Isomerization of prostaglandin H2 into prostaglandin D2 in the presence of serum albumin.

The prostaglandin endoperoxide, prostaglandin H2, decomposes in aqueous media mainly into prostaglandin E2. This paper shows that in the presence of serum albumin from a number of species prostaglandin H2 decomposes mainly into prostaglandin D2, an isomer of prostaglandin E2. The effect on endoperoxide decomposition exerted by serum albumin may well have physiological significance since intace endoperoxides can be released from tissues and since the biological properties of prostaglandins E2 and D2 are quite different.     

Radioimmunoassay of prostaglandins

The earlier radioimmunoassays were mainly intended for the measurement of prostaglandins of the E-F-A or B type in blood plasma/serum or urine. Many recent studies, however, explain the use of radioimmunoassay to measure the prostaglandin content of tissues, and many other studies are concerned with the prostaglandin production in a single cell type, or in a few cell types, rather than the whole tissue. To date, however, by far the greatest number of quantitative prostaglandin studies have been carried out on blood plasma or serum, while assay for primary prostaglandins are now fairly seldom applied to the peripheral circulation, unless it is to study the prostaglandin production in vivo. It has been proposed that prostglandins of the A type are circulating hormones in contrast to other prostglandins, and a number of laboratories have developed quantitative methods for the measurements of PGA compounds. The sensitivity and specificity of the prostaglandins radioimmunoassays have increased considerably in later years through the use of labelled ligands of better quality; on the other hand, the accuracy of many radioimmunoassays seems to be very low when they are applied to biologic materials.     

Development of a radioimmunoassay for prostaglandin D2 using an antiserum against 11-methoxime prostaglandin D2

A sensitive and specific radioimmunoassay for prostaglandin D2 has been developed using its stabilized 11-methoxime derivative, which was obtained after treatment of prostaglandin D2 with methoxamine-HCl. The antiserum was obtained after injection of prostaglandin D2-methoxamine coupled to bovine serum albumin. A (125I)-Histamide prostaglandin D2-methoxamine tracer was prepared by iodination of the corresponding histamide, followed by thin layer chromatography purification. The sensitivity of the assay was 280 femtomoles per ml at 50% displacement. The cross reactivities were 15% with prostaglandin D1-methoxamine and less than 0.20% with other prostaglandins. Determination of the half-life of prostaglandin D2 in a solution containing albumin was also carried out, since it has been shown to catalyze prostaglandin D2 destruction. The unstability of this prostaglandin is due to the presence of a beta-hydroxy ketone group, and all prostaglandins possessing this labile moiety could be stabilized by such a derivatization before developing a radioimmunoassay.     

Evidence for a role of prostaglandins in the antepartum release of relaxin in the pregnant rat

The relationship of the antepartum elevation in serum relaxin levels in pregnant rats to luteolysis was examined by determining the effects of the luteolysin prostaglandin F2 alpha (PGF2 alpha) and the prostaglandin synthetase inhibitor indomethacin on antepartum serum relaxin levels, as well as on luteolysis and birth. Intravenous administration of PGF2 alpha on the morning of Day 20 elevated serum relaxin levels approximately fourfold within 15 min. Administration of the prostaglandin synthetase inhibitor indomethacin from Day 19 until Day 23 protracted luteolysis, delayed or prevented birth, and delayed the antepartum elevation of serum relaxin levels, until after indomethacin treatment had been terminated. Collectively, these results indicate that prostaglandins, in particular PGF2 alpha, may promote the antepartum increase in serum relaxin levels, as well as luteolysis and birth in rats.     

Elevated prostaglandin synthetase activity in methylcholanthrene-transformed mouse BALB/3T3

Cell lines transformed from 3T3 spontaneously, by radiation, or by treatment with chemical carcinogens, polyoma and SV40 virus produce up to 5 times more prostaglandins than their untransformed parent line. Several aspects of prostaglandin biosynthesis by MC5-5 and 3T3 were compared. When stimulated by serum, bradykinin, or thrombin, MC5-5 produced 2-to 5-fold more prostaglandins than 3T3. With the use of cells labeled with radioactive arachidonic acid in their cellular lipids, these higher levels were shown not to be due to increased availability of the prostaglandin precursor, arachidonic acid. Prostaglandin synthetase activity in microsomal fractions prepared from MC5-5 was 6 times higher than that of microsomes of untransformed cells. The increased prostaglandin levels produced by transformed cells therefore appear to be the result of elevated prostaglandin synthetase activity.     

Determination of prostaglandins and thromboxane as their pentafluorobenzyl-trimethylsilyl derivatives by electron-capture gas chromatography

The optimization of the parameters affecting the chromatographic properties and separation of prostaglandin pentafluorobenzyl derivatives by gas chromatography using electron-capture detection is described. The effects of composition and flow-rate of carrier gas, temperatures of detector and column, and nature of stationary phases on the detector response to different pentafluoroebenzyl (both oxime and ester) trimethylsilyl ether derivatives of prostaglandins were systematically examined. The stability of some selected prostaglandin derivatives at ?20°C was also determined. After standardizing these parameters, prostaglandins and related compounds from biological samples, e.g. semen, rat aorta, dog serum and trout gill were successfully analyzed. Identification of prostaglandins was confirmed by gas chromatography—mass spectrometry.     

Biosynthesis of prostaglandins in rabbit kidney medulla. Properties of prostaglandin synthase.

A simple radioactive-substrate assay for prostaglandin synthase (EC 1.14.99.1), which uses t.l.c. to measure simultaneously different prostaglandins synthesized from one precursor substrate, was developed. Rabbit kidney-medulla prostaglandin synthase catalyses the formation of prostaglandin E2, prostaglandin F2alpha and prostaglandin D2 from arachidonic acid. Fractionation of crude homogenates indicated that the microsomal fraction possessed the highest specific activity of prostaglandin synthase, whereas the soluble fraction exhibited little enzyme activity but rather contained a heat-labile inhibitory macromolecular factor(s), which might be attributed to the serum albumin present in this fraction. The microsomal fraction possessed low intrinsic enzyme activity, but the actvity could be fully stimulated by the presence of both GSH (reduced glutathione) and a phenolic cofactor. Only cysteine could partially replace GSH, whereas other thiols were inactive and some were even inhibitory. A variety of phenolic compounds, including catecholamines, dopamine (3,4-dihydroxyphenethylamine), 5-hydroxytryptamine and quinol, were active in stimulating prostaglandin synthase. In all cases, the stimulation was reflected in the synthesis of all three prostaglandins with ratios not significantly altered by different phenolic cofactors. The synthesis of each of the different prostaglandins appeared to have similar pH optima. The enzyme system was not inhibited by thiol-group inhibitors or a variety of metal chelators except for cyanide and 8-hydroxyquinoline. Characterization of the kidney-medulla prostaglandin synthase system indicated that it exhibited properties similar to those of the enzyme system present in seminal vesicles.     

Prostaglandin generation in rabbit kidney. Hormone-activated selective lipolysis coupled to prostaglandin biosynthesis.

The endogenous release of prostaglandins and free fatty acids from the isolated perfused rabbit kidney in the absence or presence of stimulation by bradykinin or angiotensin-II was investigated. Basal (nonstimulated) release of prostaglandin-precursor arachidonic acid was 15-20-fold higher than that of prostaglandin E2 indicating a low conversion of released arachidonate to prostaglandins. Addition of bovine serum albumin to the perfusion medium caused a substantial (50-250%) increase in the release of all fatty acids except myristic and arachidonic acids, and no significant change in prostaglandin E2 generation. In contrast, administration of bradykinin (0.5 microgram) or angiotensin-II (1 microgram) caused a 10-15-fold increase in prostaglandin E2 release, and with albumin present, also a 2-3-fold selective increase in arachidonic acid release. Thus, unlike what was observed under basal conditions, arachidonic acid released following hormone stimulation is efficiently converted to prostaglandin E2. We conclude that administration of bradykinin or angiotensin-II into the perfused kidney activates a lipase which selectively releases arachidonic acid, probably from a unique lipid entity. This lipase reaction is tightly coupled to a prostaglandin generating system so that the released arachidonate is first made available to the prostaglandin cyclooxygenase, resulting in its substantial conversion to prostaglandins.     

Effect of trihydroxyoctadecadienoic acids on the blood content of prostaglandins E2 and F2 alpha and 5-hydroxyeicosatetraenoic acid in rats with alloxan diabetes

It has been demonstrated that the level of prostaglandin F2 alpha and 5-hydroxyeicosatetraenoic acid (5-HETE) in the serum of alloxan-diabetic rats is reduced by 85% and 25%, respectively, whereas that of prostaglandin E2 is increased by 34%. The administration of trihydroxyoctadecadienoic acids, that have a hypoglycemic effect, to diabetic animals brings about a rise in the level of prostaglandins F2 alpha, E2 and 5-HETE by 33%, 64% and 279%, respectively, as compared to the control.     

Prostacyclin and prostaglandin synthesis in isolated brain capillaries

The synthesis of prostacyclin and prostaglandins was examined in isolated blood-free brain capillaries of guinea-pigs and rats using 1-14C-arachidonic acid as a precursor. The main prostaglandins synthesized by guinea-pig microvessels were prostaglandin D2 and prostaglandin E2. Substantially less prostaglandin F2 alpha or the prostacyclin stable metabolite, 6-oxo-prostaglandin F1 alpha was synthesized. Rat capillary prostaglandin distribution differed substantially from that of the guinea-pigs although the principle prostaglandin was also PGD2. Total prostaglandin conversion was greater in guinea-pig capillaries than in the rat. Norepinephrine stimulated the prostaglandin forming capacity of blood free cerebral microvasculature of guinea-pigs. Prostacyclin and prostaglandins could be involved in the activity dependent regulation of regional cerebral blood flow and permeability.     

Hormonal stimulation of arachidonate release from isolated perfused organs. Relationship to prostaglandin biosynthesis

The lipids of isolated Krebs perfused rabbit kidneys and hearts were labelled with [14C]arachidonic acid. Subsequent hormonal stimulation (e.g. bradykinin, ATP) of the pre-labelled tissue resulted in dose-dependent release of [14C]prostaglandins; little or no release of the precursor [14C]arachidonic acid was observed. When fatty acid-free bovine serum albumin was added to the perfusion medium as a trap for fatty acids substantial release of [14C]arachidonic acid was detected following hormonal stimulation. The release of [14C]arachidonic acid was dose-dependent and greater than 3 fold that of [14C]prostaglandin release. Indomethacin by inhibiting the cyclo-oxygenase, completely inhibited release of [14C]prostaglandins and only slightly inhibited release of [14C]arachidonic acid. These results demonstrate that in both rabbit kidney and heart much more substrate is released by hormonal stimulation than is converted to prostaglandins. This suggests that either the deacylation reaction is not tightly coupled to the prostaglandin synthetase system or that there are two deacylation mechanisms, one which is coupled to prostaglandin synthesis while the other is non-specific. It has previously been shown that prostaglandin release due to hormones such as bradykinin is transient despite continued presence of the hormone (tachyphylaxis). By utilizing albumin to trap released fatty acid, it was found that hormone-stimulated release of arachidonic acid is also transient. This directly demonstrates that tachyphylaxis occurs at a step prior to the cyclo-oxygenase.     

Bioproduction of prostaglandins in a transgenic liverwort, Marchantia polymorpha

Prostaglandins are biologically active substances used in a wide range of medical treatments. Prostaglandins have been supplied mainly by chemical synthesis; nevertheless, the high cost of prostaglandin production remains a factor. To lower the cost of prostaglandin production, we attempted to produce prostaglandins using a liverwort, Marchantia polymorpha L., which accumulates arachidonic acid, which is known as a substrate of prostaglandins. Here we report the first bioproduction of prostaglandins in plant species by introducing a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla into the liverwort. The transgenic liverworts accumulated prostaglandin F_2α, prostaglandin E₂ and prostaglandin D₂ which were not detected in the wild-type liverwort. Moreover, we succeeded in drastically increasing the bioproduction of prostaglandins using an in vitro reaction system with the extracts of transgenic liverworts.     

Prostaglandin production by methylcholanthrene-transformed mouse BALB/3T3. Requirement for protein synthesis

Stimulation of prostaglandin synthesis in transformed mouse fibroblasts by serum, thrombin, and bradykinin was blocked by actinomycin D and cycloheximide. These RNA and protein synthesis inhibitors did not affect prostaglandin synthetase in vitro or in vivo; nor did they affect the acylation of arachidonic acid into phospholipids. Serum-stimulated release of arachidonic acid and prostaglandins from [3H]arachidonic acid-labeled cells also was inhibited by actinomycin D and cycloheximide. RNA and protein synthesis appear to be required for expression of phospholipase activity; a prerequisite for prostaglandin synthesis by these cells.     

Simple direct radioimmunoassay of the F prostaglandins

A simplified and accurate method of determining the F prostaglandins in 0.1 ml of serum without previous extraction is described. The procedure involves addition of anti-prostaglandin F_2α to serum followed by tritiated prostaglandin, equilibration for 4 hours, removal of unbound prostaglandin with dextran-coated charcoal and subsequent liquid scintillation counting of the supernatant. The mean ± S.D. concentration of prostaglandin F_2α in the serum of 15 healthy men was 90 ± 33 pg/ml and in 20 women 108 ± 43 pg/ml.     

Regulation of prostaglandin synthesis during differentiation of cultured mouse myeloid leukemia cells

Mouse myeloid leukemia cells (Ml) were induced to differentiate into mature macrophages and granulocytes by various inducers. The differentiated Ml cells synthesized and released prostaglandins, whereas untreated Ml cells did not. When the cells were prelabelled with [¹⁴C]arachidonate, the major prostaglandins released into the culture media were found to be prostaglandin E₂, D₂, and F_2α in an early stage of differentiation, but the mature cells produced predominantly prostaglandin E₂. The synthesis and release of prostaglandins were completely inhibited by indomethacin. Dexamethasone, a potent inducer of differentiation of Ml cells, did not induce production of prostaglandins in resistant Ml cells that could not differentiate even with a high concentration of dexamethasone. These results suggest that production of prostaglandins in Ml cells is closely associated with differentiation of the cells. Homogenates of dexamethasone-treated Ml cells converted arachidonate to prostaglandins, but this conversion was scarcely observed with homogenates of untreated Ml cells. Dexamethasone and the other inducers stimulated the release of arachidonate from phospholipids. Therefore, induction of prostaglandin synthesis during differentiation of Ml cells may result from induction of prostaglandin synthesis activity and stimulation of the release of arachidonate from cellular lipids. Lysozyme activity, which is a typical biochemical marker of macrophages, was induced in Ml cells by prostaglandin E₂ or D₂ alone, as well as by inducers of differentiation of the cells, but it was not induced by arachidonate or prostaglandin F_2α. These results suggest that prostaglandin synthesis is important in differentiation of myeloid leukemia cells.     

Production of Hyperalgesic Prostaglandins by Sympathetic Postganglionic Neurons

Prostaglandin E2 and prostacyclin (prostaglandin I2) produce hyperalgesia in animals and humans. Because there is evidence that prostaglandins contribute to pain maintained by sympathetic nervous system activity, we evaluated whether sympathetic postganglionic neurons synthesize these hyperalgesic prostaglandins, and whether production of prostaglandins by these neurons can contribute to sensitization of primary afferent nociceptors. Intradermal injection of arachidonic acid but not linoleic acid, in the rat hindpaw, produces a decrease in mechanical nociceptive threshold. This hyperalgesic effect is prevented by indomethacin, an inhibitor of prostaglandin synthesis or by prior surgical removal of the lumbar sympathetic chain. To test the hypothesis that sympathetic postganglionic neurons are the source of prostaglandins, we measured production of prostaglandin E2 and 6-keto-prostaglandin F1 alpha (the stable metabolite of prostacyclin) by homogenates of adult rat sympathetic postganglionic neurons from superior cervical ganglia. These homogenates produced significant amounts of prostaglandin E2 and 6-keto-prostaglandin F1 alpha, and most of this production is eliminated by neonatal administration of 6-hydroxydopamine which selectively destroys sympathetic postganglionic neurons. These results demonstrate that sympathetic postganglionic neurons produce prostaglandins, and supports further the hypothesis that the release of prostaglandins from sympathetic postganglionic neurons contributes to the hyperalgesia associated with sympathetically maintained pain.     

Properties of prostaglandin synthetase of rabbit kidney medulla.

The formation in vitro of prostaglandins E2, D2, and F2alpha from arachidonic acid by rabbit kidney medulla homogenate or microsomal fraction is markedly affected by the composition of the incubation medium employed. Optimal biosynthesis is obtained in 0.1 M potassium phosphate buffer, with the optimum pH being 8.0--8.8. Under these conditions prostaglandin formation is linear up to arachidonic acid concentration of 30 muM. The initial rate of formation of prostaglandin E2 + prostaglandin D2 is 3--4 times higher than that of prostaglandin F2alpha. Reduced glutathione (1 mM) did not affect the biosynthesis by medulla homogenate and produced only small stimulation of the biosynthesis by microsomal powder. Hydroquinone produced a small stimulation at a low concentration of 0.005 mM, and a strong inhibition at concentrations of 0.1 mM or higher. Addition of bovine serum albumin (0.1%) reduced the microsomal biosynthesis of prostaglandins by approximately 80%. Addition of boiled homogenate or boiled 140 000 X g supernatant produced small stimulation of microsomal biosynthesis while 140 000 X g supernatant (not boiled) caused small inhibition which was not dose-related. It appears that rabbit kidney prostaglandin-synthetase converts arachidonic acid to prostaglandins E2 and F2alpha in comparable amounts, without apparent need for a cytoplasmic soluble cofactor or specific reducing agents.     

Effect of indomethacin on the adrenal renin response to nephrectomy in the rat

The role of prostaglandins in the control of adrenal renin in vivo was evaluated in nephrectomized rats. Nephrectomy increased adrenal renin from 13.2 +/- 1.37 ng angiotensin I/mg protein/hr to 166.5 +/- 17.3 ng angiotensin I/mg protein/hr. Indomethacin treatment significantly suppressed the adrenal renin response to nephrectomy. (47.8 +/- 5.22 ng angiotensin I/mg protein/hr). Adrenal aldosterone was also suppressed by indomethacin. Adrenal prostaglandin E2 increased after nephrectomy and decreased after indomethacin. Plasma corticosterone and serum potassium did not change after indomethacin. These data indicate that inhibition of prostaglandin synthesis by indomethacin partially blocks the adrenal renin response to nephrectomy, suggesting that prostaglandins may play a role in the adrenal response to nephrectomy.     

Interaction of prostaglandins with blood plasma proteins. Comparative binding of prostaglandins A2, F2α and E2 to human plasma proteins

1. The binding of prostaglandin A(2) and prostaglandin F(2alpha) to human plasma proteins was investigated by DEAE-Sephadex chromatography and polyacrylamide-gel electrophoresis. Both prostaglandins, when added to human plasma in vitro, were found to become bound mainly to plasma albumin. 2. The extent of binding of prostaglandins added to human plasma in low to moderate concentrations was found to be approx. 88, 73 and 58% for prostaglandins A(2), E(2) and F(2alpha) respectively. The order of affinities for the binding of the three prostaglandins to albumin appear to be A(2)>E(2)>F(2alpha). 3. The apparent association constants for the binding of these prostaglandins to human serum albumin were estimated to be approx. 4.8x10(4), 2.4x10(4) and 0.9x10(4) litre/mol for prostaglandins A(2), E(2) and F(2alpha) respectively. The results are compared with previously reported association constants for the binding of long-chain fatty acids to both human and bovine albumins.     

Prostaglandin production by dispersed granulosa and theca interna cells from porcine preovulatory follicles

Prepubertal gilts were treated with 750 IU pregnant mares' serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa (GC) and theca interna (TIC) cells were prepared by microdissection and enzymatic digestion from follicles obtained 36, 72 and 108 h after PMSG treatment and incubated for up to 6 h in a chemically defined medium in the presence or absence of arachidonic acid, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and indomethacin. Production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. Both GC and TIC had the capacity to produce prostaglandins, with production by each cell type increasing markedly with follicular maturation. PGE was the major prostaglandin produced by both cellular compartments. Only PGE production by GC was consistently enhanced by addition of arachidonic acid to the incubation medium. Neither cell type was responsive to FSH and LH in vitro. Indomethacin inhibited the production of PGE and PGF by both cell types. These results provide convincing evidence for an intrafollicular source of prostaglandins and indicate that both cellular compartments contribute significantly to the increased production of prostaglandins associated with follicular rupture.