Effects of prostaglandins on LDL receptor activity and cholesterol synthesis in freshly isolated human mononuclear leukocytes

The effects of prostaglandin (PG) E1, PGE2, the stable prostacyclin analogue Iloprost, and PGF2 alpha on low density lipoprotein (LDL) receptor activity and cholesterol synthesis were investigated in freshly isolated human mononuclear leukocytes. Incubation of cells for up to 45 hr in a lipid-free medium resulted in an increase in the rate of cholesterol synthesis from [14C]acetate and the high affinity accumulation and degradation of 125I-labeled LDL. Addition of PGE1 in increasing concentrations to the incubation medium inhibited cholesterol synthesis and the specific accumulation and degradation of 125I-labeled LDL; at a concentration of 10 microM, the inhibitions were 61%, 70%, and 67%, respectively, after an incubation of 20 hr. The effects of PGE2 and Iloprost were similar. The action of the prostaglandins on LDL receptor activity appeared to be mediated by a decrease in the number of LDL receptors and not by a change in the binding affinity. The prostaglandins yielded sigmoidal log concentration-effect curves. In contrast, PGF2 alpha had no influence on cholesterol synthesis or LDL receptor activity up to a concentration of 10 microM. PGE1, PGE2, and Iloprost, but not PGF2 alpha, led to an increase in the concentration of intracellular cyclic AMP. Dibutyryl cyclic AMP mimicked the effects of the E-prostaglandins and Iloprost on the LDL receptor activity. The results suggest that PGE1, PGE2, and prostacyclin affect LDL receptor activity and cholesterol synthesis and, therefore, may play a role in the regulation of cholesterol homeostasis and in the development of atherosclerosis.     

Interferon-mediated inhibition of prostaglandin synthesis in human mononuclear leukocytes

In this study, the question of whether human leukocyte-derived and fibroblast-derived interferon had an effect on prostaglandin metabolism in human peripheral blood mononuclear cells has been considered. Both leukocyte- and fibroblast-derived interferon were potent inhibitors of mononuclear cell prostaglandin synthesis at low physiological concentrations. Inhibition required a minimum incubation of 1 hr. Interferon had no effect on release of arachidonic acid; synthesis of hydroxy fatty acids was slightly increased.     

A comparison of delipidated sera used in studies of sterol synthesis by human mononuclear leukocytes.

Sterol synthesis in human mononuclear leukocytes is stimulated by delipidated serum. Synthesis in media containing serum delipidated by three different methods is compared. Significant differences between subjects are shown and the differences are maximized by measuring synthesis in serum delipidated by extraction with butanol-diisopropyl ether 40:60 and diethyl ether.     

Radiation-induced DNA single-strand breaks in freshly isolated human leukocytes.

Single-strand breaks are a major form of DNA damage caused by ionizing radiation, and measurement of strand breaks has long been used as an index of overall cellular DNA damage. Most assays for DNA single-strand breaks in cells rely on measuring fractionated DNA samples following alkali denaturation. Quantification is usually achieved by prelabeling cells with radioactive DNA precursors; however, this is not possible in the situation of nondividing cells or freshly isolated tissue. It has previously been demonstrated that the alkali unwinding assay of DNA strand breaks can be quantified by blotting the recovered DNA on nylon membranes and hybridizing with radiolabeled sequence-specific probes. We report here improvements to the technique, which include hot alkali denaturation of DNA samples prior to blotting and the use of carrier DNA that is non-complementary to the radiolabeled probe. Our method allows both single- and double-stranded DNA to be quantified with the same efficiency, thereby improving the sensitivity and reproducibility of the assay, and allows calibration for determination of absolute levels of DNA strand breaks in cells. We also used this method to assay radiation-induced DNA strand breaks in freshly isolated human leukocytes and found them to have a strand break induction rate of 1815 strand breaks/cell/Gy.     

Arachidonic acid and freshly isolated human bone marrow mononuclear cells

Arachidonic acid (AA), a fatty acid found in the human bone marrow plasma, is the precursor of eicosanoids that modulate bone marrow haematopoiesis. To further our understanding of the role of AA in the bone marrow physiology, we have assessed its incorporation in human bone marrow mononuclear cells. Gas chromatography analysis indicates the presence of AA in their fatty acid composition. In bone marrow mononuclear cells, [3H]-AA is incorporated into triglycerides and is later delivered into phospholipids, a result not observed with blood mononuclear cells. Prelabelling-chase experiments indicate a trafficking of labelled AA from phosphatidylcholine to phosphatidylethanolamine. Stimulation of prelabelled bone marrow mononuclear cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) results in the release of a part of the incorporated labelled AA. Finally, exogenous AA (up to 1 microM) has no significant effect on cell growth. In conclusion, human bone marrow mononuclear cells participate to the control of marrow AA concentrations by incorporating AA into phospholipids and triglycerides. In turn, bone marrow mononuclear cells can release AA in response to the potent haematopoietic growth factor GM-CSF.     

Macrolide antibiotics inhibit prostaglandin E2 synthesis and mRNA expression of prostaglandin synthetic enzymes in human leukocytes

We investigated the action of macrolide antibiotics, which are considered to have anti-inflammatory activity, on lipopolysaccharide (LPS)-stimulated prostaglandin (PG) E2 synthesis and the expression of mRNAs for cytosolic phospholipase A2 (cPLA2), cyclooxygenase (COX)-1, and COX-2 in human leukocytes. The production of LPS-stimulated PGE2 was significantly increased in peripheral polymorphonuclear leukocytes (PMNLs) and in mononuclear leukocytes (MNLs). Amounts of mRNAs for COX-2 and cPLA2, but not for COX-1, were enhanced by LPS in PMNLs and MNLs. The LPS-enhanced PGE2 synthesis and the expression of cPLA2 and COX-2 mRNAs were inhibited by clarithromycin, azithromycin and dexamethasone in PMNLs and MNLs. The mRNA expression of COX-1 in PMNLs was decreased by clarithromycin and azithromycin. Macrolide antibiotics inhibited PGE2 synthesis in human leukocytes by suppressing cPLA2, COX-1, and COX-2 mRNA expression. These data indicate one mechanism of macrolide anti-inflammatory activity.     

Stabilization mechanism of prostacyclin by human serum: an approach by binding kinetics using a stable prostaglandin I2 analogue, iloprost

We used a gel filtration method and a stable prostaglandin I2 (prostacyclin) analogue, iloprost, to study the kinetics of prostaglandin I2 binding by human serum proteins. Binding equilibrium experiments conducted at physiological prostaglandin I2 concentration (nM) yielded a KD of 10(-9) and a capacity of approx. 50 nM for the serum binding protein(s). Kinetic measurements gave a dissociation rate constant of 10(-3) s-1. When binding equilibrium was established at various ligand concentrations ranging from nM to microM, a result indicating an unsaturable binding was obtained utilizing this method. On the other hand, saturation was achieved with a ligand concentration as high as 50-100 microM by another binding method. A KD of 7 X 10(-5) and a capacity of approx. 600 microM was obtained. This apparent discrepancy was resolved by performing parallel experiments using purified human serum albumin samples and serum. It is concluded that the large quantity of serum albumin, approx. 600 microM, in serum may compensate for its low KD (approx. 10(-5] for prostaglandin I2, thus simulating a binding protein with a KD of 10(-9) and a limited capacity. These data offer direct information regarding how prostaglandin I2 is stabilized by serum and is transported to the platelet prostaglandin I2 receptors. There is a strong implication that serum albumin is the major if not the only protein responsible for binding of prostaglandin I2.     

Purification and properties of prostaglandin E1/prostacyclin receptor of human blood platelets

Activation of platelet adenylate cyclase by prostaglandin E1 or prostacyclin is initiated through the interaction of the agonists with the same receptors on membrane. Prostaglandin E1/prostacyclin receptors of human platelets were solubilized in buffer, containing 0.05% Triton X-100 and protease inhibitors. The soluble membrane protein was chromatographed on a DEAE-cellulose column and assayed by a microfiber filter by equilibrium binding technique. The active fractions eluted at 0.7 M KCl were pooled, and the receptors were purified to homogeneity by Sephadex G-200 gel filtration with an overall recovery of 30%. The isolated receptor was 2,200-fold purified over the starting platelets. As evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the receptor showed a molecular mass of 190,000 daltons and is composed of two nonidentical subunits with molecular masses of 85,000 and 95,000 daltons. The interaction of prostaglandin E1 with the purified receptor was rapid, saturable, reversible, and highly specific. Among all prostaglandins tested, only prostacyclin was capable of displacing [3H]prostaglandin E1 bound to the receptor. Scatchard analysis of [3H]prostaglandin E1 binding to the purified receptor suggested the presence of a single class of high affinity binding sites (Kd = 9.8 nM) and a second population of low affinity binding sites (Kd = 0.7 microM) in the same protein molecule. Incubation of the purified receptor with platelets stripped of the receptor by washing with low concentrations of Triton X-100 efficiently restored the ability of prostaglandin E1 and prostacyclin to activate adenylate cyclase in these cells.     

Effects of iloprost, prostaglandin E1 (PGE1) and prostacyclin (PGI2) on chemically and electrically induced seizures in mice

The effects of iloprost (ZK 36,374), a new chemically stable analogue of prostacyclin (PGI2), on strychnine-, pentylene-tetrazol-, and maximal electroshock-induced seizures were studied in mice. The time from the beginning of the injection of the convulsant or inducing electroshock to the stage of persistent seizures was determined, and lack of tonic hindlimb extension was regarded as inhibition of convulsions. In doses of 8 micrograms--16 micrograms kg-1 iloprost already exhibited an anticonvulsant action by markedly reducing the incidence of seizures and mortality following strychnine, pentylenetetrazol or maximal electroshock. The onset of tonic seizures was also reduced by iloprost. PGE1 and PGI2 were generally effective in 7 to 13 times higher doses than iloprost. It is suggested that the anticonvulsant activity of iloprost, PGE1 and PGI2 might involve a common basic mechanism. Due to its efficacy, iloprost is a useful tool in the investigation of the anticonvulsant action of prostaglandins.     

Lovastatin treatment inhibits sterol synthesis and induces HMG-CoA reductase activity in mononuclear leukocytes of normal subjects

The mechanism by which competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase decrease serum cholesterol is incompletely understood. The few available data in humans suggest that chronic administration of the competitive inhibitor, lovastatin, decreases serum cholesterol with little or no change in total body sterol synthesis. To further define the effect of lovastatin on cholesterol synthesis in normal subjects, we investigated the effect of a single oral dose of lovastatin and a 4-week treatment period of lovastatin on mononuclear leukocyte (ML) sterol synthesis as a reflection of total body sterol synthesis. In parallel, we measured serum lipid profiles and HMG-CoA reductase activity in ML microsomes that had been washed free of lovastatin. ML sterol synthesis did not significantly decrease (23 +/- 5%, mean +/- SEM) at 3 h after a single 40-mg dose of lovastatin. With a single oral 80-mg dose, ML sterol synthesis decreased by 57 +/- 10% (P less than 0.05) and remained low for the subsequent 6 h. With both doses, total HMG-CoA reductase enzyme activity in microsomes prepared from harvested mononuclear leukocytes was induced 4.8-fold (P less than 0.01) over baseline values. Both the 20-mg bid dose and the 40-mg bid dose of lovastatin administered for a 4-week period decreased serum cholesterol by 25-34%. Lovastatin at 20 mg bid decreased ML sterol synthesis by 23 +/- 6% (P less than 0.02) and increased ML HMG-CoA reductase 3.8 times (P less than 0.001) the baseline values. Twenty four hours after stopping lovastatin, ML sterol synthesis and HMG-CoA reductase enzyme activity had returned to the baseline values. The higher dose of lovastatin (40 mg bid) decreased ML sterol synthesis by 16 +/- 3% (P less than 0.05) and induced HMG-CoA reductase to 53.7 times (P less than 0.01) the baseline value at 4 weeks. Stopping this higher dose effected a rebound in ML sterol synthesis to 140 +/- 11% of baseline (P less than 0.01), while HMG-CoA reductase remained 12.5 times baseline (P less than 0.01) over the next 3 days. No rebound in serum cholesterol was observed. From these data we conclude that in normal subjects lovastatin lowers serum cholesterol with only a modest effect on sterol synthesis. The effect of lovastatin on sterol synthesis in mononuclear leukocytes is tempered by an induction of HMG-CoA reductase enzyme quantity, balancing the enzyme inhibition by lovastatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of receptors for prostaglandin E1/prostacyclin and insulin in human erythrocytes and platelets.

Prostaglandin E1/I2 and insulin receptors of human erythrocyte and platelet are capable of modulating each other's activity. This modulation of the receptor activity and number in one system by a second receptor system in human platelet and erythrocyte seems to be beneficial. Insulin increases the PGE1 binding to platelets and thereby enhances the platelet antiaggregatory action of prostaglandin by increasing cyclic AMP levels. Similarly, PGE1 increases insulin binding to human erythrocyte, and thereby reduces the optimum concentration of insulin for a maximal reduction in membrane microviscosity. During ischemia the reduced response of platelets to the inhibitory effect of PGE1 or PGI2 relates to the impaired PGE1/I2 receptor activity. Treatment of these platelets with insulin at physiological concentrations can normalise the PGE1/I2 receptor activity. This review focuses on the relationship between the two receptor systems in human blood cells.     

Effect of prostaglandin E1 on chemiluminescence response and adherence of human polymorphonuclear leukocytes to human cultured endothelial cells of prostaglandin E1 treated polytraumatized patients

We investigated the effect of prostaglandin E1 on human polymorphonuclear leukocytes, in vivo. Polymorphonuclear leukocytes of a prostaglandin E1 and placebo study group were harvested and their function, as production of oxygen-derived metabolites and adherence to human cultured endothelial cells, was compared. Additionally, data obtained from polymorphonuclear leukocytes of a prostaglandin E1 and placebo group were compared with data obtained from polymorphonuclear leukocytes from 28 blood donors, who served as a control group. Production of oxygen-derived metabolites by polymorphonuclear leukocytes during contact with endothelial cells was measured by chemiluminescence. Chemiluminescence was significantly (p < 0.01) increased in the placebo group in comparison to the control group decreasing to values of control group after 6 d (post-trauma). Chemiluminescence response was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. Adherence of polymorphonuclear leukocytes (placebo group) to endothelial cells was significantly increased (p < 0.01) within the first 6 d post-trauma Following day 6, values were in the same range as values for the control group. Adherence was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. In conclusion, prostaglandin E1 at a dose of 20 ng/kg bw/min does not influence production of oxygenderived metabolites and adherence in polytraumatized patients in comparison to a placebo group. Additionally, production of oxygen-derived metabolites by polymorphonuclear leukocytes in response to endothelial cells is shown and it is evident that endothelial cells might influence production of oxygen derived metabolites by polymorphonuclear leukocytes.     

The hyperalgesic effects of prostacyclin and prostaglandin E2.

Hyperalgesia induced in rat paws or dog knee joints by prostacyclin (PGI2) and prostaglandin E2 was measured by a modification of the Randall-Selitto method (1) or by the degree of incapacitation (2). In both species PGI2 induced an immediate hyperalgesic effect but the effect of PGE2 had a longer latency. Low doses of PGI2 caused a short lasting effect but PGE2, large doses of PGI2 or successive administration of small doses of PGI2 caused a long lasting effect. It is suggested that prostacyclin mediates rat paw hyperalgesia induced by carrageenin. The long lasting hyperalgesic effect of PGE2 and high doses of PGI2 is possibly an indirect effect caused by stimulation of a sensory nerve sensitising mechanism.     

Characterization of the interaction between prostacyclin and human serum albumin using a fluorescent analogue, 2,6-dichloro-4-aminophenol iloprost

We synthesized a fluorescent probe, 2,6-dichloro-4-aminophenol iloprost or dichlorohydroxyphenylamide of iloprost (DCHPA-iloprost) by reacting the stable prostacyclin analog, iloprost (ZK 35 374), with 2,6-dichloro-4-aminophenol with a yield of 60%. This probe exhibited an optical spectrum which overlapped with the emission spectrum of the sole tryptophan of human serum albumin (HSA). Energy transfer from the tryptophan residue to the phenol moiety of DCHPA-iloprost was observed. We utilized this donor-quenching phenomenon to quantitate the binding stoichiometry and affinity as well as the association rate of DCHPA-iloprost binding to HSA. As DCHPA-iloprost showed similar binding characteristics similar to those of iloprost and prostacyclin and competed with iloprost for HSA binding sites, we used DCHPA-iloprost as a probe to locate the binding domain of prostacyclin (PGI2) in HSA. The distance between the tryptophan indole and the phenol group of DCHPA-iloprost was estimated to be 15-18 A. Because iloprost binding to HSA was competitive with warfarin and not with free fatty acid, we propose that PGI2 binds to the 'domain 2' of HSA was competitive with warfarin and not with free fatty acid, we propose that PGI2 binds to the 'domain 2' of HSA molecules. A possible molecular mechanism by which HSA reduces the chemical degradation of PGI2 and stabilizes its activity could be derived from this model.     

Inability to fully suppress sterol synthesis rates with exogenous sterol in embryonic and extraembyronic fetal tissues

The requirement for cholesterol is greater in developing tissues (fetus, placenta, and yolk sac) as compared to adult tissues. Here, we compared cholesterol-induced suppression of sterol synthesis rates in the adult liver to the fetal liver, fetal body, placenta, and yolk sac of the Golden Syrian hamster. Sterol synthesis rates were suppressed maximally in non-pregnant adult livers when cholesterol concentrations were increased. In contrast, sterol synthesis rates were suppressed only marginally in fetal livers, fetal bodies, placentas, and yolk sacs when cholesterol concentrations were increased. To begin to elucidate the mechanism responsible for the blunted response of sterol synthesis rates in fetal tissues to exogenous cholesterol, the ratio of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) to Insig-1 was measured in these same tissues since the ratio of SCAP to the Insigs can impact SREBP processing. The fetal tissues had anywhere from a 2- to 6-fold greater ratio of SCAP to Insig-1 than did the adult liver, suggesting constitutive processing of the SREBPs. As expected, the level of mature, nuclear SREBP-2 was not different in the fetal tissues with different levels of cholesterol whereas it was different in adult livers. These findings indicate that the suppression of sterol synthesis to exogenous sterol is blunted in developing tissues and the lack of response appears to be mediated at least partly through relative levels of Insigs and SCAP.     

The use of a prostacyclin analogue, [3H]iloprost,for studying prostacyclin-binding sites on human platelets and neuronal hybrid cells

The stable prostacyclin analogue [³H]iloprost has been used for labelling prostacyclin-binding sites on human platelets and NCB-20 neuronal hybrid cells. The ligand-binding properties of the sites have been determined and correlate well with stimulation of cAMP synthesis in NCB-20 cells and inhibition of aggregation in human platelets.     

Bradykinin stimulates prostaglandin E2 formation in isolated human osteoblast-like cells

The effect of bradykinin on prostaglandin E₂ formation in cells from human trabecular bone has been studied. The cells responded to parathyroid hormone with enhanced cyclic AMP formation and were growing as cuboidal-shaped, osteoblast-like cells. In these isolated human osteoblast-like cells, bradykinin (1 mol/l) caused a rapid (5 min) stimulation of prostaglandin E₂ formation. This finding indicates that human osteoblasts are equipped with receptors for bradykinin linked to an increase in prostaglandin formation.     

The prostacyclin analogue treprostinil blocks NFkappaB nuclear translocation in human alveolar macrophages

Primary pulmonary hypertension (PPH) is characterized by increased pulmonary arterial pressure and vascular resistance. We and others have observed that inflammatory cytokines and infiltrates are present in the lung tissue, but the significance is uncertain. Treprostinil (TRE), a prostacyclin analogue with extended half-life and chemical stability, has shown promise in the treatment of PPH. We hypothesize that TRE might exert beneficial effects in PPH by antagonizing inflammatory cytokine production in the lung. Here we show that TRE dose-dependently inhibits inflammatory cytokine (tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and granulocyte macrophage colony-stimulating factor) secretion and gene expression by human alveolar macrophages. TRE blocks NFkappaB activation, but IkappaB-alpha phosphorylation and degradation are unaffected. Moreover, TRE does not affect the formation of the NFkappaB.DNA complex but blocks nuclear translocation of p65. These results are the first to illustrate the anti-cytokine actions of TRE in down-regulating NFkappaB, not through its inhibitory component or by direct binding but by blocking nuclear translocation. These data indicate that inflammatory mechanisms may be important in the pathogenesis of PPH and cytokine antagonism by blocking NFkappaB may contribute to the efficacy of TRE therapy in PPH.     

Cyclic AMP induces synthesis of prostaglandin E1 in platelets

Although platelets are known to synthesize small amounts of prostaglandin E1 the control of the formation of this prostanoid has not been investigated. Incubation of human platelet-rich plasma with various compounds which are known to increase cyclic AMP concentration in platelets and inhibit platelet aggregation also increased intracellular prostaglandin E1 synthesis. The prostaglandin E1 was isolated by high pressure liquid chromatography and definitively identified by negative and positive ionization mass spectroscopy. The amounts of prostaglandin E1 formed were proportional to the concentration of cyclic AMP in platelets. Prostacyclin (10 nM) which is the most potent stimulator of cyclic AMP formation increased intracellular cyclic AMP by 4.6 fold and prostaglandin E1 level by 3 fold over the basal levels. Addition of theophylline, a cyclic AMP phosphodiesterase inhibitor, together with prostacyclin increased cyclic AMP concentration 8.7-fold and prostaglandin E1 level 12-fold compared to basal concentrations. Dibutyryl cyclic AMP (2 mM) and 8-bromo cyclic AMP (0.1 mM) increased prostaglandin E1 levels by 3 fold and 2 fold over the basal level, respectively. Prostaglandin D2 (3 microM) when added to platelet-rich plasma increased the cyclic nucleotide levels by 2 fold concomitant with 2 fold increase in prostaglandin E1 concentration. In contrast prostaglandin E2 or prostaglandin F2 alpha which had no effect on cyclic AMP level did not affect the prostaglandin E1 synthesis. Addition of 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, to platelet-rich plasma inhibited both the increase of intracellular prostaglandin E1 and cyclic AMP levels induced by prostacyclin.