cAMP dependent inhibition of thromboxane A2, prostacyclin and PGF2 alpha synthesis in mouse hepatocytes

The role of cAMP dependent regulation in thromboxane A2, prostacyclin and PGF2 alpha synthesis (measured by radioimmunoassay) was investigated in isolated mouse hepatocytes and in microsomal membranes prepared from these cells. In isolated hepatocytes N6,O2-dibutyryl cAMP inhibited the formation of all the three derivatives, while calcium ionophore A 23187 stimulated their synthesis. Addition of the dissociated catalytic subunit of cAMP dependent protein kinase and ATP to microsomal membranes inhibited the production of TXA2, PGI2 and PGF2 alpha by about 50% and this inhibition was counteracted by the combined addition of heat stable inhibitor protein of cAMP dependent protein kinase. It is concluded that in parenchymal liver cells cAMP dependent phosphorylation is directly involved in the inhibition of prostanoid synthesis.     

Autocrine regulation of mammary cell differentiation

Summary Milk secretion and mammary function in dairy animals are regulated by local mechanisms sensitive to the frequency or efficiency of milking. Acute local control of milk secretion occurs through autocrine feedback inhibition by a milk protein. Sustained changes in milking frequency and milk secretion are associated with longer-term adaptations in the degree of differentiation and, ultimately, the number of mammary epithelial cells. Differentiation of cultured mammary cells is suppressed by a milk fraction containing the inhibitor, suggesting that intra-mammary regulation of differentiation in vivo is elicited by the same autocrine regulator subsequent to its acute effect on milk secretion. The autocrine factor may affect mammary cell differentiation by modulating the number of cell surface hormone receptors for prolactin, thereby changing their sensitivity to circulating hormones.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Menstrual induction with PGF2 alpha and PGE2.

The "prostaglandin impact" (PGI), a massive intrauterine dose of PG, converts the refractory pregnant uterus into a reactive organ by provoking a regulatory imbalance. This regulatory conversion releases the endogenous mechanism of menstruation or abortion. During initial studies, PGI successfully provoked menstrual induction (MI) in 22 and subsequently in 65 volunteers. These results were confirmed and complemented by 2 independent trials in 14 and 36 gravidas respectively. The best clinical outcome was obtained in 20 volunteers, when a "PG-Pellet" (a mini-suppositorium) was inserted in utero, containing only 2.5 mg PGF2alpha. These 157 trials in sedated volunteers had the common features of over 90% efficiency, transient and medically acceptable side effects and infrequent complications. The present study of 542 volunteers focused upon the collection of clinical data regarding efficacy, side effects and complications of MI. All patients had committee approval for legal abortion, during the 2nd week of their missed menstrual period. They volunteered to participate because of their preference for pharmacological rather than surgical pregnancy termination. The clinical outcome of the 542 MI with 5 mg PGF2alpha (428 cases) and 1.5 mg PGE2 (114 cases) was identical. On the average, 95% of the gravidas had complete evacuation of the uterus with the clinical symptoms of delayed menstruation rather than abortion; they experienced spontaneous menstruation in 34 days after having received a single dose of PG.     

Effects of prostacyclin and 6-keto PGF1alpha on electrically induced convulsions in mice.

Intracerebroventricular administration of prostacyclin (PGI₂) was shown to block the incidence of tonic convulsions in mice. Prostacyclin was administered intracerebroventricularly (i.c.v.) to conscious mice prior to a transcorneal maximal electroshock (MES) or supra-maximal electroshock (SMES) as previously described (1). PGI₂ i.c.v. blocked the tonic hindlimb extension (THE) and protected the animals from death induced by MES with an ED₅₀ of 6.27 (2.53–11.10) μg/mouse i.c.v. The i.c.v. administration of its degradation product 6-keto PGF_1α had no effect on the incidence of tonic convulsions but did reduce the duration of THE significantly. When PGI₂ was administered intraperitoneally in doses as high as 2 mg/kg it did $\text{not}$ block the THE. However, the duration of the THE as well as mortality were reduced by doses ranging from 0.25–2.0 mg/kg i.p. Prostacyclin caused a significant dose-related (p<.001) decrease in the duration of the THE with SMES in doses of 20–140 μg/mouse i.c.v. No concomitant decrease in the incidence of tonic convulsions was found against SMES.     

Metabolism of prostacyclin. III. Urinary metabolite profile of 6-keto PGF1 alpha in rat.

The in vivo metabolism of 6-keto PGF1 alpha 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 PGF1 alpha was recovered in the urine. The metabolic pattern very closely resembles that of PGI2 in rats. Metabolites were found which represented 15-dehydrogenation, beta-oxidation, omega and omega-1-hydroxylation and oxidation. Previous work showed that 6-keto PGF1 alpha is very poorly oxidized by 15-PGDH. We administered 15-[H3]-PGI2 and 15-[H3]-6-keto PGF1 alpha to rats and measured urinary tritiated water as an index for in vivo 15-PGDH activity. The results showed that PGI2 and 6-keto PGF1 alpha were both oxidized to the 15-keto product, although the rate of oxidation of PGI2 was greater than that of 6-keto PGF1 alpha. We concluded that the administered PGI2 was oxidized by 15-PGDH before hydrolysis to 6-keto PGF1 alpha. A portion of the dose is probably hydrolzyed before 15-dehydrogenation.     

Prostaglandin F2alpha (PGF2alpha) and prolactin secretion in rats.

Plasma prolactin and F-prostaglandins (PGF) were measured anesthetized male Sprague-Dawley rats before and at 15, 30, 45 and 60 minutes following i.v. injection of either PGF2alpha (4 mg/kg), chlorpromazine, 1 mg/kg or chlorpormazine (1 mg/kg) after pretreatment with i.p. indomethacin (2 mg/kg). Following PGF2alpha administration, plasma prolactin levels increased significantly only at 15 and 30 minutes in spite of extremely high PGF levels throughout 60 minutes. Besides the expected rise in plasma prolactin, chlorpromazine caused a transient but statistically significant increase in PGF. Indomethacin blocked the chlorpormazine-induced PGF rise but not prolactin increase. Animals stressed with ether anesthesia showed elevation of plasma prolactin, which was not blocked by indomethacin although PGF concentration fell. Theese results indicate that PGF2alpha can stimulate prolactin release. This effect does not appear to be physiologic since very high PGF levels are required. Furthermore, blockade of prostaglandin synthesis by indomethacin does not prevent the release of prolactin in response to chlorpormazine or stress. Our findings do not support a possible role of PGFs as intermediaries in prolactin release. However, it is possible that PGFs may work through other mechanisms not investigated in our study.     

Conversion of PGE2 to PGF2alpha by fetal sheep blood

In an attempt to explain the differences in the concentration of primary prostaglandins in the circulation of adult and fetal sheep, we have examined the ability of whole blood from adult and fetal sheep to reduce PGE2 to PGF2alpha in vitro. PGF2alpha was the principal radioactive product formed from 3H PGE2 by both adult and fetal blood. The enzyme initial reaction velocity was significantly higher (P = 0.02) per ml of fetal than adult blood. The optimum enzyme pH (7.0 - 7.5) was similar for both adult and fetal blood. The Km of the enzyme in fetal and adult blood were 3.59 x 10(-7) M and 5.02 x 10(-7) M respectively (P greater than 0.05). There was no detectable metabolism of PGF2alpha by either adult or fetal sheep blood. The results indicate that prostaglandin 9-ketoreductase is present in both adult and fetal sheep blood. Differences in the activity of this enzyme are unlikely to explain the higher concentrations of PGE reported in the plasma of fetal lambs.     

Induction of luteolysis in mares by ultrasound-guided intraluteal treatment with PGF2alpha.

To evaluate the technique of ultrasound-guided luteal injection in mares, PGF2alpha was administered under ultrasound guidance to horse mares (n = 7 to 9 per group) on Day 9 postovulation via either a systemic (i.m.; zero, 0.01, 0.1, or 5 mg/dose) route or a local intraluteal (i.l.; zero, 0.01 or 0.1 mg/dose) route. The luteolytic efficacy of each treatment was determined based on post-treatment decreases in progesterone concentration, interval to uterine edema (IE) and interovulatory interval (IOI). Local administration of PGF2alpha directly into the CL consistently induced luteolysis, at doses up to 50-fold lower than the lowest effective systemic dose. Significant decreases in IOI and IE occurred in mares treated with 5 mg PGF2alpha i.m. or 0.1 mg PGF2alpha i.l., but did not occur in mares treated with 0.1 or 0.01 mg PGF2alpha i.m., 0.01 mg PGF i.l., vehicle i.l. or vehicle i.m.. Progesterone concentrations were reduced to less than 10% of pretreatment values by two days post treatment in mares treated with 5 mg PGF2alpha i.m. or 0.1 mg PGF2alpha i.l.. PGF2alpha doses of 0.1 mg i.m. and 0.01 mg i.l. were associated with smaller but significant progesterone decreases (to 66% and 46% of pre-treatment values, respectively) by two days post treatment. Progesterone values after administration of i.l. vehicle did not differ from pre-treatment values by two days post treatment, but were significantly lower (53% of pre-treatment values) by four days post treatment. Intramuscular treatment with vehicle or 0.01 mg of PGF2alpha did not significantly reduce progesterone concentrations below pretreatment values. Overall, the minimum effective luteolytic dose of PGF2alpha given intraluteally was between 0.01 and 0.1 mg. Based on the results of this study, ultrasound-guided i.l. injection appears to be a repeatable method for studying the direct effect of other chemicals on luteal function. However, the current procedure carries some risk, since three i.l. injections were associated with ovarian abscesses.     

Inspiratory timing regulation of PGF2 alpha in newborn pigs.

In intact and vagotomized anesthetized, spontaneously breathing piglets, we investigated the regulation of inspiratory timing evoked by i.v. administration of prostaglandin (PG) F2 alpha. The inspiratory time was evaluated from the flow trace as an index of mechanical inspiratory time (Ti) and from costal and crural diaphragmatic EMG (TiEMG) as an index of neural inspiratory time. Our results under control conditions showed that TiEMG was shorter than Ti. Vagotomy abolished the difference, inducing a change in the power spectrum without modifying the centroid frequency (Cf). PGF2 alpha lengthened TiEMG, causing a postinspiratory diaphragmatic discharge to appear, while mechanical inspiratory time decreased significantly. Postvagotomy i.v. administration of PGF2 alpha did not cause any significant changes in inspiratory time and did not evoke the postinspiratory discharge. The i.v. administration of PGF2 alpha before and after vagotomy did not change the centroid frequency in spite of recruitment of new motor units synchronized with those that are active under control conditions.     

Cytoskeletal architecture regulates cyclooxygenase-2 in human endothelial cells: Autocrine modulation by prostacyclin

Endothelium is a highly dynamic tissue that controls vascular homeostasis. This requires constant rearrangements of the shape or function of endothelial cells that cannot set aside the role of the cytoskeleton. The aim of this study was to determine the mechanisms by means of which cytoskeletal alterations induce cyclooxygenase‐2 (Cox‐2) expression in human endothelial cells using compounds that interfere with microtubule or actin architecture. Microtubule disruption by nocodazole markedly increased Cox‐2 expression and activity, and provoked paracellular gap formation, a cardinal feature of endothelial barrier dysfunction. The Cox‐2 metabolite prostacyclin down‐regulated Cox‐2 through an autocrine receptor‐mediated mechanism, and partially prevented the disassembly of endothelial monolayers. There was also an interaction between microtubules and actin filaments in nocodazole‐induced Cox‐2 expression. Nocodazole provoked the dissolution of the F‐actin cortical ring and stress fiber formation, increased actin glutathionylation, and concomitantly lowered intracellular levels of reduced glutathione. The restoration of glutathione levels by N‐acetylcysteine opposed Cox‐2 expression and preserved the integrity of endothelial monolayers. Among the signaling pathways connecting microtubule disruption with Cox‐2 up‐regulation, crucial roles are played by Src family kinase activation, serine/threonine phosphatase 2A inhibition, and the phosphorylation of mitogen activated protein kinase p38. Our findings provide a mechanistic insight into the observation that Cox‐2 is induced in endothelial cells under cytoskeleton‐perturbing conditions such as those occurring in the presence of atherogenic/inflammatory stimuli and oxidative stress. In this scenario, Cox‐2 up‐regulation by endothelia exposed to noxious conditions can be considered protective of the vasodilatory and anti‐thrombotic properties of the vessel wall. J. Cell. Physiol. 227: 3847–3856, 2012. © 2012 Wiley Periodicals, Inc.     

Blood LH after PGF2alpha in diestrous and ovariectomized cattle.

Two experiments were conducted to determine whether the increased serum LH which occurs within 12 hr after a luteolytic dose of PGF2alpha is dependent upon changes in progesterone or estradiol secretion. In the first experiment, exogenous progesterone abolished the increase in serum LH caused by a subcutaneous injection of 25 mg PGF2alpha in diestrous heifers, but not in ovariectomized heifers. In the second experiment, progesterone pessaries were removed at 6 hr after a subcutaneous injection of 25 mg PGF2alpha. LH remained at pre-PGF2alpha values while the pessaries were in place, but began to increase within 1 hr after they were removed. Blood estradiol also remained at pre-PGF2alpha values until the pessaries were removed, and began to increase at 2 hr after pessary removal. We conclude that the increase in serum LH within 12 hr after PGF2alpha treatment in diestrous cattle is dependent upon withdrawal of progesterone; it is not due to increased serum estradiol.     

PGF2 alpha and breathing pattern in newborn pigs

The effect of PGF2 alpha has been evaluated in 11 unanaesthetized unrestrained piglets and in 3 anaesthetized piglets (2-3 days old) using a barometric-plethysmographic technique. PGF2 alpha (mg 0.25/pig) was administered as aerosol for 5 min. In 3 of the unanaesthetized newborn pigs the effect of PGF2 alpha aerosol has been evaluated after indomethacin (mg 1/Kg i.v.). The vagal dependent activity of the prostaglandin was also evaluated after atropine (mg 0.08/Kg i.m.). Our results show that PGF2 alpha in newborn pigs causes hypoventilation due to a decrease in respiratory rate and to a lengthening in TE. The changes in TE are due to an increase in the incidence and duration of apneic events characterizing the respiratory activity at birth. After indomethacin PGF2 alpha does not change the breathing pattern. Atropine only partially reduces the effects of PGF2 alpha while, after anaesthesia, prostaglandin does not change the breathing pattern. Consequently our results show that PGF2 alpha in newborn animals similar to other prostaglandins acts as a depressant of respiratory activity.     

Enzymatic transformation of PGH2 to PGF2 alpha catalyzed by glutathione S-transferases

Glutathione S-transferases (GSTs) purified from both rat liver cytosol and microsomes catalyzed the direct reduction of PGH2 to PGF2 alpha. As much as 40% of the substrate was transformed into a prostanoid whose Rf value corresponded to that of PGF2 alpha. The identification of the reaction product as PGF2 alpha was confirmed by TLC and reverse-phase HPLC as well as by mass spectral analysis. In the absence of GSTs, PGH2 was found to be primarily converted to PGE2 and PGD2. Also, PGF2 alpha formation was completely abolished by decylglutathione, a potent inhibitor of both peroxidase and transferase activity associated with GSTs. These results indicate that the direct reduction of endoperoxide moiety of PGH2 to form PGF2 alpha is an enzymatic process. Interestingly, selenium-dependent glutathione peroxidase (Se-GSH-Px) showed very little PGF2 alpha formation from PGH2. However, this enzyme was very active in the reduction of PGG2 to PGH2. In contrast, GSTs were very poor in the conversion of PGG2 to PGH2. Therefore, it is possible that the relative tissue distribution of Se-GSH-Px and GSTs might play an important role in the tissue specific synthesis of PGF2 alpha.     

Luteolytic activity of a synthetic prostaglandin and PGF2alpha in heifers.

Two experiments involving 44 cycling heifers were conducted to evaluate the luteolytic activity of a synthetic prostaglandin, AY 24366, and PGF2alpha. Activity was assessed by the decline in progesterone level of peripheral blood and occurrence of estrus. Progesterone concentrations of jugular blood plasma were quantified by radioimmunoassay. In the first experiment, 36 heifers were treated during diestrus with AY 24366 (A-10mg intrauterine, B-30mg intramuscular and C-60mg im) or with PGF2alpha (D-5mg iu, E-15mg im and F-30mg im). Mean progesterone 0, 24 and 48 hours after treatment were A-6.33, 5.55 and 5.06; B-6.35, 2.79 and 3.92; C-5.23, 2.69 and 3.91; D-5.19, 1.50 and 1.51; E-4.69, 0.85 and 0.61; F-6.66, 0.80 and 0.48 ng/ml. Standing estrus was observed in 1, 1, 1, 4, 5 and 6 females in groups A, B, C, D, E and F respectively within 72 hours of treatment. PGF2alpha resulted in significantly (P less than 0.01) lower progesterone at 24 and 48 hours than AY 24366. However, in administration of the latter did significantly (P less than 0.05) lower progesterone at 24 hours. In the second trial six heifers were treated with either 120 or 180mg of AY 24366 im on day 12 of the cycle. Mean progesterone declined from 3.84 to 2.12 ng/ml (P less than 0.01) by 6 hours and to 1.59 ng/ml by 12 hours. Thereafter the decline was gradual and reached a level of 0.65 ng/ml at 72 hours. All six heifers showed standing estrus at 78 +/-2 hours and were inseminated. Two in each group conceived. Doses of 15mg PGF2alpha and 120mg AY 24366 were effective in causing luteal regression, however, the latter caused respiratory discomfort for 5 to 10 minutes post treatment.     

Serum PGF2alpha levels during human pregnancy

Guttierez Cernosek and his colleagues reported that the level of PGF2a in human serum reached a peak during the second trimester of pregnancy, the highest levels being found during the 17th-24th weeks. Earlier studies in this laboratory on late pregnancy have now been extended to earlier gestations and rather than peak levels during the 17th-24th weeks, the lowest levels were found at this time. A total of 128 samples from 106 women were studied. The levels found during the second trimester were significantly lower than those found at earlier gestations. However, the mean serum PGF2a level during the second trimester was not significantly different from the mean level during the third trimester. The discrepancy between the 2 sets of results is very marked, so that there is obviously need for much further research in this sphere.     

Effects of PGF2alpha and PGF2alpha, 1-15 lactone on the corpus luteum and on early pregnancy in the rhesus monkey.

The effects of prostaglandin (PG)F2alpha and PGF2alpha, 1-15 lactone were compared in luteal phase, non-pregnant and in early pregnant rhesus monkeys. Animals treated with either PG after pretreatment with human chorionic gonadotropin (hCG) had peripheral plasma progesterone concentrations that were not statistically different from those in animals treated with hCG and vehicle. However, menstrual cycle lengths in monkeys treated with PGF2alpha, 1-15 lactone were significantly (P less than 0.02) shorter than those in vehicle treated animals. In the absence of hCG pretreatment, plasma progesterone concentrations were significantly (P less than 0.008) lower by the second day after the initial treatment with either PGF2alpha or PGF2alpha, 1-15 lactone than in vehicle treated monkeys. Menstrual cycle lengths in monkeys treated with either PG were significantly (P less than 0.04) shorter than those in animals treated with vehicle. There were no changes in plasma progesterone concentrations in early pregnant monkeys treated with PGF2alpha, and pregnancy was not interrupted. In contrast, plasma progesterone declined and pregnancy was terminated in 5 of 6 early pregnant monkeys treated with PGF2alpha, 1-15 lactone. These data indicate that PGF2alpha, 1-15 lactone decreases menstrual cycle lengths in non-pregnant rhesus monkeys. More importantly, PGF2alpha, 1-15 lactone terminates early pregnancy in the monkey at a dose which is less than an ineffective dose of PGF2alpha.