Sites of prostaglandin E2 (PGE2) synthesis along the rabbit nephron

The purpose of this study was to establish whether the nephron segments recognized as PGE2 target sites in the rabbit, i.e. the proximal tubule, the thick ascending limb and the collecting tubule, are also sites of PGE2 production. We therefore developed a microimmunoassay sensitive enough to allow the measurement of PGE2 on microdissected tubular segments about 1 mm in length. Under the conditions used (30 min incubation at 20 degrees C), a basal rate of PGE2 production was measured in the cortical (CCT) and medullary portions of the collecting tubule, as could be expected. In the presence of 10(-4) M sodium arachidonate, it was shown that: (1) The thin descending limb (TDL) is also an active site of PGE2 formation. When expressed per mm tubule length the amounts formed were lower in TDL than in CCT (14.1 +/- 2.7 SE pg/mm, n = 5, vs. 93.5 +/- 10.7, n = 8). They were quite comparable, however, when expressed per microgram total proteins (0.70 ng in TDL vs 0.6 in CCT). (2) A slight PGE2 production was noted in the connecting tubule but it was likely due to contamination by adjacent CCT cells. (3) In the other nephron segments, only negligible amounts of PGE2 were formed, which are probably of no physiological significance.     

Stimulation of rabbit synoviocyte prostaglandin E2 synthesis by lipopolysaccharides and their subunit structures

Lipopolysaccharides (LPS) induce synoviocyte activation and may lead to destruction of synovial joint tissues. We assessed the production of prostaglandin E2 (PGE2) as a measure of synoviocyte activation by LPS and their subunit structures. Diphosphoryl lipid A was the smallest portion of lipid A tested that stimulated PGE2 production. The polysaccharide fraction of LPS, containing the O antigen, was also active. Intraarticular injections of the polysaccharide resulted in a synovitis very similar to that found in association with the intact LPS molecule. These observations suggest that both parts of LPS might be involved in gram-negative, organism-associated synovitis.     

Complementary mechanisms of enhanced oxytocin-stimulated prostaglandin E2 synthesis in rabbit amnion at the end of gestation

The up-regulation of oxytocin (OT) receptors in rabbit amnion at the end of gestation is associated with a large increase in the ability of OT to stimulate PGE2 synthesis. The purpose of these investigations was to determine what other factors contribute to this increase. OT enhanced PGE2 synthesis at several levels. The concentrations of cytosolic phospholipase A2, which generates arachidonic acid for PGE2 synthesis, and PGH endoperoxide synthases (types 1 and 2), which catalyze the conversion of arachidonic acid to prostanoids, rose substantially in rabbit amnion at term. OT stimulated translocation of cytosolic phospholipase A2 to the cell particulate fraction, presumably by a Ca2+-mediated process, and phosphorylation of cytosolic phospholipase A2 via the extracellular regulated protein kinase 2/1-mediated pathway. OT-stimulated increases in intracellular Ca2+ concentrations and extracellular regulated protein kinase 2/1 phosphorylation were both mediated by G(q/11) activation. OT also increased the expression of PGH endoperoxide synthase-2 after treatment of amnion cells in culture for 2 h; however, PGE2 release in response to OT was virtually immediate. These findings show that the rapid stimulation of PGE2 synthesis by OT occurs through cytosolic phospholipase A2 activation and PGH endoperoxide synthase-1 activity, both of which, along with OT receptor concentrations, are considerably up-regulated in the amnion at the end of gestation.     

Angiotensin peptides and prostaglandin E2 synthesis: modulation of neurogenic responses in the rabbit vas deferens

Isolated rabbit vasa deferentia were used to study the neuromodulation induced by angiotensins II and III (AII and AIII) upon both phases of the electrically stimulated contraction (ESC). AIII (10(-8)-10(-7) M) was shown to inhibit the ESC at low frequencies (2-10 Hz), while AII tended to potentiate both phases. Since AIII had no effect on contractions induced by exogenous ATP (twitch, putative transmitter), or norepinephrine (NE; tonic, neurotransmitter), AIII effects were presumed to be presynaptic. AIII neuromodulation was reversed by indomethacin, and prostaglandin E2 (PGE2) mimicked the inhibitory effects of AIII on ESC. The response to AIII was blocked by [Sar1,Ala8]AII (10(-6) M), an angiotensin antagonist. AIII (10(-9)-10(-5) M) stimulated PGE2 synthesis in a concentration-dependent manner. AII (10(-9)-10(-7) M) produced a dramatic rise in PGE2 synthesis, which declined sharply at higher AII concentrations. AII increased the overflow of [3H]NE approximately 50% (P less than 0.01; in the absence of indomethacin); similar concentrations of AIII did not affect [3H]NE release. However, 4 X 10(-8) M AIII in the presence of 26 microM indomethacin significantly (P less than 0.05) increased [3H]NE overflow. Thus, AIII inhibited ESC presynaptically by stimulating PGE2 synthesis, while AII potentiated these contractions presynaptically by enhancing NE release during nerve stimulation. Despite the greater inhibitory effect of AIII on force, AII was more potent than AIII in stimulating PGE2 production.     

Leishmania donovani-induced macrophages cyclooxygenase-2 and prostaglandin E2 synthesis

Prostaglandin E2 (PGE2) secretion during Leishmania infection has been reported. However, the signalling mechanisms mediating this response are not well understood. Since cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2) are involved in PGE2 synthesis in response to various stimuli, the implication of these enzymes was evaluated in Leishmania-infected phorbol myristate acetate-differentiated U937 human monocytic cell line. Time-course experiments showed that PGE2 synthesis increased significantly in parallel with COX-2 expression when cells were incubated in the presence of Leishmania donovani promastigotes or lipopolysaccharides (LPS). Increase in cPLA2 mRNA expression was only detected when cells were stimulated with LPS. Indomethacin, genistein, and H7, which are antagonists of COX-2, protein tyrosine kinase (PTK) and protein kinase C (PKC), respectively, inhibited PGE2 production induced by L. donovani and LPS. However, only H7 inhibited COX-2 mRNA synthesis, and there was a significant correlation between PGE2 inhibition and reduced COX-2 expression. Collectively, our results indicate that infection of U937 by L. donovani leads to the generation of PGE2 in part through a PKC-dependent signalling pathway involving COX-2 expression. They further reveal that PTK-dependent events are necessary for Leishmania-induced PGE2 generation, but not for COX-2 expression. A better understanding of the mechanisms by which Leishmania can induce PGE2 production could provide insight into the pathophysiology of leishmaniasis and may help to improve therapeutic approaches.     

Microparticles stimulate the synthesis of prostaglandin E2 via induction of cyclooxygenase 2 and microsomal prostaglandin E synthase 1

Objective

Microparticles are small vesicles that are released from activated or dying cells and that occur abundantly in the synovial fluid of patients with rheumatoid arthritis (RA). The goal of these studies was to elucidate the mechanisms by which microparticles activate synovial fibroblasts to express a proinflammatory phenotype.

Methods

Microparticles from monocytes and T cells were isolated by differential centrifugation. Synovial fibroblasts were cocultured with increasing numbers of microparticles. Gene expression was analyzed by real‐time polymerase chain reaction and confirmed by Western blotting and enzyme immunoassay. Arachidonic acid labeled with tritium was used to study the transport of biologically active lipids by microparticles. The roles of NF‐κB and activator protein 1 (AP‐1) signaling were analyzed with electrophoretic mobility shift assay and transfection with small interfering RNA and IκB expression vectors.

Results

Microparticles strongly induced the synthesis of cyclooxygenase 2 (COX‐2), microsomal prostaglandin E synthase 1 (mPGES‐1), and prostaglandin E₂ (PGE₂). In contrast, no up‐regulation of COX‐1, mPGES‐2, cytosolic PGES, or phospholipase A₂ was observed. The induction of PGE₂ was blocked by selective inhibition of COX‐2. Microparticles activated NF‐κB, AP‐1, p38, and JNK signaling in synovial fibroblasts. Inhibition of NF‐κB, AP‐1, and JNK signaling reduced the stimulatory effects. Arachidonic acid was transported from leukocytes to fibroblasts by microparticles. Arachidonic acid derived from microparticles was converted to PGE₂ by synovial fibroblasts.

Conclusion

These results demonstrate that microparticles up‐regulate the production of PGE₂ in synovial fibroblasts by inducing COX‐2 and mPGES‐1. These data provide evidence for a novel mechanism by which microparticles may contribute to inflammation and pain in RA.

     

Papillary collecting tubule synthesis of prostaglandin E2 in Dahl rats

Isolated kidneys of Dahl salt-sensitive rats (DS) excrete sodium less readily than those of Dahl salt-resistant rats (DR). The collecting tubule is an important source of papillary prostaglandin E2 and is a site of significant sodium reabsorption. We cultured renal papillary collecting tubule cells from 5-week-old, prehypertensive DS and DR on a low salt diet and also after 14 weeks of high salt feeding, and we measured prostaglandin E2 synthetic capacity. Unstimulated renal papillary collecting tubule cells from 5-week-old DS produced 62 +/- 5% less prostaglandin E2 than did comparable cells from DR (p less than 0.001). The cells from DS also synthesized less prostaglandin E2 after stimulation with the calcium ionophore A23187 (67 +/- 6% of control; p less than 0.001) or the addition of exogenous arachidonate (74 +/- 7% of control; p less than 0.01). Urinary prostaglandin E2 excretion was also diminished in the 5-week-old DS compared with their salt-resistant counterparts (18.1 +/- 1.3 vs 23.9 +/- 1.7 ng/24 hr; p less than 0.025). After high salt feeding, the DS became hypertensive but the DR remained normotensive. Renal papillary collecting tubule cells cultured from these DS continued to produce less prostaglandin E2 than those from control rats, both in the basal state (60 +/- 12% of control; p less than 0.09) and after stimulation with ionophore (62 +/- 2% of control; p less than 0.002). In these older animals, the DS continued to underexcrete prostaglandin E2 compared with the DR (29.7 +/- 3.2 vs 42.2 +/- 6.1 ng/24 hr; p less than 0.08). The underproduction of prostaglandin E2 in the papillary collecting tubule of DS may play a role in their inadequate renal natriuretic capacity and contribute to the onset and maintenance of salt-induced hypertension in this strain.     

Modulation of TIMP-1 synthesis by antiinflammatory cytokines and prostaglandin E2 in interleukin 17 stimulated human monocytes/macrophages

OBJECTIVE: To examine the regulation of tissue inhibitor of metalloproteinase 1 (TIMP-1) synthesis by interleukin 17 (IL-17) stimulated human monocytes/macrophages in primary culture in the presence of prostaglandin E2 (PGE2) and antiinflammatory cytokines, and to compare this with the regulation of matrix metalloproteinase (MMP-9) production. METHODS: IL-17 stimulated human monocytes isolated from the peripheral blood of healthy donors were cultured in the presence of PGE2, cyclic adenosine monophosphate (cAMP) mimetics (IBMX, cAMP, forskolin, cholera toxin), or antiinflammatory cytokines (IL-4, IL-10, IL-13), or with protein kinase inhibitors of diverse specificity. MMP-9 and TIMP-1 were measured using specific ELISA, while expression of specific messenger RNA was determined by Northern blotting. RESULTS: IL-17 stimulated an increased level of MMP-9 production relative to TIMP-1 production in monocytes/macrophages. Stimulation was accompanied by upregulation of specific MMP-9 mRNA expression relative to TIMP-1 mRNA. Exogenous PGE2, cAMP, and cAMP-mimetics completely inhibited both basal and IL-17 induced MMP-9 synthesis, while only IL-17 induced TIMP-1 synthesis was abrogated. The same effect was found for the antiinflammatory cytokines. Both basal and IL-17 induced production of TIMP-1 involved p42/44 and p38 kinases and nuclear factor kappaB signaling pathways. CONCLUSION: The excess of MMP-9 over TIMP-1 production, and decreased inhibition of MMP-9 activity in chronic rheumatoid diseases, may result in cartilage degradation and joint destruction.     

Role of prostaglandin E2 in cholinergic-mediated glycoprotein synthesis in canine antrum

We studied the mechanism of cholinergic stimulation of mucin synthesis in canine antral explants, including the role of PGE₂ as an intermediate messenger. Isolated antral mucosa was incubated with 10^–5 M carbachol (Cb), 10^–5 M indomethacin (IND), 10^–5 M pirenzepine (PZ), 10^–5 M Cb+10^–5 M PZ, 10^–5 M Cb+10^–5 M IND, and 10^–5 M IND +PGE₂ (10^–8, 10^–7 and 10^–6 M) in the presence or absence of [³H]glucosamine. After 24 hr, total glycoprotein synthesis was quantitated by Sepharose-4B chromatography and by 10% TCA/1%PTA precipitation with lipid extraction. PGE₂ released into the media was measured by radioimmunoassay (RIA). Cb significantly increased total glycoprotein synthesis and produced a significant increase in PGE₂ release. The increase in glycoprotein synthesis and the release of PGE₂ was blocked by the addition of muscarinic antagonist PZ. The addition of IND significantly inhibited glycoprotein synthesis and almost entirely suppressed PGE₂ secretion. IND also inhibited the effect of Cb on glycoprotein synthesis and PGE₂ release. Moreover, PGE₂ (10^–6 and 10^–7 M) significantly increased the glycoprotein synthesis in the canine stomach. This suggests the coordinate participation of PGE₂-releasing cell population in modulation of glycoprotein synthesis in gastric mucosa.Supported in part by the Research Service of the Veterans Administration and grant R01-DK37989 and Michigan Gastrointestinal Peptide Research Center, 5P30-DK34933 awarded by the PHS, DHHS.Presented in part at the annual meeting of American Gastroenterological Association in 1990 (Gastroenterology 98: A153).     

Papaverine stimulation of prostaglandin E2 production by cultured rabbit gastric mucosa

Prostaglandins (PGs) are synthesised by gastric mucosa, and have been shown to inhibit gastric acid secretion and ulcer formation in man and experimental animals. Recently exogenous PGs, mainly of the E group, have been used for the treatment of peptic ulcer disease. We therefore searched for a drug that would stimulate endogenous gastric prostaglandin E₂ (PGE₂) synthesis. Rabbit gastric mucosa slices were cultured for 22 hours at 77°C. PGE₂, measured by radioimmunoassay, was found to be linearly secreted into the culture medium. PGE₂ accumulation in the medium during 22 hours of culture was 7·9±0·5 (SE) ng/mg tissue (N=20). Addition of papaverine (100 μ/ml), a cyclic nucleotide phosphodiesterase inhibitor, resulted in a significant increase (250% of control) in PGE₂ accumulation in the medium: 24·3±1·8 ng/mg tissue (N=25). Isobutylmethylxanthine (IBMX 100 μg/ml), another phosphodiesterase inhibitor, only slightly increased PGE₂ accumulation, while 8 bromo-cyclic AMP (1 mM) had no effect. Under these conditions IBMX increased by 20-fold mucosal cyclic AMP levels: 3·9±0·3 pmol/mg tissue (N=8) as compared with control levels: 0·2±0·03 pmol/mg tissue (N=8). Papaverine, however, did not alter mucosal cyclic AMP accumulation. These results indicate that papaverine stimulates PGE₂ production by cultured rabbit gastric mucosa and that this stimulation is not related to the inhibition of phosphodiesterase activity and accumulation of mucosal cyclic AMP. Papaverine induced stimulation of PGE₂ production should be further evaluated regarding its possible beneficial effects in protecting gastric mucosa and in reducing acid secretion in peptic ulcer patients.     

Microparticles stimulate the synthesis of prostaglandin E(2) via induction of cyclooxygenase 2 and microsomal prostaglandin E synthase 1

OBJECTIVE: Microparticles are small vesicles that are released from activated or dying cells and that occur abundantly in the synovial fluid of patients with rheumatoid arthritis (RA). The goal of these studies was to elucidate the mechanisms by which microparticles activate synovial fibroblasts to express a proinflammatory phenotype. METHODS: Microparticles from monocytes and T cells were isolated by differential centrifugation. Synovial fibroblasts were cocultured with increasing numbers of microparticles. Gene expression was analyzed by real-time polymerase chain reaction and confirmed by Western blotting and enzyme immunoassay. Arachidonic acid labeled with tritium was used to study the transport of biologically active lipids by microparticles. The roles of NF-kappaB and activator protein 1 (AP-1) signaling were analyzed with electrophoretic mobility shift assay and transfection with small interfering RNA and IkappaB expression vectors. RESULTS: Microparticles strongly induced the synthesis of cyclooxygenase 2 (COX-2), microsomal prostaglandin E synthase 1 (mPGES-1), and prostaglandin E(2) (PGE(2)). In contrast, no up-regulation of COX-1, mPGES-2, cytosolic PGES, or phospholipase A(2) was observed. The induction of PGE(2) was blocked by selective inhibition of COX-2. Microparticles activated NF-kappaB, AP-1, p38, and JNK signaling in synovial fibroblasts. Inhibition of NF-kappaB, AP-1, and JNK signaling reduced the stimulatory effects. Arachidonic acid was transported from leukocytes to fibroblasts by microparticles. Arachidonic acid derived from microparticles was converted to PGE(2) by synovial fibroblasts. CONCLUSION: These results demonstrate that microparticles up-regulate the production of PGE(2) in synovial fibroblasts by inducing COX-2 and mPGES-1. These data provide evidence for a novel mechanism by which microparticles may contribute to inflammation and pain in RA.     

Human cholecystitis is associated with increased gallbladder prostaglandin I2 and prostaglandin E2 synthesis.

Microsomal prostanoid synthesis was compared in normal gallbladders removed during organ donation and inflamed gallbladders removed at cholecystectomy. Normal human gallbladder microsomes demonstrated low rates of conversion of [14C]arachidonic acid to total labeled prostanoids, which increased during 1 to 30 min of incubation. Normal human gallbladder microsomes converted labeled substrate to all primary prostaglandins without demonstration of a major product. Inflamed human gallbladder microsomes increased the rate of conversion of [14C]arachidonic acid to total labeled prostanoids two or three times over the levels demonstrated by normal gallbladder microsomes at all times of incubation (p < 0.01). The main prostanoids synthesized by the inflamed human gallbladder microsomes were prostaglandin E2 and 6-keto-prostaglandin F1 alpha, which were increased four times over the levels demonstrated by normal gallbladder microsomes (p < 0.01). These data showed that inflammation of the human gallbladder was associated with increased synthesis of gallbladder 6-keto-prostaglandin F1 alpha and prostaglandin E2.     

Enhanced prostaglandin E2 secretion by cytokine-stimulated human synoviocytes in the presence of subtherapeutic concentrations of nonsteroidal antiinflammatory drugs

Human synoviocytes were stimulated for 48 hours or 72 hours with cytokines (recombinant interleukin-1 beta and/or recombinant tumor necrosis factor alpha) in the presence or absence of selected nonsteroidal antiinflammatory drugs. The drugs were tested at subtherapeutic and clinically therapeutic levels, as follows: piroxicam 0.3 pM to 3 X 10(6) pM, sodium salicylate 30 nM to 3 X 10(6) nM, and indomethacin 3 pM to 3 X 10(6) pM. At low concentrations, all 3 drugs showed significant enhancement of prostaglandin E2 (PGE2) secretion (piroxicam, salicylate greater than indomethacin). At high, clinically therapeutic concentrations, all drugs showed suppression of PGE2 secretion (indomethacin greater than piroxicam greater than salicylate). The enhancement of PGE2 secretion may be partially responsible for the flare of arthralgia or arthritis frequently seen after termination of drug therapy.     

Substrain heterogeneity in prostaglandin E2 synthesis of human dermal fibroblasts. Differences in prostaglandin E2 synthetic capacity of substrains are not stimulus-restricted

We examined prostaglandin E2 (PGE2) biosynthetic heterogeneity in fibroblast substrains and possible mechanisms that might mediate this heterogeneity. PGE2 synthesis of fibroblast substrains, in response to phytohemagglutinin-stimulated mononuclear cell supernates, ranged from 9.0 +/- 1.0 ng/ml to 79.3 +/- 7.4 ng/ml (mean +/- SD). The phenotypic behavior of individual substrains was stable. Substrains were also heterogeneous in PGE2 response to phorbol myristate acetate, and displayed stability in this phenotype as well. Substrains which were high responders to mononuclear cell supernate also ranked high in response to phorbol myristate acetate. Similar heterogeneity was observed in response to purified interleukin-1. Arachidonic acid added exogenously did not raise interleukin-1 responsiveness of low-producer substrains to that of high producers, suggesting that differences in PGE2 synthesis among substrains did not reflect differences in substrate availability or phospholipase activity. Supernates of high- and low-responder phenotype substrains, when added to cells of the reciprocal strain or to unrelated fibroblasts, did not affect the pattern of PGE2 synthesis. The concordance of substrain responsiveness to mononuclear cell supernate and phorbol myristate acetate suggests that heterogeneity among substrains in PGE2 synthesis is related to the ability to produce PGE2, rather than to the ability to respond to a given mediator. In addition, differences in PGE2 synthesis among substrains do not appear to result from release of regulatory autokines.     

Characterization of oxytocin receptors in rabbit amnion involved in the production of prostaglandin E2.

We characterized oxytocin (OT) receptors in purified plasma membranes from amnion, decidua, and myometrium of late pregnant rabbits using an iodinated OT antagonist (OTA). Saturation studies showed similar Kd values for specific binding sites in the 100- to 250-pM range in all three tissues. OT receptor concentrations in decidua and myometrium did not change until the day of labor (day 31), when they rose about 2.5- and 18-fold, respectively. Increases in amnion receptors were first apparent on day 28 and continued to maximal levels on day 31. There was an increase of about 230-fold from day 26 to labor, reaching 9.5 pmol/mg protein. Competition studies using analogs showed that ligand specificities of amnion and decidual membranes were indistinguishable. Those of myometrial membranes were somewhat different, possibly owing to the presence of both AVP receptors and OT receptors in the myometrium. Binding of OTA corresponded to the OT-induced release of prostaglandin E2 (PGE2) by amnion cells in culture. The effects of OT were dose dependent, agonist specific, and selectively inhibited by OTA. Amnion cells from days 22 and 28 did not respond significantly to either OT or phorbol 12-myristate 13-acetate (PMA), but cells from day 30 pregnant rabbits responded strongly to both. In contrast, calcium ionophore stimulated comparable amounts of PGE2 release from cells cultured on day 22, 28, or 30. These studies show that specific, high affinity OT receptors are associated with the release of PGE2 from rabbit amnion cells. Increases in amnion OT receptor and protein kinase-C activity precede by several days the increases in receptor concentrations in decidua and myometrium, suggesting important roles for the amnion and OT in the initiation of labor in rabbits.     

Prostaglandin E1 inhibits collagenase gene expression in rabbit synoviocytes and human fibroblasts.

Cartilage breakdown, as seen in inflammatory and degenerative joint diseases, can be mediated by proteolytic enzymes, such as the metalloproteinase collagenase, the only enzyme able to digest collagen at neutral pH. In vitro collagenase gene expression can be stimulated by the phorbol ester tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. We have investigated the effect of prostaglandin E1 (PGE1) on 12-O-tetradecanoyl-phorbol-13-acetate-stimulated collagenase mRNA levels in the rabbit synoviocyte cell line HIG-82. PGE1, but not PGE2 or PGF2 alpha, was able to selectively reduce collagenase mRNA levels in a dose-dependent fashion. PGE1 markedly increased intracellular levels of cAMP, while PGE2 and PGF2 alpha had little or no effect on cAMP production in the HIG-82 synoviocytes. Agents known to increase intracellular cAMP levels, such as the adenyl cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), mimicked the effect of PGE1, on collagenase mRNA levels. PGE1, forskolin, and IBMX also decreased collagenase mRNA levels in human skin fibroblasts, demonstrating that this observation was not unique to the HIG-82 cell line. Transient transfection experiments carried out in HIG-82 cells using a 1.2-kilobase portion of the 5'-flanking region of the human collagenase gene linked to the reporter gene luciferase demonstrated that PGE1, forskolin, and IBMX exert their inhibitory effect on the promoter region of the collagenase gene.     

Modulation of bovine placental prostaglandin synthesis by an endogenous inhibitor

An endogenous, heat-labile, inhibitor of prostanoid synthesis in maternal caruncle tissue of bovine placentomes was studied. Inhibitory activity was present in caruncle extracts from days 120-250 of gestation, but was not detectable at term (260-280 days). The disappearance of inhibitory activity coincided with an increase in the secretion of prostanoids by dispersed caruncle cells in culture. Coculture of caruncle cells from placentomes of 120-day gestation with fetal cotyledon cells resulted in suppression of prostanoid synthesis by the cotyledon cells. However, this inhibition was not observed in cocultures of dispersed caruncle cells and fetal cotyledon cells from term placentomes. Our findings indicate that an endogenous inhibitor modulates bovine placental prostaglandin synthesis. A decline in the level of this inhibitor at term may be one factor triggering increased prostanoid synthesis required for parturition.     

Effects of glucosamine hydrochloride on the production of prostaglandin E2, nitric oxide and metalloproteases by chondrocytes and synoviocytes in osteoarthritis

OBJECTIVES: To determine the response of glucosamine hydrochloride on chondrocytes and synoviocytes in terms of prostaglandin E2 (PGE2), nitric oxide (NO) and matrix metalloproteases (MMPs). METHODS: Chondrocytes and synoviocytes were prepared from joint specimens of patients who underwent total knee arthroplasty for osteoarthritis (OA). Chondrocytes from patients with femoral neck fracture were served as a normal control. Culture cells were stimulated by 5 ng/ml of IL-1beta and treated with various concentration of glucosamine hydrochloride (from 1 microg/ml to 500 microg/ml). PGE2, NO, MMP-1, MMP-3, and MMP-13 levels were evaluated in the culture supernatant. Further, the expression of COX-2 mRNA was studied by semiquantitative PCR. RESULTS: With IL-1beta stimulation, the levels of these mediators increased dramatically, except for NO from synoviocytes. After stimulation, levels of these mediators in OA chondrocytes were higher than synoviocytes and normal chondrocytes, and the level of MMP-3 was higher than those of MMP-1 and MMP-13. Glucosamine hydrochloride at a concentration of 100 microg/ml suppressed PGE2 production, and partly suppressed NO production. It also suppressed the production of MMPs from normal chondrocytes and synoviocytes but not from OA chondrocytes. CONCLUSION: Glucosamine modulates the metabolism of chondrocytes and synoviocytes and its mode of action differs between cells and conditions.