Impaired activation of cytosolic phospolipase A2 in inflamed canine colonic circular muscle

Background & Aims: Arachidonic acid (AA) is a critical second messenger in several cell types. We examined whether cholinergic AA acts as a second messenger in contraction of colonic circular muscle cells and if this role is altered by inflammation. Methods: The experiments were performed on single dispersed cells. AA release was measured by high-performance liquid chromatography. Escherichia coli membranes labeled with H-3-AA were used as a substrate for determining phospholipase A(2), (PLA(2)) activity, and Western immunoblotting for protein expression. Results: Acetylcholine and the PLA(2) activator melittin induced cell contractions and AA release. Both effects were inhibited by the PLA(2) inhibitor ONO-RS-082. Cytosolic and membrane PLA(2) activities increased in response to acetylcholine. These were blocked by ONO-RS-082 and cytosolic PLA(2) 100-kilodalton antibody, but not by dithiothreitol, a secretory PLA(2), inhibitor. Acetylcholine- and melittin-stimulated release of AA and their contractile response were attenuated in inflamed cells. Immunoblotting indicated that the protein expression of cPLA(2) was suppressed during inflammation, Conclusions: AA acts as a second messenger in muscarinic receptor-activated contractions of colonic circular muscle cells. cPLA(2), is the primary enzyme that releases AA in these cells; its expression as well as activation are significantly attenuated by inflammation, The attenuated release of AA may partly account for the inhibition of colonic circular muscle tone and phasic contractions observed during inflammation.