Prostaglandin E1 and prostaglandin I2 modulation of superoxide production by human neutrophils

15(S)-15-methyl-prostaglandin E1 and prostaglandin I2 rapidly and reversibly inhibit formyl-methionyl-leucyl-phenylalanine induced superoxide production by human neutrophils. In contrast, 15(S)-15-methyl-prostaglandin E1 and prostaglandin I2 did not alter the rate or the total amount of superoxide production by human neutrophils stimulated with either phorbol myristate acetate or arachidonic acid. These data suggest that the production of superoxide anion by human neutrophils may be mediated by at least two mechanisms, one regulated by prostaglandins and intracellular cyclic adenosine monophosphate levels and a second independent of prostaglandin modulation.     

Factors regulating the production of prostaglandin E2 and prostacyclin (prostaglandin I2) in rat and human adipocytes.

The regulation of PGE2 (prostaglandin E2) and PGI2 (prostaglandin I2; prostacyclin) formation was investigated in isolated adipocytes. The formation of both PGs was stimulated by various lipolytic agents such as isoproterenol, adrenaline and dibutyryl cyclic AMP. During maximal stimulation the production of PGE2 and PGI2 (measured as 6-oxo-PGF1 alpha) was 0.51 +/- 0.04 and 1.21 +/- 0.09 ng/2 h per 10(6) cells respectively. Thus PGI2 was produced in excess of PGE2 in rat adipocytes. The production of the PGs was inhibited by indomethacin and acetylsalicylic acid in a concentration-dependent manner. The half-maximal effective concentration of indomethacin was 328 +/- 38 nM and that of acetylsalicylic acid was 38.5 +/- 5.3 microM. The PGs were maximally inhibited by 70-75% after incubation for 2 h. In contrast with their effect on PG production, the two agents had a small potentiating effect on the stimulated lipolysis (P less than 0.05). The phospholipase inhibitors mepacrine and chloroquine inhibited both PG production and triacylglycerol lipolysis and were therefore unable to indicate whether the PG precursor, arachidonic acid, originates from phospholipids or triacylglycerols in adipocytes. Angiotensin II significantly (P less than 0.05) stimulated both PGE2 and PGI2 production in rat adipocytes without affecting triacylglycerol lipolysis. Finally, it was shown that PGE2 and PGI2 were also produced in human adipocytes, although in smaller quantities than in rat adipocytes. It is concluded that the production of PGs in isolated adipocytes is regulated by various hormones. Moreover, at least two separate mechanisms for PG production may exist in adipocytes: (1) a mechanism that is activated concomitantly with triacylglycerol lipolysis (and cyclic AMP) and (2) an angiotensin II-sensitive, but lipolysis (and cyclic AMP)-independent mechanism.     

Thymic hormones and prostaglandins. I. Lack of stimulation of prostaglandin production by thymic hormones

Prostaglandins (PGs) have been implicated as possible mediators of the biological activity of thymic hormones. It has been shown that type E-PGs are able to mimic the action of several thymic hormones and that indomethacin prevents in vivo or in vitro the appearance of Thy-1⁺ antigen induced by some of these factors. We thus investigated a possible role for PGs in the mechanism of action of different thymic extracts and peptides. Attempts to modulate prostaglandin production showed that neither thymosin fraction 5 (0.01 – 100 μg/ml), nor thymosin α 1 (1–10 μg/ml), thymulin (0.001–100 ng/ml), thymopoietin II (10 – 1000 ng/ml) or TP5 (10 – 1000 ng/ml) affect PGE2, 6-keto-PGF1 α, PGF2 α and TXB2 production by spleen cells from control and thymectomized mice. These results do not support the hypothesis that prostaglandins could act as mediators of thymic hormones.     

Thymic hormones and prostaglandins. I. Lack of stimulation of prostaglandin production by thymic hormones

Prostaglandins (PGs) have been implicated as possible mediators of the biological activity of thymic hormones. It has been shown that type E-PGs are able to mimic the action of several thymic hormones and that indomethacin prevents in vivo or in vitro the appearance of Thy-1+ antigen induced by some of these factors. We thus investigated a possible role for PGs in the mechanism of action of different thymic extracts and peptides. Attempts to modulate prostaglandin production showed that neither thymosin fraction 5 (0.01-100 micrograms/ml), nor thymosin alpha 1 (1-10 micrograms/ml), thymulin (0.001-100 ng/ml), thymopoietin II (10-1000 ng/ml) or TP5 (10-1000 ng/ml) affect PGE2, 6-keto-PGF1 alpha, PGF2 alpha and TXB2 production by spleen cells from control and thymectomized mice. These results do not support the hypothesis that prostaglandins could act as mediators of thymic hormones.     

Augmentation of prostaglandin E2 production by mammalian phospholipase A2 added exogenously.

Rat group II phospholipase A2 added exogenously to A23187-activated HL-60 granulocytes augmented their production of prostaglandin E2. Human group II phospholipase A2 and porcine group I phospholipase A2 augmented the prostaglandin E2 production in a similar manner. No significant increase in prostaglandin E2 production was observed when cells were treated with purified phospholipase A2 in the absence of A23187. Extracellular phospholipase A2 at inflamed sites may contribute to the generation of pro-inflammatory lipid mediators by hydrolyzing the cellular phospholipids of activated inflammatory cells.     

Keratinocytes stimulate prostaglandin I2 synthesis by 3T3 cells and exhibit enhanced cornification when exposed to prostaglandin I2 analogues.

The predominant cyclooxygenase products of keratinocytes are prostaglandin (PG)E2 and PGF2 alpha with only trace amounts of PGI2 synthesis detected. When normal or immortal (NM1) keratinocytes were co-cultured with mitomycin C-treated 3T3 cells, increased synthesis of PGI2 was noted compared to mitomycin C-treated 3T3 cells alone. The PGI2 level in co-cultures was maximum within the first week and diminished rapidly thereafter. These results suggested keratinocytes enhance the production of PGI2 by 3T3 cells. Keratinocyte cultures incubated with Iloprost and Piriprost, stable PGI2 analogues, showed evidence of increased cornification as demonstrated by staining with rhodanile blue, decreased shedding of cells into the culture medium, and more cornified material adhering to the culture surface. The cultures appeared to be responsive between the first and second weeks after plating and the inhibition of shedding could not be reversed by changing to drug-free medium. Control and treated cultures showed identical electrophoretic protein patterns. Immunoblots showed involucrin unchanged in extracts of control and treated cultures while the 22 kd pancornulin was absent in treated cultures. The findings that keratinocytes enhance the production of PGI2 by 3T3 cells and that PGI2 analogues enhance cornification of confluent keratinocytes raise the possibility that eicosanoids may serve as autoregulatory signals together with other factors.     

Regulation of agonist-induced prostaglandin E1 versus prostaglandin E2 production. A mass analysis.

Prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2), derived by enzymatic oxidation of cellular dihomogammalinolenic acid (DHLA) and arachidonic acid (AA), respectively, have diverse and, at times, distinct biological actions. It has been suggested that PGE1 specifically inhibits a variety of inflammatory processes, and, in light of the potential therapeutic benefit of PGE1 and its fatty acid precursor in inflammatory disorders, there is growing interest in the biochemical mechanisms which determine the balance between PGE1 and PGE2 synthesis. Metabolic studies in this area have been hampered by the difficulties in measuring the extremely small masses of these prostaglandins which are generated in cell culture systems. We studied the regulation of PGE1 versus PGE2 synthesis using an essential fatty acid-deficient, PGE-producing, mouse fibrosarcoma cell line, EFD-1. Because EFD-1 cells contain no endogenous AA or DHLA, we were able to replete the cells with AA and DHLA of known specific activities; thus, the mass of both cellular AA and DHLA, and synthesized PGE1 and PGE2, could be accurately determined. The major finding of this study is that production of PGE2 was highly favored over production of PGE1 due to preferential incorporation of AA versus DHLA into, and release from, the total cellular phospholipid pool. Further, we correlated the selective release of AA versus DHLA from total cellular phospholipids with the selective incorporation of AA versus DHLA into specific phospholipid pools. In addition, we showed that conversion of DHLA to AA by delta 5 desaturase was enhanced by increasing the cellular mass of n-6 fatty acids and by increasing the cell proliferative activity. Together, these results indicate that the relative abundance of PGE2 versus PGE1 in vivo is not merely a function of the relative abundance of AA versus DHLA in tissues, but also relates to markedly different cellular metabolism of these two fatty acids.     

Inhibitory effects of interleukin 6 on prostaglandin I2 production in cultured bovine vascular endothelial cells.

The effect of interleukin 6 (IL-6) upon arachidonic acid (AA) metabolism was studied in cultured bovine vascular endothelial cells (BVECs). Although, in those BVECs pretreated with IL-6 for 48 h, there was no effect upon cell proliferation, it was discovered that IL-6 suppressed the conversion of AA to prostaglandin I2 (PGI2) in cellular homogenates. First signs of the suppression occurred 3 h after incubation, and thereafter the suppression increased with time until it leveled off at 12 h. This effect of IL-6 was concentration-dependent, ranging from 20 ng/ml to a maximum of 100 ng/ml with IL-6 at 47% of control. IL-6 had no effect upon AA conversion when exogenously added to the BVEC homogenates. To investigate the mechanism of the suppression induced by IL-6, we studied its effect upon PGI2 synthesizing enzymes. We found that IL-6 had no effect upon the release of AA from phospholipids, but inhibited cyclooxygenase, the key enzyme for PGI2 production from AA. To amplify PGI2 production, AA was added exogenously to both control and IL-6-treated BVECs, and in this case also, the conversion activity in IL-6-treated BVEC's was suppressed. Through immunoblot analysis with an antibody to cyclooxygenase, it was determined that the level of cyclooxygenase protein was lowered in IL-6-treated cells. This is the first report to confirm down expression of cyclooxygenase in addition to the first observation as to the effect of IL-6 on endothelial cells.     

Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2

Current evidence suggests that two forms of prostaglandin (PG) E synthase (PGES), cytosolic PGES and membrane-bound PGES (mPGES) -1, preferentially lie downstream of cyclooxygenase (COX) -1 and -2, respectively, in the PGE2 biosynthetic pathway. In this study, we examined the expression and functional aspects of the third PGES enzyme, mPGES-2, in mammalian cells and tissues. mPGES-2 was synthesized as a Golgi membrane-associated protein, and spontaneous cleavage of the N-terminal hydrophobic domain led to the formation of a truncated mature protein that was distributed in the cytosol with a trend to be enriched in the perinuclear region. In several cell lines, mPGES-2 promoted PGE2 production via both COX-1 and COX-2 in the immediate and delayed responses with modest COX-2 preference. In contrast to the marked inducibility of mPGES-1, mPGES-2 was constitutively expressed in various cells and tissues and was not increased appreciably during tissue inflammation or damage. Interestingly, a considerable elevation of mPGES-2 expression was observed in human colorectal cancer. Collectively, mPGES-2 is a unique PGES that can be coupled with both COXs and may play a role in the production of the PGE2 involved in both tissue homeostasis and disease.     

Effect of prostaglandin I2 on ovine placental vasculature.

The response of the placental circulations to prostaglandin I2 (maternal dose 20 microgram/kg, fetal dose 180 microgram/kg) was observed in 10 near-term sheep with chronically implanted vascular catheters. The blood flows before and 90 s after the injection of prostaglandin I2 were measured using radioactive microspheres. The injection of prostaglandin I2 to the mother decreased th blood pressure from 109 +/- 4 to 69 +/- 5 mmHg (P < 0.001) and increased the vascular resistance of the maternal cotyledons from 0.166 +/- 0.018 to 0.209 +/- 0.02 mmHg/(ml/min) (P < 0.001). The vascular bed of the non-cotyledonary uterus vasodilated as the resistance fell from 0.705 +/- 0.02 to 0.266 +/- 0.02 mmHg/(ml/min). (P < 0.001). Prostaglandin I2 caused the fetal arteriovenous pressure to fall from 37.6 +/- 1.35 to 26.0 +/- 1.6 mmHg. There was no significant change in the vascular resistance of the fetal cotyledons. We observed vasodilation in the fetal membranes as vascular resistance fell from 1.06 +/- 0.14 to 0.75 +/- 0.10 mmHg/(ml/min) (P < 0.001). The infusion of prostaglandin I2 significantly depressed the response of the placenta and uterus to norepinephrine. We have not proved that prostaglandin I2 plays a direct role in maintaining placental vascular homeostasis but it may modulate the response of this organ to exogenous vasoactive agents.     

Self-association accompanies inhibition of Ca-ATPase by thapsigargin.

Recent studies have demonstrated a relationship between the activity of the Ca-ATPase of sarcoplasmic reticulum and its state of self-association. In the present study, the effects of thapsigargin (TG), a toxin that specifically inhibits the Ca-ATPase of rabbit skeletal muscle sarcoplasmic reticulum membrane, were studied by detecting the time-resolved phosphorescence anisotropy (TPA) decay of the Ca-ATPase that had been labeled with the phosphorescent probe erythrosin-isothiocyanate (ErITC). Anisotropy decays were fit to a function that consisted of three exponential decays plus a constant background, as well as to a function describing explicitly the uniaxial rotation of proteins in a membrane. In the absence of TG, the anisotropy was best-fit by a model representing the rotation of three populations, corresponding to different-sized oligomeric species in the membrane. The addition of stoichiometric amounts of TG to the Ca-ATPase promptly decreased the overall apparent rate of decay, indicating decreased rotational mobility. A detailed analysis showed that the principal change was not in the rates of rotation but rather in the population distribution of the Ca-ATPase molecules among the different-sized oligomers. TG decreased the proportion of small oligomers and increased the proportion of large ones. Preincubation of the ErITC-SR in 1 mM Ca2+, which stabilizes the E1 conformation relative to E2, was found to protect partially against the changes in the TPA associated with the presence of the inhibitor. These results are consistent with the hypothesis that TG inhibits the Ca-ATPase by stabilizing it in an E2-like conformation, which promotes the formation of larger aggregates of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amplification of Ca2+ signaling by diacylglycerol-mediated inositol 1,4,5-trisphosphate production

Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP(3) produced is particularly critical to determining the spatio-temporally coordinated Ca(2+)-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca(2+)-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity.     

Regulation of macrophage tumor necrosis factor production by prostaglandin E2

We have studied the role of prostaglandin E2 on the modulation of tumor necrosis factor by immunologically elicited and lipopolysaccharide treated murine macrophages. Indomethacin, a potent inhibitor of prostaglandin E2 production, caused a dose dependent augmentation of lipopolysaccharide induced tumor necrosis factor production (2-3 fold at 10(-7) molar). Tumor necrosis factor was released into the extracellular environment and no activity was found to be associated with membrane or cytosolic fractions. Prostaglandin E2 added to the lipopolysaccharide treated cultures suppressed tumor necrosis factor in a dose dependent manner. In these studies, 10(-7) molar PGE2 reduced tumor necrosis factor production to basal levels. These data suggest that PGE2 may be a potent autoregulatory factor that dramatically influences tumor necrosis factor production.     

Effect of melittin on prostaglandin production by guinea-pig uterus.

Melittin, an activator of phospholipase (PL) A-2, increased the outputs of prostaglandin (PG) F-2 alpha and 6-keto-PGF-1 alpha, but not of PGE-2, from Day-7 guinea-pig uterus superfused in vitro. Reducing the extracellular calcium concentration (by omitting calcium chloride from the superfusing fluid) partially inhibited the stimulatory effect of melittin on uterine PG production. TMB-8 (an intracellular calcium antagonist) completely prevented the stimulation of PGF-2 alpha and 6-keto-PGF-1 alpha output by melittin, although the production of both PGs tended to increase after stopping the melittin and TMB-8 treatments. TMB-8 also inhibited the increases in outputs of PGF-2 alpha, 6-keto-PGF-1 alpha and PGE-2 and prevented contraction of the uterus induced by exogenous PLA-2. Trifluoperazine (a calmodulin antagonist) had no inhibitory effect on the increases in outputs of PGF-2 alpha and 6-keto-PGF-1 alpha produced by melittin; it potentiated the stimulatory effect of melittin on 6-keto-PGF-1 alpha output and allowed melittin to increase PGE-2 output. When melittin was applied twice to the superfused uterus with an interval of 1 h between each treatment, partial refractoriness of the responses to melittin was seen: the magnitudes of the increases in PGF-2 alpha and 6-keto-PGF-1 alpha outputs were 40-50% less after the second treatment than after the first treatment. These results show that melittin stimulates the synthesis of PGF-2 alpha and PGI-2 (measured as 6-keto-PGF-1 alpha) in guinea-pig uterus by mechanisms which are calcium dependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the inhibition of intracellular Ca2+ transport ATPases by thapsigargin.

The effects of thapsigargin (TG), a specific inhibitor of intracellular Ca(2+)-ATPases, were studied on vesicular fragments of sarcoplasmic reticulum (SR) membranes. Inhibition of Ca2+ transport and ATPase activity was observed following stoichiometric titration of the membrane bound enzyme with TG. When Ca2+ binding to the enzyme was measured in the absence of ATP, or when one cycle of Ca(2+)-dependent enzyme phosphorylation by ATP was measured under conditions preventing turnover, protection against TG by Ca2+ was observed. The protection by Ca2+ disappeared if the phosphoenzyme was allowed to undergo turnover, indicating that a state reactive to TG is produced during enzyme turnover, whereby a dead end complex with TG is formed. Enzyme phosphorylation with Pi, ATP synthesis, and Ca2+ efflux by the ATPase in its reverse cycling were also inhibited by TG. However, under selected conditions (millimolar Ca2+ in the lumen of the vesicles, and 20% dimethyl sulfoxide in the medium) TG permitted very low rates of enzyme phosphorylation with Pi and ATP synthesis in the presence of ADP. It is concluded that the mechanism of ATPase inhibition by TG involves mutual exclusion of TG and high affinity binding of external Ca2+, as well as strong (but not total) inhibition of other partial reactions of the ATPase cycle. TG reacts selectively with the state acquired by the ATPase in the absence of Ca2+. This state is obtained either by enzyme exposure to EGTA, or by utilization of ATP and consequent displacement of bound Ca2+ during catalytic turnover.     

Decreased prostaglandin production by cholesterol-rich macrophages

The regulation of prostaglandin production by macrophages enriched in cholesterol was examined. Mouse peritoneal macrophages were incubated for 18 h with 25 micrograms/ml of human acetyl-LDL (low density lipoprotein) and trace amounts of labeled arachidonic acid. After cholesterol enrichment, the cells were incubated with phorbol 12-myristate 13-acetate (PMA), calcium ionophore, or zymosan to stimulate endogenous arachidonic acid metabolism. A high performance liquid chromatography profile of the eicosanoids released revealed no qualitative differences between unmodified and modified macrophages. Cholesterol-rich cells, however, released less prostacyclin (PGI2) and prostaglandin E2 (PGE2) compared to unmodified cells, and products from the lipoxygenase pathway became the predominant metabolites. A decrease in the synthesis of PGI2 and PGE2 by cholesterol-rich macrophages was confirmed by radioimmunoassay and radiolabeled experiments. The activity of prostaglandin synthetase was modestly increased in the cholesterol-modified macrophages compared to controls. As an estimation of phospholipase activity, the release of labeled arachidonic acid from membrane phospholipids, however, was significantly decreased in cholesterol-rich macrophages. The phosphatidylinositol fraction was particularly resistant to arachidonate release in response to calcium ionophore and PMA in the modified cells. The measurement of membrane phospholipid fatty acid composition before and after calcium ionophore supported the observation that less arachidonate was released by cholesterol-enriched cells in response to the ionophore. Based on these observations, we propose that prostaglandin synthesis from endogenous arachidonate stores is decreased in the cholesterol-rich macrophage. A decrease in agonist-induced activation of the phospholipase activity is proposed as a mechanism for this effect.     

Histophysiological studies of prostaglandin F2alpha on isolated organs. I. Effect of prostaglandin F2alpha on the heart.

The physiological and histochemical effects of PGF2alpha on isolated rabbit hearts were examined. The results showed a positive inotropic effect. The coronary flow increased. From the histochemical studies, adenosine triphosphatase (ATP-ase) and succinic dehydrogenase activities were increased while that of alkaline phosphatase was decreased. Glycogen granules were depleted. These findings were discussed on a histophysiological basis.     

Preferential production of prostaglandin D2 by lipopolysaccharide stimulated human choriodecidual explants

It has been postulated that the progression of a pregnancy to term is, in part, the result of a relative maternal Th2 immunological state. Prostaglandins (PG) are critical mediators throughout pregnancy. Recent studies have demonstrated that one PG, PGD2, may be a mediator of a Th2 immunological state. To date, very little is known about the factors that regulate of PGD2 production by human gestational tissues. Placentae were collected from women undergoing Caesarean sections at term. Amnion was separated from the choriodecidua and choriodecidual explants established. Explants were allowed to equilibrate overnight in media containing 10% fetal calf serum. The following day, media were replaced with serum free media and then after an additional 24-h, media were collected and the wet weight of the tissues determined. Production rates of PGs were determined using radioimmunoassays. At all concentrations tested, LPS significantly enhanced PGD2 production by human choriodecidual explants compared to PGE2 and PGF2alpha production. Neutralization of TNF-alpha and IL-10 further increased the production of LPS stimulated PGD2 production. We suggest that a novel stimulatory pathway that drives the production of PGD2 has been uncovered.     

Azathioprine-induced in vitro inhibition of prostaglandin E2 production by rabbit retina

Although much work exists concerning the clinical and immunosuppressive effects of azathioprine, little is known of the mechanism of action of this drug. The present study reports that azathioprine at the concentrations of 0.1 and 0.05 micrograms/ml causes a significant suppression of in vitro prostaglandin E2 production by rabbit retinas. However, at concentrations below (0.01 microgram/ml) or above (1,10 micrograms/ml) azathioprine was not inhibitory effect. These results suggest a dose-dependent inhibitory effect of azathioprine on prostaglandin E2 production.