Effects of pro-inflammatory cytokines,lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability

Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1β and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE₂, PGF_2α) or their corresponding ethanolamides (PGE₂-EA or PGF_2α-EA) over 48 h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1β and bacterial LPS incubations in an explant setting over 20 h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10⁻⁵ M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10⁻⁴ M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE₂ transiently increased Caco-2 monolayer permeability at 24 h, while LPS (10 μg/ml) increased permeability over 24–48 h.These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE₂ transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.     

The effect of tumor necrosis factor α (TNFα), interleukin 1β (IL1β) and interleukin 6 (IL6) on endometrial PGF2α synthesis, metabolism and release in early-pregnant pigs

Cytokines produced by the porcine uterus and embryos may be involved in the regulation of endometrial prostaglandin synthesis, metabolism, and release. We studied the effect of tumor necrosis factor α (TNFα), interleukin 1β (IL1β) and interleukin 6 (IL6) on: 1) endometrial release of prostaglandin F₂α (PGF₂α), 2) expression of the terminal enzyme of PGF₂α synthesis - PGF synthase mRNA (PGFS mRNA), 3) secretion of PGF₂α metabolite - 13,14-dihydro-15-keto PGF₂α (PGFM) by the endometrium and 4) presence and activity of endometrial NAD-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The effects of cytokines were determined on days 10-11 and days 12-13, e.g., before and during maternal recognition of pregnancy, and on days 15-16, e.g., during the peri-implantation period and compared with its effect in cyclic gilts on corresponding days of the estrous cycle. TNFα did not affect endometrial release of PGF₂α in pregnant and cyclic pigs. IL1β enhanced endometrial PGF₂α release on days 12-13 and 15-16 in pregnant and cyclic pigs, respectively. IL6 increased PGF₂α release mainly on days 15-16 of pregnancy. Expression of PGFS mRNA was decreased by IL1β on days 12-13 of pregnancy (P < 0.05) and increased in response to IL1β, TNFα and IL6 on 12-13 (P < 0.05) and 15-16 (P < 0.01) of the estrous cycle. IL1β increased release of PGFM in gravid pigs on days 12-13, 15-16 and in non-gravid pigs 10-11 and 15-16 of the cycle. On days 15-16 of pregnancy TNFα and IL6 increased endometrial secretion of PGFM. We determined that in porcine endometrium NAD-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is present. In gravid pigs, the highest expression of endometrial 15-PGDH occurred during days 12-13 of pregnancy, while in non-gravid pigs during days 10-11 of the estrous cycle. These data provide new evidence that TNFα, IL1β, IL6 are involved in the regulation of endometrial synthesis, release and metabolism of PGF₂α to protect CL during early pregnancy or to facilitate its regression in cyclic females.     

Cytokine regulation of adult human osteoblast-like cell prostaglandin biosynthesis

Prostaglandin (PG) biosynthesis by cytokine stimulated normal adult human osteoblast-like (hOB) cells was evaluated by thin layer chromatography, high performance liquid chromatography, and specific immunoassays. PGE₂ was the predominant PG formed under all incubation conditions tested. Control samples produced measurable amounts of PGE₂, and the measured level of this metabolite increased by 22-fold (from 7 to 152 ng/ml) following a 20 h treatment with the combination of TGFβ and tumor necrosis factor-α(TNF). The production of 6-keto-PGF_1α (the stable metabolite of prostacyclin) and of PGF_2α were each increased by about five-fold (from about 0.5 to 2.5 ng/ml) in samples treated with the cytokines. Thus, TGFβ and TNF exerted a regulation of hOB cell PG biosynthesis that was principally directed towards an increased PGE₂ biosynthesis, with lesser effects on the production of other PG metabolites. COX-2 mRNA levels were increased within 2 h of cytokine stimulation, reached a maximum at 6–12 h, and levels had appreciably diminished by 24 h after treatment. Both TGFβ and TNF could independently increase COX-2 mRNA levels and PG biosynthesis. However, the increased production of PGE₂ resulting from TNF stimulation was blocked by the addition of an interleukin-1β (IL-1β) neutralizing antibody, suggesting that TNF regulation of hOB cell PG synthesis was secondary to its capacity to increase hOB cell IL-1β production. TGFβ regulation of PG production was not affected by the addition of the neutralizing antibody. These studies support the proposition that PGs can be important autocrine/paracrine mediators of bone biology, whose production by hOB cells is responsively regulated by osteotropic cytokines. J. Cell. Biochem. 64:618–631. © 1997 Wiley-Liss, Inc.     

Interleukin-1 binding and prostaglandin E2 synthesis by amnion cells in culture: regulation by tumor necrosis factor-α, transforming growth factor-β, and interleukin-1 receptor antagonist

Proinflammatory cytokines may promote preterm labor in the setting of intrauterine infection. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) synergistically stimulate the production of prostaglandin E₂ (PGE₂) by amnion cells. Transforming growth factor-β (TGF-β) inhibits the cytokine-stimulated PGE₂ production. In the present study, we investigated the binding of IL-1β on human amnion cells in culture. Untreated amnion cells possessed 540±60 IL-1 receptors per cell, with a dissociation constant of 1.4±0.4 nM. Cells treated with TGF-β1 (10 ng/ml) had 570±110 receptors per cell. TNF-α (50 ng/ml) increased the number of IL-1 receptors to 2930±590. TGF-β1 inhibited the receptor upregulation by TNF-α. Cells treated with TGF-β1 and TNF-α expressed 1140±590 receptors per cell. The binding affinity was not changed by the cytokines. IL-1 receptor antagonist (IL-1ra) inhibited the stimulation of amnion cell PGE₂ production by IL-1β, but not by TNF-α. Amnion cells secreted large amounts of IL-1ra (1.1±0.3 ng/10⁵ cells). Treatment of the cells with TGF-β1 or TNF-α did not affect the release of IL-1ra. We conclude that IL-1 receptor expression is an important step in the regulation of the effects of cytokines on amnion cell PGE₂ production.     

Tranilast, an orally active anti-allergic drug, up-regulates the anti-inflammatory heme oxygenase-1 expression but down-regulates the pro-inflammatory cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW264.7 macrophages

Tranilast (N-[3′,4′-dimethoxycinnamonyl] anthranilic acid), an orally active anti-allergic drug, is reported to exert the anti-inflammatory effects, but the underlying mechanisms that could explain the anti-inflammatory actions of tranilast remain largely unknown. Here, we found that tranilast induces heme oxygenase-1 (HO-1) expression through the extracellular signal-regulated kinase-1/2 (ERK1/2) pathway in RAW264.7 macrophages. Tranilast suppressed cyclooxygenase-2 (COX-2) and inducible nitric oxide (NO) synthase (iNOS) expression, and thereby reduced COX-2-derived prostaglandin E₂ (PGE₂) and iNOS-derived NO production in lipopolysaccharide (LPS)-stimulated macrophages. Similarly, tranilast diminished tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) production. Interestingly, the effects of tranilast on LPS-induced PGE₂, NO, TNF-α, and IL-1β production were partially reversed by the HO-1 inhibitor tin protoporphyrin, suggesting that tranilast-induced HO-1 expression is at least partly responsible for the resulting anti-inflammatory effects of the drug. Thus, HO-1 expression via ERK1/2 activation may be at least one of the possible mechanisms explaining the anti-inflammatory actions of tranilast.     

Eicosanoid production by placental and amnion tissues from control and non-insulin-dependent diabetic rats. Influence of oxytocin in the incubating medium

Eicosanoid production by intrauterine tissues from control and neonatal-steptozotocin induced diabetic rats during late pregnancy was evaluated. In diabetic placenta the release of 6-keto-PGF_1α was found diminished when compared to controls. In addition, LTB₄ generation was increased in diabetic placenta. No alterations in the production of TXA₂, PGE₂, PGE₁ and PGF_2α was found when diabetic and control placenta were compared. In amnion tissue a decreased generation of 6-keto-PGF_1α was observed in the diabetic group, but no alteration in any other eicosanoid evaluated was found. Oxytocin (5 mU/ml, in vitro), which increases prostaglandin synthesis in rabbit and human amnion tissues, did not modify eicosanoid generation in control rat amnion. In contrast, in diabetic amnion the presence of oxytocin further decreased the release of 6-keto-PGF_1α and diminished PGE₁ generation. The present results suggest that this mildy diabetic state induces alterations in eicosanoid production in intrauterine tissues, abnormalities probably enhanced during parturition, when endogenous concentrations of oxytocin are elevated.     

Identification of prostamides,fatty acyl ethanolamines,and their biosynthetic precursors in rabbit cornea

Arachidonoyl ethanolamine (anandamide) and pros­taglandin ethanolamines (prostamides) are biologically active derivatives of arachidonic acid. Although available through different precursor phospholipids, there is considerable overlap between the biosynthetic pathways of arachidonic acid-derived eicosanoids and anandamide-derived prostamides. Prostamides exhibit physiological actions and are involved in ocular hypotension, smooth muscle contraction, and inflammatory pain. Although topical application of bimatoprost, a structural analog of prostaglandin F_2α ethanolamide (PGF_2α-EA), is currently a first-line treatment for ocular hypertension, the endogenous production of prostamides and their biochemical precursors in corneal tissue has not yet been reported. In this study, we report the presence of anandamide, palmitoyl-, stearoyl-, α-linolenoyl docosahexaenoyl-, linoleoyl-, and oleoyl-ethanolamines in rabbit cornea, and following treatment with anandamide, the formation of PGF_2α-EA, PGE₂-EA, PGD₂-EA by corneal extracts (all analyzed by LC/ESI-MS/MS). A number of N-acyl phosphatidylethanolamines, precursors of anandamide and other fatty acyl ethanolamines, were also identified in corneal lipid extracts using ESI-MS/MS. These findings suggest that the prostamide and fatty acid ethanolamine pathways are operational in the cornea and may provide valuable insight into corneal physiology and their potential influence on adjacent tissues and the aqueous humor.     

Binding of glyceraldehyde-3-phosphate dehydrogenase to the cis-acting element of structure-anchored repression in ccn2 mRNA

Studies increasingly indicate that inflammation induced by β-amyloid (Aβ) contributes to the progression of Alzheimer’s disease (AD). How to inhibit the enhanced production of proinflammatory cytokines stimulated by Aβ is an important research subject for the treatment of AD. In this study, we investigated the inhibitory effect and the molecular mechanism of the lipoxin A₄ (LXA₄) on the production of interleukin-1β (IL-1β) and tumor necrosis factorα (TNFα) induced by β-amyloid in the cortex and hippocampus of mice, and in Aβ-stimulated BV2 cells, a mouse microglial cell line. LXA₄ down-regulated the protein expression of IL-1β and TNFα, attenuated the gene expressions of IL-1β and TNFα, inhibited the degradation of IκBα, inhibited translocation of NF-κB p65 subunit into the nucleus induced by β-amyloid in the cortex and hippocampus of mice, and in Aβ-stimulated BV2 cells, and the inhibitory effects were dose dependently elevated. Our findings suggest that LXA₄ inhibits the production of IL-1β and TNFα induced by β-amyloid in the cortex and hippocampus of mice, and in BV2 microglial cells via the NF-κB signal pathway.     

Temporal effect of prostaglandins E and F on human adrenal cAMP and cortisol output.

The $\text{in vitro}$ modulating effects of the E and F series prostaglandins upon the cAMP and cortisol output of normal human adrenal dice were evaluated with time and compared to the effects of ACTH. PGE₁ and PGE₂ significantly increased human adrenal cAMP levels; cortisol output increased in a dose related manner. Although the cortisol levels produced by E prostaglandins and ACTH were quantitatively similar, on a molar basis ACTH was greater than 10⁶ fold more effective. PGE_1α, PGF_2α, PGF_1β and PGF_2β depressed adrenal cAMP, except PGF_2α and PGF_2β at 100 μg/ml. PGF_1α and PGF_2α depressed cortisol levels at all doses. Similarly, PGF_1β and PGF_2β also depressed cortisol output, except PGF_2β at 100 μg/ml which was stimulatory. In both series of prostaglandins the temporal responses were dose related in regard to the cyclic nucleotide and steroid alterations. The findings demonstrate the E and F series prostaglandins act antagonistically in respect to cAMP and cortisol output. In addition, as the cAMP level and cortisol output are not always correlated, it appears that these prostaglandin mediated events are separable. The relationship between adrenal prostaglandins and cyclic nucleotides, therefore, invites a more sophisticated second messenger concept in terms of adrenocortical function, than currently proposed.     

Effect of estrus induction on prostaglandin content and prostaglandin synthesis enzyme expression in the uterus of early pregnant pigs

Prostaglandins (PGs) play a pivotal role in maternal recognition of pregnancy and implantation in pigs. In the present study, PGE₂, PGF_2α, and PGFM (PGF_2α metabolite) content, as well as PGE₂ synthase (mPGES-1) and PGF_2α synthase (PGFS) expression was investigated in early pregnant gilts with natural (n = 21) and PMSG/hCG-stimulated (n = 19) estrus. Endometrial tissue samples, uterine luminal flushings (ULFs), and blood serum were collected on days 10-11, 12, and 15 after insemination. Additionally, day 15 conceptuses were collected for mPGES-1 and PGFS protein expression. Effect of estrus induction was observed on day 15 of pregnancy, when the content of PGE₂ in the uterine lumen was fourfold lower in gonadotropin-stimulated gilts in comparison to controls (P < 0.001). Decreased PGE₂ content in ULFs of gonadotropin-treated pigs was preceded by lower endometrial mPGES-1 gene expression in hormonally-stimulated animals in comparison to control gilts (P < 0.01). On the other hand, estrus induction with PMSG/hCG resulted in higher PGE₂ accumulation in the endometrial tissue on day 15 of pregnancy (P < 0.01). Furthermore, PGF_2α content in the endometrium and PGFM levels in blood serum were lower in gonadotropin-treated gilts, especially on day 12 after insemination when compared to control gilts (P < 0.01). Finally, PGFS expression in day 15 conceptuses was decreased in animals with hormonally-induced estrus. We conclude that PMSG/hCG stimulation of prepubertal gilts to induce estrus results in changes of PG production and secretion during early pregnancy, which, in turn, may affect conceptus development, implantation, and the course of pregnancy.     

Lipopolysaccharide from Rhodobacter capsulatus counteracts the effects of toxic lipopolysaccharides and inhibits the release of TNF-α, IL-6, and IL-1β in human whole blood

The outcome of pathological process during sepsis caused by Gram-negative bacteria depends on the reaction of human blood cells to bacterial structural components, lipopolysaccharides (LPS). A general inflammatory response develops due to the increased production of proinflammatory cytokines. One of the current methods of prevention of inflammatory response is the inhibition of LPS binding to cellular receptors. We have studied the efficacy of antagonistic properties of LPS from Rhodobacter capsulatus on the production of TNF-α, IL-6, and IL-1β cytokines induced by toxic LPS from Salmonella typhimurium and Escherichia coli in human whole blood. LPS from R. capsulatus in concentrations of 0.1 and 1 μg/mL did not induce synthesis of TNF-α, IL-6, or IL-1β. Measurements of cytokine levels showed that LPS from R. capsulatus exerted a clear protective effect against toxic LPS. In particular, LPS from R. capsulatus fullly inhibited the production of TNF-α and IL-1β and significantly decreased the IL-6 production induced by LPS from S. typhimurium. Additionally, LPS from R. capsulatus antagonized the effects of LPS from E. coli, fully inhibiting the TNF-α production and decreasing the IL-6 and IL-1β levels by 60% and 70%, respectively. Thus, LPS from R. capsulatus acts as a potent antagonist of cell activation induced by toxic LPS.     

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.     

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.     

Estrus synchronization affects WNT signaling in the porcine reproductive tract and embryos

The purpose of the study was to investigate an effect of estrus synchronization with prostaglandin (PG) F_2α and PMSG/hCG on WNT4, WNT5A, WNT7A, β-catenin (CTNNB1) and E-cadherin (CDH1) gene expression. The weight of the uterus, morphometrical parameters of the endometrium and the number of CL were recorded. The analysis of estradiol (E₂), prostaglandin (PG) F_2α and E₂ content in the uterine luminal flushings (ULFs) and progesterone (P₄) level in the blood serum were conducted. RNA was isolated from endometrial, luteal and embryonic tissue of pregnant non-synchronized (Control; n = 15) and pregnant synchronized (PGF_2α/PMSG/hCG; n = 15) pigs. Whereas there was no change in uterine weight, differences in height of endometrial surface and glandular epithelium were found. However, height of the endometrium, number of the glands and capillaries were unaffected. The total number of the CLs was higher (P < 0.05) in animals treated with PGF_2α/PMSG/hCG. The amount of E₂ and P₄ was lower (P < 0.05, P < 0.001, respectively) in pregnant gilts administrated with PGF_2α/PMSG/hCG. The concentration of PGF_2α in ULFs was not affected by hormonal management, while PGE₂ was higher (P < 0.01) in hormonally in comparison to non-hormonally treated pigs. The content of WNT4 mRNA in conceptuses increased on particular Days studied in Control and PGF_2α/PMSG/hCG administered animals. WNT7A and CTNNB1 were affected by PGF_2α/PMSG/hCG treatment in both conceptuses (P < 0.001, P < 0.05) and endometrial tissue (P < 0.001, P < 0.01). The PGF_2α/PMSG/hCG treatment resulted in elevated expression of WNT4 (P < 0.001) and CTNNB1 (P < 0.05) in luteal tissue in comparison to the Control gilts. Moreover, luteal amount of WNT5A mRNA was higher in PGF_2α/PMSG/hCG animals in comparison to the Control group (P < 0.05). Presented data show that exogenous hormones administration can affect gene expression in the porcine reproductive tract and embryo.     

Protective effects of a thromboxane synthetase inhibitor and continuous arteriovenous hemofiltration in rat endotoxic shock

We determined the efficacy of continuous arteriovenous hemofiltration (CAVH) and a thromboxane synthetase inhibitor (TSI) on survival and their effect on TXA₂, PGI₂, TNFα, and IL-1β production in rat endotoxemia. Thirty-six endotoxemic rats were randomized to one of 4 groups: (A) no TSI, sham CAVH; (B) no TSI, CAVH; (C) TSI, sham CAVH; and (D) TSI, CAVH. Either CAVH (Group B) or pretreatment with TSI (Group C) resulted in increased survival time. CAVH did not prevent the rise in TX (Group B). TNFα levels at 2 h after LPS infusion were higher in Group D compared to Group B (26.1 ± 3.7 vs 13.2 ± 4.3 ng/mL, P < 0.05) respectively. IL-1β was detected earlier in Groups C,D when compared to Groups A,B (P < 0.02). TNFα and IL-1β were not ultrafiltered. CAVH and the inhibition of TX synthesis independently improved survival in endotoxemia, however, their beneficial effects were not additive. While TSI may improve survival by blocking TXA₂ production, the salutary effects of CAVH appear to be from removal of an undetermined TXA₂ dependent mediator. TNFα and IL-1β concentrations do not appear to influence survival times in this model.     

Estrus synchronization affects WNT signaling in the porcine reproductive tract and embryos

The purpose of the study was to investigate an effect of estrus synchronization with prostaglandin (PG) F_2α and PMSG/hCG on WNT4, WNT5A, WNT7A, β-catenin (CTNNB1) and E-cadherin (CDH1) gene expression. The weight of the uterus, morphometrical parameters of the endometrium and the number of CL were recorded. The analysis of estradiol (E₂), prostaglandin (PG) F_2α and E₂ content in the uterine luminal flushings (ULFs) and progesterone (P₄) level in the blood serum were conducted. RNA was isolated from endometrial, luteal and embryonic tissue of pregnant non-synchronized (Control; n = 15) and pregnant synchronized (PGF_2α/PMSG/hCG; n = 15) pigs. Whereas there was no change in uterine weight, differences in height of endometrial surface and glandular epithelium were found. However, height of the endometrium, number of the glands and capillaries were unaffected. The total number of the CLs was higher (P < 0.05) in animals treated with PGF_2α/PMSG/hCG. The amount of E₂ and P₄ was lower (P < 0.05, P < 0.001, respectively) in pregnant gilts administrated with PGF_2α/PMSG/hCG. The concentration of PGF_2α in ULFs was not affected by hormonal management, while PGE₂ was higher (P < 0.01) in hormonally in comparison to non-hormonally treated pigs. The content of WNT4 mRNA in conceptuses increased on particular Days studied in Control and PGF_2α/PMSG/hCG administered animals. WNT7A and CTNNB1 were affected by PGF_2α/PMSG/hCG treatment in both conceptuses (P < 0.001, P < 0.05) and endometrial tissue (P < 0.001, P < 0.01). The PGF_2α/PMSG/hCG treatment resulted in elevated expression of WNT4 (P < 0.001) and CTNNB1 (P < 0.05) in luteal tissue in comparison to the Control gilts. Moreover, luteal amount of WNT5A mRNA was higher in PGF_2α/PMSG/hCG animals in comparison to the Control group (P < 0.05). Presented data show that exogenous hormones administration can affect gene expression in the porcine reproductive tract and embryo.     

Tiludronate inhibits interleukin-6 synthesis in osteoblasts: Inhibition of phospholipase D activation in MC3T3-E1 cells

In previous studies, we have reported that PGF_2α stimulates phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP-binding protein in osteoblast-like MC3T3-E1 cells, and that PGF_2α and PGE₁ induce interleukin-6 (IL-6) synthesis via activation of protein kinase C and protein kinase A, respectively. In the present study, we investigated the effect of tiludronate, a bisphosphonate known to inhibit bone resorption, on the PGF_2α- and PGE₁-induced IL-6 synthesis in these cells. Tiludronate significantly suppressed the PGF_2α-induced IL-6 secretion in a dose-dependent manner in the range between 0.1 and 30 μM. However, the IL-6 secretion induced by PGE₁ or (Bu)₂cAMP was hardly affected by tiludronate. The choline formation induced by PGF_2α was reduced by tiludronate dose-dependently in the range between 0.1 and 30 μM. On the contrary, tiludronate had no effect on PGF_2α-induced formation of inositol phosphates. Tiludronate suppressed the choline formation induced by NaF, known as an activator of heterotrimeric GTP-binding protein. However, tiludronate had little effect on the formation of choline induced by TPA, a protein kinase C activator. Tiludronate significantly inhibited the NaF-induced IL-6 secretion in human osteoblastic osteosarcoma Saos-2 cells. These results strongly suggest that tiludronate inhibits PGF_2α-induced IL-6 synthesis via suppression of phosphatidylcholine-hydrolyzing phospholipase D activation in osteoblasts, and that the inhibitory effect is exerted at the point between heterotrimeric GTP-binding protein and phospholipase D. J. Cell. Biochem. 69:252–259, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Synthesis of Ethyl Tfa- and Tfp- Amino Acid-p-Aminobenzoates and Their Juvenile Hormone Test

Adipocytes can function as endocrine cells secreting a variety of adipocytokines including tumor necrosis factor (TNF)-α. Treatment of cultured mouse 3T3-L1 preadipocytes with TNF-α induced apoptosis, as was evident from increases in nuclear condensation and caspase-3 activity, but differentiated adipocytes during the maturation phase showed resistance to apoptosis by TNF-α. Antioxidants effectively reduced TNF-α-induced apoptosis in preadipocytes, indicating the involvement of reactive oxygen species. Exposure of preadipocytes to calcium ionophore A23187 reduced TNF-α-induced apoptosis, which was accompanied by increased production of prostaglandins (PGs) E₂ and PGF_2α. TNF-αpreferentially promoted gene expression of cyclooxygenase (COX)-2 without affecting that of COX-1. Consistently, NS-398, a COX-2 inhibitor, stimulated TNF-α-induced apoptosis, which was reversed by exogenous PGE₂ and PGF_2α. These results indicate that endogenous PGE₂ and PGF_2α synthesized by preadipocytes through the induction of COX-2 can serve as anti-apoptotic factors against apoptosis by TNF-α.     

The measurement of human endometrial prostaglandin production a comparison of two in vitro methods

Two $\text{in vitro}$ methods for measuring human endometrial prostaglandin production were compared. Endometrial samples from eight patients were incubated over eight hours by a perifusion and a superfusion technique. The collected fractions were assayed by radioimmunoassay for PGE₂ and PGF_2α.There was no significant difference between the perifusion and superfusion methods for the pattern and amount of PGE₂ and PGF₂ production with time. Significantly higher production levels of PGE₂ and PGF_2α were found in secretory phase endometria than in proliferative phase endometria. Histological examination of the tissue specimens by light and electron microscopy showed that both methods caused gross tissue damage after eight hours experimentation. The superfusion method produced more morphological damage than the perifusion method. However, no tissue damage could be detected after one hour of incubation with either method.Over an eight hour period neither the perifusion nor the superfusion technique appears to be a good indicator of $\text{in vivo}$ endometrial prostaglandin production. Either reflect the $\text{in vitro}$ situation.