Endogenous synthesis of prostaglandins E1 and I2 in 3T3 fibroblasts transformed by polyoma virus. Effects on adenosine 3':5'-monophosphate production

Prostaglandins (PG)E1, E2 and I2 were produced by polyoma virus transformed (py) 3T3 fibroblasts. The levels of PGE1, PGE2 and 6-keto-PGF1 alpha (degradation product of PGI2) were 22.7, 225 and 33.2 ng/ml medium, respectively, 72 h after medium change. The stimulatory potencies of exogenous PGE1, PGE2 and PGI2 on adenosine 3':5'-monophosphate (cyclic AMP) formation were similar. Therefore, the prostaglandin mediated increase in cyclic AMP levels observed during growth of these cells (Claesson, H.-E., Lindgren, J.A. and Hammarstr?m, S. (1977) Eur. J. Biochem. 74, 13) is largely (greater than 80%) mediated by PGE2 and to lesser extents by PGE1 and PGI2.     

Stimulatory effect of prostaglandins on the production of hexosamine-containing substances by cultured fibroblasts (4) adenosine 3′:5′-cyclic monophosphate independent stimulation by prostaglandin F2α

The mechanism of the stimulatory effect of prostaglandin (PG) F_2α on the production of hexosamine-containing substances by cultured fibroblasts was studied with special reference to adenosine 3′:5′- cyclic monophosphate (cAMP). At the stationary phase, the cells were exposed for 6 hrs to PGF_2α, E₁, cAMP or dibutyryl-cAMP in a wide range of concentrations. cAMP itself showed a slight stimulation on the production of hexosamine-containing substances, and the effect was enhanced by using the dibutyryl derivative. PGF_2α had much a greater capacity than either the exogeneous cAMP or the dibutyryl-cAMP for enhancing the production of hexosamine-containing substances. To know whether cAMP is involved in the stimulatory effect of PGF_2α, intracellular cAMP level was concomitantly measured in both PGF_2α and PGE₁ treated cultures. Although the cellular cAMP level in PGE₁ treated cultures was much higher than that in the PGF_2α treated cultures, the stimulatory effect on the production of hexosamine-containing substances in PGE₁ treated cultures was always much smaller than that in the PGF_2α treated cultures. Moreover, PGF_2α had a significant stimulatory effect on the production of hexosamine-containing substances even at a low concentration as 100 pg/ml, which is small enough not to increase any cellular cAMP level. From these results, it was concluded that the stimulatory effect of PGF_2α on the production of hexosamine-containing substances by cultured fibroblasts is not mediated by cAMP and is caused by a mechanism different from that caused by cAMP.     

Effects of intracerebroventricular administration of prostaglandins I2, E2, F2α and indomethacin on blood pressure in the rat

The effects of intraventricularly administered prostaglandins I₂ (PGI2), E₂ (PGE2), F_2α (PGF2α) and indomethacin on systemic blood pressure were investigated in conscious rats. PGI2 (1.25 – 10 g/kg) decreased blood pressure in a dose-related manner, whereas PGE2 (100 – 1000 ng/kg) dose-dependently increased blood pressure. Both PGF2α (0.31 – 20 μg/kg) and indomethacin (0.625 – 40 μg/kg) had no effects on blood pressure. These results indicate that intraventricular injection of PGI2 or PGE2 can induce significant changes in blood pressure, while endogenous prostaglandins synthesized in the brain seem to play a minor role in direct regulation of systemic blood pressure in the rat.     

Formation of adenosine 3′,5′-monophosphate from adenosine in mouse thymocytes

The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3′:5′-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E₁, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E₁ and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly wihtin 1 min and was maximal by 10 to 20 min with approx. 2 and 10 μM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E₁, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [¹⁴C] adenine exhibited dramatic increases in cyclic [¹⁴C]AMP 10 min after addition of adenosine or prostaglandin E₁ which corresponded to simultaneously determined increases in total cyclic AMP. Using [¹⁴C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.     

Effect of prostaglandins E1, E2 and F2α on neutrophil aggregation

Recently we have found that chemotactic factors stimulate neutrophils in suspension to aggregate. Because of an obvious analogy to platelet aggregation, we examined the influence of three prostaglandins on this process. Prostaglandins E₁, E₂ and F_2α alone did not cause aggregation of the neutrophils but were able to partially inhibit the aggregation response induced by the synthetic chemotactic tripeptide, formly-methionyl-leucyl-phenylalanine. The minimal inhibitory concentrations for prostaglandins E₁, E₂ and F_2α were 10⁻⁷, 10⁻⁶ and 10⁻⁵M, respectively. These results are similar to those found for the prostaglandin-induced inhibition of platelet aggregation. It may be, therefore, that neutrophil aggregation, like platelet aggregation, is modulated by intracellular prostaglandins and other products of arachidonic acid metabolism.     

The central nervous effect of prostaglandins I2, E2 and F2α on aconitine — Induced cardiac arrhythmia in rats

Intracerebroventricular administration of PGI₂ or PGE₂ reduced aconitine-induced cardiac arrhythmia in rats. PGF_2α had no antiarrhythmic effect under the same conditions. The ED₅₀ values of PGI₂ and E₂ were 0.25 μg/kg and 1.1 μg/kg, respectively. Central mechanisms may participate in the antiarrhythmic effect of these PGs.     

Interactions of prostaglandins with subpopulations of ovine luteal cells. I. Stimulatory effects of prostaglandins E1, E2 and I2

Preparations of small and large steroidogenic cells from enzymatically dispersed ovine corpora lutea were utilized to study the effects of luteinizing hormone (LH) and prostaglandins (PG) E₁, E₂ and I₂. Cells were allowed to attach to culture dishes overnight and were incubated with either LH (100 ng/ml), PGE₂, PGE₂, or PGI₂ (250 ng/ml each). The secretion of progesterone by large cells was stimulated by all prostaglandins tested (P < 0.05) while the moderate stimulation observed after LH treatment was attributable to contamination of the large cell population with small cells. Prostaglandins E₁ and E₂ had no effect on progesterone secretion by small cells, while LH was stimulatory at all times (0.5 to 4 hr) and PGI₂ was stimulatory by 4 hr. Additional studies were conducted to determine if the effects of PGE₂ upon steroidogenesis in large cells were correlated with stimulated activity of adenylate cyclase. In both plated and suspended cells PGE₂ caused an increase (P < 0.05) in the rate of progesterone secretion but had no effect upon the activity of adenylate cyclase or cAMP concentrations within cells or in the incubation media. Exposure of luteal cells to forskolin, a nonhormonal stimulator of adenylate cyclase, resulted in marked increases in all parameters of cyclase activity but had no effect on progesterone secretion. These data suggest that the actions of prostaglandins E₁, E₂ and I₂ are directed primarily toward the large cells of the ovine corpus luteum and cast doubt upon the role of adenylate cyclase as the sole intermediary in regulation of progesterone secretion in this cell type.     

On the mechanism of elevated prostaglandin E2 production in 3T3 fibroblasts transformed by polyoma virus

Polyoma-virus-transformed 3T3 fibroblasts (py 3T3 cells) produce considerably more prostaglandin E2 than regular 3T3 cells during growth in cell culture. Incubations with exogenous arachidonic acid showed no increase in prostaglandin-producing capacity in the transformed cells. The rates of degradation of prostaglandin E2 were similar in the two lines. After labeling of cells with [1-14C]arachidonic acid, py 3T3 cultures continuously released radioactivity while the release by regular 3T3 cells was almost completed after 3 h. Prostaglandin E2 production during short incubations in buffer at various times after medium change was constantly higher in the transformed cells. Furthermore, hydrocortisone completely inhibited prostaglandin synthesis by the transformed cells. These results suggest that the increased formation of prostaglandin by py 3T3 cells is due to continuously elevated activity of phospholipase A2 or another acyl hydrolase.     

Prostaglandin E2 production in 3T3 cells transformed by polyoma virus raises the intracellular adenosine 3':5'-monophosphate levels.

High cellular adenosine 3':5'-monophosphate (cyclic AMP) were found in a polyoma-virus-transformed 3T3 fibroblast line, which produces comparatively high prostaglandin E2 concentrations. Prostaglandin E2 and cyclic AMP increased with time during 6 days of growth. Both effects were prevented by three different cyclo-oxygenase inhibitors. Addition of prostaglandin E2, at a concentration which would been synthesized in the absence of inhibitor, reversed the effect of indomethacin, one of the cyclo-oxygenase inhibitors, on cyclic AMP. It is concluded that endogenous prostaglandin E2 production in these transformed cells influences their cellular cyclic AMP levels.     

Stimulation of adenosine 3′,5′-monophosphate formation in mononuclear leukocytes by toad venoms

The content of adenosine 3′,5′-monophosphate in human mononuclear leukocytes was enhanced 3–5-times by venoms obtained from African toad (Bufo africanus), American toad (Bufo americanus), Colorado river toad (Bufo arenarum) and Marine toad (Bufo marinus) at 25 μg/ml for 5 min of incubation at 37°C. The maximum stimulation was observed after 1–5 min of incubation. The half-maximal stimulation was observed at 0.1 μg/ml venom obtained from Colorado river toad (Bufo arenarum). The increased content of adenosine 3′,5′-monophosphate in the mononuclear leukocytes persisted without significant change for at least 30 min of incubation at 37°C.     

Regulation of DNA synthesis by guanosine-5′-diphosphate, cyclic guanosine-3′,5′-monophosphate, and cyclic adenosine-3′,5′-monophosphate in mouse lymphoid cells

The effect of various adenine and guanine nucleotides and nucleosides on DNA synthesis was studied in various types of mouse lymphoid cells. Two out of the ten compounds tested, namely guanosine-5′-diphosphate (GDP) and cyclic guanosine-3′,5′-monophosphate (cGMP) increased the thymidine incorporation into the DNA of the spleen cells and counteracted completely or partially the inhibitory action of cyclic adenosine-3′,5′-monophosphate (cAMP) on spleen cells stimulated by various B or T cell mitogens. GDP seems to act preferentially on thymus cells while cGMP acts better on bone marrow cells. The possible significance of the results for the mechanism of the mitogenic signal is discussed.     

Phospholipid metabolism and lysosomal enzyme secretion by leukocytes Effects of dibutyryl cyclic adenosine 3′ : 5′-monophosphate and ATP

The effects of dibutyryl cyclic adenosine 3′ : 5′-monophosphate and ATP on isotope incorporation into phospholipids and the release of β-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3′ : 5′-monophosphate (0.1–1.0 mM) reduced β-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited ³²P_i incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-³H]inositol into the phosphoinositides. Added ATP (0.1–1.0 mM), but not other nucleotides, was found to potentiate β-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-³H]inositol. The mechanism of the inhibition of the isotope incorporation into these acidic phospholipids by the two nucleotides has not been defined. Dibutyryl cyclic adenosine 3′ : 5′-monophosphate at 2–4 mM concentration was not found to appreciably alter the incorporation of [γ-³²P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.     

Adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate: concentrations in Morris hepatomas of different growth rates

Cyclic AMP and cyclic GMP levels were examined in Morris hepatoma explants in vivo. All eight tumor lines examined had significantly elevated cyclic AMP and cyclic GMP levels when compared to normal liver from tumor-bearing rats. No apparent correlation was observed between the rates of tumor growth and cyclic nucleotide levels; however, two tumor lines (3924A and 7288ctc) had very high levels of cyclic GMP.     

Interferon γ stimulates prostaglandin E2 production by mouse Kupffer cells

When mononuclear phagocytes, including Kupffer cells, are activated by various agents, they synthesize and release arachidonic acid metabolites, prostaglandins (PGs) and leukotrienes (LTs). In this study, we examined the effect of in vitro Kupffer cell activation with recombinant murine IFN gamma on PGE2 and LTB4 secretion. IFN gamma enhanced PGE2 secretion, and this effect of IFN gamma was stronger than that of IL-1 or TNF. Moreover, IFN gamma promoted LTB4 release especially in the absence of PGs. On the other hand, dexamethasone and indomethacin inhibited and, EGTA and TMB-8, which reduce intracellular Ca++ Levels, blocked IFN gamma induced PGE2 production, which suggested that the activation of phospholipase A2 and cyclooxygenase in Kupffer cells requires the elevation of intracellular Ca++ levels.     

Separation of prostaglandins E2 and F2α from their pulmonary metabolites by thin-layer chromatography

A simple thin-layer chromatographic system has been developed to separate PGE₂ and PGF_2α from 13,14-dihydro-PGF_2α and other pulmonary metabolites of prostaglandins. The separation is achieved on Kieselgel-60 F-254 plates impregnated with both silver nitrate and boric acid. The system should be of use in the purification and tentative identification of primary prostaglandins.     

The lack of involvement of central nervous system in the antiarrhythmic effects of prostaglandins E2, F2α, and I2 in cat

The role of the central nervous system (CNS) in the antiarrhythmic effects of prostaglandins (PGs) E₂, F_2α, and I₂ was studied by administering each agent into the left lateral cerebral ventricle (i.c.v. administration) of chloralose-anesthetized cats. The cardiac arrhythmias were produced by intravenous (i.v.) infusion of ouabain (1 μg/kg/min). The PGs E₂, F_2α and I₂ on i.c.v. administration in the dose range of 1 ng to 10 μg failed to inhibit ouabain-induced cardiac arrhythmias. However, when infused i.v., PGE₂ (1 μg/kg/min), PGF_2α (5 μg/kg/min), and PGI₂ (2 μg/kg/min) effectively suppressed these arrhythmias. The standard antiarrhythmic drug propanolol (0.5–8.0 mg)oni.c.v.administration also significantly reduced the ouabain-induced cardiac arrhythmias. It is suggested that the CNS is not the site of action of PGs E₂, F_2α, and I₂ in antagonising the ouabain-induced cardiotoxicity in cats.     

The actions of prostaglandins I2 and E2 on arrhythmias produced by coronary occlusion in the rat and dog

Prostaglandins E₂ and I₂ were compared with known antiarrhythmics for their actions against arrhythmias produced by occlusion of the left anterior descending coronary artery in the anaesthetised rat while PGI₂ was also examined in the dog. PGI₂ in the dog suppressed early arrhythmias produced during occlusion but did not influence those produced by occlusion-release or those occurring 24 hours after a permanent occlusion; none of the A,B,C or D series prostaglandins tested markedly reduced 24 hour arrhythmias. In the rat PGE₂ was antiarrhythmic against early occlusion arrhythmias (30 minutes occlusion) in a dose related manner (infusions of 1–4 μg/kg/min) whereas PGI₂ infusions potentiated the arrhythmogenic effect of occlusion. PGE₂ was as effective an antiarrhythmic as 10mg/kg Org. 6001 which was more effective in this test situation than d1-propranolol. No obvious mechanisms for the actions of PGE₂ or PGI₂ were apparent although both agents lowered blood pressure and reduced the size of the occluded zone produced by ligation.     

Effects of prostaglandins E2, I2 and F2α, arachidonic acid and indomethacin on pressor responses to norepinephrine in conscious rats

The effects of prostaglandins E₂ (PGE₂), I₂ (PGI₂) and F₂α (PGF₂α), arachidonic acid and indomethacin on pressor responses to norepinephrine were examined in conscious rats. Intravenously infused PGE₂ (0.3, 1.25 μg/kg/min), PGI₂ (50, 100 ng/kg/min), PGF₂α (1.8, 5.4 μg/kg/min) and arachidonic acid (0.7, 1.4 mg/kg/min) did not change the basal blood pressure. Both PGE₂ and PGI₂ significantly attenuated pressor responses to norepinephrine, whereas PGF₂α significantly potentiated them. Arachidonic acid, a precursor of the prostaglandins (PGs), significantly attenuated pressor responses to norepinephrine. Since the attenuating effect of arachidonic acid was completely abolished by the pretreatment with indomethacin (5 mg/kg), arachidonic acid is thought to exert an effect through its conversion to PGs. On the contrary, intravenously injected indomethacin (0.2–5.0 mg/kg) facilitated pressor responses to norepinephrine in a dose-related manner without any direct effect on the basal blood pressure. These results suggest that endogenous PGs may participate in the regulation of blood pressure by modulating pressor responses to norepinephrine in conscious rats.     

Endogenous prostaglandin E2 synthesis inhibits growth of polyoma virus-transformed 3T3 fibroblasts.

Indomethacin lowered the cellular content of adenosine 3 ′: 5 ′-monophosphate (cAMP) and stimulated growth of polyoma virus-transformed 3T3 fibroblasts. Exogenous prostaglandin E₂, at concentrations produced in the absence of inhibitor, reversed the effects of indomethacin on cAMP levels and cell proliferation. Therefore, endogenously produced prostaglandin E₂ decreases cell growth and raises the levels of cAMP in these cells.