Release of prostaglandins from healthy and sensitized guinea-pig lung and trachea by histamine

The effects of histamine and its antagonists on the release of prostaglandin E and F_2α (PGE and PGF_2α) and the 15-keto-13,14-dihydro PGF_2α/E (metabolites) were examined in minced and whole perfused guinea pig lung.Lung fragments released considerable amounts of prostaglandins into the incubation media with time alone: parenchyma more PGF_2α than PGE, trachea more PGE than PGF_2α. The levels of PGF_2α found in the filtrates of both tissues on per gram basis were about the same, whereas the concentrations of PGE were several fold higher in the media of incubated trachea. In contrast to lung, trachea released only trace amounts of metabolites. These differences in synthesis and turnover are probably of importance for maintenance of the adequate ventilation-perfusion ratios.The process of sensitization caused a significant increase in the outflows of PGF_2α and metabolites from the lung fragments. The PGE to PGF_2α ratio was decreased in both parenchymal and tracheal tissues. Increased spontaneous release of prostaglandins was also found in whole perfused sensitized lung. This was consistent with the hypothesis that sensitization with antigen alters the biochemical properties of the organism.Incubation of lung fragments with histamine had only a small additional effect on the liberation of prostaglandins, since the baseline release was high due to the trauma of mincing. However, histamine perfusion of whole lung caused severalfold increase in the outflows of prostaglandins. Pretreatment with pyrilamine (histamine receptor 1 antagonist) decreased the subsequent release of PGF_2α by histamine. On the other hand, pretreatment with metiamide (histamine receptor 2 antagonist) diminished the subsequent release of PGE. It is suggested that stimulation of histamine receptor 1 is predominantly (but not solely) related to the synthesis of PGF_2α, and stimulation of the receptor 2 is related to the synthesis of PGE.     

Prostaglandin release by slow reacting substance from guinea pig and human lung tissue.

Slow reacting substance (SRS) injected into the pulmonary artery released prostaglandins E (PGE) and F2alpha (PGF2alpha) and the 15-keto-13, 14-dihydro PG metabolites from non-sensitized and ovalbumin sensitized, isolated, perfused guinea pig lungs. PGs were also released from lungs incubated with SRS. Sensitized lungs released more PGs in both types of preparations. Indomethacin inhibited the effect of SRS. Passively sensitized human lung fragments, in parallel to guinea pig lung, released PGE, PGF2alpha and the metabolites when incubated with SRS or antigen. In in vivo experiments, SRS and arachidonic acid given intravenously increased the airway insufflation pressure in anesthetized quinea pigs. These effects, but not the action of injected PGF2alpha and histamine, were abolished by indomethacin. The results indicate that one of the modes of SRS action is by release of PGs, and are consistent with the hypothesis that PGs are predominantly "secondary" mediators (in the temporal sense) of the antigen-antibody reaction.     

Selective inhibition of arachidonic acid metabolite release from human lung tissue by antiinflammatory steroids

The effects of antiinflammatory steroids on arachidonic acid metabolite release from human lung fragments were analyzed. Incubation of lung fragments for 24 hr with 10(-6) M dexamethasone inhibited the net release of the prostacyclin metabolite 6-keto-PGF1 alpha, PGE2, and PGF2 alpha from lung fragments stimulated with anti-IgE but failed to inhibit the anti-IgE-induced release of PGD2, TXB2, and iLTC4. The IC50 of dexamethasone for inhibition of both spontaneous and anti-IgE-induced 6-keto-PGF1 alpha release was approximately 2 X 10(-8) M, and a 6-hr preincubation with the drug was required for 50% inhibition of prostaglandin release. Other agents were tested for activity in stimulating arachidonic acid metabolite release from human lung fragments. FMLP (fmet-leu-phe) stimulated the release of all metabolites tested (6-keto-PGF1 alpha, PGD2, PGE2, PGF2 alpha, TXB2, iLTC4); platelet-activating factor (PAF), but not lysoPAF, stimulated the release of PGD2, TXB2, and iLTC4. In contrast to the case with anti-IgE, where dexamethasone failed to inhibit net PGD2 and TXB2 release, the steroid inhibited the release of these metabolites stimulated by both FMLP and PAF. The steroid inhibited iLTC4 release induced by the highest concentration of PAF (10(-6)M) but did not inhibit iLTC4 release stimulated by either 10(-7) M PAF, FMLP, or anti-IgE. Because neither FMLP nor PAF caused the release of PGD2 or TXB2 from purified human lung mast cells, and because they also failed to induce histamine release from lung fragments, it is suggested that these stimuli produce PGD2 and TXB2 release in lung fragments through an action on a cell distinct from the mast cell. This suggestion is supported by the selective inhibition of the release of these arachidonic acid metabolites by dexamethasone. We suggest that the inhibitory action of steroids on arachidonic acid metabolite in human lung fragments contributes to their therapeutic efficacy in pulmonary diseases.     

Epinephrine stimulates prostaglandin synthesis by bullfrog lung from warm-acclimated, but not cold-acclimated, animals

Exogenous prostaglandins (PGs) have been shown to have differing effects on frog lung contractility. In this study, prostaglandin synthesis was measured in lung tissues from warm-acclimated (WA, 22 degrees C) and cold-acclimated (CA, 5 degrees C) American bullfrogs, Rana catesbeiana, incubated for 30 min at 5 degrees or 22 degrees C. Media were assayed by radioimmunoassay for PGE2, PGF2 alpha, 6-keto PGF 1 alpha (the metabolite of PGI2), and thromboxane (TX)B2 (the metabolite of TXA2). PGE2 was produced in greatest quantity by tissues from WA and CA animals, at both incubation temperatures. Epinephrine stimulated PGE2, PGF2 alpha, and TXB2 synthesis at 22 degrees C but only stimulated PGE2 production at 5 degrees C. In tissues from CA frogs, epinephrine did not stimulate prostaglandin synthesis at either incubation temperature. Ibuprofen (10(-5) M) inhibited basal and epinephrine-stimulated prostaglandin synthesis in tissues from WA frogs incubated at 22 degrees C. The beta receptor antagonist propranolol (10(-6) M) blocked the epinephrine-stimulated synthesis of PGE2, PGF2 alpha and TXB2, suggesting epinephrine stimulates prostaglandin synthesis through beta receptor activation. The absence of stimulation by epinephrine in lung from CA animals, but not in 5 degrees C incubations of tissues from WA animals, suggests that a modification of beta receptors occurs during prolonged cold exposure.     

Effects of insulin and prior exercise on prostaglandin release from perfused rat muscle. Evidence that prostaglandins do not mediate changes in glucose uptake.

Prostaglandin generation and its inter-relation to the metabolic effects of insulin and prior exercise were examined in perfused muscle of fed rats. During a 60 min perfusion of the rat hindquarter, a substantial release of the prostaglandins PGF2 alpha, PGE2 and 6-oxoPGF1 alpha was observed. Blood cells present in the perfusate released these substances in negligible amounts indicating the prostaglandins were produced by the hindquarter. Addition of insulin to the perfusate increased both glucose uptake and the generation of PGE2 and 6-oxoPGF1 alpha. At 30 min after intense treadmill exercise, glucose and alpha-aminoisobutyric acid (AIB) uptake by the hindquarter were increased in the absence of added insulin, but prostaglandin release was not increased. Insulin further increased glucose and AIB uptake; however, in contrast with its effects in non-exercised rats, insulin no longer stimulated prostaglandin generation. Indomethacin (10 microM) added to the perfusate inhibited the release of PGF2 alpha and PGE2 by 90% and the release of 6-oxoPGF1 alpha by 54%. It had no effect on the stimulation of glucose uptake by either insulin or prior exercise. The data indicate that insulin increases prostaglandin synthesis by perfused rat muscle, and that prior exercise blocks this effect. They suggest that under the conditions studied prostaglandins do not mediate the effects of insulin or prior exercise on glucose uptake.     

Production of prostaglandins by dispersed cells and fragments from bovine parathyroid glands

We studied the production of prostaglandins by fragments and dispersed cells from bovine parathyroid glands. Fragments released 138 +/- 19 (SE), 132 +/- 21, 4.3 +/- 0.5, and 13 +/- 6.6 pg/mg/h of 6-keto-PGF1 alpha, PGF2 alpha, PGE2, and thromboxane B2, respectively (n = 7 - 26), while dispersed cells released 414 +/- 110, 22 +/- 7.3, 27 +/- 3.8, and 29 +/- 11 pg/10(6) cells/h, respectively, of the same compounds (n = 6 - 25). Indomethacin (1 microgram/ml) inhibited the release of 6-keto-PGF1 alpha by 80-90% in fragments and cells, while mellitin stimulated release of this prostaglandin, suggesting de novo synthesis of prostaglandins in these preparations. Calcium stimulated production of 6-keto-PGF1 alpha by 1.3-fold in cells and 2.6-fold in fragments and also enhanced production of PGF2 alpha by 1.9-fold in fragments. Isoproterenol, on the other hand, had no effect on production of 6-keto-PGF1 alpha in either preparation. These results demonstrate that parathyroid tissue as well as parathyroid cells per se produce a variety of prostaglandins. We have previously shown that PGE2 and PGF2 alpha modulate cAMP accumulation and PTH release in dispersed bovine parathyroid cells. The role of the endogenous production of prostaglandins by the parathyroid gland in the acute or chronic regulation of parathyroid function, however, remains to be determined.     

Antiabortifacient action of dibenzyloxyindanpropionic acid in mice

To evaluate the details of the adrenergic stimulation of urinary prostaglandins in man, ten normal volunteers were given various agonists and antagonists. The effect of 4 hour IV infusions of norepinephrine (NE), NE + phentolamine (PHT), NE + phenoxybenzamine (PHB), NE + prazosin (PZ), isoproterenol (ISO), and PHT alone on urinary PGE2 and PGI2 (6 keto PGF1 alpha) were determined. PGE2 and 6 keto PGF1 alpha were measured by radioimmunoassay from 4 hour urine samples. NE stimulated both PGE2 (196 +/- 40 to 370 +/- 84 ng/4 hrs/g creatinine and 6 keto PGF1 alpha (184 +/- 30 to 326 +/- 36), both p less than 0.01. In contrast, ISO had no effect on either PGE2 or 6 keto PGF1 alpha excretion. Alpha blockade with PHT. PHB, or PZ inhibited the NE induced systemic pressor effect. However, the effect of the alpha blockers on the NE induced stimulation of PGE2 and 6 keto PGF1 alpha varied. PHT did not alter the NE stimulated PGE2 or 6 keto PGF1 alpha release (370 +/- 84 vs. 381 +/- 80) PGE2 and (326 +/- 50 vs. 315 +/- 40) 6 keto PGF1 alpha both p greater than 0.2). PHT alone stimulated only 6 keto PGF1 alpha. PHB and the specific alpha 1 antagonist PZ similarly eliminated the NE induced prostaglandin release. These results suggest that adrenergically mediated urinary prostaglandin release in man is via an alpha receptor with alpha 1 characteristics.     

Histamine alters prostaglandin output from diestrous rat uteri. Involvement of H2-receptors and 9-keto-reductase

The effects of exogenous histamine (H) on prostaglandin (PG) generation and release in uteri isolated from diestrous rats and the influences of H2-receptors blockers (cimetidine and metiamide) on the output of uterine PGs, were explored. Moreover, the action of H on the uterine 9-keto-reductase, was also studied. Histamine (10(-4) M) failed to alter the basal output of PGE1 but reduced significantly the generation and release of PGE2 and augmented the output of PGF2 alpha. On the other hand, cimetidine (10(-5) M) enhanced the basal release of PGE2 but had no action on the outputs of PGs E1 or F2 alpha. The enhancing effect of H on the production and release of PGF2 alpha was abolished in the presence of cimetidine. Also, the antagonist reversed the influence of H on the output of PGE2. Metiamide, another H2-receptor antagonist, did not alter the basal control generation and release of uterine PGs, but antagonized the augmenting influence of H on PGF2 alpha uterine output, as much as cimetidine did, and prevented the depressive action of H on the release of PGE2 from uteri. Histamine (10(-4) M) significantly stimulated uterine formation of cyclic-adenosine monophosphate, an action which was antagonized by the presence of cimetidine (10(-5) M), a blocker of H2 receptors. Also, histamine (10(-5) M) and dibutyrylcyclic-adenosine monophosphate (DB-cAMP) at 10(-3) M, enhanced significantly the formation 3H-PGF2 alpha from 3H-PGE2. Results presented herein demonstrate that H is able to diminish the generation of PGE2 in uteri from rats at diestrus augmenting the synthesis of PGF2 alpha, apparently via the activation of H2-receptors, enhancing adenylate-cyclase. These effects appear to increase uterine 9-keto-reductase activity which transforms PGE2 into PGF2 alpha. Relationships between the foregoing results and those evoked by estradiol, are also discussed.     

Characterisation of the prostanoid receptors mediating contraction of guinea-pig isolated trachea

The receptors mediating prostanoid-induced contraction of guinea-pig isolated trachea have been characterised in terms of a recently proposed general classification of prostanoid receptors. Results obtained on the trachea were compared with those obtained on guinea-pig fundus, which contains a sub-type of PGE2-sensitive (EP-) receptor termed the EP1-receptor, and guinea-pig lung strip, which contains a thromboxane-sensitive or TP-receptor. The following agonists were studied, PGE2, PGF2 alpha and the thromboxane-like agonists U-46619 and Wy17186. The antagonists studied were SC-19220 which selectively blocks EP1-receptors, and AH19437 which selectively blocks TP-receptors. On guinea-pig fundus the rank order of agonist potency was PGE2 greater than PGF2 alpha greater than Wy-17186 approximately equal to U-46619, and responses to all agonists were antagonised by SC-19220 but not by AH19437. On guinea-pig lung strip the rank order of potency was U-46619 greater than Wy17186 much greater than PGF2 alpha greater than PGE2 and responses to all agonists tested were blocked by AH19437 but not by SC-19220. On the trachea, the rank order was PGE2 = U-46619 greater than Wy17186 = PGF2 alpha. SC-19220 antagonised responses to PGE2 and PGF2 alpha, but not those to U-46619 or Wy17186. Conversely, AH19437 antagonised responses to U-46619 and Wy17186 but not those to PGE2 or PGF2 alpha. It is concluded that prostanoid-induced contractions of guinea-pig trachea can be mediated by both EP1- and TP-receptors.     

Synthesis of prostanoids and cyclic nucleotides by phagocytosing rat Kupffer cells

Rat Kupffer cells in monolayer culture were allowed to phagocytose unopsonized zymosan granules. They responded with a strongly stimulated synthesis and release of prostanoids, mainly the immunologically determined prostaglandins PGE2 and PGF2 alpha. The same response could be obtained by treatment with the calcium ionophore A23187. The effects of the ionophore and the zymosan particles were of the same magnitude but not additive. The rapid uptake of Ca2+ after contact with phagocytosable material recently described by us [(1983) Eur. J. Biochem. 131, 539-543] appears to mediate the enhanced prostaglandin synthesis. That response was suppressed not only by indomethacin but also by trifluoperazine which does not inhibit Ca2+ entry in the Kupffer cells. Similar effects by R24571 and 4-bromophenacyl bromide support the participation of calcium-calmodulin and of phospholipase A2. The calcium channel blocker Verapamil did not influence the zymosan-provoked production of prostaglandin PGE2 nor were any indications obtained for a feedback inhibition by PGE1 or PGE2. Contact with zymosan resulted in a rapid but transient rise of the intracellular levels of cAMP and cGMP: 10 nM indomethacin completely blocked the increase of both cyclic nucleotides while trifluoperazine elicited different responses in the cAMP and cGMP levels. The stimulated release of prostaglandin E2 was inhibited in a dose-dependent manner by nordihydroguaiaretic acid, an inhibitor of 5-lipoxygenase and by FPL 55712, known as a receptor antagonist for some leukotrienes. This suggests a regulatory role for its metabolites on prostaglandin synthesis.     

Prostaglandins E2 and F2 alpha affect glycogen synthase and phosphorylase in isolated hepatocytes.

Prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) inactivated glycogen synthase and activated glycogen phosphorylase in rat hepatocytes in a dose- and time-dependent manner. These effects were dependent on the presence of Ca2+ in the incubation medium. When glycogen synthase was immunoprecipitated from cells incubated with [32P]Pi and then treated with PGE2 or PGF2 alpha, there was increased phosphorylation of the 88 kDa subunit of the enzyme. This phosphorylation affected two CNBr fragments of the glycogen synthase, CB-1 and CB-2, the same fragments that are phosphorylated by different glycogenolytic hormones. No phosphorylation of glycogen synthase by prostaglandins was observed in the absence of Ca2+. Thus the effect of PGE2 and PGF2 alpha on these glycogen-metabolizing enzymes supports a role for regulation by prostaglandins of glucose metabolism in parenchymal liver cells.     

Is the epithelium-derived inhibitory factor in guinea-pig trachea a prostanoid?

To examine further the possible prostanoid involvement in the influence of the epithelium on guinea-pig tracheal smooth muscle responsiveness, we have analyzed the effects of LTD4, methacholine and histamine on the level of airway smooth muscle tone and on the amounts of PGE2, PGF2 alpha and PGI2 (determined by radioimmunoassay) in the presence and absence of the epithelium. Removal of the epithelium increased the sensitivity of guinea-pig trachea to the contractile effects of LTD4, methacholine and histamine. LTD4 (3-100 nM), methacholine (0.1-10 microM) or histamine (0.3-30 microM) did not increase prostanoid release above control values in either the presence or absence of the epithelium. The unstimulated release of PGE2 and PGF2 alpha, but not PGI2, was decreased in tissues lacking epithelium. Indomethacin (1 microM) reduced the baseline tone to a smaller extent in the absence of epithelium. In the presence but not the absence of the epithelium, indomethacin increased the sensitivity of preparations to the contractile effect of methacholine. The results support the postulate of an epithelium-derived inhibitory factor modulating guinea-pig tracheal smooth muscle responsiveness. The identity of this factor is not known but is not PGI2 and is unlikely to be PGF2 alpha or PGE2. However, the possibility remains that the basal release of PGE2 and/or PGF2 alpha derived from the epithelium may markedly affect the responsiveness of guinea-pig tracheal smooth muscle. Furthermore, the epithelium is a significant source of PGE2 and PGF2 alpha which may be involved in the maintenance of baseline tone.     

Effects of prostaglandins on LDL receptor activity and cholesterol synthesis in freshly isolated human mononuclear leukocytes

The effects of prostaglandin (PG) E1, PGE2, the stable prostacyclin analogue Iloprost, and PGF2 alpha on low density lipoprotein (LDL) receptor activity and cholesterol synthesis were investigated in freshly isolated human mononuclear leukocytes. Incubation of cells for up to 45 hr in a lipid-free medium resulted in an increase in the rate of cholesterol synthesis from [14C]acetate and the high affinity accumulation and degradation of 125I-labeled LDL. Addition of PGE1 in increasing concentrations to the incubation medium inhibited cholesterol synthesis and the specific accumulation and degradation of 125I-labeled LDL; at a concentration of 10 microM, the inhibitions were 61%, 70%, and 67%, respectively, after an incubation of 20 hr. The effects of PGE2 and Iloprost were similar. The action of the prostaglandins on LDL receptor activity appeared to be mediated by a decrease in the number of LDL receptors and not by a change in the binding affinity. The prostaglandins yielded sigmoidal log concentration-effect curves. In contrast, PGF2 alpha had no influence on cholesterol synthesis or LDL receptor activity up to a concentration of 10 microM. PGE1, PGE2, and Iloprost, but not PGF2 alpha, led to an increase in the concentration of intracellular cyclic AMP. Dibutyryl cyclic AMP mimicked the effects of the E-prostaglandins and Iloprost on the LDL receptor activity. The results suggest that PGE1, PGE2, and prostacyclin affect LDL receptor activity and cholesterol synthesis and, therefore, may play a role in the regulation of cholesterol homeostasis and in the development of atherosclerosis.     

Prostaglandin synthesis by isolated cells of rat uterus: production rates, and effects of indomethacin and histamine

Luminal epithelial and residual cells (mainly of the endometrial stromal tissue) of proestrous rat uteri have been isolated and cultured in defined medium. The prostaglandins produced during a short-term incubation (2 h) in the presence of 10 microM arachidonic acid (to optimize PG production) were determined by direct assay of the culture medium. For the epithelial cells, PGF2 alpha was produced in greatest amounts, followed by 6-keto PGF1 alpha and PGE, while low levels were synthesized by the residual cells. The synthesis of PGF2 alpha by the epithelial cells was inhibited by incorporating indomethacin into the medium and an IC50 value of 2.3 microM was obtained. Incubations performed with histamine in the absence of exogenous arachidonic acid indicated that the pathways for the production of individual prostaglandins were followed to different relative extents, with the production of 6-keto PGF1 alpha being enhanced for both groups of cells when compared to incubations with arachidonic acid.     

Requirement for prostaglandin F2 alpha in 17 beta-estradiol stimulation of DNA synthesis in rabbit endometrial cultures

We have hypothesized that two of the endogenously synthesized endometrial prostaglandins (PGs), prostaglandin F2 alpha (PGF2 alpha), and prostaglandin E1 (PGE1), play a regulatory role in growth control of the rabbit endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used to examine the possible role of these PGs in the mechanism of action of 17 beta-estradiol on DNA synthesis. Towards this end, binding, second messenger and DNA synthesis experiments were performed. 17 beta-estradiol stimulation resulted in a time dependent (optimal: approximately 6 h) and 17 beta-estradiol concentration dependent (optimal: approximately 10(-7) M 17 beta-estradiol in phenol red-containing medium) increase in [3H]PGF2 alpha binding. Scatchard type analysis of the binding data revealed an increase in receptor number while the receptor affinity for [3H]PGF2 alpha remained the same as in the control treated cultures. This 17 beta-estradiol stimulated increase in PGF2 alpha receptor allowed a suboptimal concentration of PGF2 alpha (10(-9) M) to increase intracellular levels of inositol polyphosphates, while by itself this concentration of PGF2 alpha caused no significant change in intracellular inositol polyphosphate levels. 17 beta-estradiol, alone among the several studied steroid hormones, could increase [3H]PGF2 alpha binding. Proliferation studies revealed that, in these primary cultures of rabbit endometrium, 17 beta-estradiol could increase DNA synthesis but not in the presence of indomethacin, unless PGF2 alpha was added to the medium at a concentration (10(-10) M) near or above what is normally accumulated in the medium by these cultures. In the absence of 17 beta-estradiol stimulation, addition of these same low concentrations of PGF2 alpha had no effect on DNA synthesis. Apparently, through its effect on the PGF2 alpha receptor, 17 beta-estradiol enhances the PGF2 alpha stimulated DNA synthesis response approximately 100 fold. The DNA synthesis induced by 17 beta-estradiol can be inhibited by PGE1, as can PGF2 alpha-induced DNA synthesis. We propose that 17 beta-estradiol may be mediating its mitogenic effect through an alteration of the prostaglandin agonist:antagonist control of proliferation in rabbit endometrial cultures. In addition we suggest that, if 17 beta-estradiol acts to increase PGF2 alpha, receptors as part of its mode of action, this may be of importance in other tissues possessing both prostaglandin and 17 beta-estradiol receptors.     

Relationships among mammalian gonadotropin-releasing hormone, prostaglandins, and sex steroids in the brain of the crested newt, Triturus carnifex.

The present study was carried out to evaluate the in vitro brain release of prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), androgens, and 17 beta-estradiol in male and female crested newt, Triturus carnifex, during three different periods of the annual sexual cycle; in addition, the effects of mammalian gonadotropin-releasing hormone (mGnRH), PGF2 alpha, and PGE2 on prostaglandins and steroids release by the brain were evaluated during the same periods. In brain incubations of both sexes, PGF2 alpha and estradiol were higher during postreproduction, while PGE2 and androgens were higher during reproduction. In both sexes, mGnRH increased PGF2 alpha and estradiol during postreproduction, and PGE2 during reproduction; PGF2 alpha increased estradiol secretion during postreproduction. Only in the male, did both mGnRH and PGE2 increase androgens during reproduction. It could be suggested that in Triturus carnifex, the regulation of the reproductive activity in the central nervous system (CNS) depends on the relationships among mGnRH, prostaglandins and steroids. In particular, PGF2 alpha and PGE2 seem to play different roles in the CNS of the newt: PGF2 alpha is involved in the postreproductive processes, through estradiol secretion, while PGE2 in the reproductive ones (through androgens secretion?).     

Phytoestrogens modulate prostaglandin production in bovine endometrium: cell type specificity and intracellular mechanisms

Prostaglandins (PGs) are known to modulate the proper cyclicity of bovine reproductive organs. The main luteolytic agent in ruminants is PGF2alpha, whereas PGE2 has luteotropic actions. Estradiol 17beta (E2) regulates uterus function by influencing PG synthesis. Phytoestrogens structurally resemble E2 and possess estrogenic activity; therefore, they may mimic the effects of E2 on PG synthesis and influence the reproductive system. Using a cell-culture system of bovine epithelial and stromal cells, we determined cell-specific effects of phytoestrogens (i.e., daidzein, genistein), their metabolites (i.e., equol and para-ethyl-phenol, respectively), and E2 on PGF2alpha and PGE2 synthesis and examined the intracellular mechanisms of their actions. Both PGs produced by stromal and epithelial cells were significantly stimulated by phytoestrogens and their metabolites. However, PGF2alpha synthesis by both kinds of cells was greater stimulated than PGE2 synthesis. Moreover, epithelial cells treated with phytoestrogens synthesized more PGF2alpha than stromal cells, increasing the PGF2alpha to PGE2 ratio. The epithelial and stromal cells were preincubated with an estrogen-receptor (ER) antagonist (i.e., ICI), a translation inhibitor (i.e., actinomycin D), a protein kinase A inhibitor (i.e., staurosporin), and a phospholipase C inhibitor (i.e., U73122) for 0.5 hrs and then stimulated with equol, para-ethyl-phenol, or E2. Although the action of E2 on PGF2alpha synthesis was blocked by all reagents, the stimulatory effect of phytoestrogens was blocked only by ICI and actinomycin D in both cell types. Moreover, in contrast to E2 action, phytoestrogens did not cause intracellular calcium mobilization in either epithelial or stromal cells. Phytoestrogens stimulate both PGF2alpha and PGE2 in both cell types of bovine endometrium via an ER-dependent genomic pathway. However, because phytoestrogens preferentially stimulated PGF2alpha synthesis in epithelial cells of bovine endometrium, they may disrupt uterus function by altering the PGF2alpha to PGE2 ratio.     

Anti-apoptotic roles of prostaglandin E2 and F2alpha in bovine luteal steroidogenic cells

Production of prostaglandins (PGs) and expression of their receptors have been demonstrated in bovine corpus luteum (CL). The aim of the present study was to determine whether PGE2 and PGF2alpha have roles in bovine luteal steroidogenic cell (LSC) apoptosis. Cultured bovine LSCs obtained at the midluteal stage (Days 8-12 of the cycle) were treated for 24 h with PGE2 (0.001-1 microM) and PGF2alpha (0.001-1 microM). Prostaglandin E2 (1 microM) and PGF2alpha (1 microM) significantly stimulated progesterone (P4) production and reduced the levels of cell death in the cells cultured with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG), in the presence and absence of FAS ligand (P < 0.05). Furthermore, DNA fragmentation induced by TNF/IFNG was observed to be suppressed by PGE2 and PGF2alpha. Prostaglandin E2 and PGF2alpha also attenuated mRNA expression of caspase 3 and caspase 8, as well as caspase 3 activity (P < 0.05) in TNF/IFNG-treated cells. FAS mRNA and protein expression were decreased only by PGF2alpha (P < 0.05). A specific P4 receptor antagonist (onapristone) attenuated the apoptosis-inhibitory effects of PGE2 and PGF2alpha in the absence of TNF/IFNG (P < 0.05). A PG synthesis inhibitor (indomethacin) reduced cell viability in PGE2- and PGF2alpha-treated cells (P < 0.05). A specific inhibitor of cyclooxygenase (PTGS), PTGS2 (NS-398), also reduced cell viability, whereas an inhibitor of PTGS1 (FR122047) did not affect it. The overall results suggest that PGE2 and PGF2alpha locally play luteoprotective roles in bovine CL by suppressing apoptosis of LSCs.     

A comparison of the effects of cyclooxygenase prostanoids on progesterone production by small and large bovine luteal cells

Highly purified preparations of small and large bovine luteal cells were utilized to examine the effects of prostaglandins F2 alpha (PGF2 alpha), E2 (PGE2) and I2 (PGI2) analog on progesterone production. Corpora lutea were obtained from Holstein heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. Progesterone accumulation was determined in 1 x 10(5) small and 5 x 10(3) large cells after 2 and 4 h incubations respectively. Progesterone synthesis was increased (p less than 0.05) in the small cells by the increasing levels of PGF2 alpha, PGE2, carba-PGI2 and LH. PGF2 alpha, but not PGE2 or carba-PGI2 increased (p less than 0.05) LH-stimulated progesterone production. There was no interaction of various combinations of prostaglandins on progesterone production in the small cells. In the large cells, PGF2 alpha had no effect on basal progesterone production. However, it inhibited LH-stimulated progesterone synthesis. In contrast, PGE2 and carba-PGI2 stimulated (p less than 0.05) basal progesterone production in the large cells. In the presence of LH, high levels of carba-PGI2 inhibited (p less than 0.05) progesterone synthesis. The PGE2 and PGI2-stimulated progesterone production in the large luteal cells was also inhibited in the presence of PGF2 alpha. These data suggest all of the prostaglandins used exert a luteotropic action in the small cells. In the large cells only PGE2 and carba-PGI2 are luteotropic, while PGF2 alpha exerts a luteolytic action. The effects of the prostaglandins in the small and large luteal cells suggest that their receptors are present in both cell types.     

Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle.

To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.