Biosynthesis of prostaglandin F2alpha from arachidonic acid and prostaglandin endoperoxides in the uterus

Formation of prostaglandin F2Alpha in the cow and guinea pig uterus microsomes was studied using 14C-labeled arachidonic acid and prostaglandin H2. The total conversion of arachidonic acid was of a low order and underwent fluctuations during the estrous cycle of the guinea pig, being highest towards the end of the cycle. Injections of beta-estradiol-3-benzoate also resulted in higher activity of the uterine prostaglandin synthetase. The uterine prostaglandin synthesizing system appeared to differ in several respects from that present in seminal vesicles, with regard to the proportions of the products formed and the effects of various agents, e.g. reduced glutathione. An inhibiting factor which supressed the fatty acid cyclo-oxygenase was found to be present in uterine preparations. Prostaglandin endoperoxide (prostaglandin H2) was very efficiently reduced to prostaglandin F2alpha by cow and guinea-pig uterus microsomes. Prostaglandin G2 also gave rise to prostaglandin F2alpha. Prostaglandin E2, on the other hand, was not reduced. Both the inhibiting factor and the endoperoxide reducing activity are likely to be parts of a highly specialized mechanism that modulates prostaglandin F2alpha formation in the uterus.     

Action of prostaglandin, PGF2alpha, on the uterus of the pregnant rat

In a cytogenetic study on the spermatogonia of Chinese hamster, cyclohexylamine (neutral sulphate) was evaluated for mutagenic effects in comparison with an untreated control group and a group treated with the mutagenic compound cyclophosphamide, by assessing spermatogonial metaphases of treated Chinese hamster for chromosomal structural changes. Each test group comprised 8 male hamsters selected at random. Approximately 100 metaphases from each animal were assessed. The doses were 5 X 150 mg cyclohexylamine sulphate (approx. 5 X 102 mg base) per kg body-weight orally, and 5 X 100 mg cyclophosphamide per kg body-weight orally. The individual doses were administered at intervals of 24 h. Preparations were made 24 h after the final treatment, essentially by the method of Hoo and Bowles [10]. Gaps, breaks, fragments, deletions and translocations were assessed as structural changes; frequencies were determined of the metaphases (a) with aberration(s) including gaps, (b)with aberration(s) less gaps and (c)with translocation(s). Aberrations occurred in the untreated negative control group (1.24% incl. gaps, 0.25% without gaps). Translocations were not seen in the untreated group. In the cyclochexylamine group, the frequencies of the aberrant metaphases were sometimes less than in the control group (0.87% including gaps, 0.37% without gaps). Statistically, the results were not significantly different from the control data. No translocations were seen after administration of cyclohexylamine. The positive cyclophosphamide control group clearly differed from the untreated control and from the cyclohexylamine group in the parameters (a) to (c); mainly, the results were highly significantly different from those obtained in the untreated control group. The frequencies of the aberrant metaphases were 3.41% including gaps and 1.99% without gaps. The frequency of the translocations was 0.71% (5 out of 704). Cyclohexylamine sulphate, administered 5 times at 150 mg/kg body-weight orally, had no mutagenic effect, whereas cyclophosphamide, adminstered 5 times at 100 mg/kg body-weight orally, had a chromosome-damaging effect on Chinese hamster spermatogonia.     

Inhibitory effect of prostaglandin F2 alpha on oxytocin release and on milk ejection in lactating rats

Eleven Southdown male lambs averaging 19.8 kg were randomly allotted to two groups and fed diets containing 7.7% (low-N) or 15.8% (high-N) crude protein. All of the supplemental nitrogen in the high-N diet was supplied as urea. Intake of the low-N and high-N diets averaged 372.3 g and 340.5 g/day, respectively. Findings at the end of the thirty-day trial were: (1) mean body weights unchanged for the two groups; (2) plasma urea nitrogen three-fold higher in the high-N (19.07 mg/100 ml) than the low-N (6.57) animals; (3) similar hepatic activity levels of three urea cycle enzymes (ornithine transcarbamylase, argininosuccinase, arginase) in the two groups, and (4) similar liver weights and liver protein concentration. The absence of adaptive change in enzyme levels suggests the hypothesis that addition of non-protein nitrogen to maintenance diets may cause ammonia intoxication by exceeding the liver's reserve capacity for urea synthesis.     

A possible mechanism of Leptaden action by inhibiting prostaglandin F2alpha synthesis

Forearm skin blood flow was measured during external pressure loading in normal human subjects using 133Xe washout from intracutaneous injection sites. Pressures ranging between 5 and 150 mmHg were applied through a 3-cm-diameter disc placed over the site of flow determination. The pressure was maintained constant by a servo-controlled loading mechanism. Flow decreased with pressures from 5 to 10 and 30 to 150 mmHg, but remained constant with pressures from 10 to 30 mmHg. Reactive hyperemia occurred following removal of pressures of 90 mmHg or greater, but did not occur following removal of lower pressures. The pressure-flow curve for parasacral skin of paraplegic subjects closely paralleled the pressure-flow curve of normal skin at pressures tested: 5-15 mmHg. These data are interpreted to demonstrate autoregulation of skin blood flow. Autoregulation in parasacral skin of paraplegic subjects suggests a peripheral mechanism. The occurrence of hyperemia at pressures which exceed the ability of skin to autoregulate suggests that both autoregulation and post occlusion hyperemia may have the same mechanism.     

Dexamethasone inhibits the pyrogenic activity of prostaglandin F2 alpha, but not prostaglandin E2

The effect of dexamethasone on prostaglandin (PG) E2- and PGF2 alpha-induced fever was studied in rats. Intracerebroventricular injection of PGE2 and PGF2 alpha (500 ng) induced increases in body temperature (maximal temperature rises of 0.97 +/- 0.13 degrees C and 0.78 +/- 0.18 degrees C, respectively, vs. vehicle 0.12 +/- 0.09 degrees C) of unrestrained rats maintained within the thermoneutral zone. PGE2-induced fever peaked earlier and the defervescence was faster when compared to the response induced by PGF2 alpha. Subcutaneous pre-administration of dexamethasone (0.5 mg/kg) did not affect PGE2-induced fever (maximal temperature rise of 1.00 +/- 0.08 degrees C), but completely prevented the pyrogenic activity of PGF2 alpha (maximal temperature rise of 0.16 +/- 0.16 degrees C). Neither PGE2- nor PGF2 alpha-induced fever was significantly altered (maximal temperature rises of 0.90 +/- 0.11 degrees C and 0.64 +/- 0.14 degrees C, respectively) by intraperitoneal administration of indomethacin (2 mg/kg). These results demonstrate for the first time that glucocorticoids, in addition to inhibiting endotoxin- and cytokine-induced fever, can also modulate the pyrogenic activity of some prostaglandins, possibly via suppression of the synthesis of corticotropin-releasing factor, indicating that multiple mechanisms may be involved in the antipyretic activity of these steroids.     

Intracellular Ca2+ elevation and contraction due to prostaglandin F2alpha in rat aorta

Prostaglandin F2alpha was tested to determine (a) whether its effect on intracellular Ca2+ levels ([Ca2+]i) and force in vascular smooth muscle was mediated through activation of the thromboxane A2 and/or prostaglandin receptor, and (b) the relative roles of Ca2+ influx via L-type and non-L-type Ca2+ channels in prostaglandin receptor-mediated contraction. [Ca2+]i and force were measured simultaneously in fura-2-loaded rat aortic strips. The thromboxane A2 receptor antagonist, SQ29548 ([1S]-1a,2b(5Z),3b,4a-7-(3-[2-[(phenylamino)carbonyl] hydrazinomethyl)-7-oxobicyclo-[2.2.1]hept-2-yl-5-heptenoic acid), prevented the prostaglandin F2alpha-induced plateau [Ca2+]i elevation and force by 80-90%, while abolishing these responses due to the thromboxane A2 receptor agonist, U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy prostaglandin F2alpha). Prostaglandin F2alpha (+ SQ29548)-induced plateau [Ca2+]i elevation and force were not inhibited by verapamil. Ni2+, a non-selective cation channel blocker, in the presence of verapamil, abolished the prostaglandin F2alpha (+ SQ29548)-elevated [Ca2+]i, while the contraction was only partially inhibited. These results suggest that, in rat aorta, (1) elevated [Ca2+]i and force due to high prostaglandin F2alpha concentrations largely results from thromboxane A2 receptor activation, and (2) the prostaglandin component of the prostaglandin F2alpha-induced contraction is dependent on Ca2+ influx via non-L-type channels.     

Effect of TMB-8 on the pulmonary vasoconstrictor action of prostaglandin F2 alpha and the thromboxane mimic, U 46619

1 TMB-8 (8-(N,N-diethylamino)octyl-3,4,5 trimethoxybenzoate HCl), an intracellular calcium antagonist, had no direct action on the pulmonary vasculature of the perfused canine lung lobe preparation. 2 The pulmonary pressor response to the thromboxane mimic, U46619, was not affected by TMB-8. 3 The vasopressor response to prostaglandin F2 alpha (PGF 2 alpha) was significantly attenuated but not completely blocked by TMB-8. 4 We conclude that the pulmonary pressor response to PGF 2 alpha is dependent on both intracellular and extracellular calcium pools for contraction and that U46619 facilitates either solely extracellular calcium influx or mobilizes an intracellular calcium pool not inhibited by TMB-8.     

Radioimmunoassay for urinary metabolites of prostaglandin F2alpha

Antibodies against the main urinary metabolite of PGF2alpha in the human, 5alpha, 7alpha-dihydroxy-11-ketotetranorprosta-1,16-dioic acid, were raised in rabbits. The compound was coupled selectively in the omega position to bovine serum albumin prior to injection. The resuling antibodies did not distinguish between tetranor compounds varying only in structure at the omega carbon, and thus the assay could be used also for other metabolites of PGF2alpha, e.g. the main urinary metabolite in the guinea pig, 5alpha,7alpha-dihydroxy-11-ketotretranorporstanoic acid. Labeled ligands for the assays were prepared either in vivo by injection of [17, 18-3H]-PGF2alpha into humans after several days treatment with indomethacin, or in vitro by incubation of [17, 18-3H]-15-keto-13, 14-dihydro-PGF2alpha with mitochondria from rat liver. The sensitivity of the assay was 10 pg or 4 pg with these two preparations, respectively. The assay was employed for a number of measurements: normal daily excretion in a number of humans; excretion of urinary metabolites during treatment with prostaglandin synthetase inhibitors in human subjects, or after intravenous injection of PGF2alpha; excretion during human pregnancy; and prostaglandin production in the guinea pig during normal estrous cycles and pregnancies and after estrogen treatment. The results of these studies were in several cases compared to similar measurements earlier performed using mass spectrometric methods, and were found to agree well. Thus, this radioimmunoassay provides a simple and accurate method for estimating prostaglandin production, particularly suitable for long-term studies and for cases where repeated blood sampling must be avoided.     

Plasma progestins, estradiol, and luteinizing hormone following prostaglandin F2 alpha injection

Dairy animals, ranging from days 8 to 13 of the estrous cycle, were fitted with indwelling jugular catheters 1 day prior to either intramuscular injection of prostaglandin F2alpha free acid (30 mg, n=4) or intrauterine deposition of prostaglandin F2alpha free acid (10 mg, n = 3). Blood samples were collected at 6, 4, 2, and 0 h prior to administration of prostaglandin F2alpha and at 1, 3, and every 2 h thereafter until ovulation. Progestins, estradiol, and luteinizing hormone in plasma were measured by radioimmunoassay. Hormonal changes and interrelationships within animals were evaluated by least squares analyses. Decreases in progestins of plasma within 24 h indicated prostaglandin F2alpha induced luteolysis in six of the seven animals. Estradiol increased linearly from time of injection to 52 h postinjection. Intervals from administration of prostaglandin to onset of estrus, peak of luteinizing hormone, and ovulation were 74.9 +/- 21, 78.8 +/- 21, and 99.5 +/- 19 h.     

Radioimmunoassay of main urinary metabolite of prostaglandin F2alpha

Radioimmunoassay of 5alpha, 7alpha-dihydroxy-11-keto-tetranorprosta-1,16-dioic acid, main urinary metabolite of prostaglandin F2alpha (PGF2alpha), was performed using an antiserum produced in the rabbit. The antibody in 100 mu1 of 1,600-fold diluted antiserum binds with 60 picograms of metabolite. The main urinary metabolite level fell when flufenamic acid, a prostaglandin synthetase inhibitor, was given to rats. In contrast, it was significantly elevated when PGF2alpha was administered.     

Nitric oxide synthase activity in the gravid rat uterus decreases a day before the onset of parturition

OBJECTIVE: Our purpose was to determine the timing, tissue location, and isoform of the uterine nitric oxide synthase activity decrease at term in gravid rat uteri. STUDY DESIGN: Nitric oxide synthase specific activity was assayed in rat uteri 11 through 22 days' gestation by the difference in radiolabeled arginine to citrulline conversion with and without the cofactor reduced nicotinamide adenine dinucleotide phosphate. Nitric oxide synthase isoform was assessed by calcium sensitivity and subcellular location. RESULTS: Rat uterine nitric oxide synthase activity decreased between days 15 and 21 of gestation but did not decrease further at term (day 22), before and after the onset of labor. Decidual nitric oxide synthase activity exceeded the myometrial activity at 15 days' gestation, but then the two were equal at 18 through 22 days' gestation. The nitric oxide synthase activity was calcium insensitive except for half the decidual cytosolic activity on day 15. CONCLUSION: The decrease in pregnant rat uterine nitric oxide synthase activity coincides with the preparation of the uterus for parturition rather than the final activation of labor.     

Mode of action of prostaglandin F2 alpha in human luteinized granulosa cells: role of protein kinase C

It is well documented that prostaglandin F2 alpha (PGF2 alpha) inhibits progesterone production in luteal cells, but its mode of action is uncertain. It has recently been suggested that PGF2 alpha acts by activating the calcium and phospholipid-dependent protein kinase, protein kinase C (PKC). This hypothesis has been tested by comparing the site and mode of action of PGF2 alpha, a PGF2 alpha analogue (cloprostenol) and the PKC activator phorbol myristate acetate (4 beta PMA) in human granulosa-lutein cells. PGF2 alpha and cloprostenol exerted similar concentration-dependent inhibitory actions on gonadotrophin-stimulated cyclic AMP (cAMP) accumulation and progesterone production by human granulosa-lutein cells. The similarity in the actions of PGF2 alpha and cloprostenol in human granulosa-lutein cells suggests that they can be used interchangeably to study the role of PGF2 alpha in the regulation of steroidogenesis in the human ovary. Gonadotrophin-stimulated cAMP accumulation and progesterone production was also concentration-dependently inhibited by 4 beta PMA. In addition, cloprostenol and 4 beta PMA also inhibited dibutyryl cAMP-stimulated progesterone production, suggesting that these compounds inhibit LH action at sites before and after the generation of cAMP. The pre-cAMP site of action can be localised to the stimulatory G-protein (Gs) as both compounds inhibited cholera toxin-stimulated cAMP accumulation without affecting forskolin-stimulated cAMP accumulation. The post cAMP site of action can be localised to actions on cholesterol side chain cleavage enzyme, as both cloprostenol and 4 beta PMA inhibited 22R hydroxycholesterol-supported progesterone production without affecting pregnenolone-supported progesterone production. The finding that cloprostenol and 4 beta PMA interact with the steroidogenic cascade in a similar manner is indicative of a shared common mediator of their actions in human granulosa-lutein cells, i.e. PKC. The inhibitory actions of PGF2 alpha and 4 beta PMA on hLH-stimulated progesterone production were abolished in the presence of the PKC inhibitor, staurosporine. In addition, in PKC-depleted cells (achieved by exposure to 4 beta PMA for 20 h) the inhibitory actions of PGF2 alpha and 4 beta PMA were abolished. These results support the hypothesis that the inhibitory actions of PGF2 alpha are mediated by PKC in human granulosa-lutein cells.     

Metabolism of oxytocin in rat uterus and placenta in late gestation

Concentrations of oxytocin (OT) peptide increase in rat uterine tissues at the time of parturition. We have measured the rate of OT metabolism in these tissues in late gestation to determine whether a decrease in OT catabolism is responsible for the increase in OT concentrations. Uterine and placental tissues were obtained from groups of rats at Days 16, 19, 21, 21.5, 22, and after delivery of the first pup. Delivery usually occurs in the early afternoon of Day 22. Some animals were treated with the estrogen receptor blocker tamoxifen, which will delay parturition by approximately 24 h. Cytosolic and microsomal preparations obtained using ultracentrifugation were incubated with radiolabeled OT. Metabolites were separated using HPLC, and enzyme kinetic parameters were calculated. OT was actively metabolized in both uterine and placental tissues. Total oxytocinase activity was similar in the two tissues. In uterine tissues, activity was greater in the cytosolic fractions. In placenta, activity was evenly distributed between the cytosolic and microsomal fractions. The cytosolic fractions of each tissue contained predominantly post-proline endopeptidase activity, whereas the microsomes contained predominantly aminopeptidase activity. There was a slight trend to decreasing oxytocinase activity with advancing gestation in both subcellular fractions, but this was statistically significant only in the microsomal fraction. The maximal decline in activity was only 25-50%. Tamoxifen treatment had no effect on oxytocinase activity. We conclude that rat uterine and placental tissues contain post-proline endopeptidase and aminopeptidase activities that metabolize OT. It is doubtful that changes in these activities are major factors in regulating the increase in OT concentrations measured in rat intrauterine tissues at the time of parturition.