Prostaglandin endoperoxides. VII. Novel transformations of arachidonic acid in guinea pig lung

The following labeled compounds were isolated and identified after incubation of [1-¹⁴C]arachidonic acid with guinea pig lung homogenates: 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), the hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9,12-dihydroxy-5,10-heptadecadienoic acid (PHD), 12-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), PGE₂, PGF_2α, 11-hydroxy-5,8,12,14-eicosatetraenoic acid, and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (in order of decreasing yield). Perfused guinea pig lungs released PHD (654–2304 ng), HHT (192–387 ng), HETE (66–111 ng), PGE₂ (15–93 ng), and PGF_2α (93–171 ng) following injection of 30 μg of arachidonic acid. Thus guinea pig lung homogenates as well as intact guinea pig lung converted added arachidonic acid predominantly into PHD and HHT, metabolites of the prostaglandin endoperoxide PGG₂, and to a lesser extent into the classical prostaglandins PGE₂ and PGF_2α.     

Lipoxygenase conversion of arachidonic acid in males and inseminated females of the firebrat,Thermobia domestica (Thysanura)

This is the first investigation concerning prostaglandin-like compounds in the primitive insect, Thermobia domestica. The incubation of homogenates of reproductive tissues in the presence of [U-¹⁴C]arachidonic acid yielded several compounds which have been characterized by their chromatographic mobilities as well as by the enzyme systems involved in their formation. The three major compounds (I to III) had R_f values very different from those of several prostaglandin standards (PGE₂, PGF_2α and 6-keto PGF_1α). As the addition of aspirin or indomethacin had no effect on the conversion of arachidonic acid, a cyclo-oxygenase pathway leading to prostaglandins seems to be excluded. However, another compound (noted V), present in very small quantities, could be a prostaglandin, owing to its chromatographic mobility near that of the PGE₂ standard. By contrast, compounds I and II co-migrated with 8- and 5-hydroxyeicosatetraenoic acid standards, respectively, and the addition of 4,7,10,13-eicosatetraynoic acid (ETYA) or nordihydroguaiaretic acid (NDGA) showed a pronounced and dose-dependent inhibition of arachidonic acid conversion. These data demonstrate lipoxygenase activity. Such a pathway in the metabolism of arachidonic acid had not, as yet, been reported in insects. This enzyme system can be demonstrated in the genital tract of the male and also in the seminal receptacle of the female, especially after insemination. So the enzyme system is probably transferred from male to female during mating.     

Prostaglandin metabolism during circulatory shock

The rates of metabolic degradation and the patterns of metabolite formation of tritium-labeled prostaglandins E₂ and F_2α were assessed in vitro in tissues obtained from normal rabbits and from rabbits subjected to hemorrhagic or endotoxic shock. Normal rabbit tissues metabolized prostaglandin E₂ at the following rates: renal cortex 479 ± 34, liver 389 ± 95, and lung 881 ± 93 pmol of PGE₂ metabolized/mg soluble protein per min at 37°C (mean ± S.E.). Prostaglandin F_2α metabolism proceeded in normal animal tissues at rates of 477 ± 39, 324 ± 95, and 633 ± 69 pmol of PGF_2α metabolized/mg soluble protein per min for renal cortex, liver and lung, respectively. There were no significant differences between these rates of PGE₂ and PGF_2α metabolism when compared to rates in tissues obtained from animals subjected to either hemorrhagic or endotoxic shock. In addition, no significant differences were observed between the rate of PGE₂ metabolism and that of PGF_2α metabolism for any tissue. However, the lung was able to metabolize PGE₂ and PGF_2α significantly more rapidly than the liver, and to degrade PGE₂ at a significantly greater rate than the renal cortex. Although slightly different patterns of metabolite production were observed between lung and kidney homogenates, only the liver metabolized prostaglandins almost exclusively to more polar metabolites. While hemorrhagic or endotoxic shock induced slight changes in the patterns of PGE₂ metabolite formation in all three tissues studied, PGF_2α metabolite formation patterns were not significantly altered by circulatory shock. Thus, prostaglandin metabolism is not significantly impaired during the first 2 h of hemorrhagic or endotoxic shock in rabbit tissues. Therefore, impairment of prostaglandin metabolism is not the major factor responsible for the early increase in circulating prostaglandin concentrations in these forms of shock.     

In vitro prostaglandin synthesis by human glomeruli and papillae

We have investigated in vitro prostaglandin synthesis by human isolated glomeruli and papillary homogenates and compared the results with those obtained in parallel studies using rat material. Prostaglandins were measured by two methods, namely radiometric high performance liquid chromatography after incubation with ¹⁴C arachidonic acid and radioimmunoassay. The relative abundance of various prostaglandins synthesized by glomeruli was different in man (6 keto PGF_1α > TXB₂ > PGF_2α > PGE₂) and in the rat (PGE₂ TXB₂ > 6 keto PGF_1α). Unidentified peaks eluting between 6 keto PGF_1α and TXB₂ were observed only in rat glomeruli. These peaks were suppressed by indomethacin. Direct radioimmunoassay of prostaglandins in the incubation medium of human glomeruli confirmed the predominance of 6 keto PGF_1α synthesis and showed its stimulation by arachidonic acid, its progressive decrease with time and its linear relationship with glomerular protein at low concentrations. On the contrary, the profile of prostaglandin synthesis by the papilla was similar in man and in the rat, PGE₂ and PGF_2α being the major products in both species. However, related to one mg of protein, papillary synthesis of these two prostaglandins was greater in the rat. These results show that PGI₂ is the major prostaglandin synthesized in human glomeruli and suggest a role for this prostaglandin in glomerular physiology in man.     

Analysis of A,B, E,and F prostaglandins as pentafluorobenzyl esters by electron-capture gas-liquid chromatography

A new and sensitive method is described for the simultaneous analysis of a mixture containing PGE₁, PGE₂, PGF_1α, and PGF_2α by electron-capture gas-liquid chromatography. During derivatization of the mixture, PGE₁ and PGE₂ were converted to PGB₁ and PGB₂, respectively, yielding a mixture of PGB₁, PGB₂, PGF_1α, and PGF_2α trimethylsilyl ether pentafluorobenzyl esters. Gas chromatographic resolution of all four derivatives is sufficient for quantitation of each prostaglandin. The A prostaglandins were analyzed by similar conversion to the respective B prostaglandin derivatives. Minimum detection limits for the B and F prostaglandin derivatives were 10 pg and 1 pg, respectively. Samples of rabbit kidney medulla were incubated and analyzed for A, B, E, and F prostaglandins. The results indicate that the method is capable of high recovery and reproducibility.     

Differences in types of prostaglandins produced by two MDCK canine kidney cell sublines

The pattern of prostaglandins produced from arachidonic acid by two sublines of MDCK canine kidney epithelia cells was different. In one subline designated MDCK₁, the most prevalent prostaglandin product was PGE₂, whereas the most prevalent product in the subline designated MDCK₂ was PGF_2α. This difference was observed when cells previously labeled with [1^?14C]arachidonic acid were stimulated with either bradykinin or the calcium ionophore A23187, or when prostaglandins were produced from labeled arachidonic acid added directly to the assay medium. In the latter case, the difference was maintained over a 38-fold range of extracellular arachidoante concentrations. These findings indicate the there is a persistent difference in the distribution of prostaglandins produced by the two commonly used sublines of MDCK cells.     

Biosynthesis of prostaglandins and thromboxane B2 by fetal lung homogenates

The conversion of arachidonic acid to prostaglandins (PG's) and thromboxane B₂ (TXB₂) was investigated in homogenates from fetal and adult bovine and rabbit lungs. Adult bovine lungs were very active in converting arachidonic acid (100 μg/g tissue) to both PGE₂ (10.7 μg/g tissue) and TXB₂ (6.2 μ/g tissue). Smaller amounts of PGF_2α (0.9 μ/g) and 6-oxoPGF_1α were formed. Homogenates from fetal calf lungs during the third trimester of pregnancy were quite active in converting arachidonic acid to PGE₂, but formed very little TXB₂, PGF_2α or 6-oxoPGF_1α. Homogenates from rabbit lungs converted arachidonic acid (100 μg/g) mainly to PGE₂, both before and after birth. The amount of PGE₂ formed increased during gestation to a maximum of about 6 μg/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 μg/g) which was observed 8 days after birth, followed by an increase to about 4 μg/g in older rabbits.     

Bone resorption and prostaglandin production by mouse calvaria in vitro: Response to exogenous prostaglandins and their precursor fatty acids

Mouse calvaria were maintained in organ culture for 96 h and endogenous prostaglandin production and active bone resorption (⁴⁵ Ca release) measured. After a lag phase of 12 h, active resorption increased over the 96 h period. The amounts of prostaglandins released into the culture medium (measured by radioimmunoassay) were highest in the first 24 h of culture. Unless these were removed by preculturing for 24 h, or suppressed by indomethacin, no response to exogenous PGE₂, PGF_2α or prostaglandin precursors could be demonstrated. Bone resorption was stimulated after preculture by both PGE₂ and PGF_2α in a dose-dependent manner (10^?18M – 10^?5M), with PGE₂ being the more potent. Collagen synthesis was unaffected by PGF_2α, whereas PGE₂ (10^?5M) had an inhibitory effect. Eicosatrienoic acid did not stimulate bone resorption at lower concentrations (10^?7M – 10^?5M_, but was inhibitory at 10^?4M. Arachidonic acid also inhibited resorption at 10^?4M, but at lower concentrations (10^?7M – 10^?5M0 increased active resorption. This was concomitant with a rise in PGE₂ and PGF_2α levels, PGE₂ production being significantly higher than PGF_2α. The effects of PGE₂ (10^?8M) and PGF_2α (10^∞M appeared additive: there was no evidence of synergistic or antagonistic effects when varying ratios of PGE₂ : PGF_2α were employed.     

Kinetic measurements of the release of prostaglandins from perfused rat lungs

Prostaglandins released from isolated, ventilated and perfused rat lungs were measured by a simple modification of the Vane technique using the rat stomach fundus as a continuous bioassay tissue. Exogeneously supplied arachidonic acid was converted mainly to PGF_2α which was determined by bioassay. A novel method for mixing a stream of inhibitors with the perfusate was used to determine PGF_2α in the presence of substrate amounts of arachidonic acid. Using this system the apparent K_m for PGF_2α production with arachidonic acid as the substrate was found to be 1.90 × 10⁻⁴M, while the K_i for aspirin was found to be 2.47 × 10⁻⁴M. These kinetic parameters are close to those reported for cell free systems and subcellular fractions suggesting that both substrate and inhibitor have ready access to the site of prostaglandin synthesis. The method appears to be generally useful to determine the effect of drugs and environmental factors on the release of prostaglandins by the lung.     

Nociceptive Response to Prostaglandins and Analgesic Actions of Aspirin and Morphine

VANE et al.^1–3 have proposed that aspirin and allied antiinflammatory drugs act by inhibiting the production of prostaglandins in the tissues. Because, however, prostaglandins E₁ and E₂ (PGE₁ and PGE₂) had been reported not to elicit pain in human skin at doses inducing inflammation^{4, 5}, Vane did not suggest that the inhibition of prostaglandin production fully explains the analgesic action of aspirin-like drugs. Nonetheless, PGE₁ PGE₂ or PGF_2α irritates pulmonary^{6, 7} or ocular⁸ mucous membrane and, when injected by the subcutaneous or intramuscular route, PGE₂ or PGF_2α causes pain⁹.     

PGE2 is a more potent vasodilator of the lamb ductus arteriosus than is either PGI2 or 6 keto PGFα

It has been shown in vitro that the lamb ductus arteriosus forms prostaglandins PGE₂, PGF_2α, 6 keto PGF_1α (and its unstable precursor PGI₂). In this study the relative potencies of these endogenous prostaglandins were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO₂ and indomethacin. All the prostaglandins (except PGF_2α) relaxed the vessel. This is consistent with the hypothesis that endogenous prostaglandins inhibit the tendency of the vessel to contract in response to oxygen. Only PGE₂, however, relaxed the vessel at concentrations below 10⁻⁸M. PGI₂ and 6 keto PGF_1α had approximately 0.001 and 0.0001 times the activity of PGE₂. Although PGE₂ has been observed to be a minor product of prostaglandin production in the lamb ductus arteriosus, the tissue's marked sensitivity to PGE₂ might make it the most significant prostaglandin in regulating the patency of the vessel.     

Production of prostaglandin F2α by molecular breeding of an oleaginous fungus Mortierella alpina

Cyclooxygenases are responsible for the production of prostaglandin H₂ (PGH₂) from arachidonic acid. PGH₂ can be converted into some bioactive prostaglandins, including prostaglandin F_2α (PGF_2α), a potent chemical messenger used as a biological regulator in the fields of obstetrics and gynecology. The chemical messenger PGF_2α has been industrially produced by chemical synthesis. To develop a biotechnological process, in which PGF_2α can be produced by a microorganism, we transformed an oleaginous fungus, Mortierella alpina 1S-4, rich in triacylglycerol consisting of arachidonic acid using a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla. PGF_2α was accumulated not only in the mycelia of the transformants but also in the extracellular medium. After 12 days of cultivation approximately 860 ng/g and 6421 µg/L of PGF_2α were accumulated in mycelia and the extracellular medium, respectively. The results could facilitate the development of novel fermentative methods for the production of prostanoids using an oleaginous fungus.     

Prostaglandins in rainbow trout (Oncorhynchus mykiss Walbaum, 1792) sperm biology – searching for answers

The purpose of this study was to determine the concentrations of prostaglandins E₂ and F_2α (PGE₂ and PGF_2α) in the blood, testis and seminal plasma of mature male rainbow trout and in the ovarian fluid to assess the effects of these prostaglandins on sperm motility parameters when present in activation media. Also prolonged incubation with prostaglandins on sperm motility and calcium influx were studied. The profile of PGE₂ and PGF_2α differed in concentration between blood, testicular supernatant and seminal plasma. PGE₂ was predominant in the blood sample (0.29 ng ml^?1) and testicular supernatant (3.1 ng ml^?1) whereas their level in seminal plasma was lower than PGF_2α (0.23 ng ml^?1). The concentrations of PGF_2α in blood, testis and seminal plasma were 0.04, 0.99, 1.3 ng ml^?1, respectively. In the ovarian fluid the concentrations of both prostaglandins were higher than in the male reproductive tract. Adding both prostaglandins to activation buffer (at concentrations 15 and 70 ng ml^?1) had no effect on any CASA parameters. Calcium influx related to rainbow trout sperm incubations with PGE₂, and PGF_2α was not detected. After 24 h incubation of sperm in artificial seminal plasma solution without and with prostaglandins all sperm samples increased their motility potential and intracellular calcium concentration. Therefore, this effect was not related to the presence of prostaglandins. In summary PGE₂, and PGF_2α were present in the rainbow trout male reproductive tract, and their profile varies from that of blood, testis and seminal plasma. The specific role of both prostaglandins in salmonid sperm biology remains unclear.     

Prostaglandin-mediated hypercalcemia in the VX2 carcinoma-bearing rabbit

Prostaglandin biosynthesis and metabolism were studied in the VX₂ carcinoma-bearing rabbit, an animal model of prostaglandin-mediated hypercalcemia. All the identification and quantification of the prostaglandins were done by gas chromatography-mass spectrometry. The tumor incubated $\text{in vitro}$ converted exogeneous arachidonic acid principally to PGE₂. Biosynthesis from endogenous precursor lipids yields mainly PGE₂ and PGF₂α. The 100,000 × g supernatant fluid of the tumor did not contain any metabolizing enzymes.Significant hypercalcemia developed between the first and second week after tumor implantation. The levels of the major plasma metabolite of PGE₂, 15-keto-13,14-dihydro-PGE₂, became elevated at one week, had risen 25-fold by the end of the second week, and at the fourth week were elevated to 256 times the pre-incubation levels. The concentration of 15-keto-13,14-dihydro-PGF₂α in plasma rose in parallel but to a lesser degree. 7α-hydroxy-5,11-diketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of the E prostaglandins, was elevated two weeks after tumor implantation and rose until the fifth week. Indomethacin treatment lowered both serum calcium and the plasma level of 15-keto-13,14-dihydro-PGE₂.     

Enzymic coupling of acylhydrolase and prostaglandin synthase activities in subcellular fractions from rabbit renal medulla

We have recently shown that mitochondrial and plasma-membrane fractions from kidney medulla possess Ca²⁺-stimulated acylhydrolase and prostaglandin synthase activities. The nature of the enzymic coupling between the Ca²⁺-stimulated arachidonic acid release and its subsequent conversion into prostaglandins was investigated in subcellular fractions from rabbit kidney medulla. Plasma-membrane, mitochondrial and microsomal fractions were found to have similar apparent K_m values for conversion of added exogenous arachidonate into prostaglandins. The rate of prostaglandin biosynthesis (V_max.) from added arachidonic acid in the microsomal fraction was approx. 2-fold higher than in the other subcellular fractions. In contrast, prostaglandin E₂ synthesis from endogenous arachidonate in plasma-membrane and mitochondrial fractions was 3–4-fold higher than in microsomes. Furthermore, Ca²⁺ stimulated endogenous arachidonate deacylation and prostaglandin E₂ generation in the former two fractions but not in microsomes. In mitochondrial or crude plasma-membrane fractions, in which prostaglandin biosynthesis was inhibited with aspirin, arachidonate released from these fractions was converted into prostaglandins by the microsomal prostaglandin synthase. Thus an intracellular prostaglandin generation process that involves inter-fraction transfer of arachidonic acid can operate. Prostaglandin generation by such an inter-fraction process is, however, less efficient than by an intra-fraction process, where arachidonic acid released by mitochondria or crude plasma membranes is converted into prostaglandins by prostaglandin synthase present in the same fraction. This demonstrates the presence of a tight intra-fraction enzymic coupling between Ca²⁺-stimulated acylhydrolase and prostaglandin synthase enzyme systems in both mitochondrial and plasma-membrane fractions.     

Effect of oestradiol 17 β on prostaglandin biosynthesis by the uterus of ovariectomized rat and guinea pig

$\text{In vitro}$ prostaglandin biosynthesis by uteri of ovariectomized rats and guinea pigs treated or untreated with oestradiol 17 β, administered subsutaneously, was measured by R.I.A. of PGF_2α and PGE₂. Incubations with [1-¹⁴C] arachidonic acid were also performed and labelled metabolites were analyzed by TLC. The main metabolite in rats was 6 keto PGF_1α and in decreasing order of magniture, PGF_2α and PGE₂. In guinea pig PGF_2ga was the main product. Ovariectomy in rats completely changed the pattern of synthesized prostanoids: PGI₂ production was doubled when compared to cycling rats and PGE₂ increased 10 fold. PGF_2α walues were similar to the mean value measured during the cycle. OE₂ treatment almost completely inhibited PGI₂ synthesis and reduced PGE₂ by half. Total PG synthesis in OE₂ treated animals was decreased by 5 fold when compared to spayed rats. Endogenous PGF_sα synthesis was slightly stimulated. In the guinea pig OE₂ treatment of ovariectomized animals increased the total synthesis from 50 per cent. PGF_2α was always the main metabolite. In conclusion OE₂ regulation of uterine PG synthesis is depending on the animal species and cannot be explained by a unique effect on the cyclooxyhenase, but rather by an interplay on the various enzymes of the arachidonic acid cascade.     

Measurement of prostaglandins in embryonic tissue using radioimmunoassay

Although prostaglandins appear to play an important role in numerous physiological processes in the adult, neonate, and fetus, very little is known about the role of these compounds in the embryo. This study demonstrates that rat embryo homogenates synthesized 6-oxo-PGF_1α; PGE and PGF in markedly different amounts from endogenous substrate. Synthesis was inhibited by indomethacin (10 μM) in varying degrees (70–89%) depending on the prostaglandin. The metabolite of PGF_2α, 13,14-dihydro-15-keto PGF_2α (PGF_2α-M), was produced in limited amounts in the absence of exogenous NAD. In the presence of exogenous NAD and PGF however, embryonic homogenates produced PGF_2α-M. The potential role of prostaglandins during embryogenesis is discussed.     

Prostaglandin levels in isolated brain microvessels and in normal and norepinephrine-stimulated cat brain homogenates

The levels of PGD₂, PGE₂, PGF_2α and 6-keto-PGF_1α (6KF_1α) produced from endogenous archidonic acid (AA) were quantitated in cat cerebral cortical homogenates and microvessels isolated from cat cerebral cortex using gas chromatography/mass spectrometry (GC/MS). There was a six-fold enrichment of 6KF_1α levels in isolated microvessels, compared to homogenates, suggesting that 6KF_1α is of vascular, rather than neuronal origin. In order to further understand any possible role that norepinephrine (NE)_might have on modulation of PG synthesis, we studied the effects of 0.5 mM NE on PG synthesis from endogenous AA and from ³H-PGG₂, the endoperoxide precursor of PGs. In cat cortical homogenates NE induced a 74% increase in PGD₂ and PGF_2α, a 62% increase in PGE₂, and a 36% increase in 6KF_1α, as measured by GC/MS. NE caused a twofold increase in the conversion of ³H-PGG₂ to ³h-PGG_2α, with a concomitant decrease in ³H-PGE₂ and ³H-6KF_1α formation, and no change in ³H-PGD₂ synthesis. NE had no effect on the total conversiob of ³H-PGG₂ to ³H-PGs, nor on the breakdown of ³H-PGG₂ in the absence of brain tissue. We conclude that NE stimulates extravascular synthesis of PGD₂, PGE₂ and PGF_2α by stimulation of the prostaglandin synthetase complex, in addition to NE's stimulatory effect on the conversion of PGG₂ to PGF_2α, and that the lack of effect of NE on 6KF_1α synthesis reflects either a failure to achieve an adequate concentration at the vascular tissue, or an absence of the mechanism whereby NE stimulates PG synthetase.     

A radioimmunoassay for 6-keto-prostaglandin F1α utilizing an antiserum against 6-methoxime-prostaglandin F1α: Application to study renal in vitro synthesis of prostacyclin

PGI₂ and 6-keto-PGF_1α were converted to 6-methoxime-PGF_1α (6-MeON-PGF_1α) by treatment with methoxyamine HCl in acetate buffer. The formed 6-MeON-PGF_1α was measured by radioimmunoassay. Antisera were raised in rabbits after immunization against 6-MeON-PGF_1α-BSA conjugate. Diluted 1:20.000 to bind 50% of the tracer (³H-6-MeON-PGF_1α, 100 Ci/mmol), the antiserum cross reacted 0.8% with PGE₂, 1% with PGF_2α and less than 0.2% with PGD₂, PGF_1α, PGF_2β and TXB₂. The radioimmunoassay was used to estimate release of PGI₂ and 6-keto-PGF_1α from chopped rabbit renal medulla and cortex incubated in Krebs-Ringer bicarbonate buffer (37°C, 30 min). The 6-keto-PGf_1α radioimmunoassay was validated in biological samples by mass fragmentography. The chopped medulla (n=5) released 38±9 ng/g/min and the cortex (n=5) 4.7±2.0 ng/g/min, while the release of immunoreactive PGE₂ (iPGE₂) and iPGF_2α was 171±26 and 74±13 ng/g/min from the medulla and 4.3±1.3 and 2.7±0.3 ng/g/min from the cortex, respectively. The results confirm previous findings, which indicate that in the renal medulla prostaglandin endoperoxides are mainly transformed to prostaglandins, while in the cortex transformation to PGI₂ seems to be of greater $\text{relative}$ importance.