Cyclic AMP response to prostaglandin E1 in mononuclear cells from peripheral blood and synovial fluid of patients with rheumatoid arthritis.

The cyclic AMP response to prostaglandin E1 (PGE1) was studied in peripheral blood (PB) and synovial fluid (SF) mononuclear cells from patients with rheumatoid arthritis (RA). The PGE1 induced accumulation of cyclic AMP was consistently (7 of 8 patients) less in cell suspensions derived from SF than in suspensions of equivalent numbers of mononuclear cells obtained simultaneously from PB. The high PB/SF cyclic AMP ratio was seen most clearly at the lowest concentration (10(-6)M) of PGE1 tested. There was no correlation between the patients' therapy and cyclic AMP response to PGE1. The high PB/SF cyclic AMP ratio was not accounted for by the presence of platelets in PB cell suspensions.     

Prostaglandin E1 effects on cyclic AMP and glycogen metabolism in rat liver.

Prostaglandins E₁ or E₂ (PGE₁, PGE₂)¹ stimulated adenylate cyclase(s) from particulate fractions of whole liver homogenates 5- to 6-fold, but caused only slight (1.5- to 2-fold) stimulation of the enzyme from homogeneous hepatocytes. In contrast, glucagon stimulated enzyme from hepatocytes 12- to 15-fold and enzyme from whole liver 8- to 10-fold. Accordingly, most of the total prostaglandin-sensitive adenylate cyclase in cell suspensions was recovered in fractions containing non-parenchymal cells, and most of the total glucagon-sensitive activity was recovered with hepatocytes. PGE₁ did not change adenosine-3′,5′-monophosphate (cyclic AMP) concentrations, or alter cyclic AMP increases caused by glucagon in hepatocytes. Glucagon consistently increased hepatocyte cyclic AMP concentrations and stimulated glycogenolysis by 35 to 40%. PGE₁ did not affect basal or glucagon-stimulated glycogenolysis in the intact cells.     

Studies on growth hormone secretion VI. Effects of dibutyryl cyclic AMP,prostaglandin E1, and indomethacin on growth and hormone secretion by rat pituitary tumor cells in culture

The growth of rat pituitary tumor cells (GH₁ line) maintained in monolayer culture was inhibited by dibutyryl cyclic AMP in a dose-related fashion. Neither PGE₁ (2.8 × 10^?5M) nor indomethacin (2.8 × 10^?6M) had any significant effect on cell proliferation. Release of GH into the culture medium was stimulated by the cyclic AMP derivative but not by PGE₁ or indomethacin. In short term experiments (15 min.) both in intact monolayers and in trypsin-treated cells incubated in suspension, PGE₁ caused a 2–10 fold increase in cyclic AMP levels. This response, however, appeared to be of short duration reaching a maximum in 10 minutes. It is suggested that, at least in this line of pituitary tumor cells, PGE₁ does not mimic the effect of cyclic AMP, for it probably cannot sustain the elevated intracellular levels of this nucleotide which seem to be necessary for growth inhibition and enhanced GH secretion.     

Relationship Between the Actions of Calcium Ions, Opioids and Prostaglandin E1 on the Level of Cyclic AMP in Neuroblastoma × Glioma Hybrid Cells

Abstract: Neuroblastoma × glioma hybrid cells increase their intracellular concentration of cyclic AMP in response to prostaglandin E₁ (PGE₁). This effect is inhibited by opioids. The response to PGE₁ is positively correlated with the concentration of Ca²⁺ in the incubation medium. The Ca²⁺ antagonists Co²⁺ and La³⁺, the Ca²⁺ chelator EGTA and a blocker of Ca²⁺ influx into cells, Segontin, inhibit the response to PGE₁. At low external concentrations of Ca²⁺ the response to PGE₁ is enhanced by the Ca²⁺ ionophore A23187. The effects of A23187 and Segontin point to a cytosolic site of Ca²⁺ action. Lack of Ca²⁺ reduces the level of cyclic AMP even in the absence of PGE₁ and the presence of an inhibitor of cyclic AMP phosphodiesterase. Ca²⁺ is required even for an increase in the level of cyclic AMP in cells pretreated with cholera toxin. The increases in level of cyclic AMP evoked by PGE, in a neuroblastoma and by PGE₁ or noradrenaline in a glioma cell line do not depend on Ca²⁺. The response of the hybrid cells to the opioid leucine-enkephalin appears not to rely on the presence of Ca²⁺. Even changing the intracellular concentration of Ca²⁺ by the ionophore A23187 does not alter the effect of the opioid. The analogy between opioids and lack of Ca²⁺ in the short-term (minutes) experiments mentioned holds also for long-term (hours) experiments. Cells chronically exposed to opioids or to low concentrations of Ca²⁺ display an enhanced maximal response to PGE₁.     

Loss of the PGE1 requirement for MDCK cell growth associated with a defect in cyclic AMP phosphodiesterase

Prostaglandin E₁(PGE₁), one of the components in the hormone-supplemented, serum-free medium for Madin Darby Canine Kidney (MDCK) cells (Medium K-1), is required for both long-term growth and for dome formation. Variant cells have been isolated from MDCK populations, which lack the PGE₁, requirement for long-term growth in Medium K-1. These variants will be useful in identifying the molecular events initiated by PGE₁ which are necessary for the growth response to be observed. The growth and functional properties of five independently isolated PGE₁ independent clones have been examined. Normal MDCK cells grew at an equivalent rate in Medium K-1 and in serum-supplemented medium; the growth rate was lower in Medium K-1 lacking PGE₁. In contrast, PGE₁ independent clone 1 grew at an equivalent rate in Medium K-1 minus PGE₁, and in serum-supplemented medium. When PGE₁ was added to K-1 minus PGE₁, less growth of PGE₁ independent clone 1 was observed. A similar observation was made with one other PGE₁ independent clone which was studied. A hormone deletion study indicated that PGE₁ independent clone 1 still retained growth responses to the other four supplements in Medium K-1 (insulin, transferrin, T₃, and hydrocortisone). The molecular alterations associated with loss of the PGE₁ requirement for long-term growth were examined. At confluency, all of the PGE₁ independent clones studied had higher intracellular cyclic AMP levels following PGE₁ treatment, as compared with normal MDCK cells. The increased cyclic AMP levels in the variant cells could result from a number of different types of defects, including reduced cyclic adenylic acid (cyclic AMP) efflux, an increased affinity of PGE₂ for the PGE₁ receptor, or a defect in cyclic AMP metabolism. However, in all of the variant clones studied a decreased rate of cyclic AMP degradation by cyclic AMP phosphodiesterase was observed. Thus, the increased cyclic AMP levels in the PGE₁ independent variants may result from alterations which affect cyclic AMP metabolism. The effect of PGE₁ on dome formation by the variant cells was also examined. The frequency of dome formation by PGE₁ independent clone 1 was enhanced in a dosage-dependent manner, like normal MDCK cells. This observation suggests that PGE₁ affects MDCK cell growth and dome formation by different mechanisms.     

Effects of prostaglandins and other drugs on the cyclic AMP content of cultured bone cells

Prostaglandins of the E-series (PGE₁ and PGE₂) may be involved in disease-related, localized loss of bone. E-prostaglandins increase the cyclic AMP content of many cells; and, to determine if their effects on bone are mediated by cyclic AMP, we examined the effects of E-prostaglandins and of other agents on the cyclic AMP content of cultured bone cells. PGE₂ produced a rapid, marked and dose-related increase in the cyclic AMP content of confluent monolayers of bone cells isolated from newborn rat calvaria. At 2.8 × 10⁻⁶ M, PGE₁ and PGE₂ had approximately the same effect, while the effect of PGF_2α was much less pronounced. In the presence of theophylline, PGE₂ had a more marked effect than parathyroid hormone (PTH) and the combination of PGE₂ and PTH had a synergistic effect. The divalent, cationic, ionophore, A23187, produced an increase in cellular cyclic AMP and had an additive effect in combination with PGE₂. Synthetic salmon calcitonin (CT), which inhibits the bone resorptive effect of PGE₂, increased cellular cyclic AMP and had an additive effect in combination with PGE₂. A prostaglandin antagonist, SC-19220, partially inhibited the resorptive effect of PGE₂ and reduced its effect on cellular cyclic AMP. The calcium antagonist, D600, inhibited the bone resorptive effects of PGE₂ but had no effect on increased cellular cyclic AMP produced by PGE₂.The marked effect of PGE₂ on bone cell cyclic AMP suggests that this action is involved in the mechanism of PGE₂-related bone loss. The fact that agents with different effects on PGE₂-induced increases in cellular cyclic AMP can inhibit its resorptive actions, suggests that PGE₂-induced changes in cyclic AMP may be related less to its resorptive actions than to its inhibitory effect on bone formation.     

Effect of Noradrenaline and Prostaglandin E<Subscript>1</Subscript> on Adenosine 3′,5′;-Monophosphate Formation in Isolated Pericardial Fat Cells of Man

NORADRENALINE increases the intracellular concentration of adenosine 3′,5′-monophosphate (cyclic AMP)^1,2 which, in turn, enhances glycogenosis³ and lipolysis^4,5 in adipose tissue by increasing Phosphorylase and lipase activities. Prostaglandin E₁ (PGE₁) antagonizes the induced increases in Phosphorylase activity^6,7 and glycerol release in human adipose tissues^8,9 and isolated adipocytes⁷. The finding that the stimulatory effects of the cyclic AMP analogue N⁶—O² dibutyryl cyclic AMP, which mimics the hormonal effect of noradrenaline in human fat cells, are not blocked by PGE₁⁷ suggests that noradrenaline and PGE₁ alter fat cell metabolism by acting on the adenyl cyclase system¹⁰. Whether noradrenaline and PGE₁ alter concentrations of cyclic AMP in human fat cells, however, has not been reported.     

Prostaglandin biosynthesis and stimulation of cyclic AMP in primary monolayer cultures of epithelial cells from mouse mammary gland

Cyclic AMP levels in primary monolayer cultures of epithelial cells prepared from mid-pregnant mice are stimulated by prostaglandin E₁ and E₂. Prostaglandin F_1α and F_2α have only a slight effect upon cyclic AMP levels. In the absence of phosphodiesterase inhibitors the rise in cyclic AMP produced by PGE₁ is only transient and the levels return to normal within 30 minutes. High concentrations (16 mM) of theophylline are needed to prevent this decline, suggesting that the phosphodiesterase activity of epithelial cells in culture is high. However, theophylline alone produced only a small increase in basal cyclic AMP levels even over a 2-hour period indicating that basal cyclic AMP is turned over more slowly than cyclic AMP produced in response to stimulation with PGE₁.Both PGE and PGF synthesis were monitored using radioimmunoassay procedures previously reported. The observed levels were found to decrease as cell density increased and were sensitive to the addition of agents such as collagen and naproxen.     

Cyclic AMP and platelet prostaglandin synthesis

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3′5′-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of ¹⁴C-arachidonic acid by washed platelets. Prostaglandin E₁ (PGE₁), PGE₁ with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B₂. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE₁ with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of ¹⁴C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.     

The influence of calcium on the cyclic AMP-mediated stimulation of DNA synthesis and cell proliferation by prostaglandin E 1

Prostaglandin type E₁ (PGE₁) rapidly stimulates cyclic AMP formation and the initiation of deoxyribonucleic acid (DNA) synthesis in rat thymic lymphocytes suspended in vitro by reactions which are not affected by wide variations in the extracellular calcium concentration. On the other hand, the operation of the associated reaction(s) responsible for the subsequent progression of the stimulated cells into mitosis is profoundly affected by the extracellular calcium level. If the maximum intracellular cyclic AMP concentration is in the lower range of stimulatory values (e.g., 150 × 10^?8 picomoles per cell as produced by an exposure to 0.5 μg of PGE₁ per milliliter of medium), an extracellular calcium concentration of 0.5 to 1.0 mM is needed to obtain maximum cell proliferation, but not the maximum stimulation of DNA synthesis. Contrariwise, if the cellular cyclic AMP content is raised to a much higher level (260 × 10^?8 picomoles per cell) by exposure to a greater PGE₁ concentration (5.0 μg per millilter), cell proliferation is maximally stimulated in calcium-free medium and increasing the extracellular calcium concntration above 0.2 mM actually prevents the stimulation of cell proliferation (but does not affect the stimulation of DNA synthesis). Thus, the ultimate translation of PGE₁'s early cyclic AMP-mediated reactions into increased cell proliferation is determined by both the intracellular cyclic AMP level and the extracellular calcium concentration.     

Stimulatory effects of prostaglandin E1 on rat anterior pituitary cyclic amp and luteinizing hormone release

The effect of prostaglandin E₁ (PGE₁) on rat anterior pituitary cyclic AMP accumulation and luteinizing hormone (LH) release was studied both in vivo and in vitro. Addition of PGE₁ to incubation medium over a concentration range of 10^-6 to 10^-4 M produced a graded increase in pituitary cyclic AMP. At the lowest concentration (10^-6 M) there was no significant increase in LH release, but proportional increments in LH release were seen with increasing concentrations of PGE₁.Ten minutes after intravenous administration of 5 μg of PGE₁ to adult male rats, pituitary cyclic AMP was substantially increased while serum LH levels were not changed. Administration of a higher dose of PGE₁ (20 μg) produced a greater increase in pituitary cyclic AMP; and, at this dose serum LH was significantly increased. These results suggest that the PGE₁ effect on LH release is mediated by the adenyl cyclase — cyclic AMP system.     

Parallel activation of cyclic AMP phosphodiesterase and cyclic AMP-dependent protein kinase in two human gut adenocarcinoma cells (HT 29 and HRT 18) in culture,by vasoactive intestinal peptide (VIP) and other effectors activating the cyclic AMP system

Vasoactive intestinal peptide (VIP), secretin, catecholamines and prostaglandin E₁ (PGE₁) in the presence of a cyclic nucleotide phosphodiesterase inhibitor stimulate the accumulation of cyclic AMP in two colorectal carcinoma cell lines (HT 29 and HRT 18) with subsequent activation of the cyclic AMP-dependent protein kinases. In HT 29 cells incubated without phosphodiesterase inhibitor, 10^?9 M VIP promotes a rapid and specific activation of the low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ cyclic AMP phosphodiesterase (1.7-fold); at 25°C the effect is maintained for more than 15 min, while at 37°C the activity returns to basal value within 15 min. As shown by dose-response studies, VIP is by far the most effective inducer ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 4 · 10}{}^{\mathrm{?10}}\text{M}$) of the cyclic AMP phosphodiesterase activity; partial activation of the enzyme is obtained by 3 · 10^?7 M secretin, 10^?5 M isoproterenol and 10^?5 M PGE₁; PGE₂ and epinephrine are without effect. In HRT 18 cells VIP is less active ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 2 · 10}{}^{\mathrm{?9}}\text{M}$) whereas 10^?6 M PGE₁, 10^?6 M PGE₂ and 10^?5 M epinephrine are potent inducers of the phosphodiesterase activity. The positive cell response to dibutyryl-cyclic AMP further indicates that cyclic AMP is a mediator in the phosphodiesterase activation process. The incubation kinetics and dose response effects of the various agonists on the cyclic AMP-dependent protein kinase activity determined for both cell types in the same conditions show a striking similarity to those of phosphodiesterase. Thus coordinate regulation of both enzymes by cyclic AMP was observed in all incubation conditions.     

Effect of Carbachol and 56 mM-Potassium Chloride on the Cyclic AMP-Mediated Induction of Tyrosine Hydroxylase in Neuroblastoma Cells in Culture

Abstract: Tyrosine hydroxylase (TH) activity is increased two- to threefold in neuroblastoma cell line NBP² maintained in culture for 48 h in the presence of either the inhibitor of cyclic AMP-phosphodiesterase (PDE), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO 20- 1724), or the activator of adenylate cyclase, prostaglandin E₁ (PGE₁). Cyclic AMP levels are elevated 70–80% and 30–40% throughout the 48-h treatment with RO 20-1724 and PGE₁, respectively. Carbachol does not affect either basal TH activity or cyclic AMP levels in the cells. However, the cholinergic agonist delays the induction of TH elicited by either RO 20-1724 or PGE₁. This delay is prevented by atropine. The elevation in cyclic AMP levels elicited by either RO 20-1724 or PGE₁ is blocked for 1 h or 15 min. respectively, after treatment with carbachol. Cyclic AMP levels then begin to rise until they reach those levels observed in the presence of RO 20-1724 or PGE₁ alone by 12 h or 1 h of treatment, respectively. Time course studies demonstrate that this transient inhibition of the elevation of cyclic AMP is associated with a 48-h delay in the induction of TH elicited by either RO 20-1724 or PGE₁. In contrast, the induction elicited by 8-bromo cyclic AMP is unaffected by carbachol. A depolarizing concentration (56 mM) of KCl produces a 24-h delay in the induction of TH elicited by RO 20-1724, without affecting the concomitant elevation of cyclic AMP produced by the PDE inhibitor. Furthermore, 56 mM-KCl inhibits the induction of TH elicited by 8-bromo cyclic AMP. It thus appears that carbachol delays the induction of TH by transiently inhibiting the elevation of cyclic AMP, whereas potassium depolarization delays the induction of TH by inhibiting a process with a site of action that is distal to the elevation of cyclic AMP.     

Accumulation of adenosine 3',5'-monophosphate in rat brain slices: effects of prostaglandins.

Prostaglandin E₁ and E₂ and 15(S)-15-methyl PGE₂ methyl ester stimulate the accumulation of radioactive cyclic AMP in brain slices from Sprague-Dawley rats, labelled during a prior incubation with [¹⁴C] adenine. Prostaglandins A₁ and B₁ have marginal effects and prostaglandin F_1α has no effect. Relatively high concentrations of about 80 μM PGE₁, PGE₂ and 15(S)-15-methyl PGE₂ are required to elicit a maximal 2–5 fold increase in accumulation of cyclic AMP in slices from cerebrum, but significant increases are elicited by 3.5 μM prostaglandin. Similar increases are elicited in slices from neocortex, striatum or midbrain-thalamus-hypothalamus, while lesser increases pertain in slices from cerebellum, medulla-pons or hippocampus. The accumulation of cyclic AMP elicited by PGE₁ in slices from cerebrum was not blocked by naloxone, propranololphentolamine, tetracaine, theophylline, or by nearly equimolar concentrations of either of two prostaglandin antagonists, 7-oxa-13-prostynoic acid and the dibenzoxazepine hydrazide, SC 19220. Morphine potentiated the effects of PGE₁. The combination of 85 μM PGE₁ with either isoproterenol, norepinephrine, adenosine or veratridin did not increase the accumulation of cycli AMP significantly above those elicited by the isoproterenol, norepinephrine, adenosine or veratridine alone. The combined effect of PGE₁ and norepinephrine in the presence of a β-adrenergic antagonist, sotalol, was, however, additive. The results indicate that PGE₁ stimulates cyclic AMP formation in rat brain slices, but that it either has antagonist activity with respect to accumulations of cyclic AMP-elicited by other agents or has no detectable agonist activity when cyclases are maximally stimulated by other agents.     

The effect of 7-oxa-13 prostynoic acid on the mechanism of action of bradykinin in human synovial fibroblasts

Bradykinin, a potent inflammatory mediator, induces an increment in intracellular cyclic AMP concentrations of human synovial fibroblasts and evokes the synthesis and release of ³H-arachidonic acid and ³H-E prostaglandins from these cells pre-labeled in their phospholipids. Fetal calf serum in the media also stimulates the synthesis and release of these labeled lipids from pre-labeled human synovial fibroblasts and potentiates the bradykinin-induced cyclic AMP response. The PGE₁ analogue, 7-oxa-13 prostynoic acid, completely abrogates both the bradkinin-induced cyclic AMP response and the bradykinin- and fetal calf serum-evoked release of labeled E-prostaglandins from pre-labeled cells. In serum-free media, the prostaglandin antagonist stimulated the release of ³H-arachidonic acid from pre-labeled human synovial fibroblasts and did not inhibit the bradykinin-induced release of this lipid.     

Calcium-mediated effects of calcitonin on cyclic AMP formation and lymphoblast proliferation in thymocyte populations exposed to prostaglandin E 1

The calcitonin (SCT) from salmon ultimobranchial bodies which (like mammalian calcitonins) lowers the plasma calcium concentration in mammals can also affect cyclic AMP (cyclic adenosine 3′,5′-monophosphate) metabolism and proliferation of lymphoblasts in normal and prostaglandin E₁ (PGE₁)-treated rat thymocyte populations in three different ways. In the first case, low concentrations (0.5–5.0 ng per milliliter) of SCT lower (by a calcium-mediated process) the ability of PGE₁ to transiently increase cyclic AMP synthesis, but the reduced surge of cyclic AMP production is still ample to stimulate lymphoblasts in the cell population to initiate deoxyribonucleic acid (DNA) synthesis. Secondly, these low SCT concentrations affect the eventual progression of the PGE₁-stimulated, DNA-synthesizing lymphoblasts into mitosis by a calcium-mediated process. Depending on the extracellular calcium concentration and the magnitude of the initial increment in the intracellular cyclic AMP content, SCT can either promote or inhibit the progression of the stimulated cells into mitosis. SCT's third action is a rapid (within 5 minutes), calcium-independent elevation of the cellular cyclic AMP content in otherwise untreated thymic lymphocyte populations exposed to a very high concentration (100 ng per milliliter) of the hormone. This early, transient rise in the cyclic AMP level is followed by a calcium-dependent increase in lymphoblast proliferation. An attempt is made to interrelate and explain the different actions of SCT on cyclic AMP metabolism and mitogenesis.     

Decidual adenylate cyclase and prostaglandin production in vitro

Human decidua contains an active adenylate cyclase, and a number of studies indicate that adenylate cyclase is functionally linked to increased in vitro prostaglandin synthesis. Increased decidual prostaglandin synthesis is associated with parturition, and therefore activation of adenylate cyclase may be involved in the control of human parturition. In this study, third trimester human decidual cells were preincubated for no more than 24 h prior to stimulation with a number of reagents which increase cellular cyclic AMP levels. Forskolin rapidly increased intracellular and extracellular cyclic AMP levels, but there was no increase in prostaglandin E₂ biosynthesis during incubations ranging from 5 min up to 24 h. Dibutyryl cyclic AMP or 8-bromo-cyclic AMP were also without effect on PGE₂ production, which suggests that the adenylate cyclase was not linked to the mechanisms regulating prostaglandin production. Cholera toxin increased basal cyclic AMP and PGE₂ synthesis, and was without effect on IL-1β-stimulated PGE₂ levels. PGE₂ synthesis was increased by 24 h culture with IL-1β in all the cell preparations, indicating that the cells were biologically active, and that the lack of effect of changes in cyclic AMP synthesis on PGE₂ levels could not be attributed to a defect in the prostaglandin synthetic pathway. Our findings did not agree with earlier work which showed that changes in cyclic AMP were correlated with changes in PGE₂ production by human decidual cells. It is clear that in the previous studies the decidual cells were preincubated for 4–7 days prior to stimulation, in contrast with 24 h in our investigation. We suggest that the functional link between cyclic AMP and PGE₂ synthesis reported previously may develop during culture, and not be a part of normal decidual cell function, but further studies are needed to test this hypothesis.     

Rat osteogenic sarcoma cells: Effects of some prostaglandins, their metabolites and analogues on cyclic AMP production

Cyclic AMP production by freshly isolated cells, from a ³²P-induced transplantable rat osteogenic sarcoma, was stimulated by PGE₁, PGE₂ and to a less extent by PGF_2α and PGA₂. In the case of PGE₂, the cyclic AMP content of cells was miximal within 5 min. The 13, 14-dihydro derivatives of PGE₁, PGE₂ and PGF_2α had approximately 40% of the activity of the parent prostaglandin whilst, in every case, the metabolites (15-keto and 13,14-dihydro-15-keto) had very little activity. Two prostaglandin endoperoxide analogues (U44069 and U46619) had only 10% of the activity of an equimolar dose of PGE₂. The data presented in this paper demonstrates similarities between the responses of these cells and cells derived from bony tissue in terms of the ability of prostaglandins to stimulate bone resorption in tissue culture.     

In vitro correction of antigen-induced immune suppression: effects of poly(A) poly(U) and prostaglandin E

Incubation of SJL or DBA/1 mouse spleen cells with poly(lTyr, lGlu)-polylPro—polylLys, (T, G)-Pro—L in vitro reduced the immune response potential of the cells to this immunogen as tested by adoptive transfer into irradiated, syngeneic recipients, followed by immunization with (T, G)-Pro—L in complete Freund's adjuvant. This reduction in immunocompetence was antigen-specific, since incubation with another antigen (rabbit immunoglobulin G) did not result in a suppression of responsiveness of the cells to subsequent in vivo immunization with (T, G)-Pro—L. Incubation of the spleen cell-(T, G)-Pro—L mixture in the presence of either prostaglandin E₁(PGE₁) or polyadenylic-polyuridylic acid (poly(A)·poly (U)) restored the immune response potential to the normal level. Incubation of (T, G)-Pro—L with spleen cells had no effect on cyclic AMP accumulation, whereas incubation of PGE₁ with the cells stimulated cyclic AMP production, irrespective of the presence of antigens. In contrast, the level of cyclic AMP was not affected by poly(A) · poly(U). The difference in cyclic AMP accumulation suggests that PGE₁ and poly(A) · poly(A) modify immune responsiveness by different mechanisms. The above observations were verified both in SJL and DBA/1 mice, which are the respective genetic high and low responders to (T, G) -Pro—L. This implies that the modifications of responsiveness described are not related to the genetic control of immune response to this immunogen.     

Characterisation of the prostaglandin E2-ethanolamide suppression of tumour necrosis factor-α production in human monocytic cells

Background and purpose

Prostaglandin ethanolamides or prostamides are naturally occurring neutral lipid derivatives of prostaglandins that have been shown to be synthesised in vivo following COX-facilitated oxygenation of arachidonoyl ethanolamine (anandamide). Although the actions of prostaglandins have been extensively studied, little is known about the physiological or pathophysiological effects of prostamides. Since prostaglandin E₂ has potent immunosuppressive/immunomodulating actions, the aim of the present study was to determine whether the derivative, prostaglandin E₂ ethanolamide (PGE₂-EA), could modulate the production of the pro-inflammatory cytokine tumour necrosis factor-α in human blood and human monocytic cells and indicate whether this action involved the same receptor systems/signals as PGE₂.

Experimental approach

Whole human blood, monocytes isolated from the blood or the human monocytic cell line THP-1 was incubated with LPS and the level of TNF-α produced was measured by ELISA assay. The actions of PGE₂-EA were assessed on the LPS-induced TNF-α release. In addition, in order to ascertain the receptors involved, the levels of cyclic AMP in cells were measured in monocytes and THP-1 cells in response to PGE₂-EA and directly compared to those of PGE₂. The effect of PGE₂-EA on the binding of radiolabelled PGE₂ to cells was also measured. Cells were incubated with radiolabelled arachidonic acid and ethanolamine to estimate the production of PGE₂-EA.

Key results

PGE₂-EA potently suppressed TNF-α production in blood, monocytes and the cell line THP-1 in a concentration-dependent manner. This occurred via cyclic AMP pathways as indicated by agents which interfere with these pathways and also direct ligand binding experiments. It was also shown that the cells were able to endogenously produce PGE₂-EA.

Conclusions and implications

This study reports that PGE₂-EA can downregulate the production of TNF-α by human mononuclear cells in response to an immune stimulus, i.e. LPS-activated TLR4, and that this appears to occur via a cAMP-dependent mechanism that most likely involves binding to the EP₂ receptor.