The 8-isoprostaglandins: Evidence for eight compounds in human semen

Evidence is presented for the existence of a group of 8-iso prostaglandins in human semen, comprising 8-iso PG E₁^*, 8-iso PG E₂, 8-iso PG F_1α, 8-iso PG F_2α and the four corresponding 19-hydroxy prostaglandins. The E and F compounds have been positively identified by comparison of their mass spectra and chromatographic properties with those of authentic standards. Preliminary measurements of levels of these compounds in pooled semen are presented.     

Inhibitory and stimulating effects of various types and series of prostaglandins on in vitro biosynthesis of proteochondroitin-4,6-sulfate and protein and anaerobic glycolysis in calf rib cartilage

The effect of various types of prostaglandins (PGs) have been studied in a semi-in-vitro system with cartilage slices of calf ribs. 0.1 mmol/1 PG B1, D2, E1, E2, F1 alpha, F2 alpha inhibit biosynthesis of Ch-4,6-S protein to a higher extent than 10 mumol/l; 1 and 2 series PG E and F (but not B) inhibit similarly, PG A2 inhibits twice as much. With biosynthesis of total protein 2-series PG A, B, E, F act more inhibitorily than 1-series PG. 10 mumol/l PG A2, E1, E2 produce cAMP-like effects, e.g. acceleration of biosynthesis of Ch-4,6-S protein and total protein as well as of anaerobic glycolysis; PG F2 alpha stimulates the first two anabolic processes, PG B1 only the second one. PG A1 stimulates Ch-4,6-S protein biosynthesis and anaerobic glycolysis, a cGMP-like effect. 20 mumol/l diBu-cAMP or cAMP (together with 0.1 mmol/l theophylline) produce stimulating and inhibitory effects on these three anabolic processes; both compounds produce additively positive or additively negative effects in connection with the inhibitory PG effects on these three anabolic processes.     

Effects of various concentrations and combinations of prostaglandins on in vitro migration of frozen-thawed ram spermatozoa in cervical mucus of ewes

The aim of this investigation was to study the effects of various concentrations and combinations of prostaglandins (PG) on sperm migration in cervical mucus collected from estrous ewes. Semen from six rams was pooled and extended in Tes-tris-yolk-glycerol to 500 x 10(6) sperm per ml, cooled and held at 5 degrees C for three hours. Aliquots of the semen were supplemented with various concentrations of PGF(2alpha) or PGE(1), combinations of PGF(2alpha) + E(1), or PGE(1) + E(2) + E(3) + F(1alpha) + F(2alpha). The semen was filled into 0.25-ml French straws and immediately frozen in liquid nitrogen vapor. The semen was thawed in a 35 degrees C water bath for two minutes. Cervical mucus was collected from 30 ewes, pooled and filled in the capillary tubes. Frozen-thawed ram semen with PG added was brought into contact with cervical mucus, held at 37 degrees C and evaluated for sperm migration in the capillary tube at 15, 30, 60, 120, and 180 minutes under a phase-contrast microscope. At 180 minutes, sperm numbers were counted and sperm motility was graded at 20, 30, 40, 50, and 60-mm distances in the capillary tube. Analysis of the results showed that sperm migration distance increased with time, but there was no difference between the controls and samples with PG. At 180 minutes, sperm motility and number decreased, while distance of migration in the capillary tube increased. This sperm response was similar, whether the supplementation of semen was with any of several individual PGs, or a combination of two or five PGs.     

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.     

Lipase-catalysed acylation of prostanoids.

Natural prostaglandins (PG) F2alpha and E1 as well as (+)-cloprostenol were regioselectively 11-acylated using Novozym 435 as a catalyst and vinyl acetate as an acyl donor. Unlike the above compounds the 15-OH group of PGE2 was also acylated with a significant velocity under the same conditions. The enantiospecificity of the lipase-catalysed 11-acetylation of cloprostenol was established by separate treatment of(+)- and (-)-cloprostenols.     

Basal level of human platelet prostaglandins: PGE1 is more elevated than PGE2.

Radioimmunoassays of platelet prostaglandins E1 and F1 alpha in platelet rich plasma or platelet suspension, demonstrate that both PGE1 and PGF1 alpha are present at higher concentrations than prostaglandins E2 and F2 alpha. Gas chromatography--mass spectrometry determinations of prostaglandins E1 and E2 in resting washed platelets confirm this difference. Lastly, there is a greater incorporation of [1--14C] acetate into prostaglandins E1 and F1 alpha compared to that into prostaglandins E2 and F2 alpha.     

The influence of prostaglandins E2 and F2 alpha on electrotropic and inotropic vagal effects on the isolated rabbit atrium

1. PG E2 (2.10(-8) g/ml) produces a positive inotropic effect and lengthens the duration of the action potential at the level of 50% and 90% of repolarization. 2. PG F2 alpha acts negatively inotropic and shortens the action potential at the level of 75% and 90% of repolarization. 3. PG F2 alpha (2.10(-8) g/ml) increases both the electrotropic and inotropic vagal effects, PG E2 (2.10(-8) g/ml) decreases them. 4. PG E2 improves the electromechanical coupling, PG F2 alpha reduces it.     

Interaction of prostaglandins and fatty acids with native and acetaldehyde-alkylated proteins

Using intrinsic and probe fluorescence, microcalorimetry and isotopic methods, the interactions of prostaglandins (PG) E2 and F2 alpha and some fatty acids with native and alkylated proteins (human serum albumin (HSA) and rat liver plasma membrane PG receptors), were studied. The fatty acid and PG interactions with human serum albumin (HSA) resulted in effective quenching of fluorescence of the probe, 1.8-anilinonaphthalene sulfonate (ANS), bound to the protein. Fatty acids competed with ANS for the binding sites; the efficiency of this process increased with an increase in the number of double bonds in the fatty acid molecule. PG induced a weaker fluorescence quenching of HSA-bound ANS and stabilized the protein molecule in a lesser degree compared to fatty acids. The sites of PG E2 and F2 alpha binding did not overlap with the sites of fatty acid binding on the HSA molecule. Nonenzymatic alkylation of HSA by acetaldehyde resulted in the abnormalities of binding sites for fatty acids and PG. Modification of the plasma membrane proteins with acetaldehyde sharply diminished the density of PG E2 binding sites without changing the association constants. Alkylation did not interfere with the parameters of PG F2 alpha binding to liver membrane proteins.     

Prostaglandin production in myotube cultures. Influence on protein turnover.

The production of prostaglandins (PG) E2 and F2 alpha and their possible role in regulation of protein turnover in cultured skeletal-muscle cells were examined. Primary chick myoblasts and myotubes, and L8 myotubes, produced PGE2 and PGF2 alpha from endogenous arachidonic acid. PG production by all three cell types was increased manyfold by the addition of exogenous arachidonic acid. Arachidonate-stimulated PG production was inhibited by the addition of indomethacin (0.1 mM). When L8 and chick myotubes were treated with PGE2, PGF2 alpha, arachidonic acid (0.01 mM) or indomethacin (0.1 mM), no significant alterations in rates of protein synthesis or degradation were observed. Rates of protein synthesis and degradation in these cells were responsive to the addition of 10% fetal-bovine serum under identical experimental conditions. Thus, in contrast with incubated adult skeletal muscle, it appears that the production of prostaglandin metabolites from arachidonic acid is unrelated to regulation of protein turnover in cultured muscle cells.     

Bradykinin and electrical stimulation increase prostaglandin production in the rat vas deferens

The epididymal portion of the rat vas deferens produced prostaglandins (PG) E(2), F(2alpha)and 6-keto F(1alpha). Electrical stimulation (ES, 0.1 Hz, 1 ms) increased such production by 100%, and similar results were obtained in the presence of 1.0 microM bradykinin (Bk). When both stimuli were applied simultaneously, the increases in PG production were 1100% for PGE(2), 800% for PGF(2alpha)and 400% for PG6-keto F(1alpha). Prazosin abolished the effect of ES on PG production. A selective Bk B(2)-receptor antagonist abolished the increase in PG production induced by Bk, both in non-stimulated and in ES tissues. Bk (1.0 microM) elicited contractile responses in non-stimulated as well as in ES tissues, responses that were not modified in the presence of 10 microM indomethacin. In conclusion, the effects of Bk on prostaglandin production appears to depend on the activation of B(2) receptors, while the increase in prostaglandin release induced by ES, and the effects observed with both stimuli simultaneously, should be mediated by the release of noradrenaline and the subsequent activation of alpha(1) adrenoceptors.     

Isoprostanes induce plasma extravasation in rat skin

Isoprostane E2 (8-iso PGE) and isoprostane F2 alpha (8-iso PGF) contribute to numerous vascular, proinflammatory, and nociceptive functions. The underlying mechanisms for many of their actions are still under investigation. We examined the ability of isoprostanes to promote cutaneous inflammation using the Evan's blue dye method. Our data show that 4 micrograms subcutaneously (s.c.) injected 8-iso PGE or 8-iso PGF induced plasma extravasation in glabrous rat skin. Dye extravasation was also elicited in hairy skin after injections of 8-iso PGE, but not after 8-iso PGF. Isoprostane-evoked dye extravasation can be reduced by pretreatment with both the S+ and R- isomers of the cyclooxygenase (COX)-inhibitor ibuprofen (30 mg/kg intraperitoneally), indicating perhaps a nonspecific inhibition; pretreatment with ketorolac (1 and 10 mg/kg i.v.) was without effect. Unlike isoprostane-induced cutaneous nociceptor sensitization, which is blocked in a stereospecific and dose-dependent manner by COX-inhibitors, the effect of these drugs on isoprostane-induced cutaneous plasma extravasation is less consistent. We conclude that at least a large component of the isoprostane effect on cutaneous plasma extravasation is COX-independent.     

Specific receptors for prostaglandins in airways

The relative bronchomotor activities of prostaglandins (PG) E1, E2, F2 alpha, F2 beta and I2 and of three synthetic E prostaglandin analogues (TR4161, TR4367 and TR4752) were determined on a large number of isolated preparations of guinea-pig trachea and human bronchial muscle. Each prostaglandin was capable of eliciting both contraction and relaxation, the relative incidence of these responses partly depending on concentration. TR4161 was a virtually pure relaxant; TR4367 was virtually devoid of bronchomotor activity; and TR4752 was a potent relaxant, devoid of contractant activity. The results also provided distinct rank orders of approximate potency for contraction and relaxation. Tachyphylaxis to the relaxant activities of PGE1 and TR4752 confirmed the underlying contractant activity of the two natural E prostaglandins. Antagonism with a high dose of indomethacin of the contractant actions of PGE1, PGE2 and PGF2 alpha confirmed the presence of relaxant activities in each. Inhaled aerosols of the same natural and synthetic prostaglandins were evaluated for irritant activity on the airways, using the cough response of the restrained conscious cat. All of them, except TR4161, elicited severe coughing. The rank order of potencies for irritancy differed from those for tracheobronchial contractant and relaxant activities. These findings suggest that the three responses studied arise from the activation of three distinct PG receptors in the airways. We propose the terms chi (contractant), psi (relaxant) and omega (irritant) for these putative receptors for prostaglandins or possibly other prostanoids.     

Low prostaglandin concentrations cause cardiac rhythm disturbances. Effect reversed by low levels of copper or chloroquine

In perfused male rat hearts concentrations of prostaglandins (PGs) E2 and F2alpha in the range 1 pg/ml to 10 ng/ml (2.8 X 10(-12) to 2.8 X 10(-8)M) consistently caused rhythm irregularities. Higher concentrations had no effect themselves and stabilized rhythm in hearts made unstable by lower concentrations. Copper ions (as the sulphate) at 2 X 10(-6)M stabilized hearts made unstable by PGs and when present prior to the PGs prevented PG induced disturbances. Chloroquine also reversed PG-induced rhythm changes.     

Metabolism of prostaglandins D2 and F2 alpha in primary cultures of rat hepatocytes

3H-Labeled prostaglandins D2 and F2 alpha rapidly degraded to more-polar metabolites in primary cultured rat hepatocytes. The metabolites of prostaglandins D2 and F2 alpha accumulated in the culture medium. The metabolites extracted by ethyl acetate at pH 3 were purified by silicic acid column and thin-layer chromatography of silica gel, and were analysed by gas chromatography-mass spectrometry. The major metabolites from prostaglandin D2 were identified as dinor-prostaglandin D1 (7 alpha,13-dihydroxy-9-ketodinorprost-11-enoic acid) and tetranor-prostaglandin D1 (5 alpha,11- dihydroxy-7-ketotetranorprost-9-enoic acid). Those from prostaglandin F2 alpha were identified as dinor-prostaglandin F1 alpha (7 alpha,9 alpha,13-trihydroxydinorprost-11-enoic acid), tetranor-prostaglandin F1 alpha (5 alpha,7 alpha,11-trihydroxytetranorprost-9-enoic acid) and 9 alpha,11 alpha,15-trihydroxyprost-13-ene-1,20-dioic acid. These data indicate that prostaglandins D2 and F2 alpha mainly degraded by beta-oxidation, which is the same process as reported earlier for prostaglandins E1 and E2, and that prostaglandin F2 alpha was also subjected to omega-oxidation.     

Follicular and uterine prostaglandin levels in relation to uterine contraction and the first ovulation of a sequence in the hen

Changes in the concentration of progesterone, estrone, estradiol, prostaglandins (PG) E2, 6-keto F1 alpha and 13,14-dihydro-15-keto F2 alpha (PGFM) were measured in peripheral plasma, and in venous effluent from the shell gland and the largest (F1) and the second largest (F2) preovulatory follicles. Tissue concentrations in the F1, F2 and the most recently ruptured follicle and the shell gland also were determined. Changes in these criteria were compared to changes in uterine contraction before the first ovulation of a sequence. Significant increases of PGF2 alpha and PGFM in the peripheral plasma were observed when the frequency of uterine contraction reached a maximum, about 1 h before ovulation. Relative to peripheral plasma, the concentrations in F1 plasma of progesterone, PGF2 alpha and PGFM were increased 20-fold, 150-fold and 15-fold, respectively, at the time of the maximum frequency of uterine contraction. The highest tissue concentrations of PGs were also observed in the F1 follicle. These results suggest that the largest preovulatory follicle is the major source of PG synthesis and release. These PGs may stimulate uterine contraction and may also play a role in follicular rupture and release of the ovum.     

De novo biosynthesis of prostaglandins by the Australian field cricket, Teleogryllus commodus

The accumulation and metabolism of certain polyunsaturated fatty acids by testes from the Australian field cricket, Teleogryllus commodus, are described. Testes accumulated a substantial proportion (about 16%) of label from radioactive C20:3n6 that was injected into the haemocoel. Fifty percent of the label accumulated by testes was associated with the phospholipid fraction, whereas in the remainder of the body 30% was incorporated into the phospholipid fraction. Prostaglandins (PG) E1, E2 and F2 alpha were quantified in extracts of the testes of adult insects by radioimmunoassay. Label from injected radioactive C18:2n6, C20:3n6 and C20:4n6 was recovered as prostaglandins PGE and PGF. The radioactivity from C18:2n6 that was recovered as PGE1 and PGF1 alpha indicated elongation/desaturation to C20:3n6 followed by conversion to PG. Since C18:2n6 is readily formed from acetate in T. commodus, these findings indicate the de novo biosynthesis of C20 polyunsaturated fatty acids and prostaglandins by this species.     

Prostaglandins can modify gamma-radiation and chemical induced cytotoxicity and genetic damage in vitro and in vivo

The effect of prostaglandin E1, E2, and F2 alpha on gamma-radiation, benzo(a)pyrene and diphenylhydantoin-induced cytotoxicity in vivo and genotoxicity in vitro was investigated. Prostaglandin E1 prevented both cytotoxic and genotoxic actions of all the three agents, where as both PGE2 and PGF2 alpha were ineffective. In fact, it was seen that both PGE2 and PGF2 alpha are genotoxic by themselves. Gamma-linolenic acid and dihomogamma-linolenic acid, the precursor of PGE1 were also as protective as that of PGE1, where as arachidonic acid, the precursor of 2 series PGs, has genotoxic actions to human lymphocytes in vitro. These results suggest that prostaglandins and their precursors can determine the susceptibility of cells to cytotoxic and genotoxic actions of chemicals and radiation. This study is particularly interesting since, it is known that some tumor cells contain excess of PGE2 and PGF2 alpha and many carcinogens can augment the synthesis of 2 series of PGs.     

Effects of prostaglandins on rat renal adenylate cyclase-cyclic AMP systems.

The effects of prostaglandin (PG) E1, E2, A1, F1alpha, F2alpha or D2 on the rat renal cortical, outer medullary and inner medullary adenylate cyclase-cyclic AMP systems were examined. While high concentrations (8X10-4M) of each prostaglandin stimulated adenylate cyclase activity in each area of the kidney, PGE1 was the only prostaglandin to stimulate at 10-7M. PGA's were the only prostaglandins tested besides PGE's which stimulated adenylate cyclase at less than 10-4M. This effect of PGA's was limited to the outer medulla. PGD2 was the least stimulatory. Observations with renal slices yielded qualitatively similar results. The PGE's were the most potent in each area with PGA's only stimulatory in the outer medulla. O2 deprivation (5% O2) lowered the slice cyclic AMP content in each area of the kidney. In the cortex and outer medulla, prostaglandin mediated increases in cyclic AMP content were either lower or absent at 5% O2 compared to 95% O2. However, in the inner medulla PGE stimulation was observed only at 5% O2 and not 95% O2. No other prostaglandins were found to increase inner medullary cyclic AMP content at 95% or 5% O2. These results illustrate that the adenylate cyclase-cyclic AMP system responds uniquely to prostaglandins in each area of the kidney. Consideration of these results along with correlative observations suggests that inner medullary produced PGE's may act as local modulators of inner medullary adenylate cyclase.     

Endogenous production of prostaglandins E and F2-alpha in cerebral and extracerebral vessels of cats and its change under the effect of noradrenaline]

The quantitative determination of prostaglandins (PG) E and F2 alpha has been carried out in isolated cerebral and extracerebral vessels of cats. It has been found that the basal production of PG predominates in cerebral vessels, the content of PGF2 alpha prevailing compared to PGE. The incubation of isolated vessels with noradrenaline causes a decrease in production of vasopressor PGF2 alpha with a simultaneous considerable increase in the content of vasodepressor PGE. The changes indicated are especially clearly seen in the cerebral vessels. It is suggested that the level of endogenic production of PG in the cerebral vessels may play an important role in the local regulation of the vascular tone, while the disbalance of their dynamic correlation may be responsible for cerebrovascular abnormalities.     

Chemical synthesis of tritium-labeled prostaglandins A, B and F

Formation of prostaglandins A, B and F from prostaglandin E has been studied. Synthesis of tritium-labelled prostaglandins A1, A2, B1, B2, F1 alpha, F2 alpha, F1 beta, F2 beta of high molar radioactivity from highly labelled PGE1 and PGE2 is described.