Immunofluorescent localization of constitutive and inducible prostaglandin H synthase in ovine astroglia

We have used immunofluorescent techniques to examine the distribution of prostaglandin H synthase (PGHS) in ovine astrocyte-enriched secondary cultures and in mixed cortical cells in primary culture. A battery of monoclonal and polyclonal antibodies specific for the constitutive (PGHS-1) or inducible (PGHS-2) forms of the enzyme were used to examine the cells in culture. Varying levels of PGHS-1 and PGHS-2-specific immunofluorescence were seen in astrocytes as well as in other cells. The fluorescent pattern and localization seen with antisera to both PGHS-1 and PGHS-2 were similar but were not identical. Both immunoreactive species were confined to nuclear and perinuclear regions of the cell, with no immunoreactivity evident in plasmalemma. In addition, PGHS-2-specific fluorescence was concentrated often as a homogeneous ring around the nucleus in heavily stained astrocytes. Mixed cortical glia/fibroblasts in primary culture were double labeled with antibodies to glial fibrillary acidic protein (GFAP) and to PGHS-2. GFAP and PGHS-2 were colocalized in clusters of astrocytes, but PGHS-2 was evident in GFAP- cells as well. Cells treated with the mitogenic agent phorbol dibutyrate displayed more PGHS-2+ immunofluorescence compared to either vehicle control or cells pretreated with dexamethasone. We conclude that astrocytes cultured in serum express both constitutive and inducible forms of PGHS and that PGHS-2 is induced by mitogens in this cell type.