Catabolism of an isolated, purified intermediate of prostaglandin biosynthesis by regions of the adult rat kidney.

1. A heat labile, cold-stable, stannous chloride-reducible intermediate of prostaglandin biosynthesis was formed in good yield (greater than 60%) from 3H-labeled arachidonic acid during brief incubations (30--90 s, 37 degrees C) with sheep seminal vesicle microsomes in the presence of p-hydroxymercuribenzoate (4 mM). This intermediate appears to have properties similar to one of the endoperoxides (15-hydroxyprostaglandin-9,11-endoperoxide) recently isolated by Hamberg and Samuelsson (Proc. Natl. Acad. Sci. U.S. (1973) 70, 889-903) AND Nugteren and Hazelhof (Biochem. Biophys. Acta. (1973) 326, 448-461). 2. Treatment of the purified intermediate with homogenates of rat kidney cortex, medulla and papilla resulted within 2 min (37 degrees C) in complete conversion into several compounds including prostaglandins E2 and F2alpha. The main product (40-50% yield formed by papilla homogenates was prostaglandin E2. The conversion into prostaglandin E2 was largely abolished by previous bo9ling of the homogenate whereas the conversion into prostaglandin F2alpha was not. The intermediate was stable in buffer for the same period of incubation. 3. The ratio of tritiated prostaglandins E2: F2alpha obtained were: papilla, 1.90; medulla, 0.76; cortex, 0.48. 4. These observations indicate that both types of prostaglandins can be formed by all three regions of the rat kidney and that regional differences exist in the proportion of E2 : F2alpha that is formed. Whereas prostaglandin E2 is mostly formed by an enzymatic process, prostaglandin F2alpha is not.     

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.     

Prostaglandin Biosynthesis from Endogenous Precursors in Rabbit Kidney

THIS report describes the biosynthesis of the naturally occurring renal prostaglandins E₂ (PGE₂) and F_2α (PGF_2α)^1,2 by homogenates and slices of rabbit renal medulla, from endogenous precursors. I have confirmed that rabbit renal cortex contains little prostaglandin and cannot synthesize them from endogenous lipids³. Hamberg has reported that arachidonic acid, which is converted to PGE₂ and PGF_2α by enzymes present in ram seminal vesicles⁴, can be efficiently converted to PGE₂ and PGF_2α by homogenates of rabbit renal medulla³. I have now confirmed that arachidonic acid, added to such medullary homogenates, can increase the quantities of prostaglandins synthesized. There was no evidence that the major prostaglandin biosynthesized, PGE2, was further metabolized to inactive products.     

Triacylglycerol lipase mediated release of arachidonic acid for prostaglandin synthesis in rabbit kidney medulla microsomes

The effect of triarachidonin on the synthesis of prostaglandins in rabbit kidney medulla microsomes was examined. Medulla microsomes were incubated with triarachidonin in 0.1 M--Tris/HCl buffer (pH 7.0) containing reduced glutathione and hydroquinone and the formed prostaglandin E2, prostaglandin F2 alpha and prostaglandin D2 were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization. The addition of triarachidonin (1-10 microM) stimulated prostaglandin formation in a dose-dependent manner. Under our incubation conditions rabbit kidney medulla was found to produce prostaglandin E2 mainly. When arachidonic acid, instead of triarachidonin, was added to the incubation mixture of microsomes, the identical profile of prostaglandin products was obtained. When the pH of the reaction mixture was changed from 7.0 to 8.0, the rate of triarachidonin-induced prostaglandin E2 formation was approximately 60% of that observed at pH 7.0. Studies utilizing Ca2+ and EGTA revealed that triacylglycerol lipase of kidney medulla is independent of Ca2+. The addition of epinephrine made the stimulatory effect of triarachidonin on prostaglandin E2 formation more pronounced. These results suggest that epinephrine-activated triacylglycerol lipase is present in the renomedullary microsomes, and this enzyme activity is a potential mediator of release of arachidonic acid for prostaglandin synthesis in the kidney medulla.     

Prostaglandin synthesis in human placenta and fetal membranes.

The conversion of exogenous arachidonic acid into prostaglandins was studied in human placenta and fetal membrane microsomes. Only one prostaglandin was formed, prostaglandin E2 (PGE2), in fetal membrane microsomes. In placental microsomes PGE2 was further transformed into 15 keto-PGE2. Cofactor requirements and some characteristics of the system were studied. 1 to 3% conversion of arachidonic acid into prostaglandins was observed in placental microsomes and 5 to 8% conversion in fetal membrane microsomes.     

Glucocorticosteroid regulation of prostaglandin biosynthesis in human myometrial smooth muscle cells in monolayer culture

In the present investigation, we evaluated the production of prostaglandins by human myometrial smooth muscle cells maintained in monolayer culture in the absence or presence of glucocorticosteroids. In the presence of cortisol (10(-7) M) or dexamethasone (10(-8) M), the rate of production of prostacyclin (PGI2) by these cells was decreased significantly. The glucocorticosteroid-mediated inhibition of prostaglandin production was attenuated when cortisol-21-mesylate (10(-6) M), a glucocorticosteroid antagonist, was present in the culture medium. The rate of conversion of radiolabeled arachidonic acid to radiolabeled prostaglandins as determined by use of sonicates of myometrial cells and optimal assay conditions, however, was not affected significantly by treatment with cortisol or dexamethasone in concentrations sufficient to inhibit prostaglandin formation by more than 80%. These findings are suggestive that glucocorticosteroids act in human myometrial smooth muscle cells in culture to inhibit prostaglandin formation by way of a receptor-mediated process that does not involve inhibition of enzyme activities that are involved in the biosynthesis of prostaglandins, i.e. the conversion of arachidonic acid to prostaglandin.     

Solubilization and partial purification of prostaglandin endoperoxide synthetase of rabbit kidney medulla.

The microsomes of rabbit kidney medulla converted arachidonic acid into prostaglandin E2 in the presence of hemoglobin, tryptophan and glutathione as activators. When themicrosomal suspension was treated with 1% Tween 20, a solubilized enzyme was obtained which catalyzed the conversion of arachidonic acid to prostaglandins G2 and H2. The solubilized enzyme was adsorbed to and then eluted from an omega-aminooctyl Sepharose 4B column, resulting in about 10-fold purification over the microsomes. The partially purified enzyme produced predominantly prostaglandin G2 in the presence of hemoglobin, while prostaglandin H2 was produced in the presence of both hemoglobin and tryptophan. The stimulation of prostaglandin endoperoxide formation was also observed with other heme and aromatic compounds. Prostaglandin H2 synthesis was inhibited by a variety of compounds including non-steroidal anti-inflammatory drugs, thiol compounds and prostaglandin analogues with a thiol group(s).     

Prostaglandin synthesis in human placenta and fetal membranes

The conversion of exogenous arachidonic acid into prostaglandins was studied in human placenta and fetal membrane microsomes. Only one prostaglandin was formed, prostaglandin E₂ (PGE₂), in fetal membrane microsomes. In placental microsomes PGE₂ was further transformed into 15 keto-PGE₂. Cofactor requirements and some characteristics of the system were studied. 1 to 3% conversion of arachidonic acid into prostaglandins was observed in placental microsomes and 5 to 8% conversion in fetal membrane microsomes.     

Endogenous inhibition of arachidonic acid metabolism in the endometrium of the sheep

Arachidonic acid is metabolised via the cyclo-oxygenase pathway to several biologically active metabolites. These metabolites control important reproductive functions like luteolysis of the corpus luteum. The metabolism of arachidonic acid was studied by the enzymatic conversion of [1-14C]-labelled arachidonic acid in sheep endometrial tissue. The inhibitory capacity of sheep endometrial tissue was measured by the enzymatic conversion of [1-14C]-arachidonic acid by sheep seminal vesicular gland microsomes. Endometrial microsomes converted arachidonic acid into different prostaglandins and monohydroxy acids but at a low rate. A factor(s) inhibiting both prostaglandin and monohydroxy acid synthesis was found in both the microsomal and cytosolic fractions of endometrial tissue. A very high inhibitory potency of prostaglandin and monohydroxy acid synthesis, calculated as IC50 values, was found in cytosolic fractions. For comparison IC50 values of indomethacin, mefenamic acid, carprofen and acetylsalicylic acid were also calculated in this in vitro system. These data indicate that both prostaglandin and monohydroxy acid synthesizing capacities and an inhibitory factor(s) are present in sheep endometrium and possibly regulate arachidonic acid metabolism in this tissue.     

Prostaglandins and renin release: I. Stimulation of renin release from rabbit renal cortical slices by PGI2.

Prostaglandins have been shown to be involved in the mechanism of renin secretion in a variety of situations. Both arachidonic acid and prostaglandin endoperoxide have been shown to release renin from cortical slices and to be converted to PGI2 by cortical microsomes. In the present studies PGI2 was found to cause a time dependent increase in renin release from rabbit renal cortical slices, a system isolated from any indirect effects that result from the administration of prostaglandins in vivo. The stimulation was linear up to 30 minutes and effective over a range of concentrations from 10(7 M to 10(-5) M. At similar concentrations 6-keto-prostaglandin F1alpha was not active on these slices. Thus, it is proposed that PGI2 exerts a direct effect on the release of renin from cortical cells and may be the mediator of arachidonate or prostaglandin endoperoxide stimulated renin secretion.     

Augmentation of unesterified arachidonic acid in the renal inner medulla of dexamethasone-treated rats. Relationship to altered triglyceride metabolism

The present study was designed to investigate the effect of dexamethasone treatment for 2 weeks (2.5 mg/kg/week, subcutaneously) on the level of unesterified fatty acids, particularly arachidonic acid, in the renal medulla of rats, and to relate the observed effect to changes in the tissue concentration and the fatty acid composition of renal medulla phospholipids and triglycerides. Dexamethasone treatment caused an increase in the renal inner medulla level of unesterified fatty acids, including arachidonic acid, that was associated with a reduction of triglycerides and of arachidonic acid esterified into triglycerides, and with an increase in the rate of fatty acids esterification into triglycerides. In contrast, dexamethasone treatment did not affect the renal medulla concentration of phospholipids, the arachidonic acid content of renal medulla phospholipids, or the rate of esterification of fatty acids into renal medulla phospholipids. In the face of increased fatty acid esterification into triglycerides, the finding of reduced triglyceride levels in the renal medulla of dexamethasone-treated rats suggests excessive triglyceride breakdown. If so, fatty acids including arachidonic acid liberated from triglycerides may contribute to elevation of unesterified fatty acid levels in the renal medulla during dexamethasone treatment. The increased level of free arachidonic acid in the renal medulla of dexamethasone-treated rats may explain in part the reported effect of this steroid in increasing urinary prostaglandins.     

Distribution of prostaglandins in rabbit kidney

Three prostaglandins (PGE(2), PGF(2alpha) and PGA(2)) are present in rabbit kidney medulla. An acidic lipid extract (0.165g) obtained from 2kg of frozen rabbit kidney cortex was separated by silicic acid chromatography to yield eluates containing fatty acids, possible non-polar prostaglandin metabolites, PGA, PGE and PGF compounds. Ultraviolet spectra of the eluates before and after treatment with sodium hydroxide did not yield chromophores typical of any known prostaglandins or related metabolites. By using more sensitive bioassay procedures (contraction of rabbit duodenum) weak activity equivalent to 60mug of PGE(2) and 10mug of PGF(2alpha) was detected in the PGE and PGF eluates respectively. Extraction and bioassay of fresh kidney cortex revealed no prostaglandin-like activity. Attempts to biosynthesize prostaglandins in fresh homogenates of rabbit kidney cortex from endogenous precursors and from added arachidonic acid were unsuccessful. When freshly prepared homogenates of rabbit kidney cortex were incubated with added PGE(1) no evidence of enzymic breakdown was obtained. It is concluded that rabbit kidney prostaglandins are present predominantly in the medulla and there are no cortical mechanisms for their biosynthesis or inactivation under normal conditions.     

Prostaglandin biosynthesis and its regulation in the bovine endometrium: A comparison between nonpregnant and pregnant status

Prostaglandin F(2alpha) (PGF(2alpha)), an arachidonic acid metabolism product of the prostaglandin synthetase pathway, is synthesized and released from the endometrium during luteolysis in nonpregnant animals. When proper conception occurs, the synthesis and release pattern is changed to maintain the corpus luteum (CL) function. The biosynthesis of prostaglandins in the bovine endometrium was highest in the microsomes but of low order. In nonpregnancy, the formation of prostaglandins from labelled precursor acid was higher than in pregnancy. Besides the prostaglandin synthetase, an inhibiting activity on the conversion of arachidonic acid to prostaglandins was found in both the nonpregnant and pregnant endometrium. During luteolysis (Day 17), a low inhibiting capacity was seen in comparison with other days of the estrous cycle (Days 1, 4 and 14). The inhibitory capacity was very high on Days 16 to 20, 25, and 31 of pregnancy. In the nonpregnant endometrium at Day 17, a very low inhibitor potency, calculated as IC(50) values, was found both in the cytoplasma and in the microsomes, whereas during early pregnancy (Days 17, 18, and 20) both cytoplasma and microsomes possessed very high inhibitor potency. This finding indicates that the bovine endometrium contains both prostaglandin synthetase and an unknown potent inhibitor of prostaglandin biosynthesis that regulates prostaglandin biosynthesis both during the estrous cycle and early pregnancy.     

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.     

Induction of prostacyclin formation by sodium n-butyrate in a cloned epithelial liver cell line

The effect of sodium n-butyrate on prostaglandin synthesis in cultured cells was examined. Exposure of BC-90 cells, a clone of an epithelial rat liver cell line, to 1 mM sodium n-butyrate for 40 h induced prostacyclin production. Prostacyclin synthesis was proved by demonstrating: (1) production of labeled 6-ketoprostaglandin F1 alpha by treating [14C]arachidonic acid pre-labeled cells with calcium ionophore A23187, (2) production of unstable substance that inhibited adenosine diphosphate-induced platelet aggregation, and (3) conversion of [14C]arachidonic acid to 6-ketoprostaglandin F1 alpha in homogenates of n-butyrate-treated cells. Untreated control cells showed negligible prostaglandin synthesis. Untreated cell homogenates did not convert [14C]arachidonic acid to any prostaglandins, but they converted [14C]prostaglandin H2 to prostacyclin. Induction of prostacyclin production by n-butyrate was also demonstrated with cells that had been treated with acetylsalicylic acid before n-butyrate treatment in acetylsalicylic acid-free medium. Incorporation of [3H]acetylsalicylic acid by sodium n-butyrate-treated cells increased in accordance with treatment time, while that of untreated cells did not change during culture. There was no difference in the phospholipase A2 activities of n-butyrate-treated and -untreated cells. From these findings, the possibility that n-butyrate induced prostacyclin in BC-90 cells through induction of fatty acid cyclooxygenase activity is discussed.     

Analysis of prostaglandins formed from endogenous and exogenous arachidonic acid in homogenates of human reproductive tissues

Isotope-labelled arachidonic acid has been used to study in vitro formation of prostaglandins and other products in mammalian tissue. Quantitative conclusions about cyclooxygenase activity have been drawn from such studies. However, arachidonic acid is present in all tissues, free and esterified, and therefore it can be expected that endogenous arachidonate would interfere with transformation of the radioactive exogenous substrate. (1-14C)-labelled arachidonate was, therefore, incubated with homogenates of various human tissues (amnion, chrorion, placenta and myometrium), and the two molecular forms, 12C and 14C, of arachidonic acid as well as of prostaglandin E2 and prostaglandin F2 alpha were quantitated, before and after 30 min of incubation, using gas chromatography-mass spectrometry with multiple ion detection. The results demonstrate a substantial release of arachidonic acid into the medium during incubation. There was no correlation between either the initial concentration of [12C]arachidonic acid and initial concentration of [12C]prostaglandin E2 or the percent increase of those compounds during incubation. The net formation of [12C]prostaglandin E2 and [14C]prostaglandin E2 from endogenous and exogenous precursor, respectively, were also very different. The study shows that by simply incubating (1-14C)-labelled arachidonic acid in tissue homogenates and measuring the amount of radioactivity transformed into various prostaglandins only qualitative conclusions can be drawn.     

Distribution of prostaglandin ω-hydroxylases in different tissues

The conversion of prostaglandins E₂ and F_2α to their 19- and 20-hydroxy metabolites by various tissues has been measured by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring. A number of different tissues of the pregnant rabbit possess prostaglandin 20-hydroxylase activity (lung > liver > fetal placenta > maternal placenta ≈ uterus > renal cortex > renal medulla ≈ placental membranes). With the exception of the liver, prostaglandins E₂ and F_2α are metabolized at equal rates by the 20-hydroxylases of different tissues. Only lung and liver microsomes possess high levels of prostaglandin 20-hydroxylase in non-pregnant rabbits and only liver microsomes have appreciable 19-hydroxylase activity. Pulmonary prostaglandin 20-hydroxylase is induced in male rabbits by treatment with progesterone. On the basis of substrate specificity studies and the effects of a cytochrome P-450 inhibitor, SKF-525A, the prostaglandin 20-hydroxylases of lung and liver microsomes from pregnant rabbits appear to be different enzymes. In pregnant rats and hamsters, liver and kidney are the only tissues in which we detected prostaglandin ω-hydroxylase activity.     

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.     

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.