PGE2 secretion from organ cultured gastric mucosa: correlation with cyclooxygenase activity and endogenous substrate release.

In gastrointestinal research the in vitro release of prostaglandins from incubated or cultured biopsies is a widely used method to estimate prostaglandin synthesis. We therefore investigated the rate limiting mechanisms of PGE2 release in organ cultured gastric mucosa of the rabbit, determining PGE2 secretion from organ cultured mucosal biopsies by radioimmunoassay and prostaglandin synthesizing capacity by in vitro incubation of mucosal homogenate or microsomes with [14C]-arachidonic acid. Freshly taken biopsies secreted PGE2 at an initial high rate, that decreased during the following 4 hrs of culture. This PGE2 release was dose dependently reduced by inhibitors of the prostaglandin cyclooxygenase. 5mM acetylsalicylic acid (ASA) maximally suppressed PGE2 secretion to 7% of controls, and the inhibition by ASA was quantitatively similar at every given culture period. PGE2 release was markedly increased by carbenoxolone but was only slightly activated by extracellular calcium and the Ca(++)-ionophore A23187. However, Ca++/A23187 were unable to maintain PGE2 secretion at the initial rate. PGE2 secretion was undisturbed in calcium-free medium but was reduced to 50-60% of controls by excess EDTA. The intracellular calcium chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N',-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) similarly inhibited PGE2 release to 72% of controls. In contrast, PGE2 release was unaffected by the intracellular calcium antagonist 3,4,5-trimethylene-bis(4-formylpyridinium bromide) dioxime (TMB-8), the calmodulin antagonists N-(6-aminohexyl)-1-5-chloro-1-naphthalenesulfonamide (W-7) and calmidazolium (compound R24571) or various direct inhibitors of endogenous arachidonic acid release like tetracaine, bromophenacyl bromide, neomycin or low dose quinacrine, indicating that the reduction of PGE2 release by EDTA or BAPTA may be mediated by mechanisms different from substrate release. In contrast, an inhibition of PGE2 secretion by quinacrine at high concentrations (greater than or equal to 0.8 mM) was attributed to a direct inhibition of the prostaglandin cyclooxygenase, similar to ASA. Finally, the reduction of the prostaglandin synthesizing capacity by ASA was strongly correlated with the inhibition of PGE2 secretion, also at low concentrations and minor degrees of inhibition. From these data we conclude, that the activity of the prostaglandin cyclooxygenase is rate limiting for PGE2 secretion from organ cultured mucosal biopsies rather than arachidonic acid release by a phospholipase A2. This should be considered for interpretation of studies based on prostaglandin release from cultured mucosa.