PGF2 Alpha Output Research Articles

Canine placental prostaglandin E2 synthase: expression, localization, and biological functions in providing substrates for prepartum PGF2alpha synthesis.

The prepartum output of PGF2alpha in the bitch is associated with increased placental PGE2-synthase (PTGES) mRNA levels. Contrasting with this is a decreased expression of PGF2alpha-synthase (PGFS/AKR1C3) in uteroplacental compartments during prepartum luteolysis, suggesting an involvement of alternative synthetic pathways in PGF2alpha synthesis, for example, conversion of PGE2 to PGF2alpha. However, because the expression and possible functions of the respective PTGES proteins remained unknown, no further conclusion could be drawn. Therefore, a canine-specific PTGES antibody was generated and used to investigate the expression, cellular localization, and biochemical activities of canine uteroplacental PTGES throughout pregnancy and at prepartum luteolysis. Additionally, the biochemical activities of these tissues involved in the conversion of PGE2 to PGF2alpha were investigated. The endometrial PTGES was localized in the uterine surface epithelium at preimplantation and in superficial and deep uterine glands, endothelial cells, and myometrium throughout pregnancy and at parturition. Placental signals were mostly in the trophoblast. The biochemical properties of recombinant PTGES protein were confirmed. Additionally, expression of two PGE2-receptors, PTGER2/EP2 and PTGER4/EP4, revealed their decreasing expression during luteolysis. In contrast, the uteroplacental expression of prostaglandin transporter (PGT) was strongly elevated prior to parturition. These localization patterns resembled that of PTGES. The increased expression of PTGES and PGT at parturition, together with the accompanying decreased levels of PGE2-receptors and the capability of canine uterine and placental homogenates to take part in the conversion of PGE2 to PGF2alpha, as found in this study, suggest that PGE2 could be used locally as a substrate for prepartum PGF2alpha synthesis in the dog.

Prostaglandins f and e secretion by porcine epithelial and stromal endometrial cells on different days of the oestrous cycle.

The endometrial tissue of the uterus plays a key role in reproduction and is a source of hormones and factors responsible for the proper physiological function of reproductive tract during the oestrous cycle and pregnancy. In this study, we investigated the pattern of PGF(2alpha) and PGE(2) secretion from cultured porcine endometrial cells at different days of the oestrous cycle. Epithelial and stromal cells were isolated by differential enzymatic digestion on days 6-8, 10-12 and 14-16. After attachment cells were incubated for 3 and 24 h to estimate PGF(2alpha) and PGE(2) output. The purity of culture was 85-90% for epithelial and 95-98% for stromal cells as determined by immunofluorescent staining. Release of PGF(2alpha) and PGE(2) was affected by cell type, days of the oestrous cycle and the time of incubation. After 3 h of incubation epithelial cells secreted more PGF(2alpha) than PGE(2) during all studied periods of the oestrous cycle (p < 0.01 and p < 0.001, respectively), whereas stromal cells released more PGE(2) (p < 0.01) on days 10-12 and 14-16. Longer incubation of stromal cells revealed that PGF(2alpha) output tended to overcome PGE(2) on days 10-16. The lowest secretion of prostaglandins was observed on days 6-8 in both cell types. The highest secretion of PGF(2alpha) from epithelium was measured on days 10-12 after 24 h of incubation when compared with other days studied (p < 0.001). In stromal cells, PGE(2) output increased on consecutive days studied (p < 0.001) after 3 h of incubation. The differential properties of endometrial cell types seem to play an important role in the profile of PGF(2alpha) and PGE(2) release before and during luteolysis. Described endometrial cells culture might serve as the model for further studies on the hormonal regulation of prostaglandin production in the pig.

A passage and storage system for isolated bovine endometrial epithelial and stromal cells.

To establish a storage system for isolated endometrial cells, we investigated the basal, oxytocin (OT)- and tumor necrosis factor (TNF) alpha-stimulated production of prostaglandin (PG) F(2alpha) in bovine-passaged and frozen-thawed endometrial cells. Stromal and epithelial cells obtained from cows in the early stage of the estrous cycle (Days 2-5) were frozen at -80 C or further cultured and/or passaged until passage 4 in DMEM/Ham's F-12 supplemented with 10% calf serum. A fresh-unfrozen primary culture and one-time passaged fresh-unfrozen cells were used as the control. When both unfrozen and frozen cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA and the cells were stimulated with OT (100 ng/ml) or TNFalpha (1 ng/ml) for 4 h. The passage and freezing of the endometrial cells did not affect their morphology. In primary culture of frozen and unfrozen endometrial cells, OT strongly stimulated PGF(2alpha) production in epithelial cells, and TNFalpha strongly stimulated PGF(2alpha) production in stromal cells (P<0.05). The basal output of PGF(2alpha) in frozen stromal cells was similar to that in unfrozen stromal cells. However, the basal output of PGF(2alpha) in frozen epithelial cells was significantly lower than that unfrozen cells (P<0.05). On the other hand, in passaged cells, the basal level of PGF(2alpha) production was retained until passage 1 in epithelial cells, whereas it was retained until passage 4 in stromal cells. Although epithelial cells responded to OT in PGF(2alpha) production until passage 2 (P<0.05), the stromal cells showed a significant response to TNFalpha until passage 4 (P<0.05). These results suggest that stored cells could be used for studying the physiology of bovine endometrium in vitro until passage 1 in endometrial epithelial cells, and until passage 4 in stromal cells.

Effects of epidermal growth factor, interleukin 1 and nitric oxide on prostaglandin production by guinea-pig uterus.

Initial experiments in the present study investigated the effects of epidermal growth factor (EGF), interleukin 1beta (IL-1beta) and sodium nitroprusside (a nitric oxide donor) on the output of prostaglandins from guinea-pig uterus on day 7 of the oestrous cycle. Superfusion of day 7 guinea-pig uterus in vitro with either EGF or sodium nitroprusside increased the output of PGF(2alpha) and 6-keto-PGF(1alpha), but not of PGE(2). IL-1beta had no effect on the output of these three prostaglandins. EGF still increased the output of PGF(2alpha), but did not increase the output of 6-keto-PGF(1alpha) in a calcium-depleted superfusate. Subsequent experiments investigated the effect of sodium nitroprusside on contractile activity of day 7 guinea-pig uterus. Basal spontaneous activity of both the intact uterus and isolated myometrium superfused in vitro was low. Sodium nitroprusside increased the contractile activity of these tissues two- to fourfold. EGF did not affect the contractile activity of the uterus, indicating that sodium nitroprusside-induced contractions are not due to increased prostaglandin production. Overall, the findings indicate that EGF and nitric oxide may act as mediators in the mechanism by which oestradiol acting on a progesterone-primed uterus stimulates the increase in PGF(2alpha) production by the guinea-pig uterus necessary for luteolysis. Nitric oxide may increase the spontaneous activity of the uterus when this activity is low.

Effects of epidermal growth factor, interleukin 1 and nitric oxide on prostaglandin production by guinea-pig uterus

Initial experiments in the present study investigated the effects of epidermal growth factor (EGF), interleukin 1beta (IL-1beta) and sodium nitroprusside (a nitric oxide donor) on the output of prostaglandins from guinea-pig uterus on day 7 of the oestrous cycle. Superfusion of day 7 guinea-pig uterus in vitro with either EGF or sodium nitroprusside increased the output of PGF(2alpha) and 6-keto-PGF(1alpha), but not of PGE(2). IL-1beta had no effect on the output of these three prostaglandins. EGF still increased the output of PGF(2alpha), but did not increase the output of 6-keto-PGF(1alpha) in a calcium-depleted superfusate. Subsequent experiments investigated the effect of sodium nitroprusside on contractile activity of day 7 guinea-pig uterus. Basal spontaneous activity of both the intact uterus and isolated myometrium superfused in vitro was low. Sodium nitroprusside increased the contractile activity of these tissues two- to fourfold. EGF did not affect the contractile activity of the uterus, indicating that sodium nitroprusside-induced contractions are not due to increased prostaglandin production. Overall, the findings indicate that EGF and nitric oxide may act as mediators in the mechanism by which oestradiol acting on a progesterone-primed uterus stimulates the increase in PGF(2alpha) production by the guinea-pig uterus necessary for luteolysis. Nitric oxide may increase the spontaneous activity of the uterus when this activity is low.

Is tumor necrosis factor alpha a trigger for the initiation of endometrial prostaglandin F(2alpha) release at luteolysis in cattle?

To determine the physiological significance of tumor necrosis factor alpha (TNFalpha) in the regulation of luteolytic prostaglandin (PG) F(2alpha) release by the bovine endometrium, the effect of TNF-alpha on PGF(2alpha) output by the endometrial tissues in vitro was investigated and compared with the effect of oxytocin (OT). Furthermore, the presence of specific receptors for TNFalpha in the bovine endometrium during the estrous cycle was determined. Endometrial slices (20-30 mg) taken from six stages of the estrous cycle (estrus: Day 0; early I: Days 2-3; early II: Days 5-6; mid-: Days 8-12; late: Days 15-17; and follicular: Days 19-21), as determined by macroscopic examination of the ovaries and uterus, were exposed to TNFalpha (0.06-6 nM) and/or OT (100 nM). OT stimulated PGF(2alpha) output at the follicular stage and at estrus (P < 0.001), but not at the late luteal stage. On the other hand, the stimulatory effects of TNFalpha on PGF(2alpha) output were observed not only at the follicular stage but also at the late luteal stage (P < 0.001). When the endometrial tissues at late luteal stage were simultaneously exposed to TNFalpha (0.6 nM) and OT (100 nM), the stimulatory effect on PGF(2alpha) output was higher than the effect of TNFalpha or OT alone (P < 0.05). Specific binding of TNFalpha to the bovine endometrial membranes was observed throughout the estrous cycle. The concentration of TNF-alpha receptor at the early I luteal stage was less than the concentrations at other luteal stages (P < 0.01). The dissociation constant (K(d)) values of the endometrial membranes were constant during the estrous cycle. The overall results lead us to hypothesize that TNFalpha may be a trigger for the output of PGF(2alpha) by the endometrium at the initiation of luteolysis in cattle.

Production of Prostaglandin F2α by Cultured Bovine Endometrial Cells in Response to Tumor Necrosis Factor α: Cell Type Specificity and Intracellular Mechanisms1

Tumor necrosis factor alpha (TNFalpha) has been shown to be a potent stimulator of prostaglandin (PG) F(2alpha) secretion in the bovine endometrium. The aims of the present study were to determine the cell types in the endometrium (epithelial or stromal cells) responsible for the secretion of PGF(2alpha) in response to TNFalpha, and the intracellular mechanisms of TNFalpha action. Cultured bovine epithelial and stromal cells were exposed to TNFalpha (0.006-6 nM) or oxytocin (100 nM) for 4 h. TNFalpha resulted in a dose-dependent increase of PGF(2alpha) production in the stromal cells (P < 0.001) but not in the epithelial cells. On the other hand, oxytocin stimulated PGF(2alpha) output in the epithelial cells but not in the stromal cells. When the stromal cells were incubated for 24 h with TNFalpha and inhibitors of phospholipase (PL) C or PLA(2), only PLA(2) inhibitor completely stopped the actions of TNFalpha (P < 0.001). When the stromal cells were exposed to TNFalpha and arachidonic acid, the action of TNFalpha was augmented (P < 0.001). When the stromal cells were incubated for 24 h with a nitric oxide (NO) donor (S-NAP), S-NAP stimulated the PGF(2alpha) production dose-dependently. Although an NO synthase (NOS) inhibitor (L-NAME) reduced TNFalpha-stimulated PGF(2alpha) production, an inhibitor of phosphodiesterase augmented the actions of TNFalpha and S-NAP (P < 0. 05). The overall results indicate that the target of TNFalpha for stimulation of PGF(2alpha) production in cattle is the endometrial stromal cells, and that the actions of TNFalpha are mediated via the activation of PLA(2) and arachidonic acid conversion. Moreover, TNFalpha may exert a stimulatory effect on PGF(2alpha) production via the induction of NOS and the subsequent NO-cGMP formation.

Effects of inhibitors of arachidonic acid turnover on the production of prostaglandins by the guinea-pig uterus.

The supply of free arachidonic acid from phospholipids is generally regarded as the rate-limiting step for prostaglandin (PG) synthesis by tissues. Two enzymes involved in arachidonic acid uptake into, and release from, phospholipids are acyl-CoA:lysophospholipid acyltransferase (ACLAT) and phospholipase A2 (PLA2), respectively. PGF2 alpha produced by the endometrium induces luteolysis in several species including guinea-pigs. Thimerosal, an inhibitor of ACLAT, and aristolochic acid, an inhibitor of PLA2, both reduced, in a concentration-dependent manner, the output of PGF2 alpha from guinea-pig endometrium cultured for 24 h on days 7 and 15 of the oestrous cycle. This study showed that the continual production of PGF 2 alpha by guinea-pig endometrium is not only dependent upon the activity of PLA2 for releasing free arachidonic acid for PGF2 alpha synthesis, but also on the incorporation of arachidonic acid into the phospholipid pool by the activity of ACLAT. The inhibitory effects of thimerosal and aristolochic acid on the outputs of PGE2 and 6-keto-PGF1 alpha were less marked, particularly on day 7 when the low output of PGE2 was unaffected and the output of 6-keto-PGF1 alpha was increased at the lower concentrations of thimerosal. This finding indicates that there are different pools of arachidonic acid bound as phospholipid for the syntheses of PGF2 alpha and 6-keto-PGF1 alpha by guinea-pig endometrium.

Effects of inhibitors of arachidonic acid turnover on the production of prostaglandins by the guinea-pig uterus

The supply of free arachidonic acid from phospholipids is generally regarded as the rate-limiting step for prostaglandin (PG) synthesis by tissues. Two enzymes involved in arachidonic acid uptake into, and release from, phospholipids are acyl-CoA:lysophospholipid acyltransferase (ACLAT) and phospholipase A2 (PLA2), respectively. PGF2 alpha produced by the endometrium induces luteolysis in several species including guinea-pigs. Thimerosal, an inhibitor of ACLAT, and aristolochic acid, an inhibitor of PLA2, both reduced, in a concentration-dependent manner, the output of PGF2 alpha from guinea-pig endometrium cultured for 24 h on days 7 and 15 of the oestrous cycle. This study showed that the continual production of PGF 2 alpha by guinea-pig endometrium is not only dependent upon the activity of PLA2 for releasing free arachidonic acid for PGF2 alpha synthesis, but also on the incorporation of arachidonic acid into the phospholipid pool by the activity of ACLAT. The inhibitory effects of thimerosal and aristolochic acid on the outputs of PGE2 and 6-keto-PGF1 alpha were less marked, particularly on day 7 when the low output of PGE2 was unaffected and the output of 6-keto-PGF1 alpha was increased at the lower concentrations of thimerosal. This finding indicates that there are different pools of arachidonic acid bound as phospholipid for the syntheses of PGF2 alpha and 6-keto-PGF1 alpha by guinea-pig endometrium.

Correlation of increased concentration of ovine endometrial cyclooxygenase 2 with the increase in PGE2 and PGD2 in the late luteal phase.

Ovine endometrium showed transient expression of high concentrations of the inducible isoform of cyclooxygenase, cyclooxygenase 2 (COX-2), whereas the constitutive isoform, cyclooxygenase 1 (COX-1), was expressed at much lower concentrations and did not change. In this study, the pattern of prostaglandin synthesis in endometrial luminal cells was investigated in relation to their COX-2 content. Endometrial cells from cyclic or pregnant ewes at days 9, 12, 14 and 16 were isolated and analysed for the presence of COX-1 and COX-2 proteins using western blot analysis. Freshly isolated cells were incubated with 0.5 microCi [3H]arachidonic acid ml-1. Radioactive cyclooxygenase metabolites were analysed by reverse-phase HPLC. Luminal cells produced mainly PGF2 alpha, PGE2, PGD2 and 13,14-dihydro-15-keto PGF2 alpha and to a lesser extent 6-keto PGF1 alpha, thromboxane B2 and 13,14-dihydro-15-keto PGE2. The production of PGE2 and PGD2 was proportional to the cellular concentration of COX-2. PGE2 and PGD2 release was low on day 9 when COX-2 was not expressed, whereas high concentrations of PGE2 and PGD2 were synthesized on days 12-14 when COX-2 was highly expressed, reaching 100 ng microgram-1 cellular protein. In contrast, the basal production of PGF2 alpha did not appear to be related to COX-2 concentration and was greatest on day 16. Moreover, the release of PGF2 alpha was maintained at steady state values between days 9 and 14 by the production of 13,14-dihydro-15-keto PGF2 alpha. Although PGF2 alpha output was lower at day 16 of pregnancy compared with the oestrous cycle, no difference was observed in the pattern of prostaglandin synthesis between pregnant and non-pregnant ewes.

Effects of genistein and lavendustin on reproductive processes in domestic animals in vitro

The aim of our experiments was to study the influence of genistein [tyrosine kinase (TK) inhibitor with estrogenic activity] and lavendustin A (TK inhibitor without estrogenic activity) on female reproductive processes in domestic animals in vitro. It was found that genistein (0.001–1 μg/ml) increased IGF-I release by cultured bovine and porcine granulosa cells, but decreased its secretion by rabbit granulosa cells (0.01–10 μg/ml). Genistein stimulated progesterone secretion by bovine and rabbit granulosa cells (at 0.01–10 μg/ml), estradiol output by rabbit granulosa cells (at 1 μg/ml) and porcine ovarian follicles (at 10 μg/ml), as well as cAMP production by bovine (at 0.001–1 μg/ml) and rabbit (at 1 μg/ml) granulosa cells. No effects of genistein (at 10 μg/ml) on PGF-2 alpha and progesterone release by porcine ovarian follicles were observed. Genistein significantly (P < 0.05) stimulated the reinitiation and completion of nuclear maturation of porcine oocytes (at 5 μg/ml), as well as the preimplantation development of rabbit zygotes (at 1 μg/ml). Lavendustin A (0.001–1 μg/ml) increased IGF-I release by bovine (but not by porcine) granulosa cells, cAMP release by bovine granulosa cells, and PGF-2 alpha output by porcine ovarian follicles (at 10 μg/ml). Lavendustin (at 1 μg/ml) had no significant effect on IGF-I release by porcine granulosa cells, on estradiol and cAMP output by rabbit granulosa cells, or on progesterone secretion by porcine follicles (at 10 μg/ml). Inhibitory actions of lavendustin (at 10 μg/ml) on estradiol secretion by porcine follicles were also found. Furthermore, lavendustin, like genistein, promoted the reinitiation and completion of meiosis in porcine oocytes. The present study demonstrates a predominantly stimulatory effect of TK inhibition on endocrine and generative processes in domestic animals. The majority of these effects are similar for both compounds, indirectly suggesting that their action is due to tyrosine kinase inhibition and protein kinase A-stimulation, rather than estrogenic activity.

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 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.

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)

A possible explanation for the refractoriness of uterine prostaglandin production

Arachidonic acid increased the outputs of prostaglandin (PG) F-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from the Day-7 and Day-15 guinea-pig uterus superfused in vitro. Similar increases in PG output were observed when the arachidonic acid treatment was repeated after an interval of 1, 3 or 5 h. Phospholipase (PL) A-2 increased the outputs of PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from the Day-7 guinea-pig uterus, but repeating the PLA-2 treatment 1 h later failed to stimulate PG output. The increase in outputs of PGF-2 alpha and PGE-2 caused by PLA-2 were partly restored after 3 h and were fully restored after 5 h, whereas the increase in 6-keto-PGF-1 alpha output produced by PLA-2 was only partly restored after 3 and 5 h. PLA-2 had little or no effect on PGF-2 alpha and PGE-2 outputs from the Day-15 guinea-pig uterus initially, and when repeated after 1, 3 and 5 h. This was probably due to the output of these two PGs, particularly of PGF-2 alpha, being stimulated in vivo before removal of the uterus. PLA-2 increased 6-keto-PGF-1 alpha output from the Day-15 uterus initially, but failed to cause a response when administered again 1 h later. After 3 and 5 h, the increase in 6-keto-PGF-1 alpha output from the Day-15 uterus caused by PLA-2 was partly restored. A23187 and PLC increased the outputs of PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from the Day-7 and Day-15 guinea-pig uterus. These responses to A23187 and PLC were reduced (but not abolished) when the two compounds were administered again 1 h later. After 3 and 5 h, the increases in output of PGF-2 alpha and PGE-2 produced by A23187 and PLC had returned to the initial values. The increases in output of 6-keto-PGF-1 alpha from the Day-7 and Day-15 guinea-pig uterus produced by A23187 and PLC were partly restored after 3 and 5 h, except for the response to PLC on Day 7 which was fully restored after 5 h. The results show that there is no failure with time in the mechanism which converts arachidonic acid into PGF-2 alpha in the guinea-pig uterus.(ABSTRACT TRUNCATED AT 400 WORDS)

Prostaglandin Production by the Largest Preovulatory Follicles in the Domestic Hen (Gallus Domesticus)1

An injection of 5 micrograms of gonadotropin-releasing hormone (GnRH) into hens 8 h prior to oviposition advanced the expected time of oviposition by approximately 1 h. The plasma concentration of progesterone increased approximately 1 h earlier in GnRH-injected hens in comparison to saline-injected hens. The plasma concentration of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) increased significantly (p less than 0.05) at the time of oviposition in both the GnRH- and saline-injected hens. Significantly (p less than 0.05) greater concentrations of prostaglandin F2 alpha (PGF2 alpha) were assayed in media containing the largest preovulatory follicles collected at oviposition than in media containing the second and fifth largest preovulatory follicles collected at the same time. No prostaglandin was detected in media containing small, nonhierarchial follicles. The concentration of PGF2 alpha in media containing granulosa cells from the largest preovulatory follicle was significantly greater (p less than 0.05) than in media containing 4 times as many theca cells. Ovine luteinizing hormone (oLH) alone or in combination with arachidonic acid had no effect on PGF2 alpha output from granulosa cells collected 6 h before oviposition, whereas A23187 caused a small stimulation of PGF2 alpha output. However, treating cells first with oLH and then with A23187 stimulated a 15- to 20-fold increase in PGF2 alpha. None of these stimuli enhanced the already high output of PGF2 alpha when added to incubations of granulosa cells collected within 5 min after oviposition. These data suggest that the granulosa cells of the largest preovulatory follicle are the major intraovarian source of prostaglandin and that production of PGF2 alpha is associated with the preovulatory surges of gonadotropins and steroid hormones preceding oviposition.

Investigations into the mechanism by which sodium fluoride stimulates prostaglandin production in guinea-pig uterus

Sodium fluoride (10 mM) caused a slow increase in the outputs of PGF-2 alpha, 6-keto-PGF-1 alpha and, to a lesser extent, PGE-2 from the Day-7 and Day-15 guinea-pig uterus superfused in vitro. This stimulatory action of sodium fluoride was not prevented by using calcium-free Krebs' solution. There was also a faster stimulation of 6-keto-PGF-1 alpha output from the Day-7 guinea-pig uterus produced by sodium fluoride, and this quicker response was abolished by using calcium-free Krebs' solution. TMB-8 (an intracellular calcium antagonist) inhibited the stimulatory action of sodium fluoride on the outputs of PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from the Day-7 guinea-pig uterus. W-7 and trifluoperazine (calmodulin antagonists) and neomycin (an inhibitor of phospholipase C) had no inhibitory effect on the increases in outputs of PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from the Day-7 guinea-pig uterus produced by sodium fluoride. These results indicate that sodium fluoride slowly stimulates uterine PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha synthesis in the guinea-pig uterus by mobilizing intracellular calcium by a mechanism which apparently does not involve the activation of phospholipase C or the participation of calmodulin (or a related compound). The initial, faster stimulation of 6-keto-PGF-1 alpha synthesis in the Day-7 guinea-pig uterus by sodium fluoride is dependent upon extracellular calcium.

Effects of Corticotropin-Releasing Hormone and Adrenocorticotropin on Prostaglandin Output by Human Placenta and Fetal Membranes

We have examined the possibility that corticotropin-releasing hormone (CRH) and adrenocorticotropin (ACTH) might affect prostaglandin (PG) E2 and PGF2 alpha output by term placenta, amnion and decidua during short-term (48-hour) culture, and that effects of CRH might be mediated by ACTH. In all three tissues PG output was stimulated by both CRH and ACTH. These effects were inhibited in the presence of antisera to CRH and to ACTH. Moreover, in placenta, but not in amnion or decidua, the stimulatory effects of CRH on PGE2 and PGF2 alpha output were attenuated in the presence of an antibody to ACTH. Our results support the possibility of paracrine stimulation by CRH and ACTH of PG production in intrauterine tissues, and suggest that in part the effects of CRH on placental PG output might be mediated through ACTH.

Cigarette Smoking Increases Gastric Luminal Prostaglandin F&lt;sub&gt;2α&lt;/sub&gt; and Thromboxane B&lt;sub&gt;2&lt;/sub&gt; in Healthy Smokers

Pentagastrin-stimulated gastric luminal prostaglandin E2 (PGE2), 6-keto-PGF1 alpha, PGF2 alpha and thromboxane B2 (TxB2) were measured using a second antibody solid-phase enzyme immunoassay before, during and after cigarette smoking in healthy smokers. Smoking significantly increased PGF2 alpha and TxB2 concentration and output; in contrast no significant changes were found for PGE2 and 6-keto-PGF1 alpha levels. In addition, cigarette smoking caused a significant reduction in gastric juice volume and acid output but did not alter intragastric acidity. These findings may suggest a possible role of prostanoids in the response of the stomach to cigarette smoking.

Release of cyclooxygenase products from primary cultures of tracheal epithelia of dog and human

Release of cyclooxygenase products from primary cultures of dog or human tracheal epithelium was measured by radioimmunoassay. In both species, bradykinin, platelet-activating factor (PAF), and A23187 (a calcium ionophore) caused increases in the rate of release of prostaglandin (PG) E2 and smaller increases in PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane B2 output. Isoproterenol, vasoactive intestinal peptide (VIP), methacholine, and leukotrienes C4 and D4 had no effect on release of these cyclooxygenase products. Gas chromatography-mass spectrometry showed that the ratio of PGE2 to PGD2 released from dog cells by A23187 was 30:1. Short-circuit current across dog cells was stimulated by bradykinin, A23187, PAF, VIP, methacholine, and isoproterenol. Only the responses to bradykinin and A23187 were reduced by pretreatment with indomethacin.