Stimulatory Effect of Nitric Oxide on Bicarbonate Secretion in Bullfrog Duodenums in vitro

Abstract

The effect of nitric oxide (NO) on HCO<sup>–</sup><sub>3</sub> secretion was examined in vitro using an isolated preparation of bullfrog duodenum. The tissue was bathed in unbuffered Ringer’s solution gassed with 100% O<sub>2</sub> on the mucosal side and HCO<sup>–</sup><sub>3</sub> Ringer’s solution gassed with 95% O<sub>2</sub>–5% CO<sub>2</sub> on the serosal side. The HCO<sup>–</sup><sub>3</sub> secretion was measured by the pH-stat method using 2 mmol/l HCl as the titrant to keep the mucosal pH at 7.4. (±)-(E)-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamine (NOR3) was used as a NO donor and added to the serosal solution. To analyze the NOR3 action on HCO<sup>–</sup><sub>3</sub> secretion, the effects of dibutyryl adenosine-3′,5′-cyclic monophosphate (dbcAMP), dibutyryl guanosine-3′,5′-cyclic monophosphate (dbcGMP), methylene blue, and indomethacin on the HCO<sup>–</sup><sub>3</sub> response were also examined. NOR3 (1×10<sup>–4</sup> and 3×10<sup>–4</sup> mol/l) caused an increase in HCO<sup>–</sup><sub>3</sub> secretion in a dose-dependent manner, and this effect appeared with an about 30-min time lag, reaching the level of 1.5–2.5 times greater than basal values at 1–2 h later. Both dbcAMP (1×10<sup>–3</sup> mol/l) and dbcGMP (1×10<sup>–3</sup> mol/l) also caused a significant increase in HCO<sup>–</sup><sub>3</sub> secretion in bullfrog duodenums in vitro, although the onset of the HCO<sup>–</sup><sub>3</sub> response to dbcGMP was delayed as compared to the former. The stimulatory action of NOR3 on duodenal HCO<sup>–</sup><sub>3</sub> secretion was significantly attenuated by methylene blue (5×10<sup>–5</sup> mol/l) and indomethacin (1×10<sup>–5</sup> mol/l), the latter also inhibiting the HCO<sup>–</sup><sub>3</sub> response to dbcGMP. The release of prostaglandin E<sub>2</sub> in the serosal solution was significantly increased after addition of NOR3 (3×10<sup>–4</sup> mol/l) and dbcGMP (1×10<sup>–3</sup> mol/l) in an indomethacin-sensitive manner. These results suggest that the NO donor increases duodenal HCO<sup>–</sup><sub>3</sub> secretion in vitro, and this action of NO donor is cGMP-dependent and mediated by endogenous prostaglandins. Duodenal HCO<sup>–</sup><sub>3</sub> secretion may be regulated locally by NO/cGMP in addition to prostaglandin/cAMP.