The autocoid adenosine is produced in the intestinal lumen during inflammation via the enzymatic conversion of inflammatory cell-secreted ATP, and is thought to be a factor in the diarrhoea-associated response in certain disease states (Sitaraman et al., 2002). The adenosine receptor subtype responsible has been characterized in the model colon epithelial cell line, T84, and was shown to be A_2B (Sitaraman et al., 2002). The aim of the present study was to determine which adenosine receptor subtype is responsible for fluid secretion caused by the non-selective agonist 5’-(N-ethylcarboxamido)-adenosine (NECA) in human colonic mucosa.

Human colonic mucosa samples were obtained from 15 donors (7 female, 8 male, aged between 44 and 76) post operatively (PO) with the informed consent of the donor, and with the approval of local ethics committees. Tissues were placed in PBS at 4°C and transported immediately to the laboratory; in all cases the PO delay was less than 7 hours. The smooth muscle layers were removed and sections of mucosa mounted in Ussing chambers, containing gassed Krebs solution (95% O₂ / 5% CO₂) at 37°C, for the measurement of short circuit current (Isc, indicative of electrogenic fluid secretion). After 60 minutes equilibration, carbachol (0.1mM) was applied to the tissues to assess viability. After a further period of washing, adenosine receptor antagonists selective for A 1 (DPCPX, Lohse et al., 1987), A_2A (ZM241385, Ongini et al., 1999), A_2B (MRS1754, Ji et al., 2001) and A₃ (MRS1220, Jacobson et al., 1998) were applied apically at a concentration of 100nM. After a contact period of 30 minutes NECA (0.1nM – 0.1mM) was added apically by cumulative application. After addition of the final concentration of NECA, carbachol (0.1mM) was added as a final viability check. Statistical analysis was by one-way analysis of variance.

Cumulative administration of NECA (0.1nM – 0.1mM) in the absence of antagonist resulted in a concentration-dependent increase in Isc (pEC₅₀ of 6.0 ±0.03 ), with a maximum response of 49.5±6.8µAcm^-2 (n=15). The potency of NECA was not significantly different in the presence of DPCPX (pEC₅₀ 5.8 ± 0.03, n=4), ZM241385 ( pEC₅₀ 5.6 ± 0.04, n=6), MRS1754 ( pEC₅₀ 5.8 ± 0.03, n=3), MRS1220 ( pEC₅₀ 6.4 ± 0.09, n=3), or a combination of ZM241385 and MRS1754 ( pEC₅₀ 5.8 ± 0.03, n=3).

In conclusion, contrary to published data in T84 cells, the increase in colonic mucosal fluid secretion caused by NECA in vitro does not appear to be mediated by any of the known adenosine receptor subtypes. The receptor responsible for this effect of NECA in human colonic mucosa has yet to be identified.

Jacobson, K.A. (1998). Trends Pharmacol. Sci., 19, 184-91
Ji, X., et al. (2001). Biochem. Pharmacol., 61, 657-63
~ Lohse, M.J. et al. (1987). Naunyn-Schmiedeberg’s Arch. Pharmacol., 336, 204-10
Ongini, E., et al. (1999). Naunyn-Schmiedeberg's Arch. Pharmacol., 359, 7-10
Sitaraman, S.V. (2002). J. Biol. Chem., 6;277, 33188-95