Cyclic AMP does not inhibit A23187-induced prostaglandin biosynthesis in cultured rabbit renal microvascular endothelial cells.

We have previously shown that cultured rabbit renal preglomerular microvascular endothelial cells have the ability to synthesize a number of common prostaglandins. In the present study we have examined whether endogenous cyclic AMP is involved in the regulation of PGI2 and PGE2 biosynthesis in these cultured cells. Isoproterenol and forskolin produced an increase in cyclic AMP accumulation in these cells but had no effect on PGI2 or PGE2 biosynthesis either in the presence or absence of A23187. Similar results were noted in the presence of 3-isobutyl-1-methylxanthine, a cyclic AMP-phosphodiesterase inhibitor. These studies suggested that endogenous cyclic AMP does not regulate the biosynthesis of PGI2 or PGE2 in cultured renal preglomerular microvascular endothelial cells either under basal or A23187-stimulated condition. They further suggested that the effect of 3-isobutyl-1-methylxanthine on prostaglandin biosynthesis in these cultured cells was not secondary to its effects on phosphodiesterase.     

Effect of prostaglandins on the in vitro steroidogenesis in human ovarian tissues

This is a brief report of preliminary findings from in vitro studies of the effect of PGs (prostaglandins) on progesterone formation in human corpora lutea and on the utilization of C21 steroids by the luteal and follicular compartments of the ovary. Ovaries were obtained from cyclic women undergoing ovariectomies for therapeutic purposes. The laboratory procedures employed in the study are explained. Results are tabulated. PGE2 stimulated progesterone biosynthesis in the corpus luteum as measured by tissue content and by de novo synthesis from acetate-1-14C. PGE2 also stimulated the biosynthesis of DPS (digitonin-precipitable sterols) from acetate. These results confirm findings of other researchers. In 1 of the experiments, PGF2alpha failed to demonstrate stimulation of progesterone biosynthesis in the human corpus luteum as measured by tissue progesterone content after incubation. Both PGF2alpha and PGE2 showed generally stimulatory effects on the utilization of exogenous labelled progesterone for the formation of androgens and estradiol by the human corpus luteum. In the follicular tissue, however, PGE2 showed an inhibitory effect on the formation of androgens and progesterone from exogenous labelled pregnenolone. No significant amounts of estrogens were biosynthesized in these experiments. These preliminary results must be confirmed by measurement of the endogenous steroidal concentrations in the tissues.     

Effects of inhibitors of steroid biosynthesis on prostaglandin formation in rabbit ovarian follicles

Rabbit ovarian follicles were incubated without stimulation, with LH and with LH + an inhibitor of steroid biosynthesis. Formation of prostaglandins PGE and PGF and of progesterone and estradiol was measured in these incubates. It was found that aminoglutethimide phosphate (AGP) inhibited the LH stimulated biosynthesis of both prostaglandins and steroids. However U 30870 and Metyrapone, while completely inhibiting the LH stimulated biosynthesis of progesterone and estradiol respectively, had no effect on the formation of prostaglandins. Further, the inhibition of prostaglandin formation by AGP could not be reversed by exogenous steroids. It, therefore, appears that the effect of AGP on prostaglandin biosynthesis may not be related to its effect on steroid biosynthesis. However, the response of rabbit follicles to AGP is contrary to that reported for rat follicles and indicates different control mechanisms for prostaglandin formation in the follicles of the two species.     

Effects of inhibitors of steroid biosynthesis on prostaglandin formation in rabbit ovarian follicles

Rabbit ovarian follicles were incubated without stimulation, with LH and with LH + an inhibitor or steroid biosynthesis. Formation of prostaglandins PGE and PGF and of progesterone and estradiol was measured in these incubates. It was found that aminoglutethimide phosphate (AGP) inhibited the LH stimulated biosynthesis of both prostaglandins and steroids. However U 30870 and Metyrapone, while completely inhibiting the LH stimulated biosynthesis of progesterone and estradiol respectively, had no effect on the formation of prostaglandins. Further, the inhibition of prostaglandin formation by AGP could not be reversed by exogenou steroids. It, therefore, appears that the effect of AGP on prostaglandin biosynthesis may not be related to its effect on steroid biosynthesis. However, the response of rabbit follicles to AGP is contrary to that reported for rat follicles and indicates different control mechanisms for prostaglandin formation in the follicles of the two species.     

A role for somatomedin C as a differentiating hormone and amplifier of hormone action on ovarian cells: studies with synthetically pure human somatomedin C and swine granulosa cells

Isolated swine granulosa cells incubated in chemically defined medium in vitro responded to synthetically pure human somatomedin C/IGF-I in a dose and time-dependent fashion with increased pregnenolone, progesterone and estradiol biosynthesis. These stimulatory actions were not mimicked by growth hormone, proinsulin, desoctapeptide insulin, epidermal growth factor, or fibroblast growth factor. Moreover, somatomedin C/IGF-I augmented the steroidogenic response of granulosa cells to exogenously supplied sterol substrate in the form of low-density lipoprotein, and amplified the stimulatory actions of the classical ovarian effector hormones, estradiol and follicle-stimulating hormone, in a synergistic fashion. The ability of somatomedin C/IGF-I to stimulate estradiol production on the one hand, and to act synergistically with estradiol to stimulate progesterone biosynthesis on the other hand, suggests a unique intrafollicular mechanism for amplifying progestin biosynthetic capacity in granulosa cells.     

Estradiol effect on indomethacin and prostaglandin E2-modulation of adrenergic transmission in rabbit oviduct.

The effect of prostaglandin E2 /PGE2/ and indomethacin on 3H-noradrenaline (3H-NA) release- and on contractions-evoked by field electrical stimulation (FES) was studied in vitro in oviductal isthmus of mature rabbits (untreated and treated with estradiol). FES evoked guanethidine-sensitive contractions and calcium-dependent tritium overflow, which reflected 3H-NA overflow. Marked and concentration-dependent decrease of FES-evoked contractions by PGE2 (0.1-100 nM) was observed in both groups of animals. The inhibitory effect of PGE2 was more pronounced in estradiol treated animals (IC50 1.5 nM, n = 9) than in untreated animals (IC50 18 nM, n = 6). Indomethacin, 1 microM, induced a remarkably pronounced increase of FES-evoked contractions in estradiol treated (by 57.3 +/- 6.3%, n = 8) in comparison with untreated rabbits (21.4 +/- 3.8%, n = 7). The amount of FES-evoked release of tritium was significantly higher in untreated than in estradiol treated rabbits. PGE2 decreased and indomethacin increased tritium-evoked release. The effects of PGE2 and indomethacin on tritium-evoked release showed no estradiol dependence. The competitive results of PGE2 and indomethacin on both evoked contraction and 3H-NA release suggest that endogenous prostaglandin E2 takes part in modulation of adrenergic mediated contraction and that estradiol enhanced the prostaglandin effect.     

Role of renal PGE2 in the adaptation from foetal to extrauterine life in term and preterm infants

Urinary PGE(2) concentrations were assayed using a new EIA method, in 16 preterm and 18 term neonates at birth and 3 days later, since there is evidence that PGE(2) in urine are likely to reflect their renal generation and then could be correlated with kidney maturation or renal problems. PGE(2) concentrations were not different at birth (1.50+/-1.12 vs 1.56+/-1.94 ng/day), while resulted significantly higher in preterms, compared to terms, three days after birth (2.22+/-1.23 vs 1.39+/-0.79 ng/day). This increase in daily PGE(2) excretion observed only in preterm neonates could be due to an increased renal biosynthesis as a mechanism of compensatory response to prevent further decrements in renal plasma flow, since prostanoids play an important role in protecting the immature kidney from high levels of angiotensin II. Otherwise, the passive reabsorption of PGE(2) along the distal nephron could be altered because of kidney immaturity. The measurement of PGE(2) in urine of neonates, particularly prematures, could be useful to provide a better understanding of the homeostatic function of the kidney in the phase of adaptation to extra-uterine life.     

PGF2 alpha, PGE2, and sex steroids from the abdominal gland of the male crested newt Triturus carnifex (Laur.).

Prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), progesterone, androgens, and 17 beta-estradiol in vitro release by the abdominal gland of the crested newt, Triturus carnifex (Laur.), was studied during the prereproductive, reproductive and postreproductive periods. In addition, the in vitro effects of the PGF2 alpha and/or PGE2 on progesterone, androgens and estradiol release by the abdominal gland were evaluated. PGF2 alpha, PGE2 and progesterone release was higher during the reproductive period, and in the same period, PGE2 treatment induced a progesterone increase. PGF2 alpha induced an increase of abdominal gland estradiol release at the end of the reproductive period. These results seemed to confirm the pheromonal role assigned to progesterone, and suggested a PGE2 stimulatory role in inducing progesterone release, even if pheromonal activity of PGF2 alpha and PGE2 cannot be excluded. In addition, PGF2 alpha-dependent estradiol increase at the end of reproduction could be interpreted as a mechanism for interruption of the abdominal gland activity.     

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?).     

Ectodomain shedding of pro-TGF-alpha is required for COX-2 induction and cell survival in renal medullary cells exposed to osmotic stress

In the renal medulla, cyclooxygenase (COX)-2 is induced by osmotic stress as present in this kidney region during antidiuresis. Increasing evidence suggests that EGF receptor (EGFR) signaling is involved in this process. The aim of the present study was to examine the mechanisms responsible for COX-2 expression and PGE(2) production during hypertonic conditions and to identify potential autocrine/paracrine EGFR ligands. Immunohistochemisty and Western blot analysis revealed abundant expression of the pro-EGFR ligand pro-transforming growth factor (TGF)-alpha in renal medullary cells in vivo and in cultured Madin-Darby canine kidney cells. In Madin-Darby canine kidney cells, hypertonicity rapidly increased TNF-alpha converting enzyme (TACE)-dependent ectodomain shedding of pro-TGF-alpha; phosphorylation of EGFR, p38, and ERK1/2; expression of COX-2; and production of PGE(2). Conversely, TACE inhibition prevented TGF-alpha release; EGFR, p38, and ERK1/2 activation; and COX-2 expression. Furthermore, cell survival was reduced substantially, a response that could be reversed by the addition of PGE(2). Simultaneous addition of recombinant TGF-alpha during TACE inhibition restored EGFR and MAPK phosphorylation, COX-2 expression, PGE(2) production, and cell survival during osmotic stress. These results indicate that hypertonicity induces TACE-mediated ectodomain shedding of pro-TGF-alpha, which subsequently activates COX-2 expression in an autocrine/paracrine fashion, via EGFR and MAPKs. We conclude that tonicity-induced TGF-alpha release is required for COX-2 expression, PGE(2) synthesis, and survival of renal medullary cells during osmotic stress.     

Effects of prostaglandins on erythropoiesis

A model has been presented for the role of the kidney in the physiologic and pathophysiologic control of erythropoiesis. It is postulated that an oxygen deficit created by anemia or hypobaric hypoxia results in the release of prostacyclin and its metabolite 6-keto PGE1, and the release of PGE2 with ischemic hypoxia. Prostacyclin, 6-keto-PGE1, or PGE2 activation of adenylate cyclase, an increase in cyclic AMP, activation of a protein kinase and the phosphorylation of hydrolases, which have been released from lysosomes by hypoxia, lead to increased biosynthesis of erythropoietin (Ep). The mechanism of labilization of lysosomes and the release of hydrolases from these cell organelles is postulated to be related to increases in cyclic GMP levels in a renal cell. An Ep-producing human renal carcinoma cell line grown in tissue culture has been demonstrated to produce significant amounts of PGE2. Meclofenamate, an inhibitor of prostaglandins synthesis, was found to inhibit in vitro production of PGE2, Ep, and dome formation in these renal carcinoma cells, giving support to our hypothesis that pathophysiologic production of Ep tumor cells depends upon prostaglandins production. An Ep-producing clone from this renal carcinoma cell line has been developed that contains low electron density (LED) cells after the cells reach confluency, which show a cytoplasm, with abundant and widely dilated endoplasmic reticulum, an oval nucleus, dispersed chromatin, and prominent nucleoli. These are the cells responsible for dome formation and Ep production. Non-EP-producing clones have also been produced from this renal carcinoma cell line, which did not produce domes even at high cell density and had a distinctly different cell type than the Ep-producing clone. Thus, it is postulated that prostacyclin (PGI2) and its metabolite 6-keto PGE1 play a significant role in hypoxic hypoxia stimulation of Ep production and PGE2 is involved in ischemic hypoxia and renal carcinoma cell production of Ep. A modulating effect of PGE2 and PGD2, the two primary bone marrow prostaglandins, has been proposed in Ep stimulation of the erythroid progenitor cell compartment (CFU-E and BFU-E).     

Decreased renal preglomerular microvascular PGI2 and PGE2 biosynthesis in cholesterol-fed rabbits

Although atherosclerosis is a common disease afflicting the kidney, few studies have examined the biochemical disturbances associated with this process directly in the renal microvasculature. Using a technique developed in this laboratory to isolate renal preglomerular microvessels, we have examined whether these vascular segments have a reduced capacity to synthesize vasodilator prostanoids (i.e., PGI2 and PGE2), an observation which has been made in atherosclerotic blood vessels from other vascular beds. The synthesis of these two prostanoids was assessed by radioimmunoassay in a group of white New Zealand rabbits fed a 2% cholesterol diet for 30 days and the results compared to that obtained in a similar group of rabbits fed a normal diet. The results of these studies showed the development of hypercholesterolemia in the cholesterol-fed rabbits which was associated with a 35% decrease in the biosynthesis of PGI2 and a 51% decrease in PGE2 in freshly isolated renal preglomerular microvessels. These findings are similar to those observed in non-renal major blood vessels and suggest that common biochemical mechanisms may operate in the development of atherosclerosis in many organ systems.     

Possible role of renal prostaglandin E2 in natriuresis associated with supraventricular tachycardia

We investigated the possible role of renal prostaglandin (PG) E2 in natriuresis associated with supraventricular tachycardia (SVT). In five female patients with paroxysmal tachycardia, SVT was artificially induced and then stopped 60 min later. Before, during, and after SVT, plasma levels of arginine vasopressin and atrial natriuretic peptide (ANP) and the urinary excretion of sodium and PGE2 were measured. Polyuria was observed during SVT. However, natriuresis did not occur until immediately after the termination of SVT. During SVT, the plasma levels of arginine vasopressin tended to decrease. When SVT was terminated, the vasopressin levels increased significantly (p less than 0.01). Urinary excretion of PGE2 tended to decrease during SVT and then increased significantly (p less than 0.01) after SVT ended. Urinary excretion of sodium was correlated (r = 0.699, p less than 0.001) with the urinary excretion of PGE2. Plasma ANP increased during SVT, but there was no correlation with urinary sodium excretion. These results suggest that renal PGE2, the biosynthesis of which may be stimulated by a increase in plasma vasopressin, is an important factor contributing to the natriuresis observed after the end of SVT.     

Aldosterone augments endothelin-1-induced cardiac myocyte hypertrophy with the reinforcement of the JNK pathway

Monocyte-derived dendritic cells (moDCs) are increasingly used in clinical settings to stimulate tumor immunity. Prostaglandin E2 (PGE2), which is a member of the eicosanoid family of oxygenated arachidonic acid derivatives generated through the action of cyclooxygenases (COXs), is frequently used to enhance the tumor necrosis factor-alpha-induced terminal maturation of moDCs. We show here that one effect of interleukin (IL)-4, which is used together with GM-CSF to generate moDCs, is the suppression of endogenous PGE2 production in moDCs. IL-4 inhibits the cytoplasmic form of phospholipase A2, the enzyme that specifically liberates arachidonic acid from membrane phospholipids. Although moDCs failed to mobilize endogenous arachidonic acid, they converted exogenous arachidonic acid into PGE2 in a COX-1- and COX-2-dependent fashion. IL-4-mediated suppression of PGE2 biosynthesis in human moDCs explains the previously reported maturation-enhancing effect of exogenous PGE2. The general suppression of eicosanoid biosynthesis may, however, limit the immunological efficacy of moDCs generated with IL-4.     

Progesterone and estradiol secretion by porcine luteal cells is influenced by individual and combined treatment with prostaglandins E2 and F2alpha throughout the estrus cycle.

The present experiments were conducted to test whether the ratio of PGE2:PGF2alpha affects steroid secretion by porcine luteal cells. We examined the effect of separate and combined treatment with PGE2 and PGF2alpha on progesterone and estradiol secretion. Luteal cells were collected at three different stages of the luteal phase (1-3 days after ovulation; 10-12 days after ovulation and 14-16 days after ovulation). PGE2 alone in a dose dependent manner increased progesterone production by cells collected from mature corpora lutea. On the other hand, PGF2alpha in a dose dependent manner decreased progesterone secretion by cells of the same origin. Progesterone secretion by cells isolated from mature and regressing corpora lutea and treated with both prostaglandins increased in comparison to PGF2alpha-treated cultures. However, in cells collected from regressing corpora lutea PGE2 and PGF2alpha in a ratio of 2:1 and 4:1 increased estradiol production when compared to control and both ratios increased estradiol secretion in comparison to PGF2alpha-treated cells. These data 1) confirm the luteotropic effect of PGE2 and the luteolytic effect of PGF2alpha; 2) demonstrate that when the ratio of PGE2 to PGF2alpha changed from 1:1 to 2:1 or 4:1 cells were protected against the inhibitory effects of PGF2alpha on progesterone secretion by cells collected during the mid- and late luteal phase; and 3) suggest that elevated estradiol production by luteal cells, isolated during late luteal phase, under the influence of increased doses of PGE2 may serve as an additional source of estradiol to blastocysts, during early pregnancy in the pig.     

Prostaglandin-E2: a point of divergence in estradiol-mediated sexual differentiation

The preoptic area of the mammalian forebrain is a critical substrate for the development and maintenance of many sexually dimorphic behaviors relevant to reproduction. Normal development of the male rodent brain requires completion of two processes: (1) masculinization-induction of the male phenotype, and (2) defeminization-removal of the female phenotype. Both processes, although distinct, are largely directed by the same steroid, estradiol. Whether estradiol achieves both ends via the same or separate mechanisms has been unknown. Here, we report that prostaglandin-E(2) (PGE(2)) acting downstream of estradiol, is necessary and sufficient to masculinize sexual behavior but does not affect defeminization of sexual behavior or maternal behavior. Moreover, the volume of the sexually dimorphic nucleus of the preoptic area predicts defeminization of sexual behavior, but not masculinization of sexual behavior. Another sexually dimorphic cellular endpoint regulated by estradiol, spinophilin protein expression in the mediobasal hypothalamus, was not affected by PGE(2). Thus, PGE(2) is a key divergence point in the downstream actions of estradiol to simultaneously masculinize and defeminize sexual behavior.     

Evidence that the prostaglandin E2 receptor and the anhydrolevuglandin E2 receptor in the rat uterus are the same receptor.

Anhydrolevuglandin E2 (AnLGE2) is closely related to prostaglandin E2 (PGE2) and has been found to inhibit the uterotonic activity of PGE2. The binding of PGE2 and its inhibition by AnLGE2 has been determined in rat uterine membrane fractions. AnLGE2 inhibited the binding of 3HPGE2 in a dose related fashion. 3HAnLGE2 also binds to rat uterine membrane fractions and its binding is inhibited by PGE2 in a dose related fashion. These data support previous physiological observations that AnLGE2 inhibits the actions of PGE2 by acting at the PGE2 receptor. Thus, AnLGE2 appears to be a specific inhibitor of PGE2 actions at its uterine receptors.     

The role of renal metabolism of PGE2 in determining its activity as a renal vasodilator in the dog.

Since the mammalian renal cortex avidly metabolizes prostaglandin E2 (PGE2), we examined the importance of renal metabolism of PGE2 in determining its renal vascular activity in the dog. We used 13, 14 dihydro PGE2 (DHPGE2) as a model compound to study this because DHPGE2 retains similar activity to the parent prostaglandin, PGE2, but is a poorer substrate than PGE2 for both the metabolism and the cellular uptake of the prostaglandins. Using dog renal cortical slices, we found that under similar experimental conditions, PGE2 was metabolized several-fold faster than DHPGE2. Both prostaglandins were metabolized to the 15 keto 13, 14 dihydro PGE2, which was positively identified using GC-MS. In vivo, we infused increasing concentrations of DHPGE2 into the renal artery of dogs and measured renal hemodynamic changes using radioactive microspheres. DHPGE2 was a potent renal vasodilator beginning at an infusion rate of 10(-9)g/kg/min. When compared to PGE2, DHPGE2 was about 10 times more potent in affecting renal vasodilation. The intrarenal redistribution of blood flow towards the inner cortex seen with DHPGE2 was identical to that seen with PGE2. We conclude that renal catabolism of PGE2 is very important in limiting the in vivo biological activity of PGE2, but regional differences in metabolism of PGE2 within the cortex are an unlikely determinant of the pattern of redistribution of renal blood flow.     

Expression of mouse membrane-associated prostaglandin E2 synthase-2 (mPGES-2) along the urogenital tract

Prostaglandin E(2) (PGE(2)) is the most common prostanoid and has a variety of bioactivities including a crucial role in urogenital function. Multiple enzymes are involved in its biosynthesis. Among 3 PGE(2) terminal synthetic enzymes, membrane-associated PGE(2) synthase-2 (mPGES-2) is the most recently identified, and its role remains uncharacterized. In previous studies, membrane-associated PGE(2) synthase-1 (mPGES-1) and cytosolic PGE(2) synthase (cPGES) were reported to be expressed along the urogenital tracts. Here we report the genomic structure and tissue distribution of mPGES-2 in the urogenital system. Analysis of several bioinformatic databases demonstrated that mouse mPGES-2 spans 7 kb and consists of 7 exons. The mPGES-2 promoter contains multiple Sp1 sites and a GC box without a TATA box motif. Real-time quantitative PCR revealed that constitutive mPGES-2 mRNA was most abundant in the heart, brain, kidney and small intestine. In the urogenital system, mPGES-2 was highly expressed in the renal cortex, followed by the renal medulla and ovary, with lower levels in the ureter, bladder and uterus. Immunohistochemistry studies indicated that mPGES-2 was ubiquitously expressed along the nephron, with much lower levels in the glomeruli. In the ureter and bladder, mPGES-2 was mainly localized to the urothelium. In the reproductive system, mPGES-2 was restricted to the epithelial cells of the testis, epididymis, vas deferens and seminal vesicle in males, and oocytes, stroma cells and corpus luteum of the ovary and epithelial cells of the oviduct and uterus in females. This expression pattern is consistent with an important role for mPGES-2-mediated PGE(2) in urogenital function.