Enzymatic conversion of prostaglandin H2 to prostaglandin F2 alpha by aldehyde reductase from human liver: comparison to the prostaglandin F synthetase from bovine lung

The primary structure of prostaglandin (PG) F synthetase from bovine lung shows 62% similarity with that of human liver aldehyde reductase (EC 1.1.1.2) (Watanabe, K., Fujii, Y., Nakayama, K., Ohkubo, H., Kuramitsu, S., Kagamiyama, H., Nakanishi, S., and Hayaishi, O. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 11-15). We therefore purified human liver aldehyde reductase to homogeneity and compared the immunological and catalytic properties of aldehyde reductase and PGF synthetase. Although both enzymes belong to a group of aldoketoreductases and their molecular weights are essentially identical, aldehyde reductase had no cross-reactivity to anti-PGF synthetase antiserum. Furthermore, there was a difference in the substrate specificity for reduction of PGs between the two enzymes. Aldehyde reductase catalyzed the reduction of PGJ2, delta 12-PGJ2, PGH2, or PGA2, but not that of PGB2, PGD2, or PGE2, whereas PGF synthetase reduced PGD2. The optimum pH, Km value for PGH2, and the turnover number were 6.5, 100 microM, and 3.1 min-1, respectively. The PGH2 9,11-endoperoxide reductase activity of aldehyde reductase was not affected in the presence of a substrate such as p-nitrobenzaldehyde, DL-glyceraldehyde, or 9,10-phenanthrenequinone, suggesting that PGH2 9,11-endoperoxide and other substrates are reduced at different active site(s). The reaction product formed from PGH2 by this enzyme was identified as PGF2 alpha by gas chromatography/mass spectrometry. These results suggest that aldehyde reductase is not exactly identical to PGF synthetase in terms of its immunological property and substrate specificity for PGs, but that this enzyme is also involved in the direct conversion of PGH2 to PGF2 alpha similar to PGF synthetase.     

Diamide inhibits pulmonary vasoconstriction induced by hypoxia or prostaglandin F2 alpha

Diamide oxidizes glutathione and other cellular sulfhydryl groups. It decreases calcium ATPase activity and alters mitochondrial calcium flux, probably as a result of the sulfhydryl oxidation. We examined the effect of diamide (5 mg/kg, iv) on pulmonary vascular reactivity in 12 anesthetized dogs. Diamide reversed the pulmonary vasoconstriction caused by hypoxia in seven dogs (control delta PVR + 2.5 +/- 0.6 mm Hg/liter/min; postdiamide delta PVR - 0.1 +/- 0.4 mm Hg/liter/min; P less than 0.01). The pulmonary pressor response to prostaglandin F2 alpha (5 micrograms/kg/min, iv) was also reduced (control delta PVR + 3.8 +/- 0.5 mm Hg/liter/min; postdiamide delta PVR + 1.1 +/- 0.7 mm Hg/liter/min; P less than 0.01). However, in a further five dogs, diamide had only a small effect on the pulmonary vasoconstriction caused by angiotensin II, while the pressor response to hypoxia was again inhibited. The mechanism by which diamide reverses pulmonary vasoconstriction is not certain but the effect is rapid, consistent, and reversible. Because the intravenous infusion of diamide does not produce systemic hypotension, during its period of action on the pulmonary vasculature, unlike the drugs currently available for the clinical treatment of pulmonary hypertension, further studies of its mechanism of action are indicated.     

Regulation of hysterectomy induced derangements in ovarian carbohydrate metabolism in albino rats. II. Role of prostaglandin F2 alpha.

Wistar strain albino rats were subjected to bilateral hysterectomy surgically and the ovarian carbohydrate metabolism of these animals was compared with sham operated controls. The ovarian glycogen content of hysterectomized animals was elevated with inhibition of glycogenolysis, hexose mono and diphosphate pathways and oxidative metabolism. Administration of PGF2 alpha to hysterectomized animals led to activation of ovarian glycogenolysis and other pathways of carbohydrate metabolism of hysterectomized animals was restored towards normal level after PGF2 alpha substitution.     

Role of prostaglandin F2alpha in ovulation.

Using radioimmunoassay procedures, the levels of plasma, uterine and ovarian prostaglandin (PG) F2alpha, and those of plasma estradiol and progesterone were measured in intact, hysterectomized or ovariectomized immature female rats pretreated with PMS and subsequent HCG. Occurrence of ovulation was confirmed at 8 hours after the HCG administration not only in the intact rats but also in the hysterectomzied rats. The levels of plasma estradiol and progesterone, and of uterine and ovarian PGF2alpha rose with the PMS injection alone, but they did not reach the peaks before the HCG administration. Both plasma estradiol and uterine PGF2alpha showed a peak at 2 hours after the HCG injection. These peaks were antecedent 2 or 6 hours before the peaks of ovarian and plasma PGF2alpha, respectively. However, such increase of uterine PGF2alpha does not seem to be indispensable for ovulation, because ovulation could occur in the hysterectomized rats. The levels of ovarian PGF2alpha showed a high plateau from 4 to 8 hours after the HCG injection, and then rapidly decreased after ovulation. The levels of plasma PGF2alpha peaked not only in the intact rats but also in the hysterectomized rats at 8 hours after the HCG treatment. But in the ovariectomized rats, this plasma PGF2alpha peak at 8 hours disappeared and there was no statistical change of plasma PGF2alpha throughout the PMS-HCG treatment. Plasma progesterone gradually increased and reached the maximum at 10 hours after the HCG injection. These results conclude that the main source of increased plasma PGF2alpha during the ovulatory process induced with the PMS-HCG treatment is the ovary, and it is strongly suggested that a rapid increase of PGF2alpha in the ovary may play some important role(s) in the ovulatory process.     

Fertilization rate after synchronization of the oestrous cycle in heifers with prostaglandin F2alpha.

Oestrus was synchronized in 31 heifers by the intrauterine administration of PGF₂α than salt. Nineteen were given 2 doses of 0.5mg 24 hr apart, and 10 of these received 1500 I.U. of PMSG i.m. 24 hr before the treatment with PGF₂α. The remaining 12 heifers in the experiment were given a single dose of 2mg followed at the beginning of oestrus by 1500 I.U. of HCG i.m. Of 9 heifers which received only the two doses of 0.5mg (Group 1), 7 were observed to have corpora lutea when slaughtered 56–72 hr after the onset of oestrus, and four fertilized eggs were recovered. In those which received PMSG before the double injection of PGF₂α(Group 2), 118 corpora lutea were observed at slaughter and 34 fertilized eggs were recovered. Each heifer which received a single injection of PGF₂α and HCG had a corpus luteum, and 9 fertilized eggs were recovered. Unovulated follicles were most commonly observed in the PMSG-treated heifers but they were also observed in the heifers given the double injection treatment. It was observed that in the two-injection treatments, whether or not given PMSG, time of ovulation relative to the onset of oestrus was variable, and eggs were found in the uterus before the expected time.     

Enzymatic formation of prostaglandin F2 alpha from prostaglandin H2 and D2. Purification and properties of prostaglandin F synthetase from bovine lung

Prostaglandin F synthetase from bovine lung was purified 540-fold to apparent homogeneity, as assessed by polyacrylamide gel electrophoreses and ultracentrifugation. The purified enzyme proved to be a monomeric protein with a molecular weight of about 30,500. The enzyme catalyzed not only the reduction of the 11-keto group of prostaglandin D2 but also the reduction of 9,11-endoperoxide of prostaglandin H2 and various carbonyl compounds (e.g. phenanthrenequinone). Experiments using column chromatography, polyacrylamide gel electrophoreses, immunotitration using antibody against the purified enzyme, and heat treatment indicated that three enzyme activities resided in a single protein. Although phenanthrenequinone and prostaglandin D2 competitively inhibited the prostaglandin D2 and phenanthrenequinone reductase activities, respectively, these two substrates were all but ineffective on the prostaglandin H2 (at the Km value) reductase activity up to 14-fold of those Km values. These results suggest that a single enzyme protein purified from the bovine lung catalyzes the reduction of prostaglandin D2, prostaglandin H2, and various carbonyl compounds and that prostaglandin D2 and prostaglandin H2 are metabolized at two different active sites, yielding prostaglandin F2 alpha as the reaction product.     

Histophysiological studies of prostaglandin F2alpha on isolated organs. I. Effect of prostaglandin F2alpha on the heart.

The physiological and histochemical effects of PGF2alpha on isolated rabbit hearts were examined. The results showed a positive inotropic effect. The coronary flow increased. From the histochemical studies, adenosine triphosphatase (ATP-ase) and succinic dehydrogenase activities were increased while that of alkaline phosphatase was decreased. Glycogen granules were depleted. These findings were discussed on a histophysiological basis.     

Thromboxane B2, 6-keto-prostaglandin F1 alpha, and prostaglandin F2 alpha production by contracting pregnant rate uteri in vitro

While prostaglandin production by uterine tissue has been shown to be involved in the contractile mechanism of this tissue, less attention has focused upon the involvement of other prostanoids. We have simultaneously measured in vitro isometric contractility of pregnant rat uteri with the release of prostaglandin F2 alpha (PGF), 6-keto-prostaglandin F1 alpha (6-k-PGF1 alpha) and thromboxane B2 (TXB2) into the bathing medium under various conditions. Frequency of uterine contractions and integrated contractile force (ICF) increased from 15 days of gestation and peaked at the time of parturition. Activity was generally greatest during the first 15 min of incubation except during parturition and on Day 1 postpartum when the uterine segment remained active for 1 h experimental period. Indomethacin (INDO) significantly reduced contractile activity regardless of gestational stage. PGF, TXB2, and 6-k-PGF1 alpha increased with gestational age, peaking at the time of parturition. Production was greatest during the first 15 min of incubation and INDO inhibited production of each prostanoid regardless of gestational stage. Imidazole (100 micrograms/ml) inhibited TXB2 production without affecting PGF or 6-k-PGF1 alpha levels. Frequency of contraction and ICF were not affected by imidazole treatment despite TXB2 reduction. These data demonstrate that the in vitro uterus from pregnant rats is capable of producing prostanoids other than prostaglandins and their production generally parallels uterine contractile activity. Thus, the possibility that these prostanoids are involved in physiologic changes during parturition warrants further investigation.     

Transpulmonary prostaglandin F2 alpha metabolism in sheep: an in vivo model

We investigated transpulmonary enzymatic conversion of prostaglandin F2 alpha (PGF) to the 13,14-dihydro-15-keto metabolite (PGFM) in normal and acutely lung injured sheep. PGF was infused directly into the right ventricle. Sequential, simultaneous blood samples were drawn from the pulmonary artery (PA) and aorta (A). PGF and PGFM plasma concentrations were quantitated by double antibody radioimmunoassay (RIA). The pulmonary conversion rate of PGF in normal lung was established over a wide range of concentrations in intubated, normoxic, and hemodynamically stable sheep. Both zero and first order kinetics were present. PGF had no physiological effects on either pulmonary or systemic hemodynamics at any infusion rate studied. Acute lung injury was produced by intravenous injections of oleic acid into the PA until the resting mean pulmonary artery pressure doubled. Infusions were then repeated and fractional metabolism of PGF across the lung was assessed. PGF, at infusion rates of 2 micrograms/kg/min and 8 micrograms/kg/min, was metabolized greater than 70% respectively. Thus, there was no difference between control or experimental groups in PGF conversion. We conclude that the in vivo sheep lung has an extensive substrate-dependent capacity to metabolize PGF and this mechanism is resistant to severe acute oleic acid lung injury.     

Secretion rate of prostaglandin F during induced labor in goats.

Relationships between plasma flow and plasma concentrations of prostaglandin F were examined in the utero-ovarian veins of three pregnant goats. Plasma flow, measured by veno-arterial dilution of para-Aminohippurate in two goats, was unchanged or increased slightly when PGF concentrations were elvated by short-term infusions of PGF2alpha into a uterine vein. Utero-ovarian plasma flow was measured during labor in two goats. Flow doubled during advanced labor and then decreased sharply to very low rates during the terminal expulsive phase of stage II labor. A total of 8.3 and 9.5 mg PGF was released into the utero-ovarian vein of two goats during the last 6 hours before fetal delivery and maximal release rates of approximately 100 ug. min-1 were obtained some 5-10 minutes before delivery was completed. The highest plasma concentrations of PGF were detected immediately after completion of fetal delivery when utero-ovarian plasma flows were lowest.     

Response of the 1-5 day-aged ovine corpus luteum to prostaglandin F2alpha

The hypothesis that, in the ewe, prostaglandin (PG) F2alpha administration on day 3 after ovulation is followed by luteolysis and ovulation was tested using 24 animals. The ewes were treated with a dose of a PGF2alpha analogue (delprostenate, 160 microg) on days 1 (n=8), 3 (n=8) or 5 (n=8) after ovulation, was established by transrectal ultrasonography. Daily scanning and blood sampling were performed to determine ovarian changes and progesterone serum concentrations by radioinmunoassay. The treatment induced a sharp decrease of progesterone concentrations followed by oestrus and ovulation in all ewes treated on days 3 and 5 and in one ewe treated on day 1 (8/8, 8/8, 1/8; P<0.05). Seven ewes treated on day 1 did not respond to PGF2alpha treatment and had an inter-ovulatory cycle of normal length (17.4 +/- 0.5 days). However, the profile of progesterone concentrations during the cycle of these ewes was delayed 1 day (P<0.05) compared with a control cycle. The overall interval between PGF2alpha and oestrus for the 17 responding ewes was 42.4 +/- 2.3 h. In 15 of these ewes the ovulatory follicle was originated from the first follicular wave and the ovulation occurred at 60.8 +/- 1.8 h after PGF2alpha treatment. The other two responding ewes ovulated an ovulatory follicle originated from the second follicular wave between 72 and 96 h after treatment. These results support the hypothesis and suggest that refractoriness to PGF2alpha of the recently formed corpus luteum (CL) may be restricted to the first 1-2 days post-ovulation.     

Stereospecificity of enzymatic reduction of prostaglandin E2 to F2alpha.

Antibodies directed toward PGF2beta were prepared in rabbits. The serologic specificity of the immune reaction was determined by inhibition of sodium borohydride-reduced (3H) PGE2 anti-PGF2beta binding by several prostaglandins. The antibodies to PGF2beta recognize the beta-hydroxyl configuration in the cyclopentane ring of PGF2beta. With the use of both anti-PFG2alpha and anti-PFG2beta, the product of PGE2 reduction by 9-ketoreductase purified from chicken heart was identified as PGF2alpha. Guinea pig liver and kidney homogenates were examined for PGE 9-ketoreductase activity. Although enzyme activity was present, no evidence of PGF2beta production was found.