14C-labeled arachidonic acid bioconversion in guinea pig placenta during the last third of gestation

In the present study we investigated the arachidonic acid metabolism in guinea pig placenta during the last third of gestation. Homogenates were incubated with 14C-labeled substrate, and eicosanoid formation was determined using rp HPLC. Arachidonic acid was substantially converted to cyclooxygenase products i.e 6-keto-PGF1 alpha, TxB2, PGF2 alpha, PGE2, PGD2 and 12-HHT. Lipoxygenase activity was also found but of a much lower degree and represented by the mono-hydroxy acids 12-HETE and 15-HETE. The total conversion of arachidonic acid exhibited a progressive rise from day 50 to term, due principally to the increasing part of TxB2, PGE2 and 12-HHT throughout this gestational period and in addition, near term, of 6-keto-PGF1 alpha and PGF2 alpha. These results suggest that there is an increasing concentration and/or activity of cyclooxygenase system enzymes with placental development in guinea pig, which may contribute to the augmented intrauterine availability of prostanoids near parturition. Additional experiments were performed to compare the metabolism of exogenously added 14C-arachidonic acid and endogenously present 12C-arachidonic acid during placental homogenate incubation by means of isotope dilution GC-MS. Although the 14C- and 12C-prostanoid patterns were comparable, the 14C/12C ratios of the prostanoids formed during incubation were significantly different. These data indicate that exogenous arachidonic acid and endogenous arachidonic acid in placental homogenate do not follow up exactly the same metabolic pathway so that the assumption of biochemical identity between exogenous radio-tracer and studied endogenous substrate is not quite true.     

Ultrastructural studies of the rabbit placenta in the last third of gestation.

Placental attachment and the ultrastructure of the decidua and placental labyrinth have been studied in rabbits during the final third of gestation. The placenta became progressively easier to separate from the uterine wall as gestation proceeded. This ease of separation was associated with degenerative changes in the decidual tissue, but disruption of the placental labyrinth was not observed until the last 24 hr of pregnancy. Two types of decidual cells were observed; smaller uninucleate glycogen-containing cells and larger multinucleate cells with lipid inclusions. The ageing placentae exhibited increasing decidual degeneration associated with deposition of extracellular fibrous materials. Glycogen became less widely distributed over the period of study and changed from the beta- to the alpha-configuration. In contrast to the observed disruption of the decidual tissue, the placental labyrinth maintained its integrity until the final stages of pregnancy. A dramatic increase in subcellular activity was observed in the syncytiotrophoblast after 28 days of gestation.     

Changes in permeability of fetal guinea pig skin during gestation

Permeability of fetal skin to tritiated water was measured in vitro using samples taken from the back and flanks of 21 guinea pig fetuses whose gestational age ranged from 30 to 67 days (term = 68 days). From 30 to 45 days, fetal skin was relatively permeable to water, with a permeability coefficient for unidirectional, diffusional transfer of labelled water that averaged 0.372 +/- 0.041 (SEM) X 10(-4) cm/s. Then during a 5-10 day interval, the measured permeability coefficient decreased abruptly to very low and barely detectable levels. These changes took place at the time during gestation when others have shown the skin becomes keratinized and growth of new hair follicles is completed. Thus these findings are consistent with a relatively free exchange of water between amniotic fluid and fetal interstitium across the skin during the first two-thirds of gestation and then with further maturation an abrupt functional separation between these fluid compartments during the last third of gestation.     

Metabolism of arachidonic acid by guinea pig Clara cells.

A method for the isolation of non-ciliated bronchiolar epithelial (Clara) cells from the guinea pig is described. Following digestion of the lung tissue with Type XXIV protease, the isolated lung cells showed a viability greater than 90% and contained 3% of Clara cells. Several cell populations were then separated on the basis of size using 2 centrifugal elutriations. The macrophages and endothelial cells were removed from the Clara cells enriched fractions by differential adherence on Petri dishes. The Clara cell-rich suspension was then further purified by centrifugation on Percoll non-continuous density gradients consisting of 48-52-55% Percoll solution. The lower interface and the pellet of the non-continuous gradient consisted of approximately 80% Clara cells. Identification of isolated Clara cells was confirmed by light microscopic observations after nitroblue tetrazolium staining and by ultrastructural characteristic features as observed by electron microscopy. The metabolism of arachidonic acid into prostaglandins and TxB2 by purified Clara cells was examined by enzyme immunoassay (EIA) and leukotriene formation was investigated by reverse phase high performance liquid chromatography (RP-HPLC). Enriched guinea pig Clara cells incubated with arachidonic acid released TxB2, PGE2 and 6-keto PGF1 alpha, but did not produce leukotrienes. These cells could however transform exogenous leukotriene A4 into leukotriene B4. These results suggest that guinea pig Clara cells possess the enzymes of the cyclooxygenase pathway required for TxB2, PGE2 and 6-keto-PGF1 alpha synthesis. Clara cells do not possess the 5-lipoxygenase enzyme but show some leukotriene A4 hydrolase activity since they can produce leukotriene B4 upon incubation with leukotriene A4.     

Measurement of arachidonic acid release from permeabilised myometrial cells of guinea pig uterus.

A technique has been developed for prelabelling and permeabilisation of guinea pig uterine myocytes to enable measurement of arachidonic acid release/phospholipase A2 activity in cells with intact membranes. Intact cells were prelabelled with [3H]inositol or [3H]arachidonic acid for measurement of phospholipase C and A2 respectively. In intact cells 10 nM endothelin-1 or 1 microM bradykinin stimulated both inositol polyphosphate and arachidonic acid release, whilst 1 microM oxytocin, arginine vasopressin or histamine were without effect. In Streptolysin-O permeabilised myometrial cells calcium-stimulation of inositol polyphosphate and arachidonic acid release was detected between 10 microM and 1 mM free calcium. The patterns of inositol polyphosphate and arachidonic acid release were broadly similar. Responses to 1 mM calcium were not detected in intact cells not treated with Streptolysin-O. For arachidonic acid release the K0.5 for calcium activation was about 7 microM, a level above that normally likely to be found in the uterine myocyte. Hence it is concluded that unless there are high local concentrations of calcium close to the plasma membrane, calcium is unlikely alone to be the primary regulator of arachidonic acid release and phospholipase A2.     

Testicular responsiveness to hCG of the ovine fetus in the last third of gestation

The in vivo and in vitro testicular responsiveness to hCG of hemicastrated lamb fetuses 95-99, 110-118 and 130-141 days of gestational age was studied. Basal plasma testosterone (T) levels were similar at all ages (less than 0.25 ng/ml), while the mean testicular concentrations of dehydroepiandrosterone sulfate (DHA-S), 17 alpha-hydroxyprogesterone (17-OHP) and T were higher in 95- to 99-day-fold fetuses. Plasma T levels and the concentration of T, DHA-S, 17-OHP, androstenedione (A) and cyclic adenosine 3'5'-monophosphate (cAMP) were increased by hCG in the hemicastrated animal at all ages. cAMP and T production by enriched preparations of dispersed interstitial cells from control testes was increased by hCG in all groups. In fetuses pretreated with hCG in vivo the addition of hCG in vitro failed to modify cAMP and T production. 100 micrograms of LHRH to a 130-day-old fetus increased plasma LH and T levels. From these experiments, it is suggested that the low plasma LH and T levels found throughout the last trimester of fetal life reflect a relative lack of endogenous LHRH synthesis and/or release, rather than reduced testicular steroidogenic capacity.     

Leukotriene F4 and the release of arachidonic acid metabolites from perfused guinea pig lungs in vitro

Radioimmunoassay and bioassay techniques have been used to investigate the ability of leukotriene (LT)F4 to release products of arachidonic acid metabolism from guinea pig isolated lungs perfused via the pulmonary artery. Also, the abilities of LTC4, LTD4, LTE4 and LTF4 to contract guinea pig ileal smooth muscle (GPISM) was studied. Each of the LT's contracted GPISM. The rank order of potency was LTD4 greater than LTC4 greater than LTE4 much greater than LTF4 in a ratio of 1:7:170:280 respectively. Bioassay of pulmonary effluents indicated the passage of LTF4 through the lungs caused a contraction of rabbit aorta as well as an FPL-55712 sensitive contraction of GPISM. The contractions of rabbit aorta were inhibited by pretreatment of the lungs with Indomethacin but not with the thromboxane synthetase inhibitor Dazoxiben. Radioimmunoassay of the lung effluents indicated LTF4 to cause a 70-fold increase in thromboxane B2 (TXB2), 4-fold increase in prostaglandin (PG)E2 and a 16-fold increase in 6-keto PGF1 alpha levels. The LTF4-induced increments of these immunoreactive metabolites was inhibited by pretreatment of the lungs with Indomethacin. Pretreatment of lungs with Dazoxiben inhibited the LTF4-induced increment in TXB2 and enhanced the effluent levels of PGE2 24-fold (compared with untreated lungs). There were no detectable differences in either immunoreactive LTC4 or immunoreactive LTB4 levels. It is concluded LTF4 is a relatively weak agonist on GPISM and can induce the release of cyclooxygenase products of arachidonic acid metabolism from guinea pig perfused lung.     

Inhibition of arachidonic acid-induced contraction of guinea pig lung strips

The effects of several enzyme inhibitors on arachidonic acid-induced contractions of guinea pig lung strips were studied. Varying concentrations of indomethacin, an inhibitor of cyclooxygenase, produced only a limited effect on contraction of tissue strips. By contrast, nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), and phenidone, which inhibit either lipoxygenase, or both lipoxygenase and cyclooxygenase, caused a dose-related antgonism of the arachidonic acid-induced contraction. The effects of these latter agents were similar to that of FPL 55712. Results indicate that the products of cyclooxygenase are predominantly involved in the early phase and the products of lipoxygenase are predominantly related to the late phase of arachidonic acid-induced contraction.     

Effect of fat free diet during the last week of pregnancy upon the linoleic and arachidonic acid content of fetal organ lipids and placenta lipids of the rat

Pregnant Wistar rats were fed a fatfree diet from day 16--22 of pregnancy. On day 22, the fatty acid components of cholesterol esters, triglycerides, free fatty acids and phospholipids of maternal (brain, muscle, serum, white adipose tissue, liver) and fetal (brain, carcass, serum, liver) tissues, including the placenta, were examined gaschromatographically for the participation of linoleic and arachidonic acid. In all fetal and maternal organs the linoleic acid levels in the fatty acid patterns were strongly reduced. The alterations nearly always involved all the lipid fractions of a tissue and were mostly equal within a tissue. The strongest decreases of linoleic acid occurred in the placenta, and the weakest, in the lipids of maternal muscle and maternal adipose tissue. The linoleic acid alterations were principally similar in fetal and the corresponding maternal tissues, while being less pronounced in case of maternal muscle. The participation of arachidonic acid in the fatty acid pattern is completely retained in the lipids of fetal organs, and is even enhanced in those of the placenta.