions as an important mediator of diarrhea caused by hemolysin and that PGE2 is produced primarily through a COX-2-dependent mechanism. Subsequently, we examined the relationship between PGE2, cyclic AMP (cAMP), and cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels in mouse intestinal mucosa exposed to hemolysin. Hemolysin increased the levels of cAMP in the intestinal mucosa. NS-398 inhibited the increase in cAMP production, but SC-560 did not. In addition, H-89, a cAMP-dependent protein kinase A (PKA) inhibitor, and glibenclamide, a CFTR inhibitor, inhibited fluid accumulation. Taken together, these results indicate that hemolysin activates PGE2 production via COX-2 and that PGE2 stimulates cAMP production. cAMP then activates PKA, which in turn stimulates CFTR Cl- channels and finally leads to fluid accumulation in the intestines.

FIG. 1.

Effect of hemolysin dose on PGE₂ production and the time course of PGE₂ production in a mouse intestinal loop assay. The time course (A) and dose response (B) of PGE₂ release into the intestinal fluid after exposure to hemolysin were examined. (A) Two hundred fifty nanograms of hemolysin was injected into each intestinal loop and incubated for various time periods. Control loops were inoculated with phosphate-buffered saline. The fluid accumulation ratio (○, loop injected with hemolysin; •, control loop) and PGE₂ level in accumulated fluid (□, loop injected with hemolysin; ▪, control loop) were determined. (B) Hemolysin was injected into the intestinal loops of mice. After 3 h, the fluid accumulation ratio (○) and PGE₂ level in the accumulated fluid (□) were determined. Values are expressed as the means ± standard errors of five determinations.

FIG. 2.

Effect of carbenoxolone on intestinal fluid in response to hemolysin. Indicated sample solutions were injected into the intestinal loops of mice. The amount of fluid accumulated in each loop was determined 3 h after the injection of hemolysin. Values are expressed as the means ± standard errors of eight determinations.

FIG. 3.

Effect of COX inhibitors on the intestinal fluid accumulation (A) and on luminal PGE₂ release (B) in response to hemolysin (Hemo). (A) The indicated amounts of COX inhibitors were administered subcutaneously. Forty minutes later, 125 ng of hemolysin was injected into the loop. The amount of fluid accumulated was determined 2.5 h after the injection. (B) A COX inhibitor (10 mg/kg) or vehicle solution (dimethyl sulfoxide-propylenglycol solution [1:9 {vol/vol}]) was administered subcutaneously. Forty minutes later, 125 ng of hemolysin was injected into the loop. The PGE₂ concentration in the fluid accumulated was determined 2.5 h after the injection. All values are expressed as the means ± standard errors of eight determinations. Significance (versus hemolysin alone). *, P < 0.02.

FIG. 4.

Detection of COX-2 and COX-1 in the mouse intestinal mucosa by Western blot analysis. Hemolysin (125 ng) was injected into the intestinal loops of mice. After 45 min and 150 min, the loops were immediately cut open along their lengths, and the mucosa was scraped by drawing a glass microscope slide over it. The mucosa recovered was immediately placed in 600 μl of the lysis buffer and tissue homogenates were prepared by disrupting the mucosa by repeated vortexing. Western blot analyses to detect COX-1 and COX-2 were performed as described in the text. The fluorescence intensity of the corresponding band was measured. The values obtained from five experiments were leveled and expressed as values relative to those of control. Values are expressed as the means ± standard errors of five determinations. Differences between two values are presented with P values.

FIG. 5.

Action of hemolysin to elevate the level of mucosal cAMP in mouse intestines (A) and effect of COX inhibitors on the action of hemolysin (B). (A) Hemolysin (125 ng) was injected into the intestinal loop and incubated for 30 or 180 min. (B) COX inhibitor (10 mg/kg) or vehicle solution was administered subcutaneously. Forty minutes later, 125 ng of hemolysin was injected into the loop and incubated for 2.5 h. The cAMP level in each mouse intestinal mucosa was determined using a radioimmunoassay kit. Values are expressed as the means ± standard errors of eight determinations. Significance (versus control). *, P < 0.02; **, P < 0.04.

FIG. 6.

Effects of H-89 (A) and of glibenclamide (B) on the actions of hemolysin and CT to induce fluid secretion in mouse intestinal loops. The indicated amount of H-89 (A) or glibenclamide (B) was injected into the intestinal loops of mice together with hemolysin (125 ng) or CT (500 ng). After 2.5 h, the amount of fluid accumulated in each loop was determined. Values are expressed as the means ± standard errors of eight determinations. Significance (versus hemolysin alone or CT alone). *, P < 0.03; **, P < 0.001.