Prostaglandin D2 production and identification of prostaglandin H synthase within canine mast cell granule

Abstract

We have identified the presence of functional prostaglandin H synthase (PGH synthase, E.C. 1.14.99.1, or cyclooxygenase) within canine mast cell granules by demonstrating the generation of prostaglandin (PG) D2 from isolated and purified granules incubated with substrate as arachidonic acid or stimulated with calcium ionophore, A23187. This confirms the presence of both enzyme and substrate within the granule. Localization of PGH synthase to the granule was confirmed by immunoblotting of the pure granule preparation and by immunocytochemistry using the whole cell. In functional studies, colchicine, a microtubule polymerization inhibitor, caused a fall of up to 70%, both in the amount of histamine released and in the amount of PGD2 generated. This suggests either that functional PGH synthase is closely associated and coactivated with granules or that there is an independent association of this enzyme with the microtubule system. Release of the preformed and newly formed mediators of the mast cell appear to be closely linked, and prevention of degranulation may therefore attenuate the effects of both classes of mediators.