The anti-tumour drug irinotecan clinically causes severe diarrhoea as a side effect. Diarrhoea is generally accompanied with active secretion of electrolytes, especially Cl^–. We found recently that irinotecan stimulates Cl^– secretion in isolated rat colonic mucosa via the release of thromboxane A₂ (TXA₂), and that 9,11-epithio-11,12-methano-thromboxane A₂ (STA₂), a stable TXA₂ analogue, mimics the effect of irinotecan (Sakai et al. 1997; 2002). TXA₂ receptor was cloned and found to link both the Ca²⁺-and cAMP-signalling pathways (Hirata et al. 1996). STA₂ elevates [Ca²⁺]_i specifically via TXA₂ receptor in rat colonic crypt cells (Ikari et al. 1999). But it has not been clarified whether the the cAMP pathway is involved in the TXA₂-induced Cl^– secretion in the colon. Herein we investigated if the TXA₂-induced Cl^– secretion is mediated by elevation of the intracellular cAMP level.

Rats were sacrificed rapidly by stunning and cervical dislocation. Effects of the chemicals on the Cl^– secretion were examined in isolated rat distal colon mounted on Ussing chamber. The cAMP content in the HT-29 human colonic cancer cell line was measured using an enzyme immunoassay kit. Data are shown as means ± S.E.M. Differences between groups were analysed by one-way ANOVA. Comparison between the two groups was made with paired t test.

In HT-29 colonic carcinoma cells (1 X 10⁵ cells), STA₂ (0.1 µM) increased the cAMP level by 110 ± 16 fmol in the presence of indomethacin (1 µM) (n = 3). This increase was significantly inhibited by KW-3635 (30 µM), a selective TXA₂ receptor antagonist (n = 3, P < 0.05), suggesting that the effect is mediated specifically via the receptor. In the Ussing chamber experiments using isolated rat colonic mucosa, both the STA₂ (1 µM)-and PGE₂ (0.5 µM)-induced Cl^– secretion were inhibited in a concentration-dependent manner by chromanol 293B, a cAMP-dependent K⁺ channel blocker. (IC₅₀ = 0.09 and 0.1 µM, respectively; n = 5). The STA₂-induced Cl^– secretion was significantly stimulated by pre-treatment of the mucosa with 3-isobutyl-1-methylxanthine (IBMX; 100 µM), a phosphodiesterase inhibitor, in the presence of indomethacin (1 µM) and tetrodotoxin (1 µM): that is, IBMX increased the STA₂-elicited current from 45 ± 5 to 139 ± 13 µA cm^-2 (n = 5, P < 0.01).

Our results suggest that the TXA₂-induced Cl^– secretion is mediated by the elevation of intracellular cAMP in the colonic mucosa.