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.     

Menstrual-PGF2α, PGE2 and TXA2 in normal and dysmenorrheic women and their temporal relationship to dysmenorrhea

Although it has been demonstrated that primary dysmenorrhea is associated with elevated levels of PGF_2α in the menstrual fluid, little is actually known of the menstrual-PG profiles of either dysmenorrheic or normal women. In this study, menstrual fluid from normal and dysmenorrheic women was collected from tampons and extracted for PG-like substances. The PGF_2α, PGE₂ and TXA₂ content was analyzed by RIA.This study demonstrates that dysmenorrheics have significantly higher levels/concentrations of menstrual-PGF_2α and PGe₂ than do normal women, and that there is no difference in the menstrual-PGF_2α: PGE₂ ratio between the two groups. Also, there is no significant difference in the amount/concentration of menstrual-thromboxane between dysmenorrheic and normal women. Of the parameters considered, the levels/-concentrations of menstrual-PGF_2α, PGE₂ and TXA₂, dysmenorrheic pain correlates best with the rate of menstrual-PGF_2α release.     

Effects of oestradiol, progesterone, hydrocortisone and oxytocin on prostaglandin output from the guinea-pig endometrium maintained in tissue culture

The effects of oestradiol, oxytocin, progesterone and hydrocortisone on prostaglandin (PG) output from guinea-pig endomerium, removed on days 7 and 15 of the oestrous cycle and maintained in tissue culture for 3 days, have been investigated. Oetradiol (3.7 to 3700nM) and oxytocin ( 2 to 200pM) did not stimulate endometrial PGF_2α output, thus not confirming the findings of a previous report (Leaver & Seawright, 1928), nor did they stimulate the outputs of PGE₂ and 6-keto-PGF_1α. In fact, oestradiol (3700nM) inhibited the outputs of PGF_2α, PGE₂ and, to a lesser extent, 6-keto-PGF_1α. Progesterone (3.2 to 3200nM) inhibited the outputsof PGF_2α and PGE₂; hydrocortisone (2.8 to 2800nM) had no effect on endometrial PG output. These findings indicate that the inhibitory effect of progesterone on endometrial PG synthesis and release in the guinea-pig is not due to progesterone having a glucocorticoid-like action. Furthermore, progesterone had no effect on 6-keto-PGF_1α output, suggesting that the mechanisms controlling endometrial PGI₂ synthesis (as reflected by measuring 6-keto-PGF_1α) are different from those controlling endometrial PGF_2α and PGE₂ synthesis.     

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.     

Stimulation of hepatocyte DNA synthesis by prostaglandin E2 and prostaglandin F2α additivity with the effect of norepinephrine,and synergism with epidermal growth factor

Previous data obtained in vivo and in vitro suggest that both prostaglandins (PGs) and catecholamines may have a role in promoting hepatocyte proliferation, and PGE₂ and PGFF_2α have also been implicated as mediators of the mitogenic actions of epidermal growth factor (EGF) (and transforming growth factor alpha [TGFα]). We have studied the effects of PGs and norepinephrine on DNA synthesis in serum-free primary cultures of rat hepatocytes, and compared the PG effects with those of norepinephrine. PGE₂, PGF_2α, PGD₂, and the synthetic analog dimethyl-PGE₂ markedly enhanced the DNA synthesis. A more quantitative analysis of the effects of PGE₂ and PGF_2α on the DNA synthesis, in the presence and absence of EGF, indicated that these PGs interacted in an essentially multiplicative manner with the effect of EGF. The effects of PGE₂ and PGF_2α showed almost complete additivity with the stimulation of DNA synthesis produced by maximally effective concentrations of norepinephrine. The data suggest (a) that PGE₂ and PGF_2α facilitate and synergize with, rather than mediate, the actions of EGF in hepatocytes, and (b) that this effect of the PGs occurs by mechanisms that are at least partly distinct from those of norepinephrine. © 1994 wiley-Liss, Inc.     

Determination of menstrual prostaglandin levels in non-dysmenorrheic and dysmenorrheic subjects

We have developed a method which can measure the menstrual prostaglandin (PG) activity in a single tampon specimen by bioassays. This method makes it possible to monitor the menstrual PG activity continuously during menstruation. Using this technique, we determined the menstrual PG patterns of two normal non-dysmenorrheic subjects, one subject on oral contraceptives (OC) and one subject with moderate to severe dysmenorrhea. Two to four cycles were studied per subject. We observed three menstrual patterns among the four subjects studied. Compared to the two normal controls, the subject on OC had a significantly lower menstrual fluid total and menstrual PG activity. The mean values ± S.E. per menstrual period were 33.4 g ± 1.5 vs 21.5 g ± 2.0 and 28.6 μg (PGF_2α equivalent) ± 1.5 vs 11.3 μg ± 4.2 respectively (control vs OC). The dysmenorrheic subject had a menstrual fluid total of 37.0 g ± 1.9 similar to the two normal controls. Her menstrual PG activity (49.8 μg ± 7.7), however, was nearly two times higher than the normal controls. In one cycle studied, the dysmenorrheic subject was treated with a PG synthetase inhibitor, ibuprofen (Motrin). Remarkable relief was achieved. The alleviation of symptoms was accompanied by a concomitant marked reduction in the menstrual PG activity.     

The effect of anti-inflammatory agents on human synovial fibroblast prostaglandin synthetase

Human synovial fibroblast prostaglandin synthetase activity is inhibited by many different non-steroidal anti-inflammatory agents. Aspirin, indomethacin and phenylbutazone significantly inhibit both PGE₁, PGE₂ and PGF_1α and PGF_2α synthesis; whereas penicillamine and aurothioglucose are more potent inhibitors of the F prostaglandins. Histidine and antimalarials do not inhibit, to a significant degree, human synovial prostaglandin synthetase activity. Hydrocortisone has no direct effect on prostaglandin synthetase activity. No changes in synthetase activity are observed when synovial cells are incubated with hydrocortisone, and the prostaglandin synthetase system subsequently isolated and assayed. The proposed inhibitory effects of hydrocortisone on prostaglandin production by synovium may be the result of an alteration of enzyme substrate or cofactor concentration rather than a direct effect on prostaglandin synthetase.     

Effect of prolonged prostaglandin exposure on prostaglandin synthesis by human lung fibroblasts

Pretreatment of human lung fibroblasts with PGE₂ but not PGF_2α enhanced synthesis of prostaglandins (PGs). The effect of the pretreatment on PG synthesis was related to the concentration of PGE₂ that was added to the culture medium. Pretreatment with PGE₂ at 5 × 10⁻¹²M did not enhance PG synthesis whereas pretreatment with PGE₂ at 5 × 10⁻⁶M induced a maximal effect. Production of PGs was increased following 1 day of pretreatment with PGE₂ and was increased further following 3 days of pretreatment. The PGE₂ treated cells showed only a slight increase in the bradykinin-induced release of radioactivity from cells prelabeled with [³H]arachidonic acid but showed a dramatic increase in the bradykinin-induced synthesis of radio-labeled PGs. The conversion of free arachidonate to PGs in both intact cells and in a cell-free preparation was increased by PGE₂ pretreatment. The presence of cyclohexamide during the pretreatment did not inhibit the PGE₂-induced activation of PG synthesis. Taken together, the results indicate that pretreatment of cells with PGE₂ increased PG synthesis by augmenting the conversion of arachidonate to PGs.     

Bone resorption and prostaglandin production by mouse calvaria in vitro: Response to exogenous prostaglandins and their precursor fatty acids

Mouse calvaria were maintained in organ culture for 96 h and endogenous prostaglandin production and active bone resorption (⁴⁵ Ca release) measured. After a lag phase of 12 h, active resorption increased over the 96 h period. The amounts of prostaglandins released into the culture medium (measured by radioimmunoassay) were highest in the first 24 h of culture. Unless these were removed by preculturing for 24 h, or suppressed by indomethacin, no response to exogenous PGE₂, PGF_2α or prostaglandin precursors could be demonstrated. Bone resorption was stimulated after preculture by both PGE₂ and PGF_2α in a dose-dependent manner (10^?18M – 10^?5M), with PGE₂ being the more potent. Collagen synthesis was unaffected by PGF_2α, whereas PGE₂ (10^?5M) had an inhibitory effect. Eicosatrienoic acid did not stimulate bone resorption at lower concentrations (10^?7M – 10^?5M_, but was inhibitory at 10^?4M. Arachidonic acid also inhibited resorption at 10^?4M, but at lower concentrations (10^?7M – 10^?5M0 increased active resorption. This was concomitant with a rise in PGE₂ and PGF_2α levels, PGE₂ production being significantly higher than PGF_2α. The effects of PGE₂ (10^?8M) and PGF_2α (10^∞M appeared additive: there was no evidence of synergistic or antagonistic effects when varying ratios of PGE₂ : PGF_2α were employed.     

Determination of cyclooxygenase products and prostaglandin metabolites using high-pressure liquid chromatography and radioimmunoassay.

A method is described for measurement of the cyclooxygenase products, thromboxane,prostacyclin, and prostaglandins (PG), and several prostaglandin metabolites. The procedure involves separation of the compounds by high-pressure liquid chromatography combined with identification and estimation by serologic analysis. These combined procedures have been used to identify and estimate five such products, PGE₂, PGE₁ PGF2α, PGF_1α, and 6-keto-PGF_1α, in the culture fluids of dog kidney cells stimulated by a tumor-promoting phorbol diester. The prostaglandin metabolites, 13,14-dihydro-15-keto-PGE₂, 13,14-dihydro-15-keto-PF2_2α, 13,14-dihydro-PGE₂, and 13,14-dihydro-PGF_2α, were not found in these culture fluids.     

Catechol oestrogens stimulate and direct prostaglandin synthesis

We have tested the action of a catechol oestrogen -2,3,17β- trihydroxy oestra-1,3,5 (10)-triene (2-OH oestradiol) in stimulating prostaglandin (PG) production by an homogenate of rat uterus. Marked and dose dependent stimulation was observed in PGF_2α and PGE₂ production using 20–250 μM concentrations of catechol oestrogen; a concentration of 250 μM 2-OH oestradiol resulted in a 23 fold increase in PGF_2α production with a 50% reduction in the synthesis of 6-keto PGF_1α. Tryptophan, catechol and glutathione were without effect on PGF_2α and PGE₂ production whereas adrenalin stimulated the production of all PGs, although the increase was less than that seen with 2-OH oestradiol. Oestradiol had a slight stimulatory action on PGF_2α production which reached a maximum at around 40 μM but had a more marked stimulation of 6-keto PGF_1α formation. Stimulation of prostaglandin production by oestradiol and 2-OH oestradiol showed no variation at different stages of the rat oestrous cycle. The use of 5 to 100 mg of tissue/ml gave similar product distribution although the effect of catechol oestrogen both in terms of stimulation of E and F formation (expressed per mg of tissue) and in its action on product distribution was more marked at lower concentrations of tissue.     

The prostaglandin system and insulin release. Studies with the isolated perfused rat pancreas

Using the isolated perfused rat pancreas PGE₂ (1 μM and 10 μM) had no effect on basal or glucose (10 and 20 mM)-induced insulin release (IR). PGF_2α stimulated basal IR at 1 μM and inhibited IR at 10 μM. The glucose-induced IR was unaffected by this PG. Furosemide (5 and 10 mM) led to a monophasic IR at low glucose (glu) and to a potentiation of IR at high glu. Only high indomethacin (Indo) (50 μg/ml) inhibited glu-induced IR. The stimulatory effect of furosemide on IR could not be inhibited by indomethacin. However mepacrine (0.1 mM) abolished the furosemide effect. Also glu-induced IR was inhibited by mepacrine. Acetylsalicylic acid (30 mg/100 ml) had no significant influence on glu-induced IR.These findings provide evidence that phospholipase activation rather than increased PG synthesis might primarily be involved in the secretory process of insulin.     

Seminal plasma affects prostaglandin synthesis in the porcine oviduct

Seminal fluids introduced to the female reproductive tract at mating can affect subsequent events, such as ovulation, fertilization, conception, and pregnancy. Bioactive molecules present in seminal plasma can modify the cellular composition, structure, and function of local tissues and of tissues distal to the tract. The oviduct plays a decisive role in reproduction providing a beneficial milieu for gamete maturation, fertilization, and early embryonic development. Therefore we have investigated whether intrauterine infusion of seminal plasma can modulate prostaglandin (PG) synthesis in the porcine oviduct through regulation of gene and protein expression of enzymes of prostaglandin synthesis pathway. Among several enzymes involved in the prostaglandin synthesis pathway tested in the present study PGF_2α synthase (PTGFS) and prostaglandin 9-ketoreductase (CBR1), which convert PGE₂ to PGF_2α, expression were significantly down-regulated in the oviducts on Day 1 after seminal plasma infusion into the uterine horns. The effects of the treatment were transient and by Day 5 levels of PTGFS and CBR1 were comparable in seminal plasma-treated and control animals. Additionally, increased PGE₂ to PGF_2α and PGFM to PGF_2α ratios in the oviductal tissues were indicated. Our results clearly demonstrate that seminal plasma affects prostaglandin synthesis in the porcine oviduct. Altered PTGFS and CBR1 expression in consequence changed PGE₂ to PGF_2α and PGFM to PGF_2α ratios in the porcine oviduct.     

Immunological and non-immunological synthesis and release of prostaglandins and thromboxanes from isolated guinea pig trachea

Specific radioimmunoassays were used to demonstrate the synthesis by the guinea pig trachea of 6-keto PGF_1α, TxB₂, and PGF_2α in addition to PGE₂. The rank order of both spontaneous and stimulated release was PGE₂ > PGF_2α > 6-keto PGF_1α = TxB₂. Ovalbumin-induced prostanoid release from sensitized tissue was antigen-specific. The release was unlikely to be a secondary consequence of tracheal contraction since incubations with calcium ionophore A23187, at a concentration which produces an equivalent magnitude of contraction of sensitized trachea, did not induce a significant PG or Tx production. In contrast, significantly higher prostanoid synthesis was induced by A23187 in unsensitized than sensitized trachea. Thus sensitization altered the profile of arachidonic acid metabolism evoked by the ionophore.     

Influence of angiotensins (I,II & III) on prostaglandin production by minced rat renal medulla

Minced rat renal medulla was incubated for 30 min at 37 °C in the presence of angiotensin, I, II or III (100 ng/ml) to determine the existence of a direct stimulating effect on prostaglandin (PG) production. PGE₂, PGF_2α, 6-keto PGF_1α and Thromboxane B₂ (TXB₂)_were determined by radioimmunoassay.For analysis of data variance, the results were separated according to whether the net output of PGE₂ was above or below 1.5 ng PGE₂ equivalent/mg tissue/30 min. Under low-output conditions, angiotensin I, II or III stimulated PGE₂ production significantly (p<0.02) and tended to augment PGF_2α production, while under high-output conditions no effect on PGE₂ or PGF_2α production was observed.Under either output condition, angiotensin I, II or III had no effect on 6-keto PGF_1α and TXB₂.     

Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2alpha receptors.

Pretreatment of membranes for 1 hr at 4° with up to 0.1% Triton X-100 (TX-100) and sodium desoxycholate (SDC), resulted in a greater loss of [³H] prostaglandin (PG)F₂α binding compared to E₁ binding. Lubrol WX (LWX) tended to cause a greater loss of [³H]PGF₂α than E₁ binding. However, the differential loss was not as marked as with TX-100 or SDC. Triton X-305 was relatively ineffective, but loss of [³H]PGE₁ binding was greater than for PGF₂α. Increasing concentrations of dimethylsulfoxide (DMSO) progressively inhibited PGF₂α binding without affecting PGE₁ binding. The detergent, but not DMSO, induced losses of membrane PG binding were due to solubilization of the receptors. Greater amounts of membrane protein and phospholipids were solubilized at detergent (TX-100 and SDC) concentrations that solubilized 100% of PGE₁ receptors compared to 100% solubilization of F₂α receptors. Neither the duration of preincubation nor the amount of membrane protein chosen were responsible for differential PGE₁ and F₂α receptor losses. These differential membrane PG receptor losses raise the possibility of differences in PGE₁ and F₂α receptors association with the membrane structure.     

Effect of oestradiol 17 β on prostaglandin biosynthesis by the uterus of ovariectomized rat and guinea pig

$\text{In vitro}$ prostaglandin biosynthesis by uteri of ovariectomized rats and guinea pigs treated or untreated with oestradiol 17 β, administered subsutaneously, was measured by R.I.A. of PGF_2α and PGE₂. Incubations with [1-¹⁴C] arachidonic acid were also performed and labelled metabolites were analyzed by TLC. The main metabolite in rats was 6 keto PGF_1α and in decreasing order of magniture, PGF_2α and PGE₂. In guinea pig PGF_2ga was the main product. Ovariectomy in rats completely changed the pattern of synthesized prostanoids: PGI₂ production was doubled when compared to cycling rats and PGE₂ increased 10 fold. PGF_2α walues were similar to the mean value measured during the cycle. OE₂ treatment almost completely inhibited PGI₂ synthesis and reduced PGE₂ by half. Total PG synthesis in OE₂ treated animals was decreased by 5 fold when compared to spayed rats. Endogenous PGF_sα synthesis was slightly stimulated. In the guinea pig OE₂ treatment of ovariectomized animals increased the total synthesis from 50 per cent. PGF_2α was always the main metabolite. In conclusion OE₂ regulation of uterine PG synthesis is depending on the animal species and cannot be explained by a unique effect on the cyclooxyhenase, but rather by an interplay on the various enzymes of the arachidonic acid cascade.     

The in vitro effect of indomethacin on basal bone resorption, on prostaglandin production and on the response to added prostaglandins

Mouse calvaria were maintained in organ culture without serum additives. Basal active resorption, as measured by ⁴⁵Ca and hydroxyproline release, was significantly inhibited to 74% control levels by indomethacin (1.4 × 10⁻⁷ M). Prostaglandin F and prostaglandin E₂ production, determined by radioimmunoassay, were both significantly lowered by this concentration of indomethacin. DNA, protein and hydroxyproline synthesis, as indices of cell toxicity, were unaffected by low concentrations of indomethacin, while concentrations of 1.4 × 10⁻⁶M inhibited protein synthesis (p<0.005). In the presence of indomethacin (1.4 × 10⁻⁷M) both PGE₂ and PGF_2α stimulated resorption in a dose-dependent manner, with PGE₂ being the more potent. Neither prostaglandin affected hydroxyproline synthesis at low concentrations, but PGE₂ had a marked inhibitory action at a higher concentration (10⁻⁶M). In combination, the effects of PGE₂ and PGF_2α showed no evidence of synergism or any antagonistic action. The study shows that in vitro calcium and hydroxyproline resorption in the unstimulated mouse calvaria are inhibited by indomethacin at concentrations measured in serum during human therapy. The decreased PGF and PGE₂ production associated with this decreased bone resorption in the presence of non-toxic concentrations of indomethacin would suggest a role for these prostaglandins in maintaining the basal resorption of cultured bone.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Comparative studies of prostaglandins F2α and E2 in late cyclic and early pregnant sheep: In vitro synthesis by endometrium and conceptus effects of in vivo indomethacin treatment on establishment of pregnancy

Endometrial concentrations of prostaglandins F₂α (PGF₂α) and E₂ (PGE₂) were measured by specific radioimmunoassay in sheep, on day 14 of estrous cycle or pregnancy, during luteolysis (Day 16 of the cycle), and after implantation (Day 23 of pregnancy) : concentrations observed on day 14 of cycle and pregnancy were similar. During luteolysis, on day 16 of cycle, a consistent drop was noticed. If luteal regression did not occur, as a consequence of the presence of an embryo, endometrial concentrations of PGF₂α on day 23, were twice those of day 14, and PGE₂ remained unchanged. $\text{In vitro}$ 2 hour incubations of endometrial caruncular tissue from 14 days cyclic or pregnant ewes resulted in de novo synthesis of PG which could be increased by Arachidonic Acid and inhibited by Indomethacin; during the first 30 min of incubation, the PGF₂α synthesis was comparable for both endometrial tissues, whereas PGE₂ synthesis was twice as great in pregnant endometrium. Fourteen and 23 day conceptuses had high PGF₂α and PGE₂ concentrations which were not due to maternal PG sequestration : $\text{de novo}$ PG synthesis which could be inhibited by Indomethacin was observed in incubated 14 day old embryos. Treatment of pregnant ewes from day 7 to day 22 after mating, either with Indomethacin (300 mg s.c. daily) or with Acetylsalicylic Acid (1 g I.V. daily) resulted in a sharp diminution of endometrial PG concentration and release, with no apparent effect on the establishment of pregnancy. These results tend to ascribe a less important role to PG during early pregnancy in sheep as compared with rodents, in terms of embryonic growth and implantation.