Stimulation of duodenal HCO3⁻ secretion by hydrogen sulphide in rats: relation to prostaglandins, nitric oxide and sensory neurones.

AIM: We examined the effect of H2S on duodenal HCO3⁻ secretion in rats and investigated the mechanism involved in this response. METHODS: Animals were fasted for 18 h and anaesthetized with urethane. A duodenal loop was perfused with saline, and HCO3⁻ secretion was measured at pH 7.0 using a pH stat-method. The loop was perfused at a rate of 0.2 mL min⁻¹ with NaHS (H2S donor: 0.1-1 mm) for 5 min or 10 mm HCl for 10 min. Indomethacin or l-NAME [nitric oxide (NO) synthase inhibitor) was given s.c. 30 min or 3 h, respectively, before NaHS or acidification, while glibenclamide (K(ATP) channel blocker) or propargylglycine (cystathionine-g-lyase inhibitor) was given i.p. 30 min before. RESULTS: Mucosal perfusion with NaHS dose dependently increased the HCO3⁻ secretion, and this effect was significantly attenuated by indomethacin and l-NAME as well as by sensory deafferentation, but not by glibenclamide. Mucosal prostaglandin E2 (PGE2) production and luminal release of NO were both increased by NaHS perfusion. Mucosal acidification stimulated HCO3⁻ secretion concomitant with an increase in PGE2 and NO production, and these responses were mitigated by propargylglycine. The duodenal damage induced by acid (100 mm HCl for 4 h) was aggravated by pre-treatment with propargylglycine. CONCLUSION: These results suggest that H2S increases HCO3⁻ secretion in the rat duodenum, and that this action is partly mediated by PG and NO as well as by capsaicin-sensitive afferent neurones. It is assumed that endogenous H2S is involved in the regulatory mechanism of acid-induced HCO3⁻ secretion and mucosal protection in the duodenum.

Stimulatory effect of Coca-Cola on gastroduodenal HCO3- secretion in rats.

We examined the effect of various carbonated beverages, especially Coca-Cola, on the HCO3- secretion in the rat stomach and duodenum. Under urethane anaesthesia, a chambered stomach or a proximal duodenal loop was perfused with saline, and HCO3- secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. The amount of CO2 contained in these beverages was about 4-7 g/mL. Coca-Cola topically applied to the mucosa for 10 min significantly increased the HCO3- secretion in both the stomach and the duodenum. The HCO3- response in the duodenum was totally abolished by indomethacin and also partially inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Likewise, the response in the stomach was also markedly inhibited by either acetazolamide or indomethacin. The mucosal application of Coca-Cola increased the PGE2 contents in both the stomach and the duodenum. Other carbonated beverages, such as sparkling water, Fanta Grape or cider, also increased the HCO3- secretion in these tissues. These results suggest that Coca-Cola induces HCO3- secretion in both the stomach and the duodenum, and these responses may be attributable to both the intracellular supply of HCO3- generated via carbonic anhydrase, and endogenous PGs, probably related to the acidic pH of the solution.

Involvement of cyclooxygenase-1, prostaglandin E2 and EP1 receptors in acid-induced HCO3- secretion in stomach.

We investigated the cyclooxygenase (COX) isoforms as well as prostaglandin E receptor EP subtypes responsible for acid-induced gastric HCO(3)(-) secretion in rats and EP receptor-knockout (-/-) mice. Under urethane anesthesia, a chambered stomach (in the presence of omeprazole) was perfused with saline, and HCO(3)(-) secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. Mucosal acidification was achieved by exposing the stomach for 10 min to 50 or 100 mM HCl. Acidification of the mucosa increased the secretion of HCO(3)(-) in the stomach of both rats and WT mice, in an indomethacin-inhibitable manner. The acid-induced gastric HCO(3)(-) secretion was inhibited by prior administration of indomethacin and SC-560 but not rofecoxib in rats and mice. Acidification increased the PGE(2) content of the rat stomach, and this response was significantly attenuated by indomethacin and SC-560 but not rofecoxib. This response was also attenuated by ONO-8711 (EP1 antagonist) but not AE3-208 (EP4 antagonist) in rats and disappeared in EP1 (-/-) but not EP3 (-/-) mice. PGE(2) increased gastric HCO(3)(-) secretion in both rats and WT mice, and this action was inhibited by ONO-8711 and disappeared in EP1 (-/-) but not EP3 (-/-) mice. These results support a mediator role for endogenous PGs in the gastric response induced by mucosal acidification and clearly indicate that the enzyme responsible for production of PGs in this process is COX-1. They further show that the presence of EP1 receptors is essential for the increase in the secretion of HCO(3)(-) in response to mucosal acidification in the stomach.

Mutational analysis of catalytic sites of the cell wall lytic N-acetylmuramoyl-L-alanine amidases CwlC and CwlV.

The Bacillus subtilis CwlC and the Bacillus polymyxa var. colistinus CwlV are the cell wall lytic N-acetylmuramoyl-l-alanine amidases in the CwlB (LytC) family. Deletion in the CwlC amidase from the C terminus to residue 177 did not change the amidase activity. However, when the deletion was extended slightly toward the N terminus, the amidase activity was entirely lost. Further, the N-terminal deletion mutant without the first 19 amino acids did not have the amidase activity. These results indicate that the N-terminal half (residues 1-176) of the CwlC amidase, the region homologous to the truncated CwlV (CwlVt), is a catalytic domain. Site-directed mutagenesis was performed on 20 highly conserved amino acid residues within the catalytic domain of CwlC. The amidase activity was lost completely on single amino acid substitutions at two residues (Glu-24 and Glu-141). Similarly, the substitution of the two glutamic acid residues (E26Q and E142Q) of the truncated CwlV (CwlV1), which corresponded to Glu-24 and Glu-141 of CwlC, was critical to the amidase activity. The EDTA-treated CwlV1 did not have amidase activity. The amidase activity of the EDTA-treated CwlV1 was restored by the addition of Zn2+, Mn2+, and Co2+ but not by the addition of Mg2+ and Ca2+. These results suggest that the amidases in the CwlB family are zinc amidases containing two glutamic acids as catalytic residues.

Overexpression, purification, and characterization of Bacillus subtilis N-acetylmuramoyl-L-alanine amidase CwlC.

N-acetylmuramoyl-L-alanine amidase CwlC of Bacillus subtilis was overproduced in Escherichia coli and purified 21-fold. The amidase hydrolyzed type A cell walls such as B. subtilis. The amidase bound slightly to the Microbacterium lacticum cell wall (type B), but did not entirely hydrolyze it. The presence of calcium or magnesium ion increased the resistance of the amidase to heat denaturation.