Metabolism of prostacyclin. III. Urinary metabolite profile of 6-keto PGF1α in rat

The in vivo metabolism of 6-keto PGF_1α was investigated in rats. Following continuous intravenous infusion for 14 days the urinary metabolites were isolated and identified. A substantial amount of unchanged 6-keto PGF_1α was recovered in the urine. The metabolic pattern very closely resembles that of PGI₂ in rats. Metabolites were found which represented 15-dehydrogenation, β-oxidation, ω and ω-1-hydroxylation and oxidation.Previous work showed that 6-keto PGF_1α is very poorly oxidized by 15-PGDH. We administered 15-[H³]-PGI₂ and 15-[H³]-6-keto PGF_1α to rats and measured urinary tritiated water as an index for in vivo 15-PGDH activity. The results showed that PGI₂ and 6-keto PGF_1α were both oxidized to the 15-keto product, although the rate of oxidation of PGI₂ was greater than that of 6-keto PGF_1α. We concluded that the administered PGI₂ was oxidized by 15-PGDH before hydrolysis to 6-keto PGF_1α. A portion of the dose is probably hydrolyzed before 15-dehydrogenation.     

Comparison of substrate specificities of the human placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases.

A study of the relative activity of the purified placental NAD- and NADP-linked 15-hydroxyprostaglandin dehydrogenases with various prostaglandins and thromboxane B2 (TxB2) suggests that most, if not all, oxidation in the placenta of the 15-hydroxyl group of prostaglandins of the A, E, and F series as well as PGI2 (prostacyclin) and 6-keto PGF1 alpha is catalyzed by the NAD-linked enzyme. Prostaglandin B1 is an excellent substrate for the NADP-linked enzyme. Despite the conformational similarities between PGB1 and PGI2, the latter molecule is a poor substrate for the NADP-linked enzyme. Thromboxane B2 is not oxidized by the NAD-linked enzyme and is oxidized slowly by the NADP-linked enzyme.     

Enzyme immunoassay measurement of the urinary metabolites of thromboxane A2 and prostacyclin

We have used a recently developed enzyme immunoassay (EIA) method for measuring urinary concentrations of TXB2, 6-keto PGF1 alpha, 2,3-dinor-TXB2, 2,3-dinor-6-keto PGF1 alpha and 11-dehydro-TXB2 using acetylcholinesterase from Electrophorus Electricus coupled to TXB2, 6-keto PGF1 alpha and 11-dehydro-TXB2. Urinary PGI2 and TXA2 breakdown products and their metabolites were extracted from 3-40 ml of urine corresponding to 100 mumoles creatinine. Measurements were performed after Sep-Pak extraction and thin layer chromatography separation in a system that allows separation between dinor- and parent derivatives. Because of the relatively high cross reactivity (10-15%) of the anti-TXB2 serum with 2,3-dinor TXB2 and the anti-6-keto PGF1 alpha serum with 2,3-dinor-6-keto PGF1 alpha, measurements were done using 3 antisera (anti-TXB2 and anti-6-keto PGF1 alpha diluted 1/50,000, anti 11-dehydro-TXB2 diluted 1/200,000). The reproducibility of the technique was assessed by measuring the same urine stored frozen in aliquots together with each series of samples (Coefficient of variation 6-12% (n = 20), depending on the compound). In addition, the use of a different solvent system for the thin layer chromatography did not affect the results although the migration of the compounds was modified significantly. Determination of the urinary excretion of TXB2 and prostacyclin metabolites in 17 healthy individuals by this method provided results in agreement with those obtained by other methodologies. In addition, comparisons made between EIA and gas chromatography/mass spectrometry analysis showed good correlation between the urinary metabolites as determined by each technique (r = 0.98).     

Epidermal growth factor (EGF) and tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate PG synthesis and thymidine incorporation in cultured porcine thyroid cells

This is the first report to show that epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate the production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in the thyroid gland. In cultured porcine thyroid cells, EGF and TPA stimulate PGE2 and 6-keto PGF1 alpha production; the maximum PG levels were obtained after 3-4 h incubation with EGF or TPA; the addition of as little as 10(-11) M EGF or 5 X 10(-11) M TPA resulted in increases in PGE2 and 6-keto PGF1 alpha, and the maximum levels were obtained with 10(-8)-10(-7) M EGF or TPA. This report also shows that EGF and TPA stimulate [3H] thymidine incorporation.     

Altered levels of tissue and urinary prostacyclin in rats subjected to pancreas transplantation

Significant increases of TXB2 and PGE2 are reported to occur in pancreas transplantation. These increases are prevented with scavengers of oxygen-free radicals. In this communication, we report on changes of prostacyclin metabolites such as tissue 6-keto prostaglandin F1 alpha and urinary 2,3-dinor 6-keto prostaglandin F1 alpha in rats subjected to pancreas transplantation after different periods of organ cold preservation ischemia as well as the effect of superoxide dismutase (SOD) on these changes. For this purpose, male Lewis rats were classified as follows: Group I, Control; Group II, syngenic pancreas transplantation after 15 min of organ preservation in Collins solution at 4 degrees C; Group III, same as II but with 12 hours of organ preservation; Group IV, same as III, but with SOD pretreatment. Results have shown significant posttransplantation increases of both tissue 6-keto PGF1 alpha and urinary 2, 3 dinor 6-keto PGF1 alpha, the latter being a useful marker to evaluate systemic prostacyclin (PGI2) production by rat pancreas. This effect was prevented when the organ had been exposed to SOD during the period of cold preservation ischemia. These results confirm the implication of oxygen-free radicals (OFR) in the ischemia-reperfusion process associated to rat pancreas transplantation leading to enhanced arachidonic acid metabolism.     

Prostacyclin induces a surprising long-lasting motility in quiescent uterine strips (indomethacin-treated) isolated from ovariectomized rats

Dose-response curves for several prostaglandins (PGI2; PGD2; PGF2 and PGE2); BaCl2 or prostaglandin metabolites (15-keto-PGF2 alpha; 13,14-diOH-15-keto-PGF2 alpha; 6-keto-PGF1 alpha and 6-keto PGE1 in quiescent (indomethacin-treated) uterine strips from ovariectomized rats, were constructed. All PGs tested as well as BaCl2, triggered at different concentrations, evident phasic contractions. Within the range of concentrations tested the portion of the curves for the metabolites of PGF2 alpha was shifted to the right of that for PGF2 alpha itself; the curve for 6-keto-PGF1 alpha was displaced to the right of the curve for PGI2 and that for 6-keto-PGE1 to the left. It was also demonstrated that the uterine motility elicited by 10(-5) M PGF2 alpha and its metabolites was long lasting (more than 3 hours) and so it was the activity evoked by PGI2;6-keto-PGF1 alpha and BaCl2, but not the contractions following 6-keto-PGE1, which disappeared much earlier. The contractile tension after PGF2 alpha; 15-keto-PGF2 alpha; 13,14-diOH-15-keto-PGF2 alpha and PGI2, increased as time progressed whilst that evoked by 6-keto-PGF1 alpha or BaCl2 fluctuated during the same period around more constant levels. The surprising sustained and gradually increasing contractile activity after a single dose of an unstable prostaglandin such as PGI2, on the isolated rat uterus rendered quiescent by indomethacin, is discussed in terms of an effect associated to its transformation into more stable metabolites (6-keto-PGF1 alpha, or another not tested) or as a consequence of a factor which might protects prostacyclin from inactivation.     

Metabolism of prostacyclin in rat.

Following a single intravenous administration of [11-3H]prostacyclin in rat, 77% of the administered dose was excreted within 3 days with 33% in urine and 44% in feces. Urinary metabolites were accumulated by chronic intravenous infusions of [11-3H]prostacyclin for 14 days. The drug was extensively metabolized and the structures of seven metabolites were elucidated by combined gas chromatography and mass spectrometry. The urinary products include the dinor and 19-hydroxy dinor derivatives of 6-keto-PGF1alpha and 13,14-dihydro-6,15-diketo-PGF1alpha, omega-hydroxy and omega-carboxyl dinor derivates of dihydro-6,15-diketo-PGF1alpha, and a dihydrodiketotetranordicarboxylic acid. The metabolic pathways of PGI2 in rat are similar to that of PGF2alpha.     

Urinary levels of 2,3-dinor-6-oxo-PGF1 alpha: a reliable index of the production of PGI2 in the spontaneously hypertensive rat

The urinary levels of 2,3-dinor-6-oxo-PGF1 alpha (PGI2-M), a major metabolite of PGI2, are determined by the balance between the amount of PGI2 synthesized and the extent of its further metabolic oxidation. The purpose of the present study was to determine if the urinary excretion of PGI2-M can be used as a reliable index of the in vivo production of PGI2 in both normal Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). This involved the exclusion of differences in metabolism between these two strains of rats. In order to do so, we monitored the urinary excretion of PGI2-M during paired intravenous infusions of 6-oxo-PGF1 alpha (the stable product of the spontaneous hydrolysis of PGI2) in conscious, unrestrained SHR and WKY rats aged 12-15 weeks, in doses ranging from 250 to 700 ng. In one experiment, PGI2 was infused instead of 6-oxo-PGF1 alpha. The results of these experiments indicate that SHR and WKY rats are equal with regard to the transformation of 6-oxo-PGF1 alpha and PGI2 into PGI2-M. For both groups, there is a good correlation between the amount of 6-oxo-PGF1 alpha infused and the amount of PGI2-M excreted in urine. These observations confirm the validity of using the urinary levels of 2,3-dinor-6-oxo-PGF1 alpha as an index of PGI2 production in both WKY and SHR. In addition, they support the conclusions drawn from our previous studies, namely that SHR do not produce more PGI2 than WKY rats in vivo, contrary to the situation prevailing in vitro.     

Enhanced formation of PGI2, a potent hypotensive substance, by aortic rings and homogenates of the spontaneously hypertensive rat.

Intact rings and homogenates of aorta from spontaneously hypertensive rats (SHR) contain enhanced capacity over normal rats (NR) to convert arachidonic acid into PGI2. The PGI2 synthetic system in SHR is stimulated to a greater extent than NR by norepinephrine. Indomethacin blocks this stimulation. PGE2 and PGF2alpha were detected in much smaller amounts in homogenates (undetected in rings) but their formation was not enhanced by the hypertensive tissue. The identity of PGI2 was based on 1) direct pharmacological assay on the rat blood pressure. In this system identical vasodepressor responses to PGI2 are observed after intracarotid and intrajugular administration 2) indirectly as 6-keto PGF1alpha isolated after incubation of aortic homogenates with tritiated arachidonic acid and 3) indirectly by GC-MS assay of PGE2, PGF2alpha and 6-keto PGF1alpha formed during incubation of aortic homogenates with excess unlabeled arachidonic acid. These results provide additional support to our recent hypothesis that PGI2, of aortic origin, might actively participate in the regulation of systemic blood pressure. Its enhanced formation by intact hypertensive vascular tissue reflects an increase in the number of enzyme molecules immediately available to the substrate. This could probably be an adaptive response to the elevated levels of catecholamines in the circulation.     

Thromboxane synthase inhibition: implications for prostaglandin endoperoxide metabolism. II. Testing the 'redirection hypothesis' in an acute intravenous challenge model

In the preceding paper we described the characterisation of an acute intravenous challenge model for the evaluation of the effects of thromboxane synthase inhibition (TXSI) on eicosanoid metabolism. Herein we describe the biochemical pharmacology of two TXSI and aspirin in this model. Both TXSI caused significant inhibition of plasma TXB2 in vivo without elevation of 6-oxo-PGF1 alpha levels. Similar results were obtained when combined levels of 6-oxo-PGF1 alpha,13,14 dihydro 6-oxo-PGF1 alpha,13,14 dihydro 6,15-dioxo-PGF1 alpha and 6-oxo-PGE1 were measured as an index of PGI2 biosynthesis (PGI2m). Thus no evidence of in vivo redirection of PGH2 to PGI2 was found. Ex vivo experiments performed in serum gave an apparent stimulation of immunoreactive 6-oxo-PGF1 alpha following TXSI but RPHPLC analysis of extracted serum showed that this stimulation was accounted for by increase in a product co-eluting with [3H]PGF2 alpha. The implications of these findings in relation to TXSI and receptor antagonists are discussed.     

Stimulation of ovine myometrial activity by 6-keto prostaglandin E1

6-keto prostaglandin E1 (6KE) is a metabolite of PGI2, which we have shown previously inhibits spontaneous myometrial activity. In the present study we examined the effects of 6KE on uterine electrical and mechanical activity in non-pregnant ovariectomized sheep. 6KE stimulated uterine activity in a dose-dependent fashion. The effect was enhanced by pre-treatment with estradiol (E2). It was not influenced by pre-treatment with meclofenamic acid and was not associated with significant changes in the concentrations of 13,14 dihydro 15-keto PGF2 alpha in vena cava plasma. After E2 treatment, 6KE had 0.2-0.3 of the stimulatory activity of PGF2 alpha. In the absence of E2, the uterine response to both 6KE and PGF2 alpha was decreased. In animals in which spontaneous myometrial activity was inhibited by PGI2, the uterus remained responsive to 6KE. We conclude that in the ovariectomized non-pregnant sheep 6KE stimulates uterine activity, and that the effect is independent of endogenous PG production.     

Stability of authentic PGI2 during routine extraction. Clarification of the nature and sequence of products formed by the rat stomach including the origin of the ozonolysis products reported in 1971.

Authentic PGI2 and PGI2 formed by rat stomach homogenates were carried through a simple extraction and purification procedure to explore the feasibility of isolation of this biologically active bicyclic ether product of arachidonic acid. The integrity of PGI2 was followed throughout by bioassay on the rat blood pressure. In this system we recently reported that PGI2 has very potent hypotensive actions which are easily distinguishable from those observed for PGE2 (14). Our results indicate that PGI2 survives the initial extraction steps (i.e. ethanol extraction, diethyl ether - HCl extraction and methylation) up to the step involving thin layer chromatography with an acidic developing solvent system. This latter procedure converts PGI2 entirely into a stable derivative, 6-keto-PGF1alpha (3,8--10). Oxidative ozonolysis of the methyl ester acetate derivative of authentic 6-keto PGF1alpha reveals products identical to those reported by Pace-Asciak and Wolfe in 1971 (1) which are also produced from authentic PGI2. This data sheds new light into 1) the nature of the biological product formed by stomach homogenates, 2) its transformation into 6-keto PGF1alpha during purification and 3) the origin of the ozonolysis products in the experiments reported in 1971.     

Irradiation of rats abolishes susceptibility of PGI2 synthesis in blood vessels to peroxidative agents

Thoracic and abdominal aortas were obtained from rats after irradiation and used for the estimation of the synthesis of prostacyclin (PGI2) determined as 6-keto PGF1 alpha. Twenty four h after exposure to 7.0 Gy an increase was noted in the amount of PGI2 released, and 4 weeks later its level significantly decreased. The 24 h value did not increase with the further radiation dose increment (9, 12.5, 15 Gy). Cysteine or H2O2 intensified prostacyclin synthesis in control vessels but decreased it in vessels from the animals irradiated 24 h earlier. Later after the exposure cysteine or H2O2 were no longer effective.     

Effect of alpha-adrenergic stimulation on prostacyclin and renin release in the rat kidney

The relationship between renin secretion and PGI2 production, in response to intrarenal infusion of norepinephrine, was examined in the isolated perfused rat kidney. Infusion of norepinephrine in a dose which caused substantial vasoconstriction (100 ng/min), markedly increased urinary excretion of 6-keto PGF1 alpha, the stable derivative of PGI2, without significantly altering renin secretion measured in the effluent perfusate. No change in urinary 6-keto PGF1 alpha occurred when vasoconstriction was prevented by infusing the alpha-adrenoceptor blocking drug phenoxybenzamine (2 x 10(3) ng/min) alongside norepinephrine (100 ng/min). However, under these conditions there was marked stimulation of renin secretion which, as has been demonstrated previously, is mediated by a beta-adrenoceptor. There were significant increases in urine flow rates during both vasoconstrictor and non-vasoconstrictor infusions. These findings clearly indicate that in the rat kidney prostacyclin production and renin release in response to norepinephrine are dissociated.     

Prostacyclin-independence in beta-adrenoceptor mediated renin release from dog renal cortical slices

There is some controversy regarding whether stimulation of renin release by the beta-adrenergic system is dependent on prostaglandin (PG) production. We have examined this problem in renal cortical slices of the dog and have obtained the following results: (1) Isoproterenol (4 X 10(-6) M) stimulated renin release, but had no effect on the formation of 6-keto PGF1 alpha, a stable metabolite of PGI2; (2) Indomethacin (2 X 10(-5) M) had no effect on isoproterenol stimulated renin release, but inhibited 6-keto PGF1 alpha formation; (3) Dibutyryl cyclic AMP (10(-3) M) stimulated both renin release, and 6-keto PGF1 alpha release. Indomethacin (2 X 10(-5) M) did not inhibit dibutyryl cyclic AMP-stimulated renin release, but did inhibit the production of 6 keto PGF1 alpha. These results indicate that the beta-adrenoceptor mediated renin release does not depend on the formation of PGI2, but renin release is dependent on cyclic AMP formation.     

Luteolytic action of 15-keto prostaglandin F2alpha in the rhesus monkey.

The naturally-occurring metabolite of prostaglandin F2alpha, 15-keto prostaglandin F2alpha (15-keto PGF2alpha), elicited rapid and sustained declines in serum progesterone concentrations when administered to rhesus monkeys beginning on day 22 of normal menstrual cycles. Evidence for luteolysis of a more convincing nature was obtained in studies where a single dose of 15-keto PGF2alpha was given on day 20 of ovulatory menstrual cycles in which intramuscular injections of hCG were also given on days 18-20; serum progesterone concentrations fell precipitously in monkeys within 24 hours following intramuscular administration of 15-keto PGF2alpha. However, corpus luteum function was impaired in only 4 of 11 early pregnant monkeys when 15-keto PGF2alpha was administered on days 30 and 31 from the last menses, a time when the ovary is essential for the maintenance of pregnancy. Gestation failed in 2 additional monkeys 32 and 60 days after treatment with 15-keto PGF2alpha, but progressed in an apparently normal manner in the remaining 5 animals. Two pregnant monkeys treated with 15-keto PGF2alpha on day 42 from the last menstrual period, a time when the ovary is no longer required for gestation, continued their pregnancies uneventfully. Corpus luteum function was not impaired in 9 control monkeys which received injections of vehicle or hCG at appropriate times during the menstrual cycle or pregnancy.     

Effect of prostaglandins against alloxan-induced diabetes mellitus

Previously, we observed that alloxan-induced in vitro cytotoxicity and apoptosis in an insulin secreting rat insulinoma, RIN, cells was prevented by prior exposure to prostaglandin (PG) E(1), PGE(2), PGI(2), PGF(1)(alpha), and PGF(3)(alpha) (P<0.05 compared to alloxan), whereas thromboxane B(2) (TXB(2)) and 6-keto-PGF(1)(alpha) were ineffective. In an extension of these studies, we now report that prior intraperitoneal administration of PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) prevented alloxan-induced diabetes mellitus in male Wistar rats, whereas PGI(2), TXB(2), and 6-keto PGF(1)(alpha) were not that effective. PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) not only attenuated chemical-induced diabetes mellitus but also restored the antioxidant status to normal range in red blood cells and pancreas. These results suggest that PGE(1), PGE(2), PGF(1)(alpha), and PGF(3)(alpha) can abrogate chemically induced diabetes mellitus in experimental animals and attenuate the oxidant stress that occurs in diabetes mellitus.     

Longitudinal and cross-sectional studies of the relationship between 6-keto PGF1 alpha and high density lipoproteins

Two studies were performed to examine the relationship between Prostacyclin (PGI2) and high density lipoprotein cholesterol (HDL-C). A longitudinal study examined the stable metabolite of PGI2, 6-keto PGF1 alpha, along with HDL-C, and total cholesterol (TC) before (Week 0), during (Week 4), and after (Week 9) an eight week aerobic conditioning program. 6-keto PGF1 alpha was measured by radioimmunoassay using 125I, and HDL-C and TC were spectrophotometrically analysed. Maximal oxygen uptake and resting heart rate data obtained at Week 0 confirmed that the training group (E) was not different from the sedentary group (C). Results obtained at Week 9 indicated that maximal oxygen uptake was higher (p less than 0.05) and resting heart rate lower (p less than 0.05) in the E group, while these variables remained unchanged in group C. Levels of 6-keto PGF1 alpha, HDL-C, and TC did not differ between groups E and C when Week 0 was compared with Week 9. However, within the E group, significantly lower concentrations of 6-keto PGF1 alpha (p less than 0.05) and HDL-C (p less than 0.05) were found at Week 4 compared with Week 0. A cross-sectional study involving 52 subjects examined the relationship between 6-keto PGF1 alpha and several cardiovascular disease risk factors which included HDL-C. The only significant correlation (r = 0.50 p less than 0.05) observed was between 6-keto PGF1 alpha and HDL-C.     

Effect of oestradiol 17 beta on prostaglandin biosynthesis by the uterus of ovariectomized rat and guinea pig

In vitro prostaglandin biosynthesis by uteri of ovariectomized rats and guinea pigs treated or untreated with oestradiol 17 beta, administered subcutaneously, was measured by R.I.A. of PGF2 alpha and PGE2. Incubations with [1-14C] arachidonic acid were also performed and labelled metabolites were analyzed by TLC. The main metabolite in rats was 6 keto PGF1 alpha and in decreasing order of magnitude, PGF2 alpha and PGE2. In guinea pig PGF2 alpha was the main product. Ovariectomy in rats completely changed the pattern of synthetized prostanoids : PGI2 production was doubled when compared to cycling rats and PGE2 increased 10 fold. PGF2 alpha values were similar to the mean value measured during the cycle. OE2 treatment almost completely inhibited PGI2 synthesis and reduced PGE2 by half. Total PG synthesis in OE2 treated animals was decreased by 5 fold when compared to spayed rats. Endogenous PGF2 alpha synthesis was slightly stimulated. In the guinea pig OE2 treatment of ovariectomized animals increased the total synthesis from 50 per cent. PGF2 alpha was always the main metabolite. In conclusion OE2 regulation of uterine PG synthesis is depending on the animal species and cannot be explained by a unique effect on the cyclooxygenase, but rather by an interplay on the various enzymes of the arachidonic acid cascade.     

Pancreas prostanoid production in ischemia and reperfusion.

This study was carried out to investigate the proportion of the 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) and thromboxane B2 (TXB2) alteration that is due to ischemia in pancreas transplantation against the proportion due to reperfusion. For this purpose, Lewis rats were divided in three experimental groups: Group I = Control, Group II = Donor pancreas subjected to 15 minutes of cold ischemia, Group III = Same as group II but pancreas were transplanted to the recipient individual and then subjected to reperfusion. The results indicate that increases in pancreas 6-keto PGF1 alpha occur as a consequence of cold ischemia while TXB2 remains unchanged. When blood flow was restored, 6-keto PGF1 alpha remained unchanged compared to the ischemic group while pancreatic levels of TXB2 were significantly increased. These results suggest a different induction of prostanoid metabolism during ischemia and reperfusion in pancreatic tissue.