Preliminary observations on in vitro biosynthesis of prostaglandins E in tumor cells and virus transformed cells

Biosynthesis of prostaglandins of E series in BK virus-transformed rabbit-kidney cells (RKBK) and mouse hepatoma cells cultured in vitro with and without exogenous arachidonic acid was determined by radioimmunoassay. Elevated concentrations of prostaglandin E2 were observed in both cell types; moreover, tumor and transformed cells were capable to synthesize higher levels of prostaglandin E2 than normal cells. Mouse hepatoma cells produced more prostaglandins compared to RKBK cells; in addition they seemed less sensitive to inhibition by indomethacin. These data suggest that arachidonic acid metabolism may be another one of the intrinsic biochemical properties that differentiate tumor and virus transformed cells.     

Inhibition of experimental metastasis with indomethacin: role of macrophages and natural killer cells

The metastatic capacity of murine mammary tumor line 410.4 is greatly increased by treatment of the host with asialo-GM1 antiserum (5-fold), 2-chloroadenosine (4-fold) or k-carrageenan (2.5-fold). This suggests that both NK cells and macrophages contribute to control of metastatic dissemination. The metastatic potential of these cells is associated with the synthesis of high levels of prostaglandin E2 (PGE2) (1). When line 410.4 cells are cultured in vitro in the presence of the prostaglandin synthesis inhibitor indomethacin (INDO) 1 microM) their subsequent lung colonization ability (experimental metastasis) is reduced by 50-90% as compared to solvent-treated cells. The inhibitory effect of INDO is highly dependent on the presence of asGM1 positive cells, and is compromised to a lesser extent by treatments directed towards macrophages. The INDO-mediated inhibition is neither due to differential arrest of tumor cells in the lung nor does it appear to be due to shifts in the replication cycle.     

Effects of prostaglandin E2, indomethacin and adenosine deaminase on basal and insulin-stimulated glucose metabolism in human adipocytes

The effects of prostaglandin E2 were studied on glucose metabolism (3-O-methylglucose transport, CO2 production and lipogenesis) in human adipocytes. Initially, the effects of endogenously produced adenosine and prostaglandins were indirectly demonstrated by using adenosine deaminase and indomethacin in the incubations. From these studies it was found that adenosine deaminase (5 micrograms/ml) had a pronounced effect on adipocyte glucose metabolism in vitro. In the basal (nonhormonal-stimulated) state, glucose transport, CO2 production and lipogenesis were inhibited by about 30% (P less than 0.05). Furthermore, adenosine deaminase significantly inhibited the isoproterenol- and insulin-stimulated CO2 production and lipogenesis (P less than 0.01). Indomethacin (50 microM) had a consistently inhibitory effect on the insulin-stimulated CO2 production (P less than 0.05), whereas indomethacin had no significant effects on basal or isoproterenol-stimulated glucose metabolism. In contrast to the relatively minor effect of endogenous prostaglandins, the addition of exogenous prostaglandin E2 significantly stimulated the glucose transport, glucose oxidation and lipogenesis in human adipocytes, especially in the presence of adenosine deaminase. Half-maximal stimulation was obtained at prostaglandin E2 concentrations of 2.2, 0.8 and 0.8 nM, respectively. The effect of prostaglandin E2 was specific, since the structurally related prostaglandin, prostaglandin F2 alpha, had practically no effect on glucose metabolism. The maximal effect of prostaglandin E2 (1 microM) on glucose metabolism was 30-35% of the maximal insulin (1 nM) effect. When insulin and prostaglandin E2 were added together, the effect of prostaglandin E2 on glucose metabolism was additive at all insulin concentrations tested.     

Correlation of the biosynthesis of prostaglandin and cyclic AMP in monolayer cultures of rabbit articular chondrocytes

We have utilized ionophores to test whether stimulation of chondrocyte prostaglandin biosynthesis is accompanied by an increase in cyclic nucleotide levels in these cells. Radioimmunoassay of prostaglandin E2, 6-oxo-prostaglandin F1 alpha (the stable metabolite of prostaglandin I2) and prostaglandin F2 alpha showed that synthesis of each was stimulated by the divalent-cation ionophore, A23187 after short-term incubation (1-7 min) in serum-free medium. No stimulation of thromboxane B2 was detected. Two monovalent ionophores, lasalocid and monensin failed to stimulate prostaglandin biosynthesis after short-term incubation. Ionophore A23187-stimulated prostaglandin biosynthesis was variably and partially inhibited by sodium meclofenamate, indomethacin and aspirin, but not by sodium salicylate. Ionophore A23187-stimulated prostaglandin biosynthesis was accompanied by a 7.5-fold increase in cyclic AMP levels after 15 min. Sodium meclofenamate, indomethacin and aspirin which inhibited prostaglandin E2 biosynthesis also reduced cyclic AMP levels. Exogenous prostaglandin E2 (1 microgram/ml) stimulated cyclic AMP biosynthesis, which was not inhibited by aspirin. These results indicated that prostaglandins can be considered as one of the local effectors controlling cyclic AMP production in articular cartilage.     

The influence of phenelzine and tranylcypromine on the release of prostaglandins from the rat mesenteric vascular bed

We investigated the effects of phenelzine and tranylcypromine on the release of prostacyclin, thromboxane A2, prostaglandin E2, and prostaglandin E1 from the isolated perfused rat mesenteric vascular bed. Perfusion of the preparation with phenelzine in concentrations of 15, 45, and 135 microM for 150 min led to attenuated release of all four prostaglandins measured. Inhibition generally occurred with the lowest dose used and was most prominent with the highest concentration. Tranylcypromine also decreased prostaglandin formation. However, low doses were not effective in the suppression of prostacyclin release. Both drugs had an inhibitory effect on production of prostaglandin E1, which is a metabolite of dihomo-gamma-linolenic acid, the precursor of arachidonic acid, but this was only shown to be significant with phenelzine. In this work we demonstrate that phenelzine and tranylcypromine have an inhibitory effect on the production of 2-series prostaglandins derived from arachidonic acid, and possibly a similar effect on prostaglandins of the 1-series derived from dihomo-gamma-linolenic acid.     

Endogenous prostaglandins are physiological regulators of endocrine cells in the gastroduodenal mucosa of the rat.

BACKGROUND/AIM: To investigate whether endogenous prostaglandins participate in the regulation of the gastrointestinal endocrine cell system. METHODS: Sprague-Dawley rats were treated with 1 mg/kg indomethacin subcutaneously or indomethacin subcutaneously and 500 microg/kg oral prostaglandin E2 or solvents for 2 months. Endocrine cells were visualized by using immunohistochemistry and by the Sevier-Munger silver stain on specimens from the gastroduodenal mucosa, and their total volume was estimated, using standard stereological methods. Plasma and gastrointestinal tissue concentrations of regulatory peptides were analyzed by radioimmunoassay. RESULTS: Fundic mucosa. The total volume of cells stained with the Sevier-Munger silver stain (enterochromaffin-like) was increased by indomethacin, but reduced by the administration of prostaglandin E2 (P < 0.05 vs. indomethacin). Indomethacin increased the total volume of somatostatin-immunoreactive. Similarly, rats given indomethacin and prostaglandin E2 had higher values than controls. Indomethacin increased the tissue concentration of somatostatin in the gastric fundus whereas prostaglandin E2 prevented such changes (P < 0.05 vs. indomethacin). Antral mucosa. The total volume of serotonin-immunoreactive cells was reduced by indomethacin, but increased by prostaglandin E2 (P < 0.05 vs. controls and indomethacin, respectively). Duodenal mucosa. The total volume of somatostatin-immunoreactive cells was reduced in the rats given indomethacin and prostaglandin E2 (P < 0.05 vs. controls and indomethacin). Indomethacin reduced and simultaneous administration of prostaglandin E2 increased the total volume of CCK-immunoreactive cells (P < 0.05 vs. controls and indomethacin). Indomethacin reduced the total volume of serotonin-immunoreactive cells whereas the simultaneous administration of PGE2 comparatively increased their total volumes (P < 0.05 vs. indomethacin), although they were still lower than the control values. The total volume of GIP-immunoreactive cells was slightly increased in the rats given both indomethacin and indomethacin + prostaglandin E2. The tissue concentration of somatostatin in the duodenum was reduced in rats given indometacin and prostaglandin E2 (P < 0.05 vs. controls and indomethacin). CONCLUSION: Endogenous prostaglandins, particularly prostaglandin E2, regulate CCK-, enterochromaffin-like-, somatostatin-, GIP- and enterochromaffin cells in the gastroduodenal mucosa of the rat.     

Effect of prostaglandin inhibition by indomethacin on plasma active and inactive renin concentration in men.

The effect of inhibition of prostaglandin synthesis by indomethacin on active renin and on acid-activable inactive renin was studied in nine healthy, sodium-replete men, both at rest and exercise. These volunteers were investigated after pretreatment with placebo or indomethacin, 150 mg daily for 3 days. Indomethacin induced a decrease in active (p = 0.004), total (p less than 0.001), and inactive (p = 0.02) renin at rest recumbent on average by 42, 19, and 8%, respectively, and at rest sitting on average by 45, 15, and 3%, respectively. Inhibition of prostaglandins with indomethacin reduced (p less than 0.001) active and total renin at each level of work load but not (p = 0.32) inactive renin. However, the exercise-induced stimulation (p less than 0.05) of active and total renin still occur during indomethacin. Indomethacin reduced (p less than 0.001) at rest sitting and at maximal exercise the plasma concentrations of immunoreactive prostaglandins E2 by 50 and 54%, respectively, prostaglandin F2 alpha by 36 and 39%, respectively, and 13,14-dihydro-15-keto-prostaglandin F alpha by 38 and 60%, respectively. The urinary excretion of immunoreactive prostaglandin E2 and F2 alpha was also reduced.     

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.     

Regulation of 3T3-L1 fibroblast differentiation by prostacyclin (prostaglandin I2)

Prostaglandin biosynthesis and prostaglandin-stimulated cyclic AMP accumulation were studied in 3T3-L1 fibroblasts as they differentiated into adipocytes. Incubation of 3T3-L1 membranes with [1-14C]prostaglandin H2, and subsequent radio-TLC analysis, showed that prostacyclin (prostaglandin I2) is the principal enzymatically synthesized prostaglandin in this cell line. Confirmation of the radiochemical data was obtained by demonstrating the presence of 6-keto-prostaglandin F1 alpha, the stable hydrolysis product of prostaglandin I2, by gas chromatography-mass spectrometry. In support of previous work, indomethacin, the prostaglandin endoperoxide synthetase (EC 1.14.99.1) inhibitor, accelerated 3T3-L1 differentiation. More importantly, the incubation of 3T3-L1 cells with insulin and the prostaglandin I2 synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I) also enhanced the rate of cellular differentiation, even though this compound does not inhibit the synthesis of other prostaglandins. The repeated addition of exogenous prostaglandin I2 to 3T3-L1 cells inhibited insulin- and indomethacin-mediated differentiation. When 3T3-L1 cells were exposed to various prostaglandins and the cyclic AMP levels were measured, prostaglandin I2 proved to be the most potent stimulator of cyclic AMP accumulation, followed by prostaglandin E1 greater than prostaglandin H2 much greater than prostaglandin E2, while prostaglandin D2 was inactive. As 3T3-L1 cells differentiate, the ability of prostaglandin I2 or prostaglandin H2 to stimulate cyclic AMP accumulation progressively diminishes. It is suggested that 3T3-L1 differentiation may be controlled by the rate of prostaglandin I2 synthesis and/or sensitivity of the adenylate cyclase to prostaglandin I2.     

Prostaglandin-mediated suppression of macrophage phagocytosis of Listeria monocytogenes

Suppression of macrophage phagocytosis of Listeria monocytogenes has been shown to be due to a low-molecular-weight component of spleen cell culture supernatant. The possibility that the factor could be a prostaglandin was investigated. When murine peritoneal macrophages were treated with prostaglandin E2 (PGE2), L. monocytogenes was phagocytized at a rate comparable to that phagocytized when treated with a low-molecular-weight fraction of concanavalin A-generated spleen cell culture supernatant. Suppressive activity of the spleen cell culture supernatant was abrogated when supernatant was prepared in the presence of indomethacin, a prostaglandin synthetase inhibitor. Prostaglandins were identified in supernatants with thin-layer and high-pressure liquid chromatography. These results suggest a role for prostaglandins, particularly PGE2, as a modulator of macrophage phagocytosis of L. monocytogenes.     

Alterations in lung prostaglandin synthesis and release in murine respiratory mycoplasmosis

Pathogenetic mechanisms in murine respiratory mycoplasmosis are poorly understood; however, non-specific immune responses appear to be important in controlling the growth of Mycoplasma pulmonis in vitro. To date, no study has examined the role of pulmonary prostaglandin production during the development of M. pulmonis infection. The present study was designed to determine if alterations in pulmonary prostaglandin synthesis and release occur in M. pulmonis infection and the possible role for prostaglandins in the modulation/pathogenesis of murine respiratory mycoplasmosis. Ten to 20 days after intranasal inoculation of pathogen-fee F344 rats with M. pulmonis, lung lavage concentrations of prostaglandin E (PGE) and thromboxane A2 (TxA2) were significantly elevated. To confirm a role for prostaglandins in the pathogenesis of murine mycoplasmosis we blocked the cyclo-oxygenase pathway with indomethacin. Indomethacin-treated rats had significantly lower lavage levels of PGE and TxA2 and significantly increased numbers of M. pulmonis in the lung. These data indicate that prostaglandins may be involved in the pathogenesis of murine respiratory mycoplasmosis, possibly through alteration of mycoplasmacidal and/or mycoplasmastatic mechanisms.     

Possible role of prostaglandins as negative regulators in growth stimulation by tumor necrosis factor and epidermal growth factor in human fibroblasts

Recombinant tumor necrosis factor (TNF), epidermal growth factor (EGF), and transforming growth factor beta (TGF-beta) stimulated growth of confluent human diploid fibroblasts (FS-4 cells) in the presence of fetal calf serum. TGF-beta synergistically enhanced both the TNF- and EGF-stimulated cell growth, whereas synergism between the mitogenic action of EGF and that of TNF was not observed. When indomethacin or acetylsalicylic acid, an inhibitor of prostaglandin production, was added to FS-4 cells, cell growth stimulated by EGF or TNF was increased, suggesting that prostaglandins induced by these mitogens antagonize their growth stimulatory actions. In contrast, neither indomethacin nor acetylsalicylic acid had a significant effect on the TGF-beta-induced growth of FS-4 cells. Mitogenic responses of indomethacin-treated cells to EGF, TNF, and TGF-beta were similarly suppressed by the addition of exogenous prostaglandin D2 (PGD2). Other prostaglandins such as PGE2 and PGF2 produced less inhibition of the cell growth.     

Effects of indomethacin on renal inner medullary plasma flow

The effects of the prostaglandin system on renal hemodynamics were studied by treating rats with a single intraperitoneal dose of indomethacin, an inhibitor of prostaglandin synthesis. Medullary plasma flow was significantly reduced 30–45 minutes after indomethacin, but was elevated 3–6 hours after indomethacin. These changes in medullary plasma flow correlated well with circulating levels of prostaglandins A and E. Total renal blood flow decreased following indomethacin treatment, but returned to normal levels within an hour. These results indicate that the inhibition of prostaglandin synthesis following a single intraperitoneal dose of indomethacin is short-lived and is followed by a significant elevation in prostaglandin synthesis. It is likely that prostaglandin levels play an important role in the control of renal medullary plasma flow.     

Alterations in lung prostaglandin synthesis and release in murine respiratory mycoplasmosis

Pathogenetic mechanisms in murine respiratory mycoplasmosis are poorly understood; however, non-specific immune responses appear to be important in controlling the growth of . To date, no study has examined the role of pulmonary prostaglandin production during the development of infection. The present study was designed to determine if alterations in pulmonary prostaglandin synthesis and release occur in infection and the possible role for prostaglandins in the modulation/pathogenesis of murine respiratory mycoplasmosis. Ten to 20 days after intranasal inoculation of pathogen-fee F344 rats with , lung lavage concentrations of prostaglandin E (PGE) and Thromboxane A₂ (TxA₂) were significantly elevated. To confirm a role for prostaglandins in the pathogenesis of murine mycoplasmosis we blocked the cyclo-oxygenase pathway with indomethacin. Indomethacin-treated rats had significantly lower lavage levels of PGE and TxA₂ and significantly increased numbers of in the lung. These data indicate that prostaglandins may be involved in the pathogenesis of murine respiratory mycoplasmosis, possibly through alteration of mycoplasmacidal and/or mycoplasmastatic mechanisms.     

The unfolding and attempted refolding of mitochondrial aspartate aminotransferase from pig heart.

The role of prostaglandins in the regulation of muscle protein breakdown is controversial. We examined the influence of arachidonic acid (5 microM), prostaglandin E2 (PGE2) (2.8 microM) and the prostaglandin-synthesis inhibitor indomethacin (3 microM) on total and myofibrillar protein breakdown in rat extensor digitorum longus and soleus muscles incubated under different conditions in vitro. In other experiments, the effects of indomethacin, administered in vivo to septic rats (3 mg/kg, injected subcutaneously twice after induction of sepsis by caecal ligation and puncture) on plasma levels and muscle release of PGE2 and on total and myofibrillar protein breakdown rates were determined. Total and myofibrillar proteolysis was assessed by measuring production by incubated muscles of tyrosine and 3-methylhistidine respectively. Arachidonic acid or PGE2 added during incubation of muscles from normal rats did not affect total or myofibrillar protein degradation under a variety of different conditions in vitro. Indomethacin inhibited muscle PGE2 production by incubated muscles from septic rats, but did not lower proteolytic rates. Administration in vivo of indomethacin did not affect total or myofibrillar muscle protein breakdown, despite effective plasma levels of indomethacin with decreased plasma PGE2 levels and inhibition of muscle PGE2 release. The present results suggest that protein breakdown in skeletal muscle of normal or septic rats is not regulated by PGE2 or other prostaglandins.     

Arachidonic acid metabolism by isolated epidermal basal and differentiated keratinocytes from the hairless mouse

The metabolism of arachidonic acid was studied using basal and differentiated keratinocytes as well as sebaceous cells isolated from hairless mice. These disassociated cells metabolized arachidonic acid predominantly to the prostaglandin H synthase products prostaglandins E2 and D2. 12-Hydroxyheptadecatrienoic acid (HHT), prostaglandin F2 alpha, thromboxane B2 and 6-ketoprostaglandin F1 alpha were also detected. Smaller amounts of the lipoxygenase products 5-, 12- and 15-hydroxyeicosatetraenoic acids (HETEs) were also detected. The major lipoxygenase product observed was 12-HETE. No leukotrienes or dihydroxy fatty acids were observed. The identity of the metabolites was established using several high-pressure liquid chromatography solvent systems. The biosynthesis of prostaglandins E2 and D2 was very rapid and was inhibited by the addition of indomethacin to the cells. The mixed population of keratinocytes and sebaceous cells were separated into enriched fractions by metrizamide gradients and elutriation techniques. The small, undifferentiated cells had high prostaglandin H synthase and 12-lipoxygenase activity. The basal cell-enriched fractions had the highest activity. With increasing differentiation of the cells, decreased biosynthetic activity was observed. These results indicate that undifferentiated keratinocytes, that is, the basal cells, may be an important source of prostaglandins and 12-HETE but are not a source of leukotrienes for the hairless mouse. It also suggests a role for keratinocyte-derived eicosanoids in the normal physiology of epidermal differentiation.     

Effect of epidermal growth factor on synthesis of prostaglandins and cyclic AMP by embryonic palate mesenchymal cells

The influence of epidermal growth factor (EGF) on the ability of murine embryonic palate mesenchymal (MEPM) cells to be stimulated to synthesize cAMP and prostaglandins was investigated. Preincubation of MEPM cells with EGF enhanced, in a dose-dependent fashion, (1) the responsiveness of MEPM cells to prostaglandin E1-induced elevation of intracellular levels of cAMP, and (2) the responsiveness of cells to calcium ionophore (A23187) and melittin-induced synthesis of prostaglandins E2 and F2 alpha. Hormonal responsiveness of MEPM cells to EGF, prostaglandins and cAMP has been implicated as being involved in controlling various aspects of normal oro-facial development. We show here that EGF can potentiate hormonal responsiveness of these cells and thus allows consideration of EGF as a factor which may modulate hormonally regulated craniofacial growth and differentiation.     

Inhibition of prostaglandin E2 synthesis by a blocker of epithelial chloride channels

Arginine-vasopressin (AVP) elicits a variety of responses in cultured rat mesangial cells, among them stimulation of prostaglandin biosynthesis and activation of Cl- channels. AVP produced an 11-fold increase over basal levels in prostaglandin E2 release from cultured mesangial cells. This response was completely inhibited by 25 microM indomethacin and 82 +/- 5% inhibited by 25 microM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) which is a potent blocker of epithelial Cl- channels. The IC50 for NPPB inhibition of prostaglandin E2 release was 8 microM. Indomethacin and NPPB at 25 microM also inhibited AVP-stimulated cellular accumulation of prostaglandin E2 by 98% and 79 +/- 7% respectively. The inhibitory effect of NPPB was not due to interference with the cellular response to AVP since at 50 microM it did not block AVP-stimulated release of arachidonate metabolites from cells metabolically labeled with [3H]-arachidonic acid. It is suggested that NPPB inhibition of prostaglandin E2 synthesis is at the cyclooxygenase level on the basis of its structural similarity to the fenamic acid type of cyclooxygenase inhibitors.     

Content and formation of prostaglandins and distribution of prostaglandin-related enzyme activities in the rat ocular system

The steady-state levels of prostaglandin D2, E2 and F2 alpha in the rat eye were 0.5, 0.1 and 1.0 ng/g, respectively, which increased differently among the prostaglandins after a 40-min incubation of the homogenate at 37 degrees C (to 23, 12 and 14 ng/g, respectively). When the eye was dissected into anterior uveal, scleral, and retinal complexes, prostaglandin D2 was formed in the highest degree in all the complexes, whereas prostaglandin E2 and F2 alpha formation was specific to given ocular regions. Three prostaglandin synthetase activities with similar Km values (20-40 microM) were found in the 10,000 X g supernatant of these tissues, i.e., GSH-independent and soluble D, GSH-dependent and membrane-bound E, and soluble F synthetase activities. These enzyme activities correlated well with the prostaglandin formation in each tissue. D synthetase activity being highest in all the tissues (11-25 nmol/min per g). Three types of prostaglandin-catabolizing enzyme activities were detected in the 100,000 X g supernatant of the tissues, i.e., type II 15-hydroxy dehydrogenase (Km = 10-30 microM), 9-keto (500 microM) and 11-keto reductase (2.5 mM). The activity of the dehydrogenase was low even in the retina, the tissue with the highest levels (0.51, 0.35 and 0.15 nmol/min per g for prostaglandin E2, F2 alpha and D2, respectively).     

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.