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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.

We examined effects of protein kinase C (PKC) activation by phorbol dibutyrate (PDB) on prostaglandin production in astroglia. Astroglia were cultured from sheep fetal cortex and grown in Eagle's basal media supplemented with 10% fetal calf serum (BME-C). Prostaglandin F2a (PGF2 alpha) levels in media were determined at 2-24 hours after exposure to PDB. PDB increased production of PGF2 alpha at 10(-8)M and 10(-6)M. In addition, PDB increased the ratio of membrane to cytosolic PKC. Coapplication of H7 [1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine] (10(-4)M) with PDB (10(-6)M) inhibited PDB-induced PGF2a production. To investigate the role of protein synthesis in increased prostaglandin production by PDB, astroglia were coincubated with actinomycin D (1 mg/ml) or cycloheximide (10 mg/ml). At 4 hrs, both actinomycin D and cycloheximide inhibited increases in PGF2a in response to PDB application. In addition, COX-2 mRNA levels and COX activity levels were examined. PDB increased COX-2 mRNA levels by 2 hours, and COX activity tripled after 12 hr exposure to PDB. In addition, the increase in COX activity was blocked by cycloheximide. In summary, PKC activation promotes enhanced prostaglandin production via an increase in COX synthesis.

We examined the effects of interleukin-1 alpha (IL-1 alpha) and involvement of protein kinases on prostaglandin production in cultured ovine astroglia. Ovine astroglia were exposed to media alone, or 10 ng/mL IL-1 alpha and prostaglandin F2 alpha (PGF2 alpha) levels were analyzed using enzyme immunoassay. Application of IL-1 alpha augmented the production of PGF2 alpha at 4 h. Coapplication of H-7 (10-1000 microM) and staurosporine (0.1-10 microM), inhibitors of protein kinase C (PKC), blocked IL-1 alpha-induced PGF2 alpha production. IL-1 alpha increased cyclooxygenase (COX) activity while coapplication of staurosporine prevented an increase, implying that COX activity was dependent upon PKC activation. In contrast, forskolin, sodium nitroprusside, and cyclic nucleotide analogs alone did not affect prostaglandin production significantly, excluding the involvement of cAMP/cGMP-dependent protein kinases. Coapplication of quinacrine (10 microM) and bromophenacyl bromide (100 microM), inhibitors of phospholipase A2 (PLA2), prevented the IL-1 alpha-induced increases in PGF2 alpha production. Lastly, IL-1 alpha increased labeled arachidonic acid (AA) release whereas coaddition of quinacrine (10 microM) attenuated increased AA release. Therefore, we propose that IL-1 alpha enhances prostaglandin production by ovine astroglia via steps involving activation of PKC and increased activity of COX and PLA2.

Experiments were done to determine whether interleukin 1 alpha (IL-1 alpha) induces prostaglandin (PG) synthesis in cultured astroglia and to examine the role of enhanced cyclooxygenase 2 (COX 2) synthesis in these responses. Immunologically identified fetal ovine astrocytes were exposed to IL-1 alpha or to media alone. Application of 10 ng/ml IL-1 alpha increased the production of PGF2 alpha for 2-24 h. Coincubation of IL-1 alpha with actinomycin D (1 microgram/ml) or cycloheximide (10 micrograms/ml) completely blocked the increase in PGF2 alpha. Immunoblot analyses revealed that the synthesis of COX2 was not increased by IL-1 alpha at 4 h. However, with incubation of IL-1 alpha for 8-12 h, COX2 levels were increased. These results indicate that longer periods than 4 h are necessary to increase COX2 protein, suggesting that proteins other than COX2 are involved in early IL-1 alpha-induced PG synthesis.

We examined effects of interleukin 1 alpha (IL-1 alpha) on prostaglandin production in piglet cultured astroglia. Immuno- and morphologically identified polymorphic and process-bearing astrocytes were collected from cerebral cortex and white matter from piglets (1-3 days of age). Levels of prostaglandins were determined using enzyme immunoassay. Baseline levels for prostaglandin (PG) F2 alpha were 631 +/- 332 pg/ml and increased to 1417 +/- 353 pg/ml 20 min after addition of 11 micrograms/ml IL-1 alpha (p < 0.05) and to 2280 +/- 391 pg/ml 20 min after addition of 22 micrograms/ml IL-1 alpha (p < 0.05) (n = 7). Only small amounts of 6-keto-PGF 1 alpha or PGE2 were detected in medium under baseline conditions or in the presence of IL-1 alpha. Medium alone did not change PGF2 alpha levels (n = 3). Coapplication of cycloheximide (10(-3) M) blocked the increase in PGF2 alpha (n = 3). We conclude that IL-1 alpha causes a rapid increase in prostaglandin production by astroglia, and this process involves a step requiring continued or increased protein synthesis.

We examined effects of phorbol 12,13-dibutyrate (PDB), which activates protein kinase C (PKC), on prostaglandin and leukotriene production in piglet cultured glia derived from cerebral cortex and white matter. Levels of prostaglandins were determined using enzyme immunoassay. Baseline levels in media for prostaglandin F2 alpha (PGF2 alpha) were 730 +/- 116 pg/ml and increased to 1,551 +/- 196 pg/ml at 10(-8) M PDB (P < 0.05) and to 2,182 +/- 190 pg/ml at 10(-6) M PDB (P < 0.05) (n = 16). Little or no 6-keto-prostaglandin F1 alpha, prostaglandin E2, or leukotrienes C4/D4 were produced. PGF2 alpha levels in media did not increase in the presence of the vehicle for PDB (dimethyl sulfoxide) or 4 alpha-phorbol 12,13-didecanoate (PDD; a phorbol ester that does not activate protein kinase C) or when indomethacin (10 micrograms/ml), quinacrine (10(-6) M), or isoquinolinylsulfonylmethyl piperazine (10(-4) M) (an inhibitor of PKC activation) was coadministered with PDB. We conclude that glia can be important contributors of prostaglandins to extracellular-cerebrospinal fluids where they could influence cerebrovascular tone, and that PDB probably increases prostaglandin production via liberation of arachidonic acid by PKC-induced activation of phospholipase A2.