Endogenous prostaglandin production by established cultures of neoplastic rat mammary epithelial cells

Summary The production and release of prostaglandins (PGs) into the growth medium by established cultures of neoplastic, mammary epithelial cells derived from (a)N-nitrosomethyl-urea (NMU)-induced and (b) 7,12-dimethylbenz(a) anthracene (DMBA)-induced mammary tumors, was assessed using radioimmunoassay techniques. Prostaglandin production was determined, to a considerable extent, by in vitro conditions and the tumor line analyzed. In medium supplemented with bovine calf serum (10%), NMU cells synthesized and released nanogram quantities of PGF₂, PGE₁, and PGF_2α (6.7, 4.7, and 1.7 ng/10⁶ cells per 48 h, respectively). Concentrations of the two stable protanoid metabolites, 6-keto-PGF_1α and TXB₂, were indistinguishable from controls. In cells derived from the DMBA-induced tumor (RBA cells), no net production of immunoreactive PGs was detected. In contrast, in media supplemented with fetal bovine serum (10%), both RBA and NMU cells synthesized and released nanogram quantities of PGE₂ (1 and 4 ng/10⁶ cells per 48 h, respectively). PGE₂ production by both NMU and RBA cells was inhibited by ibuprofen, an inhibitor of cyclooxygenase (EC 1. 14. 99.1). The pattern of PG inhibition by ibuprofen differed in the two cell lines. In NMU cells, a linear dose-response inhibitory pattern was discernable, whereas in RBA cells a biphasic pattern was observed; PGE₂ levels incresed at low concentration of ibuprofen and then decreased at higher concentration. At 100 μg/ml ibuprofen, PG synthesis and release was inhibited by 90 and 100% and cell growth by 64 and 66% in NMU and RBA cells, respecively. There was no obvinous dse-response relationship between ibuprofen concentration and cell growth inhibition in either cell line. These results underline the importance of the serum component of growth medium when analyzing PG production in vitro and suggest that the epithelial cell components of experimental mammary tumors are capable of producing physiogically relevant amounts of PGs. This work was supported by Grant CA 29602 from the National Cancer Institute, Bethesda, MD, and Grant PDT-208 from the American Cancer Society.     

In vitro prostaglandin release from and platelet-activating factor accumulation in isolated endometrial cells from pregnant and pseudopregnant rabbits.

Prostaglandin (PG) release from and platelet-activating factor (PAF) accumulation by enzymatically isolated endometrial epithelial and stromal cells from Day 6 pregnant and Day 6 pseudopregnant rabbits were studied in vitro, using RIA for PG measurement and a platelet aggregation assay for PAF measurement. On the first day of culture in serum-free media, PGF release into the medium was significantly higher from epithelial cells from Day 6 of pregnancy than from stromal cells from Day 6 of pregnancy or pseudopregnancy. PGE release did not differ significantly among these cell types. The addition of indomethacin (10(-5) M) to similar cultures inhibited release of both PGs from both cell types, but to a much greater extent from stromal than from epithelial cells. Significant stimulation of PG release by A23187 was achieved under all conditions on the fifth day of culture; PGE release was significantly greater than PGF release from stromal cells from Day 6 of pregnancy and pseudopregnancy, and release of both PGs from stromal cells was significantly greater from Day 6 of pregnancy than from Day 6 of pseudopregnancy. PG release from similar cells, cultured in medium containing 10% calf serum, was highest on the first or second day of culture and then, especially for PGF, declined with continued culture. PGE release was significantly higher than PGF release from stromal cells on the third and fourth days of culture. The ratios of PGF/PGE release from epithelial cells were significantly higher than those from stromal cells over the 5-day culture period for both reproductive stages. These ratios indicate the differential release of PGE and PGF from rabbit endometrial cell subpopulations and indicate a preferential release of PGE from stromal and of PGF from epithelial cells. Under basal conditions, PAF was not detected in epithelial or stromal cells cultured for 2 or 4 days, or in the associated culture media. If PAF had been released into the medium, it would have rapidly metabolized. Short exposure to calcium ionophore A23187 (10(-5) M) was able to stimulate PAF accumulation in epithelial and stroma cells in serum-free media, probably via the remodeling pathway. PAF was not detected in the medium. Intracellular PAF accumulation after exposure to A23187 (10(-5) M) for 5 min was significantly greater on the second day of culture than on the fourth day in epithelial and stromal cells from Day 6 of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of prostaglandins on hypothalamic-pituitary-testicular function in vitro.

The effect of prostaglandins (PG) A1, E1, E2 and F2 alpha in the concentration range of 10(-7)--10(-4) M were studied in vitro on a rat hypothalamic tissue, collagenase-digested isolated anterior pituitary cell and Leydig cell suspension system by measuring the testosterone production of incubated Leydig cells. PGs did not change the testosterone production and the hCG sensitivity of the Leydig cells, nor the LH secretion and the LHRH sensitivity of the anterior pituitary cells. PGE2 at concentrations of 10(-6), 10(-5) and 10(-4) M significantly increased the hypothalamic tissue-induced pituitary-testicular activation, and this stimulatory effect of PGE2 was dose dependent. PGA1, PGE1 and PGF2 alpha did not alter hypothalamic LHRH release measured in vitro. The results suggest that PGE2 has a direct stimulatory effect on hypothalamic LHRH release.     

Gonadotropic regulation of prostaglandin production by ovarian follicular cells of the pig

Prepubertal gilts were treated with 750 IU pregnant mare's serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa cells (GC) and theca interna cells (TC) from follicles of gilts 72 h (GC-72 and TC-72, respectively) and 108 h (GC-108 and TC-108 h, respectively) after PMSG treatment were cultured for 0, 12, 24, and 36 h in medium with or without luteinizing hormone (LH), dibutyryl cyclic adenosine 3',5'-monophosphate [Bu)2cAMP), calcium ionophore (A23187), and/or arachidonic acid (AA), and the production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. TC-72 was the principal source of PGs 72 h after PMSG. At 108 h, the production of PGE and PGF by GC was increased 10- and 30-fold, respectively, whereas corresponding increases by TC were 2-fold. LH and A23187 significantly stimulated PGE and PGF production by both GC-72 and TC-72, but only thecal PG production was stimulated by (Bu)2cAMP. LH had minimal or no effect on PG production by GC-108 and TC-108, but A23187 (GC-108, TC-108) and (Bu)2cAMP (TC-108) were stimulatory. Basal PG production by GC-72, GC-108, and TC-108 was stimulated by AA. However, production by GC and TC cultured in medium containing AA and LH, A23187, or (Bu)2cAMP was not different from that produced by AA alone. These findings suggested that GC and TC can synthesize PGs in vitro, but AA availability is rate-limiting in GC. After exposure to hCG in vivo, the capacity of both cell types to produce PGs is increased but is limited by AA availability.(ABSTRACT TRUNCATED AT 250 WORDS)     

The release of PGF2 alpha and PGE2 from separated cells of human endometrium and decidua

The capacity of separated glandular and stromal cells from endometrium and first trimester decidua to release prostaglandins (PGs) was studied over 48 hours in culture. Glandular preparations released more PGs than stromal preparations in all tissues. Stromal release of PGs did not alter throughout the cycle or in early pregnancy but the capacity of glandular preparations to release PGs varied considerably. Proliferative glands released most PGF2 alpha and PGE2 followed by secretory glands and decidua. Histamine (10(-5)) stimulated PG release from endometrial and decidual glands but the response of proliferative glands was greatest. Actinomycin D stimulated release of PGF2 alpha and PGE2 from glandular cells of secretory endometrium and decidua. These results suggest that in vitro release of PGs is suppressed after ovulation and is in part due to inhibition of PG release by a protein or proteins synthesized in the glandular fraction of secretory endometrium or decidua.     

Prostaglandin production by corpora lutea of rhesus monkeys: characterization of incubation conditions and examination of putative regulators

Prostaglandins (PGs) are produced by the corpus luteum (CL) of many domestic and laboratory species and may play a role in CL regulation. The production of PGs by luteal tissue of the rhesus monkey has yet to be clearly elucidated. The production of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha by CL from rhesus monkeys and the incubation conditions (time and cell number) that permit assessment of their synthesis were examined. CL (n = 3 per characterization) were surgically removed from nonpregnant monkeys during the mid-luteal phase of the menstrual cycle (approximately 8-10 days after ovulation). Luteal tissue was dissociated and the cells were incubated at varying concentrations for increasing periods of time at 37 degrees C. Subsequent to defining incubation conditions, various exogenous factors were examined for their potential to modify PG production. Indomethacin, calcium ionophore, human chorionic gonadotropin (hCG), estradiol-17 beta (E2), progesterone (P), testosterone (T), dihydrotestosterone (DHT), and 1-4-6 androstatriene-3, 17-dione (ATD) were incubated with luteal cells in increasing doses. PG and P concentrations in the medium were determined by radioimmunoassay. PGs in the medium after 6 h incubation were detectable at all cell concentrations tested (50,000, 100,000, 200,000 cells/tube). Concentrations of PGs and P increased with cell number (p less than 0.05). Luteal cells (50,000 cells/tube) were incubated for times of 0-24 h. Concentrations of P, PGE2, and PGF2 alpha in the medium were relatively low prior to incubation (0 h), increased (p less than 0.05) linearly within the first 6-12 h, and plateaued through the remaining 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity of prostaglandin biosynthetic pathways in rat pancreatic islets

Isolated pancreatic islets of the rat were either prelabeled with [3H]arachidonic acid, or were incubated over the short term with the concomitant addition of radiolabeled arachidonic acid and a stimulatory concentration of glucose (17mM) for prostaglandin (PG) analysis. In prelabeled islets, radiolabel in 6-keto-PGF1 alpha, PGE2, and 15-keto-13,14-dihydro-PGF2 alpha increased in response to a 5 min glucose (17mM) challenge. In islets not prelabeled with arachidonic acid, label incorporation in 6-keto-PGF1 alpha increased, whereas label in PGE2 decreased during a 5 min glucose stimulation; after 30-45 min of glucose stimulation labeled PGE levels increased compared to control (2.8mM glucose) levels. Enhanced labelling of PGF2 alpha was not detected in glucose-stimulated islets prelabeled or not. Isotope dilution with endogenous arachidonic acid probably occurs early in the stimulus response in islets not prelabeled. D-Galactose (17mM) or 2-deoxyglucose (17mM) did not alter PG production. Indomethacin inhibited islet PG turnover and potentiated glucose-stimulated insulin release. Islets also converted the endoperoxide [3H]PGH2 to 6-keto-PGF1 alpha, PGF2 alpha, PGE2 and PGD2, in a time-dependent manner and in proportions similar to arachidonic acid-derived PGs. In dispersed islet cells, the calcium ionophore ionomycin, but not glucose, enhanced the production of labeled PGs from arachidonic acid. Insulin release paralleled PG production in dispersed cells, however, indomethacin did not inhibit ionomycin-stimulated insulin release, suggesting that PG synthesis was not required for secretion. In confirmation of islet PGI2 turnover indicated by 6-keto-PGF1 alpha production, islet cell PGI2-like products inhibited platelet aggregation induced by ADP. These results suggest that biosynthesis of specific PGs early in the glucose secretion response may play a modulatory role in islet hormone secretion, and that different pools of cellular arachidonic acid may contribute to PG biosynthesis in the microenvironment of the islet.     

Rat mammary preadipocytes in culture produce a trophic agent for mammary epithelia-prostaglandin E2

Cell strains and cell lines rat mammary (Rama) 350-353 have been isolated from the slowly adherent stromal fraction of enzymatically digested rat mammary glands. Primary cultures of this fraction yield fat cels on extended culture. Their proportion can be increased with horse serum or growth hormone in the medium, and this increase is associated with a 100-fold or more increase in the release of radioimmunoassayable prostaglandins of the E type (PGE). The stromal cell strains and lines that are capable of yielding fat cells also secrete elevated levels (greater than 100 ng/mg/24 hr) of PGE; the fast-sticking epithelial fraction in primary cultures and the epithelial cell lines derived from it secrete 10-100 times less. Chromatography and radioisotopic labeling of the culture media from Rama 352 cells identify the PG as PGE2. PGE2 with insulin and hydrocortisone maximally stimulates [3H]DNA synthesis of epithelial cell lines and primary cultures from normal and tumorous glands by 2-4-fold at concentrations (10-20 ng/ml) well below those released by the preadipocytic stromal cells (20-100 ng/ml). Medium exposed to most cultured cells stimulates [3H]DNA synthesis of one epithelial cell line, Rama 25, by 2-4-fold. Prevention of the synthesis of PGE2 in Rama 352 cultures with indomethacin or flurbiprofen abolishes the mitogenic activity present in the culture medium, and the PG receptor antagonist polyphloretin phosphate inhibits completely the mitogenic activity for Rama 25 cells. Myoepithelial-like cell lines normally secrete moderate levels of PGE (10-100 ng/mg/24 hr) but the mitogenic activity for Rama 25 cells released from one such line, Rama 29, is not abolished by preventing the synthesis of PG's nor by PG-receptor antagonists.     

Radioimmunoassay measurement of ACTH-facilitated PGE2 and PGF2alpha release from isolated cat adrenocortical cells.

Prostaglandin (PG) biosynthesis by trypsin-dispersed cat adrenocortical cells was studied by radioimmunoassay (RIA). Parallel assays of incubation media using PGF2alpha and PGF1alpha antisera established that PGF2alpha is the primary PGF released by feline cortical cells. Following the reduction of PGE to PGF with sodium borohydride (NaBH4) these same two antisera were also used to identify PGE2 as the primary PGE released. RIA using a PGE antiserum confirmed the presence of PGE in the incubation medium. Steroidogenic concentrations of ACTH (50-250muU) enhanced PGE and PGF release, and indomethacin suppressed the ACTH-facilitated release. These studies provide additional evidence for ACTH-induced PG synthesis by feline cortical cells, and support the hypothesis that PGs play some role in the steroidogenic action of ACTH.     

Effects of prostaglandin E2 and lipopolysaccharide on osteoclastogenesis in RAW 264.7 cells

INTRODUCTION: Prostaglandins (PGs) can act on both hematopoietic and osteoblastic lineages to enhance osteoclast formation. METHODS: We examined PGE2 stimulated osteoclastogenesis in RAW 264.7 cells and the role of endogenous PGE2 in lipopolysaccharide (LPS) stimulated osteoclastogenesis. RESULTS: RANKL (1-100 ng/ml) increased formation of osteoclasts, defined as tartrate resistant acid phosphatase multinucleated cells, with peak effects at 30 ng/ml. Addition of PGE2 (0.01-1.0 microM) to RANKL (30 ng/ml) dose dependently increased osteoclast number 30-150%. Use of NS-398 (0.1 microM) or indomethacin (Indo, 1.0 micro M) to block endogenous PG synthesis had little effect on the response to RANKL alone but significantly decreased the response to PGE2. Addition of LPS (100 ng/ml) to RANKL increased osteoclast number 50%, and this response was significantly decreased by NS-398 and Indo. RANKL and PGE2 produced small, additive increases in COX-2 mRNA levels, while LPS produced a larger increase. PG release into the medium was not increased by RANKL and PGE2 but markedly increased by LPS. CONCLUSION: We conclude that RANKL stimulated osteoclastogenesis can be enhanced by PGE2 and LPS though direct effects on the hematopoietic cell lineage and that these effects may be mediated in part by induction of COX-2 and enhanced intracellular PG production.     

Uterine and placental prostaglandins and their modulation of oxytocin sensitivity and contractility in the parturient uterus

The parturient uterus develops a markedly enhanced sensitivity to the uterotonic action of oxytocin (OT). The mechanism leading to this enhanced OT sensitivity is not known. Our previous work suggested that prostaglandins (PGs) may be involved. To define the relationship between OT sensitivity and uterine PG production, we measured uterine sensitivity to OT by a quantitative dose-response procedure in rats on Days 19, 20, 21 and 22 of pregnancy and monitored uterine and placental tissue concentrations of PGF2 alpha and PGE2. In addition, we determined the effects of inhibition of endogenous PG synthesis on OT sensitivity and uterine contractility. We found that both OT sensitivity and spontaneous contractility are positively related to uterine PGF2 alpha production. An abrupt increase in OT sensitivity was observed on Days 21 and 22 of pregnancy. The increase in OT sensitivity was coincidental with the marked increase in PGF2 alpha production in the uterus on Days 21 and 22 of pregnancy. Suppression of in vivo PG synthesis caused a reduction in both spontaneous uterine contractility and OT-induced contractions. Uterine PGE2 concentrations and release were 3-5 times lower than PGF2 alpha. There were no significant fluctuations of uterine PGE2 concentration measured on these last 4 days of gestation. Placental PG levels were also found not to be related to uterine contractility. Placental PGE2 levels were higher than PGF2 alpha and may play a regulatory role in placental perfusion. However, placental PGs did not vary with gestational age.     

Prostaglandin release by zygotes and endometria of pregnant rabbits

When 4-day rabbit zygotes were incubated for 1 h at 37 degrees C in vitro, very little prostaglandin (PG) was released into the medium, and the concentration of PGs in the zygotes after incubation was also low. The release of prostaglandin E (PGE) and prostaglandin F (PGF) into the medium, and their concentration in the zygotes after incubation, increased sharply on Days 6 and 7 of pregnancy, reaching, by Day 7, values close to 200 ng of each PG released in 1 h per mg of protein. By contrast, endometrial samples on Days 4 and 5 of pregnancy released more PGF and less PGE than the zygotes of the same ages on a per mg of protein basis, and on Days 6 and 7, less of both PGs. Furthermore, endometrial concentrations of PGs after incubation, except for PGF on Day 4, were always lower than values for zygotes. Endometrial concentrations of PGs on Day 6 were lower before than after incubation. Although there was a slight upward trend in PG release by endometrial samples with increasing length of pregnancy, the changes were minimal and, in the case of PGE, none of the mean values exceeded 1 ng per mg of protein. In 7-day blastocysts, high levels of both PGF and PGE were found in the blastocoelic fluid, and these did not change during the 1-h incubation. The release of PGF and PGE during in vitro incubation of ruptured and washed Day 6 blastocysts was stimulated by arachidonic acid, and that of PGF, but not PGE, inhibited by indomethacin. The release of PGE, but not of PGF, from Day 6 blastocysts was inhibited by low temperature, and the same conditions inhibited release of both PGF and PGE from endometrial cell suspensions. It seems that both blastocysts and endometria have capability to synthesize PGs, the blastocysts being particularly active in this regard on Days 6 and 7 of pregnancy. It is hypothesized that, in vivo, Day 6 and 7 blastocysts release large quantities of PGs which trigger some of the local endometrial changes associated with pregnancy.     

Glycerylprostaglandin synthesis by resident peritoneal macrophages in response to a zymosan stimulus

Cyclooxygenase (COX)-2 oxygenates arachidonic acid (AA) and 2-arachidonylglycerol (2-AG) to endoperoxides, which are subsequently transformed to prostaglandins (PGs) and glycerylprostaglandins (PG-Gs). PG-G formation has not been demonstrated in intact cells treated with a physiological agonist. Resident peritoneal macrophages, which express COX-1, were pretreated with lipopolysaccharide to induce COX-2. Addition of zymosan caused release of 2-AG and production of the glyceryl esters of PGE2 and PGI2 over 60 min. The total quantity of PG-Gs (16 +/- 6 pmol/10(7) cells) was much lower than that of the corresponding PGs produced from AA (21,000 +/- 7,000 pmol/10(7) cells). The differences in PG-G and PG production were partially explained by differences in the amounts of 2-AG and AA released in response to zymosan. The selective COX-2 inhibitor, SC236, reduced PG-G and PG production by 49 and 17%, respectively, indicating a significant role for COX-1 in PG-G and especially PG synthesis. Time course studies indicated that COX-2-dependent oxygenation rapidly declined 20 min after zymosan addition. When exogenous 2-AG was added to macrophages, a substantial portion was hydrolyzed to AA and converted to PGs; 1 microm 2-AG yielded 820 +/- 200 pmol of PGs/10(7) cells and 78 +/- 41 pmol of PG-Gs/10(7) cells. SC236 reduced PG-G and PG production from exogenous 2-AG by 88 and 76%, respectively, indicating a more significant role for COX-2 in the utilization of exogenous substrate. In conclusion, lipopolysaccharide-pretreated macrophages produce PG-Gs from endogenous 2-AG during zymosan phagocytosis, but PG-G formation is limited by substrate hydrolysis and inactivation of COX-2.     

Tumor necrosis factor stimulates prostaglandin production and cyclic AMP levels in rat cultured mesangial cells

Human recombinant tumor necrosis factor-alpha (TNF) was found to stimulate the production of prostaglandins (PG) by cultured rat mesangial cells. This effect was demonstrable from 6 h, was dose dependent and affected the synthesis of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. It required both RNA and protein synthesis but was not associated with a modification of cell proliferation. TNF also stimulated adenosine 3'-5' cyclic monophosphate (cAMP) levels in the mesangial cell culture medium. Indomethacin suppressed the effect of TNF on PGs but only reduced that on cAMP, indicating that PG production partly mediates the increase in cAMP. These findings demonstrate that mesangial cells can be a target for TNF and that the mechanism of TNF action includes stimulation of both PG production and cAMP levels.     

Stimulatory and inhibitory effects of prostaglandins on the gonadotropin-sensitive adenylate cyclase in the monkey corpus luteum

Detailed analysis of the action of prostaglandins (PGs) on the corpus luteum in primate species is very limited. In this study we examined the response of the adenylate cyclase system to PGs in homogenates prepared from the corpus luteum of rhesus monkeys at midluteal phase of the menstrual cycle. The conversion of [alpha 32p] ATP to [32p] cyclic AMP (cAMP) was assessed in the absence (control activity; 50 microM GTP) and presence of various concentrations of seven PGs and arachidonic acid, either alone or in combination with 250 nM hCG. Cyclic AMP production increased up to three-fold in the presence of PGD2, PGE2, PGI2 or PGF2 alpha; however PGA2, PGB2, 13, 14-dihydro-15-keto PGE2 and arachidonic acid alone did not alter cAMP levels. In dose-response studies, adenylate cyclase was 10 and 100-fold more sensitive to PGD2 (Vmax at 1 X 10(-5) M) than to PGE2 or to PGI2 and PGF2 alpha, respectively. Activity in the presence of hCG plus either PGD2, PGE2, PGI2 or PGF2 alpha did not differ from that for hCG (or the PG) alone. In contrast, addition of PGA2 or arachidonate inhibited (p less than 0.05) hCG-stimulated cAMP production by 50 and 100 percent. We conclude that the gonadotropin-sensitive adenylate cyclase of the macaque corpus luteum is also modulated by several PGs. These factors may either mimic (e.g., PGD2, PGE2, PGI2) or suppress (PGA2) gonadotropin-stimulated cAMP production and possibly cAMP-mediated events in luteal cells.     

Factors regulating the production of prostaglandin E2 and prostacyclin (prostaglandin I2) in rat and human adipocytes.

The regulation of PGE2 (prostaglandin E2) and PGI2 (prostaglandin I2; prostacyclin) formation was investigated in isolated adipocytes. The formation of both PGs was stimulated by various lipolytic agents such as isoproterenol, adrenaline and dibutyryl cyclic AMP. During maximal stimulation the production of PGE2 and PGI2 (measured as 6-oxo-PGF1 alpha) was 0.51 +/- 0.04 and 1.21 +/- 0.09 ng/2 h per 10(6) cells respectively. Thus PGI2 was produced in excess of PGE2 in rat adipocytes. The production of the PGs was inhibited by indomethacin and acetylsalicylic acid in a concentration-dependent manner. The half-maximal effective concentration of indomethacin was 328 +/- 38 nM and that of acetylsalicylic acid was 38.5 +/- 5.3 microM. The PGs were maximally inhibited by 70-75% after incubation for 2 h. In contrast with their effect on PG production, the two agents had a small potentiating effect on the stimulated lipolysis (P less than 0.05). The phospholipase inhibitors mepacrine and chloroquine inhibited both PG production and triacylglycerol lipolysis and were therefore unable to indicate whether the PG precursor, arachidonic acid, originates from phospholipids or triacylglycerols in adipocytes. Angiotensin II significantly (P less than 0.05) stimulated both PGE2 and PGI2 production in rat adipocytes without affecting triacylglycerol lipolysis. Finally, it was shown that PGE2 and PGI2 were also produced in human adipocytes, although in smaller quantities than in rat adipocytes. It is concluded that the production of PGs in isolated adipocytes is regulated by various hormones. Moreover, at least two separate mechanisms for PG production may exist in adipocytes: (1) a mechanism that is activated concomitantly with triacylglycerol lipolysis (and cyclic AMP) and (2) an angiotensin II-sensitive, but lipolysis (and cyclic AMP)-independent mechanism.     

Radioimmunoassay measurement of ACTH-facilitated PGE2 and PGF2α release from isolated cat adrenocortical cells

Prostaglandin (PG) biosynthesis by trypsin-dispersed cat adrenocortical cells was studied by radioimmunoassay (RIA). Parallel assays of incubation media using PGF_2α and PGF_1α antisera established that PGF_2α is the primary PGF released by feline cortical cells. Following the reduction of PGE to PGF with sodium borohydride (NaBH₄) these same two antisera were also used to identify PGE₂ as the primary PGE released. RIA using a PGE antiserum confirmed the presence of PGE in the incubation medium. Steroidogenic concentrations of ACTH (50–250μU) enhanced PGE and PGF release, and indomethacin suppressed the ACTH-facilitated release. These studies provide additional evidence for ACTH-induced PG synthesis by feline cortical cells, and support the hypothesis that PGs play some role in the steroidogenic action of ACTH.     

Progesterone reduces erectile dysfunction in sleep-deprived spontaneously hypertensive rats

Background

The rate-limiting step in prostaglandin (PG) biosynthesis is catalyzed by phospholipase A2 (PLA2) enzymes which hydrolyze arachidonic acid from membrane phospholipids. Despite their importance in uterine PG production, little is known concerning the specific PLA2 enzymes that regulate arachidonic acid liberation in the uterine endometrium. The objectives of this study were to evaluate the expression and activities of calcium-independent Group VI and Group IVC PLA2 (PLA2G6 and PLA2G4C) and calcium-dependent Group IVA PLA2 (PLA2G4A) enzymes in the regulation of bovine uterine endometrial epithelial cell PG production.

Methods

Bovine endometrial epithelial cells in culture were treated with oxytocin, interferon-tau and the PLA2G6 inhibitor bromoenol lactone, alone and in combination. Concentrations of PGF2alpha and PGE2 released into the medium were analyzed. Western blot analysis was performed on cellular protein to determine the effects of treatments on expression of PLA2G4A, PLA2G6 and PLA2G4C. Group-specific PLA2 activity assays were performed on cell lysates following treatment with oxytocin, interferon-tau or vehicle (control), alone and in combination. To further evaluate the role of specific PLA2 enzymes in uterine cell PG biosynthesis, cells were transfected with cDNAs encoding human PLA2G6 and PLA24C, treated as described above and PG assays performed.

Results

Constitutive cell production of PGF2alpha was about two-fold higher than PGE2. Oxytocin stimulated production of both PGs but the increase of PGF2alpha was significantly greater. Interferon-tau diminished oxytocin stimulation of both PGs. The PLA2G6 inhibitor, bromoenol lactone, abolished oxytocin-stimulated production of PGF2alpha. Treatments had little effect on PLA2G4A protein expression. In contrast, oxytocin enhanced expression of PLA2G6 and this effect was diminished in the presence of interferon-tau. Expression of PLA2G4C was barely detectable in control and oxytocin treated cells but it was enhanced in cells treated with interferon-tau. Oxytocin stimulated PLA2 activity in assays designed to evaluate PLA2G6 activity and interferon-tau inhibited this response. In assays designed to measure PLA2G4C activity, only interferon-tau was stimulatory. Cells overexpressing PLA2G6 produced similar quantities of the two PGs and these values were significantly higher than PG production by non-transfected cells. Oxytocin stimulated production of both PGs and this response was inhibited by interferon-tau. Bromoenol lactone inhibited oxtocin stimulation of PGF2alpha production but stimulated PGE2 production, both in the absence and presence of oxytocin. Cells over-expressing PLA2G4C produced more PGE2 than PGF2alpha and interferon-tau stimulated PGE2 production.

Conclusion

Results from these studies indicate that oxytocin stimulation of uterine PGF2alpha production is mediated, at least in part, by up-regulation of PLA2G6 expression and activity. In addition to its known inhibitory effect on oxytocin receptor expression, interferon-tau represses oxytocin-stimulated PLA2G6 expression and activity and this contributes to diminished PGF2alpha production. Furthermore, endometrial cell PGE2 biosynthesis was associated with PLA2G4C expression and activity and interferon-tau was stimulatory to this process.     

Mechanical injury increases eicosanoid production in cultured cardiomyocytes.

The release of three stable metabolites of the arachidonic acid cascade was determined in cultures of cardiac myocytes and of non-muscle cells. In both cell types, the main product was 6-keto-PGF1 alpha much less PGE2 was released, while TXB2 was only detected in muscle cells. Preincubation with arachidonic acid increased the release of all the PGs in both types of culture. Mechanical injury had a synergistic effect on the increased PG release in AA-preincubated cells. However, TXB2 was not detected in F-cells in any experimental conditions. These results suggest that PG production serves a functional role in heart preservation during injury.