Leukotriene synthesis inhibition and anti-ige challenge of human lung parenchyma

The leukotriene (LT) synthesis inhibitors BAY x1005 and MK-886 were evaluated in human lung parenchyma challenged with an anti-IgE. The anti-IgE-induced LTE₄ release was time- and dose-dependent. Treatment of the parenchyma with indomethacin (3 μM) prior to anti- IgE challenge inhibited the 6-keto prostaglandin F_1α (6-keto PGF_1α) release and enhanced (36%) the quantities of LTE₄ detected during IgE-stimulations. BAY x1005 and MK-886 were assessed in the presence of indomethacin (3 μM) and the IC₅₀ values for both inhibitors were similar (0.13 μM). BAY x1005 (1 μM) produced the same percent of inhibition of anti-IgE-induced LTE₄ release in the presence or absence of indomethacin. BAY x1005 (1 μM) did not alter the 6-keto PGF_1α release during anti-IgE challenge. The results indicate that BAY x1005 and MK-886 are potent inhibitors of LT synthesis when human lung parenchyma were stimulated by an anti-IgE.     

Stereoselective cyclooxygenase inhibition in cellular models by the enantiomers of ketoprofen

The pharmacological activity of rac-ketoprofen and its enantiomers was investigated in vitro using different cellular models. The effect of these compounds on arachidonic acid metabolism was assessed by measuring the inhibition of prostanoid generation under the action of several agonists. Thus, we have evaluated the inhibition of (1) thromboxane B₂ synthesis in rabbit platelets and human polymorphonuclear leukocytes (PMNs), (2) prostaglandin E₂ synthesis in three cultured cells, namely human umbilical vein endothelial cells (HUVEC), human keratinocytes, and mouse macrophage-like P388D1 cells. The IC₅₀ values found for (+)-(S)-ketoprofen were in the range between 0.1 nM and 0.8 μM, being slightly lower in all models than those found for rac-ketoprofen (0.4 nM–3 μM). On the other hand, (?)-(R)-ketoprofen showed inhibition of cyclooxygenase only at concentrations two or three orders of magnitude higher than those required for the (+)-(S) enantiomer. These results, obtained with cell types of relevance for inflammatory processes and with compounds of high optical purity, demonstrate that the prostanoid biosynthesis inhibition caused by the drug rac-ketoprofen is exclusively due to its dextrorotatory enantiomer. © 1993 Wiley-Liss, Inc.     

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.     

Prostaglandin D2, a potential antineoplastic agent

Cytotoxic actions of various prostaglandins were examined on L1210 mouse leukemia and several human leukemia cell lines, and prostaglandin D₂ (PGD₂) was found most active. PGD₂ exerted a dose dependent inhibition of L1210 cell growth over 3.6 μ$\text{M}$. At 14.3 μ$\text{M}$ growth was completely inhibited, and the number of viable cells remarkably decreased during culture. Microscopically the remaining cells showed degenerative changes with many vacuoles in their cytoplasm. The IC₅₀ value of PGD₂ on L1210 cell growth was calculated to be 6.9 μ$\text{M}$ (2.4 μg/ml), and at this concentration the DNA synthesis in 24 hr cultured cells was also decreased to a half of the level in the control cells. Such growth inhibition by PGD₂ was also found at similar concentrations with several human leukemia cell lines such as NALL-1, RPMI-8226, RPMI-8402, and Sk-Ly-16. Among other prostaglandins tested, PGA₂ showed a comparable, and PGE₂ a less but significant growth inhibitory activity, while PGB₂, PGF_2α and PGI₂ had no such effects on cell proliferation at 14.3 μ$\text{M}$ concentration. These results suggest a potential antineoplastic activity of PGD₂.     

Production of prostaglandin F2α by molecular breeding of an oleaginous fungus Mortierella alpina

Cyclooxygenases are responsible for the production of prostaglandin H₂ (PGH₂) from arachidonic acid. PGH₂ can be converted into some bioactive prostaglandins, including prostaglandin F_2α (PGF_2α), a potent chemical messenger used as a biological regulator in the fields of obstetrics and gynecology. The chemical messenger PGF_2α has been industrially produced by chemical synthesis. To develop a biotechnological process, in which PGF_2α can be produced by a microorganism, we transformed an oleaginous fungus, Mortierella alpina 1S-4, rich in triacylglycerol consisting of arachidonic acid using a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla. PGF_2α was accumulated not only in the mycelia of the transformants but also in the extracellular medium. After 12 days of cultivation approximately 860 ng/g and 6421 µg/L of PGF_2α were accumulated in mycelia and the extracellular medium, respectively. The results could facilitate the development of novel fermentative methods for the production of prostanoids using an oleaginous fungus.     

Mevalonate dependency of the early cell cycle mitogenic response to epidermal growth factor and prostaglandin F2α in Swiss mouse 3T3 cells

Lovastatin (LOV), a hydroxy-methylglutaryl-coenzyme A (HMGCoA) reductase competitive inhibitor, blocks epidermal growth factor (EGF)— or prostaglandin F_2α (PGF_2α)—induced mitogenesis in confluent resting Swiss 3T3 cells. This inhibition occurs even in the presence of insulin, which potentiates the action of these mitogens in such cells. LOV exerts its effect in a 2–80 μM concentration range, with both mitogens attaining 50% inhibition at 7.5 μM. LOV exerted its effect within 0–8 h following mitogenic induction. Mevanolactone (10–80 μM) in the presence of LOV could reverse LOV inhibition within a similar time period. LOV-induced blockage of PGF_2α response is reflected in a decrease in the rate of cell entry into S phase. Neither cholesterol, ubiquinone, nor dolichols of various lengths could revert LOV blockage. In EGF- or PGF_2α-stimulated cells, LOV did not inhibit [³H]leucine or [³H]mannose incorporation into proteins, while tunicamycin, an inhibitor of N′ glycosylation, prevented this last phenomenon. Thus, it appears that LOV exerts its action neither by inhibiting unspecific protein synthesis nor by impairing the N′ glycosylation process. These findings strongly suggest that either EGF or PGF_2α stimulations generate early cell cycle signals which induce mevalonate formation, N′ glycoprotein synthesis, and proliferation. The causal relationship of these events to various mechanisms controlling the onset of DNA synthesis is also discussed. © 1995 Wiley-Liss, Inc.     

In vitro inhibition of aromatase by the enantiomers of aminoglutethimide and analogs

The in vitro aromatase activity in microsomal fractions from rat ovary and its inhibition by enantiomers of aminoglutethimide (AG), rogletimide (RG), and cyclohexylaminoglutethimide (ChAG) were studied by analysing the [³H]H₂O released when [1β-³H]androstenedione was converted to estrone. Maximum velocity (V_max) and the Michaelis-Menten constant (K_m) of the microsomal aromatase enzyme were 17.40 ± 0.45 pmol/ml/mg protein/min and 1.02 ± 0.06 μM, respectively. The IC₅₀s for the enantiomers were similar for (+)-R-AG and (?)-R-ChAG (0.86 ± 0.06 and 0.89 ± 0.15 μM, respectively). (+)S-ChA'G was most potent with IC₅₀ of 0.075 ± 0.003 μM. The IC₅₀s for (?)-S-AG, (+)-R-RG, and (?)-S-RG were in the same range (23.15 ± 2.74, 24.58 ± 2.46, and 24.43 ± 2.20 μM, respectively). © 1994 Wiley-Liss, Inc.     

Differences in the central actions of arachidonic acid and prostaglandin F2α between spontaneously hypertensive and normotensive rats

Prostaglandin F_2α (PGF_2α) is one of the most common metabolites of arachidonic acid (AA) in rat brain. When administered intracerebroventricularly (i.c.v.) to rats, both AA and PGF_2α exert dose-related hypertensive, tachycardic and hyperthermic effects. Metabolic alterations in the endogenous formation of some prostaglandins in the brain-stem of spontaneously hypertensive rats (SHR) have been reported. Therefore the central effects of AA and PGF_2α on blood pressure, heart rate and body temperature were studied both in SHR and normotensive Wistar rats (NR) under urethane-anaesthesia. The hypertensive effect of AA i.c.v. (0.01–100 μg/rat) was larger in magnitude in SHR than in NR, but there was no significant difference in the AA-induced changes of heart rate and body temperature between the groups. Pretreatment of NR with sodium meclofenamate (1 mg/rat i.c.v.) antagonised the central effects of AA indicating that these effects are not due to AA itself but to its conversion to prostaglandins. Unlike the effects of AA, the central hypertensive, tachycardic and hyperthermic responses to PGF_2α (0.5–50 μg/rat i.c.v.) were significantly attenuated in SHR. The present results obtained with AA are compatible with the previous assumption that the synthesis of prostaglandins in the brain of SHR might differ from that in NR. The results also demonstrate that the central effects of PGF_2α are reduced in SHR.     

Stimulative effect of prostaglandins on production of hexosamine-containing substances by cultured fibroblasts (2) early effect of various prostaglandins at various doses

Early effects of various prostaglandins on the production of hexosamine-containing substances by cultured fibroblasts, which were derived from a rat carrageenin granuloma, were studied. At the stationary phase, the cells were exposed for 6 h to one of the prostaglandin A₁ (PGA₁), A₂, B₁, B₂, D₂, F_1α, F_2α, E₁, E₂ or arachidonic acid in various concentrations ranging from 0.01 to 10 μg/ml for all the stimuli and from 10 pg to 10 μg/ml for PGF_2α. The activity of the cells in incorporating ³H-glucosamine into hexosamine-containing substances (acidic) glycosaminoglycans and glycoproteins) during this period was compared with that of control cells. All the stimuli tested showed more or less stimulative effect on the synthesis of hexosamine-containing substances at their specific concentrations. PGF_2α was found to be the most potent stimulant and its stimulative effect was found significant even at the low concentration of 100 pg/ml. PGD₂, F_1α and E₂ were the next potent stimuli. Their optimum dose were around 1 μg/ml but they still had significant stimulation at the concentration of 0.01 μg/ml. Effect of PGE₂ was rather mild. Stimulation by PGA₁, A₂, B₁ and B₂ or arachidonic acid was seen at high dose, and its seemed to be non-specific. The results suggested that these prostaglandins such as PGF_2α, D₂, F_1α and E₂ play some important role on regulating the production of intercellular ground substances.     

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.     

Inhibition of ADH-enhanced transepithelial urea and water movement by prostaglandins

The effects of PGF_2α and PGE₂ on transepithelial urea flux and osmotic water flow were evaluated in toad bladders. Mucosal to serosal urea flux and osmotic water flow were not changed from basal values by the addition of either prostaglandin to the serosal bath. However, treatment with either PGF_2α or PGE₂ inhibited both urea flux and osmotic water flow in response to ADH stimulation in a concentration-dependent manner. The hydrosmotic response to ADH was more sensitive to prostaglandin inhibition than was urea flux. The inhibitory effect of the prostaglandins on ADH-enhanced urea flux was not dependent upon inhibition of the hydrosmotic response, since both PGF_2α and PGE₂ decreased urea flux in the absence of a trans-epithelial osmotic gradient. Prostaglandin E₂ was a more potent inhibitor than PGE_2α of both ADH-enhanced urea flux and osmotic water flow. The PGF_2α antagonism of osmotic water flow was apparently competitive, while antagonism of urea flux was apparently non-competitive. The results are consistent with the hypothesis of the existence of a “spare” population of prostaglandin receptors that modulate water flow, but the absence of a “spare” prostaglandin receptor population with respect to the modulation of urea flux.     

Measurement of prostaglandins in embryonic tissue using radioimmunoassay

Although prostaglandins appear to play an important role in numerous physiological processes in the adult, neonate, and fetus, very little is known about the role of these compounds in the embryo. This study demonstrates that rat embryo homogenates synthesized 6-oxo-PGF_1α; PGE and PGF in markedly different amounts from endogenous substrate. Synthesis was inhibited by indomethacin (10 μM) in varying degrees (70–89%) depending on the prostaglandin. The metabolite of PGF_2α, 13,14-dihydro-15-keto PGF_2α (PGF_2α-M), was produced in limited amounts in the absence of exogenous NAD. In the presence of exogenous NAD and PGF however, embryonic homogenates produced PGF_2α-M. The potential role of prostaglandins during embryogenesis is discussed.     

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.     

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2α

The inhibition of human platelet aggregation produced by PGF_2α is not specific for thromboxane A₂ mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF_2α (8 μM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH₂). In contrast the thromboxane receptor antagonist EP 045 (up to 20 μM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC₅₀ = 0.5 μM), displaces the specific binding of [³H] 9,11-epoxymethano PGH₂ to washed human platelets.PGF_2α produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF_2α is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF_2α.We suggest that the very weak effect of PGF_2α on cyclic AMP_ production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.     

Effect of polyamines on prostaglandin synthesis in various cell-free systems

Synthesis of PGF_2α by bovine uterus and guinea pig lung microsomes and that of TXB₂ by human platelet and rat spleen microsomes were stimulated by spermine. PGE₂ synthesis by bovine seminal vesicle and porcine lung microsomes, and 6-keto-PGF_1α synthesis by bovine seminal vesicle and uterus microsomes were inhibited by spermine. When phospholipid-free prostaglandin synthetase from bovine seminal vesicle was used instead of microsomes, the inhibition of PGE₂ synthesis by spermine disappeared. The inhibition of PGE₂ synthesis by spermine gradually appeared with an increase of phospholipid added. Among phospholipids tested, phosphatidylcholine was the most effective for the inhibition of PGE₂ synthesis by spermine.     

Luteal function in sheep with ovarian autotransplants given concurrent infusions of prolactin and prostaglandin F2α into the ovarian artery

Four experimental sheep with ovarian autotransplants were infused with prolactin (10 or 100 μg/h), into the ovarian artery, for 6 hours and then prolactin together with prostaglandin F_2α (PGF_2α) (5 μg/h) for a further 6 hours. A control sheep received PGF_2α alone, for 6 hours on two separate occasions. Prolactin failed to overcome the luteolytic action of PGF_2α in 3 of the 4 experimental sheep.     

Stimulatory effect of prostaglandins on the production of hexosamine-containing substance by cultured fibroblasts (3) induction of hyaluronic acid synthetase by prostaglandin F2α

The mechanism of the stimulatory effect of prostaglandin(PG) F_2α on the production of hexosamine-containing substance by cultured fibroblasts was studied. Treatment of the cells with 1 μg/ml of PGF_2α resulted in a doubled net synthesis of acidic glycosaminoglycans during 20 hrs measured with uronic acid as index, and also resulted in 300 per cent increase of ³H-glucosamine incorporation into hexosamine-containing substances during the first 6 hrs. Fractionation of the PGF_2α-stimulated hexosamine-containing substances with double isotope technique revealed that hyaluronic acid was the most stimulated component. Prior to the increase of hyaluronic acid, hyaluronic acid synthetase activity was found to be augmented by PGF_2α as high as 4 times over the control. The augmentation of hyaluronic acid synthetase activity by PGF_2α did not take place if actinomycin D was simultaneously present in the culture medium, suggesting that PGF_2α induced the enzyme.     

Evidence that Prostaglandin E2 Can Block Calcium-Activated 86Rb Efflux from Rat Brain Synaptosomes via a Protein Kinase C-Dependent Mechanism

Abstract: The effects of prostaglandin E₂ (PGE₂) on ⁸⁶Rb efflux from rat brain synaptosomes were studied to explore its role in nerve ending potassium (K⁺) channel modulation. A selective dose-dependent inhibition of the calcium-activated charybdotoxin-sensitive component of efflux was found upon application of PGE₂. No significant effect was seen on basal and voltage-dependent components over the concentration range of 10–⁸ to 10–⁵M. The protein kinase C (PKC) inhibitors H-7 (10 μM) and staurosporine (100 nM), as well as prolonged preincubation (90 min) with 40-phorbol 12, 13-dibutyrate, which has been reported to down-regulate PKC, abolished the PGE₂-in- duced inhibition, whereas HA1004 (10 μM) and Rp-3′,5’cyclic phosphorothioate (100 nM), which are relatively more selective for protein kinase A than PKC, did not. 4β-Phorbol 12, 13-dibutyrate (100 nM), an activator of PKC, produced a similar inhibition of the Ca²⁺-dependent component of ⁸⁶Rb efflux but also had no effect on the basal and voltage-dependent components. These data suggest that PGE₂ can inhibit rat brain nerve ending calcium-activated ⁸⁶Rb efflux, and this inhibition may involve PKC activation.     

Luteal function in sheep injected with prostaglandin F2α directly into the corpus luteum

Groups of ewes received either saline or prostaglandin F_2α (PGF_2α) as an injection directly into the corpus luteum. Changes in circulating progesterone levels were measured as well as subsequent histological examination of the corpora lutea. Saline, or PGF_2α given at the two lower doses (60 and 120 μg respectively), failed to suppress progesterone levels permanently, or to induce degenerative changes in the corpora lutea. Treatment with a higher dose of PGF_2α (240 μg) was followed by a marked elevation in progesterone levels. These results are discussed in relation to reported effects of PGF_2α arriving at the ovary via the arterial circulation.     

Effect of acetaminophen on prostaglandin E2 and prostaglandin F2α synthesis in the renal inner medulla of rat

Effects of acetaminophen on the renal inner medullary production of prostaglandin E₂ and F_2α were compared with the well-known effects of aspirin on this process. Acetaminophen was found to elicit a dose-dependent inhibition of both prostaglandin E₂ and F_2α accumulation in media with a K_i of 100–200 μM. This inhibition could not be accounted for by increased accumulation of prostaglandins within slices. Acetaminophen inhibition was reversed by removal of acetaminophen during the incubation or by addition of arachidonic acid. Similar manipulations did not reverse aspirin or indomethacin-mediated inhibition of prostaglandin synthesis. Thin-layer and gas chromatographic analysis of acetaminophen following incubation with slices demonstrated that this material was identical to authentic acetaminophen. This, in addition to the lack of an effect of glutathione on inhibition, suggests that acetaminophen does not have to be metabolized to exert this inhibition. Arachidonic acid did not alter the metabolism or increase the efflux of acetaminophen. Lower levels of prostaglandin E₂ observed with 5 mM acetaminophen and 1 mM aspirin caused a corresponding decrease in cyclic AMP content. Removal of acetaminophen from the second incubation or addition of arachidonic acid caused increases in both prostaglandin E₂ and cyclic AMP. Aspirin inhibition of cyclic AMP content was not reversed by similar manipulations. In vivo inhibition of inner medullary prostaglandin E₂ and prostaglandin F_2α synthesis was observed 2 h after a 375 mg/kg, intraperitoneal injection of acetaminophen. These data suggest that acetaminophen, like aspirin, is capable of reducing tissue prostaglandin synthesis. However, the mechanisms by which these two analgesic and antipyretic agents elicit their inhibition of prostaglandin synthesis are quite different.