Calcitonin enhances production of prostaglandins by stimulated human monocytes

Stimulated monocytes produce prostaglandins that may play a role in bone resorption. We studied the effects of salmon calcitonin (sCT) on human monocyte production of various prostaglandin metabolites. Latex particle-stimulated human monocyte production of prostaglandin E2, thromboxane A2 measured as thromboxane B2, and prostacyclin measured as 6-keto-PGF1 alpha was each increased in the presence of sCT. This effect required surface stimulation, was blocked by indomethacin, was less marked with equivalent concentrations of human calcitonin, and was not seen with parathyroid hormone. Calcitonin specifically affects prostaglandin pathways in stimulated human monocytes.     

The relation between thromboxane and prostaglandin synthesis in human decidua tissue: a comparison of eicosanoid synthesis in minced tissue with that in a cell-free preparation

Radiotracer studies and radioimmunoassay measurements demonstrate that minced tissues of human decidua produce chiefly thromboxane B2 (TxB2) (70% of total eicosanoids) and small amounts of prostaglandin F2 alpha (PGF2 alpha) (13%) PGD2 (8%), 6-keto-PGF1 alpha (5%) and PGE2 (4%). Inhibition of thromboxane synthesis with a specific inhibitor (OKY-1581: sodium (E)-3-[4(-3-pyridylmethyl)-phenyl]-2-methyl propenoate) increased prostaglandin formation in general, with the main product being PGF2 alpha (38%), a nonenzymic derivative of PGH2. Crude particulate fractions prepared from the same tissue synthesized two major products from [3H]arachidonate, TxB2 and 6-keto-PGF1 alpha (54 and 30%, respectively) and some PGF2 alpha and PGE2 (8-8%). However, in the presence of reduced glutathione (GSH), PGE2 became the main product (81%) (TxB2, 15%; PGF2 alpha, 2%; and 6-keto-PGF1 alpha, 2%). Half-maximal stimulation of PGE2 synthesis occurred at 46 microM GSH. The GSH concentration of tissue samples was found to be 110 +/- 30 microM. We conclude that human first trimester decidua cells possess the key enzymes of prostaglandin and thromboxane synthesis. Apparently, the production of these compounds is controlled by a specific mechanism in the tissue, which keeps PGE and prostacyclin synthesis in a reversibly suppressed state, whereas the formation of thromboxane is relatively stimulated.     

Effects of Hypoxia and Anoxia on the Ex Vivo Release of Prostaglandins from Mouse Cortical Slices

Arachidonic acid is transiently accumulated in the brain as a result of a variety of pathological conditions. The synthesis and release of some of its metabolites, namely, prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) from cortical slices of mice were studied following exposure to 6 min of hypoxia (7% O2), 45 s of anoxia, and 5 min-4 h of reoxygenation following anoxia. Hypoxia induced a slight increase in the rate of TXB2 release and a slight decrease in the rate of PGE2 release, whereas 6-keto-PGF1 alpha was unaffected. Anoxia (45 s) followed by reoxygenation induced a transient increase in the release of PGE2 and of 6-keto-PGF1 alpha with a return to the normal rate at 30 min and 2 h of recovery, respectively. However, the rate of TXB2 synthesis and release reached its peak (twofold increase) after 1 h and remained significantly higher than the control rate even after 4 h of normal air breathing. Our results demonstrate that hypoxia and anoxia, even of short duration, selectively trigger the activity of thromboxane synthetase and that this elevated rate of synthesis and release persists long after normal oxygen supply is restored. We suggest that enhanced thromboxane synthesis, with normal prostacyclin levels, might have a role in the pathophysiology of ischemic cell damage.     

Increase in the formation of leukotriene B4 and other lipoxygenase products in peritoneal macrophages of adrenalectomized rats

The effect of adrenalectomy on the formation of cyclooxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined. After isolation, the cells were incubated with [1-14C]arachidonic acid and the calcium ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the controls are 6-keto-prostaglandin F1 alpha, thromboxane B2 and 12-HETE. One peak represents 5,12-di-HETE. Smaller amounts of prostaglandin F2 alpha, prostaglandin E2, prostaglandin D2, leukotriene B4 and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of leukotriene B4, 15-HETE and 12-HETE. The increase in the prostaglandins is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, 6-keto-prostaglandin F1 alpha and thromboxane B2 are produced in higher amounts than leukotriene B4. After adrenalectomy, the formation of leukotriene B4 is much more increased than that of 6-keto-prostaglandin F1 alpha. These effects are most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid-induced peptide with phospholipase A2 inhibitory activity in adrenalectomized animals.     

Thromboxane and Prostacyclin Levels in Fetal Rabbit Brain and Placenta After Intrauterine Partial Ischemic Episodes

The appearance of arachidonic acid (AA) oxidation products in fetal rabbit brain and placenta under normal or partial short-term ischemic episodes induced by placental blood vessel restriction was examined. Intracerebral administration of [3H]AA into close-to-term rabbit fetuses gave rise to radioactively labeled prostaglandin (PG) E2, thromboxane B2, and 6-keto-PGF1 alpha metabolites as detected by HPLC analysis. A significant increase of 20-30% of [3H]AA precursor into eicosanoids was detected in brain of fetuses after 2-h restriction. The thromboxane B2 and 6-keto-PGF1 alpha levels were determined by radioimmunoassay technique over a period of 48 h following ischemic episodes. Thromboxane B2 content in affected animals was higher by five- and twofold at 3 h over control fetal brain and placental tissue values, respectively, and remained significantly higher for 24 h. 6-Keto-PGF1 alpha levels reached a peak value that was greater by 2.5- and 1.5-fold at 6 h for the ischemic brain and placental tissue, respectively, compared with control fetuses. PGE2 levels were less affected, attaining a maximum of 1.9- and 1.1-fold in brain and placenta correspondingly. The thromboxane/prostacyclin ratio reached a maximum in the brain after approximately 3 h, while that in the placenta continued to rise even after 20 h. Persisting high levels of thromboxane are indicative of cerebral vasoconstriction and may suggest possible damaging effects.     

Effect of in vitro aging on 6-ketoprostaglandin F1 alpha-producing activity in cultured human diploid lung fibroblasts.

Prostaglandin synthesis in human diploid fibroblasts was studied by incubating [14C]-arachidonic acid with cell homogenates. The majority of prostaglandins produced in young cells was 6-ketoprostaglandin F1 alpha. The 6-ketoprostaglandin F1 alpha-producing activity of cultures declined with in vitro aging, and was almost undetectable at the senescent stage, while total production of thromboxane B2, prostaglandin F2 alpha and prostaglandin E2-like metabolites increased with in vitro aging.     

Bioconversion of arachidonic acid to prostaglandins and related compounds in human myometrium and uterine cervix

Arachidonic Acid metabolites in human myometrium and uterine cervix were studied using silicic acid column chromatography, thin layer chromatography, reversed phase partition chromatography and gas-liquid chromatography. Myometrium produced 6-ketoPGF1 alpha, PGF2 alpha, PGE2, thromboxane B2. Uterine cervix produced 6-ketoPGF1 alpha, PGF2 alpha, PGE2, thromboxane B2, and one hydroxyacid. There was no difference between the rate of conversion of prostaglandins in myometrium and cervix. But only cervix could convert arachidonic acid to hydroxyacid.     

Redirection of eicosanoid metabolism in mPGES-1-deficient macrophages

Microsomal prostaglandin E synthase (mPGES)-1 is one of several prostaglandin E synthases involved in prostaglandin H2 (PGH2) metabolism. In the present report, we characterize the contribution of mPGES-1 to cellular PGH2 metabolism in murine macrophages by studying the synthesis of eicosanoids and expression of eicosanoid metabolism enzymes in wild type and mPGES-1-deficient macrophages. Thioglycollate-elicited macrophages isolated from mPGES-1-/- animals and genetically matched wild type controls were stimulated with diverse pro-inflammatory stimuli. Prostaglandins were released in the following order of decreasing abundance from wild type macrophages stimulated with lipopolysaccharide: prostaglandin E2 (PGE2)>thromboxane B2 (TxB2)>6-keto prostaglandin F1alpha (PGF1alpha), prostaglandin F(2alpha) (PGF2alpha), and prostaglandin D2 (PGD2). In contrast, we detected in mPGES-1-/- macrophages a >95% reduction in PGE2 production resulting in the following altered prostaglandin profile: TxB2>6-keto PGF1alpha and PGF2alpha>PGE2, despite the comparable release of total prostaglandins. No significant change in expression pattern of key prostaglandin-synthesizing enzymes was detected between the genotypes. We then further profiled genotype-related differences in the eicosanoid profile using macrophages pre-stimulated with lipopolysaccharide followed by a 10-min incubation with 10 microm [3H]arachidonic acid. Eicosanoid products were subsequently identified by reverse phase high pressure liquid chromatography. The dramatic reduction in [3H]PGE2 formation from mPGES-1-/- macrophages compared with controls resulted in TxB2 and 6-keto PGF1alpha becoming the two most abundant prostaglandins in these samples. Our results also suggest a 5-fold increase in 12-[3H]hydroxyheptadecatrienoic acid release in mPGES-1-/- samples. Our data support the hypothesis that mPGES-1 induction in response to an inflammatory stimulus is essential for PGE2 synthesis. The redirection of prostaglandin production in mPGES-1-/- cells provides novel insights into how a cell processes the unstable endoperoxide PGH2 during the inactivation of a major metabolic outlet.     

Effect of bacterial cell wall and lipopolysaccharide on arachidonic acid metabolism by caruncular and allantochorionic tissues from cows that calved normally and those that retained fetal membranes

Immunoactive eicosanoids may have a role in both placental separation and uterine involution in cattle. In the present study, we examined the effects of bacterial cell wall preparation and endotoxins on in vitro prostaglandin synthesis and arachidonic acid (AA) metabolism by caruncular and allantochorionic tissues. Placentomes were obtained about 6 h post partum from cows that delivered normally (n = 10) or those with retained fetal membranes (n = 4); the tissue explants were incubated for 6 h in the presence of labeled or nonlabeled AA. Prostaglandin F(2alpha) (PGF(2alpha)) and E(2) (PGE(2)) were measured by radioimmunoassay, and labeled AA metabolites were separated by reverse phase-high pressure-liquid chromatography. There was no effect of bacterial cell wall preparations or endotoxins on in vitro caruncular PGF(2alpha) secretion. However, bacterial products increased caruncular PGE(2) secretion in both cows that delivered normally and those with retained fetal membranes. For normal delivery cows caruncular tissue, bacterial product also increased leukotriene B(4) (LTB(4)) and decreased both thromboxane B(2) (TXB(2)) and hydroxy-eicosatetranoic acids (HETE) in vitro secretion. For the allantochorion, bacterial products increased in vitro PGF(2alpha) secretion only in cows that delivered normally and increased PGE(2) secretion essentially in cows with retained fetal membranes. In general, 6 keto PGF(1alpha) was the main metabolite secreted by both allantochorionic and carucular tissues. However, in cows with retained fetal membranes, PGE(2) became the most important metabolite secreted by allantochorion, especially in the presence of endotoxin. In conclusion, these results suggest that bacteria found in the early postpartum uterus or their endotoxin affect primarily caruncular and allantochorionic PGE(2) synthesis.     

Biosynthesis of prostaglandins by human spermatozoa in vitro and their role in acrosome reaction and fertilization.

Five homogenates of human sperm cells were separately incubated with [14C]arachidonic acid in the presence of reduced glutathione, L-tryptophan, and haematin as cofactors. The cyclooxygenase products of arachidonic acid metabolism were extracted, separated, and measured for their radioactivity. The rate of formation of prostaglandin (PG)D2, PGE2, PGF2 alpha, 6-keto PGF1 alpha, and thromboxane (TX)B2 were 18.0 +/- 1.11, 10.9 +/- 0.68, 5.8 +/- 0.21, 3.9 +/- 0.13 and 6.6 +/- 0.52 pmol/10(6) cells/min, respectively. These results are discussed in relation to the hypothesis that cyclooxygenase metabolites of certain polyunsaturated fatty acids play an important part in the sperm acrosome reaction and fertilization.     

Bioconversion of arachidonic acid by human uterine cervical tissue and endocervix in late pregnancy

Prostaglandins (PGs) may play an important role on cervical ripening in late pregnancy, namely cervical dilatation and softening. To investigate this, arachidonic acid metabolites of cervical tissue and endocervix were studied. To separate and identify the metabolites, silicic acid chromatography, thin layer chromatography, reversed phase chromatography, gas-liquid chromatography and GC-MS were used. In cervical tissue, arachidonic acid was converted to 6-ketoPGF1 alpha, PGF2 alpha, PGE2, thromboxane B2, and 12-HETE. In endocervix, arachidonic acid was converted to PGF2 alpha, PGE2, thromboxane B2, 12-hydroxy-5, 8, 10-heptadecatrienoic acid, and 12-HETE. There was no relation between the arachidonic acid conversion rate and the Bishop score (points of cervical ripening).     

Glucocorticoid effect on arachidonic acid metabolism in vivo

Glucocorticoids have been shown in in vitro systems to inhibit the release of arachidonic acid metabolites, namely prostaglandins (PGs) and leukotrienes, apparently, via the induction of a phospholipase A2 inhibitory protein, called lipocortin. On the basis of these in vitro results, it has been suggested that inhibition of eicosanoid production is, at least partially, responsible for the well-known anti-inflammatory effect of glucocorticoids. There is, however, no firm evidence proving that glucocorticoids also inhibit prostaglandin or leukotriene synthesis in vivo. In a series of studies, we have investigated the effects of anti-inflammatory steroids on the production of six different cyclo-oxygenase products in vivo. Urinary prostaglandin (PG) E2(1), PGF2 alpha, thromboxane B2 (TxB2), 6-keto-PGF1 alpha, and the major urinary metabolites of the E and F PGs, PGE-M and PGF-M, respectively, were determined by radioimmunoassay and by GC-MS. Administration of pharmacological doses of dexamethasone to rabbits failed to inhibit urinary excretion rates of PGE2, TxB2, 6-keto-PGF1 alpha and that of PGE-M and PGF-M. In contrast, urinary PGF2 alpha was slightly reduced by dexamethasone. In further experiments the effect of dexamethasone was studied in humans. Urinary excretion rates of PGE2, PGE-M, PGF-M, 2,3-dinor TxB2 and 2,3-dinor 6-keto-PGF1 alpha were not suppressed by dexamethasone. Collagen-induced platelet TxB2 formation and platelet aggregation was also unaltered. To test one possible explanation for the apparent discrepancy between in vitro and in vivo effects of glucocorticoids on arachidonic acid metabolites we investigated the effects of dexamethasone in vivo on basal and on antidiuretic hormone-stimulated renal PG synthesis. Dexamethasone treatment failed to inhibit both basal and antidiuretic hormone-stimulated PGE2 and PGF2 alpha production. We conclude that glucocorticoids in vivo do not decrease the basal rate of total body, kidney and platelet prostanoid synthesis, and that dexamethasone does not inhibit renal PG production when it is elevated by antidiuretic hormone, a physiological stimulus. Thus, a differential effect of glucocorticoids on basal vs stimulated PG synthesis cannot account for the discrepancy between in vivo and in vitro effects.     

Sex differences in gallbladder prostaglandin synthesis mediated by acute inflammation

The influences of sex and acute inflammation on prostaglandin biosynthesis in rabbit gallbladder were examined by radiochromatography. Male rabbit gallbladder microsomes converted small amounts of labelled arachidonate to total prostaglandin synthesis with PGE2, 6-keto PGF1 alpha (stable metabolite of PGI2) and PGF2 alpha as the major products synthesized. Microsomes from the male rabbit gallbladder inflamed by bile duct ligation for 3 days increased total prostaglandin synthesis five-fold with 6-keto PGF1 alpha being the major prostaglandin produced. Female rabbit gallbladder microsomes converted three times more arachidonate to total prostaglandin synthesis than did microsomes from the male rabbit. Bile duct ligation did not alter total prostaglandin biosynthesis in the female rabbit gallbladder, but significantly decreased synthesis of PGE2, thromboxane B2 and PGF2 alpha and increased synthesis of 6-keto PGF1 alpha. These data suggest that although bile duct ligation had different effects on male and female gallbladder total prostaglandin synthesis, 6-keto PGF1 alpha is the major product induced by this stimulus for acute inflammation.     

Metabolism of prostaglandins, prostaglandin analogs and thromboxane B2 by lung and liver microsomes from pregnant rabbits

Liver microsomes from pregnant rabbits converted prostaglandins F2 alpha, E1, and E2 to their 20-hydroxy metabolites along with smaller amounts of the corresponding 19-hydroxy compounds. Prostaglandins E1 and E2 were also reduced to prostaglandins F1 alpha and F2 alpha, respectively, and prostaglandin E1 was isomerized to 8-isoprostaglandin E1. The above products were also identified after incubation of prostaglandins with liver microsomes from non-pregnant rabbits. In this case, the yield of 20-hydroxy metabolites was much lower. Thromboxane B2 and a number of prostaglandin F2 alpha analogs were also hydroxylated by lung and liver microsomes from pregnant rabbits. The relative rates of hydroxylation by lung microsomes were: prostaglandin E2 approximately prostaglandin F2 alpha approximately 16,16-dimethylprostaglandin F2 alpha approximately 13,14-didehydroprostaglandin F2 alpha greater than thromboxane B2 greater than 15-methylprostaglandin F2 alpha approximately 17-phenyl-18,19,-20-trinorprostaglandin F2 alpha approximately ent-13,14-didehydro-15-epiprostaglandin F2 alpha. Similar results were obtained with liver microsomes except that thromboxane B2 was a relatively poorer substrate for hydroxylation.     

Arachidonic acid metabolism and prostaglandin production by primate preimplantation blastocysts.

Preimplantation embryos of many species are known to synthesize prostaglandins. These tissue hormones are believed to influence embryonic metabolism, as well as embryo-maternal interaction during implantation although their putative role(s) remains obscure. Here, prostaglandin production by blastocysts from cynomolgus monkeys (Macaca fascicularis) was examined qualitatively during in vitro culture. Tritium labelled arachidonic acid was metabolized to 6 keto-prostaglandin F1 alpha, 2,3-dinor-prostaglandin F1 alpha and thromboxane B2, as characterized by HPLC separation. Also, 6-keto-prostaglandin F1 alpha, and thromboxane B2 as characterized by HPLC separation. Also, 6-keto-prostaglandin F1 alpha and thromboxane B2 were identified by specific RIA's. Our data suggest that the main arachidonic acid metabolites produced by blastocysts of cynomolgus monkeys are prostacyclin and thromboxane.     

Urinary excretion of prostaglandins during infancy and childhood: influence of age, sodium restriction and posture

The influences of age, sodium restriction and posture on 24-hour urinary excretion of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF 2 alpha), 6-keto-prostaglandin F1 alpha (6-keto-PGF 1 alpha) and thromboxane B2 (TXB2) were investigated in 111 healthy children and youngsters in the age between 1 day and 16 years. A considerable degree of variation was found in normal 24-hour urinary prostaglandin excretion in all age groups. There was no significant effect of age on the urinary excretion of prostaglandins when data were corrected for body surface area. In addition, sodium restriction and posture had no influence on the excretion of PGE2, PGF 2 alpha, 6-keto-PGF 1 alpha and TXB2. Our results indicate that in the first days of life the kidney already has the capacity to synthesize prostaglandins in amounts comparable to older children.     

Transitional changes in arachidonic acid metabolism by bovine embryos at different developmental stages

In order to determine the profile of arachidonic acid (AA) metabolites synthesized by bovine embryos during early developmental stages, embryos collected from superovulated beef cattle (days 6 through 17) were incubated with AA and its metabolites were analyzed by high performance liquid chromatography and radioimmunoassay (RIA). Embryos harvested and cultured before day 12 of the estrous cycle metabolized AA primarily to prostaglandin E2 (PGE2), whereas, those harvested on day 13 of the cycle metabolized AA to both PGE2 and PGF2 alpha. Furthermore, embryos collected after day 15 of the cycle metabolized AA to PGI2 in addition to PGE2 and PGF2 alpha. In view of the luteotropic properties that have been attributed to PGE2 and the vasodilatory effect of PGI2, this transitional change in prostaglandin synthesis during early stages of embryonic development may be a part of the mechanism by which the embryo exerts a luteotropic effect leading to maternal recognition of pregnancy and by which the conceptus begins preparing for subsequent implantation.     

Cyclooxygenase products formed by primary cultures of cells from human chorion laeve: influence of steroids

Cells were isolated from human chorion laeve obtained at term (38-40 weeks gestation) by elective caesarean section and were maintained in primary culture for 1 week in defined media supplemented with 10% fetal calf serum. The production of various cyclooxygenase products by the cultures was examined. Little or no prostaglandin (PG) F2 alpha, 6-keto-PGF1 alpha, thromboxane B2, or 13,14-dihydro-15-keto-PGF2 alpha was found. In contrast, the cells produced PGE2 which was low on day 0, increased during culture to a maximum on day 1 or 2, then declined to low levels. When cells were grown in the presence of media containing cortisol, dexamethasone, progesterone, and estradiol (at 10(-7) or 10(-9) M), the glucocorticoids (at 10(-7) and 10(-9) M), but not estrogen or progesterone, markedly inhibited the increase in PGE2 output. There was no difference in the protein content and thymidine incorporation of cells grown in the presence of glucocorticoids when compared with controls. This inhibitory effect was not sensitive to cycloheximide (1 microgram/mL) indicating protein synthesis may not be involved in the process. These studies indicate that PGE2 is the major prostaglandin formed by primary cultures of chorion laeve and that prostaglandin metabolism in the chorion is sensitive to glucocorticoid inhibition.     

Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype.

A functional cDNA clone for mouse EP3 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of the human thromboxane A2 receptor and cross-hybridization screening. The mouse EP3 receptor consists of 365 amino acid residues with putative seven-transmembrane domains. The sequence revealed significant homology to the human thromboxane A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGE2 binding. The binding was displaced with unlabeled PGs in the order of PGE2 = PGE1 greater than iloprost greater than PGF2 alpha greater than PGD2. The EP3-selective agonists, M&B 28,767 or GR 63799X, potently competed for the [3H]PGE2 binding, but no competition was found with EP1- or EP2-selective ligands. PGE2 and M&B 28,767 decreased forskolin-induced cAMP formation in a concentration-dependent manner in Chinese hamster ovary cells permanently expressing the cDNA. Northern blot analysis demonstrated that the EP3 mRNA is expressed abundantly in kidney, uterus, and mastocytoma P-815 cells and in a lesser amount in brain, thymus, lung, heart, stomach, and spleen.