PGF2 Alpha Synthesis Research Articles

Isoprostane 8-epi PGF2alpha, a product of oxidative stress, is synthesized in the bladder and causes detrusor smooth muscle contraction.

Isoprostane 8-epi PGF2alpha is a product of oxidative stress that causes potent smooth muscle contraction. Its production increases in conditions associated with oxidative stress such as in diabetes, smoking, and aging. The aim was to study whether the urinary bladder synthesizes isoprostane 8-epi PGF2alpha and releases to the urine and whether isoprostane 8-epi PGF2alpha causes bladder smooth muscle contraction. Urine samples were obtained transurethrally from 12 male New Zealand white rabbits for measurement of isoprostane 8-epi PGF2alpha levels. To examine whether bladder synthesizes isoprostane 8-epi PGF2alpha, both ureters were ligated, then the bladder was washed 5 times by filling and emptying with normal saline. Bladder was refilled with normal saline, and at 5 minutes a bladder washout sample was taken. After this, the bladder was contracted by nerve stimulation periodically for 30 minutes, and then another washout sample was taken. Strips of bladder tissues were processed for study of isoprostane 8-epi PGF2alpha production in tissue culture chambers and for isometric tension measurements in the organ bath. Enzyme immunoassay (EIA) revealed a remarkable amount of isoprostane 8-epi PGF2alpha in the rabbit urine. EIA of washout samples showed that the bladder synthesizes isoprostane 8-epi PGF2alpha and its production increases with nerve stimulation-induced contractions. EIA of samples from the tissue culture media showed that bladder strips synthesize isoprostane 8-epi PGF2alpha in vitro. Electrical field stimulation (EFS) significantly increased the synthesis and release of isoprostane 8-epi PGF2alpha by the bladder strips. In the organ bath, isoprostane 8-epi PGF2alpha caused concentration-dependent contraction of bladder tissue. While the threshold contraction required smaller concentration of isoprostane 8-epi PGF2alpha (3 nmol) than carbachol (10 nmol), the amplitude of contraction to carbachol was greater than isoprostane 8-epi PGF2alpha. Our studies show that the rabbit bladder synthesizes isoprostane 8-epi PGF2alpha and releases it to the urine. Production of isoprostane 8-epi PGF2alpha in the bladder increases with nerve stimulation-induced contraction. Exogenous isoprostane 8-epi PGF2alpha causes significant bladder smooth muscle contraction. Our findings necessitate further studies to evaluate the possible role of oxidative stress and increased isoprostane 8-epi PGF2alpha production in bladder dysfunction. Neurourol. Urodynam. 19:43-51, 2000.

Paracrine regulation of insulin-like growth factor I (IGF-I) an IGF-II on prostaglandins F2alpha and E2 synthesis by human corpus luteum in vitro: a possible balance of luteotropic and luteolytic effects.

The existence of a complete intraovarian insulin-like growth factor (IGF) system replete with ligands, receptors, and binding proteins has been demonstrated as well as the ability of IGF-I to positively affect steroidogenesis in human granulosa cells. Furthermore, we recently showed that IGF-I and IGF-II stimulate progesterone secretion by human luteal cells. As the PGs, PGE2 and PGF2alpha, are classically known to have luteotropic and luteolytic effects, we wanted to determine whether the IGFs could affect the human luteal phase by influencing the PG system. For this reason, human luteal cells were cultured for different times (12, 24, and 48 h) with IGF-I, IGF-II (10-100 ng/mL), and GH (100 ng/mL), and both PGs were assayed in the medium culture. We found that both IGF-I and IGF-II were able to stimulate PGE2 synthesis in a time- and dose-dependent way, whereas they both inhibited PGF2alpha production. GH, too, significantly reduced PGF2alpha synthesis; this effect was IGF-I mediated because it was reverted by increasing dilutions of an anti-IGF-I antibody. On the contrary, no GH effect was observed on PGE2 production. In conclusion, based on these data and on our previous results, we speculate that IGFs could influence luteal steroidogenesis through PG system.

Oxytocin- and aluminium fluoride-induced phospholipase C activity and prostaglandin F2 alpha secretion during the ovine luteolytic period.

A series of studies was conducted to characterize changes in components of the cell signalling cascade that mediates oxytocin-induced prostaglandin F2 alpha (PGF2 alpha) synthesis at the onset of luteolysis in sheep. In the first experiment, caruncular tissue was dissected from 20 ewes on days 12-15 of the oestrous cycle, and incubated for the measurement of phospholipase C (PLC) activity or secretion of PGF2 alpha. Activation of GTP-binding proteins with aluminium fluoride stimulated both inositol phosphate accumulation and PGF2 alpha secretion on all days examined. However, oxytocin did not stimulate PLC activity or PGF2 alpha accumulation until day 13. While the ability of oxytocin to stimulate PLC activity increased after day 13, oxytocin-induced PGF2 alpha secretion declined slightly from day 13 to 15, suggesting that cell signalling components downstream from PLC modulate the response to oxytocin after day 13. Oxytocin failed to stimulate PGF2 alpha synthesis on day 14 after oestrus. Secretion of endogenous luteal oxytocin may have rendered uterine tissues collected on day 14 refractory to oxytocin in vitro. Therefore, a second study was conducted in ovariectomized, steroid replaced ewes. Ovarian steroids were administered to mimic endogenous changes in progesterone and oestradiol. The temporal patterns of PGF2 alpha synthesis in response to oxytocin and pharmacological agents were similar to uterine tissues from cyclic ewes in the first experiment; however, the magnitude of the response was less. These data suggest that oxytocin receptors are absent or are not coupled to PLC until day 13 after oestrus.

Regulatory role of eicosanoids in extracellular matrix overproduction induced by long-term exposure to high glucose in cultured rat mesangial cells.

Accumulation of extracellular matrix in the mesangium and altered renal eicosanoid synthesis are two prominent features of diabetic glomerular disease. We investigated the relationship between eicosanoid and extracellular matrix production in rat mesangial cells cultured under high glucose vs normal glucose conditions. Long-term exposure of rat mesangial cells to high glucose, but not to iso-osmolar mannitol, significantly increased extracellular matrix accumulation and gene expression and transforming growth factor-beta (TGF-beta) mRNA levels, and decreased prostaglandin (PG) E2 synthesis without affecting production of either thromboxane (TX) B2 or PGF2 alpha, with respect to cells incubated in normal glucose. Addition of exogenous PGE2 resulted in a dose-dependent reduction of matrix protein and mRNA levels and TGF-beta gene expression in cells cultured in either normal or high glucose conditions, whereas exposure to exogenous PGF2 alpha produced a significant increment in matrix production and matrix and TGF-beta gene expression in cells grown in normal glucose, but only a slight increase in those cultured in high glucose. Stimulation of endogenous endoperoxide metabolism towards PGE2 and PGF2 alpha synthesis with FCE-22,178, a drug originally developed as TXA2 synthase inhibitor, resulted in a dose-dependent decrease in matrix accumulation and matrix and TGF-beta gene expression which was suppressed by coincubation with the cyclo-oxygenase inhibitor fenoprofen blocking the FCE-22,178-enhanced PG production. In both cell lines, the rate of synthesis of TXA2 was very low and the selective blockade of its synthesis (by two other TXA2 synthase inhibitors, OKY-046 and Ridogrel) or action (by the TXA2 receptor antagonist BM-13,177) did not alter matrix production or TGF-beta mRNA levels. These results suggest that the cyclo-oxygenase pathway is involved in the regulation of matrix changes induced by high glucose in rat mesangial cells; the reduced production of PGE2 may enhance the synthesis or potentiate the effect of stimulators of ECM formation such as TGF-beta, whereas TXA2 does not appear to be involved. These data also indicate that glucose-enhanced mesangial matrix accumulation may be prevented by exogenous PGE2 or by drugs capable of increasing endogenous PGE2 synthesis.

Metabolic alterations of zinc and prostaglandins in both human and animal colonic tumor cells.

To understand the relationship between zinc and prostaglandin (PG) metabolisms in inducing colon cancer incidence in human and animals. Human colonic tumor and normal cells were obtained from Departments of Surgery and Pathology at the Kaiser Permanente Medical Center, Los Angeles, CA and US VA Medical Center, North Hills, CA. Rat colonic tumor and normal cells were isolated from the rats that received two injections of 50 mg/kg of Azoxymethan (AOM) in 2 weeks and then kept 30 weeks in the animal facility. Then, the effects of zinc on the PGE2 synthesis and PGE2 on zinc metabolism in tumor and normal cells were determined. PGE2 concentrations in both human and AOM-induced rat colonic tumor cells increased compared to those in adjacent normal colonocytes, whereas PGF2 alpha concentrations did not change. Gene expression of inducible form of prostaglandin synthase (PGS-2) is stimulated in rat colonocytes by epidermal growth factor and by tetradecanoyl 13-phorbol acetate (a tumor promoter and mitogen) only in the presence of zinc. PGE2 binding activity of rat enterocytes was maximum at 15 microM of zinc (normal plasma zinc concentration), but PGE2 synthesis activity increased for the first 15 minutes when extracellular zinc concentrations were either higher or lower than the normal extracellular zinc concentration. However, variations in extracellular zinc concentrations did not change the rate of PGF2 alpha synthesis in the normal rat enterocytes. PGE2 significantly increased zinc uptake rates of colonic tumor cells but PGF2 alpha showed only moderate effect. These results suggest that zinc is required for PGS-2 gene expression, that maintaining an optimal zinc nutriture is important for normal PG synthesis of intestinal cells, and that only PGE2 synthesis mechanisms rather than PGS-2 gene expression are altered in colonic tumor cells resulting in the abnormal zinc nutriture of these cells.

Modulation of prostaglandin synthesis in mammalian sperm acrosome reaction.

Exogenous arachidonic acid induces the acrosome reaction and the production of the prostaglandins PGE2 and PGF2 alpha in bovine spermatozoa. Exogenous PGE2 also induces the acrosome reaction and PGF2 alpha synthesis. To understand better the role of PGE2 in the induction of PGF2 alpha synthesis through modulation of phospholipase A2, inhibitors of this enzyme were used. The effects of PGE2 were blocked by phospholipase A2 inhibitors and this inhibition was reversed by addition of arachidonic acid. These data indicate that PGE2 activates phospholipase A2 to produce arachidonic acid. To determine whether protein kinase C modulates phospholipase A2 activity in this process, staurosporin, an inhibitor of protein kinase C, was used. The effect of PGE2 on PGF2 alpha production is inhibited by staurosporin and this inhibition was reversed by addition of arachidonic acid indicating that protein kinase C is involved in phospholipase A2 activation. The effect of exogenous arachidonic acid or PGE2 on the acrosome reaction is blocked by lipoxygenase inhibitors but not by inhibitors of cyclo-oxygenase, indicating that lipoxygenase products are involved in the mechanism of the acrosome reaction. The presented data shed light on the cross-talk between cyclo-oxygenase and lipoxygenase and their involvement in the sperm acrosome reaction. It is suggested that cyclo-oxygenase products modulate the activity of lipoxygenase which is a key enzyme in the mechanism leading to the acrosome reaction. Stimulation of cyclo-oxygenase to synthesize PGE2 activates phospholipase A2 to release arachidonic acid which is the substrate for lipoxygenase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 beta induces cyclooxygenase-2 in cultured human decidual cells.

The purpose of this study was to examine the hypothesis that interleukin-1 beta (IL-1 beta)-elicited increases in decidual prostaglandin E2 and F2 alpha (PGE2 and PGF2 alpha) biosynthesis are due to the de novo expression of the inducible isoform of cyclooxygenase (i.e., COX-2). Primary human decidual cell cultures were established from term placentas delivered by cesarean section. After 8 days in vitro, when the cultures secreted immunoreactive prolactin, the cells were incubated for 24 h in serum-free medium, and then challenged with IL-1 beta from 1 to 48 h. PGE2 and PGF2 alpha content in the media were measured by specific radioimmunoassays. IL-1 beta stimulated a time-dependent enhancement in PGE2 and PGF2 alpha production, with PGF2 alpha synthesis predominating over PGE2. IL-1 beta also induced a dose-dependent increase in the output of both arachidonic acid metabolites. When Northern blots of IL-1 beta-treated and control cells were probed with cDNAs encoding either COX-1 or COX-2 isoforms or an oligonucleotide probe encoding a portion of the human beta-actin, we detected a time- and dose-dependent increase in the steady-state levels of COX-2, but not COX-1 or beta-actin mRNA transcripts. Moreover, the expression of COX-2 mRNA in IL-1 beta-stimulated cells was superinduced by preincubation with cycloheximide, but completely abolished by actinomycin D. Taken together, the data suggest that COX-2 mRNA expression is largely responsible for the robust increase in PG formation seen in IL-1 beta-treated decidual cells.

Eicosanoid synthesis in cardiomyocytes: influence of hypoxia, reoxygenation, and polyunsaturated fatty acids

The synthesis of eicosanoids was investigated in cultured rat ventricular myocytes. Under normoxia, the cardiomyocytes released 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) and prostaglandin (PG) E2 and smaller amounts of PGF2 alpha and thromboxane B2. Hypoxia enhanced the production of PGE2 and PGF2 alpha, whereas the synthesis of 6-keto-PGF1 alpha was not affected. Conversely, posthypoxic reoxygenation greatly increased the synthesis of 6-keto-PGF1 alpha, whereas the synthesis of PGF2 alpha, was not affected and that of PGE2 was reduced. The cardiomyocyte polyunsaturated fatty acid (PUFA) profile was altered by arachidonic acid or eicosapentaenoic acid and docosahexaenoic acid. Under normoxia, the eicosanoid production appeared to be roughly related to the cell phospholipid arachidonic acid content. Conversely, during posthypoxic reoxygenation, the production of eicosanoids was related to the cell phospholipid n-3 PUFA content, with the n-3-rich cells displaying a marked inhibition of the synthesis. This inhibition was mainly attributed to eicosapentaenoic acid and/or docosapentaenoic acid. Whether this inhibition occurs in vivo during postischemic reperfusion, it may contribute to the beneficial effect of n-3 PUFA on the heart.

Effects of Brief and Prolonged Ischemia on Eicosanoid Synthesis in Dog and Rat Hearts

We compared the effects of brief and prolonged myocardial ischemia on tissue prostaglandin synthesis. Regional ischemia was induced in dogs and maintained for 5 or 30 minutes. Isolated rat hearts were subjected to global ischemia in a working heart preparation for periods of 5 or 20 minutes. De novo synthesis of prostaglandins PGE2 and PGF2 alpha was measured in endocardial and epicardial explants from hearts subjected to transient ischemia. After 5 minutes of coronary ligation, PGE2 production by the ischemic canine endocardium increased by 68%, compared with non-ischemic tissue, and PGF2 alpha levels rose by 29%. In isolated rat hearts, 5-minute ischemia increased endocardial rate of PGE2 synthesis (13.2 +/- 1.7 pmol/g/h, as compared with 6.7 +/- 0.5 under normoxic conditions). Ischemic stimulation of eicosanoid production by the endocardium was no longer apparent after 30-minute regional or 20-minute global ischemia. In contrast, the epicardium showed significant changes only after 30-minute ischemia and only with respect to PGF2 alpha (12.0 +/- 6.2 in the ischemic vs 7.1 +/- 3.2 pmol/g/h in the non-ischemic tissue). Ischemia-induced differences in prostaglandin production were attenuated in the presence of arachidonic acid or aspirin.

The effect of caffeine on prostaglandin output from the guinea-pig uterus.

1. Caffeine increased the outputs of prostaglandin F2 alpha (PGF2 alpha), PGE2 and 6-keto-PGF1 alpha from the guinea-pig uterus on days 7 and 15 of the oestrous cycle. The effect on PGE2 output depended on the age of the animals and was absent in younger guinea-pigs (< 4 months). Theophylline also stimulated the outputs of PGF2 alpha and 6-keto-PGF1 alpha, but not the output of PGE2, from the day 7 guinea-pig uterus. 2. The stimulatory effects of caffeine on the outputs of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha from the guinea-pig uterus were not prevented by lack of extracellular calcium, ryanodine or ruthenium red (both inhibitors of calcium release via the ryanodine receptor), although the increase in PGF2 alpha output tended to be slower when extracellular calcium was absent. Also, ryanodine flattened and broadened the peak of increased PGF2 alpha release. 3. The calmodulin antagonists, W-7 and trifluoperazine, had no inhibitory effect on the caffeine-stimulated increases in uterine prostaglandin output. In fact, W-7 (but not trifluoperazine) greatly potentiated the action of caffeine on uterine PGF2 alpha output, but had little or no potentiating effect on the action of caffeine on uterine PGE2 and 6-keto-PGF1 alpha outputs. 4. TMB-8, an intracellular calcium antagonist, inhibited the increase in PGF2 alpha output produced by caffeine without preventing the increases in outputs of PGE2 and 6-keto-PGF1 alpha. 5. These studies suggest that caffeine stimulates uterine PGF2 alpha synthesis and release by a mechanism dependent upon intracellular calcium, but this mechanism is not mediated by activation of any of the three well-characterized ryanodine receptors or by calmodulin. Furthermore, the increases in the synthesis and release of PGE2 and 6-keto-PGFI alpha. in the guinea-pig uterus induced by caffeine appear to involve mechanism(s) different from that which stimulates PGF2 alpha production.

Effects of oestrogen on progesterone synthesis and arachidonic acid metabolism in human luteal cells.

Locally produced oestrogens and prostaglandins (PGs) are implicated in the regulation of luteal lifespan in the human ovary. This study (1) assesses direct effects of these factors on progesterone synthesis in isolated luteal cells, and (2) explores interactions between luteal age and treatment with gonadotrophin or oestrogen on the metabolism of arachidonic acid (prostaglandin precursor) by steroidogenic luteal cells in vitro. Primary monolayer cultures of human luteal cells obtained at different stages of the luteal phase were used to investigate the effect of oestradiol, catechol oestrogens (2- and 4-hydroxyoestradiol), diethylstilboestrol, PGE2 and PGF2 alpha on basal and human chorionic gonadotrophin (hCG) stimulated progesterone production in vitro. The role of PGs as modulators of luteal cell function was further investigated by studying the metabolic fate of radioactively labelled arachidonic acid in hormone treated (oestradiol and hCG) and control cultures, assessed by high performance liquid chromatography. Corpora lutea were enucleated from nine women with regular ovulatory cycles undergoing microsurgical reversal of tubal sterilization. Granulosa cell aspirates were obtained from three patients undergoing in-vitro fertilization treatment. PGE2 and PGF2 alpha at various concentrations did not have a consistent effect, whereas oestradiol, diethylstilboestrol (and 2-hydroxyoestradiol in early luteal cell cultures) significantly inhibited basal and hCG stimulated progesterone biosynthesis. Evidence for direct inhibition of 3 beta-hydroxysteroid dehydrogenase enzymic activity by oestradiol was obtained. Both major metabolic pathways of arachidonic acid (lipoxygenase and cyclo-oxygenase) were operative in steroidogenic luteal cells recovered throughout the luteal phase. The ratio of PGE2 to PGF2 alpha synthesis in vitro by human luteal cells from endogenously incorporated arachidonic acid did not change significantly with corpus luteum age, with PGE2 tending to predominate. Oestradiol treatment shifted arachidonic acid metabolism from the lipoxygenase towards the cyclooxygenase pathway in cells isolated from ageing corpora lutea. Oestradiol, at relatively high concentrations, is a potent inhibitor of basal and hCG induced luteal cell steroidogenesis in vitro. No support is provided for the concept that luteolysis is mediated by local production of PGF2 alpha. The putative luteolytic effect of oestradiol may entail reduced metabolism of arachidonic acid to lipoxygenase derived products by luteal cells rather than direct stimulation of prostaglandin production by itself.

Dietary lipids influence insulin action.

Insulin binding and insulin responsiveness are altered by dietary fat-induced changes in the fatty acid composition of the adipocyte plasma membrane. Feeding a high P/S diet increased polyunsaturated fatty acid content of major membrane phospholipids of adipocyte plasma membrane in normal and diabetic animals, increased membrane linoleic acid content, and prevented a decrease in arachidonic acid level in diabetic animals. The high P/S diet increased insulin binding in control animals. Animals fed the high P/S diet had significantly higher rates of insulin-stimulated glucose transport and lipogenesis than did animals fed the low P/S diet. Feeding a high P/S diet significantly increased the amount of glucose transported when expressed as a function of the specific amount of insulin bound. To determine if dietary fat-induced alterations in the fatty acid composition of skeletal muscle lipid alter insulin-dependent and basal muscle metabolism, contralateral epitrochlearis and extensor digitorum longus muscles were isolated and incubated in vitro. High levels of dietary omega-3 fatty acids reduced PGE2 and PGF2 alpha synthesis in extensor digitorum longus and epitrochlearis muscle. Insulin increased glucose and amino acid transport; the increase in glucose transport by insulin was significantly greater after consumption of the high omega-3 fatty acid diet. Rats fed high levels of omega-3 fatty acids showed reduced net protein degradation in the presence and absence of insulin due to decreased rates of protein degradation and synthesis. These experiments indicate that high levels of dietary omega-3 fatty acids alter muscle membrane composition, glucose transport, and metabolism of muscle protein. To determine if dietary fatty acids alter the onset of diabetes and insulin binding to liver nuclei in spontaneously diabetic rats, weanling rats were fed chow or semipurified diets containing 20% (w/w) fat of either high or low P/S ratio. Feeding a high P/S diet increased insulin binding to liver nuclei of control and diabetic animals. Although diet did not alter the onset of diabetes, insulin binding to liver nuclei is higher in animals at the onset of diabetes than in highly diabetic animals. Eight-week-old female C57 B 6J lean and ob/ob mice were fed semipurified diets containing 20% (w/w) fat of either high or low P/S ratio to investigate the effect of diet on specific binding of insulin to liver nuclei. Insulin binding was highest in nuclei from lean mice fed a high P/S diet. Specific binding of insulin to nuclei from obese mice was also increased by the high P/S diet, but to a lesser extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of onapristone, tamoxifen and ICI 182780 on uterine prostaglandin production and luteal function in nonpregnant guinea-pigs.

Onapristone (a progesterone antagonist) or ICI 182780 (an oestrogen antagonist) administered to guinea-pigs on days 11-14 of the cycle significantly reduced uterine PGF2 alpha output on day 15. Concentrations of progesterone in plasma of onapristone-treated and ICI 182780-treated guinea-pigs were still high on day 15 indicating that luteal regression had been prevented. These findings indicate that progesterone and oestradiol are necessary for increased PGF2 alpha production by the uterus towards the end of the cycle, and support the hypothesis that oestradiol acting on a progesterone-primed uterus is the physiological stimulus for increased uterine PGF2 alpha synthesis and release in guinea-pigs. The capacity of the endometrium to synthesize PGF2 alpha on day 15 was reduced by treatment with ICI 182780 and, unexpectedly, by treatment with onapristone, indicating that onapristone may also be antagonizing the release or action of oestradiol in some way. Tamoxifen was an agonist in guinea-pigs since it induced vaginal opening. It had no inhibitory effect on uterine PGF2 alpha output and did not delay luteal regression when administered between days 11 and 14 of the cycle. However, it redirected PG synthesis in homogenates of endometrium and myometrium from PGI2 (as indicated by 6-keto-PGF1 alpha) to PGF2 alpha. The output of 6-keto-PGF1 alpha from the uterus of day 15 guinea-pigs was reduced following tamoxifen treatment, but the high output of PGF2 alpha from the uterus was not affected.

Effects and interactions of prostaglandin F2 alpha, oxytocin, and cytokines on steroidogenesis of porcine luteal cells

In the porcine corpora lutea (CL), prostaglandin F2 alpha (PGF2 alpha) and oxytocin (OXT) inhibit progesterone (P) but stimulate estradiol (E2) secretion from luteal cells kept under primary culture conditions. In vivo, both compounds are reported to have luteolytic properties when administered during the late luteal phase; in young CL, however, both substances stimulate P secretion, an effect which is E2-mediated. During the late luteal phase luteal cells appear to produce cytokines, and in addition, cytokine-producing macrophages invade the CL. We tested therefore whether cytokines, particularly tumor necrosis factor-alpha (TNF), have effects on basal or human CG-stimulated steroidogenesis. Furthermore, the interactions of cytokines with PGF2 alpha and/or OXT were investigated. TNF, and less potently interleukin (IL)-1 and IL-2 but not IL-6, inhibited basal as well as human CG-stimulated release of P and E2 in both small and large luteal cells. The inhibiting effect of PGF2 alpha and OXT on P secretion was augmented by these active cytokines. The stimulatory effect of PGF2 alpha and OXT on small and large luteal cell E2 production was completely inhibited. A profound stimulatory effect of E2 and small luteal cell P secretion was completely prevented by the cytokines, with TNF being more potent than IL-1 or -2. We conclude that the cytokines, particularly TNF, have luteolytic functions by their direct inhibiting effects on luteal cell P production. In addition, the cytokines inhibit synthesis and action of PGF2 alpha- and OXT-stimulated E2 secretion. Since E2 is a potent stimulator of luteal cell P production, this luteotropic signal is eliminated by cytokines, which add to the process of luteolysis.

Effects and interactions of prostaglandin F2 alpha, oxytocin, and cytokines on steroidogenesis of porcine luteal cells.

In the porcine corpora lutea (CL), prostaglandin F2 alpha (PGF2 alpha) and oxytocin (OXT) inhibit progesterone (P) but stimulate estradiol (E2) secretion from luteal cells kept under primary culture conditions. In vivo, both compounds are reported to have luteolytic properties when administered during the late luteal phase; in young CL, however, both substances stimulate P secretion, an effect which is E2-mediated. During the late luteal phase luteal cells appear to produce cytokines, and in addition, cytokine-producing macrophages invade the CL. We tested therefore whether cytokines, particularly tumor necrosis factor-alpha (TNF), have effects on basal or human CG-stimulated steroidogenesis. Furthermore, the interactions of cytokines with PGF2 alpha and/or OXT were investigated. TNF, and less potently interleukin (IL)-1 and IL-2 but not IL-6, inhibited basal as well as human CG-stimulated release of P and E2 in both small and large luteal cells. The inhibiting effect of PGF2 alpha and OXT on P secretion was augmented by these active cytokines. The stimulatory effect of PGF2 alpha and OXT on small and large luteal cell E2 production was completely inhibited. A profound stimulatory effect of E2 and small luteal cell P secretion was completely prevented by the cytokines, with TNF being more potent than IL-1 or -2. We conclude that the cytokines, particularly TNF, have luteolytic functions by their direct inhibiting effects on luteal cell P production. In addition, the cytokines inhibit synthesis and action of PGF2 alpha- and OXT-stimulated E2 secretion. Since E2 is a potent stimulator of luteal cell P production, this luteotropic signal is eliminated by cytokines, which add to the process of luteolysis.

Ovulatory effect of interleukin-1 beta on the perfused rat ovary.

The involvement of cytokines derived from ovarian and hematopoietic cells have been suggested in the cyclic events of the ovary. In the present study the effects of two cytokines, interleukin-1 beta (IL-1 beta) and IL-2, on ovulation, steroidogenesis, and prostaglandin (PG) production were explored in rat ovaries perfused in vitro. Ovaries of equine CG (20 IU)-primed immature rats were perfused in a recirculating system for 20 h, and samples were taken for analysis of progesterone, estradiol, prostaglandin E (PGE), and PGF2 alpha. The number of ovulations was estimated by counting the number of oocytes released into the ovarian bursae. Unstimulated ovaries did not ovulate, whereas the addition of LH (100 ng/ml) resulted in 3.4 +/- 0.6 ovulations/treated ovary. The addition of human recombinant IL-1 beta (4 ng/ml) induced ovulation (1.6 +/- 0.4) and increased the LH-induced ovulation rate 3-fold (9.8 +/- 0.5). The addition of human recombinant IL-2 (40 ng/ml) did not induce ovulation and did not affect the LH-induced ovulation rate. Ovarian release of progesterone and PGF2 alpha was increased by IL-1 beta, but estradiol and PGE release was not affected. IL-2 did not affect steroidogenesis or PG release. To elucidate whether the IL-1 beta-stimulated ovarian synthesis of PGF2 alpha was crucial for the ovulatory effect of IL-1 beta, experiments were performed in the presence of indomethacin (5 micrograms/ml), an inhibitor of PG synthesis. Indomethacin (5 micrograms/ml) inhibited LH-induced ovulation almost completely (one ovulation in one of the five treated ovaries), but did not affect the IL-1 beta-induced ovulation rate (1.4 +/- 0.2). The number of ovulations in the group treated with LH and IL-1 beta was significantly reduced (3.2 +/- 0.6) in the presence of indomethacin. These data demonstrate that IL-1 beta induces ovulation in the rat ovary, and this effect may be partly mediated by the increased production of the ovulatory mediator progesterone.

Dietary omega 3 fatty acid alters prostaglandin synthesis, glucose transport and protein turnover in skeletal muscle of healthy and diabetic rats.

The present study was designed to determine if dietary-fat-induced alterations in the fatty acid composition of skeletal-muscle lipid alters insulin-dependent and basal muscle metabolism, including glucose and amino acid transport, prostaglandin (PG) synthesis and protein turnover. Rats were fed on high-fat semi-purified diets providing 19% or 1% omega 3 fatty acids in the form of fish oil, for 6 weeks. After 3 weeks, half of the rats were made diabetic by a single injection of streptozotocin (50 mg/kg body wt.). After a further 3 weeks, contralateral epitrochlearis and extensor digitorum longus (EDL) muscles from each rat were incubated in vitro. High levels of dietary omega 3 fatty acids decreased PGE2 and PGF2 alpha synthesis in EDL and epitrochlearis muscle (P less than 0.0001). Diabetes and insulin had no effect on PG synthesis. Diet did not alter basal glucose or amino acid transport in EDL muscle from healthy or diabetic rats. Insulin increased glucose and amino acid transport (P less than 0.0001); the increase in glucose transport by insulin was significantly greater in muscles of rats fed on high levels of omega 3 fatty acids (P less than 0.05). Epitrochlearis from rats fed on high levels of omega 3 fatty acids showed decreased net protein degradation in the presence and absence of insulin, owing to decreased rates of protein degradation and synthesis. The data suggest that high levels of dietary omega 3 fatty acids that alter muscle membrane composition also result in alterations in glucose transport and the metabolism of muscle protein.

Effects of p,p′-DDE and some other chlorinated hydrocarbons on the formation of prostaglandins by the avian eggshell gland mucosa

Some structurally related chlorinated hydrocarbons were investigated for their effects on the production of prostaglandins by the eggshell gland mucosa of ducks and domestic fowl. Formation of PGF2 alpha, PGE2 and TxB2 by homogenates of domestic fowl eggshell gland mucosa was significantly inhibited by in vitro addition of p,p'-DDE, Arochlor 1242 and, to a lesser extent, Arochlor 1260, but not by p,p'-DDT and o,p'-DDE. Comparatively, in duck eggshell gland mucosa homogenates, synthesis of the same prostaglandins was somewhat more sensitive to inhibition by 5 microM p,p'-DDE added in vitro. Eggshell gland mucosa synthesized significantly more PGF2 alpha, PGE2 and TxB2 than did the mucosa of the magnum and isthmus regions of the oviduct. Duck eggshell gland mucosa homogenates synthesized significantly more prostaglandins than similar homogenates from the domestic fowl, and, considering the former synthesis of PGF2 alpha was significantly higher when ducks were slaughtered at 08:00 than at 16:00 hours. In ducks, dietary administration of 40 ppm, p,p'-DDE for 45 days resulted in 21% eggshell thinning compared to the contemporary control values. This treatment also resulted in notable effects in homogenates of the eggshell gland mucosa, as compared to controls: Ca2+ uptake was reduced by 43%, synthesis of PGF2 alpha, PGE2 and TxB2 was reduced by 26%, 38% and 53%, respectively; the Ca content was increased to 145%. The role of p,p'-DDE in inhibiting prostaglandin formation in the eggshell gland is discussed as a mechanism of the eggshell thinning action of this chlorinated hydrocarbon.

Eicosanoids enhance epidermal growth factor receptor activation and proliferation in glomerular epithelial cells.

Proliferation of glomerular epithelial cells (GEC) and release of prostaglandins (PG) and thromboxane (Tx) A2 may occur in glomerular injury. We studied the relationship of eicosanoids to epidermal growth factor (EGF)-induced proliferation of rat GEC in culture. After 48 h of serum-deprivation, EGF stimulated [3H]thymidine incorporation ninefold above serum-deprived cells. Inhibition of cyclooxygenase with indomethacin or of Txsynthase with OKY-046 decreased the proliferative effect of EGF by 50 and 38%, respectively. The effect of indomethacin was reversed by addition of PGE2. Synthesis of PGE2, PGF2 alpha, and TxA2 by serum-deprived GEC was not enhanced by EGF. Scatchard analysis of 125I-EGF binding to GEC demonstrated two populations of EGF receptors; the high-affinity site had a dissociation constant (Kd) of 444 pM and 24,864 receptors/cell. EGF receptor autophosphorylation (reflecting receptor activation) was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of GEC membrane proteins with anti-phosphotyrosine antibody. EGF increased phosphorylation of a protein of approximately 170 kDa, which comigrated with proteins immunoprecipitated from [35S]methionine-labeled GEC with antibodies to EGF receptor. Indomethacin and OKY-046 decreased the EGF-dependent phosphorylation of the 170-kDa protein, and this decrease was overcome by addition of PGE2. Indomethacin and OKY-046 did not, however, reduce 125I-EGF binding. Thus, in GEC, the basal synthesis of eicosanoids enhanced EGF-induced proliferation. This effect appears to be due to enhancement of EGF receptor activation.

Effect of melittin on prostaglandin production by guinea-pig uterus

Melittin, an activator of phospholipase (PL) A-2, increased the outputs of prostaglandin (PG) F-2 alpha and 6-keto-PGF-1 alpha, but not of PGE-2, from Day-7 guinea-pig uterus superfused in vitro. Reducing the extracellular calcium concentration (by omitting calcium chloride from the superfusing fluid) partially inhibited the stimulatory effect of melittin on uterine PG production. TMB-8 (an intracellular calcium antagonist) completely prevented the stimulation of PGF-2 alpha and 6-keto-PGF-1 alpha output by melittin, although the production of both PGs tended to increase after stopping the melittin and TMB-8 treatments. TMB-8 also inhibited the increases in outputs of PGF-2 alpha, 6-keto-PGF-1 alpha and PGE-2 and prevented contraction of the uterus induced by exogenous PLA-2. Trifluoperazine (a calmodulin antagonist) had no inhibitory effect on the increases in outputs of PGF-2 alpha and 6-keto-PGF-1 alpha produced by melittin; it potentiated the stimulatory effect of melittin on 6-keto-PGF-1 alpha output and allowed melittin to increase PGE-2 output. When melittin was applied twice to the superfused uterus with an interval of 1 h between each treatment, partial refractoriness of the responses to melittin was seen: the magnitudes of the increases in PGF-2 alpha and 6-keto-PGF-1 alpha outputs were 40-50% less after the second treatment than after the first treatment. These results show that melittin stimulates the synthesis of PGF-2 alpha and PGI-2 (measured as 6-keto-PGF-1 alpha) in guinea-pig uterus by mechanisms which are calcium dependent.(ABSTRACT TRUNCATED AT 250 WORDS)