Prostaglandin 9-hydroxydehydrogenase activity in the adult rat kidney. Identification, assay, pathway, and some enzyme properties.

Prostaglandin F2alpha is converted to 15-keto-13,14-dihydroprostaglandin E2 by adult rat kidney homogenates. A variety of substrates labeled as either the 9beta position alone or at several other positions in the prostaglandin molecule were used to define the step at which the crossover from the F type to the E type prostaglandins takes place. Time course studies further confirmed that 15-keto-13,14-dihydroprostaglandin F2alpha is the immediate substrate for this enzyme which we have termed prostaglandin 9-hydroxydehydrogenase. An assay system based on specific loss of tritium from 9beta-tritiated prostaglandin F2alpha is described. Enzyme activity with prostaglandin F2alpha as substrate is linear with time up to 10 min, stimulated by NAD+, saturable at low concentrations of substrate, stable to storage at minus 25 degrees in phosphate buffer (up to 3 weeks), and has a broad pH optimum around 7.5. The product, 15-keto,13,14-dihydroprostaglandin E2 was identified by mass spectrometry through a sodium borohydride-sodium borodeuteride reduction method.     

9-Hydroxyprostaglandin dehydrogenase in rat kidney cortex converts prostaglandin I2 into 15-keto-13,14-dihydro 6-ketoprostaglandin E1

15-Keto-13,14-dihydro 6-ketoprostaglandin E1 was positively identified by gas chromatography-mass spectrometry with negative-ion chemical ionisation detection from samples of rat kidney high-speed supernatant incubated with prostaglandin I2 in the presence of NAD+. A decreased formation of this product was observed when NAD+ was substituted with NADP+ and none was observed in the absence of nucleotide or substrate prostaglandin I2. Experiments with [9 beta-3H]prostaglandin I2 showed a time- and concentration-dependent loss of tritium which appeared as tritiated water, typical of reaction of [9 beta-3H]prostaglandin substrates with the enzyme, 9-hydroxyprostaglandin dehydrogenase. Time-course measurements of the appearance of tritiated water showed similar rates with 6-keto[9 beta-3H]prostaglandin F1 alpha and 15-keto-13,14-dihydro 6-keto[9 beta-3H]prostaglandin F1 alpha as substrates. These experiments suggest that the transformation of prostaglandin I2 and 6-ketoprostaglandin F1 alpha into the 15-keto-13,14-dihydro 6-ketoprostaglandin E1 catabolite occurs in this in vitro preparation via the corresponding 15-keto-13,14-dihydro catabolite of 6-ketoprostaglandin F1 alpha.     

Uterine vein prostaglandin levels in late pregnant dogs.

We studied the uterine venous plasma concentrations of prostaglandins E2, F2 alpha, 15 keto 13,14 dihydro E2 and 15 keto 13,14 dihydro F2 alpha in late pregnant dogs in order to evaluate the rates of production and metabolism of prostaglandin E2 and F2 alpha in pregnancy in vivo. We used a very specific and sensitive gas chromatography-mass spectrometry assay to measure these prostaglandins. The uterine venous concentrations of prostaglandin E2 and 15 keto 13,14 dihydro E2 were 1.35 +/- .27 ng/ml and 1.89 +/- .37 ng/ml, respectively; however, we could not find any prostaglandin F2 alpha and very little of its plasma metabolite in uterine venous plasma. Since uterine microsomes can generate prostaglandin F2 alpha and E2 from endoperoxides, prostaglandin F2 alpha production in vivo must be regulated through an enzymatic step after endoperoxide formation. Prostaglandin E2 is produced by pregnant canine uterus in quantities high enough to have a biological effect in late pregnancy; however, prostaglandin F2 alpha does not appear to play a role at this stage of pregnancy.     

On the metabolism of prostaglandins by human gastric fundus mucosa.

1.Specific radioimmunoassays for the prostaglandins E2, F2alpha and A2 and the metabolites 13,14-dihydro-15-keto-prostaglandin E2, 15-keto-prostaglandin F2alpha and 13,14-dihydro-15-keto-prostaglandin F2alpha were used to study the metabolism of prostaglandins by gastroscopically obtained small biopsy specimens of human gastric fundus mucosa. 2.Three prostaglandin-metabolizing enzymes were found in the 100 000 X g supernatant of human gastric fundus mucosa, 15-hydroxy-prostaglandin-dehydrogenase, delta13-reductase and delta9-reductase. The specific activity was highest for 15-hydroxy-prostaglandin-dehydrogenase and lowest for delta9-reductase. 3.Formation of prostaglandin A2 (or B2) was not observed under the same conditions. 4.None of the three enzyme activities detected in the 100 000 X g supernatant was found in the 10 000 X g and 100 000 X g pellets of human gastric fundus mucosa. 5.The results indicate that high speed supernatant derived from human gastric mucosa can rapidly metabolize prostaglandin E2 and prostaglandin F2alpha to the 15-keto and 13,14-dihydro-15-keto-derivatives. Furthermore, prostaglandin E2 can be converted to prostaglandin F2alpha, the biological activity of which, on gastric functions, differs from that of prostaglandin E2.     

Metabolism of prostaglandin D2 in isolated rat lung: the stereospecific formation of 9 alpha,11 beta-prostaglandin F2 from prostaglandin D2

The metabolic transformation of exogenous prostaglandin D2 was investigated in isolated perfused rat lung. Dose-dependent formation (2-150 ng) of 9 alpha,11 beta-prostaglandin F2, corresponding to about 0.1% of the perfused dose of prostaglandin D2, was observed by specific radioimmunoassay both in the perfusate and in lung tissue after a 5-min perfusion. To investigate the reason for this low conversion ratio, we analyzed the metabolites of tritium-labeled 9 alpha,11 beta-prostaglandin F2 and prostaglandin D2 by boric acid-impregnated TLC and HPLC. By 5 min after the start of perfusion, 9 alpha,11 beta-prostaglandin F2 disappeared completely from the perfusate and the major product formed remained unchanged during the remainder of the 30-min perfusion. The major product was separated by TLC and identified as 13,14-dihydro-15-keto-9 alpha,11 beta-prostaglandin F2 by GC/MS. In contrast, pulmonary breakdown of prostaglandin D2 was slow and two major metabolites in the perfusate increased with time, each representing 56% and 11% of the total radioactivity at the end of the perfusion. The major product (56%) was identified as 13,14-dihydro-15-ketoprostaglandin D2 and the minor one (11%) was tentatively identified as 13,14-dihydro-15-keto-9 alpha,11 beta-prostaglandin F2 based on the results from radioimmunoassays, TLC, HPLC, and the time course of pulmonary breakdown. These results demonstrate that the metabolism of prostaglandin D2 in rat lung involves at least two pathways, one by 15-hydroxyprostaglandin dehydrogenase and the other by 11-ketoreductase, and that the 9 alpha,11 beta-prostaglandin F2 formed is rapidly metabolized to 13,14-dihydro-15-keto-9 alpha,11 beta-prostaglandin F2.     

The purification and properties of NADPH-dependent carbonyl reductases from rat ovary

Two carbonyl reductases have been highly purified from rat ovary to apparent homogeneity. Though they have similarities in terms of molecular weight (33,000), substrate specificities, inhibitor sensitivities, amino acid composition, and immunological properties, they differed in pI values (6.0 and 5.9). Both enzymes reduced aromatic aldehydes, ketones, and quinones at higher rates, compared to prostaglandins and 3-ketosteroids, whereas they showed higher affinity for prostaglandins and 3-ketosteroids. The enzymes also catalyzed oxidation of the 9-hydroxy group of prostaglandin F2 alpha. Moreover, they showed the remarkable characteristic of catalyzing the reduction of not only the 9-keto group of prostaglandin E2 but also the 15-keto group of 13,14-dihydro-15-keto-prostaglandin F2 alpha. Both enzymes were inhibited by SH-reagents, quercitrin, indomethacin, furosemide, and disulfiram. The results of immunoinhibition, using antibody against the purified enzymes, indicated that the enzymes were solely responsible for the overall catalytic activities of prostaglandin E series reduction, as well as 13,14-dihydro-15-keto-prostaglandin F2 alpha reduction and prostaglandin F2 alpha oxidation in rat ovarian cytosol. Western-blot analysis revealed that immunoreactive proteins were present in adrenal gland and various reproductive tissues except uterus of rats.     

Sex differences in prostaglandin metabolism.

Prostaglandin E2 (PGE2), PGF2alfa and PGD2 were synthetized from [1-¹⁴C]-arachidonic acid by rat kidney medulla microsomal fraction. The formation of each prostaglandin was significantly less in female animals than in males. The rate of inactivation of [³H]-PGF2alfa by kidney cortex cytosol was almost linear with the time of incubation during the first 30 min. The production of PGF2alfa metabolite (13,14-dihydro-15-keto PGF2alfa) was higher in male rats than in females.     

Prostaglandin production by type II alveolar epithelial cells.

Prostaglandin production was studied in fetal and adult type II alveolar epithelial cells. Two culture systems were employed, fetal rat lung organotypic cultures consisting of fetal type II cells and monolayer cultures of adult lung type II cells. Dexamethasone, thyroxine, prolactin and insulin, hormones which influence lung development, each reduced the production of prostaglandin E and F alpha by the organotypic cultures. The fetal cultures produced relatively large quantities of prostaglandin E and F alpha and smaller quantities of 6-keto-prostaglandin F1 alpha and thromboxane B2. However, prostaglandin E2 production was predominant. In contrast, the adult type II cells in monolayer culture produced predominantly prostacyclin (6-keto-prostaglandin F1 alpha) along with smaller quantities of prostaglandin E2 and F2 alpha. The type II cells were relatively unresponsive to prostaglandins. Exogenously added prostaglandin E, had no effect on cell growth, and only a minimal effect on cyclic AMP levels in the monolayer cultures.     

Specific production of prostaglandin E by human amnion in vitro

Prostaglandin production by intra-uterine human tissues has been investigated using a method of tissue superfusion. Tissues were obtained at elective Caesarean section and after spontaneous vaginal delivery. It was found that all the tissues studied (amnion, chorion, decidua and placenta) produced more prostaglandin E (PGE) and 13,14-dihydro-15-keto-prostaglandin F (PGFM — the major circulating metabolite of prostaglandin F) than prostaglandin F (PGF). Amnion produced significantly more PGE (but not PGF or PGFM) than any other tissue. Prostaglandin production by each tissue was similar whether it was taken at elective Caesarean section or after spontaneous vaginal delivery.     

Altered glomerular eicosanoid biosynthesis in uranyl nitrate-induced acute renal failure

The present studies were designed (1) to examine the pattern of changes in eicosanoid biosynthesis in isolated rat glomeruli, and (2) to correlate these changes with the previously observed alterations in renal perfusion and glomerular filtration rate which occur after uranyl nitrate administration, a model of toxin-induced acute renal failure. In the first part of this study, the in vitro and the in vivo effects of two cyclooxygenase inhibitors were examined for their ability to inhibit rat glomerular eicosanoid biosynthesis. Inhibition of prostaglandin E2 and prostaglandin F2 alpha generation by 1 mM aspirin in vitro was 76 and 82%, respectively. Similar inhibitions of 85 and 72% of biosynthesis of the above-mentioned lipids by 0.1 mM indomethacin were also noted. Intraperitoneal administration of aspirin (150 mg/kg) resulted in a significant inhibition of 88% or greater of prostaglandin E2, prostaglandin F2 alpha, 6-keto-prostaglandin F2 alpha, and thromboxane B2 biosynthesis. These results indicated that the expected alterations produced under in vivo conditions were detectable by in vitro techniques used in this study. 24 h after the administration of uranyl nitrate (25 mg/kg), significant increases in the biosynthesis of prostaglandin E2 (124%) and prostaglandin F2 alpha (88%) were observed when compared to the control values. No significant changes in prostacyclin or thromboxane formation were noted at this time. A further increase in the biosynthesis of prostaglandin E2 (248%), prostaglandin F2 alpha (262%), and a significant increase in prostacyclin (120%), measured as 6-keto-prostaglandin F1 alpha, were noted at 48 h. No changes in thromboxane B2 biosynthesis were noted. It is concluded that these data are consistent with the hypothesis that the increased glomerular biosynthesis of vasodilator eicosanoids (i.e., prostaglandin E2 and prostacyclin) may play a significant role in the homeostatic regulation of renal perfusion and glomerular filtration after acute toxic injury to the kidney.     

Radioimmunoassay of 13,14-dihydro-15-keto prostaglandin F in bovine peripheral plasma

A radioimmunoassay has been developed for 13,14-dihydro-15-keto-prostaglandin F in bovine peripheral plasma. Acidified plasma samples were extracted with diethyl ether and the dried extracts assayed for 13,14-dihydro-15-keto-prostaglandin F using antiserum raised against a 13,14-dihydro-15-keto-prostaglandin F_2α-albumin complex. The tracer used for the assay was prepared enzymatically from tritiated prostaglandin F_1α. Polyethylene glycol was employed to separate free and bound 13,14-dihydro-15-keto-prostaglandin F. The inter-assay coefficient of variation based on 9 determinations of control plasma was 13.8%. The detection limit of the assay was 25 pg 13,14-dihydro-15-keto-prostaglandin F/ml plasma.In 3 cows around estrus there was a complex series of peaks of 13,14-dihydro-15-keto-prostaglandin F concentrations coincident with luteolysis and declining progesterone concentrations. Changes in peripheral plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F in the pregnant cow near term showed a close correlation with prostaglandin F levels in utero-ovarian venous plasma. The concentration of 13,14-dihydro-15-keto-prostaglandin F in 12 men was 114±20 pg/ml plasma. It is concluded that the measurement of peripheral 13,14-dihydro-15-keto-prostaglandin F concentrations may offer a simple and convenient method for monitoring uterine prostaglandin F production in the cow.     

Radioimmunoassay of 13,14-dihydro-15-keto prostaglandin F in bovine peripheral plasma

A radioimmunoassay has been developed for 13,14-dihydro-15-keto-prostaglandin F in bovine peripheral plasma. Acidified plasma samples were extracted with diethyl ether and the dried extracts assayed for 13,14-dihydro-15-keto-prostaglandin F using antiserum raised against a 13,14-dihydro-15-keto-prostaglandin F_2α-albumin complex. The tracer used for the assay was prepared enzymatically from tritiated prostaglandin F_1α. Polyethylene glycol was employed to separate free and bound 13,14-dihydro-15-keto-prostaglandin F. The inter-assay coefficient of variation based on 9 determinations of control plasma was 13.8%. The detection limit of the assay was 25 pg 13,14-dihydro-15-keto-prostaglandin F/ml plasma.In 3 cows around estrus there was a complex series of peaks of 13,14-dihydro-15-keto-prostaglandin F concentrations coincident with luteolysis and declining progesterone concentrations. Changes in peripheral plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F in the pregnant cow near term showed a close correlation with prostaglandin F levels in utero-ovarian venous plasma. The concentration of 13,14-dihydro-15-keto-prostaglandin F in 12 men was 114±20 pg/ml plasma. It is concluded that the measurement of peripheral 13,14-dihydro-15-keto-prostaglandin F concentrations may offer a simple and convenient method for monitoring uterine prostaglandin F production in the cow.     

Secretory diarrhea in villous adenoma of rectum: effect of treatment with somatostatin and indomethacin.

The effects of treatment with the synthetic long-acting somatostatin analogue SMS-201-995 were studied in a patient with a fluid and electrolyte secreting villous adenoma of the rectum. The effects of SMS-201-995 on rectal fluid volume and electrolyte loss, and local and general prostanoid production were compared with those of treatment with indomethacin. During treatment with the somatostatin analogue iso-osmolar rectal fluid production increased about 25%; the quantity of prostaglandin E2 in the rectal fluid rose almost 20-fold. Prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha and 13,14-dihydro-15-keto-prostaglandin F2 alpha output showed similar, though less impressive increments during somatostatin treatment. The somatostatin analogue did not affect urinary prostanoid excretion except for levels of 2,3-dinor-thromboxane B2, which doubled. With indomethacin treatment diurnal rectal fluid production dropped by about 50% and all prostanoids measured in urine and rectal fluid decreased below control values. It appears that the somatostatin analogue SMS-201-995 has a marked stimulatory effect on the in vivo prostanoid production by the villous adenoma. Perhaps this stimulation is not confined to the tumor only, but also affects thromboxane synthesis.     

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.     

Stimulation of the formation of 13,14-dihydroprostaglandin F2 alpha by gonadotropin in rat ovary

Effects of pregnant mare serum gonadotropin and human chorionic gonadotropin on the formation of 13,14-dihydroprostaglandin F2 alpha, a biologically active compound, were investigated in rat ovarian homogenate. The mass number of the compound, which was formed prostaglandin F2 alpha via 13,14-dihydro-15-ketoprostaglandin F2 alpha in rat ovarian homogenate but was not produced in rat homogenate, accorded with that of the authentic 13,14-dihydroprostaglandin F2 alpha by negative ion chemical ionization mass spectrometry. In the present experiment, the radioactivity of [3H]prostaglandin F2 alpha added to ovarian homogenate was decreased linearly and immediately until the incubation time of 10 min. The formation of 13,14-dihydroprostaglandin F2 alpha was increased up to 60 min. The formation of 13,14-dihydroprostaglandin F2 alpha from prostaglandin F2 alpha was markedly increased by pregnant mare serum gonadotropin and human chorionic gonadotropin. However, there was no additive or synergistic effect of these hormones. The formation of 13,14-dihydroprostaglandin F2 alpha from 13,14-dihydro-15-ketoprostaglandin F2 alpha weas also greatly stimulated by pregnant mare serum gonadotropin and human chorionic gonadotropin. The formation of 13,14-dihydro-15-ketoprostaglandin F2 alpha steeply declined until 24 h after treatment with human chorionic gonadotropin in pregnant mare serum gonadotropin-primed rats. In contrast, the formation of 13,14-dihydroprostaglandin F2 alpha was markedly increased until 24 h after human chorionic gonadotropin treatment, and the level was about 2.5-fold higher than that at 0 h, 48 h after injection of pregnant mare serum gonadotropin.     

Formation of 20-hydroxyprostaglandins by lungs of pregnant rabbits

Homogenates or particulate fractions (1,000 to 100,000 X g) from lungs of pregnant rabbits were incubated with prostaglandins or prostaglandin metabolites and the products were purified by chromatography and identified by gas chromatography-mass spectrometry. In the presence of NADPH, particulate fractions from pregnant rabbit lungs converted prostaglandins E1, E2, and F2alpha as well as 13,14-dihydro-15-oxoprostaglandin E2 and 13, 14-dihydro-15-oxoprostaglandin F2alpha to their 20-hydroxy derivatives. In the cases of the 3 primary prostaglandins, the corresponding omega-carboxylic acids were also isolated. The omega-hydroxylation reaction occurred in the presence of the microsomal fraction. The mitochondrial fraction was much less active whereas the cytosol fraction converted prostaglandins to their 13, 14-dihydro-15-oxo derivatives. When prostaglandin F2alpha was incubated with homogenates of lungs from pregnant rabbits, omega-oxidation was combined with oxidation of the 15-hydroxyl group and reduction of the 13, 14-double bond to give 13, 14-dihydro-20-hydroxy-15-oxoprostaglandin F2alpha as well as the corresponding derivative with an omega-carboxylic acid group. Lungs from nonpregnant rabbits were much less active than lungs from pregnant rabbits in the omega-oxidation of prostaglandins.     

Plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F during pregnancy in sheep

A specific and sensitive radioimmunoassay is described for 13,14-dihydro-15-keto-prostaglandin F in ovine plasma. Using this assay it has been shown that, in sheep, jugular venous 13,14-dihydro-15-keto-prostaglandin F concentrations increase at parturition and correlate well with concentrations of prostaglandin F in the utero-ovarian vein. It is suggested that uterine prostaglandin F production under these conditions may be assessed by measuring peripheral venous 13,14-dihydro-15-keto-prostaglandin F, thereby avoiding the need for chronic utero-ovarian venous catheters.     

Prostaglandin concentrations in intra-uterine tissues from late-pregnant sheep before and after labour

Prostaglandin E (PGE), prostaglandin F (PGF) and 13,14-dihydro-15-keto-prostaglandin F (PGFM) have been measured in cotyledons and myometrium from sheep before and after labour. Fetal cotyledons contained more PGE than maternal cotyledons which in turn contained more than myometriu. The maternal cotyledon contained the highest concentrations of PGF, but the fetal cotyledon was the only tissue exhibiting a statistically significant rise in the concentration of PGF following labour. Concentrations of PGFM were closely correlated with (although usually lower than) those of PGF.     

15-Keto-13,14-dihydroprostaglandin E2- and F2 alpha-metabolite levels in blood from men and women given prostaglandin E2 orally

Prostaglandin E2 (PGE2) was administered orally in a dose of 1 mg to healthy males (n = 20) and females (n = 10). Blood levels of 15-keto-13,14-dihydroprostaglandin F2 alpha (PGF2 alpha-M) and 15-keto-13,14-dihydroprostaglandin E2 (PGE2-M), determined as the rearrangement product 11-deoxy-15-keto-13,14-dihydro-11 beta, 16-cycloprostaglandin E2 (PGE2-cyclo-M), were measured. The levels of the two PG metabolites increased already 10 minutes after ingestion of the tablet and the mean peak value for PGE2-cyclo-M in the men was 4.64 nmol/l which was reached 50 minutes after PGE2 administration. The mean peak value in women was 4.99 nmol/l which was obtained after 30 minutes. The increase in PGE2-cyclo-M concentration was significantly faster (p less than 0.05) in women than in the men. The mean plasma concentration of PGF2 alpha in males were 0.20 nmol/l prior to treatment and rose after PGE2 ingestion to mean peak level of 0.84 nmol/l after 70 minutes. The corresponding values for the females were 0.18 nmol/l and 0.88 nmol/l 50 minutes into treatment. When the data from both sexes were amalgamated PGE2-cyclo-M peak levels were reached significantly (p = 0.004) sooner than the PGF2 alpha-M peak. The two PG metabolites returned to baseline levels in 70% of the individuals after 240 minutes. The increase in PGF2 alpha-M concentration following oral administration of PGE2 indicates that part of the PGE2 was reduced to PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Responsiveness of the lamb ductus arteriosus to prostaglandins and their metabolites

The relative potencies of the prostaglandins A1, A2, E1, E2, F2alpha and their 15-keto-, 15-keto-13,14-dihydro-, and 13,14-dihydro-metabolites were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO2. All the prostaglandins (except PGF2alpha and its 15-keto-metabolites) relaxed the tissue. However, only PGE1, E2, and their 13,14-dihydro-metabolites, were effective at concentrations below 10(-8) M. Therefore, events that alter metabolism of circulating PGs in the perinatal period may have significant effects on the relative patency or closure of the ductus arteriosus.