Prostaglandin E₂ release in gastric antral mucosa of guinea‐pigs: basal PGE₂ release by cyclo‐oxygenase 2 and ACh‐stimulated PGE₂ release by cyclo‐oxygenase 1

Abstract

Prostaglandin E 2 (PGE 2 ), which is generated by two isoforms of cyclo-oxygenase (COX 1 and COX 2 ), is a key mediator in gastric mucosal defense. In the present study, antral mucosa of guineapigs was incubated with various agonists or antagonists in a medium, the PGE 2 concentration of which was measured using a PGE 2 EIA kit. Prostaglandin E 2 was released from the antral mucosa spontaneously (basal PGE 2 release) and acetylcholine (ACh, 10 μM) enhanced the PGE 2 release (ACh-stimulated PGE 2 release) was mediated via intracellular Ca 2+ concentration ([Ca 2+ ] i ). Arachidonic acid enhanced both forms of PGE 2 release, and a phospholipase A 2 inhibitor (amylcinnamoyl anthranilic acid) and COX inhibitors (acetylsalicylic acid and indomethacin) decreased them. 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazol (SC560, 100 nM, a COX 1 -selective inhibitor) inhibited ACh-stimulated PGE 2 release without any decrease in basal PGE 2 release. N -(2-Cyclohexyloxy-4-nitrophenyl) methanesulphonamide (NS398, 20 μM, a COX 2 -selective inhibitor) decreased basal PGE 2 release without any reduction of AChstimulated PGE 2 release. However, ionomycin (a Ca 2+ ionophore) increased PGE 2 release from antral mucosa in the presence of SC560 or NS398, suggesting that COX 1 and COX 2 are regulated by [Ca 2+ ] i . These findings indicate that COX 1 -containing cells have ACh receptors but COX 2 -containing cells do not. Moreover, in isolated antral epithelial cells, SC560 decreased basal and ACh-stimulated PGE 2 release, but NS398 did not. In conclusion, in antral mucosa, basal PGE 2 release is mainly maintained by COX 2 of non-epithelial cells, and ACh-stimulated PGE 2 release is maintained by COX 1 of epithelial cells.

A PKG inhibitor increases Ca2+-regulated exocytosis in guinea pig antral mucous cells: cAMP accumulation via PDE2A inhibition

“…Thus, 1 M ACh stimulation induced no increase in the amount of PGE 2 released from the antral mucosae, whereas 10 M ACh stimulation induced a significant increase in it. Similar results have already been reported in the previous report (17).…”

“…PGE 2 released under the basal condition may activate the PGE 2 autocrine mechanism. Our previous study demonstrated that, in antral mucosae, PGE 2 released under the basal condition are from nonepithelial cells via COX2, those during ACh stimulation are from epithelial cells via COX1, and that, in isolated epithelial cells, the amount of PGE 2 released under the basal condition is negligibly small (17). Because we used isolated antral mucous cells for observing exocytosis, the amount of PGE 2 released under the basal condition is negligibly small.…”

“…The microtube containing the sample was immediately cooled on ice and stored at Ϫ30°C until the PGE 2 concentration measurements. PGE2 concentrations were measured using a PGE2 EIA kit (Cayman Chemical), and PGE2 contents were expressed as micrograms per gram weight of tissue (4,16,17). The amount of PGE2 released was calculated from the difference between the values before and 15 min after ACh stimulation.…”

<b>PPARα induced NOS1 phosphorylation via PI3K/Akt in guinea pig antral mucous cells: NO-enhancement in Ca</b>^<b>2+</b><b>-regulated </b><b>exocytosis </b>

“…In antral mucous cells, the Ca 2+ -regulated exocytosis which is activated by ACh (acetylcholine) is the main mechanism for mucin release (8,14,15,19) and is modulated by many substances, such as cAMP, NO, cGMP, intracellular Cl − , and arachidonic acid (AA) (8,14,19,(21)(22)(23)(24). NO is synthesized by nitric oxide synthase 1 (NOS1) and enhances the Ca 2+ -regulated exocytosis mediated via cGMP accumulation in antral mucous cells (28).…”

“…Guinea pigs were sacrificed by intraperitoneal injection of pentobarbital sodium (100 mg/kg), and then, the stomach was removed from the animal. The procedures for the cell preparation have been previously described (8,9,14,15,19,(22)(23)(24). Briefly, the gastric antrum was excised from the stomach, and the mucosal layer was stripped from the muscle layer in cold phosphate buffered saline (4°C) using glass slides.…”