Inhibitors of phospholipase A2 block the stimulation of protein synthesis by insulin in L6 myoblasts.

Insulin at a concentration close to the physiological range (100 mu-units/ml) stimulated protein synthesis in L6 myoblasts by 17%. Pre-treatment with the phospholipase A2 inhibitors mepacrine or dexamethasone prevented this stimulation and decreased the release of prostaglandin F2 alpha, implicating the action of phospholipase A2 and the subsequent metabolism of arachidonic acid to prostaglandins in the stimulation of protein synthesis by physiological doses of insulin. Higher concentrations of insulin (500-1000 mu-units/ml) stimulated protein synthesis in the presence of mepacrine or dexamethasone, suggesting that an alternative pathway may become important in insulin action when phospholipase A2 is inhibited.     

Dexamethasone-induced catabolism and insulin resistance in L6 myoblasts are reversed by the removal of serum.

1. One hundred nanomolar dexamethasone reduced protein synthesis by 16% and also decreased the accretion of protein and RNA in L6 myoblasts when foetal calf serum was present; these effects were reversed when serum was omitted from the medium. 2. Insulin (100 microU/ml) increased protein synthesis, protein accretion and RNA accretion both in the presence and the absence of serum. 3. Dexamethasone inhibited the effects of 100 microU insulin/ml in the presence of serum and induced insulin resistance; in the presence of 25 or 100 nM dexamethasone insulin was ineffective at concentrations below 250 microU and 1 mU/ml respectively.     

Synthesis of type C virus particles from murine-cultured cells induced by iododeoxyuridine. V. Effect of interferon and its interaction with dexamethasone.

Previous studies have shown that in certain cell systems dexamethasone may enhance the production of type C viruses. Conversely, interferon has been shown to inhibit their production. Both appear to exert their influence late in the viral replication cycle rather than on the synthesis of viral-specific RNA. In this report dexamethasone and interferon have been used to study some aspects of the mechanisms involved in the synthesis of type C viruses in murine K-BALB cells following induction of virus production by iododeoxyuridine. Interferon inhibited production of xenotropic type C virus induced by iododeoxyuridine from K-BALB cells both in the absence and presence of dexamethasone, but it did not affect production of N-tropic type C virus. Exposure of the cells to interferon for longer than 12 h was required for maximum effect. Two types of inhibitory effects were observed: one diminished by dexamethasone when the steroid was added 24 h after interferon removal, and the second resistant to dexamethasone. The concentration of intracellular group-specific antigen was diminshed after interferon and increased after dexamethasone exposure. When induced cells were treated with both interferon and dexamethasone, the intracellular group-specific protein concentration was slightly increased, but virus production was reduced 10-fold compared with induced cells treated with dexamethasone alone. We conclude that interferon and dexamethasone may affect both the synthesis of viral proteins and the assembly or release of virus particles and that dexamethasone can partially nullify the inhibitory activity of interferon. The results also support previous conclusions that the regulatory mechanisms for synthesis of viral proteins and for the release of viral particles may differ and that controls for xenotropic and ecotropic virus formation may not be identical.     

Dexamethasone-induced catabolism and insulin resistance in L6 myoblasts are reversed by the removal of serum

1. One hundred nanomolar dexamethasone reduced protein synthesis by 16% and also decreased the accretion of protein and RNA in L6 myoblasts when foetal calf serum was present; these effects were reversed when serum was omitted from the medium.2. Insulin (100 μU/ml) increased protein synthesis, protein accretion and RNA accretion both in the presence and the absence of serum.3. Dexamethasone inhibited the effects of 100 μU ulin/ml in the presence of serum and induced insulin resistance; in the presence of 25 or 100 nM dexamethasone insulin was ineffective at concentrations below 250 μU and 1 mU/ml respectively.     

Dexamethasone stimulates arachidonic acid conversion to prostaglandin E2 in human amnion cells

The purpose of this investigation was to study the mechanism of stimulation of PGE2 output from human amnion epithelial cells by the synthetic glucocorticoid dexamethasone. Cells incubated in serum-free pseudo-amniotic fluid produced very low levels of PGE2, even when arachidonic acid (1 microM) was present. Pretreatment of cells with dexamethasone (50 nM) for 21 h increased the PGE2 output 6- to 7-fold in 2-h incubations only in the presence of arachidonic acid. The RNA synthesis inhibitor, actinomycin D (1 microgram/ml), and the protein synthesis inhibitor, cycloheximide (40 micrograms/ml), each blocked dexamethasone-stimulated arachidonic acid conversion to PGE2. The time course of these events suggests that dexamethasone first initiates RNA synthesis. Acetylsalicylic acid, a specific and irreversible blocker of prostaglandin endoperoxide H synthase (cyclooxygenase), was used to determine whether dexamethasone could stimulate new enzyme synthesis. Cells treated first with acetylsalicylic acid (30 min) then dexamethasone (22 h) produced as much PGE2 in response to 1 microM arachidonate as did cells exposed to dexamethasone only. Exposing cells to acetylsalicylic acid after dexamethasone completely eliminated PGE2 output. These data suggest that dexamethasone stimulates the synthesis of prostaglandin endoperoxide H synthase.     

Effect of Dexamethasone on Prostaglandin Synthesis in Various Areas of the Rat Brain

Glucocorticoid hormones are known to inhibit the production of prostaglandins in many cell types and tissues. The effect of these hormones on the biosynthesis of brain tissue is not yet clear. In the present study we investigated the effect of dexamethasone on the release of prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and 6-keto-PGF1 alpha from various brain areas of male rats. Slices from cortex, hippocampus, hypothalamus, and striatum taken from rats pretreated with dexamethasone 4 mg/kg body weight or vehicle, 18 h and 2 h prior to killing, were incubated in Krebs-Ringer-bicarbonate for 1 h. The accumulation of PGs in the medium was determined by radioimmunoassay. Pretreatment with dexamethasone significantly reduced the release of all PGs from the cortex by 40-50%. In the striatum and hippocampus only TXB2 was reduced by approximately 40%. In the hypothalamus the effect of dexamethasone was not significant. When slices of the same brain areas from intact rats were incubated for 1 h in the presence of 40 microM dexamethasone, only the release of PGE2 from the cortex was reduced (by 30%). These results suggest that glucocorticoids can inhibit PG synthesis in brain tissue, and that the cortex is the most sensitive area to the inhibitory effect of the hormone.     

Dexamethasone inhibition of cyclooxygenase expression in bovine term placenta.

Since both prostaglandin (PG) F2 alpha and corticosteroids are elevated in mammals before the onset of parturition, we studied the effect of the synthetic corticosteroid dexamethasone on PGF2 alpha accumulation and cyclooxygenase (prostaglandin synthase, PGS) expression in the bovine fetal placenta. Cultures were prepared from cotyledons at different stages of gestation. The effect of dexamethasone on PGF2 alpha accumulation and PGS expression was determined by radioimmunoassay and [35S]methionine metabolic labeling followed by immunoprecipitation with specific anti-cyclooxygenase antibodies, respectively. Data demonstrate that in fetal placental cells at term, both PGF2 alpha accumulation and cyclooxygenase expression are significantly inhibited after 18 hours of dexamethasone treatment (150 nM). In contrast, neither first nor second trimester cells were sensitive to dexamethasone treatment. Dexamethasone inhibition of PGF2 alpha synthesis in fetal cells at term was abolished in the presence of RNA or protein synthesis inhibitors (actinomycin D or puromycin, 10 micrograms/ml each). Neither progesterone nor 17 beta-estradiol accumulation were affected by dexamethasone treatment at any stage of gestation. Data suggest that corticosteroids play a role in parturition through PGF2 alpha synthesis regulation by fetal placental cells. Since abnormalities during parturition e.g. retained placenta, are common following dexamethasone induction of labor in cows, we postulate that the local inhibition of PGF2 alpha accumulation by cotyledon cells after corticosteroid administration, may be involved in placental retention.     

Functional disturbances in brain following injury

It was shown previously that local cerebral glucose utilization is less than 50% of normal in all cortical areas of rat brain 3 days following a focal freeze-lesion and that this effect of trauma is significantly diminished by dexamethasone (0.25 mg/Kg/day), and by indomethacin (7.5 mg/Kg single dose). To elucidate the mechanism of action of steroids and non-steroidal antiinflammatory drugs in traumatized brain, the effects of dexamethasone and indomethacin on arachidonic acid release, malondialdehyde production and prostaglandin synthesis in the lesion area were investigated. Five seconds after a freezing lesion arachidonic acid was significantly increased in the lesion area of untreated animals. Neither dexamethasone nor indomethacin had any effect on this release. The thiobarbituric acid reaction, as an estimate of malondialdehyde and non-enzymatic free radical lipoperoxide formation from unsaturated free fatty acids showed no change in the control and lesion areas of untreated and both dexamethasone and indomethacin treated groups. There was a marked increase in PGF2 alpha, PGE2, PGD2 in the lesion area of untreated animals. Indomethacin prevented the formation of prostaglandins by more than 90% while dexamethasone had no effect. These results suggest that some components of the arachidonic acid metabolism must be involved in functional disturbances resulting from trauma while steroid action is mediated in injured brain independently from the prostaglandin cascade.     

Oxysterol activation of arachidonic acid release and prostaglandin E2 biosynthesis in NRK 49F cells is partially dependent on protein kinase C activity [corrected]

We previously demonstrated that the oxysterol potentiation of arachidonic acid release and prostaglandin biosynthesis induced by foetal calf serum activation of normal rat kidney (NRK) cells (fibroblastic clone 49F) was not related to a direct effect of oxysterols on cell free Ca²⁺ level. Since both Ca²⁺ variations and protein C are involved in arachidonic acid release in some models, we looked for a possible modulation by protein C in the oxysterol effect on arachidonic acid release. We show that when the phorbol ester 12-O-tetradecanoyl-phorbol-13acetate (TPA), a protein kinase C activator, was added to the culture medium, the oxyterol effect on arachidonic acid release and prostaglandin synthesis clearly increased. Moreover, the effect of TPA was dose-dependent and TPA EC₅₀ (4 × 10⁻⁹ M) was unchanged in the presence of the oxysterol. Preincubation of cells with TPA for 24 h prevented the arachidonic acid release induced by TPA alone, whereas the oxysterol effect was decreased but not abolished. In the absence of serum, TPA and ionomycin added together induced the same noticeable (arachidonic acid) release and PGE₂ synthesis as serum alone. Nevertheless, the potentiating effect of cholest-5-ene-3β,25-diol was much higher when serum itself was used to activate NRK cells than it was in the present serum-mimicking experimental conditions. Thus, the presence of growth factors is probably required to obtain a full oxysterol effect. We conclude that the oxysterol effect was synergistic with, but not fully dependent on, protein kinase C and Ca²⁺ ion fluxes, therefore oxysterols could affed earlier events triggered by serum growth factor binding to their cell membrane receptors.     

The possible involvement of prostaglandin F2 alpha in the stimulation of muscle protein synthesis by insulin

The addition of insulin (8 ng/ml) in vitro to muscles from fasted rabbits increased protein synthesis (+80%) to a value similar to that found in muscles from fed donors. The addition of either indomethacin or meclofenamate completely blocked this effect of insulin. Muscles from fasted rabbits released less prostaglandin (PG)F2 alpha into the medium and the presence of insulin increased and indomethacin and meclofenamate reduced PGF2 alpha release. Other conditions (work load and leucocyte pyrogen) which increase protein synthesis in muscle also stimulate PGF2 alpha release. As both arachidonic acid and PGF2 alpha in themselves increase protein synthesis we suggest that accelerated phospholipolysis and PG synthesis have a general role in the control of muscle protein turnover.     

A23187 and protein kinase C activators stimulate phosphatidylinositol metabolism and prostaglandin synthesis in a human lung cancer cell line

Activation of cell phospholipase, release of arachidonic acid and stimulation of prostaglandin synthesis were studied in a newly described human tumor cell line (Lu-65). In the Lu-65 tumor cell line, the calcium ionophore A23187 (2 microM) caused a 100% increase in the release of 3H-arachidonic acid and a 7-fold increase in the synthesis of prostaglandin E2. 1-oleoyl, -2-acetyl-glycerol (100 microM) increased arachidonate release and prostaglandin E2 synthesis by 100%. A23187 and the protein kinase C activators, 1,2-dioctanoyl-glycerol and 1-oleoyl, -2-acetyl-glycerol, decreased the specific radioactivity of 3H-arachidonate in phosphatidylinositol by 37% and 57%, respectively. The effects of A23187 were blocked in Ca2+-free media or in the presence of the phospholipase A2 inhibitor, p-bromophenacyl bromide, while those of 1-oleoyl, -2-acetyl-glycerol were not. The data provide evidence in a human tumor cell line for calcium/phospholipase A2-dependent and independent pathways for arachidonic acid release, both of which preferentially hydrolyze phosphatidylinositol.     

Glucocorticoid effect on arachidonic acid metabolism in vivo

Glucocorticoids have been shown in in vitro systems to inhibit the release of arachidonic acid metabolites, namely prostaglandins (PGs) and leukotrienes, apparently, via the induction of a phospholipase A2 inhibitory protein, called lipocortin. On the basis of these in vitro results, it has been suggested that inhibition of eicosanoid production is, at least partially, responsible for the well-known anti-inflammatory effect of glucocorticoids. There is, however, no firm evidence proving that glucocorticoids also inhibit prostaglandin or leukotriene synthesis in vivo. In a series of studies, we have investigated the effects of anti-inflammatory steroids on the production of six different cyclo-oxygenase products in vivo. Urinary prostaglandin (PG) E2(1), PGF2 alpha, thromboxane B2 (TxB2), 6-keto-PGF1 alpha, and the major urinary metabolites of the E and F PGs, PGE-M and PGF-M, respectively, were determined by radioimmunoassay and by GC-MS. Administration of pharmacological doses of dexamethasone to rabbits failed to inhibit urinary excretion rates of PGE2, TxB2, 6-keto-PGF1 alpha and that of PGE-M and PGF-M. In contrast, urinary PGF2 alpha was slightly reduced by dexamethasone. In further experiments the effect of dexamethasone was studied in humans. Urinary excretion rates of PGE2, PGE-M, PGF-M, 2,3-dinor TxB2 and 2,3-dinor 6-keto-PGF1 alpha were not suppressed by dexamethasone. Collagen-induced platelet TxB2 formation and platelet aggregation was also unaltered. To test one possible explanation for the apparent discrepancy between in vitro and in vivo effects of glucocorticoids on arachidonic acid metabolites we investigated the effects of dexamethasone in vivo on basal and on antidiuretic hormone-stimulated renal PG synthesis. Dexamethasone treatment failed to inhibit both basal and antidiuretic hormone-stimulated PGE2 and PGF2 alpha production. We conclude that glucocorticoids in vivo do not decrease the basal rate of total body, kidney and platelet prostanoid synthesis, and that dexamethasone does not inhibit renal PG production when it is elevated by antidiuretic hormone, a physiological stimulus. Thus, a differential effect of glucocorticoids on basal vs stimulated PG synthesis cannot account for the discrepancy between in vivo and in vitro effects.     

Dexamethasone modulates interleukin-12 production by inducing monocyte chemoattractant protein-1 in human dendritic cells

Glucocorticoids have long been used as first-line immunosuppressants, although their precise mechanism of action has not been fully elucidated yet. This study evaluated the gene and protein expression of monocyte chemoattractant protein-1 (MCP-1), and its relationship with interleukin-12 and interleukin-10 synthesis, in human monocyte-derived dendritic cells exposed to dexamethasone. Dendritic cells were differentiated in the presence or in the absence of dexamethasone and then activated by IFN-gamma+soluble CD40 ligand; the gene and protein expression of target cytokines was measured by real-time PCR and ELISA, respectively. Our results showed that dexamethasone-primed mature dendritic cells expressed low levels of interleukin-12, and, at the opposite, high levels of interleukin-10 and MCP-1. Transfection experiments confirmed the ability of dexamethasone to activate MCP-1 gene promoter. Dexamethasone increased also MCP-2, but not MCP-3 synthesis, and the gene expression of CC chemokine receptor-2 by mature dendritic cells. The addition of anti-MCP-1 blocking antibody depressed MCP-1 release, and increased interleukin-12 production in dexamethasone-treated dendritic cells, thus demonstrating that interleukin-12 downregulation is largely dependent on MCP-1 overexpression. Our findings suggest that the induction of MCP expression in human dendritic cells by dexamethasone, and the amplification of cell response via the upregulation of the chemokine cognate receptor, may be critical to inhibit type 1 T-helper-biased immune response and, possibly, to favor type 2 T-helper-skewed response.     

Dissociation of tumour-promoter-induced effects on prostaglandin release, polyamine synthesis and cell proliferation of 3T3 cells.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate induces tumour promotion, inflammation, cell proliferation and prostaglandin release. Recent reports suggest that the prostaglandins released by 12-O-tetradecanoylphorbol 13-acetate (TPA) initiate a cascade of events leading to polyamine synthesis and cell proliferation. In experiments designed to test this contention, it was found that addition of TPA (1 microM to 1 nM) to confluent mouse 3T3 fibroblasts successively caused the release of prostaglandins E2 and I2, induction of the enzyme ornithine decarboxylase (EC 4.1.1.17), stimulation of [3H]thymidine incorporation into DNA, and cell proliferation. Pretreatment of the cells with the anti-inflammatory steroid dexamethasone (1 microM) or the non-steroidal anti-inflammatory drug indomethacin (1 microM) inhibited TPA-induced prostaglandin release. However, dexamethasone enhanced the other effects of TPA, whereas indomethacin was ineffective. Addition of prostaglandin E2 to the cultures did not induce ornithine decarboxylase activity and cell proliferation. Pretreatment of the cells with 1,3-diaminopropane (1 mM) or alpha-methylornithine (5 mM), inhibitors of polyamine synthesis, decreased TPA-induced ornithine decarboxylase activity without affecting DNA synthesis. TPA stimulated [3H]thymidine incorporation into DNA, even when the ornithine decarboxylase activity was completely blocked. These data suggest that the proliferative effect of TPA on 3T3 cells is independent of prostaglandin release and polyamine synthesis.     

Induction of refractoriness of cyclic AMP responses to prostaglandin E1 and epinephrine by prior exposure of guinea pig macrophages to lipopolysaccharide

Prior exposure of guinea pig macrophages to LPS (lipopolysaccharide) resulted in reduced cAMP-generating responses to prostaglandin E1 and epinephrine. LPS-induced refractoriness was diminished when LPS treatment was carried out in the presence of an inhibitor of prostaglandin synthesis, hydrocortisone, or indomethacin, or an inhibitor of protein synthesis, cycloheximide. The release of arachidonic acid and its metabolites, especially prostaglandin E2 and thromboxane B2, increased during incubation of macrophages with LPS. These increases were efficiently antagonized by hydrocortisone, indomethacin, or cycloheximide. Preincubation of macrophages with prostaglandin E1 greatly reduced the subsequent responses of cAMP generation to prostaglandin E1 and unexpectedly also to epinephrine. Thus, increased production of prostaglandins during the LPS treatment is likely to be responsible for decreased cAMP responses to subsequent addition of prostaglandin E1 and epinephrine.     

Effects of protein synthesis inhibition on PGE2 production in rabbit colonic explants cultured in vitro

Colonic mucosal biopsies cultured for 6 h in the presence of cycloheximide (CH) showed a dose-dependent inhibition of protein synthesis but a biphasic PGE2 production pattern with an increase in both basal and A23187 stimulated PGE2 release at 0.2 microM. At 10 microM CH both protein synthesis as well as basal and PMA induced PGE2 production was inhibited by 90% whereas A23187 stimulated release showed a 50% decrease. At a dose of 100 microM, CH totally blocked also A23187 stimulated PGE2 release without much further decrease in protein synthesis. The effects of 10 microM CH were time-dependently reversible. In biopsies loaded with 3H-arachidonic acid (AA), 10 microM CH had no apparent effect on phospholipase A2 activity, nor could exogenous AA overcome the CH inhibition of basal PGE2 release. No inhibition of prostaglandin synthetase (PS) activity was found in homogenates of biopsies treated with 10 microM CH for 6 h. No direct effect of CH (up to 1 mM) was seen in control homogenates. It is concluded that at least one step in the PGE2 production is protein synthesis dependent. The effect is however not due to a limitation in the enzymes of the major PS system but more likely to one of its co-factors. This factor only plays a role in the intact cell and its importance seems to be reduced during A23187 conditions possibly due to altered cell status and/or other sources of PS. Commonly used high doses (100 microM) of CH give unspecific effects unrelated to inhibition of protein synthesis.     

Prostaglandin E2 and muscle protein turnover inPseudomonas aeruginosa sepsis

Rats were injected intraperitoneally withPseudomonas aeruginosa (septic group) or sterile 0.9% NaCl (controls). Soleus muscles were excised 7 h later, and muscle prostaglandin E₂ release and tyrosine release were measured in vitro. Muscles of septic rats exhibited 226–326% higher release of prostaglandin E₂ and 54–84% higher net proteolysis than muscles of controls. Inclusion of aspirin or indomethacin in the incubation medium almost completely inhibited prostaglandin E₂ production, but had no effect on net proteolysis in muscles from either group. Inclusion of cycloheximide, a protein synthesis inhibitor, increased tyrosine release of control muscles by 42%, whereas no statistically significant increase was observed in muscles from infected rats. However, total proteolytic rate, indexed by tyrosine release in the presence of cycloheximide, was 22% higher in muscles of septic rats compared to that of control animals. Concomitantly, inclusion of cycloheximide inhibited prostaglandin E₂ release by muscles of infected rats by 91% and that of controls by 65%. It is concluded that (a) muscles of septic animals exhibit a pronounced stimulation of prostaglandin E₂ release and net proteolysis, combined with a small increase in total proteolytic rate, (b) the stimulation of net proteolysis is mainly due to inhibition of protein synthesis, (c) the increases in net and total proteolysis appear to be independent of prostaglandin E₂ production, (d) cycloheximide has a previously unrecognized inhibitory effect on muscle prostaglandin E₂ production.     

Comparison of inhibition of bone resorption and escape with calcitonin and dibutyryl 3',5' cyclic adenosine monophosphate

Both parathyroid hormone (PTH) and calcitonin (CT) can increase the concentration of cyclic 3',5' adenosine monophosphate (cAMP) in fetal rat bone in organ culture. Moreover, dibutyryl cAMP (dbcAMP) can both stimulate and inhibit 45Ca release from such bones depending on dose and experimental conditions. In this study we compared dbcAMP and CT for their effects on bones pretreated with PTH. Both compounds produced transient inhibition of bone resorption followed by escape. Escape from dbcAMP was independent of prostaglandin synthesis, since it occurred both in the presence and absence of indomethacin, a prostaglandin cyclo-oxygenase inhibitor.     

Prostaglandin formation in feline cerebral microvessels: effect of endotoxin and interleukin-1

The pathogenesis of fever involves the appearance of interleukin-1 in the circulation in response to appropriate noxae (e.g., endotoxin) and subsequent generation of prostaglandin E2 in the CNS. The present study was undertaken to determine whether the cerebral microvasculature may function as a source of the fever-producing prostaglandin E2. Microvessels, consisting predominantly of capillaries, were isolated from the cat forebrain by selective sieving and glass bead elutriation. Preparations contained enzymes for the synthesis of 6-keto-prostaglandin F1 alpha (hence prostaglandin I2), prostaglandin E2, and possibly prostaglandin F2 alpha. No prostaglandin D2 was detected, nor was evidence obtained for the formation of 6-keto-prostaglandin E1. Intact microvessels released prostaglandin E2 and 6-keto-prostaglandin F1 alpha under basal conditions, the latter compound exceeding the former by about sevenfold. Endotoxin stimulated prostaglandin E2 release without significantly altering 6-keto-prostaglandin F1 alpha release. In contrast, monocyte-derived interleukin-1 reduced the release of both compounds, while recombinant interleukin-1 was ineffective. Endotoxin stimulation is likely directed on the cleavage of substrate arachidonic acid from precursor lipids, while inhibition from monocyte-derived interleukin-1 is ascribed to the presence of an interfering substance. This substance, like endotoxin, is thought to act prior to the cyclooxygenase cascade and its identity remains to be ascertained. We conclude that the cerebral microvasculature does not lend itself to an active role in the genesis of fever by being the site at which blood-borne interleukin-1 promotes prostaglandin E2 synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucocorticoid stimulation of amnion cell prostaglandin synthesis: suppression by protein kinase C inhibitors and independence of phorbol ester-sensitive protein kinase C.

Glucocorticoids stimulate the prostaglandin E2 production of confluent amnion cell cultures, but have no stimulatory effect on the PGE2 output of freshly isolated human amnion cells. Since protein phosphorylation may modify the responsiveness of target cells to steroids, and activators of protein kinase C (PKC), as well as corticosteroids, promote amnion cell PGE2 output by stimulating the synthesis of prostaglandin endoperoxide H synthase (PGHS), we investigated the possibility that PKC is involved in the glucocorticoid-induction of PGE2 synthesis in cultured amnion cells. The dexamethasone-induced PGE2 output of arachidonate-stimulated cells was blocked by the protein kinase inhibitors staurosporine, K-252a, H7, HA1004, and sphinganine, in a manner consistent with their effect on PKC. However, dexamethasone increased the PGE2 production of cultures treated with maximally effective concentrations of the PKC-activator compound TPA. Moreover, dexamethasone stimulated PGE2 synthesis in cultures which were desensitized to TPA-stimulation by prolonged phorbol ester treatment. Concentration-dependence studies showed that staurosporine completely (greater than 95%) blocked glucocorticoid-provoked PGE2 synthesis at concentrations which did not inhibit TPA-stimulated prostaglandin output, and that K-252a inhibited the effect of TPA by more than 95% at concentrations which decreased the effect of dexamethasone only moderately (approximately 40%). Dibutyryl cyclic AMP had no influence on the basal- or dexamethasone-stimulated PGE2 production, and on the staurosporine inhibition of the steroid effect. These results show that glucocorticoids and phorbol esters control amnion PGE2 production by separate regulatory mechanisms. It is suggested that the response of human amnion cells to glucocorticoids is modulated by protein kinase(s) other than phorbol ester-sensitive PKC and cyclic AMP-dependent protein kinase.