Arachidonic acid metabolism in growth control of A549 human lung adenocarcinoma cells

The role of individual eicosanoids of the arachidonic acid (AA) cascade in the growth control of A549 human lung adenocarcinoma cells has been studied. Cyclooxygenase and lipoxygenase metabolites of [¹⁴C]AA incorporated were actively synthesized in the cultures of tumor cells with full confluence unaccomplished. In such cultures inhibitors of AA metabolism (indomethacin and esculetin) and also a lipoxygenase metabolite of AA, 15-hydroxyeicosatetraenoic acid (15-HETE), significantly suppressed the incorporation of [³H]thymidine and biosynthesis of prostaglandin E₂(PGE₂). Other lipoxygenase metabolites of AA (5-HETE and 12-HETE) had no effect on these parameters. The basic fibroblast growth factor (bFGF) had practically no affect on the growth of A549 cells and the PGE₂ production in cultures with 5% fetal calf serum (FCS); however, in the presence of 0.5% FCS this factor significantly increased the number of tumor cells. The growth-stimulating effect of bFGF was completely abolished by a cyclooxygenase inhibitor indomethacin. The data suggest a key role of PGE₂ in the growth control of A549 cells with an active synthesis of cyclooxygenase and lipoxygenase metabolites of AA, its importance in realization of the mitogenic effect of bFGF, and specific features of 15-HETE as a down-regulator of the PGE₂-dependent proliferation.     

Arachidonic acid metabolic pathway of the rabbit placenta

Placenta microsomes prepared from animals late in gestation (29 days) efficiently metabolize arachidonic acid into PGE2, PGF2 alpha, PGD2, TxA2 and little or no prostacyclin. In contrast to the late gestation placenta, the early (17 day) placental microsomes synthesize primarily PGE2. The cytosolic (100,000 X g supernatant) fraction from early or late gestation placentae converted arachidonic acid, with a calcium dependent enzyme, into non-polar metabolites whose synthesis was inhibited by ETYA but not indomethacin. These metabolites were purified by HPLC and GC-MS analysis indicated the presence of 12-hydroxy-, 15-hydroxy-, and 11-hydroxy-eicosatetraenoic acid. The mitochondrial (8,000 X g pellet) produced PGE2; PGF2 alpha; 12-, 11-, 15-HETE; the C-17 fragment HHT; and the unusual cyclooxygenase metabolite 15-keto-PGE2. These biologically active metabolites may play a vital role in the reproductive function of the placenta.     

Arachidonic Acid Oxidation by Brain and Placenta Preparations from Normal and Placental Insufficient Fetal Rabbit

Cytosolic (100,000 g) fractions of fetal rabbit brain and placenta tissue convert [1-14C]arachidonic acid into several oxidation products identified with the lipoxygenase [12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE] and cyclooxygenase [prostaglandin E2 (PGE2)] pathways. Formation of 12-HETE and 15-HETE by fetal brain is time-dependent, reaching a plateau after 40 min and is linear with protein concentration. An apparent affinity constant of 0.06 mM and a Vmax of 0.1 mumol/h/g wet weight are presumably responsible for the excessive accumulation of 12-HETE and 15-HETE in comparison to PGE2 (Km = 0.5 mM). The latter is synthesized by the placenta particulate fraction but almost exclusively by the brain cytosol. Compared to brain, the activity of the placenta tissue is exceedingly higher and in addition to 12-HETE and 15-HETE there is a substantial formation of 12-L-hydroxyheptadecatrienic acid. Formation of 12-HETE and 15-HETE at 21 days is as effective as at 31 days gestation and is strongly inhibited by nordihydroguaiaretic acid (93%), BW755c (99%), and AA861 (84%) but not by indomethacin. Placenta and brain tissues of intrauterine growth retarded fetuses after ligation of placental blood vessels fail to convert arachidonic acid into other eicosanoids. Loss of enzymatic activity also observed in normal tissue after prolonged storage cannot be restored by the addition of several SH agents, ascorbate, or ferric iron.     

Placental synthesis of estrogens at parturition and during placental retention in the cow

Nine cows were submitted to lutectomy at 250 or 270 d of pregnancy and catheters were implanted in the jugular, carotid, uterine artery and uterine vein to determine endocrine changes following lutectomy and throughout parturition. Blood samples were collected at 8-h intervals and assayed for estrogens. Fetal and maternal placental tissues were also collected at parturition and 3 d postpartum for incubation studies on estrogen synthesis. Based on plasma concentrations, the uterus is able to secrete considerable quantities of unconjugated and conjugated estrone (E1) and estradiol 17 μ (E2α) at both 250 and 270 d of gestation. In vitro conversion of androstendione to total estrogens averaged 32.4% and 16.8% for fetal and maternal tissues at parturition, respectively. Incubation of placental tissues collected from animals with placental retention on Day 3 postpartum resulted in conversion of 3.2 and 4.6% of androstendione by fetal and maternal tissues, respectively. One cow which retained the placenta was sampled until 3 d postpartum and assay of the plasma estrogen content indicated that there was always a higher concentration of estrogen in the uterine vein than in the uterine artery, supporting the in vitro incubation data.     

Immunohistochemical localization of prostaglandin H synthase in the sheep placenta from early pregnancy to term.

Prostaglandin H synthase (PGHS) activity within intrauterine tissues is considered to catalyze a critical step in prostaglandin (PG) biosynthesis at parturition. In sheep, the placenta is a major site of PG production throughout pregnancy, but little information is available concerning the cells that are responsible. Therefore we determined the distribution of immunoreactive (IR-) PGHS in ovine placental tissue obtained at different times of pregnancy using immunohistochemical techniques. In placentomes from early pregnancy (Days 30-54), IR-PGHS was present in maternal epithelial syncytium, but was not detectable in trophoblast cells. Between Day 54 and Day 100, the number of cells that stained positive for PGHS declined in the maternal epithelial layer in the body of the placenta, but IR-PGHS was present in maternal epithelial cells overlying the vascular cones of the placental hemophagous zone. It was also present in the chorionic fibroblasts, but remained undetectable from all classes of trophoblast cells. IR-PGHS was first detectable in the trophoblastic epithelium by Day 114. Between Day 119 and term the trophoblast mononuclear epithelial cells were intensely immunopositive for PGHS, although immunonegative binucleate cells were present. The maternal epithelium was immunonegative except during the last 7-10 days of pregnancy when PGHS immunostaining appeared in both basal and apical regions of the placenta. Thus, the cellular localization of IR-PGHS changes during ovine pregnancy, from predominantly maternal during the first half of gestation to undetectable and then to predominantly trophoblastic between Day 114 and term, suggesting a gestation-dependent change in sites of PG production during ovine pregnancy. Appearance of IR-PGHS in the trophoblast precedes activation of the fetal hypothalamic-pituitary-adrenal axis, generally considered to provide the trigger to the onset of parturition in sheep, and would therefore appear to be regulated through alternative pathways or mechanisms.     

Late but not early gestational maternal growth hormone treatment increases fetal adiposity in overnourished adolescent sheep

In the overnourished adolescent sheep, maternal tissue synthesis is promoted at the expense of placental growth and leads to a major decrease in lamb birth weight at term. Maternal growth hormone (GH) concentrations are attenuated in these pregnancies, and it was recently demonstrated that exogenous GH administration throughout the period of placental proliferation stimulates uteroplacental and fetal development by Day 81 of gestation. The present study aimed to determine whether these effects persist to term and to establish whether GH affects fetal growth and body composition by increasing placental size or by altering maternal metabolism. Adolescent recipient ewes were implanted with singleton embryos on Day 4 postestrus. Three groups of ewes offered a high dietary intake were injected twice daily with recombinant bovine GH from Days 35 to 65 of gestation (high intake plus early GH) or from Days 95 to 125 of gestation (high intake plus late GH) or remained untreated (high intake only). A fourth moderate-intake group acted as optimally nourished controls. Pregnancies were terminated at Day 130 of gestation (6 per group) or were allowed to progress to term (8-10 per group). GH administration elevated maternal plasma concentrations of GH, insulin, glucose, and nonesterified fatty acids during the defined treatment windows, while urea concentrations were decreased. At Day 130, GH treatment had reduced the maternal adiposity score, percentage of fat in the carcass, and internal fat depots and leptin concentrations, predominantly in the high-intake plus late GH group. Placental weight was lower in high-intake vs. control dams but independent of GH treatment. In contrast, fetal weight was elevated by late GH treatment, and these fetuses had higher relative carcass fat content, perirenal fat mass, and liver glycogen concentrations than all other groups. Expression of leptin mRNA in fetal perirenal fat and fetal plasma leptin concentrations were not significantly altered by maternal nutritional intake or GH. In pregnancies proceeding to term, the duration of gestation, fetal placental mass, and lamb birth weight were reduced in high-intake compared with control dams but were not significantly affected by GH treatment. In conclusion, exogenous GH has profound effects on maternal endocrinology, metabolism, and body composition when administered during early and late pregnancy. Treatment during late pregnancy has a modest effect on fetal growth independent of placental size and a profound effect on fetal adiposity, which may have implications beyond the fetal period.     

Steroid synthesis by the fetal part of the bovine placenta of late pregnancy in vitro: Effect of a low dose of dexamethasone in vivo

Pregnant cows were treated with 5 mg dexamethasone (DEX) (n = 5) or 0.9% saline (n = 3) on Day 255 of gestation. Placentomes were removed on Day 250, Day 260 by laparotomy, and immediately after parturition. Fetal placental tissue (500 mg) was homogenized and frozen for the determination of initial tissue content of progesterone (P(4)) and estrone (E(1)) by radioimmunoassay (RIA). Tissue samples (500 mg) were incubated for 1, 5, or 9 h in the presence of 0, 10, or 100 ng/ml androstenedione (A) or pregnenolone (P(5)). After incubation, tissue was homogenized in the incubation medium (HOM) and P(4) and E(1) content assessed by RIA. Finally, tissue was incubated in medium containing (3)H-A or (3)H-P(5) with or without cortisol (70 ng/ml) to measure conversion of precursors into estrogens (E). Overall initial tissue concentration of P(4) decreased as pregnancy progressed (P < 0.05). An interaction between DEX treatment and stage of gestation (P < 0.05) indicated that P(4) decreased only in tissue from DEX-treated cows, whereas P(4) in tissue from control cows remained constant. Tissue concentrations of E(1) increased from Day 250 to parturition (P < 0.05) and were not influenced by in vivo DEX treatment. Progesterone concentration in HOM increased during incubation indicating P(4) synthesis (P < 0.001). Addition of 100 ng/ml P(5) stimulated P(4) production (P < 0.05). There was a larger increase in P(4) in HOM from DEX-treated cows than in HOM from control cows (P < 0.05). Concentration of E(1) decreased during incubation (P < 0.0001) and was not influenced by the addition of A or P(5). Estrogen synthesis was indicated by the conversion of A or P(5) into E (pmol/g/h). The conversion of precursors (A or P(5)) into E increased during gestation and was not influenced by in vivo DEX treatment. Cortisol application in vitro did not affect E synthesis. It is concluded that treatment with 5 mg DEX in vivo on Day 255 of pregnancy does not seem to influence placental E synthesis in vitro. However, the decrease in initial P(4) tissue concentration and the increase in P(4) synthesis by fetal placental tissue from DEX-treated cows in vitro suggest that DEX treatment in vivo stimulates P(4) synthesis with increased metabolism of P(4) into other metabolites.     

Studies on the equine placenta II. Ultrastructure of the placental barrier.

In early pregnancy the equine placenta consists of a simple apposition of fetal and maternal epithelia, but it becomes more complex with the formation of microcotyledons between 75 and 100 days of gestation. Although the placental barrier maintains an epitheliochorial arrangement throughout the course of pregnancy, a thinning of the maternal epithelium and a progressive indentation of the chorionic epithelium by fetal capillaries shortens the length of the diffusion pathway and reduces the amount of placental tissue between fetal and maternal bloodstreams. These structural modifications may reflect the changing requirements of the fetus for O2 and other metabolites as gestation proceeds. During the first 200 days of pregnancy there is evidence of intense pinocytotic activity by the cells of the trophoblast. From the 100th day of pregnancy there is a pronounced development of smooth endoplasmic reticulum, while rough endoplasmic reticulum and irregular, dense, membrane-bound bodies are a prominent feature of the paranuclear cytoplasm from Day 200. These changes suggest that the cells of the trophoblast become more highly involved in synthetic processes with increasing gestational age.     

Reduced progesterone metabolites in human late pregnancy

In this review, we focused on the intersection between steroid metabolomics, obstetrics and steroid neurophysiology to give a comprehensive insight into the role of sex hormones and neuroactive steroids (NAS) in the mechanism controlling pregnancy sustaining. The data in the literature including our studies show that there is a complex mechanism providing synthesis of either pregnancy sustaining or parturition provoking steroids. This mechanism includes the boosting placental synthesis of CRH with approaching parturition inducing the excessive synthesis of 3beta-hydroxy-5-ene steroid sulfates serving primarily as precursors for placental synthesis of progestogens, estrogens and NAS. The distribution and changing activities of placental oxidoreductases are responsible for the activation or inactivation of the aforementioned steroids, which is compartment-specific (maternal and fetal compartments) and dependent on gestational age, with a tendency to shift the production from the pregnancy-sustaining steroids to the parturition provoking ones with an increasing gestational age. The fetal and maternal livers catabolize part of the bioactive steroids and also convert some precursors to bioactive steroids. Besides the progesterone, a variety of its 5alpha/beta-reduced metabolites may significantly influence the maintenance of human pregnancy, provide protection against excitotoxicity following acute hypoxic stress, and might also affect the pain perception in mother and fetus.     

Identification of a Feed-Forward Loop Between 15(S)-HETE and PGE2 in Human Amnion at Parturition

Human parturition is associated with massive arachidonic acid (AA) mobilization in the amnion, indicating that large amounts of AA-derived eicosanoids are required for parturition. Prostaglandin E2 (PGE2) synthesized from the cyclooxygenase (COX) pathway is the best characterized AA-derived eicosanoid in the amnion which plays a pivotal role in parturition. The existence of any other pivotal AA-derived eicosanoids involved in parturition remains elusive. Here, we screened such eicosanoids in human amnion tissue with AA-targeted metabolomics and studied their role and synthesis in parturition by using human amnion fibroblasts and a mouse model. We found that lipoxygenase (ALOX) pathway-derived 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and its synthetic enzymes ALOX15 and ALOX15B were significantly increased in human amnion at parturition. Although 15(S)-HETE is ineffective on its own, it potently potentiated the activation of NF-κB by inflammatory mediators including lipopolysaccharide, interleukin-1β, and serum amyloid A1, resulting in the amplification of COX-2 expression and PGE2 production in amnion fibroblasts. In turn, we determined that PGE2 induced ALOX15/15B expression and 15(S)-HETE production through its EP2 receptor-coupled PKA pathway, thereby forming a feed-forward loop between 15(S)-HETE and PGE2 production in the amnion at parturition. Our studies in pregnant mice showed that 15(S)-HETE injection induced preterm birth with increased COX-2 and PGE2 abundance in the fetal membranes and placenta. Conclusively, 15(S)-HETE is identified as another crucial parturition-pertinent AA-derived eicosanoid in the amnion, which may form a feed-forward loop with PGE2 in parturition. Interruption of this feed-forward loop may be of therapeutic value for the treatment of preterm birth.     

Lipoxygenases in rat embryo tissue

It has previously been reported that rat embryonic tissue produces various prostanoids. This report demonstrates that rat embryo homogenates synthesized various lipoxygenase metabolites, including 12-hydroxyeicosatetraenoic acid (12-HETE) as the major metabolite, 5-HETE, and 15-HETE. The cyclooxygenase product 11-HETE was also formed. Product identification was based on radioimmunoassay and comparison of reverse-phase- and straight-phase-high-pressure liquid chromatography retention times with authentic standards. Additional evidence was the observation that the lipoxygenase inhibitor nordihydroguaiaretic acid inhibited HETE formation. It appears that, under the same (though not necessarily optimal) experimental conditions, lipoxygenase metabolites predominate quantitatively over cyclooxygenase pathway products and that 11-day embryonic tissue produces more HETEs than either 12-day or 13-day embryo homogenates.     

Placental growth, angiogenic gene expression, and vascular development in undernourished adolescent sheep

Limiting maternal nutrient intake during ovine adolescent pregnancy progressively depleted maternal body reserves, impaired fetal nutrient supply, and slowed fetal soft tissue growth. The present study examined placental growth, angiogenic gene expression, and vascular development in this undernourished adolescent model at Days 90 and 130 of gestation. Singleton pregnancies were established, and ewes were offered an optimal control (C; n = 14) or low (L [0.7 x C]; n = 21) dietary intake. Seven ewes receiving L intakes were switched to C intakes on Day 90 of gestation (L-C). Fetal body weight (P < 0.01) and glucose concentrations (P < 0.03) were reduced in L versus C pregnancies by Day 130, whereas L-C group values were intermediate. Placental cellular proliferation, gross morphology, and mass were independent of maternal nutrition at both Day 90 and 130. In contrast, capillary area density in the maternal caruncular portion of the placentome was reduced by 20% (P < 0.001) at both stages of gestation in L compared with C groups. Caruncular capillary area density was equivalent in the L and L-C groups at Day 130. Placental mRNA expression of five key angiogenic ligands or receptors increased (P < 0.001) between Days 90 and 130 of gestation. VEGFA mRNA expression was higher (P < 0.04) in L compared with C and L-C pregnancies at Day 130, but otherwise gene expression of the remaining angiogenic factors and receptors analyzed was unaffected by maternal intake. Undernourishing the pregnant adolescent dam restricts fetal growth independently of changes in placental mass. Alterations in maternal placental vascular development may, however, play a role in mediating the previously reported reduction in maternal and hence fetal nutrient supply.     

Effects of lipoxygenase metabolites of arachidonic acid on the growth of human mononuclear marrow cells and marrow stromal cell cultures.

The effects of various lipoxygenase metabolites of arachidonic acid (AA) were investigated on the growth of freshly isolated human bone marrow mononuclear cells and marrow stromal cell cultures. LTB4, LXA4, LXB4, 12-HETE and 15-HETE (1 microM) decreased [3H]-thymidine incorporation on marrow stromal cell cultures without affecting cell number. Only 12-HETE showed a dose-response effect on [3H]-thymidine incorporation. While LTB4 (1 microM) decreased thymidine incorporation on marrow mononuclear cells, LTC4, LXA4, LXB4, 12-HETE and 15-HETE had no effect. The lipoxygenase inhibitor NDGA had no effect on both cell types suggesting no role of endogenous lipoxygenase metabolites on cell growth. These results suggest no important role of lipoxygenase metabolites of AA on the proliferation of human marrow mononuclear cells and marrow stromal cell cultures.     

Pre-parturition profile of steroids and prostaglandin in cows with or without foetal membrane retention

Retained foetal membranes in cattle is one of the most common complications associated to the reduction in milk yield and impaired fertility in dairy cattle. In order to determine some endocrine mechanisms controlling parturition and delivery of foetal membranes, plasma concentrations of steroids and prostanoids were determined in 20 healthy Holstein cows. Samples were taken within the interval of 5 days pre-parturition to 12h after calving. Progesterone (P4) levels were similar in cows with (PR) and without (NPR) placental retention. While the estradiol-17beta (E2) peak at parturition was lower in PR than in NPR cows, cortisol levels were greater in PR cows 12 and 24h pre-parturition. The Prostaglandin F2alpha metabolite (PGFM) levels were higher at parturition in NPR compared with the PR group, but 12h later, these levels in the PR group increased so that concentrations were greater as compared with NPR cows. The Prostaglandin E2 metabolite (PGEM), 24, 48 and 72 h pre-parturition, were higher in PR cows. However, the PGFM:PGEM ratio was greater in cows up NPR at all time when included, indicating the importance of higher levels of Prostaglandin F2alpha (PGF2alpha) than Prostaglandin E2 (PGE2) for normal placental delivery. In conclusion, placental retention was related to both estrogen and PGF2alpha deficiency, which may be a consequence of metabolic stress leading to PGE2 and maternal cortisol synthesis before parturition.     

Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells

Arachidonic acid is metabolized via the cyclooxygenase pathway to several potent compounds that regulate important physiological functions in the cardiovascular system. The proaggregatory and vasoconstrictive thromboxane A2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of prostacyclin (prostaglandin I2) synthesized by blood vessels. Furthermore, arachidonic acid is metabolized by lipoxygenase enzymes to different isomeric hydroxyeicosatetraenoic acids (HETE's). This metabolic pathway of arachidonic acid was studied in detail in endothelial cells obtained from bovine aortae. It was found that this tissue produced 6-ketoprostaglandin F1 alpha as a major cyclooxygenase metabolite of arachidonic acid, whereas prostaglandins F2 alpha and E2 were synthesized only in small amounts. The monohydroxy fatty acids formed were identified as 15-HETE, 5-HETE, 11-HETE and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The latter two compounds were produced by cyclooxygenase activity. Nordihydroguaiaretic acid (NDGA), a rather selective lipoxygenase inhibitor and antioxidant blocked the synthesis of 15- and 5-HETE. It also strongly stimulated the cyclooxygenase pathway, and particularly the formation of prostacyclin. This could indicate that NDGA might exert its effect on prostacyclin levels by preventing the synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a potent inhibitor of prostacyclin synthetase. 15-HPETE could therefore act as an endogenous inhibitor of prostacyclin production in the vessel wall.     

Amniotic fluid lipoxygenase metabolites during spontaneous labor and after RU486 treatment during late pregnancy in rhesus macaques

To determine changes in amniotic fluid (AF) lipoxygenase metabolites prior to spontaneous labor and after RU486 administration, we implanted AF and vascular catheters and myometrial electromyographic (EMG) electrodes in 8 rhesus macaques at 120-130 days of pregnancy (term = 167 days). Four animals had AF samples taken serially until they delivered their infants normally at term. The other four animals received RU486 (20 mg/kg/day) for 3 days. AF samples were collected every 2-3 days and at 12 hour intervals for 72 hours before and after treatment with RU486. Uterine activity was monitored continuously. LTB4, 5-HETE and 15-HETE were measured by radioimmunoassay. In untreated animals, LTB4 and 5-HETE concentrations in AF increased significantly (P less than 0.05) 4 days before delivery with no change in 15-HETE. After RU486, mean levels of LTB4 and 5-HETE were increased although the difference was not statistically significant. No change in 15-HETE levels was observed. In conclusion, LTB4 and 5-HETE increase in AF before the onset of spontaneous labor. Progesterone receptor blockade by RU486 does not reproduce the changes in AF lipoxygenase metabolites observed during normal parturition.     

Lipoxygenase Pathway of Arachidonic Acid Metabolism in Growth Control of Tumor Cells of Different Type

The influence of inhibitors of different lipoxygenases (LOX) on the growth of human tumor cells with different profiles of synthesized eicosanoids was studied. The studied LOX inhibitors had virtually no influence on the growth of A549 cells actively synthesizing cyclooxygenase and lipoxygenase metabolites of arachidonic acid (AA). The inhibitor of 12-LOX, baicalein, significantly inhibited proliferation in cultures of A431 epidermoid carcinoma cells with a characteristic domination of the major lipoxygenase metabolite of AA, 12-hydroxyeicosatetraenoic acid (12-HETE), in the profile of synthesized eicosanoids and reduced to 70% the incorporation of [3H]thymidine into DNA. Treatment of these cultures with 12-HETE virtually restored the growth potential of the tumor cells. The findings suggest that the lipoxygenase metabolite of AA, 12-HETE, is a growth-limiting factor for tumor cells of definite type.     

Alterations of intrauterine eicosanoid production in pregnancy-induced hypertension: Decreased production of 12-hydroxyeicosatetraenoic acid in the placenta

The important role of eicosanoids in pregnancy-induced hypertension is generally accepted. Because of the lack of innervation of the uteroplacental vessels, humoral vasoactive factors are important for the regulation of vascular tone. Until now, mainly the balance of vasodilatative and vasoconstrictive prostaglandins has been studied. We were able to confirm their intrauterine imbalance in hypertensive pregnancies. In addition, the placental production of less known lipoxygenase metabolites has been analyzed in this study. Intrauterine tissues (30–100mg wet weight) were examined for their release of eicosanoids. Short term tissue cultures were performed in Hanks balanced salts solution (HBSS) at 37°C in an atmosphere of 95% air/5% CO₂ with and without incorporation of tritiated arachidonic acid. The arachidonate metabolites in culture media were analyzed by High Performance Liquid Chromatography (HPLC) with radioactivity detection or by enzymeimmunoassays or radioimmunoassays, respectively. All intrauterine tissues released more lipoxygenase metabolites than cyclooxygenase metabolites with 12-hydroxyeicosatetraenic acid (12-HETE) as their main metabolite. The placental release of 12-HETE was significantly decreased in hypertensive pregnancies. In hypertensive pregnancies the ratio TXB₂/6-keto-PGF_1α synthesis was increased.Lipoxygenase metabolites, especially 12-HETE, seem to have impotant physiological and pathophysiological functions in the intrauterine compartment. Their biological role in this context needs further investigation.     

The production of 5-HETE and leukotriene B in rat neutrophils from carrageenan pleural exudates

Rat neutrophils isolated from three-hour carrageenan pleural exudates actively metabolize arachidonic acid into three major metabolites, HHT, 11-HETE and 15-HETE. However, in the presence of the calcium ionophore, A23187, or the non-ionic detergent, BRIJ 56, these cells also produce 5-HETE and LTB. The production of these lipoxygenase products is calcium dependent. While non-steroidal anti-inflammatory drugs do not affect 5-HETE or LTB production, BW 755C and ETYA inhibit formation of these metabolites from exogenously added arachidonic acid.     

Measurement of plasma and tissue relaxin concentrations in the pregnant hamster and fetus using a homologous radioimmunoassay

A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 +/- 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 +/- 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 +/- 0.001 microg/g tissue), increased to Day 15 (6.96 +/- 0.86 microg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.