The effect of cortisol on the synthesis of prostaglandins (PGF2 alpha, PGE2) by human endometrial fibroblasts in vitro with and without addition of estradiol-17 beta or progesterone

Cortisol is known as a potent inhibitor of phospholipase A2 activity in several tissues. In fibroblast monolayer cell cultures from proliferative human endometrium cortisol alone does not affect the basal PGF2 alpha or PGE2 synthesis. After stimulation of PGF2 alpha production by 10(-7) mol/l estradiol-17 beta increasing concentrations of cortisol up to 10(-5) mol/l dosedependently reduce the PGF2 alpha production. Also the progesterone (10(-4) mol/l) stimulated increase of PGF2 alpha and PGE2 synthesis is inhibited by cortisol (10(-7) mol/l).     

Okadaic acid and dinophysistoxin-1, non-TPA-type tumor promoters, stimulate prostaglandin E2 production in rat peritoneal macrophages

Okadaic acid and dinophysistoxin-1 isolated from a black sponge, Halichondria okadai are non-12-O-tetrade-canoylphorbol 13-acetate (non-TPA)-type tumor promoters of mouse skin. Okadaic acid at concentrations of 10-100 ng/ml stimulated prostaglandin E2 production in rat peritoneal macrophages. Dinophysistoxin-1 (35-methylokadaic acid) stimulated prostaglandin E2 production as strong as okadaic acid, but okadaic acid tetramethyl ether, an inactive compound as a tumor promoter, did not. Okadaic acid at 10 ng/ml (12.4 nM) stimulated prostaglandin E2 production as strongly as TPA at 10 ng/ml (16.2 nM) 20 h after incubation. Unlike TPA-type tumor promoters, okadaic acid required a lag phase before stimulation. The duration of this lag phase was dependent on the concentration of okadaic acid. Indomethacin inhibited okadaic acid-induced preostaglandin E2 production in a dose-dependent manner, and its inhibition was more strongly observed in okadaic acid-induced prostaglandin E2 production. Cycloheximide inhibited okadaic acid-induced release of radioactivity from [3H]arachidonic acid-labeled macrophages and prostaglandin E2 production dose dependently, suggesting that protein synthesis is a prerequisite for the stimulation of arachidonic acid metabolism. These results support our idea that tumor promoters, at very low concentrations, are able to stimulate arachidonic acid metabolism in rat peritoneal macrophages.     

H2O2 increases production of constrictor prostaglandins in smooth muscle leading to enhanced arteriolar tone in Type 2 diabetic mice

Our previous study showed that arteriolar tone is enhanced in Type 2 diabetes mellitus (T2-DM) due to an increased level of constrictor prostaglandins. We hypothesized that, in mice with T2-DM, hydrogen peroxide (H(2)O(2)) is involved in the increased synthesis of constrictor prostaglandins, hence enhanced basal tone in skeletal muscle arterioles. Isolated, pressurized gracilis muscle arterioles ( approximately 100 microm in diameter) of mice with T2-DM (C57BL/KsJ-db(-)/db(-)) exhibited greater basal tone to increases in intraluminal pressure (20-120 mmHg) than that of control vessels (at 80 mmHg, control: 25 +/- 5%; db/db: 34 +/- 4%, P < 0.05), which was reduced back to control level by catalase (db/db: 24 +/- 4%). Correspondingly, in carotid arteries of db/db mice, the level of dichlorofluorescein-detectable and catalase-sensitive H(2)O(2) was significantly greater. In control arterioles, exogenous H(2)O(2) (0.1-100 micromol/l) elicited dilations (maximum, 58 +/- 10%), whereas in arterioles of db/db mice H(2)O(2) caused constrictions (-28 +/- 8%), which were converted to dilations (maximum, 16 +/- 5%) by the thromboxane A(2)/prostaglandin H(2) (TP) receptor antagonist SQ-29548. In addition, arteriolar constrictions in response to the TP receptor agonist U-46619 were not different between the two groups of vessels. Endothelium denudation did not significantly affect basal tone and H(2)O(2)-induced arteriolar responses in either control or db/db mice. Also, in arterioles of db/db mice, but not in controls, 3-nitrotyrosine staining was detected in the endothelial layer of vessels. Thus we propose that, in mice with T2-DM, arteriolar production of H(2)O(2) is enhanced, which leads to increased synthesis of the constrictor prostaglandins thromboxane A(2)/prostaglandin H(2) in the smooth muscle cells, which enhance basal arteriolar tone. These alterations may contribute to disturbed regulation of skeletal muscle blood flow in Type 2 diabetes mellitus.     

Microtubule disruption enhances prostaglandin E2 production in osteoblastic cells

Prostaglandins have been implicated in the response of bone to mechanical stimuli. To explore the potential role of the cytoskeleton in the control of prostaglandin production, we examined the effect of cytoskeleton disrupting agents on arachidonic acid metabolism in rat calvaria osteoblastic cells. We found that microtubule disrupting agents increase prostaglandin E production 4-5-fold. Stimulation was first detectable at 4 h and rose sharply between 4 and 8 h. 2 h exposure to 1 microM colchicine was sufficient to produce the maximum effect. Cytochalasin B at concentrations which caused marked shape changes had no effect on prostaglandin E production or on its stimulation by colchicine. Taxol, a stabilizer of microtubules, reduced the colchicine effect. The increase in prostaglandin E production was associated with enhanced conversion of arachidonic acid to prostaglandin E2 rather than enhanced release of arachidonic acid from phospholipids. This increase in enzymatic activity was not abolished by cycloheximide treatment at concentrations which inhibited 90% of protein synthesis in the cells.     

Cortisol production by dispersed guinea-pig adrenal cells; a specific, sensitive and reproducible response to ACTH....and its fragments

Naturally occurring steroids and peptide hormones, tested at supraphysiological concentrations, were without effect on basal and human (h) 1-39 ACTH (NIBSC code 74/555, 25 ng/l (5.5 X 10(-12) mol/l] stimulated cortisol production. Further, low concentrations of angiotensin II, N-pro-opiocortin (N terminal fragment 1-76) and gamma-MSH all of which have been reported to synergise with ACTH with regard to cortisol production, were without significant effect alone or in combination with ACTH over the range 2.2 X 10(-13) to 5.5 X 10(-12) mol/l. The activity of h 1-39 was compared with that of the ACTH related peptides 1-24, 1-18, 1-17, 1-16, 1-13-NH2 (alpha MSH), 1-10 and 4-10. The dose responses were parallel and the same maximal cortisol output was observed with all the peptides except the 1-10 fragment. Half maximal stimulation occurred at 3.1 X 10(-12) (1-24), 4.4 X 10(-12) (h 1-39), 1.5 X 10(-11) (1-39), 3.3 X 10(-10) (1-18), 5 X 10(-9) (1-13-NH2), 8 X 10(-9) (1-17), 2 X 10(-7) (1-16) and 1 X 10(-5) (4-10) mol/l respectively. Interference by the above ACTH-derived peptides in cortisol secretion by the cells in response to 5.5 X 10(-12) mol/l h 1-39 ACTH was minimal over the range 5.2 X 10(-12)-2.2 X 10(-6) mol/l. The sensitivity of the adrenal cells to h 1-39 ACTH was such that 2 ng/l (4.4 X 10(-13) mol/l) provoked cortisol secretion over the control (P less than 0.05, n = 17). The coefficient of variation within assay for each dose on the full standard curve (2.2 X 10(-13)-1.1 X 10(-10) mol/l) was less than 10% (n = 6). Half maximal stimulation was given by 14.5 ng/l (3.2 X 10(-12) mol/l). Between control and 1.1 X 10(-10) mol/l ACTH there was a 32 +/- 8 (mean +/- SD, n = 9) fold change in cortisol production.     

Acute hormonal control of acetyl-CoA carboxylase. The roles of insulin, glucagon, and epinephrine

Acetyl-CoA carboxylase, purified from rapidly freeze-clamped livers of rats maintained on a normal laboratory diet and given 0-5 units of insulin shortly before death, gives a major protein band (Mr 265,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase from untreated rats has relatively low activity (0.8 unit/mg protein when assayed in the absence of citrate) and high phosphate content (8.5 mol of Pi/mol of subunit), while the enzyme from livers of rats that received 5 units of insulin has higher activity (2.0 units/mg protein) and lower phosphate content (7.0 mol of Pi/mol of subunit). Addition of citrate activates both preparations with half-maximal activation (K0.5) at 1.0 and 0.6 mM citrate, respectively. The enzyme from rats that did not receive insulin is mainly in the octameric state (Mr approximately 2 x 10(6)), while that from rats that received insulin is mainly in the polymeric state (Mr approximately 10 x 10(6)). Thus, short-term administration of insulin results in activation of acetyl-CoA carboxylase, lowering of its citrate requirement, and dephosphorylation and polymerization of the protein. The insulin-induced changes in the carboxylase are probably due to dephosphorylation of the protein since similar changes are observed when the enzyme from rats that did not receive insulin is dephosphorylated by the Mn2(+)-dependent [acetyl-CoA carboxylase]-phosphatase 2. The effect of glucagon or epinephrine administration on acetyl-CoA carboxylase was also investigated. The carboxylase from fasted/refed rats has a relatively high specific activity (3.4 units/mg protein in the absence of citrate), lower phosphate content (4.9 mol of Pi/mol of subunit), and is present mainly in the polymeric state (Mr approximately 10 x 10(6)). Addition of citrate activates the enzyme with K0.5 = 0.2 mM citrate. Glucagon or epinephrine injection of fasted/refed rats yielded carboxylase with lower specific activity (1.4 or 1.9 units/mg, respectively, in the absence of citrate), higher phosphate content (6.4 or 6.7 mol of Pi/mol of subunit, respectively), and mainly in the octameric state (Mr approximately 2 x 10(6)). Treatment of these preparations with [acetyl-CoA carboxylase]-phosphatase 2 reactivated the enzyme (specific activity approximately 8 units/mg protein in the absence of citrate) and polymerized the protein (Mr approximately 10 x 10(6]. These observations indicate that insulin and glucagon, by altering the phosphorylation state of the acetyl-CoA carboxylase, play antagonistic roles in the acetyl-control of its activity and therefore in the regulation of fatty acid synthesis.     

Upregulation of cyclooxygenase-2 and thromboxane A2 production mediate the action of tumor necrosis factor-alpha in isolated rat myenteric ganglia

Intact myenteric ganglia from 4- to 10-day-old rats were isolated from the small intestine. The preparations were cultured overnight, and drugs were applied within this time frame (20 h). Whole cell patch-clamp technique was used to measure basal membrane potential and carbachol-induced depolarization at neurons within these ganglia. Pretreatment with TNF-alpha (100 ng/ml) hyperpolarized the membrane (from -31.0 +/- 2.7 mV under control conditions to -61.2 +/- 3.2 mV in the presence of the cytokine) and potentiated the depolarization induced by carbachol (from 5.2 +/- 0.7 mV under control conditions to 27.5 +/- 2.0 mV in the presence of the cytokine). These effects were mimicked by carbocyclic thromboxane A2 (10(-6) mol/l), a stable thromboxane A2 agonist. The TNF-alpha action was inhibited by 1-benzylimidazole (2 x 10(-4) mol/l), a thromboxane synthase inhibitor, and BAY U 3405 (5 x 10(-4) mol/l), an inhibitor of thromboxane receptors. Measurements of thromboxane production in the supernatant of the culture revealed an increased concentration of thromboxane B2, the stable metabolite of thromboxane A2, after exposure to TNF-alpha. Immuncytochemical staining for cyclooxygenase-2 (COX-2) and the neuronal marker microtubule-associating protein-2 revealed an upregulation of COX-2 in myenteric neurons after exposure to the cytokine. These results demonstrate the involvement of COX-2 and the subsequent production of thromboxane A2 in the presence of TNF-alpha.     

Interleukin-1 beta and transforming growth factor-beta 2 enhance cytosolic high-molecular-mass phospholipase A2 activity and induce prostaglandin E2 formation in rat mesangial cells.

Interleukin-1 beta induces gene expression and secretion of group-II phospholipase A2 and release of prostaglandin E2 from rat mesangial cells. The interleukin-1 beta-induced synthesis of group-II phospholipase A2 is prevented by transforming growth factor-beta 2, whereas transforming growth factor-beta 2 potentiated the interleukin-1 beta-evoked prostaglandin E2 production. Transforming growth factor-beta 2 itself did not induce synthesis of group-II phospholipase A2, although it stimulated prostaglandin E2 formation. Here we describe the effect of interleukin-1 beta and transforming growth factor-beta 2 on a cytosolic phospholipase A2 activity and prostaglandin E2 formation in rat mesangial cells. Based on the resistance to dithiothreitol and migration profiles on a Mono-Q anion-exchange column and a Superose 12 gel-filtration column, the cytosolic phospholipase A2 activity was assigned to a high-molecular-mass phospholipase A2. Measured with 1-stearoyl-2-[1-14C]arachidonoylglycero-phosphocholine as substrate, both interleukin-1 beta and transforming growth factor-beta 2 enhanced the high-molecular-mass phospholipase A2 activity. The stimulation of rat mesangial cells with interleukin-1 beta and transforming growth factor-beta 2 was time- and dose-dependent with maximal cytosolic phospholipase A2 activities at 10 nM and at 10 ng/ml respectively, after 24 h of stimulation. Under these conditions, interleukin-1 beta and transforming growth factor-beta 2 enhanced the cytosolic phospholipase A2 activity 2.2 +/- 0.6-fold and 2.5 +/- 0.6-fold, respectively. These results strongly suggest that an enhanced cytosolic high-molecular-mass phospholipase A2 activity is involved in the formation of prostaglandin E2 mediated by transforming growth factor-beta 2. Whether interleukin-1 beta induced group-II phospholipase A2 and/or interleukin-1 beta-enhanced cytosolic phospholipase A2 activity is involved in prostaglandin E2 formation in rat mesangial cells is discussed.     

Prostaglandin E2, prostaglandin F2 alpha and endothelin-1 production by cow oviductal epithelial cell monolayers: effect of progesterone, estradiol 17 beta, oxytocin and luteinizing hormone

The optimal oviductal environment, including contractile activity for gamete transport, fertilization and early embryonic development, is mediated by physiological and anatomical changes in the oviduct during the estrous cycle. Oviductal epithelial cell culture was utilized to investigate the effect of ovarian steroids (progesterone [P4] and estradiol 17 beta [E2]), oxytocin (OT) and luteinizing hormone (LH) on the local production of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha) and endothelin-1 (ET-1) in the cow oviduct. Epithelial cells isolated from oviducts collected during the follicular phase were cultured in M199 under standard culture conditions until monolayer formation. Then the cells were trypsinized and plated at a density of 3 x 10(4)/mL/well and cultured again until subconfluency, at which time the cells were incubated for 4 or 24 h with M199 only (control), high P4 (H-P4; 1 microgram/mL), low P4 (L-P4; 10 ng/mL), E2 (1 ng/mL), LH (10 ng/mL), OT (10(-9) M) ET-1 (10(-9) M), PGE2 (10(-8) M) PGF2 alpha (10(-9) M) or their combination (H-P4 + E2, L-P4 + E2, LH + E2, ET-1 + E2, L-P4 + E2 + LH and H-P4 + E2 + LH). The production of both PG and ET-1 was increased by E2 + low P4 and LH + E2 + low P4 (P < 0.05), while LH + E2 enhanced the production of PGF2 alpha and ET-1 (P < 0.05). Moreover, E2 + ET-1 stimulated PG production (P < 0.05). However, OT had no effect on the production of any of these substances. These results suggest that the preovulatory LH surge, together with locally re-circulated high levels of E2 from the Graafian follicle and basal P4 from regressing corpus luteum (CL), induces the maximum stimulatory effect on oviductal PGE2, PGF2 alpha and ET-1 production during the periovulatory period. Consequently, the elevated local ET-1 concentration during periovulatory period may induce the high contractile activity of the oviduct and, at the same time, the stimulation of PG production. Thus, ET-1 may act as a local amplifier for oviductal PG production stimulated by LH and ovarian steroids.     

Cholinergic stimulation of isolated rat parietal cells: role of calcium, calmodulin and protein kinase C

We studied the cholinergic stimulation of isolated and enriched rat parietal cells. H+ production was indirectly measured by the uptake of 14C-aminopyrine into the parietal cells. Stimulation by carbachol required the presence of extracellular Ca2+ not only in the initial phase but also during the sustained phase of a 100-min incubation period. The response to carbachol was prevented by the Ca2+ entry blocker lanthanum IC50: 1.5 X 10(-7) mol/l). Furthermore, the dependence on Ca2+ influx of cholinergic stimulation was demonstrated by a 269% increase in total intracellular Ca2+ in response to carbachol, as determined by optical emission spectrometry. The naphthalene sulfonamides W7 and W5 which bind calmodulin and thus block the intracellular transduction of Ca2+ effects also inhibited a carbachol-induced H+ production. In the following experiments we studied the effect of agents which activate the protein kinase C, an enzyme which is supposed to play a key role in intracellular signal transduction of Ca2+-dependent effects. Phospholipase C is supposed to activate protein kinase C via induction of the phosphoinositol breakdown. In our preparation of isolated rat parietal cells, phospholipase C (4-100 mU/ml) exerted inhibition instead of amplification of the response to 10(-4) mol/l carbachol. Similarly, the direct activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate or by 1-oleoyl-2-acetyl-sn-glycerol (both tested at 10(-7) to 10(-5) mol/l) reduced the submaximal and maximal response to 10(-5) or 10(-4) mol/l carbachol. We conclude that the cholinergic stimulation of rat parietal cells is dependent on the influx of extracellular Ca2+. Calmodulin seems to mediate intracellular Ca2+ effects during cholinergic stimulation. The activation of protein kinase C impairs carbachol-induced H+ production instead of augmenting the response. This might be due to an already maximal activation of protein kinase C by carbachol alone or to autoregulatory down-regulation by the protein kinase C of muscarinic parietal-cell receptors.     

Role of NOX2 in the regulation of afferent arteriole responsiveness

NADPH oxidases (NOX) are the major source of reactive oxygen species (ROS) in the vasculature and contribute to the control of renal perfusion. The role of NOX2 in the regulation of blood pressure and afferent arteriole responsiveness was investigated in NOX2(-/-) and wild-type mice. Arteriole constrictions to ANG II (10(-14)-10(-6) mol/l) were weaker in NOX2(-/-) compared with wild types. N(omega)-nitro-l-arginine methyl ester (l-NAME; 10(-4) mol/l) treatment reduced basal diameters significantly more in NOX2(-/-) (-18%) than in wild types (-6%) and augmented ANG II responses. Adenosine (10(-11)-10(-4) mol/l) constricted arterioles of wild types but not of NOX2(-/-). However, simultaneous inhibition of adenosine type-2 receptors induced vasoconstriction, which was stronger in NOX2(-/-). Adenosine (10(-8) mol/l) enhanced the ANG II response in wild type, but not in NOX2(-/-). This sensitizing effect by adenosine was abolished by apocynin. Chronic ANG II pretreatment (14 days) did not change the ANG II responses in NOX2(-/-), but strengthened the response in wild types. ANG II pretreatment augmented the l-NAME response in NOX2(-/-) (-33%), but not in wild types. Simultaneous application of l-NAME and ANG II caused a stronger constriction in the NOX2(-/-) (-64%) than in wild types (-46%). Basal blood pressures were similar in both genotypes, however, chronic ANG II infusion elevated blood pressure to a greater extent in wild-type (15 +/- 1%) than in NOX2(-/-) (8 +/- 1%) mice. In conclusion, NOX2 plays an important role in the control of afferent arteriole tone and is involved in the contractile responses to ANG II and/or adenosine. NOX2 can be activated by elevated ANG II and may play an important role in ANG II-induced hypertension. NOX2-derived ROS scavenges nitric oxide, causing subsequent nitric oxide-deficiency.     

Bradykinin stimulates the production of prostaglandin E2 and interleukin-6 in human osteoblast-like cells.

The effect of bradykinin (BK) on proteinase activity, prostaglandin synthesis, and the production of interleukin-6 (IL-6) was investigated in cultures of human osteoblast-like cells. Bradykinin had no effect on stromelysin activity and plasminogen activator activity produced by human osteoblast-like cells. However, BK stimulated the production of prostaglandin E2, an effect that was markedly enhanced by pre-incubation with recombinant interleukin-1 alpha (rhIL-1 alpha), but was apparently unaffected by BK receptor antagonists types 1 and 2. Bradykinin stimulated the intracellular accumulation of total inositol phosphates suggesting that its effects were mediated by stimulation of phosphoinositide metabolism. Bradykinin within the dose range of 10(-11)-10(-5) M also significantly stimulated the production of IL-6. Bradykinin may, therefore, mediate a variety of responses in bone under both physiological and pathological conditions.     

Tetrodotoxin-resistant relaxation to transmural nerve stimulation in canine saphenous veins: a possible neural origin

Transmural nerve stimulation following sympathetic (guanethidine 10(-4) mol/L, phenoxybenzamine 2 X 10(-5) mol/L, propanolol 2 X 10(-6) mol/L) and muscarinic blockade (atropine 5 X 10(-5) mol/L) produces a relaxatory response in canine saphenous veins contracted with prostaglandin F2 alpha. This relaxatory response was shown previously to be resistant to tetrodotoxin. Transmural nerve stimulation (10 V, 1.0 ms) was applied as intermittent trains of stimuli of 30 s duration at frequencies of 1-32 Hz. The veins showed a frequency dependent relaxation (maximum 2.65 +/- 0.20 g). The stimulations were repeated in the presence of lignocaine (10(-3) mol/L), apamin (10(-8) mol/L), ascorbic acid (10(-4) mol/L), or catalase (50 micrograms/mL). The relaxatory response was unaffected by apamin, scorpion toxin, superoxide dismutase, ascorbic acid, and catalase (p greater than 0.05). However, lignocaine (10(-3) mol/L) reduced significantly the relaxatory response to transmural nerve stimulation in this preparation (p less than 0.05). In a separate group of veins, lignocaine (10(-3) mol/L)l abolished the contractile response to transmural nerve stimulation with little effect upon the contractile response to exogenous noradrenaline and the relaxatory responses to isoprenaline and sodium nitrite. These findings support the proposition that the nonadrenergic, noncholinergic tetrodotoxin-resistant relaxatory response observed with transmural nerve stimulation in the canine saphenous vein is mediated by a neural mechanism.     

Specific inhibition by prostaglandin D2 and its metabolites of lysozyme synthesis in mouse macrophage-like cell line, Mm-1

The cultured mouse macrophage-like cell line Mm-1 synthesizes and secretes lysozyme continuously like normal macrophages. Culture of the cells in the presence of prostaglandin D2 for 3 days strongly inhibited their production of lysozyme activity. Prostaglandin D2 caused dose-dependent inhibition of the activity: 1 X 10(-6) M prostaglandin D2 caused about 50% inhibition. Inhibition by prostaglandin D2 was not related to cytotoxicity and was reversible. The rate of synthesis of lysozyme protein was measured by culturing Mm-1 cells with radioactive amino acids and then immunoprecipitating the protein. At the concentrations used, prostaglandin D2 inhibited the synthesis of lysozyme dose-dependently, but did not suppress the synthesis of total protein. Of the various types of prostaglandin, only prostaglandin D2 inhibited the production of lysozyme in Mm-1 cells. Moreover, prostaglandin D2 did not inhibit the production of other lysosomal enzymes, such as acid proteinase, acid phosphatase and beta-glucuronidase, and did not affect Fc receptors on the cell surface, adherence of cells to the culture dish or the cell morphology. These results indicate that prostaglandin D2 specifically inhibits the synthesis of lysozyme in Mm-1 cells. When Mm-1 cells were cultured for 3 days in the presence of the ethyl acetate extract from the culture medium in which Mm-1 cells had been cultured with prostaglandin D2 for 3 days, the production of lysozyme activity of Mm-1 cells was also markedly inhibited by the extract. After the incubation of prostaglandin D2 for 3 days with Mm-1 cells, less than 10% of the initial prostaglandin D2 remained and two major metabolites appeared. These results suggest that the metabolites of prostaglandin D2 were involved in the inhibitory action of prostaglandin D2 in Mm-1 cells.     

Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)gamma agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1beta-stimulated rat chondrocytes: evidence for PPARgamma-independent inhibition by 15-deoxy-Delta12,14prostaglandin J2

Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF1alpha and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1beta, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)gamma agonists. Real-time PCR analysis showed that IL-1beta induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1alpha and PGE2 peaked 24 hours after stimulation with IL-1beta; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Delta12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 microM), with more potency on PGE2 level than on 6-keto-PGF1alpha level (-90% versus -66% at 10 microM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 microM. Inhibitory effects of 10 microM 15d-PGJ2 were neither reduced by PPARgamma blockade with GW-9662 nor enhanced by PPARgamma overexpression, supporting a PPARgamma-independent mechanism. EMSA and TransAM analyses demonstrated that mutated IkappaBalpha almost completely suppressed the stimulating effect of IL-1beta on mPGES-1 expression and PGE2 production, whereas 15d-PGJ2 inhibited NF-kappaB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-kappaB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARgamma through inhibition of the NF-kappaB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2.     

Rapid,fluorimetric-liquid chromatographic determination of malondialdehyde in biological samples

Capillary zone electrophoresis was employed for the determination of lactate using end-column amperometric detection at a carbon fiber bundle microdisk electrode. The optimum conditions of separation and detection are 3.6 x 10(-3) mol/l Na(2)HPO(4)-1.4 x 10(-3) mol/l NaH(2)PO (pH 7.2) for the buffer solution, 18 kV for the separation voltage and 1.60 V versus the saturated calomel electrode for the detection potential. The limit of detection is 7.6 x 10(-7) mol/l or 1.7 fmol (S/N=3) and the linear range is 1.7 x 10(-6)-8.2 x 10(-4) mol/l for the injection voltage of 6 kV and injection time of 5 s. The RSD is 1.8% for the migration time and 3.3% for the electrophoretic peak current. The method was applied to the determination of lactate in human saliva. The recovery of the method is between 95 and 109%.     

The effect of CdCl2 on the respiration of rat small intestine mucosa

Process of oxygen consumption of rat small intestine have already been studied at the level of whole intestinal wall or mucosa only by means of manometric or polarographic methods. The influence of cadmium on mucosal respiration of three sections of rat small intestine was determined because of its toxicological importance. Oxygen consumption was measured polarographically with Clark electrode at 38 degrees C in Krebs Ringer phosphate medium with 0.011 mol/l glucose. Control as well as cadmium affected respiration values were measured one after the other on the same mucosa. High cadmium concentration (applied as CdCl2) that is 2.2 and 1.4 . 10(-2) mol/l significantly inhibited the respiration in duodenum, jejunum and ileum. Observed inhibition of 80 and 50% on the average approximately for these two concentrations was related (similarly as following effects) to 100% value of control respiration. Concentrations 7.8 and 4.5. 10(-3) mol/l caused mostly lower inhibition of oxygen consumption (with the exception of significant 53% inhibition of jejunal respiration for 7.8. 10(-3) mol/l CdCl2). Low concentrations 10(-4) - 10(-6) mol/l CdCl2 had either no effect or stimulated oxygen uptake in intestinal mucosa. In older rats (weight 300-350 g) the respiration of duodenal and jejunal mucosa was inhibited with cadmium chloride concentration of 10(-6) mol/l.     

Cadmium stimulates prostaglandin E2 synthesis in osteoblast-like cells, MC3T3-E1

The effect of Cd on prostaglandin E2 production in osteoblasts was studied using cloned osteoblast-like cells, MC3T3-E1, which were established from new-born mouse calvaria. Treatment of the cells with Cd caused a dose- (0-10 microM) and time- (0-24 h) dependent increase in the release of prostaglandin E2 from the cells into the culture medium. A lag time of 4 h was required for the onset of the phenomenon. The release of [14C]arachidonic acid from prelabeled cell membrane was little influenced by the Cd treatment, while conversion of [14C]arachidonic acid to prostaglandin E2 by the homogenate of the cells treated with Cd was enhanced as compared to that by untreated cells. The stimulatory effect of Cd on prostaglandin E2 production was abolished in the presence of cycloheximide (100 ng/ml). By Western blot analysis with polyclonal rabbit anti-cyclooxygenase antibody, it was revealed that Cd treatment augmented the amount of immunoreactive cyclooxygenase in the cells. These results strongly suggest that Cd stimulates prostaglandin E2 production through the induction of cyclooxygenase in MC3T3-E1 cells. This effect of Cd may be involved in the mechanisms of Cd-induced bone injury.     

Effects of protein kinase C activation on prostaglandin production and cyclooxygenase mRNA levels in ovine astroglia

We examined effects of protein kinase C (PKC) activation by phorbol dibutyrate (PDB) on prostaglandin production in astroglia. Astroglia were cultured from sheep fetal cortex and grown in Eagle's basal media supplemented with 10% fetal calf serum (BME-C). Prostaglandin F_2a (PGF_2α) levels in media were determined at 2–24 hours after exposure to PDB. PDB increased production of PGF_2α at 10⁻⁸M and 10⁻⁶M. In addition, PDB increased the ratio of membrane to cytosolic PKC. Coapplication of H7 [1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine] (10⁻⁴M) with PDB (10⁻⁶M) inhibited PDB-induced PGF_2a production. To investigate the role of protein synthesis in increased prostaglandin production by PDB, astroglia were coincubated with actinomycin D (1 mg/ml) or cycloheximide (10 mg/ml). At 4 hrs, both actinomycin D and cycloheximide inhibited increases in PGF_2a in response to PDB application. In addition, COX-2 mRNA levels and COX activity levels were examined. PDB increased COX-2 mRNA levels by 2 hours, and COX activity tripled after 12 hr exposure to PDB. In addition, the increase in COX activity was blocked by cycloheximide. In summary, PKC activation promotes enhanced prostaglandin production via an increase in COX synthesis.     

Acetyl glyceryl ether phosphorylcholine (platelet-activating factor) mediates heightened metabolic activity in macrophages: Studies on PGE, TXB2 and O2 production, spreading, and the influence of calmodulin-inhibitor W-7

The phospholipid mediator AGEPC (acetyl glyceryl ether phosphorylcholine) was examined for its effects on guinea pig peritoneal macrophages. At a concentration of 10⁻⁹ -10⁻⁶ M, AGEPC evoked release of prostaglandin E (PGE) and thromboxane B₂ (TXB₂) from albumin-elicited macrophages. It also triggerd generation of O⁻₂ by Corynebacterium parvum-induced cells. Moreover, it caused augmented spreading of macrophages. The calmodulin antagonis W-7 attenuated AGEPC-mediated O⁻₂ production and cell spreading whereas prostanoid synthesis was enhanced. These novel actions of AGEPC on the major cellular component of the inflammatory process attest to its role as a potent mediator of immunoinflammatory responses.